Patent Publication Number: US-2022220514-A1

Title: Compositions and methods for robust dynamic metabolic control

Description:
CROSS-REFERENCE 
     This application is a continuation of U.S. Application Ser. No. 16/487,542, filed Aug. 21, 2019, which is a National Stage Entry of PCT/US 18/19040, filed Feb. 21, 2018 which claims the benefit of U.S. Provisional Application No. 62/461,436, filed Feb. 21, 2017, which application is incorporated herein by reference in its entirety. 
    
    
     STATEMENT AS TO FEDERALLY SPONSORED RESEARCH 
     This invention was made with Government support under Federal Grant Nos. HRI 1-14-C-0075 awarded by DOD/DARPA, 12043956 and N00014-16-1-2558 awarded by NAVY/ONR, and 1445726 awarded by NSF. The Government has certain rights to this invention. 
    
    
     REFERENCE TO A SEQUENCE LISTING 
     The instant application contains a Sequence Listing which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 21, 2018, is named 52240_702_601_SL.txt and is 81,697 bytes in size. 
     BACKGROUND OF THE INVENTION 
     Biotechnology-based fermentation processes have been successfully developed to produce everything from biologics and small molecule therapies to specialty, bulk and commodity chemicals, and even next generation biofuels. These processes have made rapid advancements in recent years due to technology developments in the fields of fermentation science and synthetic biology, as well as metabolic and enzyme engineering. Despite these substantial advances, most successful examples of rational and directed engineering approaches have also greatly relied on numerous and often lengthy cycles of trial and error. The present disclosure provides a strategy that simultaneously reduces the complexity of the problem (as well as the size of the relevant design space), while also minimizing metabolic responses to environmental conditions, increasing robustness and scalability of engineered strains. 
     SUMMARY OF THE INVENTION 
     The present disclosure provides, in part, a high-throughput engineering platform that enables the rapid development of microbial production strains. 
     In one aspect, the present disclosure provides a cell for generating a product, wherein the cell comprises: a heterologous polynucleotide for controlled reduction of expression of an enzyme of a metabolic pathway, wherein the controlled reduction of expression of the enzyme induces a stationary phase of the cell; and a heterologous production polynucleotide for mediating controlled increase in expression of a production enzyme for generation of the product; wherein a rate of production of the product during the stationary phase is reduced less in response to a change of an environmental condition as compared to a cell lacking the enzyme. 
     In some embodiments, the heterologous polynucleotide reduces flux through the metabolic pathway. In some embodiments, the enzyme is selected from the group consisting of enoyl-ACP/CoA reductase, glucose-6-phosphate dehydrogenase, lipoamide dehydrogenase, citrate synthase, soluble transhydrogenase, and NADH-dependent glyceraldehyde-3-phosphate dehydrogenase. In some embodiments, the production enzyme is selected from the group consisting of NADPH-dependent alanine dehydrogenase, an alanine exporter, and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase. In some embodiments, the change of an environmental condition comprises increasing or decreasing a concentration of a sugar in a culture medium contacting the cell. In some embodiments, the sugar is glucose. In some embodiments, the change of an environmental condition comprises increasing or decreasing oxygenation of a culture medium contacting the cell. In some embodiments, the product comprises 3-hydroxypropionic acid. 
     In some embodiments, the product comprises an amino acid. In some aspects, the amino acid comprises alanine. In some aspects, the cell is grown in a culture, and a rate of production of the alanine by the culture is at least 0.5 g/L/hour. In some aspects, the rate of production of the alanine is at least 1.0 g/L/hour. In some aspects, the rate of production of the alanine is at least 1.5 g/L/hour. In some aspects, the rate of production of the alanine is at least 1.6 g/L/hour. In some aspects, the culture produces at least 80 g/L of the alanine. In some aspects, the culture produces at least 100 g/L of the alanine. In some aspects, the culture produces at least 120 g/L of the alanine. In some aspects, the culture produces at least 140 g/L of the alanine. In some aspects, the production polynucleotide encodes an alanine exporter. In some aspects, the alanine exporter is alaE. 
     In some embodiments, the product comprises mevalonic acid. In some embodiments, the cell is grown in a culture, and a rate of production of the mevalonic acid by the culture is at least 0.5 g/L/hour. In some embodiments, the rate of production of the mevalonic acid is at least 1.0 g/L/hour. In some embodiments, the rate of production of the mevalonic acid is at least 1.2 g/L/hour. In some embodiments, the rate of production of the mevalonic acid is at least 1.25 g/L/hour. In some aspects, the cell is grown in a culture, and the culture produces at least 50 g/L of the mevalonic acid. In some embodiments, the culture produces at least 70 g/L of the mevalonic acid. In some embodiments, the culture produces at least 90 g/L of the mevalonic acid. In some embodiments, the culture produces at least 95 g/L of the mevalonic acid. In some embodiments, the heterologous polynucleotide is selected from the group consisting of: a silencing polynucleotide for repressing transcription of a gene encoding the enzyme; and a degradation polynucleotide for mediating cellular degradation of the enzyme. 
     In some aspects, the heterologous polynucleotide comprises a silencing polynucleotide, and the silencing polynucleotide comprises a guide RNA (gRNA) comprising a gRNA sequence that recognizes a promoter of a gene encoding the enzyme. In some aspects, the heterologous polynucleotide encodes a CRISPR enzyme, and the CRISPR enzyme specifically binds to the promoter sequence when bound to the gRNA. In some aspects, the CRISPR enzyme is catalytically inactive. In some aspects, the heterologous polynucleotide comprises a degradation polynucleotide, wherein the degradation polynucleotide comprises a sequence encoding a degradation tag, wherein the degradation tag mediates degradation of the enzyme. In some embodiments, expression of the heterologous polynucleotide is regulated by phosphate availability in the cell. In some embodiments, expression of the production polynucleotide is regulated by phosphate availability in the cell. In some embodiments, the cell is an  E. coli  cell. 
     In another aspect, disclosed herein is a method comprising: culturing independently a plurality of strains of a cell, wherein each strain comprises (i) a heterologous polynucleotide for mediating controlled reduction of expression of an enzyme of a metabolic pathway, wherein the controlled reduction of expression of the enzyme induces a stationary phase of the cell; and (ii) a heterologous production polynucleotide for mediating controlled increase in expression of a production enzyme for generation of the product; wherein each strain of the plurality of strains differs from another strain in a sequence of at least one of the heterologous polynucleotide or the heterologous production polynucleotide; growing the plurality of strains to stationary phase; and selecting a strain of the plurality of strains based on a level of the product produced by the selected strain during the stationary phase. 
     In some embodiments, the method comprises determining the level of the product. In some embodiments, the method comprises growing the selected strain. In some embodiments, the selected strain is grown in a bioreactor. In some embodiments, a culture medium comprising the selected strain has a volume of at least 500 ml. In some embodiments, the culture medium has a volume of at least 1 L. In some embodiments, the heterologous polynucleotide is selected from the group consisting of: a silencing polynucleotide for repressing transcription of a gene encoding the enzyme; and a degradation polynucleotide for mediating cellular degradation of the enzyme. In some embodiments, a first and second strain of the plurality of strains comprises a silencing polynucleotide. In some embodiments, the silencing polynucleotide comprises a guide RNA (gRNA) comprising a gRNA sequence that recognizes a promoter sequence of a gene encoding the enzyme. In some embodiments, the gRNA sequence differs between the first and second strains. In some embodiments, the first and second strain of the plurality of strains comprise a degradation polynucleotide. In some embodiments, the degradation polynucleotide differs between the first and second strains. In some embodiments, the enzyme is selected from the group consisting of enoyl-ACP/CoA reductase, glucose-6-phosphate dehydrogenase, lipoamide dehydrogenase, citrate synthase, soluble transhydrogenase, and NADH-dependent glyceraldehyde-3-phosphate dehydrogenase. In some embodiments, the production enzyme is selected from the group consisting of NADPH-dependent alanine dehydrogenase, an alanine exporter, and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase. In some embodiments, the product is selected from the group consisting of mevalonic acid, 3-hydroxypropionic acid, and an amino acid. 
     In some embodiments, the product is an amino acid and the amino acid is alanine. In some embodiments, the cell of the selected strain a rate of production of the product during the stationary phase is reduced less in response to a change of an environmental condition as compared to a cell lacking the heterologous polynucleotide. In some embodiments, the change of an environmental condition comprises a change in concentration of a sugar of a culture medium contacting the cell. In some embodiments, the change of an environmental condition comprises a change in oxygenation of a culture medium contacting the cell. 
     In another aspect, disclosed herein is a method of generating a cellular product comprising: culturing a heterologous cell in a culture medium, wherein the heterologous cell comprises: (i) a heterologous polynucleotide for mediating controlled reduction of expression of an enzyme of a metabolic pathway, wherein the controlled reduction of expression of the enzyme induces a stationary phase of the cell; and (ii) a heterologous production polynucleotide for mediating controlled increase in expression of a production enzyme for generation of the product; wherein a rate of production of the product during the stationary phase is reduced less in response to a change of an environmental condition as compared to a cell lacking the enzyme. 
     In one embodiment, the method further comprises changing the environmental condition. In one embodiment, the environmental condition comprises a concentration of a sugar of the culture medium, and changing the environmental condition comprises increasing or decreasing the concentration. In some embodiments, the sugar is glucose. In some embodiments, the environmental condition comprises an oxygen concentration of the culture medium, and changing the environmental condition comprises increasing or decreasing the oxygen concentration. In some embodiments, the culturing is performed in a bioreactor. In some embodiments, the culture medium has a volume of at least 500 ml. In some embodiments, the culture medium has a volume of at least 1 L. In some embodiments, the product comprises 3-hydroxypropionic acid. In some embodiments, the product comprises an amino acid. In some embodiments, the amino acid comprises alanine. In some embodiments, the rate of production of the alanine is at least 0.5 g/L/hour. In some embodiments, the rate of production of the alanine is at least 1.0 g/L/hour. In some embodiments, the rate of production of the alanine is at least 1.5 g/L/hour. In some embodiments, the rate of production of the alanine is at least 1.6 g/L/hour. In some embodiments, the production polynucleotide encodes an alanine exporter. In some embodiments, the alanine exporter is alaE. 
     In some embodiments, the product comprises mevalonic acid. In some embodiments, the rate of production of the mevalonic acid is at least 0.5 g/L/hour. In some embodiments, the rate of production of the mevalonic acid is at least 1.0 g/L/hour. In some embodiments, the rate of production of the mevalonic acid is at least 1.2 g/L/hour. In some embodiments, the rate of production of the mevalonic acid is at least 1.25 g/L/hour. In some embodiments, the heterologous polynucleotide is selected from the group consisting of: a silencing polynucleotide for repressing transcription of a gene encoding the enzyme; and a degradation polynucleotide for mediating cellular degradation of the enzyme. In some embodiments, the heterologous polynucleotide comprises a silencing polynucleotide, and the silencing polynucleotide comprises a guide RNA (gRNA) comprising a gRNA sequence that recognizes a promoter sequence of a gene encoding the enzyme. In some embodiments, the heterologous polynucleotide encodes a CRISPR enzyme, wherein the CRISPR enzyme specifically binds to the promoter sequence when bound to the gRNA. In some embodiments, the CRISPR enzyme is catalytically inactive. In some embodiments, the heterologous polynucleotide comprises a degradation polynucleotide, wherein the degradation polynucleotide comprises a sequence encoding a degradation tag, wherein the degradation tag mediates degradation of the enzyme. In some embodiments, the expression of the heterologous polynucleotide is regulated by phosphate availability in the cell. In some embodiments, the expression of the production polynucleotide is regulated by phosphate availability in the cell. In some embodiments, the cell is an  E. coli  cell. 
     In another aspect, disclosed herein is a cell for production of alanine, wherein the cell comprises: (i) a heterologous polynucleotide for controlled reduction of expression of an enzyme of a metabolic pathway, wherein the enzyme is selected from the group consisting of enoyl-ACP/CoA reductase, glucose-6-phosphate dehydrogenase, lipoamide dehydrogenase (lpd), citrate synthase (gltA), soluble transhydrogenase, and NADH-dependent glyceraldehyde-3-phosphate dehydrogenase; and (ii) an alanine exporter, wherein the alanine exporter is expressed at increased levels as compared to a wildtype cell. 
     In some embodiments, the alanine exporter is encoded by an alaE gene. In some embodiments, the controlled reduction of expression of the enzyme induces a stationary phase of the cell. In some embodiments, the cell further comprises a heterologous production polynucleotide for controlled increase in expression of a production enzyme for generation of the alanine. In some embodiments, the production enzyme is selected from the group consisting of NADPH-dependent alanine dehydrogenase and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase. In some embodiments, the heterologous polynucleotide is selected from the group consisting of: a silencing polynucleotide for mediating transcriptional repression of a gene encoding the enzyme; and a degradation polynucleotide for mediating cellular degradation of the enzyme. In some embodiments, the heterologous polynucleotide comprises a silencing polynucleotide, and the silencing polynucleotide comprises a guide RNA (gRNA) comprising a gRNA sequence that recognizes a promoter sequence of a gene encoding the enzyme. In some embodiments, the polynucleotide further encodes a CRISPR enzyme, wherein the CRISPR enzyme specifically binds to the promoter sequence when bound to the gRNA. In some embodiments, the CRISPR enzyme is catalytically inactive. In some embodiments, the heterologous polynucleotide comprises a degradation polynucleotide, wherein the degradation polynucleotide comprises a sequence encoding a degradation tag, wherein the degradation tag mediates degradation of the enzyme. In some embodiments, the polynucleotide is regulated by phosphate availability in the cell. In some embodiments, the production polynucleotide is regulated by phosphate availability in the cell. In some embodiments, the cell is an  E. coli  cell. 
     In some embodiments, a culture comprises the cell. In some embodiments, a rate of production of the alanine by the culture is at least 0.5 g/L/hour. In some embodiments, a rate of production of the alanine by the culture is at least 1.0 g/L/hour. In some embodiments, a rate of production of the alanine by the culture is at least 1.5 g/L/hour. In some embodiments, a rate of production of the alanine by the culture is at least 1.6 g/L/hour. In some embodiments, the culture produces at least 100 g/L of the alanine. In some embodiments, the culture produces at least 120 g/L of the alanine. In some embodiments, the culture produces at least 140 g/L of the alanine. 
     In some aspects, disclosed herein is a method of production of alanine comprising growing in a culture medium a cell comprising (i) a heterologous polynucleotide for controlled reduction of expression of a enzyme of a metabolic pathway, wherein the enzyme is selected from the group consisting of enoyl-ACP/CoA reductase, glucose-6-phosphate dehydrogenase, lipoamide dehydrogenase, citrate synthase, soluble transhydrogenase, and NADH-dependent glyceraldehyde-3-phosphate dehydrogenase; and (ii) an alanine exporter, wherein the alanine exporter is expressed at increased levels as compared to a wildtype cell. 
     In some embodiments, the controlled reduction of expression of the enzyme induces a stationary phase of the cell. In some embodiments, the method further comprises decreasing an oxygenation level or a sugar concentration of the culture medium during the stationary phase, wherein a rate of production of the cellular product is reduced less in response to the decreasing as compared to a cell lacking the heterologous polynucleotide. In some embodiments, the sugar is glucose. In some embodiments, the alanine exporter is encoded by an alaE gene. In some embodiments, the cell further comprises a heterologous production polynucleotide for controlled increase in expression of a production enzyme for generation of the alanine. In some embodiments, the production enzyme is selected from the group consisting of: NADPH-dependent alanine dehydrogenase and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase. In some embodiments, the heterologous polynucleotide is selected from the group consisting of: a silencing polynucleotide for mediating transcriptional repression of a gene encoding the enzyme; and a degradation polynucleotide for mediating cellular degradation of the enzyme. In some embodiments, the heterologous polynucleotide comprises a silencing polynucleotide, and the silencing polynucleotide comprises a guide RNA (gRNA) comprising a gRNA sequence that recognizes a promoter sequence of a gene encoding the enzyme. In some embodiments, the heterologous polynucleotide encodes a CRISPR enzyme, wherein the CRISPR enzyme specifically binds to the promoter sequence when bound to the gRNA. In some embodiments, the CRISPR enzyme is catalytically inactive. In some embodiments, the heterologous polynucleotide comprises a degradation polynucleotide, wherein the degradation polynucleotide comprises a sequence encoding a degradation tag, wherein the degradation tag mediates degradation of the enzyme. 
     In some embodiments, the expression of the heterologous polynucleotide is regulated by phosphate availability in the cell. In some embodiments, the production polynucleotide is regulated by phosphate availability in the cell. In some embodiments, the cell is an  E. coli  cell. In some embodiments, a rate of production of the alanine is at least 0.5 g/L/hour. In some embodiments, a rate of production of the alanine is at least 1.0 g/L/hour. In some embodiments, a rate of production of the alanine is at least 1.5 g/L/hour. In some embodiments, a rate of production of the alanine is at least 1.6 g/L/hour. 
     INCORPORATION BY REFERENCE 
     All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: 
         FIG. 1A  depicts an overview of dynamic metabolic control in 2-stage fermentations. 
         FIG. 1B  depicts strain and bioprocess optimization. 
         FIGS. 2A-D  depict an example of implementation of 2-stage Synthetic Metabolic Valves (SMVs) in  E. coli.    
         FIGS. 3A-K  depict an example of alanine production in  E. coli  utilizing 2-stage dynamic control. 
         FIGS. 4A-F  depict example robustness comparison between 2-stage and growth associated approaches. 
         FIGS. 5A-J  depict example comparisons of “Valve” and growth associated alanine production in micro-fermentations and 1 L fermentation. 
         FIG. 6A-H  depict an example of mevalonate production in  E. coli  utilizing 2-stage dynamic control. 
         FIG. 7  depicts an example of phosphate depletion promoter characterization. 
         FIG. 8  depicts an example of insulated phosphate depletion promoter characterization. 
         FIG. 9  depicts an example of insulated constitutive promoter characterization. 
         FIG. 10  depicts an example of metabolic modeling results for optimal 3-HP flux in two stage fermentations. 
         FIG. 11  depicts examples of chromosomal modifications. 
         FIG. 12  depicts an example of average maximal growth rates of starting host strains in 1 L FGM10 minimal medium fermentations, n=2. 
         FIG. 13A-E  depict examples of distribution of glucose utilized during the growth phase of starting host strains in 1 L standard minimal medium fermentations. 
         FIG. 14  depicts pCASCADE-control plasmid construction scheme. 
         FIGS. 15A-B  depict pCASCADE construction scheme. 
         FIGS. 16A-C  depict an overview of micro-fermentation process. 
         FIG. 17  depicts micro-fermentation for L-alanine production using different insulated phosphate promoters in DLF_0025 strain. 
         FIG. 18  depicts Heatmap for L-alanine production by gapN/gapA strains. 
         FIGS. 19A-D  depict alanine production in response to different OTR and glucose concentration in micro-fermentation for 4 strains evaluated for robustness. 
         FIGS. 20A-D  depict alanine production in response to different OTR and glucose concentration in micro-fermentation for 4 strains evaluated for robustness. 
         FIGS. 21A-D  depict alanine production in response to different OTR and glucose concentration in micro-fermentation for 4 strains evaluated for robustness. 
         FIGS. 22A-D  depict alanine production in response to different OTR and glucose concentration in micro-fermentation for 4 strains evaluated for robustness. 
         FIGS. 23A-D  depict alanine production in response to different OTR and glucose concentration in micro-fermentation for 4 strains evaluated for robustness. 
         FIGS. 24A-D  depict alanine production in response to different OTR and glucose concentration in micro-fermentation for 4 strains evaluated for robustness. 
         FIGS. 25A-D  depict alanine production in response to different OTR and glucose concentration in micro-fermentation for 4 strains evaluated for robustness. 
         FIGS. 26A-D  depict alanine production in response to different OTR and glucose concentration in micro-fermentation for 4 strains evaluated for robustness. 
         FIGS. 27A-D  depict alanine production in response to different OTR and glucose concentration in micro-fermentation for 4 strains evaluated for robustness. 
         FIGS. 28A-D  depict alanine production in response to different OTR and glucose concentration in micro-fermentation for 4 strains evaluated for robustness. 
         FIGS. 29A-D  depict alanine production in response to different OTR and glucose concentration in micro-fermentation for 4 strains evaluated for robustness. 
         FIGS. 30A-D  depict alanine production in response to different OTR and glucose concentration in micro-fermentation for 4 strains evaluated for robustness. 
         FIGS. 31A-D  depict alanine production in response to different OTR and glucose concentration in micro-fermentation for 4 strains evaluated for robustness. 
         FIG. 32  depicts alanine production in response to different OTR and glucose concentration in micro-fermentation for one strain evaluated for robustness. 
         FIGS. 33A-B  depict growth profile for all valve and growth associated strains at 1 L scale evaluated in this paper. 
         FIG. 34  depicts specific Productivity (SP) comparison for strain with highest mevalonate titer from literature and mevalonate strain 1 evaluated in this work. 
         FIG. 35  depicts alanine standard curve from MS measurement. Average and standard deviation for mass spec response from triplicate standard measurement were plotted. 
         FIGS. 36A-B  depict glucose and ethanol standard curves from RI measurement. 
         FIG. 37  depicts 3-Hydroxypropionic acid standard curve from TUV measurement. 
         FIGS. 38A-D  depict TUV standard curves for L-alanine, D-alanine, mevalonic acid, and mevalonolactone. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Definitions 
     As used in the specification and the claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to an “expression vector” includes a single expression vector as well as a plurality of expression vectors, either the same (e.g., the same operon) or different; reference to “microorganism” includes a single microorganism as well as a plurality of microorganisms; and the like. 
     As used herein, “reduced enzymatic activity,” “reducing enzymatic activity.” and the like is meant to indicate that a microorganism cell&#39;s, or an isolated enzyme, exhibits a lower level of activity than that measured in a comparable cell of the same species or its native enzyme. That is, enzymatic conversion of the indicated substrate(s) to indicated product(s) under known standard conditions for that enzyme is at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, or at least 90 percent less than the enzymatic activity for the same biochemical conversion by a native (non-modified) enzyme under a standard specified condition. This term also can include elimination of that enzymatic activity. A cell having reduced enzymatic activity of an enzyme can be identified using any method known in the art. For example, enzyme activity assays can be used to identify cells having reduced enzyme activity. See, for example, Enzyme nomenclature, Academic Press, Inc., New York 2007. 
     The term “heterologous DNA,” “heterologous nucleic acid sequence,” and the like as used herein refers to a nucleic acid sequence wherein at least one of the following is true: (a) the sequence of nucleic acids foreign to (i.e., not naturally found in) a given host microorganism; (b) the sequence may be naturally found in a given host microorganism, but in an unnatural (e.g., greater than expected) amount; or (c) the sequence of nucleic acids comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, regarding instance (c), a heterologous nucleic acid sequence that is recombinantly produced will have two or more sequences from unrelated genes arranged to make a new functional nucleic acid, such as a nonnative promoter driving gene expression. 
     The term “synthetic metabolic valve,” and the like as used herein refers to either the use of controlled proteolysis, gene silencing or the combination of both proteolysis and gene silencing to alter metabolic fluxes. 
     The term “heterologous” is intended to include the term “exogenous” as the latter term is generally used in the art. With reference to the host microorganism&#39;s genome prior to the introduction of a heterologous nucleic acid sequence, the nucleic acid sequence that codes for the enzyme is heterologous (whether or not the heterologous nucleic acid sequence is introduced into that genome). 
     As used herein, the term “gene disruption,” or grammatical equivalents thereof (and including “to disrupt enzymatic function,” “disruption of enzymatic function.” and the like), is intended to mean a genetic modification to a microorganism that renders the encoded gene product as having a reduced polypeptide activity compared with polypeptide activity in or from a microorganism cell not so modified. The genetic modification can be, for example, deletion of the entire gene, deletion or other modification of a regulatory sequence required for transcription or translation, deletion of a portion of the gene which results in a truncated gene product (e.g., enzyme) or by any of various mutation strategies that reduces activity (including reducing activities to no detectable activity level) the encoded gene product. A disruption may broadly include a deletion of all or part of the nucleic acid sequence encoding the enzyme, and also includes, but is not limited to other types of genetic modifications, e.g., introduction of stop codons, frame shift mutations, introduction or removal of portions of the gene, and introduction of a degradation signal, those genetic modifications affecting mRNA transcription levels and/or stability, and altering the promoter or repressor upstream of the gene encoding the enzyme. 
     Bio-production or fermentation, as used herein, may be aerobic, microaerobic, or anaerobic. 
     When the genetic modification of a gene product, e.g., an enzyme, is referred to herein, including the claims, it is understood that the genetic modification is of a nucleic acid sequence, such as or including the gene, that normally encodes the stated gene product, e.g., the enzyme. 
     As used herein, the term “metabolic flux” and the like refers to changes in metabolism that lead to changes in product and/or byproduct formation, including production rates, production titers and production yields from a given substrate. 
     Species and other phylogenic identifications are according to the classification known to a person skilled in the art of microbiology. 
     Enzymes are listed here within, with reference to a Universal Protein Resource (Uniprot) identification number, which would be well known to one skilled in the art (Uniprot is maintained by and available through the UniProt Consortium). 
     Where methods and steps described herein indicate certain events occurring in certain order, those of ordinary skill in the art will recognize that the ordering of certain steps may be modified and that such modifications are in accordance with the variations of the invention. Additionally, certain steps may be performed concurrently in a parallel process when possible, as well as performed sequentially. 
     The meaning of abbreviations is as follows: “C” means Celsius or degrees Celsius, as is clear from its usage, DCW means dry cell weight, “s” means second(s), “min” means minute(s), “h”, “hr,” or “hrs” means hour(s), “psi” means pounds per square inch, “nm” means nanometers, “d” means day(s), “μL” or “uL” or “ul” means microliter(s), “mL” means milliliter(s), “L” means liter(s), “mm” means millimeter(s), “nm” means nanometers, “mM” means millimolar, “μM” or “uM” means micromolar, “M” means molar, “mmol” means millimole(s), “μmol” or “uMol” means micromole(s), “g” means gram(s), “μg” or “ug” means microgram(s) and “ng” means nanogram(s), “PCR” means polymerase chain reaction, “OD” means optical density. “OD 600 ” means the optical density measured at a photon wavelength of 600 nm, “kDa” means kilodaltons, “g” means the gravitation constant, “bp” means base pair(s). “kbp” means kilobase pair(s), “% w/v” means weight/volume percent, “% v/v” means volume/volume percent, “IPTG” means isopropyl-μ-D-thiogalactopyranoiside, “aTc” means anhydrotetracycline, “RBS” means ribosome binding site, “rpm” means revolutions per minute, “HPLC” means high performance liquid chromatography, and “GC” means gas chromatography. 
     Overview 
     Provided herein is a high-throughput metabolic engineering platform enabling the rapid optimization of microbial production strains. The platform, which bridges a gap between current in vivo and in vitro bio-production approaches, relies on dynamic minimization of the active metabolic network. Dynamic metabolic network minimization can be accomplished using combinations of CRISPR interference and controlled proteolysis to reduce the activity of multiple enzymes in essential central metabolism. Minimization can be implemented in the context of standardized 2-stage bio-processes. This approach not only can result in a design space with greatly reduced complexity, but also in increased metabolic fluxes and production rates as well as in strains which are robust to environmental conditions. Robustness can lead to predictable scalability from high-throughput small-scale screens, or “micro-fermentations”, to fully instrumented bioreactors. Predictive high-throughput approaches may be critical for metabolic engineering programs to truly take advantage of the rapidly increasing throughput and decreasing costs of synthetic biology. The examples provided herein have not only demonstrated proof of principle for this approach in the common industrial microbe:  E. coli , and has validated this approach with the rapid optimization of  E. coli  strains producing two important industrial chemicals: alanine and mevalonic acid, at commercially meaningful rates, titers (147 g/L and 97 g/L, respectively), and yields. 
     Also provided herein are systems and methods to rapidly optimize a microorganism for chemical productions in a high-throughput fashion. 
     Also provided herein are microorganisms that can be used with the disclosed platform and/or methods for chemical productions. 
     Synthetic Metabolic Valves (SMVs) 
     The current disclosure describes the construction of synthetic metabolic valves (SMVs) comprising one or more or a combination of the following: controlled gene silencing and controlled proteolysis. It is appreciated that one well skilled in the art is aware of several methodologies for gene silencing and controlled proteolysis. 
     The development of platform microbial strains that utilize SMVs can decouple growth from product formation. These strains enable the dynamic control of metabolic pathways, including those that when altered have negative effects on microorganism growth. Dynamic control over metabolism is accomplished via a combination of methodologies including but not limited to transcriptional silencing and controlled enzyme proteolysis. These microbial strains are utilized in a multi-stage bioprocess encompassing as least two stages, the first stage in which microorganisms are grown and metabolism can be optimized for microbial growth and at least one other stage in which growth can be slowed or stopped, and dynamic changes can be made to metabolism to improve production of desired product, such as a chemical or fuel. The transition of growing cultures between stages and the manipulation of metabolic fluxes can be controlled by artificial chemical inducers or preferably by controlling the level of key limiting nutrients. In addition, genetic modifications may be made to provide metabolic pathways for the biosynthesis of one or more chemical or fuel products. Also, genetic modifications may be made to enable the utilization of a variety of carbon feedstocks including but not limited sugars such as glucose, sucrose, xylose, arabinose, mannose, and lactose, oils, carbon dioxide, carbon monoxide, methane, methanol and formaldehyde. 
     This approach allows for simpler models of metabolic fluxes and physiological demands during a production phase, turning a growing cell into a stationary phase biocatalyst. These synthetic metabolic valves can be used to turn off essential genes and redirect carbon, electrons and energy flux to product formation in a multi-stage fermentation process. One or more of the following enables these synthetic valves: 1) transcriptional gene silencing or repression technologies in combination with 2) inducible enzyme degradation and 3) nutrient limitation to induce a stationary or non-dividing cellular state. SMVs are generalizable to any pathway and microbial host. These synthetic metabolic valves allow for novel rapid metabolic engineering strategies useful for the production of renewable chemicals and fuels and any product that can be produced via whole cell catalysis. 
     In various cases, one SMV can refer to the manipulation of one gene (or its protein product). The manipulation can be controlled silencing of the gene and/or controlled degradation of its protein product. In certain cases, combination of SMVs can lead to improved production in yields, rate and/or robustness, which includes manipulation of two genes (or their protein products). In some cases, an engineered microorganism comprises at least one SMV. In some cases, an engineered microorganism comprises more than one SMV. In some cases, an engineered microorganism comprises two, three, four, five, six, seven, eight, nine, or ten, or more SMVs. 
     Method and Systems for Bio-Production 
     Provided herein are methods or systems for robust large scale production of molecules from biologics and small molecule therapeutics to specialty, bulk and commodity chemicals, and biofuels. The methods or systems provided herein comprise using engineered microorganism which comprises a limited set of metabolic enzymes. In some embodiments, the engineered microorganism comprises at least one metabolic enzyme that has reduced level or activity. In some embodiments, the engineered microorganism comprises two, three, four, five, six, seven, eight, nine, or ten, or more metabolic enzymes that have reduced level or activity. The methods and systems provided herein can reduce metabolic responses to environmental conditions and can be easily transferred from small scale (e.g. mgs) production to large scale (e.g. kgs) production. The methods and systems provided herein can reduce the time and costs associated with transitioning from small scale (e.g. mgs) to large scale (e.g. kgs) production. 
     Within the scope of the current disclosure are genetically modified microorganism, wherein the microorganism is capable of producing a product derived from any key metabolic intermediate including but not limited to malonyl-CoA, pyruvate, oxaloacetate, erthyrose-4-phosphate, xylulose-5-phosphate, alpha-ketoglutarate and citrate at a specific rate selected from the rates of greater than 0.05 g/gDCW-hr, 0.08 g/gDCW-hr, greater than 0.1 g/gDCW-hr, greater than 0.13 g/gDCW-hr. greater than 0.15 g/gDCW-hr, greater than 0.175 g/gDCW-hr, greater than 0.2 g/gDCW-hr, greater than 0.25 g/gDCW-hr. greater than 0.3 g/gDCW-hr, greater than 0.35 g/gDCW-hr, greater than 0.4 g/gDCW-hr, greater than 0.45 g/gDCW-hr. or greater than 0.5 g/gDCW-hr. 
     In various embodiments, the invention includes a culture system comprising a carbon source in an aqueous medium and a genetically modified microorganism, wherein said genetically modified organism is present in an amount selected from greater than 0.05 gDCW/L, 0.1 gDCW/L, greater than 1 gDCW/L, greater than 5 gDCW/L, greater than 10 gDCW/L, greater than 15 gDCW/L or greater than 20 gDCW/L, such as when the volume of the aqueous medium is selected from greater than 5 mL, greater than 100 mL, greater than 0.5 L, greater than 1 L, greater than 2 L, greater than 10 L, greater than 250 L, greater than 1000 L, greater than 10,000 L, greater than 50,000 L, greater than 100,000 L or greater than 200,000 L, and such as when the volume of the aqueous medium is greater than 250 L and contained within a steel vessel. 
     Carbon Sources 
     Bio-production media, which is used in the present invention with recombinant microorganisms must contain suitable carbon sources or substrates for both growth and production stages. Suitable substrates may include, but are not limited to glucose, sucrose, xylose, mannose, arabinose, oils, carbon dioxide, carbon monoxide, methane, methanol, formaldehyde and glycerol. It is contemplated that all of the above mentioned carbon substrates and mixtures thereof are suitable in the present invention as a carbon source(s). 
     Microorganism 
     Features as described and claimed herein may be provided in a microorganism selected from the listing herein, or another suitable microorganism, that also comprises one or more natural, introduced, or enhanced product bio-production pathways. Thus, in some embodiments the microorganism(s) comprise an endogenous product production pathway (which may, in some such embodiments, be enhanced), whereas in other embodiments the microorganism does not comprise an endogenous product production pathway. 
     The examples describe specific modifications and evaluations to certain bacterial and fungal microorganisms. The scope of the invention is not meant to be limited to such species, but to be generally applicable to a wide range of suitable microorganisms. 
     Suitable host cells or host microorganisms for bio-production can be either prokaryotic or eukaryotic. Suitable host cells or host microorganisms can be bacteria such as  Citrobacter, Enterobacter, Clostridium, Klebsiella, Aerobacter, Lactobacillus, Aspergillus, Saccharomyces. Schizosaccharomyces, Zygosaccharomyces, Pichia. Kluyveromyces. Candida, Hansenula, Debaryomyces, Mucor, Torulopsis, Methylobacter, Escherichia, Salmonella, Bacillus, Streptomyces , and  Pseudomonas . In some embodiments, a host cell or an engineered cell is  E. coli . In some embodiments, a host cell or an engineered cell is  S. cerevisiae.    
     In certain aspects, provided herein is a microorganism genetically modified to comprise: a production pathway comprising at least one enzyme for the biosynthesis of a product, and a combination of multiple synthetic metabolic valves to controllably reduce or eliminate flux through multiple metabolic pathways. In some embodiments, each of the multiple synthetic metabolic valves comprises one or more genes for (i) controlled silencing of gene expression of at least one gene or (ii) the controlled proteolytic inactivation of at least one protein. In some embodiments, a rate of the biosynthesis of the product is increased in a productive stationary phase upon a depletion of a nutrient, wherein the depletion of the nutrient induces the multiple synthetic metabolic valves. In some cases, the controlled silencing of gene expression is accomplished by RNA interference. CRISPR interference or transcriptional repression. In some cases, the controlled proteolytic inactivation is accomplished by protein cleavage by a specific protease or targeted degradation by specific peptide tags. In some cases, the nutrient is phosphate, nitrogen, sulfur, magnesium, or a combination thereof. 
     In certain aspects, provided herein is a genetically modified microorganism comprising: a production pathway comprising at least one enzyme for the biosynthesis of a product from one of the following metabolites: pyruvate, acetolactate, acetyl-CoA, acetoacetyl-CoA or malonyl-CoA; and a combination of multiple synthetic metabolic valves, wherein each of the multiple synthetic metabolic valves comprises one of a fabI, gltA, lpd, zuf or udhA gene for (i) controlled silencing of gene expression of a corresponding one of said fabI, gltA, lpd, zwf or udhA genes or (ii) controlled proteolytic inactivation of a protein encoded by a corresponding one of said fabI, gltA, lpd, zwf or udhA genes. In some embodiments, a rate of the biosynthesis of the product is increased in a productive stationary phase upon a depletion of a nutrient, wherein the depletion of the nutrient induces the multiple synthetic metabolic valves. In some embodiments, the product is alanine or a derivative thereof. In some embodiments, the product is mevalonate or a derivative thereof. In some embodiments, the product is malonic acid or a derivative thereof. In some embodiments, the nutrient is phosphate, nitrogen, sulfur, magnesium, or a combination thereof. 
