# Patent

## Title
Acetyl-coA carboxylases

## Abstract

The present invention provides various combinations of genetic modifications to a transformed host cell that provide increase conversion of carbon to a chemical product. The present invention also provides methods of fermentation and methods of making various chemical products.

## Background
This application claims priority to U.S. Provisional Patent Application No. 61/852,387, filed on Mar. 15, 2013; which is hereby incorporated herein by reference in its entirety.


SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 3, 2014, is named 34246-786-201-Seqlist.txt and is 324 Kilobytes in size.
BACKGROUND OF THE INVENTION
There is a need for alternative production methods of industrial chemicals used for various consumer products and fuels that are currently made from petroleum. One alternative method is the use of engineered microorganisms to produce industrial chemicals. Currently, in the field of bioproduced chemicals there is a need to improve microbial enzyme performance, enhanced production rate in order to reach the goal of becoming an at-cost replacement basis for petro-based chemicals.
A common challenge faced in field of bio-produced chemicals in microorganisms is that any one modification to a host cell may require coordination with other modifications in order to successfully enhance chemical bioproduction.
The current invention provides methods, systems of fermentation, genetically modified microorganisms, modified enhanced enzymes for chemical production, all of which may be used in various combinations to increase chemical production of a desired chemical product.
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.
SUMMARY OF THE INVENTION
The present invention relates to genetically modified organisms capable of producing an industrial chemical product of interest, wherein the genetic modification includes introduction of nucleic acid sequences coding for polynucleotides encoding one or more of the following: (1) an acetyl-CoA carboxylase gene with one or more of its subunits fused together in the genetic structure of the organism; (2) an acetyl-CoA carboxylase gene having a predefined stoichiometric ratio of each of the four ACCase subunits relative to one another; (3) a monofunctional malonyl-CoA reductase gene capable of catalyzing the conversion of malonyl-CoA to malonate semialdehyde and one or more genes encoding one or more of the following enzymes: ydfG, mmsB, NDSD, rutE, and nemA; (4) a monofunctional malonyl-CoA reductase gene capable of catalyzing the conversion of malonyl-CoA to malonate semialdehyde and one or more genes encoding one or more enzymes capable of converting malonate semialdehyde keto form to 3-HP, and one or more genes encoding one or more enzymes capable of converting either the malonate semialdehyde enol form or the malonate semialdehyde hydrated form to 3-HP; (5) a monofunctional malonyl-CoA reductase enzyme fused to a dehydrogenase enzyme that is either: (a) primarily not NADPH-dependent, (b) primarily NADH-dependent, (c) primarily flavin-dependent, (d) less susceptible to 3-HP inhibition at high concentration, and/or (e) catalyzes a reaction pathway to 3-HP that is substantially irreversible; (6) a monofunctional malonyl-CoA reductase enzyme fused to one or more malonate semialdehyde dehydrogenase enzymes; (7) a malonyl-CoA reductase gene that is mutated to enhance its activity at lower temperatures; (8) salt-tolerant enzymes; (9) a gene that facilitates the exportation of a chemical product of interest or the export of an inhibitory chemical from within the cell to the extracellular media; and/or (10) a gene that facilitates the importation from the extracellular media to within the cell of a reactant, precursor, and/or metabolite used in the organism's production pathway for producing a chemical product of interest.
The present invention further relates to methods of producing a chemical product using the genetically modified organisms of the invention. The present invention further includes products made from these methods. In accordance with certain embodiments that product is acetyl-CoA, malonyl-CoA, malonate semialdehyde, 3-hydroxypropionic acid (3-HP), acrylic acid, 1,3 propanediol, malonic acid, ethyl 3-HP, propiolactone, acrylonitrile, acrylamide, methyl acrylate, a polymer including super absorbent polymers and polyacrylic acid, or a consumer product.
The present invention further relates to a method of producing a chemical product from a renewable carbon source through a bioproduction process that comprises a controlled multi-phase production process wherein the initiation and/or completion of one or more phases of the production process is controlled by genetic modifications to the organism producing the chemical product and/or is controlled by changes made to the cell environment. In accordance with this aspect of the invention, the bioproduction process may include two or more of the following phases: (1) growth phase; (2) induction phase; and (3) production phase. The present invention further includes products made from these methods.



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. 1 Depicts some embodiments of the metabolic pathways to produce 3-hydroxypropionic acid.
FIG. 2 Depicts some embodiments of the of various equilibrium states in the malonate semialdehyde to 3-HP reaction in a cell environment.
FIG. 3 Depicts some embodiments of the reaction catalyzed by acetyl-CoA carboxylase (ACCase)
FIG. 4 Shows the inhibition of ACCase enzyme activity by high salt concentration
FIG. 5 Depicts some embodiments of the fusion ACCase subunit gene constructs overexpressed in E. coli. 
FIG. 6 Show improved production of 3-HP by genetically modified organism with DA fusion ACCase
FIG. 7 Shows improved production of 3-HP by genetically modified organism with overexpression of rhtA exporter.
FIG. 8 Shows various embodiments of the genetic modules used for optimizing expression in host cells.
FIG. 9 Shows various chemical products that can made from various embodiments of the invention.