     In certain aspects, provided herein is a genetically modified microorganism comprising: a production pathway to produce alanine from pyruvate; and a combination of multiple synthetic metabolic valves, wherein each of the multiple synthetic metabolic valves comprises one of a fabI, gltA, lpd, zwf or udhA gene for (i) controlled silencing of gene expression of a corresponding one of said fabI, gltA, lpd, zwf or udhA genes or (ii) controlled proteolytic inactivation of a protein encoded by one of said fabI, gltA, lpd, zuf or udhA genes. In some embodiments, a rate of the biosynthesis of alanine is increased in a productive stationary phase upon a depletion of a nutrient, wherein the depletion of the nutrient induces the multiple synthetic metabolic valves. In some embodiments, the nutrient is phosphate, nitrogen, sulfur, magnesium, or a combination thereof. 
     In some cases, a genetically modified microorganism is a heterologous cell. In some cases, provided herein is a heterologous cell for generating a product. In some cases, a heterologous cell comprises an engineered valve polynucleotide for mediating controlled reduction of expression of a valve enzyme acting in a metabolic pathway. In certain cases, a controlled reduction of expression of a valve enzyme reduces flux through a metabolic pathway, wherein the controlled reduction of expression of the valve enzyme induces a stationary phase of the heterologous cell. In some cases, a heterologous cell further comprises an engineered production polynucleotide for mediating controlled increase in expression of a production enzyme for generation of the product. In some situations, a heterologous cell comprises an engineered valve polynucleotide for mediating controlled reduction of expression of a valve enzyme acting in a metabolic pathway, wherein a rate of production of a product during a stationary phase is reduced less in response to a change of an environmental condition as compared to a cell lacking the controlled reduction of expression of the valve enzyme. 
     In some cases, provided herein is a heterologous cell for generating a product, wherein said cell comprises: an engineered valve polynucleotide for mediating controlled reduction of expression of a valve enzyme acting in a metabolic pathway, wherein said controlled reduction of expression of said valve enzyme reduces flux through said metabolic pathway, wherein said controlled reduction of expression of said valve enzyme induces a stationary phase of said cell; and an engineered production polynucleotide for mediating controlled increase in expression of a production enzyme for generation of said product; wherein a rate of production of said product during said stationary phase is reduced less in response to a change of an environmental condition as compared to a cell lacking said controlled reduction of expression of said valve enzyme. 
     In some cases, provided herein is a cell comprising a reduced expression or activity of a valve enzyme, wherein the valve enzyme comprises an enzyme selected from the group consisting of enoyl-ACP/CoA reductase (fabI), glucose-6-phosphate dehydrogenase (zwf), lipoamide dehydrogenase (lpd), citrate synthase (gltA), soluble transhydrogenase (udhA), NADH-dependent glyceraldehyde-3-phosphate dehydrogenase (gapA), and a combination thereof. 
     In some cases, provided herein is a cell comprising a production enzyme, wherein the production enzyme comprises an enzyme selected from the group consisting of NADPH-dependent alanine dehydrogenase (aid), alanine exporter (alaE), NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (gapN), and a combination thereof. 
     Environmental Conditions 
     Environmental conditions can comprise medium and culture conditions. Environmental factors that may influence production can be temperature, pH, acidity, ethanol, sulfite, and availability of nutrients. 
     In addition to an appropriate carbon source, such as selected from one of the herein disclosed types, bio-production media may contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the enzymatic pathway necessary for chemical product bio-production under the present disclosure. Another aspect of the invention regards media and culture conditions that comprise genetically modified microorganisms of the invention and optionally supplements. 
     Typically cells are grown at a temperature in the range of about 25° C. to about 40° C. in an appropriate medium, as well as up to 70° C. for thermophilic microorganisms. Suitable growth media are well characterized and known in the art. 
     Suitable pH ranges for the bio-production are between pH 2.0 to pH 10.0, where pH 6.0 to pH 8.0 is a typical pH range for the initial condition. However, the actual culture conditions for a particular embodiment are not meant to be limited by these pH ranges. 
     Bio-productions may be performed under aerobic, microaerobic or anaerobic conditions with or without agitation. 
     In some cases, a change of an environmental condition comprises a change in sugar concentration of a culture medium contacting a cell. In some cases, a change in sugar concentration of a culture medium is an increase of sugar concentration. In some other cases, a change in sugar concentration is a decrease of sugar concentration. In some situations, an increase of sugar concentration is from 1% to 2%, from 2% to 3%, from 3% to 4%, from 4% to 5%, from 5% to 10%, from 10% to 15%, from 15% to 20%, from 20% to 30%, from 30% to 40%, from 40% to 50%, from 50% to 60%, from 60% to 70%, from 70% to 80%, from 80% to 90%, or from 90% to 100% more sugar compared with the original sugar concentration in the culture medium. In some situations, a decrease of sugar concentration is from 1% to 2%, from 2% to 3%, from 3% to 4%, from 4% to 5%, from 5% to 10%, from 10% to 15%, from 15% to 20%, from 20% to 30%, from 30% to 40%, from 40% to 50%, from 50% to 60%, from 60% to 70%, from 70% to 80%, from 80% to 90%, or from 90% to 100% less sugar compared with the original sugar concentration in the culture medium. 
     In some cases, a change of an environmental condition comprises a change in oxygenation of a culture medium contacting a cell. In some cases, a change in oxygenation of a culture medium is an increase of oxygenation. In some other cases, a change in oxygenation of a culture medium is a decrease of oxygenation. In some situations, an increase of oxygenation is the addition of oxygen from 1% to 2%, from 2% to 3%, from 3% to 4%, from 4% to 5%, from 5% to 10%, from 10% to 15%, from 15% to 20%, from 20% to 30%, from 30% to 40%, from 40% to 50%, from 50% to 60%, from 60% to 70%, from 70% to 80%, from 80% to 90%, or from 90% to 100% more than the original amount of oxygen added in a culture medium. In some situations, a decrease of oxygenation is the addition of oxygen from 1% to 2%, from 2% to 3%, from 3% to 4%, from 4% to 5%, from 5% to 10%, from 10% to 15%, from 15% to 20%, from 20% to 30%, from 30% to 40%, from 40% to 50%, from 50% to 60%, from 60% to 70%, from 70% to 80%, from 80% to 90%, or from 90% to 100% less than the original amount of oxygen added in a culture medium. 
     Bio-production Reactors and Systems 
     Fermentation systems utilizing methods and/or compositions according to the invention are also within the scope of the invention. 
     Any of the recombinant microorganisms as described and/or referred to herein may be introduced into an industrial bio-production system where the microorganisms convert a carbon source into a product in a commercially viable operation. The bio-production system includes the introduction of such a recombinant microorganism into a bioreactor vessel, with a carbon source substrate and bio-production media suitable for growing the recombinant microorganism, and maintaining the bio-production system within a suitable temperature range (and dissolved oxygen concentration range if the reaction is aerobic or microaerobic) for a suitable time to obtain a desired conversion of a portion of the substrate molecules to a selected chemical product. Bio-productions may be performed under aerobic, microaerobic, or anaerobic conditions, with or without agitation. Industrial bio-production systems and their operation are w-ell-known to those skilled in the arts of chemical engineering and bioprocess engineering. The amount of a product produced in a bio-production media generally can be determined using a number of methods known in the art, for example, high performance liquid chromatography (HPLC), gas chromatography (GC), or GC/Mass Spectroscopy (MS). 
     Genic Modifications, Nucleotide Sequences, and Amino Acid Sequences 
     Embodiments of the present disclosure may result from introduction of an expression vector into a host microorganism, wherein the expression vector contains a nucleic acid sequence coding for an enzyme that is, or is not, normally found in a host microorganism. 
     The ability to genetically modify a host cell is essential for the production of any genetically modified (recombinant) microorganism. The mode of gene transfer technology may be by electroporation, conjugation, transduction, or natural transformation. A broad range of host conjugative plasmids and drug resistance markers are available. The cloning vectors are tailored to the host organisms based on the nature of antibiotic resistance markers that can function in that host. Also, as disclosed herein, a genetically modified (recombinant) microorganism may comprise modifications other than via plasmid introduction, including modifications to its genomic DNA. 
     More generally, nucleic acid constructs can be prepared comprising an isolated polynucleotide encoding a polypeptide having enzyme activity operably linked to one or more (several) control sequences that direct the expression of the coding sequence in a microorganism, such as  E. coli , under conditions compatible with the control sequences. The isolated polynucleotide may be manipulated to provide for expression of the polypeptide. Manipulation of the polynucleotide&#39;s sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotide sequences utilizing recombinant DNA methods are well established in the art. 
     The control sequence may be an appropriate promoter sequence, a nucleotide sequence that is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present disclosure. The promoter sequence may contain transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any nucleotide sequence that shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. The techniques for modifying and utilizing recombinant DNA promoter sequences are well established in the art. 
     For various embodiments of the invention the genetic manipulations may be described to include various genetic manipulations, including those directed to change regulation of, and therefore ultimate activity of, an enzyme or enzymatic activity of an enzyme identified in any of the respective pathways. Such genetic modifications may be directed to transcriptional, translational, and post-translational modifications that result in a change of enzyme activity and/or selectivity under selected and/or identified culture conditions and/or to provision of additional nucleic acid sequences such as to increase copy number and/or mutants of an enzyme related to product production. Specific methodologies and approaches to achieve such genetic modification are well known to one skilled in the art. 
     In various embodiments, to function more efficiently, a microorganism may comprise one or more gene deletions. For example, in  E. coli , the genes encoding the lactate dehydrogenase (ldhA), phosphate acetyltransferase (pta), pyruvate oxidase (poxB), pyruvateformate lyase (pflB), methylglyoxal synthase (mgsA), acetate kinase (ackA), alcohol dehydrogenase (adhE), the clpXP protease specificity enhancing factor (sspB), the ATPdependent Lon protease (Ion), the outer membrane protease (ompT), the arcA transcriptional dual regulator (arcA), and the iclR transcriptional regulator (iclR) may be disrupted, including deleted. Such gene disruptions, including deletions, are not meant to be limiting, and may be implemented in various combinations in various embodiments. Gene deletions may be accomplished by numerous strategies well known in the art, as are methods to incorporate foreign DNA into a host chromosome. 
     In various embodiments, to function more efficiently, a microorganism may comprise one or more synthetic metabolic valves, composed of enzymes targeted for controlled proteolysis, expression silencing or a combination of both controlled proteolysis and expression silencing. In some embodiments, a microorganism may comprise two, three, four, five, six, seven, eight, nine, or ten, or more synthetic metabolic valves. For example, one enzyme encoded by one gene or a combination of numerous enzymes encoded by numerous genes in  E. coli  may be designed as synthetic metabolic valves to alter metabolism and improve product formation. Representative genes in  E. coli  may include but are not limited to the following: fabI, zwf gltA, ppc, udhA, lpd, sucD, aceA, pfkA, lon, rpoS, tktA or tktB. It is appreciated that it is well known to one skilled in the art how to identify homologues of these genes and or other genes in additional microbial species. 
     For all nucleic acid and amino acid sequences provided herein, it is appreciated that conservatively modified variants of these sequences are included, and are within the scope of the invention in its various embodiments. Functionally equivalent nucleic acid and amino acid sequences (functional variants), which may include conservatively modified variants as well as more extensively varied sequences, which are well within the skill of the person of ordinary skill in the art, and microorganisms comprising these, also are within the scope of various embodiments of the invention, as are methods and systems comprising such sequences and/or microorganisms. 
     Accordingly, as described in various sections above, some compositions, methods and systems of the present disclosure comprise providing a genetically modified microorganism that comprises both a production pathway to make a desired product from a central intermediate in combination with synthetic metabolic valves to redistribute flux. 
     Aspects of the invention also regard provision of multiple genetic modifications to improve microorganism overall effectiveness in converting a selected carbon source into a selected product. Particular combinations are shown, such as in the Examples, to increase specific productivity, volumetric productivity, titer and yield substantially over more basic combinations of genetic modifications. In addition to the above-described genetic modifications, in various embodiments genetic modifications, including synthetic metabolic valves also are provided to increase the pool and availability of the cofactor NADPH and/or NADH which may be consumed in the production of a product. 
     More generally, and depending on the particular metabolic pathways of a microorganism selected for genetic modification, any subgroup of genetic modifications may be made to decrease cellular production of fermentation product(s) other than the desired fermentation product, selected from the group consisting of acetate, acetoin, acetone, acrylic, malate, fatty acid ethyl esters, isoprenoids, glycerol, ethylene glycol, ethylene, propylene, butylene, isobutylene, ethyl acetate, vinyl acetate, other acetates, 1,4-butanediol, 2,3-butanediol, butanol, isobutanol, sec-butanol, butyrate, isobutyrate, 2-OH-isobutyrate, 3-OHbutyrate, ethanol, isopropanol, D-lactate, L-lactate, pyruvate, itaconate, levulinate, glucarate, glutarate, caprolactam, adipic acid, propanol, isopropanol, fusel alcohols, and 1,2-propanediol, 1,3-propanediol, formate, fumaric acid, propionic acid, succinic acid, valeric acid, maleic acid and poly-hydroxybutyrate. Gene deletions may be made as disclosed generally herein, and other approaches may also be used to achieve a desired decreased cellular production of selected fermentation products other than the desired products. 
     VI.A Gene Silencing 
     In particular the invention describes the use of controlled gene silencing to help enable the control over metabolic fluxes in controlled multi-stage fermentation processes. There are several methodologies known in the art for controlled gene silencing, including but not limited to mRNA silencing or RNA interference, silencing via transcriptional repressors and CRISPR interference. 
     In some cases, a valve polynucleotide comprises a polynucleotide selected from the group consisting of: a silencing polynucleotide for repressing transcription of a gene encoding said valve enzyme; a degradation polynucleotide for mediating cellular degradation of said valve enzyme; and a combination thereof. 
     In some cases, a valve polynucleotide comprises a silencing polynucleotide, and said silencing polynucleotide comprises a guide RNA (gRNA) comprising a gRNA sequence that recognizes a promoter of a gene encoding said valve enzyme. 
     In some cases, a valve polynucleotide further encodes a CRISPR enzyme, wherein said CRISPR enzyme specifically binds to said promoter sequence when bound to said gRNA. In some cases, a CRISPR enzyme is catalytically inactive. 
     In some cases, a valve polynucleotide comprises a degradation polynucleotide, wherein said degradation polynucleotide comprises a sequence encoding a degradation tag, wherein said degradation tag mediates degradation of said valve enzyme. In some cases, the expression of a valve polynucleotide is regulated by phosphate availability in a cell. In some cases, the expression of a production polynucleotide is regulated by phosphate availability in a cell. In certain cases, the cell is an  E. coli  cell. 
     Controlled Proteolysis 
     In particular the current disclosure describes the use of controlled protein degradation or proteolysis to help enable the control over metabolic fluxes in controlled multi-stage fermentation processes. There are several methodologies known in the art for controlled protein degradation, including but not limited to targeted protein cleavage by a specific protease and controlled targeting of proteins for degradation by specific peptide tags. Systems for the use of the  E. coli  clpXP protease for controlled protein degradation can be used. This methodology relies upon adding a specific C-terminal peptide tag such as a DAS4 (or DAS+4) tag. Proteins with this tag are not degraded by the clpXP protease until the specificity enhancing chaperone sspB is expressed, sspB induces degradation of DAS4 tagged proteins by the clpXP protease. In additional numerous site specific protease systems are well known in the art. Proteins can be engineered to contain a specific target site of a given protease and then cleaved after the controlled expression of the protease. In some embodiments the cleavage can be expected lead to protein inactivation or degradation. For example, an N-terminal sequence can be added to a protein of interest to enable clpS dependent clpAP degradation. In addition, this sequence can further be masked by an additional N-terminal sequence, which can be controllable cleaved such as by a ULP hydrolase. This allows for controlled N-rule degradation dependent on hydrolase expression. It is therefore possible to tag proteins for controlled proteolysis either at the N-terminus or C-terminus. 
     The preference of using an N-terminal vs. C-terminal tag will largely depend on whether either tag affects protein function prior to the controlled onset of degradation. The invention describes the use of controlled protein degradation or proteolysis to help enable the control over metabolic fluxes in controlled multi-stage fermentation processes, in  E. coli . There are several methodologies known in the art for controlled protein degradation in other microbial hosts, including a wide range of gram-negative as well as gram-positive bacteria, yeast and even archaea. In particular, systems for controlled proteolysis can be transferred from a native microbial host and used in a non-native host. 
     Synthetic Metabolic Valve Control 
     In particular the current disclosure describes the use of synthetic metabolic valves to control metabolic fluxes in multi-stage fermentation processes. There are numerous methodologies known in the art to induce expression that can be used at the transition between stages in multistage fermentations. These include but are not limited to artificial chemical inducers including: tetracycline, anhydrotetracycline, lactose, IPTG (isopropyl-beta-D-1-thiogalactopyranoside), arabinose, raffinose, tryptophan and numerous others. Systems linking the use of these well known inducers to the control of gene expression silencing and/or controlled proteolysis can be integrated into genetically modified microbial systems to control the transition between growth and production phases in multi-stage fermentation processes. 
     In addition, it may be desirable to control the transition between growth and production in multi-stage fermentations by the depletion of one or more limiting nutrients that are consumed during growth. Limiting nutrients can include but are not limited to: phosphate, nitrogen, sulfur and magnesium. Natural gene expression systems that respond to these nutrient limitations can be used to operably link the control of gene expression silencing and/or controlled proteolysis to the transition between growth and production phases in multi-stage fermentation processes. 
     Products 
     In some embodiments, provided herein is a microorganism or a cell for producing a product. In some cases, the product comprises 3-hydroxypropionic acid. In some cases, the product comprises an amino acid. In some cases, the amino acid comprises alanine. In some cases, the alanine is L-alanine. In some cases, the alanine is D-alanine. In some cases, a rate of production of alanine is at least 0.1 g/L/hr, 0.2 g/L/hr, 0.3 g/L/hr, 0.4 g/L/hr, 0.5 g/L/hr, 0.6 g/L/hr, 0.7 g/L/hr, 0.8 g/L/hr, 0.9 g/L/hr, 1.0 g/L/hr, 1.1 g/L/hr, 1.2 g/L/hr, 1.3 g/L/hr, 1.4 g/L/hr, 1.5 g/L/hr, 1.6 g/L/hr, 1.7 g/L/hr, 1.8 g/L/hr, 1.9 g/L/hr, 2.0 g/L/hr, 2.5 g/L/hr, 3.0 g/L/hr, 3.5 g/L/hr, 4.0 g/L/hr, 4.5 g/L/hr, 5.0 g/L/hr, 5.5 g/L/hr, 6.0 g/L/hr, 7.0 g/L/hr, 8.0 g/L/hr, 9.0 g/L/hr, or at least 10 g/L/hr. 
     In some cases, the alanine titers after 24 hours can be from 0 to 0.5 g/L, 0.5 g/L to 1 g/L, 1 g/L to 1.5 g/L, 1.5 g/L to 2 g/L, 2 g/L to 2.5 g/L, 2.5 g/L to 3 g/L, 3 g/L to 3.5 g/L, 3.5 g/L to 4 g/L, 4 g/L to 4.5 g/L, 4.5 g/L to 5 g/L, or from 5 g/L to 10 g/L. The dynamic range of alanine production offered by SMVs can be up to a 4-fold increase compared to that offered by solely altering the expression level of the production pathway enzymes (by changing the promoter). In some cases, the dynamic range of alanine production offered by SMVs can be up to a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold increase compared to that offered by solely altering the expression level of the production pathway enzymes. 
     In some cases, a production polynucleotide in the microorganism encodes an alanine exporter. In some cases, the alanine exporter is alaE. 
     In some cases, the product comprises mevalonic acid. In some cases, a rate of production of mevalonic acid is at least 0.1 g/L/hr, 0.2 g/L/hr, 0.3 g/L/hr, 0.4 g/L/hr, 0.5 g/L/hr, 0.6 g/L/hr, 0.7 g/L/hr, 0.8 g/L/hr, 0.9 g/L/hr, 1.0 g/L/hr, 1.1 g/L/hr, 1.2 g/L/hr, 1.3 g/L/hr, 1.4 g/L/hr, 1.5 g/L/hr, 1.6 g/L/hr, 1.7 g/L/hr, 1.8 g/L/hr, 1.9 g/L/hr, 2.0 g/L/hr, 2.5 g/L/hr, 3.0 g/L/hr, 3.5 g/L/hr, 4.0 g/L/hr, 4.5 g/L/hr, 5.0 g/L/hr, 5.5 g/L/hr, 6.0 g/L/hr, 7.0 g/L/hr, 8.0 g/L/hr, 9.0 g/L/hr, or at least 10 g/L/hr. 
     Methods 
     Provided herein are methods for producing a product in an engineered microorganism in a large scale. Also provided herein are methods for engineering microorganisms for large-scale production of a product in a high-throughput fashion. 
     In some cases, provided herein is a method, comprising: culturing a plurality of strains of a cell, wherein each strain of said plurality of strains comprises (i) an engineered valve polynucleotide for mediating controlled reduction of expression of a valve enzyme acting in a metabolic pathway, wherein said controlled reduction of expression of said valve enzyme reduces flux through said metabolic pathway; and (ii) an engineered production polynucleotide for mediating controlled increase in expression of a production enzyme for generation of said product; wherein each strain of said plurality of strains differs from another strain in a sequence of at least one of said engineered valve polynucleotide or said engineered production polynucleotide; measuring a level of said product generated by each of said plurality of strains; and selecting a strain based on said level of said product. In some embodiments, the method further comprises growing said selected strain in a bioreactor. In some embodiments, a culture medium comprising said selected strain has a volume of at least 100 ml, 200 ml, 300 ml, 400 ml, 500 ml, 60 ml, 700 ml, 800 ml, 900 ml, or at least 1000 ml. In some embodiments, a culture medium has a volume of at least 1 L. 
     In some embodiments, a valve polynucleotide comprises a polynucleotide selected from the group consisting of: a silencing polynucleotide for repressing transcription of a gene encoding said valve enzyme; a degradation polynucleotide for mediating cellular degradation of said valve enzyme; and a combination thereof. In some embodiments, a first and a second strain of said plurality of strains comprise a silencing polynucleotide. In some embodiments, a silencing polynucleotide comprises a guide RNA (gRNA) comprising a gRNA sequence that recognizes a promoter of a gene encoding said valve enzyme. In some embodiments, a gRNA sequence differs between said first and second strains. In some embodiments, a promoter recognized by said gRNA differs between said first and second strains. In some embodiments, a first strain comprises said silencing polynucleotide and said degradation polynucleotide, and a second strain comprises said silencing polynucleotide but does not comprise said degradation polynucleotide. In some embodiments, a level of product is greater in said second strain than said first strain. In some embodiments, a level of product is greater in said first strain than said second strain. In some embodiments, a valve enzyme comprises an enzyme selected from the group consisting of enoyl-ACP/CoA reductase (fabI), glucose-6-phosphate dehydrogenase (zwf), lipoamide dehydrogenase (lpd), citrate synthase (gltA), soluble transhydrogenase (udhA). NADH-dependent glyceraldehyde-3-phosphate dehydrogenase (gapA), and a combination thereof. In some embodiments, a production enzyme comprises an enzyme selected from the group consisting of NADPH-dependent alanine dehydrogenase (aid), alanine exporter (alaE), NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (gapN), and a combination thereof. 
     In some embodiments, a product is selected from the group consisting of mevalonic acid, 3-hydroxypropionic acid, an amino acid, and a combination thereof. In some embodiments, the amino acid is alanine. In some embodiments, the alanine is L-alanine. In some embodiments, the alanine is D-alanine. 
     In some embodiments, a rate of production of the product during said stationary phase is reduced less in response to a change of an environmental condition as compared to a cell lacking said controlled reduction of expression of said valve enzyme. 
     In some embodiments, a change of an environmental condition comprises a change in a sugar concentration of a culture medium contacting said cell. 
     In some embodiments, a change of an environmental condition comprises a change in oxygenation of a culture medium contacting said cell. 
     In some cases, provided herein is a method of generating a cellular product comprising: culturing a heterologous cell in a culture medium, wherein said heterologous cell comprises: (i) an engineered valve polynucleotide for mediating controlled reduction of expression of a valve enzyme acting in a metabolic pathway, wherein said controlled reduction of expression of said valve enzyme reduces flux through said metabolic pathway, wherein said controlled reduction of expression of said valve enzyme induces a stationary phase of said cell; and (ii) an engineered production polynucleotide for mediating controlled increase in expression of a production enzyme for generation of said product; wherein a rate of production of said product during said stationary phase is reduced less in response to a change of an environmental condition as compared to a cell lacking said controlled reduction of expression of said valve enzyme. In some embodiments, the method further comprises changing said environmental condition. In some embodiments, the environmental condition comprises a sugar concentration of said culture medium, and changing said environmental condition comprises increasing or decreasing said sugar concentration. In some cases, said sugar is glucose, sucrose, lactose, maltose, xylose, mannitol, or a combination thereof. In some cases, said sugar is glucose. In some cases, the environmental condition comprises an oxygen concentration of said culture medium, and changing said environmental condition comprises increasing or decreasing said oxygen concentration. In some cases, said culturing is performed in a bioreactor. 
     In some cases, said culture medium has a volume of at least 100 ml, 200 ml, 300 ml, 400 ml, 500 ml, 600 ml, 700 ml, 800 ml, 900 ml, or at least 1000. In some cases, said culture medium has a volume of at least 1 L. In some case, said product comprises 3-hydroxypropionic acid. In some cases, said product comprises an amino acid. In some cases, said amino acid comprises alanine. 
     In some cases, a rate of production of said alanine is at least 0.1 g/L/hr, 0.2 g/L/hr, 0.3 g/L/hr, 0.4 g/L/hr, 0.5 g/L/hr, 0.6 g/L/hr, 0.7 g/L/hr, 0.8 g/L/hr, 0.9 g/L/hr, 1.0 g/L/hr, 1.1 g/L/hr, 1.2 g/L/hr, 1.3 g/L/hr, 1.4 g/L/hr, 1.5 g/L/hr, 1.6 g/L/hr, 1.7 g/L/hr, 1.8 g/L/hr, 1.9 g/L/hr, 2.0 g/L/hr, 2.5 g/L/hr, 3.0 g/L/hr, 3.5 g/L/hr, 4.0 g/L/hr, 4.5 g/L/hr, 5.0 g/L/hr, 5.5 g/L/hr, 6.0 g/L/hr, 7.0 g/L/hr, 8.0 g/L/hr, 9.0 g/L/hr, or at least 10 g/L/hr. In some cases, said production polynucleotide encodes an alanine exporter. In some cases, said alanine exporter is alaE. In some cases, said culturing occurs for less than 20 hours, 30 hours, 40 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, or less than 100 hours. In some cases, said culturing occurs for less than 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, or less than 45 hours. 
     In some cases, said culturing occurs for less than 30 hours. 
     In some cases, said product comprises mevalonic acid. In some cases, a rate of production of said mevalonic acid is at least 0.1 g/L/hr, 0.2 g/L/hr, 0.3 g/L/hr, 0.4 g/L/hr, 0.5 g/l/hr, 0.6 g/L/hr, 0.7 g/L/hr, 0.8 g/L/hr, 0.9 g/L/hr, 1.0 g/L/hr, 1.1 g/L/hr, 1.2 g/L/hr, 1.3 g/L/hr, 1.4 g/L/hr, 1.5 g/L/hr, 1.6 g/L/hr, 1.7 g/L/hr, 1.8 g/L/hr, 1.9 g/L/hr, 2.0 g/L/hr, 2.5 g/L/hr, 3.0 g/L/hr, 3.5 g/L/hr, 4.0 g/L/hr, 4.5 g/L/hr, 5.0 g/L/hr, 5.5 g/L/hr, 6.0 g/L/hr, 7.0 g/l/hr, 8.0 g/L/hr, 9.0 g/L/hr, or at least 10 g/L/hr. In some cases, said culturing occurs for less than 20 hours, 30 hours, 40 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, or less than 100 hours. In some cases, said culturing occurs for less than 80 hours. 
     In some embodiments, a valve polynucleotide comprises a polynucleotide selected from the group consisting of: a silencing polynucleotide for repressing transcription of a gene encoding said valve enzyme; a degradation polynucleotide for mediating cellular degradation of said valve enzyme, and a combination thereof. In some cases, a valve polynucleotide comprises a silencing polynucleotide, and said silencing polynucleotide comprises a guide RNA (gRNA) comprising a gRNA sequence that recognizes a promoter of a gene encoding said valve enzyme. In some cases, a valve polynucleotide further encodes a CRISPR enzyme, wherein said CRISPR enzyme specifically binds to said promoter sequence when bound to said gRNA. In some cases, a CRISPR enzyme is catalytically inactive. In some case, a valve polynucleotide comprises a degradation polynucleotide, wherein said degradation polynucleotide comprises a sequence encoding a degradation tag, wherein said degradation tag mediates degradation of said valve enzyme. In some cases, an expression of said valve polynucleotide is regulated by phosphate. In some cases, an expression of said production polynucleotide is regulated by phosphate. In some cases, said cell is an  E. coli  cell. 
     Optimization of Bio-Production 
     Biotechnology based fermentation processes have been successfully developed to produce everything from biologics and small molecule therapeutics to specialty, bulk and commodity chemicals, and even next generation biofuels 1-3 . These processes have made rapid advancements in recent years due to numerous technology developments 4, 5 . It has never been easier to produce new molecules using synthetic biology. Despite these advances, a major challenge remains in taking molecules from proof of concept (POC) to commercially meaningful levels. Strain optimization, or overcoming the “mg” to “kg” hurdle has remained a key barrier to the successful commercialization of bio-processes. After the demonstration of POC, successful bio-process development routinely requires lengthy iterations of both microbial strain and fermentation optimization 6-8  ( FIG. 1B ). These optimization efforts are often specific to the product or host strain of interest. The throughput of synthetic biology has outpaced that of metabolic engineering, partly due to a lack of broadly useful tools to perform meaningful and standardized optimization of engineered microbial strains in a high-throughput manner 9 . 
     There are numerous challenges in strain optimization and moving past POC levels, not the least of which are the size and complexity of the potential design space. In contrast to simpler gene circuits, amenable to electrical circuit models 10-12 , metabolic networks are highly interconnected. Each metabolite and/or enzyme can interact with endless others. This combinatorial complexity results in a huge potential design space, which is intractable to the kinds of systematic experimentation required for the development of standardized design principles (Supplemental Materials, Table 1). The challenges in addressing such a large design space have persisted despite the dramatic advances in, and decreased costs of, reading and writing DNA that have led to new high-throughput DNA assembly and microbial strain construction methods 13-16 . It is not surprising that new synthetic biology technologies involving strain engineering are often demonstrated with easily screened or selected phenotypes 13, 17-19 . Most of these are limited to a focus on optimizing a limited set of pathway specific enzymes. 
     One approach to overcome the complexity of this challenge is the use of in vitro systems for bio-production, which comprise a limited set of metabolic enzymes. However, these approaches have challenges in replicating key advantages of in vivo systems, including cofactor recycling and energy generation 20, 21 . Another approach to deal with this complexity is to develop faster screening methods for strain evaluation 22 . However, increased throughput alone can never evaluate the full complexity of the potential design space. In addition, results obtained from high-throughput studies often do not translate, even in the same microbe, to a different environment 20, 23, 24 . Small scale screens do not readily translate to larger scale production processes, leading to iterations of process optimization on top of strain optimization ( FIG. 1B ). This is because metabolism is highly regulated and can respond, sometimes dramatically, to changes in environmental conditions 25 20, 26-28 . A lack of environmental robustness is traditionally one factor making the scale up of fermentation based processes difficult. This issue has led to the development of specialized complex micro-reactor systems for scale down offering only modest improvements in throughput 20, 29-31 . 
     There remains a significant need for broadly applicable, rapid and robust approaches to greatly reduce the time and costs transitioning from “mgs” to “kgs”. Ideally, approaches should be amenable to multiple products and production hosts. Provided herein is the development of a generalizable, high-throughput strain optimization approach that enables the use of truly scalable, standardized fermentation processes. This approach, as outlined in  FIG. 1B , panel b, involves the dynamic minimization of the active metabolic network 32 , which combines the benefits of a smaller design space common to in vitro approaches while maintaining the benefits of in vivo biosynthesis 20 . We can isolate and focus on the minimal metabolic networks required for production. Utilizing combinations of synthetic metabolic valves (SMVs) 32, 33  ( FIGS. 2A-D ) we can dynamically minimize the metabolic network and redirect metabolic flux in the context of a standardized 2-stage fermentation process 20 . 
     This approach can reduce the complexity of the problem and the size of the relevant design space, greatly speeding up optimization. In various embodiments, it is demonstrated herein that dynamic metabolic network minimization can improve pathway fluxes beyond those achievable with production pathway modifications alone ( FIGS. 3A-K  and  6 A-H). 
     Simultaneously, we demonstrate that dynamic network minimization reduces metabolic responses to environmental conditions, which increases the robustness and scalability of engineered strains ( FIGS. 3A-K  and SA-J). 
     Examples 
     2-Stage Synthetic Metabolic Valves is  E. coli    
     We first developed improved synthetic metabolic valves (SMVs) in  E. coli  that are capable of the dynamic reduction of protein levels in a 2-stage process. These SMVs can be used to reduce levels of key metabolic enzymes (or reduce enzymatic activities of key metabolic enzymes) and rely on controlled proteolysis or CRISPR-based gene silencing or both proteolysis and silencing in combination ( FIGS. 2A-D ) 32-35 . Cell growth and dynamic metabolic control can be implemented using phosphate depletion as an environmental trigger. Phosphate can be an ideal candidate as a trigger, as one of the costliest components of minimal media. In addition, stationary phases induced in  E. coli  by phosphate depletion have retained glycolytic uptake as well as increased protein expression 31, 36 . Numerous promoter systems responding to phosphate are well characterized in  E. coli  as well as other microbes including  S. cerevisiae   37 . Phosphate responsive promoter variants were evaluated (Supplemental Materials, Section 1) and subsequently used for 2-stage control. 
     SMVs were implemented in  E. coli  using the native Type I-E Cascade CRISPR system for induced gene silencing 34,38 , while controlled proteolysis was induced by incorporating C-terminal degron tags on target proteins, both as previously demonstrated 63,33  ( FIG. 2A ). These systems were introduced into a host strain initially engineered for minimal byproduct formation and high biomass yields and growth rates ( E. coli  strain DLF_0025, Supplemental Materials, Section 3) 24, 27, 28, 39 . Using this approach, as  FIGS. 2A-D  demonstrate, protein levels can be controlled in 2-stage processes, as exemplified by turning “ON” GFPuv and “OFF” mCherry fluorescent proteins with phosphate depletion in minimal medium. The combination of gene silencing with proteolysis results in the largest rates of protein degradation ( FIGS. 2C-D ). The specific impact of gene silencing and proteolysis on decay rates will likely vary depending on the host, target gene/enzyme, and its specific natural turnover rates and expression levels. 
     Metabolic Network Minimization Leads to Improved Fluxes 
     With the successful demonstration of dynamic control of protein levels in a 2-stage process, we turned to investigate the dynamic control of metabolic fluxes in  E. coli  through controlled reduction of key central metabolic enzymes alone and in combination. Reducing fluxes through thermodynamically favored “committed” reactions in the network is expected to lead to increases in network metabolite pools (Supplemental Materials Section 5), and as a result, changes in pathway fluxes. Enzymes in key committed steps in central metabolic pathways were identified and chosen as initial SMV targets and alanine was chosen as an initial test product ( FIGS. 3A-K ). A set of strains were constructed for alanine production ( FIG. 3A ), comprising an NADPH-dependent alanine dehydrogenase (ald*) 42 . Variants with multiple combinations of SMVs in central metabolic enzymes were made, with either modifications to induce proteolysis or gene silencing or both in combination. (Supplemental Materials, Section 3). Together the set of strains having SMVs evaluated in 2-stage processes are identified as “Valve” strains. A panel of alanine “Valve” strains (˜500 strains in total) were evaluated for alanine production in standardized, 2-stage, 96-well plate based micro-fermentations (Supplemental Materials, Section 7). Alanine titers after 24 hours of production are given in  FIGS. 3B-C . Briefly, alanine titers after 24 hours ranged from ˜0 g/L to ˜4.7 g/L, and as expected, varied significantly with respect to the number and combination of SMVs; most SMV combinations lead to improved performance when compared to the control with no SMVs and the alanine pathway alone. In some cases, the alanine titers after 24 hours can be from 0 to 0.5 g/L, 0.5 g/L to 1 g/L, 1 g/L to 1.5 g/L, 1.5 g/L to 2 g/L, 2 g/L to 2.5 g/L, 2.5 g/L to 3 g/L, 3 g/L to 3.5 g/L, 3.5 g/L to 4 g/L, 4 g/L to 4.5 g/L, 4.5 g/L to 5 g/L, or from 5 g/L to 10 g/L. The dynamic range of alanine production offered by SMVs can be up to a 4-fold increase compared to that offered by solely altering the expression level of the production pathway enzymes (by changing the promoter) (Supplemental Materials. Section 7). In some cases, the dynamic range of alanine production offered by SMVs can be up to a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold increase compared to that offered by solely altering the expression level of the production pathway enzymes. Importantly, the use of proteolysis or silencing alone and/or in combination had significant impacts on production, indicating that for each enzyme the fine tuning of activity using SMVs is critical. One of the best performing strains from the micro-fermentations was then evaluated in a minimal medium, 2-stage, 1 L fermentation with 10 gdcw/L of biomass ( FIG. 3F ), which resulted in 80 g/L 100% L-alanine after 48 hours of production with a yield of 0.8 gig. Further engineering of this strain by overexpressing an alanine exporter (encoded by the  E. coli  alaE gene 43 ) resulted in 147 g/L 100% L-alanine after 27 hours of production with a yield within error of theoretical yield ˜1 g/g, ( FIG. 3G ). 
     Micro-fermentation Robustness 
     A central hypothesis was that by restricting metabolism in the production stage, strain performance could not only be improved, but would be more robust to environmental (process) conditions. Simply put, carbon flow is restricted through a minimized metabolic network, which can no longer adapt via cellular responses to the environment. To test this hypothesis, strains were evaluated under different “micro-fermentation” process conditions. Glucose concentration and oxygen transfer rate (key process variables impacting strain performance in traditional fermentations 26 ) were varied ( FIG. 3D , Supplemental Materials, Section 8), and alanine production measured. A robustness score (RS) was developed to quantify environmental robustness. Larger RS scores indicate more robust strains. Whereas relative standard deviation (RSD) is one metric for robustness, we wanted to incorporate a stricter measure of robustness which also incorporates the maximal deviation (Max Dev) a strain has under all process conditions (RS, Equation (1)). 
     
       
         
           
             
               
                 
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     Robustness scores for a subset of 48 alanine “Valve” strains are given in  FIG. 3E . Results from these experiments studies are tabulated in Supplemental Materials, Section 8. A Chi 2  analysis using a cutoff of RS&gt;0.6 for robustness was used to identify key SMVs which statistically contribute to process robustness. The proteolytic degradation of fabI was a primary contributor to robustness (Chi 2 =13.85, P value &lt;0.001) and as a result. “Valve” strains with proteolytic degradation of fabI were used in further studies. In addition, the “Valve” strains with proteolytic degradation of gltA and/or the combination of the proteolytic degradation of fabI and gltA were found to also be significant contributors of robustness, albeit with a large P value . 
     2-Stage “Value” Strains Compared to Traditional Growth Associated Strains 
     To compare the 2-stage approach enabled by SMVs to more traditional growth associated processes, we constructed 5 strains, with constitutively expressed alanine dehydrogenase (ald*), capable of the growth associated production of alanine. These growth associated strains varied in the strength of the promoter used to drive ald* expression 44  (Supplemental Materials, Section 2), yet utilized the same common no-valve control host strain.  FIG. 5  illustrates the results of a direct comparison of “Valve” strains in a 2-stage process compared to “Growth Associated (GA)” strains in a traditional fermentation at the microtiter ( FIGS. 5A-D ) and 1 L ( FIGS. 5E-J ) scales. In micro-fermentations, 2-stage “Valve” strains outperformed GA strains with respect to titer and process robustness. The most robust GA strain from the micro-fermentation analysis (also with the highest production level) was compared to a robust “Valve” strain in 1 L fermentations with varied process conditions. The “Valve” strains showed consistent performance in all process conditions evaluated ( FIG. 5E ), consistent with results from micro-fermentations, where the GA strain had significant performance variability dependent on process. We hypothesized that the increased environmental robustness observed in both “micro-” and 1 L scale fermentations for “Valve” strains would lead to predictable scale up, where strains with improved performance in high-throughput micro-fermentations would reliably have improved performance in controlled bioreactors. To evaluate the scalability of the system. “Valve” alanine strains with statistically differentiated performance in micro-fermentations (P-value&lt;0.001) were evaluated in standardized 2-stage 1 L fermentations and compared to all GA strains. Statistically different performances observed in “micro-fermentations” have scaled predictably to 1 L fermentations for 2-stage “Valve” strains. This contrasts with results obtained with GA strains where no correlation between micro-fermentation and 1 L performance was observed ( FIGS. 5G-H ). 
     Product Flexibility 
     With the successful and predictable scale-up of alanine strains into 1 L fully instrumented fermentations, we moved to validate the technology platform for an additional product: mevalonic acid. To this end, additional dynamic production pathways were constructed for mevalonic acid biosynthesis ( FIG. 6A ). A set of two-gene production pathway plasmids encoding three enzymatic functions was constructed for mevalonic acid production, consisting of the  E. faecalis  mvaE and mvaS genes encoding a bifunctional acetyl-CoA acetyltransferase, NADPH dependent HMG-CoA reductase, and HMG-CoA synthase respectively. A mutant mvaS gene, mvaS(A1110G) with higher activity was used 45, 46 . Production plasmids were initially evaluated for mevalonate production in the control strain ( FIG. 6B ). The best producing plasmid was then introduced into a variety of engineered “Valve” strains and evaluated in micro-fermentations ( FIG. 6C ). A subset of statistically differentiated strains were then evaluated in 1 L fermentations to assess scalability ( FIG. 6D ), which, as in the case of alanine, was predictive. In some cases, a performing strain produced meaningful titers and yields, 97 g/L in 78 hrs of production with a yield of 0.46 g/g (84% of theoretical yield) ( FIG. 6E ). Specific productivity for this mevalonate strain is over 4-fold higher than the best previously reported results 47  (Supplemental Materials, Section 9). 
     Discussion 
     Historically some of the most successful efforts to metabolically engineer the production of small molecules have leveraged the power of anaerobic metabolism to couple product formation with growth. This has allowed for the classical design and selection of industrial strains to produce many products including ethanol, succinic acid, lactate and isobutanol, which have leveraged the power of evolution and selection to reach optimal metabolic fluxes in engineered networks 48, 49 _ENREF_2. While growth associated production is not strictly linked to anaerobic metabolism, growth association greatly limits the number and variety of different molecules that can be made using synthetic biology. A generic, robust and accessible non-growth associated platform would greatly simplify the optimization and scale up of a diverse number of products. 
     In contrast to most existing 2-stage processes, which have relied on natural metabolic responses to environmental triggers for production improvement, we have taken the next step in actively minimizing the essential metabolic network and redirecting metabolites to products of interest. Many of the targeted essential central metabolic pathways in this work have traditionally been off limits to engineering strategies, as deleting essential enzymes is incompatible with growth and growth associated production in traditional fermentation. The dynamically minimized metabolic network also results in enhanced robustness to environmental variables enabling the faithful translation of high-throughput small-scale studies to larger instrumented fermentations. A current paradigm in the field is to improve the throughput of relevant strain evaluations by developing small-scale, custom-designed micro-reactors for enhanced process control. In contrast, our approach is a move in a new direction involving engineering microbial metabolism to be less sensitive to process changes, simplifying high-throughput experimentation. 
     Beyond robustness, we have demonstrated that combinatorial modifications to essential enzymes in minimal metabolic networks can lead to significant improvements in production, particularly when compared to altering production pathway expression levels alone. These large variations in performance are due to changes in a limited subset of key central metabolic nodes, likely resulting in altered metabolite levels. Compared to previous approaches to dynamically control enzyme levels, we demonstrate improved potential for fine tuning of protein levels with a combination of gene silencing and proteolysis 50 . As stationary phase cells cannot dilute existing proteins with cell division, this dual approach makes sense. The specific control of the level of any given enzyme will of course also depend on natural turnover mechanisms. At first glance, it may still be surprising that the combination of both gene silencing and proteolysis together does not always result in improved performance. i.e. “more is not always better”. Future efforts may be needed to explain these results, which could either be due to a requirement of maintaining minimal fluxes in the larger network or a consequence of changes in the levels of key regulatory metabolites that are not part of the minimal network, yet influence network activity. 
     While the approach as demonstrated can address many issues common to most bio-production processes, many product specific challenges remain. The toxicity of a product or pathway metabolite may limit titers or production rates. A minimal network that may be optimal at a low titer, may not be optimal at elevated titers. In addition, the engineering of improved enzymes is often a challenge in many “mg” to “kg” projects. 
     Feasibility of adapting this approach to other microbial hosts is expected. Key requirements for new hosts include a rapid and robust growth phase, the ability to engineer dynamic control over protein levels, and a metabolically active stationary phase. Numerous microbes have well characterized nutrient triggers for productive stationary phase metabolism 36 , for example nitrogen limitation in  Ralstonia  species,  Yarrowia  species and others 51 52 . Even when these requirements are not naturally met, they can be engineered into the host such as  S. cerevisiae  or other microbes, with each potential host presenting unique challenges and corresponding solutions. 
     Future efforts can be aimed at applying this platform for molecules with more complex production pathways. This approach can offer a tractable route for rapid optimization to metabolic engineers and synthetic biologists, who wish to move past POC levels and begin to tackle problems at more industrially relevant rates, titers and yields. 
     Methods 
     Reagents and Media 
     Unless otherwise stated, all materials and reagents were of the highest grade possible and purchased from Sigma (St. Louis, Mo.). C13 labeled Alanine (2,3-13C2, 99%) (Item #CLM-2734-PK) was purchased from Cambridge Isotope Laboratories. Inc. (Tewksbury, Mass.). Luria Broth was used for routine strain and plasmid propagation and construction. Working antibiotic concentrations were as follows: ampicillin (100 μg/mL), kanamycin (35 μg/mL), chloramphenicol (35 μg/mL), spectinomycin (100 μg/mL), zeocin (50 μg/mL), gentamicin (10 μg/mL), blasticidin (100 μg/mL), puromycin (150 μg/mL), tetracycline (5 μg/mL). Luria broth with low salt (Lennox formulation) was used to select for zeocin, blasticidin and puromycin resistant clones. In addition, for puromycin selection, phosphate buffer (pH=8.0) was added to LB Lennox to a final concentration of 50 mM. Media formulations including stock solutions are described in Supplemental Materials. Section 7. 
       E. coli  Strain Construction 
     Oligonucleotides and synthetic linear DNA (Gblocks™) used for strain construction and confirmation are all given in Supplemental Materials, Section 3, and they were obtained from Integrated DNA Technologies (IDT, Coralville, Iowa). Strain BW25113 was obtained from the Yale Genetic Stock Center (CGSC http://cgsc.biology.yale.edu/). Strain BWapldf was a kind gift from George Chen (Tsinghua University) 62 . Chromosomal modifications were made using standard recombineering methodologies 63  either with direct antibiotic cassette integration in the case of C-terminal DAS+4 tags carrying antibiotic resistance cassettes, or through scarless tet-sacB selection and counterselection, strictly following the protocols of Li et al 64 . The recombineering plasmid pSIM5 and the tet-sacB selection/counterselection marker cassette were kind gifts from Donald Court (NCI, https://redrecombineering.ncifcrf.gov/court-lab.html). Briefly, the tet-sacB selection/counterselection cassette was amplified using the appropriate oligos supplying ˜50 bp flanking homology sequences using Econotaq (Lucigen Middleton, Wis.) according to manufacturer&#39;s instructions, with an initial 10 minutes denaturation at 94° C., followed by 35 cycles of 94° C., for 15 seconds, 52° C. for 15 seconds, and 72° C. for 5 minutes. Cassettes used for “curing” of the tet-sacB cassette or direct integration (when an antibiotic marker is present) were obtained as gBlocks from IDT. In the case of the sspB gene deletion, the open reading frame deletion replaced with a kanamycin resistance was amplified from the Keio Collection strain, JW3197-1 65 , and moved to the appropriate background strain using standard methodologies. The kanamycin resistance cassette was cured using the pCP20 plasmid, leaving an frt scar 63,65 . Chromosomal modifications were confirmed by PCR amplification and sequencing (Eton Biosciences) using paired oligonucleotides, either flanking the entire region, or in the case of DAS+4 tag insertions an oligo 5′ of the insertion and one internal to the resistance cassette. 
       E. coli  Plasmid Construction 
     Primers used for the design and construction of CASCADE guides arrays were listed in Supplemental Materials, Section 6. Gene silencing guide arrays were expressed from a series of pCASCADE plasmids. The pCASCADE-control plasmid was prepared by swapping the pTet promoter in pcrRNA.Tet 73  with an insulated low phosphate induced ugpB promoter 74 . Promoter sequences for all genes were obtained from EcoCyc database (https://ecocyc.org/). In order to design CASCADE guide array. CASCADE PAM sites near the −35 or −10 box of the promoter of interest were identified, 30 bp at the 3′ end of PAM site was selected as the guide sequence and cloned into pCASCADE plasmid using Q5 site-directed mutagenesis (NEB, MA) following manufacturer&#39;s protocol, with the modification that 5% v/v DMSO was added to the Q5 PCR reaction. PCR cycles were as follows: amplification involved an initial denaturation step at 98° C. for 30 second followed by cycling at 98° C. for 10 second, 72° C. for 30 second, and 72° C. for 1.5 min (the extension rate was 30 second/kb) for 25 cycles, then a final extension for 2 min at 72° C. 2 μL of PCR mixture was used for 10 μL KLD reaction, which proceeded under room temperature for 1 hour, after which, 1 μL KLD mixture was used for electroporation. 
     The pCASCADE guide array plasmids were prepared by sequentially amplifying complementary halves of each smaller guide plasmid by PCR, followed by subsequent DNA assembly. The pCASCADE-control vector was used as template, pCASCADE plasmids with arrays of two or more guides were prepared using Q5 High-Fidelity 2× Master Mix (NEB, MA). PCR cycles were as follows: amplification involved an initial denaturation step at 98° C. for 30 second followed by cycling at 98° C. for 10 second, 66° C. for 30 second, and 72° C. for 45 second (the extension rate was 30 second/kb) for 35 cycles, then a final extension for 2 min at 72° C. PCR product was purified by gel-extraction, 20 μL ultrapure water was used to elute 50 μL PCR reaction purification, 1 μL of each eluted PCR product was used for 10 μL of Gibson Assembly (NEB, MA), which was completed by incubation at 50° C. for 15 min. 1 μL Gibson Assembly mix was used for electroporation. 
     Production pathways enzymes were expressed from high copy plasmids via low phosphate inducible promoters. Production pathway gene sequences were codon optimized using the Codon Optimization Tool from the IDT website, phosphorylated G-blocks™ were designed and purchased from IDT for each pathway. Plasmids were assembled using NEBuilder® HiFi DNA Assembly Master Mix following manufacturer&#39;s protocol (NEB, MA), pSMART-HC-Kan (Lucigen, WI) was used as backbone for all pathway plasmids. All plasmid sequences were confirmed by DNA sequencing (Eton Bioscience, NC) and deposited with Addgene. 
       E. coli  BioLector 
     Single colonies of each strain were inoculated into 5 mL LB with appropriate antibiotics and cultured at 37° C. 220 rpm for 9 hours or until OD600 reached &gt;2. 500 μL of the culture was inoculated into 10 mL SM10 medium with appropriate antibiotics, and cultured in a square shake flask (CAT#: 25-212, Genesee Scientific, Inc. San Diego, Calif.) at 37° C., 220 rpm for 16 hours. Cells were pelleted by centrifugation and the culture density was normalized to OD600 5 using FGM3 media. Growth and fluorescence measurements were obtained in a Biolector (m2p labs, Baesweiler, Germany) using a high mass transfer FlowerPlate (CAT#: MTP-48-B, m2p-labs, Germany), 40 μL of the OD normalized culture was inoculated into 760 μL of FGM3 medium with appropriate antibiotics. Biolector settings were as follows: RFP gain=100, GFP gain=20, Biomass gain=20, shaking speed=1300 rpm, temperature=37° C. humidity=85%. Every strain was analyzed in triplicate. 
       E. coli  Micro-Fermentations 
     Plasmids were transformed into host strains by electroporation using ECM 630 High Throughput Electroporation System (Harvard Apparatus. Inc. Holliston, Mass.) following manufacturer&#39;s protocol or using individual electroporation cuvettes. Glycerol stocks were prepared for each transformation plate by adding equal volume of sterile 20% glycerol, and 3 μL were used to inoculate overnight culture in 150 μL SM10++ medium with appropriate antibiotics. Plates were covered with sandwich covers (Model #CR15% obtained from EnzyScreen, Haarlam, The Netherlands). These covers ensured minimal evaporative loss during incubation. Unless otherwise stated, 96 well plates were cultured at 37° C., 404 rpm for 16 hours, shaker orbit is 25 mm. This combination of orbit and minimal shaking speed is required to obtain needed mass transfer coefficient and enable adequate culture oxygenation. 
     After 16 hours of growth, cells were pelleted by centrifugation, excess media was removed and cells were resuspended in 150 μL of FGM3 Wash solution. Subsequently cells were once again pelleted and again excess media was removed, pellet was resuspended in 50 μL FGM3 No Phosphate media containing appropriate antibiotics, 5 μL of the resuspended culture was added to 195 μL of water for OD600 measurement using standard flat bottom 96 well plate. OD600 for production was normalized to OD600=1, using FGM3 No Phosphate media containing appropriate antibiotics, in a total volume of 150 μL using standard 96 well plate. Plates were covered with sandwich covers (Model #CR1596 obtained from EnzyScreen. Haarlam, The Netherlands) and 96 well plate cultures were incubated at 37° C., 400 rpm for 24 hours. After 24 hours of production, all samples from each well were pelleted by centrifugation and the supernatant collected for subsequent analytical measurement. Triplicate micro-fermentations were performed for each strain. 
     For growth associated alanine micro-fermentations, glycerol stock preparation and 16 hour overnight culture in SM10++ proceeded as described above. After 16 hours of growth in SM10++ medium, 5 μL of overnight culture was inoculated into 150 μL FGM3 with 40 mM phosphate containing appropriate antibiotic. Plates were covered with sandwich covers (Model #CR1596 obtained from EnzyScreen, Haarlam, The Netherlands) and 96 well plate cultures were incubated at 37° C., 400 rpm for 24 hours. After 24 hours of production, OD600 was recorded, all samples from each well were then pelleted by centrifugation and the supernatant collected for subsequent analytical measurement. Triplicate micro-fermentations were performed for each strain. 
     Micro-fermentation robustness evaluations were conducted as described in Supplemental Materials, Section 8. 
     1 L Fermentation Seeds 
     Single colony from transformation plate was inoculated into 5 mL LB with appropriate antibiotics and cultured at 37° C., 220 rpm for 16 hours, 500 μL of the LB culture was inoculated into 50 mL SM10 media with appropriate antibiotics in square shake flask (CAT#: 25-214, Genesee Scientific, Inc. San Diego, Calif.), the culture was incubated at 37° C. with a shaking speed of 220 rpm for 24 hours, at which time OD600 is usually between 3 and 10, the culture was harvested by centrifugation at 4000 rpm for 15 min, supernatant was discarded and cell culture was normalized to OD600=10 using SM10 media. For 1 L fermentation seed, 6 mL of normalized OD600=10 culture was added to 1.5 mL of 50% glycerol in cryovials, and stored at −80° C. 
     1 L Fermentations 
     An Infors-HT Multifors (Laurel, Md., USA) parallel bioreactor system was used to perform 1 L fermentations, including three gas connection mass flow controllers configured for air, oxygen and nitrogen gases. Vessels used had a total volume of 1400 mL and a working volume of up to 1 L. Online pH and pO2 monitoring and control were accomplished with Hamilton probes. Offgas analysis was accomplished with a multiplexed Blue-in-One BlueSens gas analyzer (BlueSens. Northbrook. Ill., USA). Culture densities were continually monitored using Optek 225 mm OD probes, (Optek, Germantown, Wis., USA). The system used was running IrisV6.0 command and control software and integrated with a Seg-flowy automated sampling system (Flownamics, Rodeo, Calif., USA), including FISP cell free sampling probes, a Segmod 4800 and FlowFraction 96 well plate fraction collector. 
     For the standardized 2-stage process with ˜10 gcdw/L biomass, tanks were filled with 800 mL of FGM10 medium, with enough phosphate to target a final  E. coli  biomass concentration ˜10 gcdw/L. Antibiotics were added as appropriate. Frozen seed vials were thawed on ice and 7.5 mL of seed culture was used to inoculate the tanks. After inoculation, tanks were controlled at 37° C. and pH 6.8 using 5 M ammonium hydroxide and 1 M hydrochloric acid as titrants. 10 M ammonium hydroxide was used for  FIG. 3G  fermentation run. The following oxygen control scheme was used to maintain the desired dissolved oxygen set point. First gas flow rate was increased from a minimum of 0.3 L/min of air to 0.8 L/min of air, subsequently, if more aeration was needed, agitation was increased from a minimum of 300 rpm to a maximum of 1000 rpm. Finally, if more oxygen was required to achieve the set point, oxygen supplementation was included using the integrated mass flow controllers. Starting glucose concentration was 25 g/L. A constant concentrated sterile filtered glucose feed (500 g/L) was added to the tanks at specified rate, i.e. 2 g/h, once agitation reached 800 rpm. In cases where feed rate or dissolved oxygen content needed to be varied for robustness study, changes were made after cells entered stationary phase. Fermentation runs were extended for up to ˜50 hours after entry into stationary phase and samples automatically withdrawn every 3 hours. Samples were saved for subsequent analytical measurement. 
     In the case of growth associated fermentation processes, tanks were filled with 800 mL of FGM10 medium with 40 mM phosphate, which was in great excess and ensured phosphate depletion doesn&#39;t happen for growth associated fermentation processes. Antibiotics were added as appropriate. Frozen seed vials were thawed on ice and 7.5 mL of seed culture was used to inoculate the tanks. After inoculation, tanks were controlled at 37° C. and pH 6.8 using 5 M ammonium hydroxide and 1 M hydrochloric acid as titrants. The following oxygen control scheme was used to maintain the desired dissolved oxygen set point. First gas flow rate was increased from a minimum of 0.3 L/min of air to 0.8 L/min of air, subsequently, if more aeration was needed, agitation was increased from a minimum of 300 rpm to a maximum of 1000 rpm. Finally, if more oxygen was required to achieve the set point, oxygen supplementation was included using the integrated mass flow controllers. Starting glucose concentration was 25 g/L. A constant concentrated sterile filtered glucose feed (500 g/L) was added to the tanks at specified rate, i.e. 2 g/h, once agitation reached 800 rpm. Feed rate and dissolved oxygen concentration was set to desired values in the beginning, and maintained throughout the fermentation process. Fermentation runs were continued for up to˜50 hours and samples automatically withdrawn every 3 hours. Samples were saved for subsequent analytical analysis. 
     Analytical Methods 
     Sample standard curves for all compounds quantified are shown in Supplemental Materials. Section 10. 
     Glucose and Ethanol Quantification: A UPLC-R1 method was developed for the simultaneous quantification of glucose and ethanol concentrations, using an Acquity H-Class UPLC integrated with a Waters 2414 Refractive Index (RI) detector (Waters Corp., Milford, Mass. USA). Chromatographic separation was performed using a Bio-Rad Fast Acid Analysis HPLC Column (100×7.8 mm, 9 μm particle size: CAT#: #1250100. Bio-Rad Laboratories, Inc., Hercules, Calif.) at 65° C. 5 mM sulfuric acid was used as the eluent. The isocratic elution was as follows: 0-0.1 min, flow rate increased from 0.4 mL/min to 0.42 mL/min, 0.1-12 min flow rate at 0.48 mL/min. Sample injection volume was 10 μL. UPLC method development was carried out using standard aqueous stock solutions of analytes. Peak integration and further analysis was performed using MassLynx v4.1 software. The linear range used for glucose was 1-10 g/L, for ethanol was 1-20 g/L. Samples were diluted as needed to be within the accurate linear range. Dilution was performed using ultrapure water. 
     Alanine Quantification: A reverse phase UPLC-MS/MS method was developed for alanine. Chromatographic separation was performed using a Restek Ultra AQ C18 column (150 mm×2.1 i.d., 3 μm; CAT#: 9178362, Restek Corporation, Bellefonte, Pa.) at 70° C. The following eluents were used: solvent A: H 2 O, 0.2% formic acid and 0.05% ammonium (v/v); solvent B: MeOH, 0.2% formic acid and 0.05% ammonium (v/v). The gradient elution was as follows: 0-0.1 min isocratic 5% B, flow rate increased from 0.65 mL/min to 0.75 mL/min; 0.1-0.3 min, linear from 5% to 95% B at 0.75 mL/min; 0.3-0.9 min isocratic 95% B at 0.75 mL/min; and 0.9-1.2 min linear from 95% to 5% B at 0.75 mL/min; 1.2-1.3 min isocratic 5% B at 0.75 mL/min. Sample injection volume was 5 μL. UPLC method development was carried out using standard aqueous stock solutions of analyte. Separations were performed using an Acquity H-Class UPLC integrated with a Xevo™ TQD Mass spectrometer (Waters Corp., Milford. Mass. USA). MS/MS parameters including MRM transitions were tuned for each analyte and are listed in Table 22. Alanine (2.3-13C2, 99%) was used as internal standard for alanine at a concentration of 5 mg/L. Peak integration and further analysis was performed using MassLynx v4.1 software. The linear range for alanine was 1-100 mg/L. Samples were diluted as needed to be within the accurate linear range. Dilution was performed using ultrapure water, and the final 10-fold dilution was performed using solvent A, with 5 mg/l of C13 alanine (2,3-13C2, 99%). 
     Mevalonic Acid Quantification: A reverse phase UPLC-TUV method was developed for the simultaneous quantification of mevalonic acid and mevalonolactone. Chromatographic separation was performed using a Restek Ultra AQ C18 column (150 mm×2.1 i.d., 3 μm; CAT#: 9178362, Restek Corporation, Bellefonte, Pa.) at 30° C. 20 mM phosphoric acid was used as the eluent. The isocratic elution was as follows: 0-3 min isocratic at 1 mL/min. Sample injection volume was 10 μL. Absorbance was monitored at 210 nm. UPLC method development was carried out using standard aqueous stock solutions of analytes. Separations were performed using an Acquity H-Class UPLC (Waters Corp., Milford, Mass. USA). Peak integration and further analysis was performed using MassLynx v4.1 software. The linear range for mevalonic acid and mevalonolactone were 0.01-0.1 g/L. Samples were diluted as needed to be within the accurate linear range. Mevalonic acid diluted in 20 mM phosphoric acid would spontaneously convert to mevalonolactone 80 , thus, quantification of both mevalonic acid and mevalonolactone was necessary for fermentation samples. Mevalonic acid and mevalonolactone standards were prepared fresh each time, and ran immediately on UPLC. Dilution was performed using ultrapure water, and the final 10-fold dilution was performed using 20 mM phosphoric acid. 
     Alanine Stereoisomer Quantification: A reverse phase UPLC-TUV method was developed for the simultaneous quantification and differentiation of L-/D-alanine. Chromatographic separation was performed using a Chirex 3126 (D)-penicillamine column (150×4.6 mm, 5 μm; Phenomenex Inc., Torrance, Calif.) at 50° C. 2 mM Copper Sulfate was used as the eluent. The isocratic elution was as follows: 0-10 min at 0.75 mL/min. Sample injection volume was 10 μL. Absorbance was monitored at 254 nm. UPLC method development was carried out using standard aqueous stock solutions of analytes. Separations were performed using an Acquity H-Class UPLC (Waters Corp., Milford, Mass. USA). Peak integration and further analysis was performed using MassLynx v4.1 software. The linear range for L-/D-alanine was 0.1-1 g/L. Samples were diluted as needed to be within the accurate linear range. Dilution was performed using ultrapure water. 
     Supplemental Materials 
       
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Combinatorial complexity of metabolic networks. 
               
            
           
           
               
               
            
               
                   
                 Number of Experiments 
               
            
           
           
               
               
               
            
               
                   
                 Entire  E.   coli   
                   
               
               
                 Combination # 
                 Gene Network 
                 Reduced Central Metabolism Network 
               
               
                   
               
               
                 1 
                 4500 
                 ~45 (Glycolysis, TCA, PPP and ETC genes only) 
               
               
                 2 
                 1.0 × 10 6   
                    990 
               
               
                 3 
                  1.5 × 10 10   
                  14,190 
               
               
                 4 
                  1.7 × 10 13   
                 148,995 
               
               
                 5 
                  1.5 × 10 16   
                 1.2 × 10 6   
               
               
                   
               
            
           
         
       
     
     Section 1: Phosphate Promoters 
     Phosphate promoter sequences were obtained from the EcoCyc database 81  for PhoB regulated promoters (https://ecocyc.org/, Table 2). We sought to evaluate not only the relative strength of promoters previously characterized to respond to phosphate depletion, but in addition the relative leakiness in phosphate rich conditions. To this aim we constructed a set of fluorescent reporter plasmids. We cloned the ultraviolet excitable GFPuv gene behind a set of 12 phosphate dependent promoters, in the pSMART-HC-Kan (Lucigen. WI) backbone. These reporter strains were evaluated in a 2-stage micro-fermentation protocol in an m2p-labs Biolector™. Results are illustrated in  FIG. 7 . The ugpB gene promoter was often chosen for high level tightly controlled expression when expression cassettes were chromosomally integrated or for the inducible expression of guide arrays. 
     Insulators 82  were added to both 5′ and 3′ end of a subset of phosphate promoters (Table 3) to help with consistent performance in different sequence contexts. To reduce read-through transcription, a unique terminator was added to the 3′ end of each insulated promoter. Terminator sequences were from http://parts.igem.org/Terminators/Catalog. Insulated phosphate promoters were similarly characterized using GFPuv expression in a m2p-labs Biolector™ ( FIG. 8 ). 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Phosphate inducible promoter sequences evaluated; the ribosomal binding site 
               
               
                 is underlined, and the start codon of the gene (GFPuv) is shown in green. 
               