Table 1 Lists the accession numbers for genes encoding ACCase subunits from Halomonas elongate. 
Table 2 Depicts some embodiments of the RBS sequences used to enhance expression of H. elongate ACCase subunits.
Table 3 Shows the improvement in 3-HP production by RBS-optimized expression of H. elongata ACCase subunits.
Table 4 Shows some embodiments of the ACCase subunit fusions that increase and ACCase enzyme complex activity.
Table 5 Shows some of the genetic modifications of a host cell for increase chemical production.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
The term “homology” refers to the optimal alignment of sequences (either nucleotides or amino acids), which may be conducted by computerized implementations of algorithms. “Homology”, with regard to polynucleotides, for example, may be determined by analysis with BLASTN version 2.0 using the default parameters. “Homology”, with respect to polypeptides (i.e., amino acids), may be determined using a program, such as BLASTP version 2.2.2 with the default parameters, which aligns the polypeptide or fragments (and can also align nucleotide fragments) being compared and determines the extent of amino acid identity or similarity between them. It will be appreciated that amino acid “homology” includes conservative substitutions, i.e. those that substitute a given amino acid in a polypeptide by another amino acid of similar characteristics. Typically seen as conservative substitutions are the following replacements: replacements of an aliphatic amino acid such as Ala, VaI, Leu and He with another aliphatic amino acid; replacement of a Ser with a Thr or vice versa; replacement of an acidic residue such as Asp or GIu with another acidic residue; replacement of a residue bearing an amide group, such as Asn or GIn, with another residue bearing an amide group; exchange of a basic residue such as Lys or Arg with another basic residue; and replacement of an aromatic residue such as Phe or Tyr with another aromatic residue. For example, homologs can have at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% overall amino acid or nucleotide identity to the gene or proteins of the invention; or can have 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% amino acid or nucleotide to the essential protein functional domains of the gene or proteins of the invention; or at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% overall amino acid or nucleotide to the essential binding amino acids within an essential functional domain of the gene or proteins of the invention.
The above descriptions and methods for sequence homology are intended to be exemplary and it is recognized that this concept is well-understood in the art. Further, it is appreciated that nucleic acid sequences may be varied and still provide a functional enzyme, and such variations are within the scope of the present invention. The term “enzyme homolog” can also mean a functional variant.
The term “Functional homolog” means a polypeptide that is determined to possess an enzymatic activity and specificity of an enzyme of interest but which has an amino acid sequence different from such enzyme of interest. A corresponding “homolog nucleic acid sequence” may be constructed that is determined to encode such an identified enzymatic functional variant.
The term “3-HP” means 3-hydroxypropionic acid.
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 is 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. Embodiments of the present invention 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. With reference to the host microorganism's genome prior to the introduction of the heterologous nucleic acid sequence, then, the nucleic acid sequence that codes for the enzyme is heterologous (whether or not the heterologous nucleic acid sequence is introduced into that genome). The term “heterologous” is intended to include the term “exogenous” as the latter term is generally used in the art as well as “endogenous”.
As used in the specification and the appended 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.
I. Introduction
The present invention relates to various genetically modified microorganisms, methods for making the same, and use of the same in making industrial products. Any and all of the microorganisms herein may include a combination of genetic alterations as described herein. The present invention contemplates, for example, a genetically modified microorganism having one or more of the following genetic modifications (i) an alteration that affects the stoichiometric ratio, expression or production of one or more ACCase enzyme genes (ii) a recombinant ACCase gene having at least 80% sequence homology to an ACCase gene from a salt tolerant organism (iii) a genetic alteration in one or more non-ACCase genes (iv) one or more genetic alterations that encodes for one or more exporters capable of exporting 3-HP out of a cell (v) new hybrid molecules or co-expressed of a mono-functional malonyl-CoA reductase enzyme with various 3-HP dehydrogenase proteins that: (a) exhibit less inhibition by high 3-HP concentrations (b) that is less reversible or irreversible (c) enzymes that utilized NADH (d) enzymes that utilized flavin (vi) one or more genetic alterations that can be used to switch the carbon in the standard metabolic pathways of the cells to a pathway engineered to produce a chemical. More details about each of the above modifications and how the modification are used together to increase chemical production in a host cell is described below.
The present invention also relates to methods of fermentation. The genetically modified microorganisms are cultured under conditions that optimized a host cell for increase chemical production. The bio-production process may include two or more of the following phases of fermentation: (1) growth phase where the culture organism replicates itself and the carbon intermediate product is built up; (2) the induction phase, where the expression of key enzymes critical to the chemical production is induced and the enzymes accumulate within the organism to carry out the engineered pathway reactions required to further produce the chemical product (3) production phase is where the organism expresses proteins that provide for continuously production the desired chemical product. The above phases are further controlled by (1) addition and amount of the initiating reactant added to the reaction vessel (2) key enzymes engineered into the organism using promoters that are sensitive to (e.g., activated by) the depletion of the initiating reactant. Addition details about the fermentation process of the invention are disclosed below.
II. Acetyl-CoA Carboxylase
Malonyl-CoA Flux
One of the steps in the biosynthesis of 3-HP involves the reaction catalyzed by acetyl-CoA carboxylase (ACCase) enzyme. ACCase is a primary control point in the 3-HP pathway shown in FIG. 1 (previously described in) for the converting acetyl-CoA to malonyl-CoA and hence to malonate semialdehyde and 3-HP. The present invention contemplates the use of genetic modifications that increase activity of ACCase complex enzymes to thereby increase 3-HP production in a host cell.
Fused Subunits
The acetyl-CoA carboxylase complex (ACCase) is a multi-subunit protein. Prokaryotes and plants have multi-subunit ACCs composed of several polypeptides encoded by distinct genes. However, humans and most other eukaryotes, such as yeast, have evolved an ACC with CT and BC catalytic domains and biotin carboxyl carrier domains on a single polypeptide. The biotin carboxylase (BC) activity, biotin carboxyl carrier protein (BCCP), and carboxyl transferase (CT) activity are each contained on a different subunit. In E. coli the ACCase complex is derived from multi polypeptide transcribed by distinct, separable protein components known as accA, accB, accC, and accD.
Acetyl-CoA carboxylase is a biotin-dependent enzyme that catalyzes the irreversible carboxylation of acetyl-CoA to produce malonyl-CoA through its two catalytic activities, biotin carboxylase (BC) and carboxyltransferase (CT). The first reaction is carried out by BC and involves the ATP-dependent carboxylation of biotin with bicarbonate. The carboxyl group is transferred from biotin to acetyl-CoA to form malonyl-CoA in the second reaction, which is catalyzed by CT. The main function of ACCase complex in the cell is to provide the malonyl-CoA substrate for the biosynthesis of fatty acids.
The conversion of acetyl-CoA to malonyl-CoA is an important step in the bioconversion of a renewable carbon source (such as, for example, sugar or natural gas) to a useful industrial chemical (such as, for example, 3-hydroxypropionic acid (3-HP)). In certain organisms, such as E. coli or yeast, the native ACCase expression from the chromosome alone is insufficient to enable the organism to produce chemicals such as 3-HP at a rate to support a commercial scale operation. Overexpression of the ACCase complex has been shown to provide some advantage [U.S. Ser. Nos. 12/891,760 12/891,790, 13/055,138].
Applicants have discovered that the introduction of an acetyl-CoA carboxylase gene with one or more of its subunits fused is beneficial to the production of a chemical product in a host cell. In certain aspects of the invention, fusion is the two gene products produced from a single polypeptide controlled by a single promoter, will further enhance an organism's bioproduction of an industrial chemical. In certain aspects of the invention, fusion is the two gene products produced by at least one promoter, will further enhance an organism's bioproduction of an industrial chemical. In certain aspects of the invention, fusion is the two gene products produced from a single polypeptide controlled by at least one inducible promoter, will further enhance an organism's bioproduction of an industrial chemical. Keeping components of the ACCase complex fused together in the genetic structure of an organism can be advantageous because it enhances the stability of the non-native ACCase genetic modification and it facilitates equimolar expression of the fused acc subunits.
In particular, the subunit-fused ACCase may be an accA-accB, accA-accC, accA-accD, accB-accC, accB-accD, accC-accD, accA-accB-accC, accA-accB-accD, accA-accC-accD, accB-accC-accD or accA-accB-accC-accD fused subunit that have having at least 80% sequence homology to E. coli accA, accB, accC and accD or is a functional homolog thereof. In addition, the organism may include any combination of these fused subunits, or any combination of these fused subunits together with one or more of the four non-fused subunits. When such combinations are used, the subunits (fused and non-fused) may be expressed on the same plasmid or on different plasmids or on the chromosome of the organism.
In accordance with a preferred embodiment, an accA-accD fused subunit is introduced into an organism either alone or in combination with the accB-accC fused subunit, the accB gene, and/or the accC gene. In accordance with a preferred embodiment, the organism is a bacteria, and preferably E. coli or Cupriavidus necator. 
Composition Stoichiometry
Composition stoichiometry is the quantitative relationships among elements that comprise a compound. A stoichiometric ratio of a reagent is the optimum amount or ratio where, assuming that the reaction proceeds to completion. Although stoichiometric terms are traditionally reserved for chemical compounds, theses theoretical consideration of stoichiometry are relevant when considering the optimal function of heterologous multi-subunit protein in a host cell.
In accordance with another aspect of the invention, the stoichiometric ratio of each of the four ACCase subunits relative to one another is important, and each such ratio can be between 0 and about 10, and preferably between about 0.5 to about 2 or about 7 to about 9. In accordance with a preferred embodiment the ratios for the protein subunits are accA:accB:accC:accD are 1:2:1:1. In accordance with a preferred embodiment, an organism is genetically modified to include an accA-accD fused subunit, an accB non-fused subunit, and an accC non-fused subunit, with the molar ratios of the accDA fusion:accB:accC being about 1:2:1, which is close to the optimum for enzymatic activity.
In certain embodiments where an organism is engineered to make 3-HP, in order to get optimal function in a host cell of a heterologous ACCase enzyme complex it is important to engineer the stoichiometry of these subunits in such a way that provides maximal production of 3-HP such that the subunit can make a more stable enzyme complex when overexpressed in the cell.
In certain aspects the invention provides for the controlled expression of the natural accA, accB, accC, and accD subunits of E. coli or having at least 80% sequence homology to E. coli accA, accB, accC and accD. In certain aspects the invention provides for the inducible expression of the natural accA, accB, accC, and accD subunits of E. coli or having at least 80% sequence homology to E. coli accA, accB, accC, and accD. In certain aspects the invention provides for the low, medium, high and/or inducible expression of the natural accA, accB, accC, and accD subunits of E. coli or having at least 80% sequence homology to E. coli accA, accB, accC and accD.
In certain aspects the invention provides for the expression of the natural accC and accD subunits of E. coli or having at least 80% sequence homology to E. coli accA, accB, accC and accD in low, medium, high or inducible expression. In certain aspects the invention provides for the expression of the natural accB and accA subunits of E. coli or having at least 80% sequence homology to E. coli accA, accB, accC, and accD in low, medium, high or inducible expression. In certain aspects the invention provides for the expression of the natural accC and accD subunits with the accA subunit of E. coli or having at least 80% sequence homology to E. coli accA, accB, accC, and accD in low, medium, high or inducible expression. In certain aspects the invention provides for the expression of the natural accC and accD subunits with the accB subunit of E. coli or having at least 80% sequence homology to E. coli accA, accB, accC, and accD in low, medium, high or inducible expression.
In certain aspects the invention provides for the expression of a fusion of two, three, or all of the four ACCase subunits in one polypeptide in low, medium, high or inducible expression. Such fusion may include any of the following combinations of the ACCase subunits: accA-accB, accA-accC, accA-accD, accB-accC, accB-accD, accC-accD, accA-accB-accC, accA-accB-accD, accA-accC-accD, accB-accC-accD, and accA-accB-accC-accD have having at least 80% sequence homology to E. coli accA, accB, accC and accD or is a functional homolog thereof.
In certain aspects the invention provides for ACC complex in the stoichiometry of these subunits of the accCB and accDA in a 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 2:1, 2:3, 2:4, 2:5, 2:6, 2:7, 2:8, 3:1, 3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 3:1, 3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 4:1, 4:3, 4:4, 4:5, 4:6, 4:7, 4:8, 5:1, 5:3, 5:4, 5:5, 5:6, 5:7, 5:8, 6:1, 6:3, 6:4, 6:5, 6:6, 6:7, 6:8, 7:1, 7:3, 7:4, 7:5, 7:6, 7:7, 7:8, 8:1, 8:3, 8:4, 8:5, 8:6, 8:7, or 8:8 in low, medium, high or inducible expression. In certain aspects the invention provides for ACC complex in the stoichiometry of these subunits of the accDA and accCB in a 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 2:1, 2:3, 2:4, 2:5, 2:6, 2:7, 2:8, 3:1, 3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 3:1, 3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 4:1, 4:3, 4:4, 4:5, 4:6, 4:7, 4:8, 5:1, 5:3, 5:4, 5:5, 5:6, 5:7, 5:8, 6:1, 6:3, 6:4, 6:5, 6:6, 6:7, 6:8, 7:1, 7:3, 7:4, 7:5, 7:6, 7:7, 7:8, 8:1, 8:3, 8:4, 8:5, 8:6, 8:7, or 8:8 in low, medium, high or inducible expression.
In certain aspects the invention provides for the stoichiometry of the accD-A subunits in a 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 2:1, 2:3, 2:4, 2:5, 2:6, 2:7, 2:8, 3:1, 3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 3:1, 3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 4:1, 4:3, 4:4, 4:5, 4:6, 4:7, 4:8, 5:1, 5:3, 5:4, 5:5, 5:6, 5:7, 5:8, 6:1, 6:3, 6:4, 6:5, 6:6, 6:7, 6:8, 7:1, 7:3, 7:4, 7:5, 7:6, 7:7, 7:8, 8:1, 8:3, 8:4, 8:5, 8:6, 8:7, or 8:8 in low, medium, high or inducible expression. In certain aspects the invention provides for the stoichiometry of the accC-B subunits in a 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 2:1, 2:3, 2:4, 2:5, 2:6, 2:7, 2:8, 3:1, 3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 3:1, 3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 4:1, 4:3, 4:4, 4:5, 4:6, 4:7, 4:8, 5:1, 5:3, 5:4, 5:5, 5:6, 5:7, 5:8, 6:1, 6:3, 6:4, 6:5, 6:6, 6:7, 6:8, 7:1, 7:3, 7:4, 7:5, 7:6, 7:7, 7:8, 8:1, 8:3, 8:4, 8:5, 8:6, 8:7, or 8:8 in low, medium, high or inducible expression. In certain aspects the invention provides for the stoichiometry of the accC-A subunits in a 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 2:1, 2:3, 2:4, 2:5, 2:6, 2:7, 2:8, 3:1, 3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 3:1, 3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 4:1, 4:3, 4:4, 4:5, 4:6, 4:7, 4:8, 5:1, 5:3, 5:4, 5:5, 5:6, 5:7, 5:8, 6:1, 6:3, 6:4, 6:5, 6:6, 6:7, 6:8, 7:1, 7:3, 7:4, 7:5, 7:6, 7:7, 7:8, 8:1, 8:3, 8:4, 8:5, 8:6, 8:7, or 8:8 in low, medium, high or inducible expression. In certain aspects the invention provides for the stoichiometry of the accC-B subunits in a 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 2:1, 2:3, 2:4, 2:5, 2:6, 2:7, 2:8, 3:1, 3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 3:1, 3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 4:1, 4:3, 4:4, 4:5, 4:6, 4:7, 4:8, 5:1, 5:3, 5:4, 5:5, 5:6, 5:7, 5:8, 6:1, 6:3, 6:4, 6:5, 6:6, 6:7, 6:8, 7:1, 7:3, 7:4, 7:5, 7:6, 7:7, 7:8, 8:1, 8:3, 8:4, 8:5, 8:6, 8:7, or 8:8 in low, medium, high or inducible expression.
III. Conversion of Malonyl-CoA to Malonate Semialdehyde
One of the steps in the biosynthesis of 3-HP involves the conversion of malonyl-CoA (MCA) to malonate semialdehyde (MSA) and the conversion of malonate semialdehyde (MSA) to 3-HP (WO2011/038364). In accordance with another aspect of the present invention, the present invention contemplates the use of novel enzymes and/or combinations of enzymes to catalyze the reaction in a microorganism from MCA to MSA, which results in enhanced cellular bioproduction of 3-HP in the host cell.
In certain aspects the invention provides novel enzyme compositions or co-expression of a combinations of enzyme compositions to catalyze the conversion of malonyl-CoA to 3-HP. A general overview of the enzymes and the relevant reaction pathways methods are shown in FIG. 1.
In accordance with this aspect of the invention, malonyl-CoA is converted to malonate semialdehyde by a malonyl-CoA reductase and malonate semialdehyde is converted to 3-HP through either or both of two alternative pathways.
In accordance with one aspect of the invention, malonyl-CoA is converted to malonate semialdehyde by a monofunctional malonyl-CoA reductase that catalyzes the malonyl-CoA conversion, but does not catalyze the malonate semialdehyde conversion.
In one embodiment, the microorganism herein comprise a genetic modification that include the monofunctional malonyl-CoA reductase may be derived from Sulfolobus tokodaii (stMCR) (SEQ ID NO. 15 nucleic acid, SEQ ID NO. 16 protein sequence) or a functional homolog of stMCR or a homolog with at least 80% identity.
In some embodiments, the microorganism herein comprise a genetic modification that include the bi-functional malonyl-CoA reductase comprised of two protein fragments with one fragment having malonyl-CoA reductase activity and the other fragment having malonate semialdehyde dehydrogenase activity may be derived from Chloroflexus aurantiacus (caMCR).
MCR-Dehydrogenase Enzymes for Conversion of 3-HP Ions
Following the conversion of the malonyl-CoA to malonate semialdehyde, the malonate semialdehyde is converted to 3-HP through either or both of two alternative pathways. Malonate semialdehyde may exist in at least three states; the keto form, the enol form, and hydrate form, as shown in FIG. 2. Malonate semialdehyde in the enol form, which will stabilize this form when compared to other aldehydes where the enol form is highly unfavored in the equilibrium among the three forms.
The malonate semialdehyde keto form is converted to 3-HP utilizing a 3-hydroxy acid dehydrogenase enzyme (ydfG SEQ ID NO. 21 nucleic acid, SEQ ID NO. 22 protein), a 3-hydroxyisobutyrate dehydrogenase enzyme (Pseudomonas aeruginosa mmsB, SEQ ID No 23 nucleic acid, SEQ ID NO. 24 protein), and/or NAD+-dependent serine dehydrogenase (Pseudomonas NDSD, SEQ ID NO. 25 nucleic acid, SEQ ID NO. 26 protein). In accordance with a preferred embodiment, Pseudomonas mmsB, Pseudomonas NDSD, and E. coli ydfG are used. The gene, ydfG from E. coli is largely NADPH dependent, whereas mmsB and NDSD from Pseudomonas can utilize either NADPH or NADH.
The malonate semialdehyde enol form is converted to 3-HP utilizing an N-ethylmaleimide reductase (nemA, SEQ ID NO. 17 nucleic acid, SEQ ID NO. 18 protein), and/or a malonic semialdehyde reductase (rutE, SEQ ID NO.19 nucleic acid, SEQ ID NO. 20 protein) from E. coli. These enzymes does not directly utilize NADPH or NADH. Instead, these enzymes utilize a flavin mononucleotide that is cycled between oxidized and reduced states by NADPH or NADH. The enol pathway also has advantages over the keto pathway in that equilibrium between the malonate semialdehyde enol form and 3-HP significantly favors 3-HP, making the reaction much less reversible, and essentially irreversible.
The malonate semialdehyde hydrated form may also be converted to 3-HP by either the 3-HP dehydrogenase or malonate semialdehyde reductase enzymes, although the hydrated form is more likely to be converted to the enol form as the equilibrium continuously readjusts.
In one embodiment, the microorganism herein comprise a genetic modification that include (i.e., microorganism) includes a polynucleotide encoding: (1) a monofunctional malonyl-CoA reductase gene capable of catalyzing the conversion of malonyl-CoA to malonate semialdehyde; and (2) one or more genes encoding one or more of the following enzymes: ydfG, mmsB, NDSD, rutE, and nemA or a functional homolog or a homolog with at least 80% identity.
In accordance with another aspect of the invention, there is provided an organism that is genetically modified to make 3-HP, wherein the genetic modification includes a polynucleotide encoding: (1) a monofunctional malonyl-CoA reductase gene capable of catalyzing the conversion of malonyl-CoA to malonate semialdehyde; (2) one or more genes encoding one or more enzymes capable of converting malonate semialdehyde keto form to 3-HP; and (3) one or more genes encoding one or more enzymes capable of converting either the malonate semialdehyde enol form or the malonate semialdehyde hydrated form to 3-HP.
In certain aspects the invention provides monofunctional malonyl-CoA reductase enzyme fused to a dehydrogenase enzyme that is either: (1) primarily not NADPH-dependent; (2) primarily NADH-dependent; (3) primarily flavin-dependent; (4) less susceptible to 3-HP inhibition at high concentration; and/or (5) catalyzes a reaction pathway to 3-HP that is substantially irreversible.
In certain aspects the invention also provides monofunctional malonyl-CoA reductase enzyme fused to a dehydrogenase enzyme that is NADPH-dependent.
Suitable 3-HP dehydrogenase enzymes that are largely NADH-dependent that can be used with the claimed invention include, but are not limited to, mmsB or NDSD. Suitable malonate reductase enzymes that are flavin-dependent include, but are not limited to, rutE and nemA. Suitable 3-HP dehydrogenase enzymes that are less susceptible 3-HP inhibition at high concentration that can be used with the claimed invention include, but are not limited to, ydfG and NDSD. Suitable 3-HP dehydrogenase or malonate semialdehyde dehydrogenase enzymes that catalyze a reaction pathway to 3-HP that is substantially irreversible are rutE and nemA.
In certain aspects the invention provides monofunctional malonyl-CoA reductase enzyme fused to one or more dehydrogenase enzymes. Malonate semialdehyde, which is the intermediate product in the conversion of malonyl-CoA to 3-HP can be very reactive. Therefore, it is advantageous to have a reaction pathway wherein the residence time of malonate semialdehyde within the cell is minimized, and its conversion to 3-HP occurs quickly. By fusing the malonyl-CoA reductase with the malonate semialdehyde dehydrogenase to create a multi-domain protein (e.g., two domain protein) and having the MCR and dehydrogenase domains adjacent in the sequence, when the themalonate semialdehyde is quickly is quickly converted to 3-HP.
In certain aspects the invention provides first monofunctional malonyl-CoA reductase enzyme fused to a first dehydrogenase enzyme of one type and second monofunctional malonyl-CoA reductase enzyme fused to a dehydrogenase enzyme of a different type than the first dehydrogenase enzyme. Suitable different dehydrogenase enzymes include, but are not limited to, enzymes that function on the different forms of malonate semialdehyde.
In certain aspects the invention provides for microorganisms comprising a genetic modification that include but are not limited to the malonyl-CoA reductase from S. tokadaii is fused to ydfG, mmsB, NDSD, rutE, or nemA (or some combination thereof). The fused enzyme may include any of the following configurations: mcr-ydfG, mcr-mmsB, mcr-NDSD, mcr-rutE, mcr-nemA, mcr-ydfG-mmsB, mcr-ydfG-NDSD, mcr-ydfG-rutE, mcr-ydfG-nemA, mcr-mmsB-ydfG, mcr-mmsB-NDSD, mcr-mmsB-rutE, mcr-mmsB-nemA, mcr-NDSD-ydfG, mcr-NDSD-mmsB, mcr-NDSD-rutE, mcr-NDSD-nemA, mcr-rutE-ydfG, mcr-rutE-mmsB, mcr-rutE-NDSD, mcr-rutE-nemA, mcr-nemA-ydfG, mcr-nemA-mmsB, mcr-nemA-NDSD, or mcr-nemA-rutE or functional homolog or homolog with 80% sequence identity thereof.
In certain aspects the invention provides for microorganisms comprising a genetic modification that include but are not limited to the malonyl-CoA reductase from C. aggregans is fused to ydfG, mmsB, NDSD, rutE, or nemA (or some combination thereof). The fused enzyme may include any of the following configurations: mcr-ydfG, mcr-mmsB, mcr-NDSD, mcr-rutE, mcr-nemA, mcr-ydfG-mmsB, mcr-ydfG-NDSD, mcr-ydfG-rutE, mcr-ydfG-nemA, mcr-mmsB-ydfG, mcr-mmsB-NDSD, mcr-mmsB-rutE, mcr-mmsB-nemA, mcr-NDSD-ydfG, mcr-NDSD-mmsB, mcr-NDSD-rutE, mcr-NDSD-nemA, mcr-rutE-ydfG, mcr-rutE-mmsB, mcr-rutE-NDSD, mcr-rutE-nemA, mcr-nemA-ydfG, mcr-nemA-mmsB, mcr-nemA-NDSD, or mcr-nemA-rutE or functional homolog or homolog with 80% sequence identity thereof.
In certain aspects the invention provides for microorganisms comprising a genetic modification that include but are not limited to the malonyl-CoA reductase from O. trichoides is fused to ydfG, mmsB, NDSD, rutE, or nemA (or some combination thereof). The fused enzyme may include but are not limited to any of the following configurations: mcr-ydfG, mcr-mmsB, mcr-NDSD, mcr-rutE, mcr-nemA, mcr-ydfG-mmsB, mcr-ydfG-NDSD, mcr-ydfG-rutE, mcr-ydfG-nemA, mcr-mmsB-ydfG, mcr-mmsB-NDSD, mcr-mmsB-rutE, mcr-mmsB-nemA, mcr-NDSD-ydfG, mcr-NDSD-mmsB, mcr-NDSD-rutE, mcr-NDSD-nemA, mcr-rutE-ydfG, mcr-rutE-mmsB, mcr-rutE-NDSD, mcr-rutE-nemA, mcr-nemA-ydfG, mcr-nemA-mmsB, mcr-nemA-NDSD, or mcr-nemA-rutE or functional homolog or homolog with 80% sequence identity thereof.
Enhanced Mutated Monofunctional MCR for Bioproduction
In certain aspects the invention provides for microorganisms comprising a genetic modification that include mutated form of stMCR that has enhanced activity at about 20° C. to about 44° C., about 30° C. to about 37° C., or about 32° C. to about 38° C. Such mutate forms may be designed based on the crystal structure now available for stMCR [Demmer et al., J. Biol. Chem. 288:6363-6370, 2013].
It is also contemplated the carboxylase domains of the malonyl-CoA reductase derived from Chloroflexus aggregans, Oscillochloris trichoides can be enhanced by mutations in the carboxylase binding domain to provide increased 3-HP production over the natural occurring enzyme.
The carboxylase activity of the malonyl-CoA reductase derived from Chloroflexus aurantiacus can be enhanced activity. In certain aspects the invention provides for mutated form of it carboxylase domain to provide increased 3-HP production over the natural occurring enzyme.
In certain aspects the invention provides for microorganisms comprising a genetic modification that include carboxylase domains of the malonyl-CoA reductase derived from C. aggregans is fused to ydfG, mmsB, NDSD, rutE, or nemA (or some combination thereof). It is contemplated that the any of the enhanced MCR by mutation, as provide above, may be fused in any of the following configurations including but not limited to mcr-ydfG, mcr-mmsB, mcr-NDSD, mcr-rutE, mcr-nemA, mcr-ydfG-mmsB, mcr-ydfG-NDSD, mcr-ydfG-rutE, mcr-ydfG-nemA, mcr-mmsB-ydfG, mcr-mmsB-NDSD, mcr-mmsB-rutE, mcr-mmsB-nemA, mcr-NDSD-ydfG, mcr-NDSD-mmsB, mcr-NDSD-rutE, mcr-NDSD-nemA, mcr-rutE-ydfG, mcr-rutE-mmsB, mcr-rutE-NDSD, mcr-rutE-nemA, mcr-nemA-ydfG, mcr-nemA-mmsB, mcr-nemA-NDSD, or mcr-nemA-rutE or functional homolog or homolog with 80% sequence identity thereof.
IV. Salt-Tolerant Enzymes
The growth of engineered microorganism for enhanced production of a chemical product, such as E. coli is severely inhibited by high salt concentrations accumulated when the chemical product is produced at high rate within the organism.
Dose-dependent studies with increasing amounts of NaCl and Na-3-HP show that salt has inhibitory effects on ACCase activity which is essential to fatty acid biosynthesis of membranes required for growth and propagation and for the production of 3-HP (see EXAMPLE 1). Thus, the use of salt-tolerant enzymes in 3-HP production should increase 3-HP production in a host cell.
A. Enzymes from Halophilic Organisms
Halophiles are characterized as organisms with a great affinity for salt. In some instances a halophilic organism is one that requires at least 0.05M, 0.1M, 0.2M, 0.3M or 0.4M concentrations of salt (NaCl) for growth. Halophiles live in hypersaline environments that are generally defined occurring to their salt concentration of their habitats. Halophilic organisms that are defined as “Slight salt affinity” have optimal growth at 2-5% NaCl, moderate halophiles have optimal growth at 5-20% NaCl and extreme halophiles have optimal growth at 20-30% NaCl.
Depending on the conditions of that the genetically engineered microorganism is under one might use homologous enzymes of the invention specifically, for example, from a moderate halophiles or an extreme halophiles depending on the engineered cell's environment.
In certain aspects the invention provides for microorganisms comprising a genetic modification that includes enzymes of the invention provided herein from slight halophiles organisms. In certain aspects the invention provides for microorganisms comprising a genetic modification that includes enzymes of the invention provided herein from moderate halophiles organisms. In certain aspects the invention provides for microorganisms comprising a genetic modification that includes homologous enzymes of the invention provided herein from extreme halophiles organisms.
Homology with genes provided by the invention may be determined by analysis with BLASTN version 2.0 provided through the NCBI website. Homology with proteins provided by the invention may be determined by analysis with BLASTP version 2.2.2 provided through the NCBI website. This program with aligns the disclosed fragments being compared and determines the extent of identity or similarity between them.
To date there are many sequenced halophilic organisms which can be used with the claimed invention. Examples of some sequenced halophilic organisms include but are not limited to Chromohalobacter salexigens, Flexistipes sinusarabici strain (MAS10T), Halobacterium sp. NRC-1, Haloarcula marismortui, Natronomonas pharaonis, Haloquadratum walsbyi, Haloferax volcanii, Halorubrum lacusprofundi, Halobacterium sp. R-1, Halomicrobium mukohataei, Halorhabdus utahensis, Halogeometricum borinquense, Haloterrigena turkmenica, Natronobacterium gregoryi SP2, Halalkalicoccus jeotgali, Natrialba magadii, Haloarcula hispanica, Halopiger xanaduensis, Halophilic archaeon DL31, Haloferax mediterranei, Halovivax ruber, Natronococcus gregoryi, and Natronococcus occultus. 
Examples of suitable moderate halophilic organisms in which homologous enzymes of the invention can be derived from include but are not limited to eukaryotes such as crustaceans (e.g. Artemia salina), insects (e.g. Ephydra hians), certain plants from the genera Salicornia spp, algae (e.g. Dunaliella viridis), fungi, and protozoa (e.g. Fabrea salina), phototrophic organisms, such as planktonic and microbial mat-formers cyanobacteria as well as other anaerobic red and green sulphur bacteria from the genera Ectothiorhodospira spp.) and non-sulphur bacteria from the genera Chromatium spp.; gram-negative anaerobic bacteria, for example from the genera Haloanaerobacter spp. some of which are methanogenic, for example from the genera Methanohalophilus spp. and either aerobic or facultative such as species from the genera Halomonas, Chromohalobacter, Salinovibrio, Pseudomonas, for example (e.g. Halomonase elongate); gram-positive bacteria from genera such as Halobacillus, Bacillus, Marinococcus, etc. as well as some actinomycetes, for example, Actinopolyspora halophila. 
Genomic and Proteomic Hallmarks of Halophilic Organisms
Comparative genomic and proteomic studies of halophiles and non-halophiles reveal some common trends in the genomes and proteomes of halophiles. At the protein level, halophilic organisms are characterized by low hydrophobicity, over-representation of acidic residues, especially Asp, under-representation of Cys, lower propensities for helix formation and higher propensities for coil structure.
At the DNA level, halophilic organisms are characterized by the dinucleotide abundance profiles of halophilic genomes bear some common characteristics, which are quite distinct from those of non-halophiles, and hence may be regarded as specific genomic signatures for salt-adaptation. The synonymous codon usage in halophiles also exhibits similar patterns regardless of their long-term evolutionary history.
In certain aspects the invention provides for microorganisms comprising a genetic modification that the proteins provided by the invention that are modified for salt tolerance such that they has low hydrophobicity, over-representation of acidic residues, especially Asp, under-representation of Cys, lower propensities for helix formation and higher propensities for coil structure.
Suitable salt-tolerant enzymes can include enzymes from salt-tolerant organisms. Salt-tolerant organisms (such as, for example, halophiles) include any living organism that are adapted to living in conditions of high salinity. Suitable salt-tolerant enzymes can include enzymes from salt-tolerant organism that are homologs of the following enzymes: Sucrose-6-phosphate hydrolase (cscA from E. coli), glucose-6-phosphate isomerase (pgi from E. coli), fructokinase (cscK from E. coli), fructose-1,6-bisphosphatase (yggF from E. coli), fructose 1,6-bisphosphatase (ybhA from E. coli), fructose 1,6-bisphosphatase II (glpX from E. coli), fructose-1,6-bisphosphatase monomer (fbp from E. coli), 6-phosphofructokinase-1 monomer (pfkA from E. coli), 6-phosphofructokinase-2 monomer (pfkB from E. coli), fructose bisphosphate aldolase monomer (fbaB from E. coli), fructose bisphosphate aldolase monomer (fbaA from E. coli), triose phosphate isomerase monomer (tpiA), glyceraldehyde 3-phosphate dehydrogenase-A monomer (gapA from E. coli), phosphoglycerate kinase (pgk), 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (gpmM from E. coli), 2,3-bisphosphoglycerate-dependent or tdcE (from E. coli), phosphoglycerate mutase (gpmA), enolase (eno from E. coli), phosphoenolpyruvate carboxylase (ppc from E. coli), malate dehydrogenase (mdh), fumarase A (fum from E. coli), fumarase B (fumB), fumarase C (fumC from E. coli), phosphoenolpyruvate synthetase (ppsA from E. coli), pyruvate kinase I monomer (pykF from E. coli), pyruvate kinase II monomer (pykA from E. coli), fumarate reductase (frdABCD from E. coli), lipoamide dehydrogenase (lpd from E. coli), pyruvate dehydrogenase (aceE from E. coli), pyruvate dehydrogenase (aceF from E. coli), pyruvate formate-lyase (pflB from E. coli), acetyl-CoA carboxylase (accABCD from E. coli), malonyl CoA reductase (mcr), 3HP dehydrogenase (mmsB, NDSD, or ydfG), malonate semialdehyde reductase (nemA, rutE from E. coli), or a combination thereof.