            
           
           
               
               
               
            
               
                 Promoter 
                   
                 SEQ 
               
               
                 Name 
                 Sequence 
                 ID NO 
               
               
                   
               
               
                 ugpBp 
                 TCTTTCTGACACCTTACTATCTTACAAATGTAACAAAAAAGTTATTTTTCTGTAATTCGA 
                  1 
               
               
                   
                 GCATGTCATGTTACCCCGCGAGCATAAAACGCGTGTGT AGGAGGA TAATCTATG 
                   
               
               
                   
               
               
                 yibDp 
                 GTGCGTAATTGTGCTGATCTCTTATATAGCTGCTCTCATTATCTCTCTACCCTGAAGTGAC 
                  2 
               
               
                   
                 TCTCTCACCTGTAAAAATAATATCTCACAGGCTTAATAGTTTCTTAATACAAAGCCTGTA 
                   
               
               
                   
                 AAACGTCAGGATAACTTCTGTGT AGGAGGA TAATCTATG 
                   
               
               
                   
               
               
                 phoAp 
                 CGATTACGTAAAGAAGTTATTGAAGCATCCTCGTCAGTAAAAAGTTAATCTTTTCAACA 
                  3 
               
               
                   
                 GCTGTCATAAAGTTGTCACGGCCGAGACTTATAGTCGCTTTGTTTTTATTTTTTAATGTAT 
                   
               
               
                   
                 TTGTAGTGTAGGAGGATAATCTATGGCTAGCAAAGG AGAAGAA CTTTTCACATG 
                   
               
               
                   
               
               
                 phoBp 
                 GCCACGGAAATCAATAACCTGAAGATATGTGCGACGAGCTTTTCATAAATCTGTCATAA 
                  4 
               
               
                   
                 ATCTGACGCATAATGACGTCGCATTAATGATCGCAACCTATTTATTGTGTAGGAGGATA 
                   
               
               
                   
                 ATCTATGGCTAGCAAAGG AGAAGAA CTTTTCACATG 
                   
               
               
                   
               
               
                 amnp 
                 AGACAGTCAACGCGCTTGATAGCCTGGCGAAGATCATCCGATCTTCGCCTTACACTTTTG 
                  5 
               
               
                   
                 TTTCACATTTCTGTGACATACTATCGGATGTGCGGTAATTGTAT AGGAGGA TAATCTATG 
                   
               
               
                   
               
               
                 ydfHp 
                 GCTATGCCGGACTGAATGTCCACCGTCAGTAATTTTTATACCCGGCGTAACTGCCGGGTT 
                  6 
               
               
                   
                 ATTGCTTGTCACAAAAAAGTGGTAGACTCATGCAGTTAACTCACTGTGT AGGAGGA TAA 
                   
               
               
                   
                 TCTATG 
                   
               
               
                   
               
               
                 mipAp 
                 CATCCATAAATTTTGCATAATTAATGTAAAGACCAGGCTCGCCAGTAACGCTAAATTCA 
                  7 
               
               
                   
                 TTTGGCTGTAAGCGCGGTGTCATCCGCGTCAGGAAAATTAAACAGTTACTTTAAAAAAT 
                   
               
               
                   
                 GAAAACGTAAAAAGGTTGGGTTTCGATGTATTGACGGGTAAACTTTGTCGCCCGCTAAA 
                   
               
               
                   
                 CATTTGTTTGTGT AGGAGGA TAATCTATG 
                   
               
               
                   
               
               
                 phoHp 
                 AATCCTGCTGAAAGCACACAGCTTTTTTCATCACTGTCATCACTCTGTCATCTTTCCAGT 
                  8 
               
               
                   
                 AGAAACTAATGTCACTGAAATGGTGTTTTATAGTTAAATATAAGTAAATATATTGTTGCA 
                   
               
               
                   
                 ATAAATGCGAGATCTGTTGTACTTATTAAGTAGCAGCGGAAGTTCGTGT AGGAGGA TAA 
                   
               
               
                   
                 TCTAT 
                   
               
               
                   
               
               
                 yhjCp 
                 CTACAGAGATGACGTGTAGAAAATAGTTACCGATATAAATAGTTACAGCTAAACGCCTG 
                  9 
               
               
                   
                 AAATTACATGTCGAGGGCACTATTTAAAACAATTTTGAGGATTTCCTTATATTGGTGGTT 
                   
               
               
                   
                 AGTACGCATGCAATTAAAAATGAAATTCCGCGACCACAAGCCAAAATAACAAACGGCA 
                   
               
               
                   
                 AGGAGACAAAAATAAGCACAAATAGCCAACACGTCCTCTGTTCACTTTAAAGGGAATCG 
                   
               
               
                   
                 CTGAAAAATACGCTCTGTTTAAGGGGATTCACCTTTCTCAGAAAGCTATTCCGCCCTTTT 
                   
               
               
                   
                 CCTGCTGAGAAATCGCCACATTCGGCATGACAACATTGTGAAAGTGT AGGAGGA TAATC 
                   
               
               
                   
                 TATG 
                   
               
               
                   
               
               
                 phoUp 
                 ACCGAACTGAAGCAGGATTACACCGTGGTGATCGTCACCCACAACATGCAGCAGGCTGC 
                 10 
               
               
                   
                 GCGTTGTTCCGACCACACGGCGTTTATGTACCTGGGCGAATTGATTGAGTTCAGCAACA 
                   
               
               
                   
                 CGGACGATCTGTTCACCAGTGT AGGAGGA TAATCTATG 
                   
               
               
                   
               
               
                 pstSp 
                 AAGACTTTATCTCTCTGTCATAAAACTGTCATATTCCTTACATATAACTGTCACCTGTTTG 
                 11 
               
               
                   
                 TCCTATTTTGCTTCTCGTAGCCAACAAACAATGCTTTATGAGTGTAGGAGGATAATCTAT 
                   
               
               
                   
                 GGCTAGCAAAGG AGAAGAA CTTTTCACATG 
                   
               
               
                   
               
               
                 phoEp 
                 AGCATGGCGTTTTGTTGCGCGGGATCAGCAAGCCTAGCGGCAGTTGTTTACGCTTTTATT 
                 12 
               
               
                   
                 ACAGATTTAATAAATTACCACATTTTAAGAATATTATTAATCTGTAATATATCTTTAACA 
                   
               
               
                   
                 ATCTCAGGTTAAAAACTTTCCTGTTTTCAACGGGACTCTCCCGCTGGTGT AGGAGGA TAA 
                   
               
               
                   
                 TCTATG 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Insulated promoter sequences. Insulator sequences are italicized. -35 and -10 
               
               
                 boxes are highlighted in bold and underlined. 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ 
               
               
                 Insulated Promoter 
                 Sequence 
                 ID NO 
               
               
                   
               
               
                 BBa_B0015_IN_yibDp 
                 CCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTT 
                 13 
               
               
                   
                 TTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCT 
                   
               
               
                   
                 TCGGGTGGGCCTTTCTGCGTTTAT ACACAGCTAACACCACGTCGTCCCTATCTG   
                   
               
               
                   
                   CTGCCCTAGGTCTATGAGTGGTTGCTGGATAAC GTGCGTAATTGTGCTGATCTC 
                   
               
               
                   
                 TTATATAGCTGCTCTCATTATCTCTCTACCCTGAA   GTGACT   CTCTCACCTGTA 
                   
               
               
                   
                 AAAATAATATCTCACAGGCT   TAATA   GTTTCTTAATACAAAGCCTGTAAAACG 
                   
               
               
                   
                 TCAGGATAACTTCT ATATTCAGGGAGACCACAACGGTTTCCCTCTACAAATAATTT   
                   
               
               
                   
                 
                   TGTTTAACTTT 
                 
                   
               
               
                   
               
               
                 BBa_B1002_IN_phoBp 
                 CGCAAAAAACCCCGCTTCGGCGGGGTTTTTTCGCACCGTCTCCATCGCTTGCC 
                 14 
               
               
                   
                 CAAGTTGTGAAG CACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCT   
                   
               
               
                   
                   ATGAGTGGTTGCTGGATAAC GCCACGGAAATCAATAACCTGAAGATATGTGCCG 
                   
               
               
                   
                 ACGAGCTT   TTCATA   AATCTGTCATAAATCTGACG   CATAAT   GACGTCGCATTA 
                   
               
               
                   
                 ATGATCGCAACCTATTTATTA TATTCAGGGAGACCACAACGGTTTCCCTCTACCA   
                   
               
               
                   
                 
                   ATAATTTTGTTTAACTTT 
                 
                   
               
               
                   
               
               
                 BBa_B1004_IN_mipAp 
                 CGCCGAAAACCCCGCTTCGGCGGGGTTTTGCCGCACGTCTCCATCGCTTGCC 
                 15 
               
               
                   
                 CAAGTTGTGAAG CACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCT   
                   
               
               
                   
                   ATGAGTGGTTGCTGGATAAC CATCCATAAATTTTGCATAATTAATGTAAAGAC 
                   
               
               
                   
                 CAGGCTCGCCAGTAACGCTAAATTCATTTGGCTGTAAGCGCGGTGTCATCCG 
                   
               
               
                   
                 CGTCAGGAAAATTAAACAGTTACTTTAAAAAATGAAAACGTAAA   AAGGTT   G 
                   
               
               
                   
                 GGTTTCGATGTATTGACGG   GTAAAC   TTTGTCGCCCGCTAAACATTTGTTT ATA   
                   
               
               
                   
                 
                   TTCAGGGAGACCACAACGGTTTCCCTCTACAAATAATTTTGTTTAACTTT 
                 
                   
               
               
                   
               
               
                 BBa_B1006_IN_phoUp 
                 AAAAAAAAACCCCGCCCCTGACAGGGCGGGGTTTTTTTTACGTCTCCATCGC 
                 16 
               
               
                   
                 TTGCCCAAGTTGTGAAG CACAGCTAACACCACGTCGTCCCTATGCTGCCCTA   
                   
               
               
                   
                   GGTCTATGAGTGGTTGCTGGATAAC ACCGAACTGAAGCAGGATTACACCGTGG 
                   
               
               
                   
                 TGATCGTCACCCACAACATGCAGCAGGCTGCGCGTTGTTCCGACCACA   CGG     
                   
               
               
                   
                     CGT   TTATGTACCTGGGCGAATT   GATTGA   GTTCAGCAACACGGACGATCTGTT 
                   
               
               
                   
                 CACCA ATATTCAGGGAGACCACAACGGTTTCCCTCTACAAATAATTTTGTTTAACTT   
                   
               
               
                   
                 
                   T 
                 
                   
               
               
                   
               
               
                 BBa_B1010_IN_phoHp 
                 CGCCGCAAACCCCGCCCCTGACAGGGCGGGGTTTCGCCGCACGTCTCCATCG 
                 17 
               
               
                   
                 CTTGCCCAAGTTGTGAAG CACAGCTAACACCACGTCGTCCCTATCTGCTGCCCT   
                   
               
               
                   
                   AGGTCTATGAGTGGTTGCTGGATAAC AATCCTGCTGAAAGCACACAGCTTTTTT 
                   
               
               
                   
                 CATCACTGTCATCACT   CTGTCA   TCTTTCCAGTAGAAAC   TAATGT   CACTGAAA 
                   
               
               
                   
                 TGGTGTTTTATAGTTAAATATAAGTAAATATATTGTTGCAATAAATGCGAGA 
                   
               
               
                   
                 TCTGTTGTACTTATTAAGTAGCAGCGGAAGTTC ATATTCAGGGAGACCACAAC   
                   
               
               
                   
                 
                   GGTTTCCCTCTACAAATAATTTTGTTTAACTTT 
                 
               
               
                   
               
            
           
         
       
     
     Section 2: Constitutive Promoters 
     A set of constitutive insulated promoters of varying strength were used for constitutive expression and taken directly from Davis et al., including the proA, proB, proC, proD promoters 82  and HCEp promoter 83 . Insulator was added to 5′ and 3′ of HCEp promoter. Similar to insulated phosphate promoters, a unique terminator was added to the 5′ end of constitutive promoters. These were used to drive constitutive pathway expression in growth associated production strains as well as to make strain modifications where constitutive heterologous gene expression was appropriate. These promoter sequences are given in Table 4 below and promoter characterized using GFPuv expression ( FIG. 9 ). 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Constitutive promoter sequences. 
               
            
           
           
               
               
               
            
               
                 Promoter 
                 Sequence 
                 SEQ ID NO 
               
               
                   
               
               
                 BBa_B1004_proA 
                 CGCCGAAAACCCCGCTTCGGCGGGGTTTTGCCGCACGTC 
                 18 
               
               
                   
                 TCCATCGCTTGCCCAAGTTGTGAAGCACAGCTAACACCA 
                   
               
               
                   
                 CGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTG 
                   
               
               
                   
                 CTGGATAACTTTACGGGCATGCATAAGGCTCGTAGGCTA 
                   
               
               
                   
                 TATTCAGGGAGACCACAACGGTTTCCCTCTACAAATAAT 
                   
               
               
                   
                 TTTGTTTAACTTT 
                   
               
               
                   
               
               
                 BBa_B1006_proB 
                 AAAAAAAAACCCCGCCCCTGACAGGGCGGGGTTTTTTTT 
                 19 
               
               
                   
                 ACGTCTCCATCGCTTGCCCAAGTTGTGAAGCACAGCTAA 
                   
               
               
                   
                 CACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGT 
                   
               
               
                   
                 GGTTGCTGGATAACTTTACGCGCATGCATAAGGCTCGTA 
                   
               
               
                   
                 ATATATATTCAGGGAGACCACAACGGTTTCCCTCTACAA 
                   
               
               
                   
                 ATAATTTTGTTTAACTTT 
                   
               
               
                   
               
               
                 BBa_B1010_proC 
                 CGCCGCAAACCCCGCCCCTGACAGGGCGGGGTTTCGCC 
                 20 
               
               
                   
                 GCACGTCTCCATCGCTTGCCCAAGTTGTGAAGCACAGCT 
                   
               
               
                   
                 AACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGA 
                   
               
               
                   
                 GTGGTTGCTGGATAACTTTACGGGCATGCATAAGGCTCG 
                   
               
               
                   
                 TATGATATATTCAGGGAGACCACAACGGTTTCCCTCTAC 
                   
               
               
                   
                 AAATAATTTTGTTTAACTTT 
                   
               
               
                   
               
               
                 BBa_B1002_proD 
                 CGCAAAAAACCCCGCTTCGGCGGGGTTTTTTCGCACGTC 
                 21 
               
               
                   
                 TCCATCGCTTGCCCAAGTTGTGAAGCACAGCTAACACCA 
                   
               
               
                   
                 CGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTG 
                   
               
               
                   
                 CTGGATAACTTTACGGGCATGCATAAGGCTCGTATAATA 
                   
               
               
                   
                 TATTCAGGGAGACCACAACGGTTTCCCTCTACAAATAAT 
                   
               
               
                   
                 TTTGTTTAACTTT 
                   
               
               
                   
               
               
                 BBa_B0015_IN_HCEp 
                 CCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGAC 
                 22 
               
               
                   
                 TGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTC 
                   
               
               
                   
                 TCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTT 
                   
               
               
                   
                 TCTGCGTTTATACACAGCTAACACCACGTCGTCCCTATC 
                   
               
               
                   
                 TGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACCTCC 
                   
               
               
                   
                 TTCACAGATTCCCAATCTCTTGTTAAATAACGAAAAAGC 
                   
               
               
                   
                 ATCAATTAAAACCCATGTCTTTCTATATTCCAGCAATGT 
                   
               
               
                   
                 TTTATAGGGGACATATTGATGAAGATGGGTATCACCTTA 
                   
               
               
                   
                 GTGAATTGCTATAAGCTGCTCTTTTTTGTTCGTGATATAC 
                   
               
               
                   
                 TGATAAATTGAATTTTCACACTTCATATTCAGGGAGACC 
                   
               
               
                   
                 ACAACGGTTTCCCTCTACAAATAATTTTGTTTAACTTT 
               
               
                   
               
            
           
         
       
     
     Section 3: Chromosomally Modified Host Strains 
       FIG. 11  depicts each chromosomal modification Strains utilized and/or constructed for this study are listed in Table 5. Tables 6 and 7 lists oligonucleotides and synthetic DNA sequences used for strain construction and/or confirmation.  FIG. 12  and  FIG. 13A-E  show growth rates and glucose distribution during growth for control strains in 1 L fermentation. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 List of chromosomally modified strains. 
               
            
           
           
               
               
               
            
               
                 Strain 
                 Genotype 
                 Source 
               
               
                   
               
               
                 BW25113 (wt) 
                 F-, λ-, Δ(araD-ataB)567, lacZ4787(del)(::rrnB-3), rph-1, 
                 CGSC 
               
               
                   
                 Δ(rhaD-rhaB)568, hsdR514 
                   
               
               
                 JW3197-1 
                 BW25113, sspB756(del)::.kan 
                 
                   53 
                 
               
               
                 Bwapldf 
                 BW25113, ΔackA-pta, ΔpoxB, ΔpflB, ΔldhA, ΔadhE 
                 
                   39 
                 
               
               
                 DLF_0001  
                 BWapldf, ΔiclR, ΔarcA 
                 this study 
               
               
                 DLF_0002  
                 BWapldf, ΔiclR, ΔarcA, ΔsspB::frt 
                 this study 
               
               
                 DLF_0025  
                 DLF_0002, Δcas3::tm-ugpb-sspB-pro-casA(N2S) 
                 this study 
               
               
                 DLF_0028  
                 DLF_0025, fabl-DAS + 4-gentR 
                 this study 
               
               
                 DLF_0031  
                 DLF_0025, lpd-DAS + 4-gentR 
                 this study 
               
               
                 DLF_0038  
                 DLF_0025, fabI-DAS + 4-gentR, udhA-DAS + 4-bsdR 
                 this study 
               
               
                 DLF_0039  
                 DLF_0025, fabI-DAS + 4-gentR, gltA-DAS + 4-zeoR 
                 this study 
               
               
                 DLF_0040  
                 DLF_0025, fabI-DAS + 4-gentR, zwf-DAS + 4-bsdR 
                 this study 
               
               
                 DLF_0041  
                 DLF_0025, lpd-DAS + 4-gentR, gltA-DAS + 4-zeoR 
                 this study 
               
               
                 DLF_0042  
                 DLF_0025, lpd-DAS + 4-gentR, udhA-DAS + 4-bsdR 
                 this study 
               
               
                 DLF_0043  
                 DLF_0025, gltA-DAS + 4-zeoR 
                 this study 
               
               
                 DLF_0044  
                 DLF_0025, gltA-DAS + 4-zeoR, zwf-DAS + 4-bsdR 
                 this study 
               
               
                 DLF_0045  
                 DLF_0025, gltA-DAS + 4-zeoR, udhA-DAS + 4-bsdR 
                 this study 
               
               
                 DLF_0046  
                 DLF_0025, fabI-DAS + 4-gentR, gltA-DAS + 
                 this study 
               
               
                   
                 4-zeoR, zwf-DAS + 4-bsdR 
                   
               
               
                 DLF_0047  
                 DLF_0025, fabI-DAS + 4-aentR, gltA-DAS + 
                 this study 
               
               
                   
                 4::zeoR, udhA-DAS + 4-bsdR 
                   
               
               
                 DLF_0048  
                 DLF_0025, lpd-DASH + 4-gentR, gltA-DAS + 
                 this study 
               
               
                   
                 4-zeoR, zwf-DAS + 4-bsdR 
                   
               
               
                 DLF_0049  
                 DLF_0025, lpd-DAS + 4-gentR, gltA-DAS + 
                 this study 
               
               
                   
                 4-zeoR, udhA-DAS + 4-bsdR 
                   
               
               
                 DLF_0165  
                 DLF_0025, lpd-DAS + 4-gentR, zwf-DAS + 4-bsdR 
                 this study 
               
               
                 DLF_0763  
                 DLF_0025, udhA-DAS + 4-bsdR 
                 this study 
               
               
                 DLF_01002 
                 DLF_0025, zwf-DAS + 4-bsdR 
                 this study 
               
               
                 DLF_01517 
                 DLF_0012, Δcas3::pro-casA(N2S) 
                 this study 
               
               
                 DLF_01530 
                 DLF_0025, fabI-DAS + 4-gentR, udhA-DAS + 
                 this study 
               
               
                   
                 4-bsdR, zeoR-proDp-gapN-zeoR 
                   
               
               
                 DLF_01531 
                 DLF_0025, fabI-DAS + 4-gentR, udhA-DAS + 
                 this study 
               
               
                   
                 4-bsdR, gltA-DAS + 4-purR 
                   
               
               
                 DLF_01532 
                 DLF_0025, fabI-DAS + 4-gentR, udhA-DAS + 
                 this study 
               
               
                   
                 4-bsdR, gapA-DAS + 4-zeoR-proDp-gapN 
                   
               
               
                 DLF_01533 
                 DLF_0025, fabI-DAS + 4-gentR, udhA-DAS + 4-bsdR, 
                 this study 
               
               
                   
                 gapA-DAS + 4-zeoR-proDp-gapN, gltA-DAS + 4-purR 
                   
               
               
                 DLF_01536 
                 DLF_0025, fabI-DAS + 4-gentR, udhA-DAS + 
                 this study 
               
               
                   
                 4-bsdR, zeoR-proDp-gapN, gltA-DAS + 4-purR 
                   
               
               
                 DLF_01537 
                 DLF_0025, fabI-DAS + 4-gentR, udhA-DAS + 
                 this study 
               
               
                   
                 4-bsdR, gapA-DAS + 4-zeoR 
                   
               
               
                 DLF_01538 
                 DLF_0025, fabI-DAS + 4-gentR, gltA-DAS + 
                 this study 
               
               
                   
                 4-zeoR, udhA-DAS + 4-bsdR, gapA-DAS + 4-zeoR 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Oligonucleotides utilized for strain construction 
               
            
           
           
               
               
               
            
               
                 Oligo 
                 Sequence 
                 SEQ ID NO 
               
               
                   
               
               
                 ilcR_tetA_F 
                 TAACAATAAAAATGAAAATGATTTCCACGATACAGAAA 
                 23 
               
               
                   
                 AAAGAGACTGTCATCCTAATTTTTGTTGACACTCTATC 
                   
               
               
                   
               
               
                 ilcR_sacB_R 
                 TGCCACTCAGGTATGATGGGCAGAATATTGCCTCTGCCC 
                 24 
               
               
                   
                 GCCAGAAAAAGATCAAAGGGAAAACTGTCCATATGC 
                   
               
               
                   
               
               
                 iclR_500up 
                 CCCGACAGGGATTCCATCTG 
                 25 
               
               
                   
               
               
                 iclR_500dn 
                 TATGACGACCATTTTGTCTACAGTTC 
                 26 
               
               
                   
               
               
                 arcA_tetA_F 
                 GGACTTTTGTACTTCCTGTTTCGATTTAGTTGGCAATTTA 
                 27 
               
               
                   
                 GGTAGCAAACTCCTAATTTTTGTTGACACTCTATC 
                   
               
               
                   
               
               
                 arcA_sacB_R 
                 ATAAAAACGGCGCTAAAAAGCGCCGTTTTTTTTGACGGT 
                 28 
               
               
                   
                 GGTAAAGCCGAATCAAAGGGAAAACTGTCCATATGC 
                   
               
               
                   
               
               
                 arcA_500up 
                 CCTGACTGTACTAACGGTTGAG 
                 29 
               
               
                   
               
               
                 arcA_500dn 
                 TGACTTTTATGGCGTTCTTTGTTTTTG 
                 30 
               
               
                   
               
               
                 sspB_kan_F 
                 CTGGTACACGCTGATGAACACC 
                 31 
               
               
                   
               
               
                 sspB_kan_R 
                 CTGGTCATTGCCATTTGTGCC 
                 32 
               
               
                   
               
               
                 sspB_conf_F 
                 CAATCAGAGCGTTCCGACCC 
                 33 
               
               
                   
               
               
                 sspB_conf_R 
                 GTACGCAGTTTGCCAACGTG 
                 34 
               
               
                   
               
               
                 cas3_tetA_F 
                 AATAGCCCGCTGATATCATCGATAATACTAAAAAAAACAG 
                 35 
               
               
                   
                 GGAGGCTATTATCCTAATTTTTGTTGACACTCTATC 
                   
               
               
                   
               
               
                 cas3_sacB_R 
                 TACAGGGATCCAGTTATCAATAAGCAAATTCATTTGTTCT 
                 36 
               
               
                   
                 CCTTCATATGATCAAAGGGAAAACTGTCCATATGC 
                   
               
               
                   
               
               
                 cas3_conf_F 
                 CAAGACATGTGTATATCACTGTAATTC 
                 37 
               
               
                   
               
               
                 cas3_500dn 
                 GCGATTGCAGATTTATGATTTGG 
                 38 
               
               
                   
               
               
                 fabl_conf_F 
                 GCAAAATGCTGGCTCATTG 
                 39 
               
               
                   
               
               
                 gapA_conf_F 
                 GAACTGAATGGCAAACTGACTG 
                 40 
               
               
                   
               
               
                 gapA_500dn 
                 TGGGGATGATCGACCACA 
                 41 
               
               
                   
               
               
                 gltA_conf_F 
                 TATCATCCTGAAAGCGATGG 
                 42 
               
               
                   
               
               
                 lpd_conf_F 
                 ATCTCACCGTGTGATCGG 
                 43 
               
               
                   
               
               
                 udhA_conf_F 
                 CAAAAGAGATTCTGGGTATTCACT 
                 44 
               
               
                   
               
               
                 zwf_conf_F 
                 CTGCTGGAAACCATGCG 
                 45 
               
               
                   
               
               
                 zwf_500dn 
                 AGAGCATGTCGTTATAGGAGGTGAT 
                 46 
               
               
                   
               
               
                 ampR_intR 
                 AGTACTCAACCAAGTCATTCTG 
                 47 
               
               
                   
               
               
                 bsdR_intR 
                 GAGCATGGTGATCTTCTCAGT 
                 48 
               
               
                   
               
               
                 gentR_intR 
                 GCGATGAATGTCTTACTACGGA 
                 49 
               
               
                   
               
               
                 purR_intR 
                 GTCGCTGGGTAATCTGCAA 
                 50 
               
               
                   
               
               
                 tetA_intR 
                 ATCAACGCATATAGCGCTAGCAG 
                 51 
               
               
                   
               
               
                 zeoR_intR 
                 ACTGAAGCCCAGACGATC 
                 52 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Synthetic DNA utilized for strain construction. 
               
            
           
           
               
               
            
               
                   
                 SEQ ID 
               
               
                   
                 NO 
               
               
                   
               
               
                 tetA-sacB Cassette 
                   
               
               
                 TCCTAATTTTTGTTGACACTCTATCATTGATAGAGTTATTTTACCACTCCCTA 
                 53 
               
               
                 TCAGTGATAGAGAAAAGTGAAATGAATAGTTCGACAAAGATCGCATTGGTA 
                   
               
               
                 ATTACGTTACTCGATGCCATGGGGATTGGCCTTATCATGCCAGTCTTGCCAA 
                   
               
               
                 CGTTATTACGTGAATTTATTGCTTCGGAAGATATCGCTAACCACTTTGGCGT 
                   
               
               
                 ATTGCTTGCACTTTATGCGTTAATGCAGGTTATCTTTGCTCCTTGGCTTGGAA 
                   
               
               
                 AAATGTCTGACCGATTTGGTCGGCGCCCAGTGCTGTTGTTGTCATTAATAGG 
                   
               
               
                 CGCATCGCTGGATTACTTATTGCTGGCTTTTTCAAGTGCGCTTTGGATGCTGT 
                   
               
               
                 ATTTAGGCCGTTTGCTTTCAGGGATCACAGGAGCTACTGGGGCTGTCGCGGC 
                   
               
               
                 ATCGGTCATTGCCGATACCACCTCAGCTTCTCAACGCGTGAAGTGGTTCGGT 
                   
               
               
                 TGGTTAGGGGCAAGTTTTGGGCTTGGTTTAATAGCGGGGCCTATTATTGGTG 
                   
               
               
                 GTTTTGCAGGAGAGATTTCACCGCATAGTCCCTTTTTTATCGCTGCGTTGCTA 
                   
               
               
                 AATATTGTCACTTTCCTTGTGGTTATGTTTTGGTTCCGTGAAACCAAAAATAC 
                   
               
               
                 ACGTGATAATACAGATACCGAAGTAGGGGTTGAGACGCAATCGAATTCGGT 
                   
               
               
                 ATACATCACTTTATTTAAAACGATGCCCATTTTGTTGATTATTTATTTTTCAG 
                   
               
               
                 CGCAATTGATAGGCCAAATTCCCGCAACGGTGTGGGTGCTATTTACCGAAA 
                   
               
               
                 ATCGTTTTGGATGGAATAGCATGATGGTTGGCTTTTCATTAGCGGGTCTTGG 
                   
               
               
                 TCTTTTACACTCAGTATTCCAAGCCTTTGTGGCAGGAAGAATAGCCACTAAA 
                   
               
               
                 TGGGGCGAAAAAACGGCAGTACTGCTCGGATTTATTGCAGATAGTAGTGCA 
                   
               
               
                 TTTGCCTTTTTAGCGTTTATATCTGAAGGTTGGTTAGTTTTCCCTGTTTTAATT 
                   
               
               
                 TTATTGGCTGGTGGTGGGATCGCTTTACCTGCATTACAGGGAGTGATGTCTA 
                   
               
               
                 TCCAAACAAAGAGTCATCAGCAAGGTGCTTTACAGGGATTATTGGTGAGCC 
                   
               
               
                 TTACCAATGCAACCGGTGTTATTGGCCCATTACTGTTTGCTGTTATTTATAAT 
                   
               
               
                 CATTCACTACCAATTTGGGATGGCTGGATTTGGATTATTGGTTTAGCGTTTTA 
                   
               
               
                 CTGTATTATTATCCTGCTATCGATGACCTTCATGTTAACCCCTCAAGCTCAGG 
                   
               
               
                 GGAGTAAACAGGAGACAAGTGCTTAGTTATTTCGTCACCAAATGATGTTATT 
                   
               
               
                 CCGCGAAATATAATGACCCTCTTGATAACCCAAGAGCATCACATATACCTGC 
                   
               
               
                 CGTTCACTATTATTTAGTGAAATGAGATATTATGATATTTTCTGAATTGTGAT 
                   
               
               
                 TAAAAAGGCAACTTTATGCCCATGCAACAGAAACTATAAAAAATACAGAGA 
                   
               
               
                 ATGAAAAGAAACAGATAGATTTTTTAGTTCTTTAGGCCCGTAGTCTGCAAAT 
                   
               
               
                 CCTTTTATGATTTTCTATCAAACAAAAGAGGAAAATAGACCAGTTGCAATCC 
                   
               
               
                 AAACGAGAGTCTAATAGAATGAGGTCGAAAAGTAAATCGCGCGGGTTTGTT 
                   
               
               
                 ACTGATAAAGCAGGCAAGACCTAAAATGTGTAAAGGGCAAAGTGTATACTT 
                   
               
               
                 TGGCGTCACCCCTTACATATTTTAGGTCTTTTTTTATTGTGCGTAACTAACTT 
                   
               
               
                 GCCATCTTCAAACAGGAGGGCTGGAAGAAGCAGACCGCTAACACAGTACAT 
                   
               
               
                 AAAAAAGGAGACATGAACGATGAACATCAAAAAGTTTGCAAAACAAGCAA 
                   
               
               
                 CAGTATTAACCTTTACTACCGCACTGCTGGCAGGAGGCGCAACTCAACCGTT 
                   
               
               
                 TGCGAAAGAAACGAACCAAAAGCCATATAAGGAAACATACGGCATTTCCCA 
                   
               
               
                 TATTACACGCCATGATATGCTGCAAATCCCTGAACAGCAAAAAAATGAAAA 
                   
               
               
                 ATATCAAGTTCCTGAGTTCGATTCGTCCACAATTAAAAATATCTCTTCTGCA 
                   
               
               
                 AAAGGCCTGGACGTTTGGGACAGCTGGCCATTACAAAACGCTGACGGCACT 
                   
               
               
                 GTCGCAAACTATCACGGCTACCACATCGTCTTTGCATTAGCCGGAGATCCTA 
                   
               
               
                 AAAATGCGGATGACACATCGATTTACATGTTCTATCAAAAAGTCGGCGAAA 
                   
               
               
                 CTTCTATTGACAGCTGGAAAAACGCTGGCCGCGTCTTTAAAGACAGCGACA 
                   
               
               
                 AATTCGATGCAAATGATTCTATCCTAAAAGACCAAACACAAGAATGGTCAG 
                   
               
               
                 GTTCAGCCACATTTACATCTGACGGAAAAATCCGTTTATTCTACACTGATTT 
                   
               
               
                 CTCCGGTAAACATTACGGCAAACAAACACTGACAACTGCACAAGTTAACGT 
                   
               
               
                 ATCAGCATCAGACAGCTCTTTGAACATCAACGGTGTAGAGGATTATAAATC 
                   
               
               
                 AATCTTTGACGGTGACGGAAAAACGTATCAAAATGTACAGCAGTTCATCGA 
                   
               
               
                 TGAAGGCAACTACAGCTCAGGCGACAACCATACGCTGAGAGATCCTCACTA 
                   
               
               
                 CCTTAGAAGATAAAGGCCACAAATACTTAGTATTTGAAGCAAACACTGGAAC 
                   
               
               
                 TGAAGATGGCTACCAAGGCGAAGAATCTTTATTTAACAAAGCATACTATGG 
                   
               
               
                 CAAAAGCACATCATTCTTCCGTCAAGAAAGTCAAAAACTTCTGCAAAGCGA 
                   
               
               
                 TAAAAAACGCACGGCTGAGTTAGCAAACGGCGCTCTCGGTATGATTGAGCT 
                   
               
               
                 AAACGATGATTACACACTGAAAAAAGTGATGAAACCGCTGATTGCATCTAA 
                   
               
               
                 CACAGTAACAGATGAAATTGAACGCGCGAACGTCTTTAAAATGAACGGCAA 
                   
               
               
                 ATGGTACCTGTTCACTGACTCCCGCGGATCAAAAATGACGATTGACGGCATT 
                   
               
               
                 ACGTCTAACGATATTTACATGCTTGGTTATGTTTCTAATTCTTTAACTGGCCC 
                   
               
               
                 ATACAAGCCGCTGAACAAAACTGGCCTTGTGTTAAAAATGGATCTTGATCCT 
                   
               
               
                 AACGATGTAACCTTTACTTACTCACACTTCGCTGTACCTCAAGCGAAAGGAA 
                   
               
               
                 ACAATGTCGTGATTACAAGCTATATGACAAACAGAGGATTCTACGCACTACA 
                   
               
               
                 AACAATCAACGTTTGCGCCAAGCTTCCTGCTGAACATCAAAGGCAAGAAAA 
                   
               
               
                 CATCTGTTGTCAAAGACAGCATCCTTGAACAAGGACAATTAACAGTTAACA 
                   
               
               
                 AATAAAAACGCAAAAGAAAATGCCGATATTGACTACCGCAAGCAGTGTGAC 
                   
               
               
                 CGTGTGCTTCTCAAATGCCTGATTCAGGCTGTCTATGTGTGACTGTTGAGCT 
                   
               
               
                 GTAACAAGTTGTCTCAGGTGTTCAATTTCATGTTCTAGTTGCTTTGTTTTACT 
                   
               
               
                 GGTTTCACCTGTTCTATTAGGTGTTACATGCTGTTCATCTGTTACATTGTCGA 
                   
               
               
                 TCTGTTCATGGTGAACAGCTTTAAATGCACCAAAAACTCGTAAAAGCTCTGA 
                   
               
               
                 TGTATCTATCTTTTTTACACCGTTTTCATCTGTGCATATGGACAGTTTTCCCTT 
                   
               
               
                 TGAT 
                   
               
               
                   
               
               
                 ΔiclR-cure 
                   
               
               
                 AAATGATTTCCACGATACAGAAAAAAGAGACTGTCATGGGCAGAATATTGC 
                 54 
               
               
                 CTCTGCCCGCCAGAAAAAG 
                   
               
               
                   
               
               
                 ΔarcA-cure 
                   
               
               
                 CTGTTTCGATTTAGTTGGCAATTTAGGTAGCAAACTCGGCTTTACCACCGTC 
                 55 
               
               
                 AAAAAAAACGGCGCTTTT 
                   
               
               
                   
               
               
                 Δcas3-pro-casA 
                   
               
               
                 CAAGACATGTGTATATCACTGTAATTCGATATTTATGAGCAGCATCGAAAAA 
                 56 
               
               
                 TAGCCCGCTGATATCATCGATAATACTAAAAAAACAGGGAGGCTATTACCA 
                   
               
               
                 GGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTA 
                   
               
               
                 TCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCG 
                   
               
               
                 GGTGGGCCTTTCTGCGTTTATATCTTTCTGACACCTTACTATCTTACAAATGT 
                   
               
               
                 AACAAAAAAGTTATTTTTCTGTAATTCGAGCATGTCATGTTACCCCGCGAGC 
                   
               
               
                 ATAAAACGCGTGTGTAGGAGGATAATCTTTGACGGCTAGCTCAGTCCTAGGT 
                   
               
               
                 ACAGTGCTAGCCATATGAAGGAGAACAAATGAATTTGCTTATTGATAACTG 
                   
               
               
                 GATCCCTGTACGCCCGCGAAACGGGGGGAAAGTCCAAATCATAAATCTGCA 
                   
               
               
                 ATCGCTATAC 
                   
               
               
                   
               
               
                 Δcas3::ugBp-sspB-pro-casA 
                   
               
               
                 CAAGACATGTGTATATCACTGTAATTCGATATTTATGAGCAGCATCGAAAAA 
                 57 
               
               
                 TAGCCCGCTGATATCATCGATAATACTAAAAAAACAGGGAGGCTATTACCA 
                   
               
               
                 GGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTA 
                   
               
               
                 TCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCG 
                   
               
               
                 GGTGGGCCTTTCTGCGTTTATATCTTTCTGACACCTTACTATCTTACAAATGT 
                   
               
               
                 AACAAAAAAGTTATTTTTCTGTAATTCGAGCATGTCATGTTACCCCGCGAGC 
                   
               
               
                 ATAAAACGCGTGTGTAGGAGGATAATCTATGGATTTGTCACAGCTAACACC 
                   
               
               
                 ACGTCGTCCCTATCTGCTGCGTGCATTCTATGAGTGGTTGCTGGATAACCAG 
                   
               
               
                 CTCACGCCGCACCTGGTGGTGGATGTGACGCTCCCTGGCGTGCAGGTTCCTA 
                   
               
               
                 TGGAATATGCGCGTGACGGGCAAATCGTACTCAACATTGCGCCGCGTGCTGT 
                   
               
               
                 CGGCAATCTGGAACTGGCGAATGATGAGGTGCGCTTTAACGCGCGCTTTGGT 
                   
               
               
                 GGCATTCCGCGTCAGGTTTCTGTGCCGCTGGCTGCCGTGCTGGCTATCTACG 
                   
               
               
                 CCCGTGAAAATGGCGCAGGCACGATGTTTGAGCCTGAAGCTGCCTACGATG 
                   
               
               
                 AAGATACCAGCATCATGAATGATGAAGAGGCATCGGCAGACAACGAAACC 
                   
               
               
                 GTTATGTCGGTTATTGATGGCGACAAGCCAGATCACGATGATGACACTCATC 
                   
               
               
                 CFGACGATGAACCTCCGCAGCCACCACGCGGTGGTCGACCGGCATTACGCG 
                   
               
               
                 TTGTGAAGTAATTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCCATATG 
                   
               
               
                 AAGGAGAACAAATGAATTTGCTTATTGATAACTGGATCCCTGTACGCCCGCG 
                   
               
               
                 AAACGGGGGGAAAGTCCAAATCATAAATCTGCAATCGCTATAC 
                   
               
               