Suitable salt-tolerant enzyme homologs that can be used with the claimed invention can have at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80%, overall amino acid or nucleotide identity to the above enzymes. Suitable salt-tolerant enzyme homologs that can be used with the claimed invention can have at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90% 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80%, amino acid or nucleotide to the essential protein function domains of the enzymes above. Suitable salt-tolerant enzyme homologs that can be used with the claimed invention can have at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% overall amino acid or nucleotide to the essential binding amino acids within an essential protein function domain of the enzymes above.
In accordance with a preferred embodiment of the invention, suitable salt-tolerant enzyme homologs are enzymes from one of the following organisms: Halomonas elongata, Salinibacter rubur, or Halobacterium species (Archaea).
In accordance with a preferred embodiment of the present invention, there is provided a non-salt-tolerant organism that is genetically modified to make 3-HP, wherein the genetic modification includes a polynucleotide encoding an acetyl-CoA carboxylase from a salt-tolerant organism. In accordance with a preferred embodiment, the acetyl-CoA carboxylase subunits accA, accB, accC and accD is from Halomonas elongata. 
V. Chemical Transporter
In accordance with another aspect of the present invention, any of the microorganisms herein may be genetically modified to introduce a nucleic acid sequence coding for a polypeptide that: (1) facilitates the exportation of the chemical of interest or the export of an inhibitory chemical from within the cell to the extracellular media; and/or (2) facilitates the importation from the extracellular media to within the cell of a reactant, precursor, and/or metabolite used in the organism's production pathway for producing the chemical of interest.
3-HP Exporter
In accordance with a preferred embodiment, this invention relates to the bioproduction of 3-HP using a genetically modified E. coli organism. Thus, the present invention contemplates of a host cell genetically modified to express or increase expression of an exporter that can function to transfer 3HP from the cellular environment extracellularly.
Bacterial cells, such as E. coli, have at least five different types of exporters: the major facilitator superfamily (MFS); the ATP-binding cassette superfamily (ABC); the small multidrug resistance family (SMR); the resistance-nodulation-cell division superfamily (RND); and the multi antimicrobial extrusion protein family (MATE). In addition, amino acid exporters, which are common to almost all host cells, are likely to export 3-HP. Additionally, solvent tolerance transporters, for example bromoacetate, butanol, isobutanol and the alike may be used to export 3-HP.
In certain aspects the invention provides a host cell with a recombinant exporter wherein the exporter is an MFS exporter, ABC exporter, SMR exporter, RND exporter, MATE exporter, amino acid exporter, solvent tolerance transporter or a combination thereof.
Suitable exporters that can be used with the s herein invention include but are not limited to acrD, bcr, cusA, dedA, eamA, eamB, eamH, emaA, emaB, emrB, emrD, emrKY, emrY, garP, gudP, hsrA, leuE, mdlB, mdtD, mdtG, mdtL, mdtM, mhpT, rhtA, rhtB, rhtC, thtB, yahN, yajR, ybbP, ybiF, ybjJ, ycaP, ydcO, yddG, ydeD, ydgE, yddG, ydhC, ydhP, ydiN, ydiM, ydjE, ydjI, ydjK, yeaS, yedA, yeeO, yegH, yggA, yfcJ, yfiK, yhjE, yidE, yigK, yigJ, yijE, yjiI, yjiJ, yjiO, ykgH, ypjD, ytfF, ytfL or functional homolog or homolog with 80% sequence identity thereof. Other potential transporter proteins may be identified using topology analysis as illustrated in [Daley et al., Science 308: 1321-1323, 2005].
In certain aspects the invention provides the exporter to be improved for binding to 3-HP. In certain aspects the invention provides the exporters named to be further enhance by genetic modification of the predicted cytoplasmic domain to increase 3-HP binding. In certain aspects the invention provides the exporter to be improved for binding to 3-HP. In certain aspects the invention provides the exporters named to be further enhance by genetic modification of the predicted transmembrane binding domain to increase 3-HP binding or incorporation into the host cell membrane.
Suitable exporter homologs that can be used with the claimed invention can have at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% overall amino acid or nucleotide identity to the above exporters. Suitable exporter homologs that can be used with the claimed invention can have 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% amino acid or nucleotide to the essential protein function domains of the exporters above. Suitable exporter homologs that can be used with the claimed invention can have at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% overall amino acid or nucleotide to the essential binding amino acids within an essential exporter domain of the enzymes above.
In certain aspects the invention provides for at least of the exporters provided herein to be expressed in a host cell to increase the chemical production of 3-HP in a host cell. In certain aspects the invention provides for at least of the exporters provided herein to be expressed in a host cell and with a genetic modification of tig to increase the chemical production of 3-HP in a host cell.
In certain aspects the invention provides for one exporter to be further modified by on one more genetic modulates so that the expression level and timing of expression of the exporter can be controlled in the host cell. In certain aspects the invention provides for one exporter to be further modified by an inducible promoter, RBS, high, multicopy plasmid or combination thereof, as provide herein, in order to control its expression in the host cell.
In certain aspects the invention provides exporters provide herein to be expressed in a host cell in equal ratio. In certain aspects the invention provides exporters provide herein to be expressed in a host cell in equal 1:2 ratio. In certain aspects the invention provides exporters provide herein to be expressed in a host cell in equal 1:3 ratio. In certain aspects the invention provides exporters provide herein to be expressed in a host cell in equal 1:4 ratio. In certain aspects the invention provides exporters provide herein to be expressed in a host cell in equal 2:3 ratio.
In certain aspects the invention provides for the exporter to maintain the host cell at pH 7.0-7.4 during the continuous production phase. In certain aspects the invention provides for the exporter and the means for importing a base inside the cell in order to maintain the host cell at pH 7.0-7.4 during the continuous production phase. In certain aspects the invention provides for the exporter maintain the host cell at pH 3.0 to pH 4.0, pH 4.0 to pH 5.0, pH 5.0 to pH 6.0, pH 6.0 to pH 7.0, pH 7.0 to pH 8.0, pH 8.0 to pH 9.0, or pH 9.0 to pH 10.0 pH 7.0-7.3 during the continuous production phase. In certain aspects the invention provides for the exporter and the means for importing a base inside the cell in order to maintain the host cell at pH 3.0 to pH 4.0, pH 4.0 to pH 5.0, pH 5.0 to pH 6.0, pH 6.0 to pH 7.0, pH 7.0 to pH 8.0, pH 8.0 to pH 9.0, or pH 9.0 to pH 10.0 pH 7.0-7.3 during the continuous production phase.
In accordance with this aspect of the present invention, addition modifications to the host cell may be made to further enhance the transporter's function. In particular, deletion of the tig gene from the genome of the host cell may enhance expression and total activity of integral membrane proteins such as exporters and importers.
Bicarbonate Importer
One of the key steps in the conversion of biomass to 3-HP is the conversion of acetyl-CoA to malonyl-CoA, which is illustrated in FIG. 3.
As shown in FIG. 3, this reaction is catalyzed by the acetyl-CoA carboxylase, and bicarbonate is a reactant needed to drive the reaction. One of the primary sources of bicarbonate to drive this reaction is carbon dioxide within the cell. Carbon dioxide is readily diffusible through a cell's membrane, and a natural equilibrium will be reached between the intracellular and extracellular carbon dioxide. As a cell produces carbon dioxide it migrates through the cell, and since it is not very soluble in the media, it will bubble out of the system and more intracellular carbon dioxide will migrate out of the cell to maintain the equilibrium. This process impedes the production of 3-HP since bicarbonate (which is in equilibrium with the dissolved carbon dioxide in the form of carbonic acid) is needed to drive the acetyl-CoA→malonyl-CoA reaction, and the intracellular carbon dioxide is the primary source for intracellular bicarbonate.
In accordance with one aspect of the present invention, an organism is provided that includes a heterologous gene encoded therein that acts as a carbon dioxide importer (i.e., it enhances the importation of carbon dioxide into the cell or inhibits the exportation of carbon dioxide from the cell), which results in increased intracellular carbon dioxide. Use of CO2 an importer mitigates against the natural outflow of carbon dioxide.
In accordance with this aspect of the invention, there is provided an organism that is genetically modified, wherein the genetic modification includes a polynucleotide encoding a gene capable of importing extracellular carbon dioxide from the media to within the cell membrane or inhibiting the exportation of intracellular carbon dioxide from within the cell membrane to the media. In accordance with a preferred embodiment of the present invention, a microorganism is genetically modified to encode one or more of the following heterologous genes: bicA from Synechococcus species, ychM gene product of E. coli, yidE gene product of E. coli, any of the bicarbonate transporters as described in [Felce and Saier, J. Mol. Microbiol. Biotechnol. 8: 169-176, 2004 or any amino acid sequences homologous thereof (e.g., at least 80%, 85%, 90%, 95%, or 99% homologous to the amino acids sequences of the CO2 importer/exporters described herein].
Bioproduction Methods
In some applications of the invention the host cell is genetically modified for increased malonyl-CoA flux by at least one heterologous ACCase complex, such as Table 4 to further increase chemical bio-production in host cell. In some applications of the invention the host cell is genetically modified with heterologous salt tolerant enzymes, such as Table 5 to increase chemical bio-production in a host cell. In some applications of the invention the host cell is genetically modified with heterologous 3-HP exporters to further increase chemical bio-production in a host cell.
In some applications of the invention the host cell is genetically modified by at least one heterologous gene and/or salt tolerant heterologous gene of FIG. 1 or Table 5 and at least one 3-HP exporter provided herein to further increase chemical bioproduction in a host cell.
In some applications of the invention the host cell is genetically modified with a heterologous gene for increased malonyl-CoA flux, 3-HP export, at least one heterologous and/or salt tolerant heterologous gene, as provided herein, to increase chemical bio-production in a host cell. In some applications of the invention the host cell is genetically modified for increased malonyl-CoA flux, 3-HP export, at least one heterologous gene and/or salt tolerant heterologous gene and the host cell is genetically modified by at least one gene, as provided herein to increase chemical bioproduction in a host cell.
When utilizing certain organisms to create certain products, it may be advantageous to control each phase discretely. For example, depending on the pathway involved, reactions, reactants, intermediates and byproducts created during cell growth can inhibit enzyme induction and/or the organism's ability to produce the desired chemical product. Similarly, reactions, reactants, intermediates and byproducts created as part of the production pathway can impact cell growth, and even the increased concentration of the chemical product as it is produced can impede cell replication.
Table. 5
VI. Multi-Phase Fermentation
In accordance with another aspect of the present invention, there is provided a method of producing a chemical product from a carbon source through a bioproduction process that comprises a controlled multi-phase production process. The multi-phase production process includes an initiation and/or completion of one or more phases of the production process is controlled by genetic modifications to the organism producing the chemical product and/or is controlled by changes made to the cell environment.
In accordance with this aspect of the invention, the bioproduction process may include two or more of the following phases: (1) growth phase; (2) induction phase; and (3) production phase. During the growth phase, the organism replicates itself and the biocatalyst needed to produce the chemical product is built up. During the induction phase, expression of key enzymes critical to the production of the chemical is induced and the enzymes accumulate within the biocatalyst to carry out the reactions required to produce the product. During the production phase organism produces the desired chemical product.
The initiation and/or completion of the growth, induction and/or production phases are controlled. In accordance with the present invention, the growth phase is dependent on the presence of a critical external reactant that will initiate growth. The initiation and completion of the growth phase is controlled by the addition and amount of the initiating reactant added to the reaction vessel.
In accordance with certain preferred embodiments of the present invention, the chemical product is 3-HP and the production organism is E. coli or yeast. The critical external reactant may be phosphate, which is needed for replication of E. coli cells. In accordance with a preferred embodiment, the growth phase is initiated by the addition of phosphate to a reaction vessel (together with a carbon source such as sugar and the E. coli cells), and the duration of the growth phase is controlled by the amount of phosphate added to the system.
The induction phase is controlled by genetic modifications to the producing organism. The key enzymes triggered during this phase are engineered into the organism using promoters that are sensitive to (e.g., activated by) the depletion of the initiating reactant. As a result, once the initiating reactant is depleted, the growth phase ends, the key enzymes are activated and the induction phase begins.
In accordance with a preferred embodiment, the induction phase is controlled by key genes that encode for enzymes in the biosynthetic pathway for the product into the production organism using promoters that are activated by phosphate depletion. In one embodiment where the chemical product is 3-HP and the production organism is E. coli, the key genetic modifications may include one or more of the following: mcr, mmsB, ydfG, rutE, nemA and NDSD; genes that encode individual or fused subunits of ACCase, such as accA, accB, accC, accD, accDA fusion, and accCB fusion, and the promoters may include one or more of the promoters that direct expression of the following E. coli genes: amn, tktB, xasA, yibD, ytfK, pstS, phoH, phnC, or other phosphate-regulated genes as described in [Baek and Lee, FEMS Microbiol Lett 264: 104-109, 2006]. In accordance with this embodiment, once the phosphate is depleted, expression of the key enzymes is activated by their promoters and the induction phase begins.
The production phase may also be controlled by genetic modifications. For example, the organism can be engineered to included mutated forms of enzymes critical to the initiation of production of the chemical product. These initiation enzymes may facilitate initiation of production either by: (1) becoming active and serving a key function in the production pathway; and/or (2) becoming inactive and thereby turning off a branch pathway or other competitive pathway that prevents or limits the production pathway leading to the chemical product. In accordance with a preferred embodiment, initiation enzymes are mutated to form temperature sensitive variants of the enzymes that are either activated by or deactivated at certain temperatures. As a result, the production phase is initiated by changing the changing the temperature within the reaction vessel.
In one embodiment, the production phase is controlled by genetically modifying the microorganism with a heterologous nucleotide sequence encoding i one or more of the following temperature sensitive enzymes: fabIts (SEQ ID NO. 27), fabBts (SEQ ID NO.28) and fabDts (SEQ ID NO. 29). These enzymes are deactivated or shut-off at the desired temperature for production of the chemical product. These enzymes play a key role shuttling carbon atoms into the fatty acid synthesis pathway. Although fatty acid synthesis pathway is critical during the growth phase, it inhibits production of the chemical product. Once the reaction vessel temperature is changed, the temperature sensitive enzymes are deactivated and the fatty acid synthesis pathway shuts down thereby allowing the production pathway of the chemical product to ramp up.
In accordance with the present invention, the growth phase can last be between 10 to 40 hours, or about 15 to about 35 hours, or about 20 to about 30 hours. The induction phase may be for about 1 to about 6 hours, about 1 to about 5 hours, or about 2 to about 4 hours. The production phase may be greater than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 hours depending on the amount of chemical product that is desired.
In accordance with the present invention, the growth phase and induction phase are conducted at a temperature of about 25° C. to about 35° C., about 28° C. to about 32° C., or about 30° C. The production phase is conducted at a temperature of about 35° C. to about 45° C., about 35° C. to about 40° C., or about 36° C. to about 38° C. Preferably, the production phase temperature is higher than the induction phase temperature, and the increase in temperature that initiates the production phase occurs over a period of about 1 to about 5 hours, about 1 to about 3 hours, about 2 hours, or about 1 hour.
In accordance with the present invention, there is provided a method of producing a chemical product from a renewable carbon source through a bioproduction process comprising:

    
    
        (1) constructing a genetically modified organism capable of converting said renewable carbon source to said chemical product, wherein said genetically modified organism requires inorganic phosphate for growth and comprises: (a) at least one heterologous gene whose expression is regulated by a promoter sensitive to inorganic phosphate levels within a culture system, wherein said gene provides a critical function in converting said carbon source to said chemical product and is not required for the genetically modified organism to replicate; and (b) a gene encoding a temperature-sensitive enzyme;
        (2) forming a culture system comprising said carbon source in an aqueous medium and said genetically modified microorganism;
        (3) maintaining the culture system under conditions that allow the genetically modified microorganism to replicate comprising maintaining a sufficient level of inorganic phosphate within said culture system;
        (4) allowing the inorganic phosphate to deplete thereby triggering the expression of the gene regulated by a promoter sensitive to inorganic phosphate levels; and
        (5) changing the temperature of the culture system thereby activating or deactivating said temperature-sensitive enzyme and initiating the production of said chemical product.
    
    


In accordance with the present invention, there is provided a method of producing 3-hydropropionic acid (3-HP) from a renewable carbon source, comprising:

    
    
        (1) constructing a genetically modified organism capable of converting said renewable carbon source to 3-HP, wherein said genetically modified organism requires inorganic phosphate for growth and comprises: (a) at least one heterologous gene whose expression is regulated by a promoter sensitive to inorganic phosphate levels within a culture system, wherein said gene is selected from the group consisting of mcr, mmsB, ydfG, rutE, nemA, NDSD, accA, accB, accC, accD, accDA fusion, and accCB fusion; and (b) a gene encoding a temperature-sensitive enzyme selected from the group consisting of fabI, fabB and fabD;
        (2) forming a culture system comprising said carbon source in an aqueous medium, phosphate and said genetically modified microorganism, and thereby initiating a growth phase during which the genetically modified microorganism replicates;
        (3) maintaining a sufficient level of inorganic phosphate within said culture system until the desired level of cell growth is achieved;
        (4) allowing the inorganic phosphate to deplete thereby initiating an induction phase which begins the expression of said gene regulated by a promoter sensitive to inorganic phosphate levels; and
        (5) changing the temperature of the culture system thereby activating or deactivating said temperature-sensitive enzyme and initiating a growth phase during which said genetically modified microorganism produces 3-HP.