                   
               
               
                 fabI-DAS+4-gentR 
                   
               
               
                 CTATTGAAGATGTGOGTAACTCTGCGCCATTCCTGTGCTCCGATCTCTCTGC 
                 58 
               
               
                 CGOTATCTCCGGTGAAGTGGTCCACGTTGACGGCGGTTTCAGCATTGCTGCA 
                   
               
               
                 ATGAACGAACTCGAACTGAAAGCGGCCAACGATGAAAACTATTCTGAAAAC 
                   
               
               
                 TATGCGGATGCGTCTTAATAGGAAGTTCCTATTCTCTAGAAAGTATAGGAAC 
                   
               
               
                 TTCCGAATCCATGTGGGAGTTTATTCTTGACACAGATATTTATGATATAATA 
                   
               
               
                 ACTGAGTAAGCTTAACATAAGGAGGAAAAACATATGTTACGCAGCAGCAAC 
                   
               
               
                 GATGTTACGGAGCAGGGCAGTCGCCCTAAAACAAAGTTAGGTGGCTCAAGT 
                   
               
               
                 ATGGGCATCATTCGCACATGTAGGCTCGGCCCTGACCAAGTCAAATCCATGC 
                   
               
               
                 GGGCTGCTCTTGATCTTTTCGGTCGTGAGTTCGGAGACGTAGCCACCTACTC 
                   
               
               
                 CCAACATCAGCCGGACTCCGATTACCTCGGGAACTTGCTCCGTAGTAAGACA 
                   
               
               
                 TTCATCGCGCTTGCTGCCTTCGACCAAGAAGCGGTTGTTGGCGCTCTCGCGG 
                   
               
               
                 CTTACGTTCTGCCCAAGTTTGAGCAGCCGCGTAGTGAGATCTATATCTATGA 
                   
               
               
                 TCTCGCAGTCTCCGGCGAGCACCGGAGGCAGGGCATTGCCACCGCGCTCAT 
                   
               
               
                 CAATCTCCTCAAGCATGAGGCCAACGCGCTTGGTGCTTATGTGATCTACGTG 
                   
               
               
                 CAAGCAGATTACGGTGACGATCCCGCACTGGCTCTCTATACAAAGTTGGGC 
                   
               
               
                 ATACGGGAAGAAGTGATGCACTTTGATATCGACCCAAGTACCGCCACCTAA 
                   
               
               
                 GAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCCGTTCTGTTGGTAAAGAT 
                   
               
               
                 GGGCGGCGTTCTGCCGCCCGTTATCTCTGTTATACCTTTCTGATATTTGTTAT 
                   
               
               
                 CGCCGATCCGTCTTTCTCCCCTTCCCGCCTTGCGTCAGG 
                   
               
               
                   
               
               
                 gapA-DAS+4-zeoR-proDp-gapN 
                   
               
               
                 TCTCCAAAGCGGCCAACGATGAAAACTATTCTGAAAACTATGCGGATGCGT 
                 59 
               
               
                 CTTGATTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCAACTTTAAAATT 
                   
               
               
                 AAAGAGGTATATATTAATGACTAACCAATATAAGAATTACGTAAATGGGGA 
                   
               
               
                 GTGGAAGCTTTCGGAGAATGAAATTAAGATCTATGAACCAGCCAGTGGGGC 
                   
               
               
                 GGAATTGGGGTCAGTCCCGGCAATGTCCACTGAAGAAGTFGACTATGTCTAC 
                   
               
               
                 GCCTCGGCCAAAAAAGCGCAGCCAGCATGGCGCTCGCTTTCCTATATTGAGC 
                   
               
               
                 GTGCGGCTTATTTGCACAAAGTCGCAGACATCCTGATGCGTGACAAGGAGA 
                   
               
               
                 AAATTGGAGCGGTATTGTCCAAGGAAGTAGCGAAAGGCTACAAATCCGCAG 
                   
               
               
                 TATCGGAGGTCGTCCGCACCGCCGAGATTATTAATTATGCGGCCGAAGAAG 
                   
               
               
                 GGCTTCGCATGGAGGGTGAGCTTCTTGGAGGGCGGCAGTTTTGAGGCGGCAT 
                   
               
               
                 CCAAGAAAAAAATCGCTGTCGTCCGTCGCGAGCCGGTGGGACTTCTGCTTG 
                   
               
               
                 CTATTAGTCCGTTCAATTACCCCGTGAATCTGGCCGGCTCCAAGATTCCCC 
                   
               
               
                 TGCACTGATCGCGGGCAATGTAATCGCTTTTAAACCACCGACCCAAGGATCG 
                   
               
               
                 ATTAGTGGACTTCTTTTAGCGGAGGCGTTTGCGGAGGCAGGTCTTCCAGCCG 
                   
               
               
                 GCGTATTCAATACCATCACGGGGCGTGGAAGTGAAATCGGGGATTACATCG 
                   
               
               
                 TGGAGCACCAGGCAGTAAATTTCATCAACTTCACGGGTTCCACGGGGATCG 
                   
               
               
                 GGGAGCGTATCGGTAAGATGGCTGGGATGCGTCCGATCATGTTGGAACTTG 
                   
               
               
                 GCGGCAAGGATAGTGCGATTGTGCTGGAAGACGCAGACTTGGAATTCACAG 
                   
               
               
                 CTAAAAACATTATCGCTGGAGCCTTCGGGTATAGTGGTCAACGTTGGACGGC 
                   
               
               
                 AGTTAAGCGCGTTCTTGTTATGGAAAGTGTCGCGGATGAATTGGTCGAGAA 
                   
               
               
                 GATTCGCGAGAAAGTGTTAGCTCTTACGATTGGAAATCCAGAGGACGATGC 
                   
               
               
                 TGACATCACTCCATTGATCGACACGAAATCCGCGGATTACGTCGAGGGGCT 
                   
               
               
                 GATCAACGACGCGAACGATAAGGGAGCAGCGGCTTTGACCGAGATCAAACG 
                   
               
               
                 CGAGGGGAACCTGATCTGCCCGATTCTTTTTGACAAAGTCACAACTGACATG 
                   
               
               
                 CGCTTGGCATGGGAAGAACCCTTCGGCCCAGTCTTGCCTATTATCCGCGTTA 
                   
               
               
                 CTAGCGTAGAGGAAGCAATTGAAATTTCCAATAAATCCGAATATGGGTTGC 
                   
               
               
                 AAGCGAGTATCTTTACTAACGATTTTCCACGTGCCTTTGGTATTGCGGAACA 
                   
               
               
                 GTTAGAAGTCGGGACAGTTCACATCAACAACAAGACGCAGCGCGGGACAGA 
                   
               
               
                 TAACTTCCCCTTTTTGGGAGCAAAGAAGTCTGGGGCTGGAATCCAAGGGGT 
                   
               
               
                 GAAATACTCCATCGAAGCCATGACGACGGTGAAGAGCGTTGTTTTTGACATC 
                   
               
               
                 AAGTAAAACATAAGGAGGAAAAACAGATGGCGAAACTGACCTCGGCGGTT 
                   
               
               
                 CCGGTTCTGACGGCACGTGATGTGGCGGGCGGGGTTGAATTTTGGACGGATC 
                   
               
               
                 GTCTGGGCTTCAGTCGTGATTTTGTGGAAGATGACTTCGCAGGCGTGGTTCG 
                   
               
               
                 CGATGACGTCACCCTGTTTATTTCCGCAGTTCAGGATCAAGTCGTGGCGGAC 
                   
               
               
                 AACACGCTGGCTTGGGTGTGGGTTCGTGGCCTGGATGAACTGTATGCGGAAT 
                   
               
               
                 GGAGCGAAGTTGTCTCTACCAATTTCCGTGACGCGAGCGGTCCGGCCATGAC 
                   
               
               
                 GGAAATCGGCGAACAGCCGTGGGCTCGCGAATTTGCTCTGCGTGACCCGGC 
                   
               
               
                 TGGCAACTGTGTCCATTTCGTGGCTGAAGAACAAGATTGAGTTGAGATGAC 
                   
               
               
                 ACTGTGATCTAAAAAGAGCGACTTCGGTCGCTCTTTTTTTTACCTGA 
                   
               
               
                   
               
               
                 gapA-zeoR-proDp-gapN 
                   
               
               
                 ACGAAACCGGTTACTCCAACAAAGTTCTGGACCTGATCGCTCACATCTCCAA 
                 60 
               
               
                 ATGATTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCAACTTTAAAATTA 
                   
               
               
                 AAGAGGTATATATTAATGACTAAGCAATATAAGAATTACGTAAATGGGGAG 
                   
               
               
                 TGGAAGCTTTCGGAGAATGAAATTAAGATCTATGAACCAGCCAGTGGGGCG 
                   
               
               
                 GAATTGGGGTCAGTCCCGGCAATGTCCACTGAAGAAGTTGACTATGTCTACG 
                   
               
               
                 CCTCGGCCAAAAAAGCGCAGCCAGCATGGCGCTCGCTTTCCTATATTGAGCG 
                   
               
               
                 TGCGGCTTATTTGCACAAAGTCGCAGACATCCTGATGCGTGACAAGGAGAA 
                   
               
               
                 AATTGGAGCGGTATTGTCCAAGGAAGTAGCGAAAGGCTACAAATCCGCAGT 
                   
               
               
                 ATCGGAGGTCGTCCGCACCGCCGAGATTATTAATTATGCGGCCGAAGAAGG 
                   
               
               
                 GCTTCGCATGGAGGGTGAGGTCTTGGAGGGCGGCAGTTTTGAGGCGGCATC 
                   
               
               
                 CAAGAAAAAAATCGCTGTCGTCCGTCGCGAGCCGGTGGGACTTGTGCTTGCT 
                   
               
               
                 ATTAGTCCGTTCAATTACCCCGTGAATCTGGCCGGCTCCAAGATTGCCCCTG 
                   
               
               
                 CACTGATCGCGGGCAATGTAATCGCTTTTAAACCACCGACCCAAGGATCGAT 
                   
               
               
                 TAGTCGACTTCTTTTAGCGGAGGCGTTTGCGGAGGCAGGTCTTCCAGCCGGC 
                   
               
               
                 GTATTCAATACCATCACGGGGCGTGGAAGTGAAATCGGGGATTACATCGTG 
                   
               
               
                 GAGCACCAGGCAGTAAATTTCATCAACTTCACGGGTTCCACGGGGATCGGG 
                   
               
               
                 GAGCGTATCGGTAAGATGGCTGGGATGCGTCCGATCATGTTGGAACTTGGC 
                   
               
               
                 GGCAAGGATAGTGCGATTGTGCTGGAAGACGCAGACTTGGAATTGACAGCT 
                   
               
               
                 AAAAACATTATCGCTGGAGCCTTCGGGTATAGTGGTCAACGTTGCACGGCA 
                   
               
               
                 GTTAAGCGCGTTCTTGTTATGGAAAGTGTCGCGGATGAATTGGTCGAGAAG 
                   
               
               
                 ATTCGCGAGAAAGTGTTAGCTCTTACGATTGGAAATCCAGAGGACGATGCT 
                   
               
               
                 GACATCACTCCATTGATCGACACGAAATCCGCGGATTACGTCGAGGGGCTG 
                   
               
               
                 ATCAACGACGCGAACGATAAGGGAGCAGCGGCTTTGACCGAGATCAAACGC 
                   
               
               
                 GAGGGGAACCTGATCTGCCCGATTCTTTTTGACAAAGTCACAACTGACATGC 
                   
               
               
                 GCTTGGCATGGGAAGAACCCTTCGGCCCAGTCTTGCCTATTATCCGCGTTAC 
                   
               
               
                 TAGCGTAGAGGAAGCAATTGAAATTTCCAATAAATCCGAATATGGGTTGCA 
                   
               
               
                 AGCGAGTATCTTTACTAACGATTTTCCACGTGCCTTTGGTATTGCGGAACAG 
                   
               
               
                 TTAGAAGTCGGGACAGTTCACATCAACAACAAGACGCAGCGCGGGACAGAT 
                   
               
               
                 AACTTCCCCTTTTTGGGAGCAAAGAAGTCTGGGGCTGGAATCCAAGGGGTG 
                   
               
               
                 AAATACTCCATCGAAGCCATGACGACGGTCAAGAGCGTTGTTTTTGACATCA 
                   
               
               
                 AGTAAAACATAAGGAGGAAAAACAGATGGCGAAACTGACCTCGGCGGTTCC 
                   
               
               
                 GGTTCTGACGGCACGTGATGTGGCGGCCGCCGTTGAATTTTGGACGGATCGT 
                   
               
               
                 CTGGGCTTCAGTCGTGATTTTGTGGAAGATGACTTCGCAGGCGTGGTTCGCG 
                   
               
               
                 ATGACGTCACCCTGTTTATTTCCGCAGTTCAGGATCAAGTCGTGCCGGACAA 
                   
               
               
                 CACGCTGGCTTGGGTGTGGGTTCGTGGCCTGGATGAACTGTATGCGGAATGG 
                   
               
               
                 AGCGAAGTTGTCTCTACCAATTTCCGTGACGCGAGCGGTCCGGCCATGACGG 
                   
               
               
                 AAATCGGCGAACAGCCGTGGGGTCGCGAATTTGCTCTGCGTGACCCGGCTG 
                   
               
               
                 GCAACTGTGTCCATTTCGTGGCTGAACAACAAGATTGAGTTGAGATGACACT 
                   
               
               
                 GTGATCTAAAAAGAGCGACTTCGGTCGCTCTTTTTTTTACCTGA 
                   
               
               
                   
               
               
                 gapA-DAS+4-zeoR 
                   
               
               
                 TCTACCGATTTCAACGGCGAAGTTTGCACTTCCGTGTTCGATGCTAAAGCTG 
                 61 
               
               
                 GTATCGCTCTGAACGACAACTTCGTGAAACTGGTATCCTGGTACGACAACGA 
                   
               
               
                 AACCGGTTACTCCAACAAAGTTCTGGACCTGATCGCTCACATCTCCAAAGCG 
                   
               
               
                 GCCAACGATGAAAACTATTCTGAAAACTATGCGGATGCGTCTTGATCCTGAC 
                   
               
               
                 GGATGGCCTTTTTGCGTTTCTACAAACTCTTTTTGTTTATTTTTCTAAATACAT 
                   
               
               
                 TCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAAT 
                   
               
               
                 ATTGAAAAAGGAAGAGTAATGGCGAAACTGACCTCGCCGGTTCCGGTTCTG 
                   
               
               
                 ACGGCACGTGATGTGGCGGGCGCGGTTGAATTTTGGACGGATCGTCTGGGC 
                   
               
               
                 TTGAGTCGTGATTTTGTGGAAGATGACTTCGCAGGCGTGGTTCGCGATGACG 
                   
               
               
                 TCACCCTGTTTATTTCCGCAGTTCAGGATCAAGTCGTGCCGGACAACACGCT 
                   
               
               
                 GGCTTGGGTGTGGGTTCGTGGCCTGGATGAACTGTATGCGGAATGGAGCGA 
                   
               
               
                 AGTTGTCTCTACCAATTTCCGTGACTCGAGCGGTCCGGCCATGACGGAAATC 
                   
               
               
                 GGCGAACAGCCGTGGGGTCGCGAATTTGCTCTGCGTGACCCGGCTGGGAAC 
                   
               
               
                 TGTGTCCATTTCGTGGCTGAAGAACAAGATTGAGTTGAGATGACACTGTGAT 
                   
               
               
                 CTAAAAAGAGCGACTTCGGTCGCTCTTTTTTTTACCTGATAAAATGAAGTTA 
                   
               
               
                 AAGGACTGCGTCATGATTAAGAAAATTTTTGCCCTTCCGGTCATCGAACAAA 
                   
               
               
                 TCTCCCCTGTCCTCTCCCGTCGTAAACTGGATGAACTGGACCTCATTGTGGTC 
                   
               
               
                 GATCATCCGCAGGTAAAAGCCTCT 
                   
               
               
                   
               
               
                 gltA-DAS+4-ampR 
                   
               
               
                 GTATTCCGTCTTCCATGTTCACCGTCATTTTCGCAATGGCACGTACCGTTGGC 
                 62 
               
               
                 TGGATCGCCCACTGGAGCGAAATGCACAGTGACGGTATGAAGATTGCCCGT 
                   
               
               
                 CCGCGTCAGCTGTATACAGGATATGAAAAACGCGACTTTAAAAGCGATATC 
                   
               
               
                 AAGCGTGCGGCCAACGATGAAAACTATTCTGAAAACTATGCGGATGCGTCT 
                   
               
               
                 TAATAGTCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTTGTTTAT 
                   
               
               
                 TTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATA 
                   
               
               
                 AATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGT 
                   
               
               
                 GTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCA 
                   
               
               
                 GAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTG 
                   
               
               
                 GGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCC 
                   
               
               
                 CCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGC 
                   
               
               
                 GGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACA 
                   
               
               
                 CTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTT 
                   
               
               
                 ACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGT 
                   
               
               
                 GATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAG 
                   
               
               
                 CTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTT 
                   
               
               
                 GGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACG 
                   
               
               
                 ATGCCTACAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTA 
                   
               
               
                 CTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAA 
                   
               
               
                 GTTGCAGGACCACTTCTGCGCTCGGCCTTTCCGGCTGGCTGGTTTATTGCTG 
                   
               
               
                 ATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGG 
                   
               
               
                 GGCCAGATGGTAAGCCCTCCCGTATCGTACTTTATCTACACGACGGGGAGTC 
                   
               
               
                 AGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCAC 
                   
               
               
                 TGATTAAGCATTGGTAACTGTCAGACTAATGGTTGATTGCTAAGTTGTAAAT 
                   
               
               
                 ATTTTAACCCGCCGTTCATATGGCGGGTTGATTTTTATATGCCTAAACACAA 
                   
               
               
                 AAAATTGTAAAAATAAAATCCATTAACAGACCTATATAGATATTTAAAAAG 
                   
               
               
                 AATAGAACAGCTCAAATTATCAGCAACCCAATACTTTCAATTAAAAACTTCA 
                   
               
               
                 TGGTAGTCGCATTTATAACCCTATGAAA 
                   
               
               
                   
               
               
                 gltA-DAS+4-purR 
                   
               
               
                 ACCGTCATTTTCGCAATGGCACGTACCGTTGGCTGGATCGCCCACTGGAGCG 
                 63 
               
               
                 AAATGCACAGTGACGGTATGAAGATTGCCCGTCCGCGTCAGCTGTATACAG 
                   
               
               
                 GATATGAAAAACGCGACTTTAAAAGCGATATCAAGCGTGCGGCCAACGATG 
                   
               
               
                 AAAACTATTCTGAAAACTATGCGGATGCGTCTTAATCCTGACGGATGGCCTT 
                   
               
               
                 TTTGCGTTTCTACAAACTCTTTTTGTTTATTTTTCTAAATACATTCAAATATGT 
                   
               
               
                 ATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAA 
                   
               
               
                 GGAAGAGTATGACTGAATACAAGCCCACGGTACGCTTGGCGACGCGCGACG 
                   
               
               
                 ATGTTCCCCGCGCTGTTCGTACATTAGCTGCGGCCTTTGCAGATTACCCAGC 
                   
               
               
                 GACGCGCCATACGGTCGATCCGGACCGCCATATCGAGCGTGTCACAGAATT 
                   
               
               
                 GCAGGAACTTTTCTTAACTCGCGTGGGCCTTGACATCGGAAAGGTCTGGGTG 
                   
               
               
                 GCTGACGATGGCGCTGCACTGGCTGTTTGGACCACTCCGGAGAGTGTAGAG 
                   
               
               
                 GCTGGTGCAGTGTTCGCCGAAATTGGTCCTCGTATGGCCGAATTAAGTGGAA 
                   
               
               
                 GTCGTCTGGCAGCCCAACAACAAATGGAAGGGTTGCTTGCGCCCCACCGTC 
                   
               
               
                 CGAAAGAACCCGCGTGGTTCCTTGCCACCGTTGGAGTAAGCCCAGATCACC 
                   
               
               
                 AGGGGAAGGGTTTAGGATCTGCCGTAGTTTTACCAGGTGTGGAGGCAGCAG 
                   
               
               
                 AACGTGCGGGAGTTCCGGCCTTCCTTGAGACGTCGGCGCCGCGCAATTTACC 
                   
               
               
                 GTTTTACGAACGTCTTGGATTCACCGTTACGGCGGACGTGGAGGTGCCGGAG 
                   
               
               
                 GGACCCCGTACTTGGTGTATGACTCGTAAACCGGGAGCCTGATAATGGTTGA 
                   
               
               
                 TTGCTAAGTTGTAAATATTTTAACCCGCCGTTCATATGGCGGGTTGATTTTTA 
                   
               
               
                 TATGCCTAAACACAAAAAATTGTAAAAATAAAATCCATTAACAGACCTATA 
                   
               
               
                 TAGATATTTAAAAAGAATAGAACAGCTCAAATTATCAGCAACCCA 
                   
               
               
                   
               
               
                 gltA-DAS+4-zeoR 
                   
               
               
                 GTATTCCGTCTTCCATGTTCACCGTCATTTTCGCAATGGCACGTACCGTTGGC 
                 64 
               
               
                 TGGATCGCCCACTGGAGCGAAATGCACAGTGACGGTATGAAGATTGCCCGT 
                   
               
               
                 CCGCGTCAGCTGTATACAGGATATGAAAAACGCGACTTTAAAAGCGATATC 
                   
               
               
                 AAGCGTGCGGCCAACGATGAAAACTATTCTGAAAACTATGCGGATGCGTCT 
                   
               
               
                 TAATAGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGA 
                   
               
               
                 CTCACTATAGGAGGGCCATCATGGCCAAGTTGACCAGTGCCGTTCCGGTGCT 
                   
               
               
                 CACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGG 
                   
               
               
                 GTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGAC 
                   
               
               
                 GTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCGGACAACACC 
                   
               
               
                 CTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCG 
                   
               
               
                 GAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAG 
                   
               
               
                 ATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGC 
                   
               
               
                 AACTGCGTGCACTTTGTGGCAGAGGAGCAGGACTGAGGATAAGTAATGGTT 
                   
               
               
                 GATTGCTAAGTTGTAAATATTTTAACCCGCCGTTCATATGGCGGGTTGATTTT 
                   
               
               
                 TATATGCCTAAACACAAAAAATTGTAAAAATAAAATCCATTAACAGACCTA 
                   
               
               
                 TATAGATATTTAAAAAGAATAGAACAGCTCAAATTATCAGCAACCCAATAC 
                   
               
               
                 TTTCAATTAAAAACTTCATGGTAGTCGCATTTATAACCCTATGAAA 
                   
               
               
                   
               
               
                 lpd-DAS+4-gentR 
                   
               
               
                 GCGGCGAGCTGCTGGGTGAAATCGGCCTGGCAATCGAAATGGGTTGTGATG 
                 65 
               
               
                 CTGAAGACATCGCACTGACCATCCACGCGCACCCGACTCTGCACGAGTCTGT 
                   
               
               
                 GGGCCTGGCGGCAGAAGTGTTCGAAGGTAGCATTACCGACCTGCCGAACCC 
                   
               
               
                 GAAAGCGAAGAAGAAGGCGGCCAACGATGAAAACTATTCTGAAAACTATG 
                   
               
               
                 CGGATGCGTCTTAATAGCGAATCCATGTGGGAGTTTATTCTTGACACAGATA 
                   
               
               
                 TTTATGATATAATAACTGAGTAAGCTTAACATAAGGAGGAAAAACATATGT 
                   
               
               
                 TACGCAGCAGCAACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGT 
                   
               
               
                 TAGGTGGCTCAAGTATGGGCATCATTCGCACATGTAGGCTCGGCCCTGACCA 
                   
               
               
                 AGTCAAATCCATGCGGGCTGCTCTTGATCTTTTCGGTCGTGAGTTCGGAGAC 
                   
               
               
                 GTAGCCACCTACTCCCAACATCAGCCGGACTCCGATTACCTCGGGAACTTGC 
                   
               
               
                 TCCGTAGTAAGACATTCATCGCGCTTGCTGCCTTCGACCAAGAAGCGGTTGT 
                   
               
               
                 TGGCGCTCTCGCGGCTTACGTTCTGCCCAAGTTTGAGCAGCCGCGTAGTGAG 
                   
               
               
                 ATCTATATCTATGATCTCGCAGTCTCCGGCGAGCACCGGAGGCAGGGCATTG 
                   
               
               
                 CCACCGCGCTCATCAATCTCCTCAAGCATGAGGCCAACGCGCTTGGTGCTTA 
                   
               
               
                 TGTGATCTACGTGCAAGCAGATTACGGTGACGATCCCGCAGTGGCTCTCTAT 
                   
               
               
                 ACAAAGTTGGGCATACGGGAAGAAGTGATGCACTTTGATATCGACCCAAGT 
                   
               
               
                 ACCGCCACCTAATTTTTCGTTTGCCGGAACATCCGGCAATTAAAAAAGCGGC 
                   
               
               
                 TAACCACGCCGCTTTTTTTACGTCTGCAATTTACCTTTCCAGTCTTCTTGCTC 
                   
               
               
                 CACGTTCAGAGAGACGTTCGCATACTGCTGACCGTTGCTCGTTATTCAGCCT 
                   
               
               
                 GACAGTATGGTTACTGTC 
                   
               
               
                   
               
               
                 udhA-DAS+4-bsdR 
                   
               
               
                 TCTGGGTATTCACTGCTTTGGCGAGCGCGCTGCCGAAATTATTCATATCGGT 
                 66 
               
               
                 CAGGCGATTATGGAACAGAAAGGTGGCGGCAACACTATTGAGTACTTCGTC 
                   
               
               
                 AACACCACCTTTAACTACCCGACGATGGCGGAAGCCTATCGGGTAGCTGCG 
                   
               
               
                 TTAAACGGTTTAAACCGCCTGTTTGCGGCCAACGATGAAAACTATTCTGAAA 
                   
               
               
                 ACTATGCGGATGCGTCTTAATAGTTGACAATTAATCATCGGCATAGTATATC 
                   
               
               
                 GGCATAGTATAATACGACTCACTATAGGAGGGCCATCATGAAGACCTTCAA 
                   
               
               
                 CATCTCTCAGCAGGATCTGGAGCTGGTGGAGGTCGCCACTGAGAAGATCAC 
                   
               
               
                 CATGCTCTATGAGGACAACAAGCACCATGTCGGGGCGGCCATCAGGACCAA 
                   
               
               
                 GACTGGGGAGATCATCTCTGCTGTCCACATTGAGGCCTACATTGGCAGGGTC 
                   
               
               
                 ACTGTCTGTGCTGAAGCCATTGCCATTGGGTCTGCTGTGAGCAACGGGCAGA 
                   
               
               
                 AGGACTTTGACACCATTCTGGCTGTCAGCCACCCCTACTCTGATGAGGTGGA 
                   
               
               
                 CAGATCCATCAGGGTGGTCAGCCCCTGTGGCATGTGCAGAGAGCTCATCTCT 
                   
               
               
                 GACTATGCTCCTGACTGCTTTGTGCTCATTGAGATGAATGGCAAGCTGGTCA 
                   
               
               
                 AAACCACCATTGAGGAACTCATCCCCCTCAAGTACACCAGGAACTAAAGTA 
                   
               
               
                 AAACTTTATCGAAATGGCCATCCATTCTTGCGCGGATGGCCTCTGCCAGCTG 
                   
               
               
                 CTCATAGCGGCTGCGCAGCGGTGAGCCAGGACGATAAACCAGGCCAATAGT 
                   
               
               
                 GCGGCGTGGTTCCGGCTTAATGCACGG 
                   
               
               
                   
               
               
                 zwf-DAS+4-bsdR 
                   
               
               
                 GAAGTGGAAGAAGCCTGGAAATGGGTAGACTCCATTACTGAGGCGTGGGCG 
                 67 
               
               
                 ATGGACAATGATGCGCCGAAACCGTATCAGGCCGGAACCTGGGGACCCGTT 
                   
               
               
                 GCCTCGGTGGCGATGATTACCCGTGATGGTCGTTCCTGGAATGAGTTTGAGG 
                   
               
               
                 CGGCCAACGATGAAAACTATTCTGAAAACTATGCGGATGCGTCTTAATAGTT 
                   
               
               
                 GACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACTCACTAT 
                   
               
               
                 AGGAGGGCCATCATGAAGACCTTCAACATCTCTCAGCAGGATCTGGAGCTG 
                   
               
               
                 GTGGAGGTCGCCACTGAGAACATCACCATGCTCTATGAGGACAACAAGCAC 
                   
               
               
                 CATGTCGGGGCGGCCATCAGGACCAAGACTGGGGAGATCATCTCTGCTGTC 
                   
               
               
                 CACATTGAGGCCTACATTGGCAGGGTCACTGTCTGTGCTGAAGCCATTGCCA 
                   
               
               
                 TTGGGTCTGCTGTGAGCAACGGGCAGAAGGACTTTGACACCATTGTGGCTGT 
                   
               
               
                 CAGCCACCCCTACTCTGATGAGGTGGACAGATCCATCAGGGTGGTCAGCCC 
                   
               
               
                 CTGTGGCATCTGCAGAGAGCTCATCTCTGACTATGCTCCTGACTGCTTTGTG 
                   
               
               
                 CTCATTGAGATGAATGGCAAGCTGGTCAAAACCACCATTGAGGAACTCATC 
                   
               
               
                 CCCCTCAAGTACACCAGGAACTAAAGTAATATCTGCGCTTATCCTTTATGGT 
                   
               
               
                 TATTTTACCGGTAACATGATCTTGCGCAGATTGTAGAACAATTTTTACACTTT 
                   
               
               
                 CAGGCCTCGTGCGGATTCACCCACGAGGCTTTTTTTATTACACTGACTGAAA 
                   
               
               
                 CGTTTTTGCCCTATGAGCTCCGGTTACAGGCGTTTCAGTCATAAATCCTCTGA 
                   
               
               
                 ATGAAACGCGTTGTGAATC 
                   
               
               
                   
               
               
                 dadX-DAS+4-purR 
                   
               
               
                 GCGTGCGCACCATGACGGTGGGGACCGTCTCGATGGATATGCTAGCGGTCG 
                 68 
               
               
                 ATTTAACGCCTTGCCCGCAGGCGGGTATTGGTACGCCGGTTGAGCTGTGGGG 
                   
               
               
                 CAAGGAGATCAAAATTGATGATGTCGCCGCCGCTGCCGGAACGGTGGGCTA 
                   
               
               
                 TGAGTTGATGTGCGCGCTGGCGCTACGCGTCCCGGTTGTGACGGTGGCGGCC 
                   
               
               
                 AACGATGAAAACTATTCTGAAAACTATGCGGATGCGTCTTAATCCTGACGG 
                   
               
               
                 ATGGCCTTTTTGCGTTTCTACAAACTCTTTTTGTTTATTTTTCTAAATACATTC 
                   
               
               
                 AAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATAT 
                   
               
               
                 TGAAAAAGGAAGAGTATGACTGAATACAAGCCCACGGTACGCTTGGCGACG 
                   
               
               
                 CGCGACGATGTTCCCCGCGCTGTTCGTACATTAGCTGCGGCCTTTGCAGATT 
                   
               
               
                 ACCCAGCGACGCGCCATACGGTCGATCCGGACCGCCATATCGACCGTCTCA 
                   
               
               
                 CAGAATTGCAGGAACTTTTCTTAACTCGCGTGGGCCTTGACATCGGAAAGGT 
                   
               
               
                 CTGGGTGGCTGACGATGGCGCTGCAGTGGCTGTTTGGACCACTCCGGAGAG 
                   
               
               
                 TGTAGAGGCTGGTGCAGTGTTCGCCGAAATTGGTCCTCGTATGGCCGAATTA 
                   
               
               
                 AGTGGAAGTCGTCTGGCAGCCCAACAACAAATGGAAGGGTTGCTTGCGCCC 
                   
               
               
                 CACCGTCCGAAAGAACCCGCGTGGTTCCTTGCCACCGTTGGAGTAAGCCCA 
                   
               
               
                 GATCACCAGGGGAAGGGTTTAGGATCTGCCGTAGTTTTACCAGGTGTGGAG 
                   
               
               
                 GCAGCAGAACGTGCGGGAGTTCCGGCCTTCCTTGAGACGTCGGCGCCGCGC 
                   
               
               
                 AATTTACCGTTTTACGAACGTCTTGGATTCACCGTTACGGCGGACGTGGAGG 
                   
               
               
                 TGCCGGAGGGACCCCGTACTTGGTGTATGACTCGTAAACCGGGAGCCTGAT 
                   
               
               
                 AACTTGTTGTAAGCCGGATCGGAGGCAACGTCTTCTGGGTGCAAAAAAATC 
                   
               
               
                 ATCCATCCGGGTGGTCAGCAACTGTAGTTGTTAATGTGACAGAGCCATTGCC 
                   
               
               
                 CATGATAGTGTCCATTAAAAGGATGGACACTATTTCCCCGGAACCTGAACTC 
                   
               
               
                 ACCGCACAGGCGTTCTACATAAAACGCTTACGCTTCATTGTTGACTC 
               
               
                   
               
            
           
         
       
     
     Section 4: Dynamics Control Over Protein Levels. 
     Plasmids expressing fluorescent proteins and silencing guides were transformed into the corresponding hosts strain listed in Table 8. Strains were evaluated in triplicate in an m2p-labs Biolector™, which simultaneously measures fluorescence including GiFPuv and mCherry levels, as well as biomass levels. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Strains used for Dynamic Control over protein levels 
               
            
           
           
               
               
               
               
            
               
                   
                 Synthetic 
                   
                   
               
               
                   
                 Metabolic 
                   
                   
               
               
                 Microbe 
                 Valves 
                 Plasmid 
                 Host Strain 
               
               
                   
               
               
                 
                   E. 
                   coli 
                 
                 RFP-control 
                 pCDF- 
                 DLF_0002  
               
               
                   
                   
                 mcherry1 + PSMART- 
                   
               
               
                   
                   
                 IN:yibDp-GFPuv 
                   
               
               
                   
                 Proteolysis 
                 pCDF- 
                 DLF_0025  
               
               
                   
                   
                 mcherry2 + PSMART- 
                   
               
               
                   
                   
                 IN:yibDp-GFPuv 
                   
               
               
                   
                 Silencing 
                 pCDF- 
                 DLF_01517 
               
               
                   
                   
                 mcherry1 + pCASCADE- 
                   
               
               
                   
                   
                 proD + pSMART- 
                   
               
               
                   
                   
                 IN:yibDp-GFPuv 
                   
               
               
                   
                 Proteolysis + 
                 pCDF- 
                 DLF_0025  
               
               
                   
                 Silencing 
                 mcherry2 + pCASCADE- 
                   
               
               
                   
                   
                 proD + pSMART- 
                   
               
               
                   
                   
                 IN:yibDp-GFPuv 
               
               
                   
               
            
           
         
       
     
     OD600 readings were corrected using the formula below, where OD600 refers to an offline measurement. OD600* refers to Biolector biomass reading, t0 indicates the start point, and if indicates the final point. 
     