Fermentation Conditions

    
    


Depending on the host cell fermentation may be performed under aerobic, microaerobic, or anaerobic conditions, with or without agitation. The operation of culture systems to achieve aerobic, microaerobic and anaerobic conditions are well known to those of ordinary skill in the art.
Suitable pH ranges for fermentation depend on the multiple factors such as the host cell. In some applications of the invention fermentation can occur between various pH ranges for example, pH 3.0 to pH 4.0, pH 4.0 to pH 5.0, pH 5.0 to pH 6.0, pH 6.0 to pH 7.0, pH 7.0 to pH 8.0, pH 8.0 to pH 9.0, or pH 9.0 to pH 10.0. However, the actual pH conditions for a particular application are not meant to be limited by these ranges and can be between the expressed pH ranges if it provides more optimal production of the fermentation process, such as increased 3-HP production.
VII. Genes and Proteins for the Bioproduction of Chemicals
An overview of the engineered pathways provided by the invention in a host cell is shown in FIG. 1. Various combinations of the pathways shown can be carried out by various combinations of genetic modifications to key enzymes either in the intrinsic pathways or supplied through the transformation of a heterologous gene.
In some applications of the genetically modified microorganism of the invention may comprise a single genetic modification, or one or more genetic modifications. Various types of genetic modifications that can be used with the invention are disclosed herein.
In some embodiments the genetic modified organism of the invention can comprise a genetic modification to the following gene/proteins or a homolog with at least 80% identity to or a functional homolog of: bifunctional malonyl-CoA reductase (MCR from Chloroflexus aurantiacus), monofunctional malonyl-CoA reductase (caMCR from Chloroflexus aurantiacus), malonyl-CoA reductase (stMCR from Sulfolobus tokodaii.), Enzyme: malonyl-CoA reductase (cgMCR from Chloroflexus aggregans), Enzyme: malonyl-CoA reductase (otMCR from Oscillochloris trichoides), Polypeptide: host restriction; endonuclease R (hsdR from E. coli), lactose metabolism (lac from E. coli), L-rhamnulose kinase (rhaB from E. coli), rhamnulose-1-phosphate aldolase (rhaD from E. coli), Enzyme: β-galactosidase (lacZ from E. coli), L-ribulose 5-phosphate 4-epimerase (araD from E. coli), L-ribulokinase (araB from E. coli), Enzyme: D-lactate dehydrogenase-fermentative (ldhA from E. coli), enzyme: pyruvate formate-lyase (pflB from E. coli), Enzyme: phosphate acetyltransferase/phosphate propionyltransferase (pta from E. coli), Enzyme: pyruvate oxidase (poxB from E. coli), Enzyme: methylglyoxal synthase (mgsA from E. coli), enzyme: Acetate kinase (ackA from E. coli), enzymes: phosphotransacetylase-acetate kinase (pta-ack from E. coli), Enzyme: enoyl-[acyl-carrier-protein] reductase (fabI from E. coli), Protein: zeocin binding protein (zeoR from Streptoalloteichus Hindustanus), Enzyme: carboxytransferase moiety of acetyl-CoA carboxylase (accAD from E. coli), Enzyme: triose phosphate isomerase (tpiA from E. coli), Enzyme: biotoin carboxylase moiety of acetyl-CoA carboxylase (accBC from E. coli), Enzyme: transhydrogenase (pntAB from E. coli), Polypeptide: Lad DNA-binding transcriptional repressor (lacI from E. coli), Enzyme: β-ketoacyl-ACP synthases I (fabB from E. coli), Enzyme: β-ketoacyl-ACP synthases II (fabF from E. coli), Enzyme: malonyl-CoA-ACP transacylase (fabD from E. coli), Enzyme: pantothenate kinase (coaA from E. coli), Enzyme: pyruvate dehydrogenase complex (aceEF from E. coli), Enzyme: 3-hydroxyisobutyrate/3-HP dehydrogenase (mmsB from Pseudomonas aeruginosa), Enzyme: lipoamide dehydrogenase (lpd from E. coli), Enzyme: γ-glutamyl-γ-aminobutyraldehyde dehydrogenase (puuC from E. coli), Enzyme: malate synthase A (aceB from E. coli), Enzyme: isocitrate lyase (aceA from E. coli), Enzyme: isocitrate dehydrogenase phosphatase/kinase (aceK from E. coli), Enzyme: 3-hydroxy acid dehydrogenase (ydfG from E. coli), Enzyme: acetyl CoA carboxylase (accADBC from E. coli), Polypeptide: predicted transcriptional regulator (yieP from E. coli), Blastocyin resistance gene (BSD from Schizosaccharomyces pombe), Enzyme: pyridine nucleotide transhydrogenase (udha from E. coli), Protein: Cra DNA-binding transcriptional dual regulator (fruR from E. coli), (SCB from E. coli), enzyme: aldehyde dehydrogenase B (aldB from E. coli), Enzyme: carbonic anhydrase (cynT from E. coli), Enzyme: cyanase (cynS from E. coli), DNA gyrase toxin-antitoxin system (ccdAB from E. coli), Enzyme: phosphoglycerate mutase (pgi from E. coli), ArcA transcriptional dual regulator or Aerobic respiration control (arcA from E. coli), Enzyme: 6-phosphofructokinase (pfk from E. coli), Enzyme: glyceraldehyde 3-phosphate dehydrogenase-A complex (gapA from E. coli), aldehyde dehydrogenase A (alda from E. coli), Enzyme: glutamate dehydrogenase (gdhA from E. coli), Enzyme: NADH-dependent serine dehydrogenase (NDSD from Pseudomonas aeruginosa), Protein: threonine/homoserine efflux transporter (rhtA from E. coli), Enzyme: glyceraldehyde 3-phosphate dehydrogenase (gapN from E. coli), Phosphotransferase system (pts from E. coli), Enzyme: 6-phosphofructokinase II (pfkB from E. coli), Enzyme: methylmalonate-semialdehyde dehydrogenase (mmsA from Pseudomonas aeruginosa), Oxaloacetate:beta-alanine aminotransferase (OAT-1 from Bacillus cereus), Enzyme: aspartate 1-decarboxylase (panD from E. coli), Gene that confers resistance to valine (ValR from E. coli), Enzyme: glucokinase (glk from E. coli), Polypeptide: 30 S ribosomal sununit protein S12 (rpsL from E. coli), Polypeptide: CynR DNA-binding transcriptional repressor (cynR from E. coli), Transporter: galactose:H+ symporter (galP from E. coli), aspartate aminotransferase (aspC from E. coli), Enzyme: alpha-ketoglutarate reductase (serA from E. coli), Enzyme: 6-phosphofructokinase I (pfkA from E. coli), Enzyme: phosphoenolpyruvate carboxylase (ppc from E. coli), Enzyme: succinate-semialdehyde dehydrogenase (NADP+) (gabD from E. coli), Enzyme: pyruvate kinase (pyk from E. coli), Enzyme: oxaloacetate 4-decarboxylase (OAD from Leuconostoc mesenteroides), Enzyme: trigger factor; a molecular chaperone involved in cell division (tig from E. coli), Transcription Unit (ptsHIcrr from E. coli), Enzyme: acetyl-CoA acetaldehyde dehydrogenase/alcohol dehydrogenase (adhE from E. coli), Enzyme: fattyacyl thioesterase I (tesA from E. coli), Enzyme: guanosine 3′-diphosphate 5′-triphosphate 3′-diphosphatase (spoT from E. coli), combination of genes encoding accABCD subunits (from E. coli and Halomonas elongata), pol (from E. coli), Enzyme: GDP pyrophosphokinase/GTP pyrophosphokinase (relA from E. coli), [Enzyme Name] (me from E. coli), Enzyme: citrate synthase (gltA from E. coli), Polypeptide: DNA gyrase, subunit A (gyrA from E. coli), Enzyme: multifunctional 2-keto-3-deoxygluconate 6-phosphate aldolase and 2-keto-4-hydroxyglutarate aldolase and oxaloacetate decarboxylase (eda from E. coli), thiamin biosynthesis (thi from E. coli), Polypeptide: acetolactate synthase II (ilvG from E. coli), acetyl CoA carboxylase (accDACB from E. coli), Citrate synthase (ArCS from Arthrobacter aurescens), Acetyl-CoA carboxylase from Corynebacter glutamicum (CgACC from Corynebacter glutamicum), Polypeptide: ferrichrome/phage/antibiotic outer membrane porin FhuA (fhuA from E. coli), Transporter: phosphate:H+ symporter PitA (pitA from E. coli), Transporter: uracil:H+ symporter (uraA from E. coli), Enzyme: uracil phosphoribosyltransferase (upp from E. coli), Enzyme: acylphosphatase (yccX from E. coli), acetyl-CoA synthetase (acsA from E. coli), Polypeptide: restriction of methylated adenine (mrr from E. coli), Protein: TrpR transcriptional repressor (trpR from E. coli), Enzymes: glutamate 5-semialdehyde dehydrogenase/gamma-glutamyl kinase (proAB from E. coli), methylcytosine restriction system (mcrBC from E. coli), Protein: citrate lyase, citrate-ACP transferase component (citF from E. coli), Enzyme: thioesterase II (tesB from E. coli), Enzyme: DNA-specific endonuclease I (endA from E. coli), Enzyme: phosphate acetyltransferase (eutD from E. coli), Enzyme: propionate kinase (tdcD from E. coli), tRNA: tRNA glnV (supE from E. coli), Enzyme: DNA-binding, ATP-dependent protease La (lon from E. coli), Polypeptide: DNA strand exchange and recombination protein with protease and nuclease activity (recA from E. coli), Transcription Unit: restriction endonuclease component of EcoKI restriction-modification system (hsdRMS from E. coli), Enzyme: restriction of DNA at 5-methylcytosine residues (mcrA from E. coli) araD (from E. coli), araB (from E. coli), rhaD (from E. coli), rhaB (from E. coli), ack (from E. coli), fruR (from E. coli), gapA (from E. coli), lad (from E. coli), lacZ (from E. coli), ldhA (from E. coli), mgsA (from E. coli), pfkA (from E. coli), pflB (from E. coli), pgi (from E. coli), poxB (from E. coli), pta-ack (from E. coli), ptsH (from E. coli), glut1 (from E. coli) and/or ack (from E. coli) or any combination thereof.
The use of genetic modifications in genetic elements, genes, proteins or the use of compounds, such as siRNA technology, anti-sense technology, and small molecule inhibitors supplied to the host cell that modulate the expression of gene and proteins provided by the present invention are also contemplated.
In some embodiments the genetic modified organism of the invention uses genetic elements such as siRNA ect, genes, proteins or compounds supplied to the host cell to modulate one or more of the following: bifunctional malonyl-CoA reductase (MCR from Chloroflexus aurantiacus), monofunctional malonyl-CoA reductase (caMCR from Chloroflexus aurantiacus), malonyl-CoA reductase (stMCR from Sulfolobus tokodaii.), Enzyme: malonyl-CoA reductase (cgMCR from Chloroflexus aggregans), Enzyme: malonyl-CoA reductase (otMCR from Oscillochloris trichoides), Polypeptide: host restriction; endonuclease R (hsdR from E. coli), lactose metabolism (lac from E. coli), L-rhamnulose kinase (rhaB from E. coli), rhamnulose-1-phosphate aldolase (rhaD from E. coli), Enzyme: β-galactosidase (lacZ from E. coli), L-ribulose 5-phosphate 4-epimerase (araD from E. coli), L-ribulokinase (araB from E. coli), Enzyme: D-lactate dehydrogenase-fermentative (ldhA from E. coli), enzyme: pyruvate formate-lyase (pflB from E. coli), Enzyme: phosphate acetyltransferase/phosphate propionyltransferase (pta from E. coli), Enzyme: pyruvate oxidase (poxB from E. coli), Enzyme: methylglyoxal synthase (mgsA from E. coli), enzyme: Acetate kinase (ackA from E. coli), enzymes: phosphotransacetylase-acetate kinase (pta-ack from E. coli), Enzyme: enoyl-[acyl-carrier-protein] reductase (fabI from E. coli), Protein: zeocin binding protein (zeoR from Streptoalloteichus Hindustanus), Enzyme: carboxytransferase moiety of acetyl-CoA carboxylase (accAD from E. coli), Enzyme: triose phosphate isomerase (tpiA from E. coli), Enzyme: biotoin carboxylase moiety of acetyl-CoA carboxylase (accBC from E. coli), Enzyme: transhydrogenase (pntAB from E. coli), Polypeptide: Lad DNA-binding transcriptional repressor (lacI from E. coli), Enzyme: β-ketoacyl-ACP synthases I (fabB from E. coli), Enzyme: β-ketoacyl-ACP synthases II (fabF from E. coli), Enzyme: malonyl-CoA-ACP transacylase (fabD from E. coli), Enzyme: pantothenate kinase (coaA from E. coli), Enzyme: pyruvate dehydrogenase complex (aceEF from E. coli), Enzyme: 3-hydroxyisobutyrate/3-HP dehydrogenase (mmsB from Pseudomonas aeruginosa), Enzyme: lipoamide dehydrogenase (lpd from E. coli), Enzyme: γ-glutamyl-γ-aminobutyraldehyde dehydrogenase (puuC from E. coli), Enzyme: malate synthase A (aceB from E. coli), Enzyme: isocitrate lyase (aceA from E. coli), Enzyme: isocitrate dehydrogenase phosphatase/kinase (aceK from E. coli), Enzyme: 3-hydroxy acid dehydrogenase (ydfG from E. coli), Enzyme: acetyl CoA carboxylase (accADBC from E. coli), Polypeptide: predicted transcriptional regulator (yieP from E. coli), Blastocyin resistance gene (BSD from Schizosaccharomyces pombe), Enzyme: pyridine nucleotide transhydrogenase (udha from E. coli), Protein: Cra DNA-binding transcriptional dual regulator (fruR from E. coli), (SCB from E. coli), enzyme: aldehyde dehydrogenase B (aldB from E. coli), Enzyme: carbonic anhydrase (cynT from E. coli), Enzyme: cyanase (cynS from E. coli), DNA gyrase toxin-antitoxin system (ccdAB from E. coli), Enzyme: phosphoglycerate mutase (pgi from E. coli), ArcA transcriptional dual regulator or Aerobic respiration control (arcA from E. coli), Enzyme: 6-phosphofructokinase (pfk from E. coli), Enzyme: glyceraldehyde 3-phosphate dehydrogenase-A complex (gapA from E. coli), aldehyde dehydrogenase A (alda from E. coli), Enzyme: glutamate dehydrogenase (gdhA from E. coli), Enzyme: NADH-dependent serine dehydrogenase (NDSD from Pseudomonas aeruginosa), Protein: threonine/homoserine efflux transporter (rhtA from E. coli), Enzyme: glyceraldehyde 3-phosphate dehydrogenase (gapN from E. coli), Phosphotransferase system (pts from E. coli), Enzyme: 6-phosphofructokinase II (pfkB from E. coli), Enzyme: methylmalonate-semialdehyde dehydrogenase (mmsA from Pseudomonas aeruginosa), Oxaloacetate:beta-alanine aminotransferase (OAT-1 from Bacillus cereus), Enzyme: aspartate 1-decarboxylase (panD from E. coli), Gene that confers resistance to valine (ValR from E. coli), Enzyme: glucokinase (glk from E. coli), Polypeptide: 30 S ribosomal sununit protein S12 (rpsL from E. coli), Polypeptide: CynR DNA-binding transcriptional repressor (cynR from E. coli), Transporter: galactose:H+ symporter (galP from E. coli), aspartate aminotransferase (aspC from E. coli), Enzyme: alpha-ketoglutarate reductase (serA from E. coli), Enzyme: 6-phosphofructokinase I (pfkA from E. coli), Enzyme: phosphoenolpyruvate carboxylase (ppc from E. coli), Enzyme: succinate-semialdehyde dehydrogenase (NADP+) (gabD from E. coli), Enzyme: pyruvate kinase (pyk from E. coli), Enzyme: oxaloacetate 4-decarboxylase (OAD from Leuconostoc mesenteroides), Enzyme: trigger factor; a molecular chaperone involved in cell division (tig from E. coli), Transcription Unit (ptsHIcrr from E. coli), Enzyme: acetyl-CoA acetaldehyde dehydrogenase/alcohol dehydrogenase (adhE from E. coli), Enzyme: fattyacyl thioesterase I (tesA from E. coli), Enzyme: guanosine 3′-diphosphate 5′-triphosphate 3′-diphosphatase (spoT from E. coli), combination of genes encoding accABCD subunits (from E. coli and Halomonas elongata), pol (from E. coli), Enzyme: GDP pyrophosphokinase/GTP pyrophosphokinase (relA from E. coli), [Enzyme Name] (me from E. coli), Enzyme: citrate synthase (gltA from E. coli), Polypeptide: DNA gyrase, subunit A (gyrA from E. coli), Enzyme: multifunctional 2-keto-3-deoxygluconate 6-phosphate aldolase and 2-keto-4-hydroxyglutarate aldolase and oxaloacetate decarboxylase (eda from E. coli), thiamin biosynthesis (thi from E. coli), Polypeptide: acetolactate synthase II (ilvG from E. coli), acetyl CoA carboxylase (accDACB from E. coli), Citrate synthase (ArCS from Arthrobacter aurescens), Acetyl-CoA carboxylase from Corynebacter glutamicum (CgACC from Corynebacter glutamicum), Polypeptide: ferrichrome/phage/antibiotic outer membrane porin FhuA (fhuA from E. coli), Transporter: phosphate:H+ symporter PitA (pitA from E. coli), Transporter: uracil:H+ symporter (uraA from E. coli), Enzyme: uracil phosphoribosyltransferase (upp from E. coli), Enzyme: acylphosphatase (yccX from E. coli), acetyl-CoA synthetase (acsA from E. coli), Polypeptide: restriction of methylated adenine (mrr from E. coli), Protein: TrpR transcriptional repressor (trpR from E. coli), Enzymes: glutamate 5-semialdehyde dehydrogenase/gamma-glutamyl kinase (proAB from E. coli), methylcytosine restriction system (mcrBC from E. coli), Protein: citrate lyase, citrate-ACP transferase component (citF from E. coli), Enzyme: thioesterase II (tesB from E. coli), Enzyme: DNA-specific endonuclease I (endA from E. coli), Enzyme: phosphate acetyltransferase (eutD from E. coli), Enzyme: propionate kinase (tdcD from E. coli), tRNA: tRNA glnV (supE from E. coli), Enzyme: DNA-binding, ATP-dependent protease La (lon from E. coli), Polypeptide: DNA strand exchange and recombination protein with protease and nuclease activity (recA from E. coli), Transcription Unit: restriction endonuclease component of EcoKI restriction-modification system (hsdRMS from E. coli), Enzyme: restriction of DNA at 5-methylcytosine residues (mcrA from E. coli). In some embodiment the genetic modifications listed above are modified further with the genetic modules provided herein.
In some embodiment the genetic modification of the genes, proteins and enzymes of the invention can be for the method of bioproduction of various chemicals which can be used to make various consumer products described herein.
In some embodiment the genetic modification of the genes, proteins and enzymes of the invention can be for the bioproduction of 1,4-butanediol (1,4-BDO) (U.S. Pub. No. 20110190513). In some embodiment the genetic modification of the genes, proteins and enzymes of the invention can be for the bioproduction of butanol (U.S. application Ser. No. 13/057,359). In some embodiment the genetic modification of the genes, proteins and enzymes of the invention can be for the bioproduction of isobutanol (U.S. application Ser. No. 13/057,359)
In some embodiment the genetic modification of the genes, proteins and enzymes of the invention can be for the bioproduction of 3-HP such and its aldehyde metabolites (U.S. application Ser. No. 13/062,917).
In some embodiment the genetic modification of the genes, proteins and enzymes of the invention can be for the bioproduction of polyketide chemical products (U.S. application Ser. No. 13/575,581).
In some embodiment the genetic modification of the genes, proteins and enzymes of the invention can be for the bioproduction of fatty acid methyl esters (U.S. Pub. No. 20110124063). In some embodiment the genetic modification of the genes, proteins and enzymes of the invention can be for the bioproduction of C4-C18 fatty acids (U.S. App No. 61/682,127).
Genetic Modifications
Various methods to achieve such genetic modification in a host strain are well known to one skilled in the art. Example of genetic modification that can be used by the claimed invention include, but are not limited to, increasing expression of an endogenous genetic element; increasing expression of an exogenous genetic element; decreasing functionality of a repressor gene; increasing functionality of a repressor gene; increasing functionality of a activator gene; decreasing functionality of a activator gene; introducing a genetic change or element integrated in the host genome, introducing a heterologous genetic element permanently, by integration into the genome or transiently by transformation with plasmid; increasing copy number of a nucleic acid sequence encoding a polypeptide catalyzing an enzymatic conversion step; mutating a genetic element to provide a mutated protein to increase specific enzymatic activity; mutating a genetic element to provide a mutated protein to decrease specific enzymatic activity; over-expressing of gene; reduced the expression of a gene; knocking out or deleting a gene; altering or modifying feedback inhibition; providing an enzyme variant comprising one or more of an impaired binding sites or active sites; increasing functionality of a siRNA, decreasing functionality of a siRNA, increasing functionality of a antisense molecule, decreasing functionality of a antisense molecule, addition of genetic modules such as RBS, ′3 UTR elements to increase mRNA stability or translation; deletion of genetic modules such as RBS, ′3 UTR elements to decrease mRNA stability or translation; addition or modification of genetic modules such as ′5 UTR elements to increase transcription; deletion or modification of genetic modules such as ′5 UTR and elements to increase transcription. In addition other genetic modules, provide herein, such a multicopy plasmids and various promoters can be used to further modify of the genetic modifications provide herein. Additionally, as known to those of ordinarily skill in the art compounds such as siRNA technology, anti-sense technology, and small molecule in inhibitors can be used to alter gene expression in the same manner as a genetic modification.
Screening methods, such as SCALE in combination with random mutagenesis may be practiced to provide genetic modifications that provide a benefit to increased production of 3-HP in a host cell. Examples of random mutagenesis can include insertions, deletions and substitutions of one or more nucleic acids in a nucleic acid of interest. In various embodiments a genetic modification results in improved enzymatic specific activity and/or turnover number of an enzyme. Without being limited, changes may be measured by one or more of the following: KM; Kcat, Kavidity, gene expression level, protein expression level, level of a product known to be produced by the enzyme, 3-HP tolerance, or by 3-HP production or by other means.
Host Cells
In some applications of the invention the host cell can be a gram-negative bacterium. In some applications of the invention the host cell can be from the genera Zymomonas, Escherichia, Pseudomonas, Alcaligenes, or Klebsiella. In some applications of the invention the host cell can be Escherichia coli, Cupriavidus necator, Oligotropha carboxidovorans, or Pseudomonas putida. In some applications of the invention the host cell is one or more an E. coli strains.
In some applications of the invention the host cell can be a gram-positive bacterium. In some applications of the invention the host cell can be from the genera Clostridium, Salmonella, Rhodococcus, Bacillus, Lactobacillus, Enterococcus, Paenibacillus, Arthrobacter, Corynebacterium, or Brevibacterium. In some applications of the invention the host cell is Bacillus licheniformis, Paenibacillus macerans, Rhodococcus erythropolis, Lactobacillus plantarum, Enterococcus faecium, Enterococcus gallinarium, Enterococcus faecalis, or Bacillus subtilis. In some applications of the invention the host cell is B. subtilis strain.
In some applications of the invention the host cell is yeast. In some applications of the invention the host cell can be from the genera Pichia, Candida, Hansenula or Saccharomyces. In some applications of the invention the host cell is Saccharomyces cerevisiae. In some applications of the invention the host cell is Saccharomyces pombe. 
In some applications of the invention the host cell is an alga. In some applications of the invention the host cell is a halophile. In some applications of the invention the host cell is an alga. In some applications of the invention the host cell is a chemolithotrophic bacterium.
In some applications of the invention the host cell is comprised of multiple host cell types. In some applications of the invention the host cell is comprised of one host cell type. In some applications of the invention the host cell is comprised of one more species or strain of a host cell type.
Downstream Consumer Products Chemicals
3-HP purified according to the methods provided in this disclosure may be converted to various other products having industrial uses including, but not limited to, acrylamide, acrylic acid, esters of acrylic acid, 1,3-propanediol, and other chemicals, collectively referred to as “downstream chemical products” or “downstream products.” In some instances the conversion is associated with the separation and/or purification steps. These downstream chemical products are useful for producing a variety of consumer products which are described in more detail below. The methods of the present invention include steps to produce downstream products of 3-HP.
As a C3 building block, 3-HP offers much potential in a variety of chemical conversions to commercially important intermediates, industrial end products, and consumer products. For example, 3-HP may be converted to acrylic acid, acrylates (e.g., acrylic acid salts and esters), 1,3-propanediol, malonic acid, ethyl-3-hydroxypropionate, ethyl ethoxy propionate, propiolactone, acrylamide, or acrylonitrile.
Additionally, 3-HP may be oligomerized or polymerized to form poly(3-hydroxypropionate) homopolymers, or co-polymerized with one or more other monomers to form various co-polymers. Because 3-HP has a single stereoisomer, polymerization of 3-HP is not complicated by the stereo-specificity of monomers during chain growth. This is in contrast to (S)-2-hydroxypropanoic acid (also known as lactic acid), which has two (D, L) stereoisomers that should be considered during its polymerizations.
As will be further described, 3-HP can be converted into derivatives starting (i) substantially as the protonated form of 3-hydroxypropionic acid; (ii) substantially as the deprotonated form, 3-hydroxypropionate; or (iii) as mixtures of the protonated and deprotonated forms. Generally, the fraction of 3-HP present as the acid versus the salt will depend on the pH, the presence of other ionic species in solution, temperature (which changes the equilibrium constant relating the acid and salt forms), and, to some extent, pressure. Many chemical conversions may be carried out from either of the 3-HP forms, and overall process economics will typically dictate the form of 3-HP for downstream conversion.
Acrylic acid obtained from 3-HP purified by the methods described in this disclosure may be further converted to various polymers. For example, the free-radical polymerization of acrylic acid takes place by polymerization methods known to the skilled worker and can be carried out, for example, in an emulsion or suspension in aqueous solution or another solvent. Initiators, such as but not limited to organic peroxides, are often added to aid in the polymerization. Among the classes of organic peroxides that may be used as initiators are diacyls, peroxydicarbonates, monoperoxycarbonates, peroxyketals, peroxyesters, dialkyls, and hydroperoxides. Another class of initiators is azo initiators, which may be used for acrylate polymerization as well as co-polymerization with other monomers. U.S. Pat. Nos. 5,470,928; 5,510,307; 6,709,919; and 7,678,869 teach various approaches to polymerization using a number of initiators, including organic peroxides, azo compounds, and other chemical types, and are incorporated by reference for such teachings as applicable to the polymers described herein.
Accordingly, it is further possible for co-monomers, such as crosslinkers, to be present during the polymerization. The free-radical polymerization of the acrylic acid obtained from dehydration of 3-HP, as produced herein, in at least partly neutralized form and in the presence of crosslinkers is practiced in certain embodiments. This polymerization may result in hydrogels which can then be comminuted, ground and, where appropriate, surface-modified, by known techniques.
An important commercial use of polyacrylic acid is for superabsorbent polymers. This specification hereby incorporates by reference Modern Superabsorbent Polymer Technology, Buchholz and Graham (Editors), Wiley-VCH, 1997, in its entirety for its teachings regarding superabsorbent polymers components, manufacture, properties and uses. Superabsorbent polymers are primarily used as absorbents for water and aqueous solutions for diapers, adult incontinence products, feminine hygiene products, and similar consumer products. In such consumer products, superabsorbent materials can replace traditional absorbent materials such as cloth, cotton, paper wadding, and cellulose fiber. Superabsorbent polymers absorb, and retain under a slight mechanical pressure, up to 25 times or more their weight in liquid. The swollen gel holds the liquid in a solid, rubbery state and prevents the liquid from leaking. Superabsorbent polymer particles can be surface-modified to produce a shell structure with the shell being more highly cross-linked than the rest of the particle. This technique improves the balance of absorption, absorption under load, and resistance to gel-blocking. It is recognized that superabsorbent polymers have uses in fields other than consumer products, including agriculture, horticulture, and medicine.
Superabsorbent polymers are prepared from acrylic acid (such as acrylic acid derived from 3-HP provided herein) and a crosslinker, by solution or suspension polymerization. Exemplary methods include those provided in U.S. Pat. Nos. 5,145,906; 5,350,799; 5,342,899; 4,857,610; 4,985,518; 4,708, 997; 5,180,798; 4,666,983; 4,734,478; and 5,331,059, each incorporated by reference for their teachings relating to superabsorbent polymers.
Among consumer products, a diaper, a feminine hygiene product, and an adult incontinence product are made with superabsorbent polymer that itself is made substantially from acrylic acid converted from 3-HP made in accordance with the present invention.
Diapers and other personal hygiene products may be produced that incorporate superabsorbent polymers made from acrylic acid made from 3-HP which is produced and purified by the teachings of the present application. The following provides general guidance for making a diaper that incorporates such superabsorbent polymer. The superabsorbent polymer first is molded into an absorbent pad that may be vacuum formed, and in which other materials, such as a fibrous material (e.g., wood pulp) are added. The absorbent pad then is assembled with sheet(s) of fabric, generally a nonwoven fabric (e.g., made from one or more of nylon, polyester, polyethylene, and polypropylene plastics) to form diapers.
More particularly, in one non-limiting process, multiple pressurized nozzles, located above a conveyer belt, spray superabsorbent polymer particles (e.g., about 400 micron size or larger), fibrous material, and/or a combination of these onto the conveyer belt at designated spaces/intervals. The conveyor belt is perforated and under vacuum from below, so that the sprayed on materials are pulled toward the belt surface to form a flat pad. In various embodiments, fibrous material is applied first on the belt, followed by a mixture of fibrous material and the superabsorbent polymer particles, followed by fibrous material, so that the superabsorbent polymer is concentrated in the middle of the pad. A leveling roller may be used toward the end of the belt path to yield pads of uniform thickness. Each pad thereafter may be further processed, such as to cut it to a proper shape for the diaper, or the pad may be in the form of a long roll sufficient for multiple diapers. Thereafter, the pad is sandwiched between a top sheet and a bottom sheet of fabric (one generally being liquid pervious, the other liquid impervious), for example on a conveyor belt, and these are attached together, for example by gluing, heating or ultrasonic welding, and cut into diaper-sized units (if not previously so cut). Additional features may be provided, such as elastic components, strips of tape, etc., for fit and ease of wearing by a person.
The ratio of the fibrous material to polymer particles is known to affect performance characteristics. In some cases, this ratio is between 75:25 and 90:10 (see e.g., U.S. Pat. No. 4,685,915, incorporated by reference for its teachings of diaper manufacture). Other disposable absorbent articles may be constructed in a similar fashion, such as absorbent articles for adult incontinence, feminine hygiene (sanitary napkins), tampons, etc. (see, for example, U.S. Pat. Nos. 5,009,653; 5,558,656; and 5,827,255 incorporated by reference for their teachings of sanitary napkin manufacture).
Low molecular weight polyacrylic acid has uses for water treatment, and as a flocculant and thickener for various applications including cosmetics and pharmaceutical preparations. For these applications, the polymer may be uncrosslinked or lightly cross-linked, depending on the specific application. The molecular weights are typically from about 200 to about 1,000,000 g/mol. Preparation of these low molecular weight polyacrylic acid polymers is described in U.S. Pat. Nos. 3,904,685; 4,301,266; 2,798,053; and 5,093,472, each of which is incorporated by reference for its teachings relating to methods to produce these polymers.
Acrylic acid may be co-polymerized with one or more other monomers selected from acrylamide, 2-acrylamido-2-methylpropanesulfonic acid, N,N-dimethylacrylamide, N-isopropylacrylamide, methacrylic acid, and methacrylamide, to name a few. The relative reactivities of the monomers affect the microstructure and thus the physical properties of the polymer. Co-monomers may be derived from 3-HP, or otherwise provided, to produce co-polymers. Ullmann's Encyclopedia of Industrial Chemistry, Polyacrylamides and Poly(Acrylic Acids), WileyVCH Verlag GmbH, Wienham (2005), is incorporated by reference herein for its teachings of polymer and co-polymer processing.
Acrylic acid can in principle be copolymerized with almost any free-radically polymerizable monomers including styrene, butadiene, acrylonitrile, acrylic esters, maleic acid, maleic anhydride, vinyl chloride, acrylamide, itaconic acid, and so on. End-use applications typically dictate the co-polymer composition, which influences properties. Acrylic acid also may have a number of optional substitutions and, after such substitutions, may be used as a monomer for polymerization, or co-polymerization reactions. As a general rule, acrylic acid (or one of its co-polymerization monomers) may be substituted by any substituent that does not interfere with the polymerization process, such as alkyl, alkoxy, aryl, heteroaryl, benzyl, vinyl, allyl, hydroxy, epoxy, amide, ethers, esters, ketones, maleimides, succinimides, sulfoxides, glycidyl and silyl (see e.g., U.S. Pat. No. 7,678,869, incorporated by reference above, for further discussion). The following paragraphs provide a few non-limiting examples of copolymerization applications.
Paints that comprise polymers and copolymers of acrylic acid and its esters are in wide use as industrial and consumer products. Aspects of the technology for making such paints can be found in e.g., U.S. Pat. Nos. 3,687,885 and 3,891,591, incorporated by reference for their teachings of such paint manufacture. Generally, acrylic acid and its esters may form homopolymers or copolymers among themselves or with other monomers, such as amides, methacrylates, acrylonitrile, vinyl, styrene and butadiene. A desired mixture of homopolymers and/or copolymers, referred to in the paint industry as “vehicle” (or “binder”) are added to an aqueous solution and agitated sufficiently to form an aqueous dispersion that includes sub-micrometer sized polymer particles. The paint cures by coalescence of these vehicle particles as the water and any other solvent evaporate. Other additives to the aqueous dispersion may include pigment, filler (e.g., calcium carbonate, aluminum silicate), solvent (e.g., acetone, benzol, alcohols, etc., although these are not found in certain no VOC paints), thickener, and additional additives depending on the conditions, applications, intended surfaces, etc. In many paints, the weight percent of the vehicle portion may range from about nine to about 26 percent, but for other paints the weight percent may vary beyond this range.
Acrylic-based polymers are used for many coatings in addition to paints. For example, for paper coating latexes, acrylic acid is used from 0.1-5.0%, along with styrene and butadiene, to enhance binding to the paper and modify rheology, freeze-thaw stability and shear stability. In this context, U.S. Pat. Nos. 3,875,101 and 3,872,037 are incorporated by reference for their teachings regarding such latexes. Acrylate-based polymers also are used in many inks, particularly UV curable printing inks. For water treatment, acrylamide and/or hydroxy ethyl acrylate are commonly co-polymerized with acrylic acid to produce low molecular-weight linear polymers. In this context, U.S. Pat. Nos. 4,431,547 and 4,029,577 are incorporated by reference for their teachings of such polymers. Co-polymers of acrylic acid with maleic acid or itaconic acid are also produced for water-treatment applications, as described in U.S. Pat. No. 5,135,677, incorporated by reference for that teaching. Sodium acrylate (the sodium salt of glacial acrylic acid) can be co-polymerized with acrylamide (which may be derived from acrylic acid via amidation chemistry) to make an anionic co-polymer that is used as a flocculant in water treatment.
For thickening agents, a variety of co-monomers can be used, such as those described in U.S. Pat. Nos. 4,268,641 and 3,915,921, incorporated by reference for their description of these co-monomers. U.S. Pat. No. 5,135,677 describes a number of co-monomers that can be used with acrylic acid to produce water-soluble polymers, and is incorporated by reference for such description.
In some cases, conversion to downstream products may be made enzymatically. For example, 3-HP may be converted to 3-HP-CoA, which then may be converted into polymerized 3-HP with an enzyme having polyhydroxy acid synthase activity (EC 2.3.1.-). Also, 1,3-propanediol can be made using polypeptides having oxidoreductase activity or reductase activity (e.g., enzymes in the EC 1.1.1.-class of enzymes). Alternatively, when creating 1,3-propanediol from 3-HP, a combination of (1) a polypeptide having aldehyde dehydrogenase activity (e.g., an enzyme from the 1.1.1.34 class) and (2) a polypeptide having alcohol dehydrogenase activity (e.g., an enzyme from the 1.1.1.32 class) can be used. Polypeptides having lipase activity may be used to form esters. Enzymatic reactions such as these may be conducted in vitro, such as using cell-free extracts, or in vivo.
Thus, various embodiments described in this disclosure, such as methods of making a chemical, include conversion steps to any downstream products of microbially produced 3-HP, including but not limited to those chemicals described herein, in the incorporated references, and known in the art. For example, in some cases, 3-HP is produced and converted to polymerized-3-HP (poly-3-HP) or acrylic acid. In some cases, 3-HP or acrylic acid can be used to produce polyacrylic acid (polymerized acrylic acid, in various forms), methyl acrylate, acrylamide, acrylonitrile, propiolactone, ethyl 3-HP, malonic acid, 1,3-propanediol, ethyl acrylate, n-butyl acrylate, hydroxypropyl acrylate, hydroxyethyl acrylate, isobutyl acrylate, 2-ethylhexyl acrylate, and acrylic acid or an acrylic acid ester to which an alkyl or aryl addition may be made, and/or to which halogens, aromatic amines or amides, and aromatic hydrocarbons may be added.
a) Reactions that form downstream compounds such as acrylates or acrylamides can be conducted in conjunction with use of suitable stabilizing agents or inhibiting agents reducing the likelihood of polymer formation. See, for example, U.S. Publication No. 2007/0219390, incorporated by reference in its entirety. Stabilizing agents and/or inhibiting agents include, but are not limited to, e.g., phenolic compounds (e.g., dimethoxyphenol (DMP) or alkylated phenolic compounds such as di-tert-butyl phenol), quinones (e.g., t-butyl hydroquinone or the monomethyl ether of hydroquinone (MEHQ)), and/or metallic copper or copper salts (e.g., copper sulfate, copper chloride, or copper acetate). Inhibitors and/or stabilizers can be used individually or in combinations as will be known by those of skill in the art.
In some cases, the one or more downstream compounds are recovered at a molar yield of up to about 100 percent, or a molar yield in the range from about 70 percent to about 90 percent, or a molar yield in the range from about 80 percent to about 100 percent, or a molar yield in the range from about 90 percent to about 100 percent. Such yields may be the result of single-pass (batch or continuous) or iterative separation and purification steps in a particular process.
The methods described in this disclosure can also be used to produce downstream compounds derived from 3-HP, such as but not limited to, polymerized-3-HP (poly-3-HP), acrylic acid, polyacrylic acid (polymerized acrylic acid, in various forms), copolymers of acrylic acid and acrylic esters, acrylamide, acrylonitrile, propiolactone, ethyl 3-HP, malonic acid, and 1,3-propanediol. Also, among esters that are formed are methyl acrylate, ethyl acrylate, n-butyl acrylate, hydroxypropyl acrylate, hydroxyethyl acrylate, isobutyl acrylate, and 2-ethylhexyl acrylate. These and/or other acrylic acid and/or other acrylate esters may be combined, including with other compounds, to form various known acrylic acid-based polymers. Numerous approaches may be employed for such downstream conversions, generally falling into enzymatic, catalytic (chemical conversion process using a catalyst), thermal, and combinations thereof (including some wherein a desired pressure is applied to accelerate a reaction). For example, without being limiting, acrylic acid may be made from 3-HP via a dehydration reaction, methyl acrylate may be made from 3-HP via dehydration and esterification, the latter to add a methyl group (such as using methanol), acrylamide may be made from 3-HP via dehydration and amidation reactions, acrylonitrile may be made via a dehydration reaction and forming a nitrile moiety, propiolactone may be made from 3-HP via a ring-forming internal esterification reaction, ethyl-3-HP may be made from 3-HP via esterification with ethanol, malonic acid may be made from 3-HP via an oxidation reaction, and 1,3-propanediol may be made from 3-HP via a reduction reaction. Additionally, it is appreciated that various derivatives of the derivatives of 3-HP and acrylic acid may be made, such as the various known polymers of acrylic acid and its derivatives. Production of such polymers is considered within the scope of the present invention. Copolymers containing acrylic acid and/or esters have been widely used in the pharmaceutical formulation to achieve extended or sustained release of active ingredients, for example as coating material. Downstream compounds may also be converted to consumer products such as diapers, carpet, paint, and adhesives.
Another important product, acrylamide, has been used in a number of industrial applications. Acrylamide may be produced from 3-HP, for example, without being limiting, via an esterification-amidation-dehydration sequence. Refluxing an alcohol solution of 3-HP in the presence of an acid or Lewis acid catalyst described herein would lead to a 3-HP ester. Treatment of the 3-HP ester with either an ammonia gas or an ammonium ion could yield 3-HP amide. Finally, dehydration of the 3-HP amide with dehydration reagents described elsewhere in this disclosure could produce acrylamide. The steps mentioned herein may be rearranged to produce the same final product acrylamide. Polymerization of acrylamide can be achieved, for example, and without being limiting, by radical polymerization. Polyacrylamide polymers have been widely used as additives for treating municipal drinking water and waste water. In addition, they have found applications in gel electrophoresis, oil-drilling, papermaking, ore processing, and the manufacture of permanent press fabrics.
VIII. Expression Systems General Concepts
The following general concepts are applicable to embodiments of the invention described above.
Multicopy Plasmids
The researcher is faced with a myriad of genetic module options when designing a plasmid for expression of a heterologous protein in a host cell. How to optimize an expression plasmid system often depends on the downstream use of the expressed protein.
Different cloning vectors or plasmids are maintained at different copy numbers, dependent on the replicon of the plasmid. Most general cloning plasmids can carry a DNA insert up to around 15 kb in size.
Multicopy plasmids can be used for the expression of recombinant genes in Escherichia coli. Examples of include multicopy plasmids include high-copy, medium-copy and low-copy plasmids (see FIG. 8). The high copy number is generally desired for maximum gene expression. However, the metabolic burden effects can result from multiple plasmid copies could prove to be detrimental for maximum productivity in certain metabolic engineering applications by adding significant metabolic burden to the system.
The low-copy plasmids for example, pBR322 is based on the original ColE1 replicon and thus has a copy number of 15-20. The pACYC series of plasmids are based on the p15A replicon, which has a copy number of 18-22, whereas pSC101 has even a lower copy number around 5, and BACs are maintained at one copy per cell. Such low copy plasmids may be useful in metabolic engineering applications, particularly when one or more of the substrates used in the recombinant pathway are required for normal cellular metabolism and can be toxic to the cell at high levels.
However, the used of high-copy plasmids may be useful in enhanced cellular metabolism contexts. The mutant ColE1 replicon, as found in the pUC series of plasmids produces a copy number of 500-700 as a result of a point mutation within the RNAII regulatory molecule.
There are transcription and translation vectors. Transcription vectors are utilized when the DNA to be cloned has an ATG start codon and a prokaryotic ribosome-binding site. Translation vectors contain an efficient ribosome-binding site and, therefore, it is not necessary for the target DNA to contain one. This is particularly useful in cases where the initial portion of the gene may be cleaved in an effort to improve solubility. Another consideration when choosing a transcription or translation vector is the source of the DNA to be expressed. Prokaryotic genes usually have a ribosome-binding site that is compatible with the host E. coli translation machinery, whereas eukaryotic genes do not. Normal prokaryotic gene expression may be enhanced by use of an engineered promoter and ribosome-binding site.
Promoters
A promoter is a region of DNA that initiates transcription of a particular gene. In bacteria, transcription is initiated by the promoter being recognized by RNA polymerase and an associated sigma factor, which are often brought to the promoter site by an activator protein's binding to its own DNA binding site located by the promoter.
Promoter selection is an important factor when designing an expression plasmid system. A promoter is located upstream of the ribosome-binding site. Owing to the fact that many heterologous protein products are toxic to the cell, the promoter can be regulated so that the heterologous protein is expressed at the appropriate amount and time to reduced the burden on the cell host.
Historically, the most commonly used promoters have been the lactose (lac) and tryptophan (trp) promoters. These two promoters were combined to create the hybrid promoters tac and trc that are also commonly used. Other common promoters are the phage lambda promoters, the phage T7 promoter (T7), and the alkaline phosphatase promoter (phoA).
Promoters can be constitutive and inducible. Constitutive promoter is active in all circumstances in the cell, while regulated or inducible promoter become active in response to specific stimuli. In addition the strength of the promoter can also differ. A strong promoter has a high frequency of transcription and generates the heterologous protein as 10-30% of the total cellular protein production (for examples see FIG. 8). Chemically-inducible promoters that can be used in various aspects of the invention include but are not limited to promoters whose transcriptional activity is regulated by the presence or absence of alcohol, tetracycline, steroids, metal and other compounds. Physically-inducible promoters that can be used in various aspects of the invention include but are not limited to including promoters whose transcriptional activity is regulated by the presence or absence of light and low or high temperatures.
In order to be an inducible promoter, the promoter should be initially be completely repressed to transcription and then transcription induced with the addition of an inducer to allow expression at the time that expression is desired in the host cell. Alternatively, an inducible promoter may be responsive to the lack of a substance, such as inorganic phosphate, such that the absence of inorganic phosphate will allow expression at the time that expression is desired in the host cell (for examples see FIG. 8).
Ribosome Binding Sites
A Ribosome Binding Sites (RBS) is an RNA sequence upstream of the start codon that affects the rate at which a particular gene or open reading frame (ORF) is translated. One can tailor an RBS site to a particular gene. Ribosome Binding Sites (RBSs) are typically short sequences, often less than 20 base pairs. Various aspects of RBS design are known to affect the rate at which the gene is translated in the cell. The RBS module can influences the translation rate of a gene largely by two known mechanisms. First, the rate at which ribosomes are recruited to the mRNA and initiate translation is dependent on the sequence of the RBS. Secondly, the sequence of the RBS can also affect the stability of the mRNA in the cell, which in turn affects the number of proteins. Through the use of genetic expression modules the expression of desired genes, such as genes encoding enzymes in the biosynthetic pathway for 3-HP, can be tailored activity either at the transcriptional and translational level.
One can access the registry RBS collection as a starting point for designing an RBS <<http://partsregistry.org/Ribosome_Binding_Sites/Catalog>>. The Registry has collections of RBSs that are recommended for general protein expression in E. coli and other prokaryotic hosts. In addition, each family of RBSs has multiple members covering a range of translation initiation rates. There are also several consensus RBS sequence for E. coli have been described. However, it is important to keep in mind the known RBS functions and mechanisms in a larger context. For example, in certain contexts the RBS can interact with upstream sequences, such as sequence that comprise the promoter or an upstream ORF. In other contexts, the RBS may interact with downstream sequences, for example the ribosome enzyme binds jointly to the RBS and start codon at about the same time. These potential interactions should be considered in the overall RBS sequence design. The degree of secondary structure near the RBS can affect the translation initiation rate. This fact can be used to produce regulated translation initiation rates.
The Shine-Dalgarno portion of the RBS is critical to the strength of the RBS. The Shine-Dalgarno is found at the end of the 16s rRNA and is the portion that binds with the mRNA and includes the sequence 5′-ACCUCC-3′. RBSs will commonly include a portion of the Shine-Dalgarno sequence. One of the ribosomal proteins, S1, is known to bind to adenine bases upstream from the Shine-Dalgarno sequence. As a result, the RBS can be made stronger by adding more adenines in the sequence upstream of the RBS.
When considering the design of the spacing between the RBS and the start codon, it is important to think of the aligned spacing rather than just the absolute spacing. While the Shine-Dalgarno portion of the RBS is critical to the strength of the RBS, the sequence upstream of the Shine-Dalgarno sequence is also important. Note that the promoter may add some bases onto the start of the mRNA that may affect the strength of the RBS by affecting S1 binding.
Computer programs that design RBS sequence to match protein coding sequences, desired upstream sequences including regulatory mRNA sequences, and account of secondary structure are known [Sans, Mirsky, and Voight, Nature Biotechnology 27: 946-950, 2009] and were used to optimize RBSs for the ACCase subunit genes as described in (see EXAMPLE 3).
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art 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.
EXAMPLES
Example 1
Salt Inhibition Studies in E. coli 
The activity of ACCase complex, a critical enzyme in the conversion of acetyl-CoA to malonyl-CoA, the immediate precursor for 3-HP, is severely inhibited by salt. Dose-dependent effects on ACCase activity was observed in the presence of NaCl, NH4Cl, Na-3-HP, or NH4-3-HP such that salt levels near 0.44M resulted in decreasing the activity of the ACCase enzyme by approximately 80%, while salts of 3-HP levels near 0.66M decreased the activity of the ACCase enzyme by approximately 80% relative to control (FIG. 4). Levels of greater than 0.66M (60 g/L) are expected to be present for commercially viable commercial production of 3-HP.
Example 2
ACCase from Halophilic Organism
Halophilic organisms, such as Halomonas elongata, are found in environments with high salt concentrations and, in general, have a salt internal concentration >2.5-3M. It is hypothesized that enzymes derived from any salt-tolerant species should be more resistant to enzyme inhibition by salts, such as 3-HP. Further, these enzymes that have greater salt tolerance should in turn have extended production under high salt conditions than enzymes with lower salt tolerance.
Accordingly, the genes encoding the accA, accB, accC, accD of H. elongata described in Table 1 were synthesized for expression in E. coli using codons optimized for this organism and supplied individually on pUC57 plasmids without promoters. Synthetic operons comprising the subunits were assembled using the Gibson assembly method.