       
         
           
             
               
                 
                   
                     OD 
                     ⁢ 
                     
                         
                     
                     ⁢ 
                     
                       600 
                       t 
                     
                   
                   = 
                   
                     
                       
                         ( 
                         
                           
                             OD 
                             ⁢ 
                             
                                 
                             
                             ⁢ 
                             
                               600 
                               t 
                               * 
                             
                           
                           - 
                           
                             OD 
                             ⁢ 
                             
                                 
                             
                             ⁢ 
                             
                               600 
                               
                                 t 
                                 ⁢ 
                                 
                                     
                                 
                                 ⁢ 
                                 0 
                               
                               * 
                             
                           
                         
                         ) 
                       
                       * 
                       
                         
                           ( 
                           
                             
                               OD 
                               ⁢ 
                               
                                   
                               
                               ⁢ 
                               
                                 600 
                                 tf 
                               
                             
                             - 
                             
                               OD 
                               ⁢ 
                               
                                   
                               
                               ⁢ 
                               
                                 600 
                                 
                                   t 
                                   ⁢ 
                                   
                                       
                                   
                                   ⁢ 
                                   0 
                                 
                               
                             
                           
                           ) 
                         
                         
                           ( 
                           
                             
                               OD 
                               ⁢ 
                               
                                   
                               
                               ⁢ 
                               
                                 600 
                                 tf 
                                 * 
                               
                             
                             - 
                             
                               OD 
                               ⁢ 
                               
                                   
                               
                               ⁢ 
                               
                                 600 
                                 
                                   t 
                                   ⁢ 
                                   
                                       
                                   
                                   ⁢ 
                                   0 
                                 
                                 * 
                               
                             
                           
                           ) 
                         
                       
                     
                     + 
                     0.25 
                   
                 
               
               
                 
                   Equation 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   S 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   1 
                 
               
             
           
         
       
     
     Section 5: Metabolic Control 
     Near Equilibrium Reactions 
     The impact of Valves on metabolite pools for near equilibrium reactions is illustrated using the G6P node as an example. Abbreviations: Gluc, glucose; G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; 6PGI, 6-phosphate-gluconolactone. 
     G6P Node without Valves 
                         Steady State Mass balance  J   1   =J   2   +J   3   Equation S2:
 
     
       
         
           
             
               
                 
                   
                     Net 
                     ⁢ 
                     
                         
                     
                     ⁢ 
                     Flux 
                   
                   = 
                   
                     
                       J 
                       i 
                     
                     = 
                     
                       
                         e 
                         
                           
                             - 
                             dG 
                           
                           RT 
                         
                       
                       - 
                       1 
                     
                   
                 
               
               
                 
                   Equation 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   S3 
                 
               
             
             
               
                 
                   
                     
                       e 
                       
                         
                           
                             - 
                             dG 
                           
                           ⁢ 
                           
                               
                           
                           ⁢ 
                           1 
                         
                         RT 
                       
                     
                     - 
                     1 
                   
                   = 
                   
                     
                       e 
                       
                         
                           
                             - 
                             dG 
                           
                           ⁢ 
                           
                               
                           
                           ⁢ 
                           2 
                         
                         RT 
                       
                     
                     - 
                     1 
                     + 
                     
                       e 
                       
                         
                           
                             - 
                             dG 
                           
                           ⁢ 
                           
                               
                           
                           ⁢ 
                           3 
                         
                         RT 
                       
                     
                     - 
                     1 
                   
                 
               
               
                 
                   Equation 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   S4 
                 
               
             
             
               
                 
                   
                     e 
                     
                       
                         
                           - 
                           dG 
                         
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         1 
                       
                       RT 
                     
                   
                   = 
                   
                     
                       e 
                       
                         
                           
                             - 
                             dG 
                           
                           ⁢ 
                           
                               
                           
                           ⁢ 
                           2 
                         
                         RT 
                       
                     
                     + 
                     
                       e 
                       
                         
                           
                             - 
                             dG 
                           
                           ⁢ 
                           
                               
                           
                           ⁢ 
                           3 
                         
                         RT 
                       
                     
                     - 
                     1 
                   
                 
               
               
                 
                   Equation 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   S5 
                 
               
             
             
               
                 
                   
                     Keq 
                     ⁢ 
                     
                         
                     
                     ⁢ 
                     1 
                   
                   = 
                   
                     
                       Keq 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       2 
                     
                     + 
                     
                       Keq 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       3 
                     
                     - 
                     1 
                   
                 
               
               
                 
                   Equation 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   S6 
                 
               
             
             
               
                 
                   
                     
                       Keq 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       1 
                     
                     + 
                     1 
                   
                   = 
                   
                     
                       Keq 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       2 
                     
                     + 
                     
                       Keq 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       3 
                     
                   
                 
               
               
                 
                   Equation 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   S7 
                 
               
             
             
               
                 
                   
                     
                       
                         [ 
                         
                           G 
                           ⁢ 
                           
                               
                           
                           ⁢ 
                           6 
                           ⁢ 
                           P 
                         
                         ] 
                       
                       
                         [ 
                         Gluc 
                         ] 
                       
                     
                     + 
                     1 
                   
                   = 
                   
                     
                       
                         [ 
                         
                           F 
                           ⁢ 
                           
                               
                           
                           ⁢ 
                           6 
                           ⁢ 
                           P 
                         
                         ] 
                       
                       
                         [ 
                         
                           G 
                           ⁢ 
                           
                               
                           
                           ⁢ 
                           6 
                           ⁢ 
                           P 
                         
                         ] 
                       
                     
                     + 
                     
                       
                         [ 
                         
                           6 
                           ⁢ 
                           PGl 
                         
                         ] 
                       
                       
                         [ 
                         
                           G 
                           ⁢ 
                           
                               
                           
                           ⁢ 
                           6 
                           ⁢ 
                           P 
                         
                         ] 
                       
                     
                   
                 
               
               
                 
                   Equation 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   S8 
                 
               
             
             
               
                 
                   
                     
                       
                         [ 
                         
                           G 
                           ⁢ 
                           
                               
                           
                           ⁢ 
                           6 
                           ⁢ 
                           P 
                         
                         ] 
                       
                       
                         [ 
                         Gluc 
                         ] 
                       
                     
                     + 
                     1 
                   
                   = 
                   
                     
                       
                         [ 
                         
                           F 
                           ⁢ 
                           
                               
                           
                           ⁢ 
                           6 
                           ⁢ 
                           P 
                         
                         ] 
                       
                       + 
                       
                         [ 
                         
                           6 
                           ⁢ 
                           PGl 
                         
                         ] 
                       
                     
                     
                       [ 
                       
                         G 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         6 
                         ⁢ 
                         P 
                       
                       ] 
                     
                   
                 
               
               
                 
                   Equation 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   S9 
                 
               
             
             
               
                 
                   
                     
                       
                         
                           [ 
                           
                             G 
                             ⁢ 
                             
                                 
                             
                             ⁢ 
                             6 
                             ⁢ 
                             P 
                           
                           ] 
                         
                         2 
                       
                       
                         [ 
                         Gluc 
                         ] 
                       
                     
                     + 
                     
                       [ 
                       
                         G 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         6 
                         ⁢ 
                         P 
                       
                       ] 
                     
                   
                   = 
                   
                     
                       [ 
                       
                         F 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         6 
                         ⁢ 
                         P 
                       
                       ] 
                     
                     + 
                     
                       [ 
                       
                         6 
                         ⁢ 
                         PGl 
                       
                       ] 
                     
                   
                 
               
               
                 
                   Equation 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   S10 
                 
               
             
             
               
                 
                   
                     [ 
                     
                       F 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       6 
                       ⁢ 
                       P 
                     
                     ] 
                   
                   = 
                   
                     
                       
                         
                           [ 
                           
                             G 
                             ⁢ 
                             
                                 
                             
                             ⁢ 
                             6 
                             ⁢ 
                             P 
                           
                           ] 
                         
                         2 
                       
                       
                         [ 
                         Gluc 
                         ] 
                       
                     
                     + 
                     
                       [ 
                       
                         G 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         6 
                         ⁢ 
                         P 
                       
                       ] 
                     
                     - 
                     
                       [ 
                       
                         6 
                         ⁢ 
                         PGl 
                       
                       ] 
                     
                   
                 
               
               
                 
                   Equation 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   S 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   11 
                 
               
             
           
         
       
     
     G6P Node with Valves 
     When zwf valve is in effect, J 3 ≈0. 
                         Steady State Mass balance  J   1   =J   2   Equation S12:
 
     
       
         
           
             
               
                 
                   
                     Net 
                     ⁢ 
                     
                         
                     
                     ⁢ 
                     Flux 
                   
                   = 
                   
                     
                       J 
                       i 
                     
                     = 
                     
                       
                         e 
                         
                           
                             - 
                             dG 
                           
                           RT 
                         
                       
                       - 
                       1 
                     
                   
                 
               
               
                 
                   Equation 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   S13 
                 
               
             
             
               
                 
                   
                     
                       e 
                       
                         
                           
                             - 
                             dG 
                           
                           ⁢ 
                           
                               
                           
                           ⁢ 
                           1 
                         
                         RT 
                       
                     
                     - 
                     1 
                   
                   = 
                   
                     
                       e 
                       
                         
                           
                             - 
                             dG 
                           
                           ⁢ 
                           
                               
                           
                           ⁢ 
                           2 
                         
                         RT 
                       
                     
                     - 
                     1 
                   
                 
               
               
                 
                   Equation 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   S14 
                 
               
             
             
               
                 
                   
                     Keq 
                     ⁢ 
                     
                         
                     
                     ⁢ 
                     1 
                   
                   = 
                   
                     Keq 
                     ⁢ 
                     
                         
                     
                     ⁢ 
                     2 
                   
                 
               
               
                 
                   Equation 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   S15 
                 
               
             
             
               
                 
                   
                     
                       [ 
                       
                         G 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         6 
                         ⁢ 
                         P 
                       
                       ] 
                     
                     
                       [ 
                       Gluc 
                       ] 
                     
                   
                   = 
                   
                     
                       [ 
                       
                         F 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         6 
                         ⁢ 
                         P 
                       
                       ] 
                     
                     
                       [ 
                       
                         G 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         6 
                         ⁢ 
                         P 
                       
                       ] 
                     
                   
                 
               
               
                 
                   Equation 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   S16 
                 
               
             
             
               
                 
                   
                     [ 
                     
                       F 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       6 
                       ⁢ 
                       P 
                     
                     ] 
                   
                   = 
                   
                     
                       
                         [ 
                         
                           G 
                           ⁢ 
                           
                               
                           
                           ⁢ 
                           6 
                           ⁢ 
                           P 
                         
                         ] 
                       
                       2 
                     
                     
                       [ 
                       Gluc 
                       ] 
                     
                   
                 
               
               
                 
                   Equation 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   S17 
                 
               
             
           
         
       
     
     Impact of Valves 
     
       
         
           
             
               
                 
                   
                     
                       [ 
                       
                         F 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         6 
                         ⁢ 
                         P 
                       
                       ] 
                     
                     ⁢ 
                     
                         
                     
                     ⁢ 
                     network 
                   
                   = 
                   
                     
                       
                         
                           [ 
                           
                             G 
                             ⁢ 
                             
                                 
                             
                             ⁢ 
                             6 
                             ⁢ 
                             P 
                           
                           ] 
                         
                         2 
                       
                       
                         [ 
                         Gluc 
                         ] 
                       
                     
                     + 
                     
                       [ 
                       
                         G 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         6 
                         ⁢ 
                         P 
                       
                       ] 
                     
                     - 
                     
                       [ 
                       
                         6 
                         ⁢ 
                         PGl 
                       
                       ] 
                     
                   
                 
               
               
                 
                   Equation 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   S11 
                 
               
             
             
               
                 
                   
                     
                       [ 
                       
                         F 
                         ⁢ 
                         
                             
                         
                         ⁢ 
                         6 
                         ⁢ 
                         P 
                       
                       ] 
                     
                     ⁢ 
                     
                         
                     
                     ⁢ 
                     value 
                   
                   = 
                   
                     
                       
                         [ 
                         
                           G 
                           ⁢ 
                           
                               
                           
                           ⁢ 
                           6 
                           ⁢ 
                           P 
                         
                         ] 
                       
                       2 
                     
                     
                       [ 
                       Gluc 
                       ] 
                     
                   
                 
               
               
                 
                   Equation 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   S 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   17 
                 
               
             
           
         
       
         
         
           
             Since close to equilibrium [6PGI]&gt;[G6P] 
           
         
       
    
       [ F 6 P ]valve&gt;=[ F 6 P ]network         The removal of thermodynamically favored reactions near equilibrium from the network will result in increased metabolite pools.       
     Section 6: Gene Silencing Arrays &amp; Pathway Expression Constructs 
     The design and construction of CASCADE guides and guide arrays is illustrated below in  FIG. 14  and  FIG. 15A-B . The pCASCADE-control plasmid was prepared by swapping the pTet promoter in pcrRNA.Tet 88  with an insulated low phosphate induced ugpB promoter 82 . Two promoters were responsible for regulating gltA gene, and sgRNA was designed for both promoters, resulting in guide gltA1 (G1) and gltA2 (G2). 89  Four promoters were responsible for regulating gapA gene, and sgRNA was designed for the first promoter, since during exponential phase of growth, gapA mRNAs were mainly initiated at the highly efficient gapA P1 promoter and remained high during stationary phase compared to the other three gapA promoters. 90  Multiple promoters upstream of lpd gene w % ere involved in lpd regulation (https://ecocyc.org/gene?orgid=ECOLI&amp;id=EG10543#tab=showAll), thus design of unique and effective sgRNA for lpd only was not possible. Promoter sequences for fabI, udhA and zwf were obtained from EcoCyc database (https://ecocyc.org/). To design CASCADE guide array, CASCADE PAM sites near the −35 or −10 box of the promoter of interest were identified, 30 bp at the 3′ end of PAM site was selected as the guide sequence and cloned into pCASCADE plasmid using Q5 site-directed mutagenesis (NEB, MA) following manufacturer&#39;s protocol, with the modification that 5% v/v DMSO was added to the Q5 PCR reaction. The pCASCADE-control vector was used as template, pCASCADE plasmids with arrays of two or more guides were prepared as illustrated in  FIG. 15A-B . The pCASCADE guide array plasmid was prepared by sequentially amplifying complementary halves of each smaller guide plasmid by PCR, followed by subsequent DNA assembly. Table 9 lists sgRNA guide sequences and primers used to construct them. All pCASCADE silencing plasmids are listed in Table 10 below and are available at Addgene. 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 List of sgRNA guide sequences and primers used to construct them. Spacers 
               
               
                 are italicized. 
               
            
           
           
               
               
               
               
            
               
                 sgRNA/Primer Name 
                 Sequence 
                 SEQ ID NO 
                 Template 
               
               
                   
               
               
                 fabI 
                 
                   TCGAGTTCCCCGCGCCAGCGGG 
                 
                  69 
                   
               
               
                   
                   GATAAACCG TTGATTATAATAA 
                   
                   
               
               
                   
                 CCGTTTATCTGTTCGTA TCGAG   
                   
                   
               
               
                   
                 
                   TTCCCCGCGCCAGCGGGGATAA 
                 
                   
                   
               
               
                   
                 
                   ACCG 
                 
                   
                   
               
               
                   
               
               
                 fabI-FOR 
                 GTTTATCTGTTCGTA TCGAGTT   
                  70 
                 pCASCADE control 
               
               
                   
                 
                   CCCCGCGCCAGCGGGGATAAAC 
                 
                   
                   
               
               
                   
                   CG AAAAAAAAACCCC 
                   
                   
               
               
                 fabI-REV 
                 GGTTATTATAATCACGGTTTA 
                  71 
                   
               
               
                   
                 TCCCCGCTGGCGCGGGGAACT 
                   
                   
               
               
                   
                 CGAGGTGGTACCAGAT 
                   
                   
               
               
                   
               
               
                 gapAP1 
                 
                   TCGAGTTCCCCGCGCCAGCGGG 
                 
                  72 
                   
               
               
                   
                   GATAAACCG GTTTTTGTAATTTT 
                   
                   
               
               
                   
                 ACAGGCAACCTTTTAT TCGAGT   
                   
                   
               
               
                   
                 
                   TCCCCGCGCCAGCGGGGATAAA 
                 
                   
                   
               
               
                   
                 
                   CCG 
                 
                   
                   
               
               
                   
               
               
                 gapAP1-FOR 
                 CAGGCAACCTTTTAT TCGAGTT   
                  73 
                 pCASCADE control 
               
               
                   
                 
                   CCCCGCGCCAGCGGGGATAAAC 
                 
                   
                   
               
               
                   
                   CG AAAAAAAAACCCC 
                   
                   
               
               
                 gapAP1-REV 
                 TAAAATTACAAAAACCGGTTT 
                  74 
                   
               
               
                   
                 ATCCCCGCTGGCGCGGGGAAC 
                   
                   
               
               
                   
                 TCGAGGTGGTACCAGATC 
                   
                   
               
               
                   
               
               
                 gltA1 
                 
                   TCGAGTTCCCCGCGCCAGCGGG 
                 
                  75 
                   
               
               
                   
                   GATAAACCG AAAAGCATATAAT 
                   
                   
               
               
                   
                 GCGTAAAAGTTATGAAGT TCG   
                   
                   
               
               
                   
                 
                   AGTTCCCCGCGCCAGCGGGGAT 
                 
                   
                   
               
               
                   
                 
                   AAACCG 
                 
                   
                   
               
               
                   
               
               
                 gltA1-FOR 
                 GCGTAAAAGTTATGAAGT TCG   
                  76 
                 pCASACADE control 
               
               
                   
                 
                   AGTTCCCCGCGCCAGCGGGGAT 
                 
                   
                   
               
               
                   
                   AAACCG AAAAAAAAACCCCC 
                   
                   
               
               
                 gltA1-REV 
                 ATTATATGCTTTTCGGTTTATC 
                  77 
                   
               
               
                   
                 CCCGCTGGCGCGGGGAACTCG 
                   
                   
               
               
                   
                 AGGTGGTACCAGATCT 
                   
                   
               
               
                   
               
               
                 gltA2 
                 
                   TCGAGTTCCCCGCGCCAGCGGG 
                 
                  78 
                   
               
               
                   
                   GATAAACCG TATTGACCAATTC 
                   
                   
               
               
                   
                 ATTCGGGACAGTTATTAGT TCG   
                   
                   
               
               
                   
                 
                   AGTTCCCCGCGCCAGCGGGGAT 
                 
                   
                   
               
               
                   
                 
                   AAACCG 
                 
                   
                   
               
               
                   
               
               
                 gltA2-FOR 
                 GGGACAGTTATTAGT TCGAGTT   
                  79 
                 pCASCADE control 
               
               
                   
                 
                   CCCCGCGCCAGCGGGGATAAAC 
                 
                   
                   
               
               
                   
                   CG AAAAAAAAACCCC 
                   
                   
               
               
                 gltA2-REV 
                 GAATGAATTGGTCAATACGGT 
                  80 
                   
               
               
                   
                 TTATCCCCGCTGGCGCGGGGA 
                   
                   
               
               
                   
                 ACTCGAGGTGGTACCAGATCT 
                   
                   
               
               
                   
               
               
                 proD 
                 
                   TCGAGTTCCCCGCGCCAGCGGG 
                 
                  81 
                   
               
               
                   
                   GATAAACCG AGTGGTTCGCTGGA 
                   
                   
               
               
                   
                 TAACTTTACGGGCATGC TCGAG   
                   
                   
               
               
                   
                 
                   TTCCCCGCGCCGCGGGGATAA 
                 
                   
                   
               
               
                   
                 
                   ACCG 
                 
                   
                   
               
               
                   
               
               
                 proD-FOR 
                 AACTTTACGGGCATGC TCGAGT   
                  82 
                 pCASCADE control 
               
               
                   
                 
                   TCCCCGCGCCAGCGGGGATAAA 
                 
                   
                   
               
               
                   
                   CCG AAAAAAAAACCCC 
                   
                   
               
               
                 proD-REV 
                 ATCCAGCAACCACTCGGTTTAT 
                  83 
                   
               
               
                   
                 CCCCGCTGGCGCGGGGAACTC 
                   
                   
               
               
                   
                 GAGGTGGTACCAGATCT 
                   
                   
               
               
                   
               
               
                 udhA 
                 
                   TCGAGTTCCCCGCGCAGCGGG 
                 
                  84 
                   
               
               
                   
                   GATAAACCG TTACCATTCTGTT 
                   
                   
               
               
                   
                 GCTTTTATGTATAAGAA TCGAG   
                   
                   
               
               
                   
                 
                   TTCCCCGCGCCAGCGGGGATAA 
                 
                   
                   
               
               
                   
                 
                   ACCG 
                 
                   
                   
               
               
                   
               
               
                 udhA-FOR 
                 TTTTATGTATAAGAA TCGAGTT   
                  85 
                 pCASCADE control 
               
               
                   
                 
                   CCCCGCGCCAGCGGGGATAAAC 
                 
                   
                   
               
               
                   
                   CG AAAAAAAAACCCC 
                   
                   
               
               
                 udhA-REV 
                 GCAACAGAATGGTAACGGTTT 
                  86 
                   
               
               
                   
                 ATCCCCGCTGGCGCGGGGAAC 
                   
                   
               
               
                   
                 TCGAGGTGGTACCAGATC 
                   
                   
               
               
                   
               
               
                 zwf 
                 
                   TCGAGTTCCCCGCGCCAGCGGG 
                 
                  87 
                   
               
               
                   
                   GATAAACCG CTCGTAAAAGCAG 
                   
                   
               
               
                   
                 TACAGTGCACCGTAAGA TCGA   
                   
                   
               
               
                   
                 
                   GTTCCCCGCGCCAGCGGGGATA 
                 
                   
                   
               
               
                   
                 
                   AACCG 
                 
                   
                   
               
               
                   
               
               
                 zwf-FOR 
                 CAGTGCACCGTAAGA TCGAGTT   
                  88 
                 control 
               
               
                   
                 
                   CCCCGCGCCAGCGGGGATAAAC 
                 
                   
                   
               
               
                   
                   CG AAAAAAAAACCCC 
                   
                   
               
               
                 zwf-REV 
                 TACTGCTTTTACGAGCGGTTTA 
                  89 
                   
               
               
                   
                 TCCCCGCTGGCGCGGGGAACT 
                   
                   
               
               
                   
                 CGAGGTGGTACCAGATC 
                   
                   
               
               
                   
               
               
                 FG1 
                 
                   TCGAGTTCCCCGCGCCAGCGGG 
                 
                  90 
                   
               
               
                   
                   GATAAACCG TTGATTATAATAA 
                   
                   
               
               
                   
                 CCGTTTATCTGTTCGTA TCGAG   
                   
                   
               
               
                   
                 
                   TTCCCCGCGCCAGCGGGGATAA 
                 
                   
                   
               
               
                   
                   ACCG AAAAGCATATAATGCGT 
                   
                   
               
               
                   
                 AAAAGTTATGAAGT TCGAGTTC   
                   
                   
               
               
                   
                 
                   CCCGCGCCAGCGGGGATAAACC 
                 
                   
                   
               
               
                   
                 
                   G 
                 
                   
                   
               
               
                   
               
               
                 gltA-FOR 
                 GCGCCAGCGGGGATAAACCG A   
                  91 
                 pCASCADE-gltA1 
               
               
                   
                   AAAGCATATAAT GCG 
                   
                   
               
               
                 pCASCADE-REV 
                 CTTGCCCGCCTGATGAATGCTC 
                  92 
                   
               
               
                   
                 ATCCGG 
                   
                   
               
               
                   
               
               
                 pCASCADE-FOR 
                 CCGGATGAGCATTCATCAGGC 
                  93 
                 pCASCADE-fabI 
               
               
                   
                 GGGCAAG 
                   
                   
               
               
                 fabI-REV 
                 CGGTTTATCCCCGCTGGCGCG 
                  94 
                   
               
               
                   
                 GGGAACTCGATACGAACAGAT 
                   
                   
               
               
                   
                 AAACGGTTATTATAATC 
                   
                   
               
               
                   
               
               
                 FG2 
                 
                   TCGAGTTCCCCGCGCCAGCGGG 
                 
                  95 
                   
               
               
                   
                   GATAAACCG TTGATTATAATAA 
                   
                   
               
               
                   
                 CCGTTTATCTGTTCGTA TCGAG   
                   
                   
               
               
                   
                 
                   TTCCCCGCGCCAGCGGGGATAA 
                 
                   
                   
               
               
                   
                   ACCG TATTGACCAATTCATTCG 
                   
                   
               
               
                   
                 GGACAGTTATTAGT TCGAGTTC   
                   
                   
               
               
                   
                 
                   CCCGCGCCAGCGGGGATAAACC 
                 
                   
                   
               
               
                   
                 
                   G 
                 
                   
                   
               
               
                   
               
               
                 gltA2-FOR 
                 GCGCCAGCGGGGATAAACCG T   
                  96 
                 pCASCADE-gltA2 
               
               
                   
                 
                   ATTGACCAATTCATTC 
                 
                   
                   
               
               
                 pCASCADE-REV 
                 CTTGCCCGCCTGATGAATGCTC 
                  97 
                   
               
               
                   
                 ATCCGG 
                   
                   
               
               
                   
               
               
                 pCASCADE-FOR 
                 CCGGATGAGCATTCATCAGGC 
                  98 
                 pCASCADE-fabI 
               
               
                   
                 GGGCAAG 
                   
                   
               
               
                 fabI-REV 
                 CGGTTTATCCCCGCTGGCGCG 
                  99 
                   
               
               
                   
                 GGGAACTCGATACGAACAGAT 
                   
                   
               
               
                   
                 AAACGGTTATTATAATC 
                   
                   
               
               
                   
               
               
                 FU 
                 
                   TCGAGTTCCCCGCGCCAGCGGG 
                 
                 100 
                   
               
               
                   
                   GATAAACCG TTGATTATAATAA 
                   
                   
               
               
                   
                 CCGTTTATCTGTTCGTA TCGAG   
                   
                   
               
               
                   
                 
                   TTCCCCGCGCCAGCGGGGATAA 
                 
                   
                   
               
               
                   
                   ACCG TTACCATTCTGTTGCTTT 
                   
                   
               
               
                   
                 TATGTATAAGAA TCGAGTTCCC   
                   
                   
               
               
                   
                 
                   CGCGCCAGCGGGGATAAACCG 
                 
                   
                   
               
               
                   
               
               
                 udhA-FOR 
                 GCGCCAGCGGGGATAAACCGT 
                 101 
                 pCASCADE-udhA 
               
               
                   
                 TACCATTCTGTTG 
                   
                   
               
               
                 pCASCADE-REV 
                 CTTGCCCGCCTGATGAATGCTC 
                 102 
                   
               
               
                   
                 ATCCGG 
                   
                   
               
               
                   
               
               
                 pCASCADE-FOR 
                 CCGGATGAGCATTCATCAGGC 
                 103 
                 pCASCADE-fabI 
               
               
                   
                 GGGCAAG 
                   
                   
               
               
                 fabI-REV 
                 CGGTTTATCCCCGCTGGCGCG 
                 104 
                   
               
               
                   
                 GGGAACTCGATACGAACAGAT 
                   
                   
               
               
                   
                 AAACGGTTATTATAATC 
                   
                   
               
               
                   
               
               
                 FZ 
                 
                   TCGAGTTCCCCGCGCCAGCGGG 
                 
                 105 
                   
               
               
                   
                   GATAAACCG TTGATTATAATAA 
                   
                   
               
               
                   
                 CCGTTTATCTGTTCGTA TCGAG   
                   
                   
               
               
                   
                 
                   TTCCCCGCGCCAGCGGGGATAA 
                 
                   
                   
               
               
                   
                   ACCG CTCGTAAAAGCAGTACA 
                   
                   
               
               
                   
                 GTGCACCGTAAGA TCGAGTTCC   
                   
                   
               
               
                   
                 
                   CCGCGCCAGCGGGGATAAACCG 
                 
                   
                   
               
               
                   
               
               
                 zwf-FOR 
                 GCGCCAGCGGGGATAAACCGC 
                 106 
                 pCASCADE-zwf 
               
               
                   
                 TCGTAAAAG 
                   
                   
               
               
                 pCASCADE-REV 
                 CTTGCCCGCCTGATGAATGCTC 
                 107 
                   
               
               
                   
                 ATCCGG 
                   
                   
               
               
                   
               
               
                 pCASCADE-FOR 
                 CCGGATGAGCATTCATCAGGC 
                 108 
                 pCASCADE-fabI 
               
               
                   
                 GGGCAAG 
                   
                   
               
               
                 fabI-REV 
                 CGGTTTATCCCCGCTGGCGCG 
                 109 
                   
               
               
                   
                 GGGAACTCGATACGAACAGAT 
                   
                   
               
               
                   
                 AAACGGTTATTATAATC 
                   
                   
               
               
                   
               
               
                 G1G2 
                 
                   TCGAGTTCCCCGCGCCAGCGGG 
                 
                 110 
                   
               
               
                   
                   GATAAACCG AAAAGCATATAAT 
                   
                   
               
               
                   
                 GCGTAAAAGTTATGAAGT TCG   
                   
                   
               
               
                   
                 
                   AGTTCCCCGCGCCAGCGGGGAT 
                 
                   
                   
               
               
                   
                   AAACCG TATTGACCAATTCATT 
                   
                   
               
               
                   
                 CGGGACAGTTATTAGT TCGAGT   
                   
                   
               
               
                   
                 
                   TCCCCGCGCCAGCGGGGATAAA 
                 
                   
                   
               
               
                   
                 
                   CCG 
                 
                   
                   
               
               
                   
               
               
                 gltA2-FOR 
                 GCGCCAGCGGGGATAAACCG T   
                 111 
                 pCASCADE-gltA2 
               
               
                   
                 
                   ATTGACCAATTCATTC 
                 
                   
                   
               
               
                 pCASCADE-REV 
                 CTTGCCCGCCTGATGAATGCTC 
                 112 
                   
               
               
                   
                 ATCCGG 
                   
                   
               
               
                   
               
               
                   
                 CCGGATGAGCATTCATCAGGC 
                 113 
                 pCASCADE-gltA1 
               
               
                   
                 GGGCAAG 
                   
                   
               
               
                 gltA1-REV 
                 CGGTTTATCCCCGCTGGCGCG 
                 114 
                   
               
               
                   
                 GGGAACTCGAACTTCATAACT 
                   
                   
               
               
                   
                 TTTAC 
                   
                   
               
               
                   
               
               
                 G1U 
                 
                   TCGAGTTCCCCGCGCCAGCGGG 
                 
                 115 
                   
               
               
                   
                   GATAAACCGAAAAGCATATAAT G 
                   
                   
               
               
                   
                 CGTAAAAGTTATGAAGT TCGA   
                   
                   
               
               
                   
                 
                   GTTCCCCGCGCCAGCGGGGATA 
                 
                   
                   
               
               
                   
                   AACCG TTACCATTCTGTTGCTT 
                   
                   
               
               
                   
                 TTATGTATAAGAA TCGAGTTCC   
                   
                   
               
               
                   
                 
                   CCGCGCCAGCGGGGATAAACCG 
                 
                   
                   
               
               
                   
               
               
                 udhA-FOR 
                 GCGCCAGCGGGGATAAACCGT 
                 116 
                 pCASCADE-udhA 
               
               
                   
                 TACCATTCTGTTG 
                   
                   
               
               
                 pCASCADE-REV 
                 CTTGCCCGCCTGATGAATGCTC 
                 117 
                   
               
               
                   
                 ATCCGG 
                   
                   
               
               
                   
               
               
                 pCASCADE-FOR 
                 CCGGATGAGCATTCATCAGGC 
                 118 
                 pCASCADE-gltA1 
               
               
                   
                 GGGCAAG 
                   
                   
               
               
                 gltA1-REV 
                 CGGTTTATCCCCGCTGGCGCG 
                 119 
                   
               
               
                   
                 GGGAACTCGAACTTCATAACT 
                   
                   
               
               
                   
                 TTTAC 
                   
                   
               
               
                   
               
               
                 G1Z 
                 
                   TCGAGTTCCCCGCGCCAGCGGG 
                 
                 120 
                   
               
               
                   
                   GATAAACCG AAAAGCATATAAT 
                   
                   
               
               
                   
                 GCGTAAAAGTTATGAAGT TCG   
                   
                   
               
               
                   
                 
                   AGTTCCCCGCGCCAGCGGGGAT 
                 
                   
                   
               
               
                   
                   AAACCG CTCGTAAAAGCAGTA 
                   
                   
               
               
                   
                 CAGTGCACCGTAAGA TCGAGTT   
                   
                   
               
               
                   
                 
                   CCCCGCGCCAGCGGGGATAAAC 
                 
                   
                   
               
               
                   
                 
                   CG 
                 
                   
                   
               
               
                   
               
               
                 zwf-FOR 
                 GCGCCAGCGGGGATAAACCGC 
                 121 
                 pCASCADE-zwf 
               
               
                   
                 TCGTAAAAG 
                   
                   
               
               
                 pCASCADE-REV 
                 CTTGCCCGCCTGATGAATGCTC 
                 122 
                   
               
               
                   
                 ATCCGG 
                   
                   
               
               
                   
               
               
                   
               
               
                 pCASCADE-FOR 
                 CCGGATGAGCATTCATCAGGC 
                 123 
                 pCASCADE-gltA1 
               
               
                   
                 GGGCAAG 
                   
                   
               
               
                 gltA1-REV 
                 CGGTTTATCCCCGCTGGCGCG 
                 124 
                   
               
               
                   
                 GGGAACTCGAACTTCATAACT 
                   
                   
               
               
                   
                 TTTAC 
                   
                   
               
               
                   
               
               
                 G2U 
                 
                   TCGAGTTCCCCGCGCCAGCGGG 
                 
                 125 
                   
               
               
                   
                 
                   GATAAACCGTATTGACCAATTCA 
                 
                   
                   
               
               
                   
                   TTC GGGACAGTTATTAGT TCGA   
                   
                   
               
               
                   
                 
                   GTTCCCCGCGCCAGCGGGGATA 
                 
                   
                   
               
               
                   
                   AACCG TTACCATTCTGTTGCTT 
                   
                   
               
               
                   
                 TTATGTATAAGAA TCGAGTTCC   
                   
                   
               
               
                   
                 
                   CCGCGCCAGCGGGGATAAACCG 
                 
                   
                   
               
               
                   
               
               
                 udhA-FOR 
                 GCGCCAGCGGGGATAAACCGT 
                 126 
                 pCASCADE-udhA 
               
               
                   
                 TACCATTCTGTTG 
                   
                   
               
               
                 pCASCADE-REV 
                 CTTGCCCGCCTGATGAATGCTC 
                 127 
                   
               
               
                   
                 ATCCGG 
                   
                   
               
               
                   
               
               
                 pCASCADE-FOR 
                 CCGGATGAGCATTCATCAGGC 
                 128 
                 pCASCADE-gltA2 
               
               
                   
                 GGGCAAG 
                   
                   
               
               
                 gltA2-REV 
                 CGGTTTATCCCCGCTGGCGCG 
                 129 
                   
               
               
                   
                 GGGAACTCGAACTAATAACTG 
                   
                   
               
               
                   
                 TC 
                   
                   
               
               
                   
               
               
                 G2Z 
                 
                   TCGAGTTCCCCGCGCCAGCGGG 
                 
                 130 
                   
               
               
                   
                 
                   GATAAACCGTATTGACCAATTCA 
                 
                   
                   
               
               
                   
                   TTC GGGACAGTTATTAGT TCGA   
                   
                   
               
               
                   
                 
                   GTTCCCCGCGCCAGCGGGGATA 
                 
                   
                   
               
               
                   
                   AACCG CTCGTAAAAGCAGTAC 
                   
                   
               
               
                   
                 AGTGCACCGTAAGA TCGAGTTC   
                   
                   
               
               
                   
                 
                   CCCGCGCCAGCGGGGATAAACC 
                 
                   
                   
               
               
                   
                 
                   G 
                 
                   
                   
               
               
                   
               
               
                 zwf-FOR 
                 GCGCCAGCGGGGATAAACCGC 
                 131 
                 pCASCADE-zwf 
               
               
                   
                 TCGTAAAAG 
                   
                   
               
               
                 pCASCADE-REV 
                 CTTGCCCGCCTGATGAATGCTC 
                 132 
                   
               
               
                   
                 ATCCGG 
                   
                   
               
               
                   
               
               
                 pCASCADE-FOR 
                 CCGGATGAGCATTCATCAGGC 
                 133 
                 pCASCADE-gltA2 
               
               
                   
                 GGGCAAG 
                   
                   
               
               
                 gltA2-REV 
                 CGGTTTATCCCCGCTGGCGCG 
                 134 
                   
               
               
                   
                 GGGAACTCGAACTAATAACTG 
                   
                   
               
               
                   
                 TC 
                   
                   
               
            
           
           
               
            
               
                 UZ 
               
            
           
           
               
               
               
               
            
               
                   
                 
                   TCGAGTTCCCCGCGCCAGCGGG 
                 
                 135 
                   
               
               
                   
                   GATAAACCG TTACCATTCTGTT 
                   
                   
               
               
                   
                 GCCTTTTATGTATAAGAA TCGAG   
                   
                   
               
               
                   
                 
                   TTCCCCGCGCCAGCGGGGATAA 
                 
                   
                   
               
               
                   
                   ACCG CTCGTAAAAGCAGTACA 
                   
                   
               
               
                   
                 GTGCACCGTAAGAT CGAGTTCC   
                   
                   
               
               
                   
                 
                   CCGCGCCAGCGGGGATAAACCG 
                 
                   
                   
               
               
                   
               
               
                 zwf-FOR 
                 GCGCCAGCGGGGATAAACCGC 
                 136 
                 pCASCADE-zwf 
               
               
                   
                 TCGTAAAAG 
                   
                   
               
               
                 pCASCADE-REV 
                 CTTGCCCGCCTGATGAATGCTC 
                 137 
                   
               
               
                   
                 ATCCGG 
                   
                   
               
               
                   
               
               
                 pCASCADE-FOR 
                 CCGGATGAGCATTCATCAGGC 
                 138 
                 pCASCADE-udhA 
               
               
                   
                 GGGCAAG 
                   
                   
               
               
                 udhA-REV 
                 CGGTTTATCCCCGCTGGCGCG 
                 139 
                   
               
               
                   
                 GGGAACTCGATTCTTATACAT 
                   
                   
               
               
                   
                 AAAAGC 
                   
                   
               
               
                   
               
               
                 FG1G2 
                 
                   TCGAGTTCCCCGCGCCAGCGGG 
                 
                 140 
                   
               
               
                   
                   GATAAACCG TTGATTATAATAA 
                   
                   
               
               
                   
                 CCGTTTATCTGTTCGTA TCGAG   
                   
                   
               
               
                   
                 
                   TTCCCCGCGCCAGCGGGGATAA 
                 
                   
                   
               
               
                   
                   ACCG AAAAGCATATAATGCGT 
                   
                   
               
               
                   
                 AAAAGTTATGAAGT TCGAGTTC   
                   
                   
               
               
                   
                 
                   CCCGCGCCAGCGGGGATAAACC 
                 
                   
                   
               
               
                   
                   G TATTGACCAATTCATTCGGG 
                   
                   
               
               
                   
                 ACAGTTATTAGT TCGAGTTCCC   
                   
                   
               
               
                   
                 
                   CGCGCCAGCGGGGATAAACCG 
                 
                   
                   
               
               
                   
               
               
                 gltA2-FOR 
                 GCGCCAGCGGGGATAAACCG T   
                 141 
                 pCASCADE-gltA2 
               
               
                   
                 
                   ATTGACCAATTCATTC 
                 
                   
                   
               
               
                 pCASCADE-REV 
                 CTTGCCCGCCTGATGAATGCTC 
                 142 
                   
               
               
                   
                 ATCCGG 
                   
                   
               
               
                   
               
               
                 pCASCADE-FOR 
                 CCGGATGAGCATTCATCAGGC 
                 143 
                 pCASCADE-FG1 
               
               
                   
                 GGGCAAG 
                   
                   
               
               
                 gltA1-REV 
                 CGGTTTATCCCCGCTGGCGCG 
                 144 
                   
               
               
                   
                 GGGAACTCGAACTTCATAACT 
                   
                   
               
               
                   
                 TTTAC 
                   
                   
               
               
                   
               
               
                 G1G2A 
                 
                   TCGAGTTCCCCGCGCCAGCGGG 
                 
                 145 
                   
               
               
                   
                   GATAAACCG AAAAGCATATAAT 
                   
                   
               
               
                   
                 GCGTAAAAGTTATGAAGT TCG   
                   
                   
               
               
                   
                 
                   AGTTCCCCGCGCCAGCGGGGAT 
                 
                   
                   
               
               
                   
                   AAACCGT ATTGACCAATTCATT 
                   
                   
               
               
                   
                 CGGGACAGTTATTAGT TCGAGT   
                   
                   
               
               
                   
                 
                   TCCCCGCGCCAGCGGGGATAAA 
                 
                   
                   
               
               
                   
                   CCG GTTTTTGTAATTTTACAGG 
                   
                   
               
               
                   
                 CAACCTTTTAT TCGAGTTCCCC   
                   
                   
               
               
                   
                 
                   GCGCCAGCGGGGATAAACCG 
                 
                   
                   
               
               
                   
               
               
                 gapAP1-FOR 
                 GCGCCAGCGGGGATAAACCGG 
                 146 
                 pCASCADE-gpaAP1 
               
               
                   
                 TTTTTGTAATTTTACAGGC 
                   
                   
               
               
                 pCASCADE-REV 
                 CTTGCCCGCCTGATGAATGCTC 
                 147 
                   
               
               
                   
                 ATCCGG 
                   
                   
               
               
                   
               
               
                 pCASCADE-FOR 
                 CCGGATGAGCATTCATCAGGC 
                 148 
                 pCASCADE-G1G2 
               
               
                   
                 GGGCAAG 
                   
                   
               
               
                 gltA2-REV 
                 CGGTTTATCCCCGCTGGCGCG 
                 149 
                   
               
               
                   
                 GGGAACTCGAACTAATAACTG 
                   
                   
               
               
                   
                 TC 
                   
                   
               
               
                   
               
               
                 G1G2U 
                 
                   TCGAGTTCCCCGCGCCAGCGGG 
                 
                 150 
                   
               
               
                   
                   GATAAACCG AAAAGCATATAAT 
                   
                   
               
               
                   
                 GCGTAAAAGTTATGAAGT TCG   
                   
                   
               
               
                   
                 
                   AGTTCCCCGCGCCAGCGGGGAT 
                 
                   
                   
               
               
                   
                   AAACCG TATTGACCAATTCATT 
                   
                   
               
               
                   
                 CGGGACAGTTATTAGT TCGAGT   
                   
                   
               
               
                   
                 
                   TCCCCGCGCCAGCGGGGATAAA 
                 
                   
                   
               
               
                   
                   CCG TTACCATTCTGTTGCTTTT 
                   
                   
               
               
                   
                 ATGTATAAGAA TCGAGTTCCCC   
                   
                   
               
               
                   
                 
                   GCGCCAGCGGGGATAAACCG 
                 
                   
                   
               
               
                   
               
               
                 udhA-FOR 
                 GCGCCAGCGGGGATAAACCGT 
                 151 
                 pCASCADE-udhA 
               
               
                   
                 TACCATTCTGTTG 
                   
                   
               
               
                 pCASCADE-REV 
                 CTTGCCCGCCTGATGAATGCTC 
                 152 
                   
               
               
                   
                 ATCCGG 
                   
                   
               
               
                   
               
               
                 pCASCADE-FOR 
                 CCGGATGAGCATTCATCAGGC 
                 153 
                 pCASCADE-G1G2 
               
               
                   
                 GGGCAAG 
                   
                   
               
               
                 gltA2-REV 
                 CGGTTTATCCCCGCTGGCGCG 
                 154 
                   
               
               
                   
                 GGGAACTCGAACTAATAACTG 
                   
                   
               
               
                   
                 TC 
                   
                   
               
               
                   
               
               
                 G1G2Z 
                 
                   TCGAGTTCCCCGCGCCAGCGGG 
                 
                 155 
                   
               
               
                   
                   GATAAACCG AAAAGCATATAAT 
                   
                   
               
               
                   
                 GCGTAAAAGTTATGAAGT TCG   
                   
                   
               
               
                   
                 
                   AGTTCCCCGCGCCAGCGGGGAT 
                 
                   
                   
               
               
                   
                   AAACCG TATTGACCAATTCATT 
                   
                   
               
               
                   
                 CGGGACAGTTATTAGT TCGAGT   
                   
                   
               
               
                   
                 
                   TCCCCGCGCCAGCGGGGATAAA 
                 
                   
                   
               
               
                   
                   CCG CTCGTAAAAGCAGTACAG 
                   
                   
               
               
                   
                 TGCACCGTAAGA TCGAGTTCCC   
                   
                   
               
               
                   
                 
                   CGCGCCAGCGGGGATAAACCG 
                 
                   
                   
               
               
                   
               
               
                 zwf-FOR 
                 GCGCCAGCGGGGATAAACCGC 
                 156 
                 pCASCADE-zwf 
               
               
                   
                 TCGTAAAAG 
                   
                   
               
               
                 pCASCADE-REV 
                 CTTGCCCGCCTGATGAATGCTC 
                 157 
                   
               
               
                   
                 ATCCGG 
                   
                   
               
               
                   
               
               
                 pCASCADE-FOR 
                 CCGGATGAGCATTCATCAGGC 
                 158 
                 pCASCADE-G1G2 
               
               
                   
                 GGGCAAG 
                   
                   
               
               
                 gltA2-REV 
                 CGGTTTATCCCCGCTGGCGCG 
                 159 
                   
               
               
                   
                 GGGAACTCGAACTAATAACTG 
                   
                   
               
               
                   
                 TC 
                   
                   
               
               
                   
               
               
                 FG1G2A 
                 
                   TCGAGTTCCCCGCGCCAGCGGG 
                 
                 160 
                   
               
               
                   
                   GATAAACCG TTGATTATAATAA 
                   
                   
               
               
                   
                 CCGTTTATCTGTTCGTA TCGAG   
                   
                   
               
               
                   
                 
                   TTCCCCGCGCCAGCGGGGATAA 
                 
                   
                   
               
               
                   
                   ACCG AAAAGCATATAATGCGT 
                   
                   
               
               
                   
                 AAAAGTTATGAAGT TCGAGTTC   
                   
                   
               
               
                   
                 
                   CCCGCGCCAGCGGGGATAAACC 
                 
                   
                   
               
               
                   
                   G TATTGACCAATTCATTCGGG 
                   
                   
               
               
                   
                 ACAGTTATTAGT TCGAGTTCCC   
                   
                   
               
               
                   
                   CGCGCCAGCGGGGATAAACCG G 
                   
                   
               
               
                   
                 TTTTTGTAATTTTACAGGCAAC 
                   
                   
               
               
                   
                 CTTTTAT TCGAGTTCCCCGCGC   
                   
                   
               
               
                   
                 
                   CAGCGGGGATAAACCG 
                 
                   
                   
               
               
                   
               
               
                 gapAP1-FOR 
                 GCGCCAGCGGGGATAAACCGG 
                 161 
                 pCASCADE-gapAP1 
               
               
                   
                 TTTTTGTAATTTTACAGGC 
                   
                   
               
               
                 pCASCADE-REV 
                 CTTGCCCGCCTGATGAATGCTC 
                 162 
                   
               
               
                   
                 ATCCGG 
                   
                   
               
               
                   
               
               
                 pCASCADE-FOR 
                 CCGGATGAGCATTCATCAGGC 
                 163 
                 pCASCADE-FG1G2 
               
               
                   
                 GGGCAAG 
                   
                   
               
               
                 gltA2-REV 
                 CGGTTTATCCCCGCTGGCGCG 
                 164 
                   
               
               
                   
                 GGGAACTCGAACTAATAACTG 
                   
                   
               
               
                   
                 TC 
                   
                   
               
               
                   
               
               
                 FG1G2U 
                 
                   TCGAGTTCCCCGCGCCAGCGGG 
                 
                 165 
                   
               
               
                   
                   GATAAACCG TTGATTATAATAA 
                   
                   
               
               
                   
                 CCGTTTATCTGTTCGTAT CGAG   
                   
                   
               
               
                   
                 
                   TTCCCCGCGCCAGCGGGGATAA 
                 
                   
                   
               
               
                   
                   ACCG AAAAGCATATAATGCGT 
                   
                   
               
               
                   
                 AAAAGTTATGAAGTT CGAGTTC   
                   
                   
               
               
                   
                 
                   CCCGCGCCAGCGGGGATAAACC 
                 
                   
                   
               
               
                   
                   G TATTGACCAATTCATTCGGG 
                   
                   
               
               
                   
                 ACAGTTATTAGT TCGAGTTCCC   
                   
                   
               
               
                   
                   CGCGCCAGCGGGGATAAACCG T 
                   
                   
               
               
                   
                 TACCATTCTGTTGCTTTTATGT 
                   
                   
               
               
                   
                 ATAAGAA TCGAGTTCCCCGCGC   
                   
                   
               
               
                   
                 
                   CAGCGGGGATAAACCG 
                 
                   
                   
               
               
                   
               
               
                 gltA2-FOR 
                 GCGCCAGCGGGGATAAACCG T   
                 166 
                 pCASCADE-udhA 
               
               
                   
                 
                   ATTGACCAATTCATTC 
                 
                   
                   
               
               
                 pCASCADE-REV 
                 CTTGCCCGCCTGATGAATGCTC 
                 167 
                   
               
               
                   
                 ATCCGG 
                   
                   
               
               
                   
               
               
                 pCASCADE-FOR 
                 CCGGATGAGCATTCATCAGGC 
                 168 
                 pCASCADE-FG1G2 
               
               
                   
                 GGGCAAG 
                   
                   
               
               
                 gltA1-REV 
                 CGGTTTATCCCCGCTGGCGCG 
                 169 
                   
               
               
                   
                 GGGAACTCGAACTTCATAACT 
                   
                   
               
               
                   
                 TTTAC 
                   
                   
               
               
                   
               
               
                 FG1G2Z 
                 
                   TCGAGTTCCCCGCGCCAGCGGG 
                 
                 170 
                   
               
               
                   
                   GATAAACCG TTGATTATAATAA 
                   
                   
               
               
                   
                 CCGTTTATCTGTTCGTA TCGAG   
                   
                   
               
               
                   
                 
                   TTCCCCGCGCCAGCGGGGATAA 
                 
                   
                   
               
               
                   
                   ACCG AAAAGCATATAATGCGT 
                   
                   
               
               
                   
                 AAAAGTTATGAAGT TCGAGTTC   
                   
                   
               
               
                   
                 
                   CCCGCGCCAGCGGGGATAAACC 
                 
                   
                   
               
               
                   
                   G TATTGACCAATTCATTCGGG 
                   
                   
               
               
                   
                 ACAGTTATTAGT TCGAGTTCCC   
                   
                   
               
               
                   
                   CGCGCCAGCGGGGATAAACCG C 
                   
                   
               
               
                   
                 TCGTAAAAGCAGTACAGTGCA 
                   
                   
               
               
                   
                 CCGTAAGA TCGAGTTCCCCGCG   
                   
                   
               
               
                   
                 
                   CCAGCGGGGATAAACCG 
                 
                   
                   
               
               
                   
               
               
                 gltA2-FOR 
                 GCGCCAGCGGGGATAAACCG T   
                 171 
                 pCASCADE-zwf 
               
               
                   
                 
                   ATTGACCAATTCATTC 
                 
                   
                   
               
               
                 pCASCADE-REV 
                 CTTGCCCGCCTGATGAATGCTC 
                 172 
                   
               
               
                   
                 ATCCGG 
                   
                   
               
               
                   
               
               
                 pCASCADE-FOR 
                 CCGGATGAGCATTCATCAGGC 
                 173 
                 pCASCADE-FG1G2 
               
               
                   
                 GGGCAAG 
                   
                   
               
               
                 gltA1-REV 
                 CGGTTTATCCCCGCTGGCGCG 
                 174 
                   
               
               
                   
                 GGGAACTCGAACTTCATAACT 
                   
                   
               
               
                   
                 TTTAC 
                   
                   
               
               
                   
               
               
                 G1G2UA 
                 
                   TCGAGTTCCCCGCGCCAGCGGG 
                 
                 175 
                   
               
               
                   
                   GATAAACCG AAAAGCATATAAT 
                   
                   
               
               
                   
                 GCGTAAAAGTTATGAAGT TCG   
                   
                   
               
               
                   
                 
                   AGTTCCCCGCGCCAGCGGGGAT 
                 
                   
                   
               
               
                   
                   AAACCG TATTGACCAATTCATT 
                   
                   
               
               
                   
                 CGGGACAGTTATTAGT TCGAGT   
                   
                   
               
               
                   
                 
                   TCCCCGCGCCAGCGGGGATAAA 
                 
                   
                   
               
               
                   
                   CCG TTACCATTCTGTTGCTTTT 
                   
                   
               
               
                   
                 ATGTATAAGAA TCGAGTTCCCC   
                   
                   
               
               
                   
                   GCGCCAGCGGGGATAAACCG GT 
                   
                   
               
               
                   
                 TTTTGTAATTTTACAGGCAAC 
                   
                   
               
               
                   
                 CTTTTAT TCGAGTTCCCCGCGC   
                   
                   
               
               
                   
                 
                   CAGCGGGGATAAACCG 
                 
                   
                   
               
               
                   
               
               
                 gapA1-FOR 
                 GCGCCAGCGGGGATAAACCGG 
                 176 
                 pCASCADE-AP1 
               
               
                   
                 TTTTTGTAATTTTACAGGC 
                   
                   
               
               
                 pCASCADE-REV 
                 CTTGCCCGCCTGATGAATGCTC 
                 177 
                   
               
               
                   
                 ATCCGG 
                   
                   
               
               
                   
               
               
                 pCASCADE-FOR 
                 CCGGATGAGCATTCATCAGGC 
                 178 
                 pCASCADE-G1G2U 
               
               
                   
                 GGGCAAG 
                   
                   
               
               
                 udhA-REV 
                 CGGTTTATCCCCGCTGGCGCG 
                 179 
                   
               
               
                   
                 GGGAACTCGATTCTTATACAT 
                   
                   
               
               
                   
                 AAAAGC 
                   
                   
               
               
                   
               
               
                 G1G2UZ 
                 
                   TCGAGTTCCCCGCGCCAGCGGG 
                 
                 180 
                   
               
               
                   
                   GATAAACCG AAAAGCATATAAT 
                   
                   
               
               
                   
                 GCGTAAAAGTTATGAAGT TCG   
                   
                   
               
               
                   
                 
                   AGTTCCCCGCGCCAGCGGGGAT 
                 
                   
                   
               
               
                   
                   AAACCG TATTGACCAATTCATT 
                   
                   
               
               
                   
                 CGGGACAGTTATTAGT TCGAGT   
                   
                   
               
               
                   
                 
                   TCCCCGCGCCAGCGGGGATAAA 
                 
                   
                   
               
               
                   
                   CCG TTACCATTCTGTTGCTTTT 
                   
                   
               
               
                   
                 ATGTATAAGAA TCGAGTTCCCC   
                   
                   
               
               
                   
                   GCGCCAGCGGGGATAAACCG CT 
                   
                   
               
               
                   
                 
                   CGTAAAAGCAGTACAGTGCAC 
                 
                   
                   
               
               
                   
                 CGTAAGA TCGAGTTCCCCGCGC   
                   
                   
               
               
                   
                 
                   CAGCGGGGATAAACCG 
                 
                   
                   
               
               
                   
               
               
                 zwf-FOR 
                 GCGCCAGCGGGGATAAACCGC 
                 181 
                 pCASCADE-zwf 
               
               
                   
                 TCGTAAAAG 
                   
                   
               
               
                 pCASCADE-REV 
                 CTTGCCCGCCTGATGAATGCTC 
                 182 
                   
               
               
                   
                 ATCCGG 
                   
                   
               
               
                   
               
               
                 pCASCADE-FOR 
                 CCGGATGAGCATTCATCAGGC 
                 183 
                 pCASCADE-G1G2U 
               
               
                   
                 GGGCAAG 
                   
                   
               
               
                 udhA-REV 
                 CGGTTTATCCCCGCTGGCGCG 
                 184 
                   
               
               
                   
                 GGGAACTCGATTCTTATACAT 
                   
                   
               
               
                   
                 AAAAGC 
                   
                   
               
               
                   
               
               
                 FG1G2UA 
                 
                   TCGAGTTCCCCGCGCCAGCGGG 
                 
                 185 
                   
               
               
                   
                   GATAAACCG TTGATTATAATAA 
                   
                   
               
               
                   
                 CCGTTTATCTGTTCGTA TCGAG   
                   
                   
               
               
                   
                 
                   TTCCCCGCGCCCAGCGGGGATAA 
                 
                   
                   
               
               
                   
                   ACCG AAAAGCATATAATGCGT 
                   
                   
               
               
                   
                 AAAAGTTATGAAGT TCGAGTTC   
                   
                   
               
               
                   
                 
                   CCCGCGCCAGCGGGGATAAACC 
                 
                   
                   
               
               
                   
                   G TATTGACCAATTCATTCGGG 
                   
                   
               
               
                   
                 ACAGTTATTAGT TCGAGTTCCC   
                   
                   
               
               
                   
                   CGCGCCAGCGGGGATAAACCG T 
                   
                   
               
               
                   
                 TACCATTCTGTTGCTTTTATGT 
                   
                   
               
               
                   
                 ATAAGAA TCGAGTTCCCCGCGC   
                   
                   
               
               
                   
                   CAGCGGGGATAAACCG GTTTTT 
                   
                   
               
               
                   
                 GTAATTTTACAGGCAACCTTT 
                   
                   
               
               
                   
                 TAT TCGAGTTCCCCGCGCCAGC   
                   
                   
               
               
                   
                 
                   GGGGATAAACCG 
                 
                   
                   
               
               
                   
               
               
                 gapAP1-FOR 
                 GCGCCAGCGGGGATAAACCGG 
                 186 
                 pCASCADE-gpaAP1 
               
               
                   
                 TTTTTGTAATTTTACAGGC 
                   
                   
               
               
                 pCASCADE-REV 
                 CTTGCCCGCCTGATGAATGCTC 
                 187 
                   
               
               
                   
                 ATCCGG 
                   
                   
               
               
                   
               
               
                 pCASCADE-FOR 
                 CCGGATGAGCATTCATCAGGC 
                 188 
                 pCASCADE- 
               
               
                   
                 GGGCAAG 
                   
                 FG1G2U 
               
               
                 udhA-REV 
                 CGGTTTATCCCCGCTGGCGCG 
                 189 
                   
               
               
                   
                 GGGAACTCGATTCTTATACAT 
                   
                   
               
               
                   
                 AAAAGC 
                   
                   
               
               
                   
               
               
                 FG1G2UZ 
                 
                   TCGAGTTCCCCGCGCCAGCGGG 
                 
                 190 
                   
               
               
                   
                   GATAAACCG TTGATTATAATAA 
                   
                   
               
               
                   
                 CCGTTTATCTGTTCGTAT CGAG   
                   
                   
               
               
                   
                 
                   TTCCCCGCGCCAGCGGGGATAA 
                 
                   
                   
               
               
                   
                   ACCG AAAAGCATATAATGCGT 
                   
                   
               
               
                   
                 AAAAGTTATGAAGT TCGAGTTC   
                   
                   
               
               
                   
                 
                   CCCGCGCCAGCGGGGATAAACC 
                 
                   
                   
               
               
                   
                   G TATTGACCAATTCATTCGGG 
                   
                   
               
               
                   
                 ACAGTTATTAGT TCGAGTTCCC   
                   
                   
               
               
                   
                   CGCGCCAGCGGGGATAAACCG T 
                   
                   
               
               
                   
                 TACCATTCTGTTGCTTTTATGT 
                   
                   
               
               
                   
                 ATAAGAA TCGAGTTCCCCGCGC   
                   
                   
               
               
                   
                   CAGCGGGGATAAACCG CTCGTA 
                   
                   
               
               
                   
                 AAAGCAGTACAGTGCACCGTA 
                   
                   
               
               
                   
                 AGA TCGAGTTCCCCGCGCCAGC   
                   
                   
               
               
                   
                 
                   GGGGATAAACCG 
                 
                   
                   
               
               
                   
               
               
                 zwf-FOR 
                 GCGCCAGCGGGGATAAACCGC 
                 191 
                 pCASCADE-zwf 
               
               
                   
                 TCGTAAAAG 
                   
                   
               
               
                 pCASCADE-REV 
                 CTTGCCCGCCTGATGAATGCTC 
                 192 
                   
               
               
                   
                 ATCCGG 
                   
                   
               
               
                   
               
               
                 pCASCADE-FOR 
                 CCGGATGAGCATTCATCAGGC 
                 193 
                 pCASCADE- 
               
               
                   
                 GGGCAAG 
                   
                 FG1G2U 
               
               
                 udhA-REV 
                 CGGTTTATCCCCGCTGGCGCG 
                 194 
                   
               
               
                   
                 GGGAACTCGATTCTTATACAT 
                   
                   
               
               
                   
                 AAAAGC 
                   
                   
               
               
                   
               
               
                 FG1G2UZA 
                 
                   TCGAGTTCCCCGCGCCAGCGGG 
                 
                 195 
                   
               
               
                   
                   GATAAACCG TTGATTATAATAA 
                   
                   
               
               
                   
                 CCGTTTATCTGTTCGTA TCGAG   
                   
                   
               
               
                   
                 
                   TTCCCCGCGCCCAGCGGGGATAA 
                 
                   
                   
               
               
                   
                   ACCG AAAAGCATATAATGCGT 
                   
                   
               
               
                   
                 AAAAGTTATGAAGT TCGAGTTC   
                   
                   
               
               
                   
                 
                   CCCGCGCCAGCGGGGATAAACC 
                 
                   
                   
               
               
                   
                   G TATTGACCAATTCATTCGGG 
                   
                   
               
               
                   
                 ACAGTTATTAGT TCGAGTTCCC   
                   
                   
               
               
                   
                   CGCGCCAGCGGGGATAAACCG T 
                   
                   
               
               
                   
                 TACCATTCTGTTGCTTTTATGT 
                   
                   
               
               
                   
                 ATAAGAA TCGAGTTCCCCGCGC   
                   
                   
               
               
                   
                   CAGCGGGGATAAACCG CTCGTA 
                   
                   
               
               
                   
                 AGA TCGAGTTCCCCGCGCCAGC   
                   
                   
               
               
                   
                   GGGGATAAACCG GTTTTTGTAA 
                   
                   
               
               
                   
                 TTTTACAGGCAACCTTTTAT TC   
                   
                   
               
               
                   
                 
                   GAGTTCCCCGCGCCCAGCGGGGA 
                 
                   
                   
               
               
                   
                 
                   TAAACCG 
                 
                   
                   
               
               
                   
               
               
                 gapAP1-FOR 
                 GCGCCAGCGGGGATAAACCGG 
                 196 
                 pCASCADE-gapA1 
               
               
                   
                 TTTTTGTAATTTTACAGGC 
                   
                   
               
               
                 pCASCADE-REV 
                 CTTGCCCGCCTGATGAATGCTC 
                 197 
                   
               
               
                   
                 ATCCGG 
                   
                   
               
               
                   
               
               
                 pCASCADE-FOR 
                 CCGGATGAGCATTCATCAGGC 
                 198 
                 pCASCADE- 
               
               
                   
                 GGGCAAG 
                   
                 FG1G2UZ 
               
               
                 zwf-REV 
                 CGGTTTATCCCCGCTGGCGCG 
                 199 
                   
               
               
                   
                 GGGAACTCGATCTTACGGTGC 
                   
                   
               
               
                   
                 ACTGTC 
                   
                   
               
               
                   
               
               
                 UZ 
                 
                   TCGAGTTCCCCGCGCCAGCGGG 
                 
                 200 
                   
               
               
                   
                   GATAAACCG TTACCATTCTGTT 
                   
                   
               
               
                   
                 GCTTTTATGTATAAGAA TCGAG   
                   
                   
               
               
                   
                 
                   TTCCCCGCGCCAGCGGGGATAA 
                 
                   
                   
               
               
                   
                   ACCG CTCGTAAAAGCAGTACA 
                   
                   
               
               
                   
                 GTGCACCGTAAGA TCGAGTTCC   
                   
                   
               
               
                   
                 
                   CCGCGCCAGCGGGGATAAACCG 
                 
                   
                   
               
               
                   
               
               
                 zwf-FOR 
                 GCGCCAGCGGGGATAAACCGC 
                 201 
                 pCASCADE-zwf 
               
               
                   
                 TCGTAAAAG 
                   
                   
               
               
                 pCASCADE-REV 
                 CTTGCCCGCCTGATGAATGCTC 
                 202 
                   
               
               
                   
                 ATCCGG 
                   
                   
               
               
                   
               
               
                 pCASCADE-FOR 
                 CCGGATGAGCATTCATCAGGC 
                 203 
                 pCASCADE-udhA 
               
               
                   
                 GGGCAAG 
                   
                   
               
               
                 udhA-REV 
                 CGGTTTATCCCCGCTGGCGCG 
                 204 
                   
               
               
                   
                 GGGAACTCGATTCTTATACAT 
                   
                   
               
               
                   
                 AAAAGC 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 10 
               
               
                   
               
               
                 List of plasmids used in this study. 
               
               
                   
               
             
            
               
                 Plasmid Utilized in this Study 
               
            
           
           
               
               
               
            
               
                 Plasmid 
                 Purpose 
                 Source 
               
               
                   
               
               
                 pSIM5 
                 Recombineering and Strain Construction 
                 Court Lab 54   
               
               
                 pCP20 
                 FRT kanamycin cassette curing 
                 Court Lab 54   
               
               
                 pSMART-HC-Kan 
                 Backbone Vector 
                 Lucigen 
               
               
                 pcrRNA.Tet 
                 pCASCADE-control backbone 
                 Beisel Lab 34   
               
               
                   
               
            
           
           
               
            
               
                 Plasmid Constructed in this Study 
               
            
           
           
               
               
               
            
               
                 Piasmid 
                 Plasmid Name 
                 Addgene ID 
               
               
                   
               
               
                 pSMART-Ala2 
                 pSMART-HCKanIN:yibDp-ald* 
                  71326 
               
               
                 pSMART-Ala3 
                 pSMART-HCKan-IN:phoBp-ald* 
                  71327 
               
               
                 pSMART-Ala4 
                 pSMART-HCKan-IN:phoBp-ald* 
                  71328 
               
               
                 pSMART-Ala5 
                 pSMART-HCKan-IN:mipAp-ald* 
                  71329 
               
               
                 pSMART-Ala11 
                 pSMART-HCKan-proA-ald* 
                  87172 
               
               
                 pSMART-Ala12 
                 pSMART-HCKan-proC-ald* 
                  87173 
               
               
                 pSMART-Ala13 
                 pSMART-HCKan-proD-ald* 
                  87174 
               
               
                 pSMART-Ala14 
                 pSMART-HCKan-proB-ald* 
                 101079 
               
               
                 pSMART-Ala15 
                 pSMART-HCKan-HCEp-ald* 
                 101080 
               
               
                 pSMART-Mev2 
                 pSMART-IN:yibDp1-mvaE-IN:phoBp2-mvaS(A110G) 
                  66642 
               
               
                 pSMART-Mev3 
                 pSMART-IN:yibDp1-mvaE-IN:mipAp2-mvaS(A110G) 
                 102761 
               
               
                 pSMART-Mev4 
                 pSMART-IN:yibDp1-mvaE-IN:phoHp2-mvaS(A110G) 
                 102762 
               
               
                 pSMART-Mev5 
                 pSMART-IN:mipAp1-mvaE-IN:yibDp2-mvaS(A110G) 
                 102763 
               
               
                 pSMART-3HP 
                 pSMART-3HP-NADPH-rhtA 
                  87143 
               
               
                 pCDF-mcherry1 
                 pCDF-proD-mcherry 
                  87144 
               
               
                 pCDF-mcherry2 
                 pCDF-proD-mcherry-DAS4 
                  87145 
               
               
                 pSMART-GFPuv 
                 pSMART-IN:yibDp-GFPuv 
                  65822 
               
               
                 pSMART-GFPuv2 
                 pSMART-IN:phoBp-GFPuv 
                  71517 
               
               
                 pSMART-GFPuv3 
                 pSMART-IN:phoUp-GFPuv 
                  71518 
               
               
                 pSMART-GFPuv4 
                 pSMART-IN:phoHp-GFPuv 
                  71519 
               
               
                 pSMART-GFPuv5 
                 pSMART-IN:mipAp-GFPuv 
                  71520 
               
               
                 pCASCADE-control 
                 pCASCADE 
                  65821 
               
               
                 pCASCADE-proD 
                 pCASCADE-proD 
                  65820 
               
               
                 pCASCADE-gapAP1 
                 pCASCADE-gapAP1 
                  87146 
               
               
                 pCASCADE-fabI 
                 pCASCADE-fabI 
                  66635 
               
               
                 pCASCADE-FG1 
                 pCASCADE-fabI-gltA1 
                  71340 
               
               
                 pCASCADE-FG1G2 
                 pCASCADE-fabI-gltA1-gltA2 
                  71342 
               
               
                 pCASCADE-FG1G2A 
                 pCASCADE-fabI-gltA1-gltA2-gapA 
                  87147 
               
               
                 pCASCADE-FG1G2U 
                 pCASCADE-fabI-gltA1-gltA2-udhA 
                  66637 
               
               
                 pCASCADE-FG1G2UA 
                 pCASCADE-fabI-gltA1-gltA2-udhA-gapA 
                  87154 
               
               
                 pCASCADE-FG1G2UZ 
                 pCASCADE-fabI-gltA1-gltA2-udhA-zwf 
                  87148 
               
               
                 pCASCADE-FG1G2UZA 
                 pCASCADE-fabI-gltA1-gltA2-udhA-zwf-gapA 
                  87149 
               
               
                 pCASCADE-FG1G2Z 
                 pCASCADE-fabI-gltA1-gltA2-zwf 
                  66638 
               
               
                 pCASCADE-FG2 
                 pCASCADE-fabI-gltA2 
                  71341 
               
               
                 pCASCADE-FU 
                 pCASCADE-fabI-udhA 
                  66636 
               
               
                 pCASCADE-FZ 
                 pCASCADE-fabI-zwf 
                  71335 
               
               
                 pCASCADE-G1G2 
                 pCASCADE-gltA1-gltA2 
                  71348 
               
               
                 pCASCADE-G1G2A 
                 pCASCADE-gltA1-gltA2-gapA 
                  87150 
               
               
                 pCASCADE-G1G2U 
                 pCASCADE-gltA1-gltA2-udhA 
                  71343 
               
               
                 pCASCADE-G1G2UA 
                 pCASCADE-gltA1-gltA2-udhA-gapA 
                  87151 
               
               
                 pCASCADE-G1G2UZ 
                 pCASCADE-gltA1-gltA2-udhA-zwf 
                  87152 
               
               
                 pCASCADE-G1G2Z 
                 pCASCADE-gltA1-gltA2-zwf 
                  71347 
               
               
                 pCASCADE-G1U 
                 pCASCADE-gltA1-udhA 
                  71339 
               
               
                 pCASCADE-G1Z 
                 pCASCADE-gltA1-zwf 
                  71337 
               
               
                 pCASCADE-G2U 
                 pCASCADE-gltA2-udhA 
                  65819 
               
               
                 pCASCADE-G2Z 
                 pCASCADE-gltA2-zwf 
                  71338 
               
               
                 pCASCADE-gltA1 
                 pCASCADE-gltA1 
                  71334 
               
               
                 pCASCADE-gltA2 
                 pCASCADE-gltA2 
                  65817 
               
               
                 pCASCADE-udhA 
                 pCASCADE-udhA 
                  65818 
               
               
                 pCASCADE-UZ 
                 pCASCADE-udhA-zwf 
                  87153 
               
               
                 pCASCADE-zwf 
                 pCASCADE-zwf 
                  65825 
               
               
                   
               
            
           
         
       
     
     Section 7: 2-Stage Micro-Fermentations 
       E. coli  Media Stock Solutions
         10× concentrated Ammonium-Citrate 30 salts (1 L), mix 30 g of (NH 4 ) 2 SO 4  and 1.5 g citric acid in water with stirring, adjust pH to 7.5 with 10 M NaOH. Autoclave and store at room temperature (RT).   10× concentrated Ammonium-Citrate 90 salts (1 L), mix 90 g of (NH 4 ) 2 SO 4  and 2.5 g citric acid in water with stirring, adjust pH to 7.5 with 10 M NaOH. Autoclave and store at RT.   1 M Potassium 3-(N-morpholino) propanesulfonic Acid (MOPS), adjust to pH 7.4 with 50% KOH. Filter sterilize (0.2 μm) and store at RT.   0.5 M potassium phosphate buffer, pH 6.8, mix 248.5 mL of 1.0 M K 2 HPO 3  and 251.5 mL of 1.0 M KH 2 PO 4  and adjust to a final volume of 1000 mL with ultrapure water. Filter sterilize (0.2 μm) and store at RT.   2 M MgSO 4  and 10 mM CaSO 4  solutions. Filter sterilize (0.2 μm) and store at RT.   50 g/L solution of thiamine-HCl. Filter sterilize (0.2 μm) and store at 4° C.   500 g/L solution of glucose, dissolve by stirring with heat. Cool, filter sterilize (0.2 μm), and store at RT.   100 g/L yeast extract, autoclave, and store at RT.   100 g/L casamino acid, autoclave, and store at RT.   500× Trace Metal Stock: Prepare a solution of micronutrients in 1000 mL of water containing 10 mL of concentrated H 2 SO 4 , 0.6 g COSO 4 .7H 2 0, 5.0 g CuSO 4 .5H 2 O, 0.6 g ZnSO 4 .7H 2 O, 0.2 g Na 2 MoO 4 .2H 2 O, 0.1 g H 3 BO 3 , and 0.3 g MnSO 4 .H 2 O. Filter sterilize (0.2 μm) and store at RT in the dark.   Prepare a fresh solution of 40 mM ferric sulfate heptahydrate in water, filter sterilize (0.2 μm) before preparing media each time.       
     Media Components 
     Prepare the final working medium by aseptically mixing stock solutions based on the following tables in the order written to minimize precipitation, then filter sterilize (with a 0.2 μm filter). 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Seed Media, pH 6.8: 
               
            
           
           
               
               
               
               
            
               
                 Ingredient 
                 Unit 
                 SM10 
                 SM10++ 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 (NH 4 ) 2 SO 4   
                 g/L 
                 9 
                 9 
               
               
                 Citric Acid 
                 g/L 
                 0.25 
                 0.25 
               
               
                 Potassium Phosphate 
                 mM 
                 5 
                 5 
               
               
                 CoSO 4  · 7H 2 O 
                 g/L 
                 0.0048 
                 0.0048 
               
               
                 CuSO 4  · 5H 2 O 
                 g/L 
                 0.04 
                 0.04 
               
               
                 ZnSO 4  · 7H 2 O 
                 g/L 
                 0.0048 
                 0.0048 
               
               
                 Na 2 MoO 4  · 2H 2 O 
                 g/L 
                 0.0016 
                 0.0016 
               
               
                 H 3 BO 3   
                 g/L 
                 0.0008 
                 0.0008 
               
               
                 MnSO 4  · 7H 2 O 
                 g/L 
                 0.0024 
                 0.0024 
               
               
                 FeSO 4  · 7H 2 O 
                 g/L 
                 0.044 
                 0.044 
               
               
                 MgSO 4   
                 mM 
                 2.5 
                 2.5 
               
               
                 CaSO 4   
                 mM 
                 0.06 
                 0.06 
               
               
                 Glucose 
                 g/L 
                 45 
                 45 
               
               
                 MOPS 
                 niM 
                 200 
                 200 
               
               
                 Thiamine-HCl 
                 g/L 
                 001 
                 0.01 
               
               
                 Yeast Extract 
                 g/L 
                 1 
                 2.5 
               
               
                 Casarnino Acids 
                 g/L 
                 0 
                 2.5 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Production/Wash Media, pH 6.8: 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                 FGM3 No 
                 FGM3 
                 FGM3 + 40 mM 
                   
               
               
                 Ingredient 
                 Unit 
                 FGM3 
                 Phosphate 
                 Wash 
                 phosphate 
                 FGM10 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 (NH 4 ) 2 SO 4   
                 g/L 
                 3 
                 3 
                 3 
                 3 
                 9 
               
               
                 Citric Acid 
                 g/L 
                 0.15 
                 0.15 
                 0.15 
                 0.15 
                 0.25 
               
               
                 Potassium 
                 mM 
                 1.8 
                 0 
                 0 
                 40 
                 5 
               
               
                 Phosphate 
                   
                   
                   
                   
                   
                   
               
               
                 CoSO 4 •7H 2 O 
                 g/L 
                 0.0024 
                 0.0024 
                 0 
                 0.0024 
                 0.0048 
               
               
                 CuSO 4 •5H 2 O 
                 g/L 
                 0.02 
                 0.02 
                 0.00 
                 0.02 
                 0.04 
               
               
                 ZnSO 4 •7H 2 O 
                 g/L 
                 0.0024 
                 0.0024 
                 0 
                 0.0024 
                 0.0048 
               
               
                 Na 2 MoO 4 •2H 2 O 
                 g/L 
                 0.0008 
                 0.0008 
                 0 
                 0.0008 
                 0.0016 
               
               
                 H 3 BO 3   
                 g/L 
                 0.0004 
                 0.0004 
                 0 
                 0.0004 
                 0.0008 
               
               
                 MnSO 4 •H 2 O 
                 g/L 
                 0.0012 
                 0.0012 
                 0 
                 0.0012 
                 0.0024 
               
               
                 FeSO 4 •7H 2 O 
                 g/L 
                 0.022 
                 0.022 
                 0 
                 0.022 
                 0.044 
               
               
                 MgSO 4   
                 mM 
                 2 
                 2 
                 0 
                 2 
                 2.5 
               
               
                 CaSO 4   
                 mM 
                 0.05 
                 0.05 
                 0 
                 0.05 
                 0.06 
               
               
                 Glucose 
                 g/L 
                 45 
                 25 
                 0 
                 45 
                 25 
               
               
                 MOPS 
                 mM 
                 200 
                 200 
                 0 
                 200 
                 0 
               
               
                 Thiamine-HCl 
                 g/L 
                 0.01 
                 0.01 
                 0 
                 0.01 
                 0.01 
               
               
                   
               
            
           
         
       
     
     Micro-Fermentations 
     An overview of the micro-fermentation protocol is illustrated in  FIG. 16A-C . Strains were evaluated for production in 96 well plate micro-fermentations, wherein cells were initially grown to mid-log phase, harvested, washed, resuspended and normalized in a phosphate free production medium to an OD600=1, for a 24 hour production stage. The success of the micro-fermentations required: (1) syncing strains up by harvesting all strains in exponential phase; (2) the use of low biomass levels, so that batch sugar could be kept low while enabling significant potential product accumulation; and (3) a method to supply adequate mixing and aeration, while minimizing evaporative losses. To address the final requirement, commercially available microplate sandwich covers and clamps from EnzyScreen™ as used, which greatly reduce evaporative losses while enabling high levels of mixing and aeration in standard 25 mm orbit shakers operating at 400 rpm 92-93 . Micro-fermentation results for alanine production with different insulated phosphate promoters are shown in  FIG. 17 . Micro-fermentation results for strains evaluated with gapA and gapN gene alterations are given in  FIG. 18 . 
     Section 8: Micro-Fermentations Robustness Evaluation 
     During micro-fermentation oxygen robustness studies, production culture volume was varied to achieve desired oxygen transfer rate (OTR) values as previously reported (http://www.enzyscreen.com/oxygen_transfer_rates.htm) 92-93  and as listed below in Table 14. Batch glucose levels during the production stage were altered to assess robustness to glucose. Strains utilized in the robustness experiments at the micro-fermentation scale are listed in Table 15. Results from the micro-fermentation robustness studies are given in  FIGS. 19A-D .  FIGS. 20A-D ,  FIGS. 21A-D ,  FIGS. 22A-D ,  FIGS. 23A-D ,  FIGS. 24A-D .  FIGS. 25A-D ,  FIGS. 26A-D .  FIGS. 27A-D ,  FIGS. 28A-D ,  FIGS. 29A-D .  FIGS. 30A-D ,  FIGS. 31A-D , and  FIG. 32 . 
     
       
         
           
               
             
               
                 TABLE 14 
               
             
            
               
                   
               
               
                 Culture conditions for different OTR values. 
               
               
                 25 mm orbit shaker 
               
            
           
           
               
               
               
            
               
                 Max OTR 
                 Shaking Speed 
                 Fill Volume 
               
               
                 (mmol/L-hr) 
                 (rpm) 
                 (μL) 
               
               
                   
               
               
                 25 
                 400 
                 100 
               
               
                 20 
                 400 
                 150 
               
               
                 15 
                 400 
                 200 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 15 
               
             
            
               
                   
               
               
                 List of strains used for micro-fermentation 
               
               
                 robustness evaluations and their RS scores. 
               
            
           
           
               
               
               
               
               
            
               
                 Strain # 
                 Silencing 
                 Proteolysis 
                 Plasmid 
                 RS 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                  1 
                 gltA1 
                 FU 
                 pSMART-Ala2  
                 89.6 
               
               
                  2 
                 gltA1 
                 F 
                 pSMART-Ala2  
                 89.5 
               
               
                  3 
                 gltA1 
                 GU 
                 pSMART-Ala2  
                 89.4 
               
               
                  4 
                 FG1G2 
                 None 
                 pSMART-Ala2  
                 89.3 
               
               
                  5 
                 G1G2 
                 GU 
                 pSMART-Ala2  
                 88.8 
               
               
                  6 
                 FG1G2 
                 G 
                 pSMART-Ala2  
                 88.2 
               
               
                  7 
                 G1G2 
                 F 
                 pSMART-Ala2  
                 83.4 
               
               
                  8 
                 gltA2 
                 FGU 
                 pSMART-Ala2  
                 83.4 
               
               
                  9 
                 gltA1 
                 FGU 
                 pSMART-Ala2  
                 83.1 
               
               
                 10 
                 G1G2 
                 FGU 
                 pSMART-Ala2  
                 82.3 
               
               
                 11 
                 gltA2 
                 U 
                 pSMART-Ala2  
                 82.2 
               
               
                 12 
                 gltA2 
                 F 
                 pSMART-Ala2  
                 80.6 
               
               
                 13 
                 FG1G2 
                 FG 
                 pSMART-Ala2  
                 80.5 
               
               
                 14 
                 None 
                 G 
                 pSMART-Ala2  
                 79.9 
               
               
                 15 
                 gltA2 
                 GU 
                 pSMART-Ala2  
                 77.9 
               
               
                 16 
                 fabI 
                 FGU 
                 pSMART-Ala2  
                 75.7 
               
               
                 17 
                 None 
                 FG 
                 pSMART-Ala2  
                 75.4 
               
               
                 18 
                 G1G2 
                 FU 
                 pSMART-Ala2  
                 75.3 
               
               
                 19 
                 None 
                 FGU 
                 pSMART-Ala2  
                 73.4 
               
               
                 20 
                 None 
                 FU 
                 pSMART-Ala2  
                 73.3 
               
               
                 21 
                 gltA1 
                 U 
                 pSMART-Ala2  
                 72.9 
               
               
                 22 
                 fabI 
                 FG 
                 pSMART-Ala2  
                 69.1 
               
               
                 23 
                 FG1G2 
                 FU 
                 pSMART-Ala2  
                 67.6 
               
               
                 24 
                 gltA2 
                 FU 
                 pSMART-Ala2  
                 67.5 
               
               
                 25 
                 None 
                 F 
                 pSMART-Ala2  
                 65.6 
               
               
                 26 
                 gltA2 
                 FG 
                 pSMART-Ala2  
                 67.1 
               
               
                 27 
                 FG1G2 
                 F 
                 pSMART-Ala2  
                 61.1 
               
               
                 28 
                 fabI 
                 GU 
                 pSMART-Ala2  
                 59.9 
               
               
                 29 
                 fabI 
                 F 
                 pSMART-Ala2  
                 59.6 
               
               
                 30 
                 gltA1 
                 FG 
                 pSMART-Ala2  
                 58.1 
               
               
                 31 
                 gltA1 
                 None 
                 pSMART-Ala2  
                 57.1 
               
               
                 32 
                 None 
                 None 
                 pSMART-Ala2  
                 55.5 
               
               
                 33 
                 G1G2 
                 None 
                 pSMART-Ala2  
                 54.1 
               
               
                 34 
                 fabI 
                 U 
                 pSMART-Ala2  
                 53.9 
               
               
                 35 
                 gltA2 
                 G 
                 pSMART-Ala2  
                 52.8 
               
               
                 36 
                 fabI 
                 None 
                 pSMART-Ala2  
                 50.3 
               
               
                 37 
                 fabI 
                 FU 
                 pSMART-Ala2  
                 48.4 
               
               
                 38 
                 gltA2 
                 None 
                 pSMART-Ala2  
                 47.8 
               
               
                 39 
                 FG1G2 
                 FGU 
                 pSMART-Ala2  
                 44.6 
               
               
                 40 
                 None 
                 GU 
                 pSMART-Ala2  
                 42.9 
               
               
                 41 
                 None 
                 U 
                 pSMART-Ala2  
                 39.3 
               
               
                 42 
                 fabI 
                 G 
                 pSMART-Ala2  
                 39.2 
               
               
                 43 
                 gltA1 
                 G 
                 pSMART-Ala2  
                 34.7 
               
               
                 44 
                 G1G2 
                 FG 
                 pSMART-Ala2  
                 32.8 
               
               
                 45 
                 FG1G2 
                 U 
                 pSMART-Ala2  
                 29.4 
               
               
                 46 
                 FG1G2 
                 GU 
                 pSMART-Ala2  
                 24.3 
               
               
                 47 
                 G1G2 
                 G 
                 pSMART-Ala2  
                 24.1 
               
               
                 48 
                 G1G2 
                 U 
                 pSMART-Ala2  
                 −25.3 
               
               
                 49 
                 None 
                 None 
                 pSMART-Ala13 
                 55.7 
               
               
                 50 
                 None 
                 None 
                 pSMART-Ala12 
                 −31.5 
               
               
                 51 
                 None 
                 None 
                 pSMART-Ala15 
                 −103.2 
               
               
                 52 
                 None 
                 None 
                 pSMART-Ala11 
                 −114.1 
               
               
                 53 
                 None 
                 None 
                 pSMART-Ala14 
                 −441.5 
               
               
                   
               
            
           
         
       
     
     Section 9: Standardized 2-Stage Fermentation 
     A standardized phosphate limited 2-stage fermentation protocol was utilized for evaluation of all valve strains. This protocol yields highly reproducible growth stage results, with minimal strain to strain variability even with strains making different products. More significant variability was observed during the production stage as a result of differing feed rates and base utilization by different strains.  FIG. 33A  gives the growth curves for all valve strains with a 10 g·cdw/L biomass level in 1 L fermentations performed in this study. This consistency is contrasted to the more variable growth of growth associated production strains, given in  FIG. 33B . 
     
       
         
           
               
             
               
                 TABLE 16 
               
             
            
               
                   
               
               
                 Strains used for rnevalonic acid scalability. 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Strain # 
                 Silencing 
                 Proteolysis 
                 Plasmid 
               
               
                   
                   
               
               
                   
                 1 
                 FG1G2 
                 FU 
                 pSMART-Mev2 
               
               
                   
                 2 
                 G2Z 
                 FGUA 
                 pSMART-Mev2 
               
               
                   
                 3 
                 FG1G2A 
                 FUN 
                 pSMART-Mev2 
               
               
                   
                 4 
                 UZ 
                 FGUA 
                 pSMART-Mev2 
               
               
                   
                   
               
            
           
         
       
     
     Section 10: Analytical Methods 
       
     
       
         
           
               
             
               
                 TABLE 17 
               
             
            
               
                   
               
               
                 UPLC-MS/MS parameters 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Retention 
                 ESI 
                 MRM 
                 Cone 
                 Collision 
               
               
                 Analyte 
                 Time (min) 
                 Mode 
                 Transition(s) 
                 Voltage 
                 Energy 
               
               
                   
               
               
                 Alanine 
                 0.5 
                 + 
                 89.95 → 44.08 
                 15 
                 9 
               
               
                 C13-Alanine 
                 0.5 
                 + 
                 91.95 → 46.06 
                 15 
                 9 
               
               
                   
               
            
           
         
       
     
     DETAILED DESCRIPTION OF FIGURES 
       FIG. 1A : An Overview of Dynamic Metabolic Control in 2-Stage Fermentations. Metabolic engineering involves optimizing a metabolic pathway to a desired product to the existing metabolic network of a host, converting feedstocks to a desired product. Filled circles indicate metabolites and lines indicate enzymatic reactions. Traditional optimization in metabolic engineering, often involves three key steps (a) the deletion of competing non-essential metabolic pathways including those leading to undesired byproducts and the overexpression of enzymes in the pathway converting feedstock molecules to the product (indicated by thicker lines) and potentially (b) attenuating enzymes in essential metabolism (indicated by orange lines) to further increase production. This process is iterated to optimize the yield to the desired product (pie charts). By contrast, dynamic metabolic network minimization can be used to fully unlock the potential of commonly used 2-stage fermentation processes (c-d). In the first stage of these processes (c) biomass growth and yield are optimized, while in the second stage (d) product formation is optimized, which is well suited for a 2-stage process (e) in which biomass levels accumulate and consume a limiting nutrient (in this case inorganic phosphate), which when depleted triggers entry into a productive stationary phase. Synthetic metabolic valves utilizing CRISPRi based gene silencing and/or controlled proteolysis can be used (f and g) to greatly reduce the pertinent metabolic network upon the transition to the production stage, (f) and array of silencing guides can be induced, processed by the CASCADE complex into individual guides and used to silencing target multiple genes of interest (GOI). (g) If C-terminal DAD+4 lags are added to enzymes of interest (EOI) through chromosomal modification, they can be inducibly degraded by the clpXP protease in the present of and inducible sspB chaperone. (h) Dynamic control over protein levels in  E. coli  using 2 stage dynamic control with inducible proteolysis and CRISPRi silencing. As cells grow phosphate is depleted, and cells “turn off mCherry and “turn on” GFPuv. Shaded areas represent one standard deviation from the mean, n=3. (i) Relative impact of proteolysis and gene silencing alone and in combination on mCherry degradation, with (j) decays rates. 
       FIG. 1B : Strain and Bioprocess Optimization. (a) Conventional approaches for strain and process optimization in metabolic engineering often involves deletion of competing non-essential metabolic pathways and overexpression of pathway enzymes (Filled circles: metabolites; lines: enzymatic reactions, green indicated a production pathway). (a-i) Strain variants are evaluated at screening scale (microtiter plates, shake flasks, etc), (a-ii) the best strains are assessed in larger scale instrumented bioreactors. Numerous design-build-test cycles (a-vi-vii) are used to iteratively optimize both the production strain and process, including the often-critical optimization of environmental (process) variables (a-vii). (a-iii) The best performing strains and associated optimized process conditions are scaled to industrially relevant levels. (b) Rapid strain and bioprocess optimization using 2-stage dynamic metabolic control. The metabolic network in the cell is dynamically minimized to only the steps essential for product formation. This is accomplished in a standardized 2-stage bioprocess (c), where a biomass accumulating growth stage is followed by a production stage, with only a minimal metabolic network. The limitation of a macronutrient can be used to “switch” cellular metabolism from growth to production. The approach results in a smaller subset of potential strain variants for screening (b-i). Metabolic network minimization helps increase relevant metabolite levels (d) and thus production levels, it also enhances process robustness (e), and as a result process and strain scalability (f). The best producers identified from screening are predictably and rapidly scaled to (b-ii) larger instrumented bioreactors, and (b-iii) subsequently to industrially relevant levels. If needed, limited design-build-test cycles (b-iv) are incorporated to guide improvements. Product independent, standardized protocols are followed for strain evaluation at all scales, eliminating the need for intensive process optimization. 
       FIGS. 2A-D : Implementation of 2-stage Synthetic Metabolic Valves (SMVs) in  E. coli .  FIG. 2A  depicts SMVs utilizing CRISPRi based gene silencing and/or controlled proteolysis were constructed. (Top) Silencing: An array of inducible silencing guide RNAs (i) can be used to silence expression of multiple genes of interest (GOI) when the native  E. coli  CRISPR/Cascade machinery is expressed, which can process guide arrays into individual guides (ii). (Bottom) Proteolysis: When C-terminal DAS+4 tags are added to enzymes of interest (EOI) (through chromosomal modification), they can be degraded by the clpXP protease (iv) upon the controlled induction of the sspB chaperone (iii).  FIG. 2B  depicts dynamic control over protein levels in  E. coli  using inducible proteolysis and CRISPRi silencing. As cells grow phosphate is depleted, cells “turn OFF” mCherry and “turn ON” GFPuv. Shaded areas represent one standard deviation from the mean, r.f.u, relative fluorescence units.  FIG. 2C  depicts relative impact of proteolysis and gene silencing alone and in combination on mCherry degradation, n.f.u, normalized fluorescence units (normalized to maximal fluorescence).  FIG. 2D  depicts relative impact of proteolysis and gene silencing alone and in combination on observed mCherry fluorescence decays rates (per hour). 
       FIGS. 3A-K : Alanine Production in  E. coli  utilizing 2-stage Dynamic Control.  FIG. 3A  depicts strain variant design. Primary pathways in central metabolism are shown including: Glycolysis, the Pentose Phosphate Pathway, the Citric Acid Cycle (TCA), Fatty Acid Biosynthesis, and the Soluble Transhydrogenase. Key valve candidate enzymes/genes that are “turned OFF” to reduce flux through central metabolism can include: glucose-6-phosphate dehydrogenase (zwf-“Z”), lipoamide dehydrogenase (lpd-“L”), citrate synthase (gltA-“G”), enoyl-ACP reductase (fabI-“F”), and the soluble transhydrogenase (udhA-“U”). Importantly, dynamic elimination of fabI has been previously demonstrated to increase intracellular malonyl-CoA pools as well as malonyl-CoA flux 55 . Enzymes that are dynamically “turned ON” can include the metabolic pathways to produce the products of interest, in this case alanine. Specific pathway enzymes include an NADPH-dependent alanine dehydrogenase (ald*) and an alanine exporter (alaE). Additionally, as the alanine production pathway utilizes NADPH as a cofactor, the NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase encoded by the gapN gene 56  from  S. mutans  was turned on alone and in combination with turning off the native gapA-“A” gene (NADH dependent glyceraldehyde dehydrogenase). Abbreviation: PTS—glucose phosphotransferase transport system, P—phosphate, BP—bisphosphate, OAA—oxaloacetate, DHAP—dihydroxyacetone phosphate, GA3P—glyceraldehyde-3-phosphate, 1,3-BPG—1,3 bisphosphoglycerate, 3-PG—3-phosphoglycerate, 2-PG—2-phosphoglycerate. PEP—phosphoenolpyruvate, MSA—malonate semialdehyde, ACP acyl carrier protein, Ru—ribulose, Xu—xylulose, E—erthryose, Ri—ribose, S—sedoheptulose. Strains were engineered with SMVs for the dynamic control of all combinations of valve genes/enzymes, either through gene silencing alone, proteolysis alone, or the combination of both. These strains were evaluated for alanine production in standardized micro-fermentations.  FIG. 3B  depicts rank order plot for average alanine titer (black) of all valve strains examined in 2-stage micro-fermentation, grey area represents standard deviation. Alanine production in the control strain was colored in red.  FIG. 3C  depicts average alanine titer in 2-stage production in response to different proteolysis and silencing combinations, from 0 g/L (purple) to 5 g/L (red).  FIG. 3D  depicts average alanine titer in response to different oxygen transfer rates (OTR) and glucose concentrations evaluated for a single “Valve” alanine strain (Silencing of gltA1 (“G”), Proteolysis of fabI and udhA (“FU”)). The results of this surface were used to calculate a strain-specific robustness score (RS) (refer to text), this strain has the highest RS score.  FIG. 3E  depicts a heat map of the robustness score for a subset of 48 “Valve” strains evaluated across multiple process conditions.  FIG. 3F  depicts scale up of one of the best producing strain from micro-fermentations (Silencing of fabI-gltA1-gltA2 (“FG1G2”), Proteolysis of fabI, gltA and udhA (“FGU”)) to 1 L bioreactors results in a titer of 80 g/L after 48 hrs of production, with a yield of 0.8 gig.  FIG. 3G  depicts overexpression of the alaE alanine exporter in this strain (Panel f) results in significantly improved production, reaching 147 g/L in 27 hrs of production, with a yield of ˜1 g/g. (Refer to Supplemental Materials, Section 3 for additional details).  FIG. 3H  depicts strains selected for robustness evaluation in micro-fermentations.  FIG. 31  depicts robustness and titer for the most robust “Valve” alanine strain (Silencing_gltA1, Proteolysis_FU). Bottom surface shows heat map for the alanine titer normalized to the median of all process conditions assessed, upper surface shows alanine tiler under all process conditions, the same color scale (alanine titer in g/L) was used for both panels.  FIG. 3J  depicts RS3 scores for the selected strains.  FIG. 3K  depicts process reproducibility heat map for all conditions evaluated, the same grayscale was used for  FIG. 3J  and  FIG. 3K . 
       FIGS. 4A-F : Robustness Comparison Between 2-Stage and Growth Associated Approaches.  FIG. 4A  depicts rank order of the RS3 scores for all alanine strains evaluated, red bars indicate valve alanine strains, and blue bars indicate growth associated (GA) alanine strains.  FIG. 4B  depicts average RS3 score for “Valve” alanine strains with proteolysis “F” valve, and growth associated alanine strains.  FIG. 4C  depicts max titer plot for a representative “Valve” alanine (Proteolysis_FGU, Silencing_gltA1), and growth associated alanine strains in micro-fermentation of all conditions evaluated.  FIG. 4D  depicts process reproducibility for growth associated alanine strains under all conditions evaluated.  FIG. 4E  depicts robustness and titer for a representative robust “Valve” alanine (Proteolysis_FGU, Silencing_gltA1).  FIG. 4F  depicts robustness and titer for the GA2 strain. Bottom surface, heat map for the alanine titer normalized to the median of all process conditions assessed, upper surface, alanine titer under all process conditions, the same color scale (alanine titer in g/L) was used for both panels. 
       FIGS. 5A-J : Comparisons of “Valve” and growth associated alanine production in micro-fermentations ( FIGS. 5A-D ) and 1 L fermentation ( FIGS. 5E-J ). Average alanine titer ( FIG. 5A ) and robustness score ( FIG. 5B ) for all strains used for robustness analysis. Average alanine titer in response to different OTR and glucose concentrations for selected “Valve” ( FIG. 5C ) and growth associated ( FIG. 5D ) alanine strains. Strains marked by asterisk in ( FIG. 5B ) were used for this analysis. These two strains were selected for 1 L performance comparison. Figure SE and Figure SF depicts 1 L performance metrics evaluated, including average specific productivity (SP, g/gdcw-h), average glucose uptake rate (GUR, g/gcdw-h), max titer (g/L), and max yield (gig). Figure G and Figure H depicts μL to 1 L scalability. 1 L data was standardized to the maximal titer within 50 hours of production. Adequate feed was used for growth associated strains to avoid glucose depletion.  FIG. 5I  and  FIG. 5J  depicts 1 L production profiles for all strains used in scalability plot  FIG. 5G  and  FIG. 5H  respectively, darker symbols represent growth curves, lighter symbols represent production curves, shape of symbols encode the same strains in  FIG. 5G  or  FIG. 5H . 
       FIG. 6A-E : Mevalonate Production in  E. coli  utilizing 2-stage Dynamic Control.  FIG. 6A  depicts Metabolic Pathways and SMVs for mevalonate production.  FIG. 6B  depicts mevalonate production using several production pathway plasmid variants with varied promoter combinations in the control strain.  FIG. 6C  depicts micro-fermentation results for a subset of “Valve” strains producing mevalonate, using the best production pathway from FIG.  6 B, along with combinations of proteolytic and silencing SMVs.  FIG. 6D  depicts μL to 1 L scalability for a subset of mevalonate strains evaluated at the 1 L scale, n=3 for μL data and n=1 for 1 L data. The maximal liter within 50 hours of production time was used for the correlation.  FIG. 6E  depicts production of the best mevalonate strain from  FIG. 6D  (Silencing of fabI-gltA1-gltA2 (“FG1G2”). Proteolysis of fabI and udhA (“FU”)) in 1 L bioreactors. A titer of 97 g/L was observed in 78 hrs of production. Yields during the production stage reached 0.46 g/g (84% of theoretical yield). (Refer to Supplemental Materials, Section 9 for additional details).  FIG. 6F  depicts micro-fermentation results for a subset of strains producing 3-HP.  FIG. 6G  depicts μL to 1 L scalability for a subset of 3-HP strains evaluated at the 1 L scale (Supplemental Materials Tables S21 and S22).  FIG. 6H  depicts production performance for the best 3-HP strains in the 1 L systems, squares, 3-HP/mevalonic acid titer; circles, OD600. Yields during the production stage reached for the 0.46 g/g for mevalonic acid and 0.63 g/g for 3-HP in the highest producers. 
       FIG. 7 : Phosphate depletion promoter characterization. A set of GFP reporter vectors were constructed to assess the expression level of 12 previously identified phosphate regulated promoters. Strains were evaluated continuously for GFP expression in the Biolector™ using a standardized protocol wherein in minimal medium limited for phosphate is used. After Biomass levels reach a peak (not shown for clarity). GFP expression begins. Importantly the current set of promoters enables a large range of expression levels. 
       FIG. 8 : Insulated phosphate depletion promoter characterization. A set of GFP reporter vectors were constructed to assess the expression level of five insulated phosphate regulated promoters in FGM3 media. Strains were evaluated continuously for GFP expression in the Biolector™ using a standardized protocol wherein in minimal medium limited for phosphate is used. After Biomass levels reach a peak (not shown for clarity). GFP expression begins. Importantly the current set of promoters enables a large range of expression levels. 
       FIG. 9 : Insulated constitutive promoter characterization. A set of GFP reporter vectors were constructed to assess the expression level of five insulated constitutive promoters in FGM3 with 40 mM phosphate media. Shaded area represents standard deviations, n=3. Strains were evaluated continuously for GFP expression in the Biolector™. GFP expression n as observed only for promoters proA, proB and proD. 
       FIG. 10 : Metabolic modeling results for optimal 3-HP flux in two stage fermentations. LEFT: Optimized fluxes during the growth stage where biomass production was used as the objective function. RIGHT: Optimized fluxes during the 3-HP production stage where 3-HP production was used as the objective function (biomass production was set to 0). Fluxes are listed as relative ratios or moles of flux through a given reaction per 100 moles of glucose utilized. 
       FIG. 11 : Chromosomal modifications. 
       FIG. 12 : Average maximal growth rates of starting host strains in 1 L FGM10 minimal medium fermentations, n=2. 
       FIG. 13A-E : Distribution of glucose utilized during the growth phase of starting host strains in 1 L standard minimal medium fermentations. Mid exponential and final growth period results are given for DLF 0025 as “production” begins in mid-late exponential phase. Results are averages of duplicate fermentations.  FIG. 13A . BW25113;  FIG. 13B . BWapldf;  FIG. 13C , DLF_0001;  FIG. 13D , DLF_0025 at mid-exponential;  FIG. 13E , DLF 0025 at end of growth phase. Unit was gram glucose. 
       FIG. 14 : pCASCADE-control plasmid construction scheme. 
       FIG. 15A-B : pCASCADE construction scheme.  FIG. 15A , single sgRNA cloning;  FIG. 15B , double sgRNA. 
       FIG. 16A-C : Micro-fermentation process overview. (A) An overview of the high throughput micro-fermentation protocol. Freezer stocks (alternatively colonies may be used) are used to inoculate into SM10++ in 96 well plates. Cultures are grown overnight for 16 hours, harvested by centrifugation, washed with no-phosphate medium and resuspended in no-phosphate medium at target biomass levels. (OD600 nm=1.0). EnzyScreen™ covers and clamps are used to reduce evaporation and enable high oxygen transfer rates. The protocol is implemented with a Tecan Evo liquid handler. (B) Representative overnight growth in a 96 well plates culture, distribution of OD600 for overnight culture was plotted. (C) Representative OD600 distribution after normalization using Tecan Evo liquid handler. 
       FIG. 17 : Micro-fermentation for L-alanine production using different insulated phosphate promoters in DLF_0025 strain. 
       FIG. 18 : Heatmap for L-alanine production by gapN/gapA strains. 
       FIGS. 19A-D : Alanine production in response to different OTR and glucose concentration in micro-fermentation for 4 strains evaluated for robustness. 
       FIGS. 20A-D : Alanine production in response to different OTR and glucose concentration in micro-fermentation for 4 strains evaluated for robustness. 
       FIGS. 21A-D : Alanine production in response to different OTR and glucose concentration in micro-fermentation for 4 strains evaluated for robustness. 
       FIGS. 22A-D : Alanine production in response to different OTR and glucose concentration in micro-fermentation for 4 strains evaluated for robustness. 
       FIGS. 23A-D : Alanine production in response to different OTR and glucose concentration in micro-fermentation for 4 strains evaluated for robustness. 
       FIGS. 24A-D : Alanine production in response to different OTR and glucose concentration in micro-fermentation for 4 strains evaluated for robustness. 
       FIGS. 25A-D : Alanine production in response to different OTR and glucose concentration in micro-fermentation for 4 strains evaluated for robustness. 
       FIGS. 26A-D : Alanine production in response to different OTR and glucose concentration in micro-fermentation for 4 strains evaluated for robustness. 
       FIGS. 27A-D : Alanine production in response to different OTR and glucose concentration in micro-fermentation for 4 strains evaluated for robustness. 
       FIGS. 28A-D : Alanine production in response to different OTR and glucose concentration in micro-fermentation for 4 strains evaluated for robustness. 
       FIGS. 29A-D : Alanine production in response to different OTR and glucose concentration in micro-fermentation for 4 strains evaluated for robustness. 
       FIGS. 30A-D : Alanine production in response to different OTR and glucose concentration in micro-fermentation for 4 strains evaluated for robustness. 
       FIGS. 31A-D : Alanine production in response to different OTR and glucose concentration in micro-fermentation for 4 strains evaluated for robustness. 
       FIG. 32 : Alanine production in response to different OTR and glucose concentration in micro-fermentation for one strain evaluated for robustness. 
       FIGS. 33A-B : Growth profile for all ( FIG. 33A ) valve and ( FIG. 33B ) growth associated strains at 1 L scale evaluated in this paper. Growth curves were synced to account for any variations in lag time. Valve strains growth curves were synced to the same mid-exponential point. Growth associated strains growth curves were synced to the same take-off point. 
       FIG. 34 : Specific Productivity (SP) comparison for strain with highest mevalonate titer from literature and mevalonate strain 1 evaluated in this work. 
       FIG. 35 : Alanine standard curve from MS measurement. Average and standard deviation for mass spec response from triplicate standard measurement were plotted. 
       FIGS. 36A-B : Glucose ( FIG. 36A ) and ethanol ( FIG. 368 ) standard curves from RI measurement. Average and standard deviation for peak area from triplicate standard measurement were plotted. 
       FIG. 37 : 3-Hydroxypropionic acid standard curve from TUV measurement. Average and standard deviation for peak area from duplicate standard measurement were plotted. 
       FIGS. 38A-D : TUV standard curves for ( FIG. 38A ) L-alanine, ( FIG. 38B ) D-alanine, ( FIG. 38C ) mevalonic acid, and ( FIG. 38D ) mevalonolactone. Average and standard deviation for peak area from triplicate standard measurement were plotted. 
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     While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the an that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.