TABLE 1







Accession numbers for genes encoding ACCase


subunits from Halomonas elongata











Gene
Accession number
SEQ ID NO.







accA
YP_003898857.1
SEQ ID NO. 1, 2



accB
YP_003897250.1
SEQ ID NO. 3, 4



accC
YP_003897249.1
SEQ ID NO. 5, 6



accD
YP_003897309.1
SEQ ID NO. 7, 8










Each gene was amplified by PCRs with Pfu Ultra II HS using the manufacturer's instructions, and the PCR products were purified using the Zymo PCR Cleanup kit. Concentrations of products were measured using the Nanodrop spectophotometer. The Gibson Assembly kit (NEB) was used to construct plasmids expressing the ACCase subunit genes as directed by the manufacturer. The effect of NH4-3-HP and NH4Cl on H. elongata ACCase was tested and compared to the E. coli ACCase. As shown in FIG. 4, whereas the E. coli ACCase is significantly inhibited by the salts, the ACCase from the halophile is less affected by either NH4-3-HP or by NH4Cl. This result indicated that use of the H. elongata ACCase in 3-HP production strains would in beneficial in relieving 3-HP inhibition of the conversion of acetyl-CoA to malonyl-CoA, a critical step in the pathway.

Example 3
RBS-Optimized Genes
Enzyme expression is regulated at transcriptional and translational levels in prokaryotes. Ribosome Binding Sites (RBS) are 15 nucleotide segments which are known to control the level of protein expression in microorganisms. To enhance H. elongata ACCase expression various customized RBS were constructed and optimized for E. coli translation expression. Table 2 shows the RBS sequences used to increase expression of the individual subunits.







TABLE 2







Table 2 RBS sequences used to enhance expression of


H. elongate ACCase subunits.











H. elongata






ACC












expression
Modified RBS sequences preceeding ATG (underlined)











plasmid
He_accD
He_accA
He_accC
He_accB





Parent 2-4
5′-
5′-
5′-
5′-



GCGTAGTAAAGGA
CAATTTATTTAAGGA
GAAATTTCATACC
GGAAGAACAAGGG



GGTAACATATG
GGACTCTTAAGATG
ACAGGCGAAGGAG
GTGTACATG



(SEQ ID NO: 110)
(SEQ ID NO: 111)
GAAAAACCATG
(SEQ ID NO: 113)





(SEQ ID NO: 112)



 


B2
Same as 2-4
Same as 2-4
Same as 2-4
5′-






ggaagaattaagg






gggacaaggggga






ataATG






(SEQ ID NO: 114)


 


13A
5′-
Same as 2-4
Same as 2-4




gcgtagtagccggg






tgataaggagccgt






aacATG






(SEQ ID NO: 115)





 


14C
5′-
Same as 2-4
Same as 2-4
Same as 2-4



gcgtagtagctgat






ataaaaggaggtaa






cggATG






(SEQ ID NO: 116)





 


15C
Same as 2-4
5′-
Same as 2-4
Same as 2-4




caatttatttttgtt






cacccaaggagtatt






gctaATG






(SEQ ID NO: 117)




 


17C
Same as 2-4
5′-
Same as 2-4
Same as 2-4




caatttatttaccga






aataaaaggagggat






gcgaATG






(SEQ ID NO: 118)




 


35C
5′-
5′-
Same as 2-4
Same as 2-4



gcgtagtagccggg
caatttatttttgtt





tgataaggagccgt
cacccaaggagtatt





aacATG
gctaATG





(SEQ ID NO: 115)
(SEQ ID NO: 117)




 


36C
5′-
5′-
Same as 2-4
Same as 2-4



gcgtagtagccggg
caatttatttaccga





tgataaggagccgt
aataaaaggagggat





aacATG
gcgaATG





(SEQ ID NO: 115)
(SEQ ID NO: 118)




 


36C-8
5′-
5′-
Same as 2-4
5′-



gcgtagtagccggg
caatttatttaccga

ggaagaattaagg



tgataaggagccgt
aataaaaggagggat

gggacaaggggga



aacATG
gcgaATG

ataATG



(SEQ ID NO: 115)
(SEQ ID NO: 118)

(SEQ ID NO: 114)


 


72B
5′-
5′-
5′-
5′-



gcgtagtagccggg
caatttatttaccga
TCTTCCCACAACA
GAAATTTCATACC



tgataaggagccgt
aataaaaggagggat
CTGGCGGACTCCA
ACAGGCGAAGGAG



aacATG
gcgaATG
TCATG
GAAAAACCATG



(SEQ ID NO: 115)
(SEQ ID NO: 118)
(SEQ ID NO: 119)
(SEQ ID NO: 112)


 


105F
5′-
5′-
5′-
5′-



gcgtagtagccggg
caatttatttttgtt
TCTTCCCACAACA
GAAATTTCATACC



tgataaggagccgt
cacccaaggagtatt
CTGGCGGACTCCA
ACAGGCGAAGGAG



aacATG
gctaATG
TCATG
GAAAAACCATG



(SEQ ID NO: 115)
(SEQ ID NO: 117)
(SEQ ID NO: 119)
(SEQ ID NO: 112)









The expression performance of the RBS-optimized H. elongata ACCases was evaluated by 3-HP production in a 96-well format, each in triplicate wells, and the averaged results shown in Table 3. Specific 3HP production is shown as g/L per OD600. As may be seen in Table 3, enhancing the efficiency of the RBS in strains B2, 35C, and 72 B clearly resulted in increased malonyl-CoA production leading to increased 3-HP production. It is evident from these results that combinations of enhanced RBS's before each of the individual genes accA, accB, accC, and accD may result in strains with even higher ACCase expression and activity.







TABLE 3







Improvement in 3-HP production by RBS-optimized


expression of H. elongata ACCase subunits.










H. elongata ACCase
3HP



expression plasmid
(g/I.OD)







Parent 2-4
0.06



B2
0.81



13A
0.01



14C
0.54



15C
0.14



17C
0.08



35C
0.68



36C
0.31



36C-8
0.31



72B
0.57



105F
0.19










Example 4
Coordinated Expression by Subunit Fusions
In nature ACCase subunit genes from prokaryotes such as E. coli and H. elongata have been shown to have a quaternary structure: accA2:accD2:accB4:accC2. However, the intrinsic levels of the ACCase subunit genes are too low for optimal production. Therefore, for optimal production it is ideal to have overexpression to be coordinated in a similar manner.
Expression of the genes encoding each ACCase subunit is regulated at transcriptional and translational levels. Coordinated overexpression of the ACCase subunit genes, accA, accB, accC, accD should give better ACCase activity. Examples of fusions of accC-B proteins exist in bacteria. It is hypothesized that coordinated overexpression may be achieved by fusion of subunit genes should ensures equimolar expression of the subunit genes at the optimal time.
The following ACCase subunit gene fusion were constructed and the constructs overexpressed in E. coli: (A) Control ABCD, (B) fusion of accC-B (SEQ ID NO. s 9, 10) subunit genes as seen in bacteria, (C) fusion of accD-A subunit genes using a flexible 15-amino acid linker (Linker sequence LSGGGGSGGGGSGGGGSGGGGSAAA (SEQ ID NO. 120); SEQ ID NO. s 11, 12) as depicted in FIG. 5.
The performance of the ACC fusions were tested for their ACCase activity and for various 3-HP production metrics in Table 4. ACCase activity was determined in cell lysates using an assay for malonyl-CoA production as described in [Kroeger, Zarzycki, and Fuchs, Analytical Biochem. 411:100-105, 2011]. Production of 3-HP was determined in cells co-transformed with a plasmid bearing the genes encoding the malonyl-CoA reductase from S. tokadaii and E. coli ydfG providing a 3-HP dehydrogenase to complete the metabolic pathway from malonyl-CoA to 3HP. These results show that the strain with the fused accDA genes had higher average specific productivity of 3-HP compared to the parental strain in which the overexpressed ACCase is not fused. FIG. 6 shows that the benefit of the accDA fusion were also manifested in 3-HP production in fermentors with environmental controls of nutrient feed, pH, aeration, and temperature.







TABLE 4







Table 4: ACC Fusions and ACCase activity













Avg
Avg
ACCase




specific
specific
specific




prodn rate
prodn rate
activity


Strain

(g/gDCW · h)
(g/gDCW · hr)
at TS + 6


designation
Plasmid
at TS + 6
at TS + 20
(U/mg)





BX3_783
Parent
0.160
0.146
0.057



(unfused



ACCase)


BX3_829
No ACC
0.069
0.062
0.000


BX3_837
EC ACC DA
0.209
0.201
0.054



fusion









Example 5
3-HP Exporter
Growth inhibition has been demonstrated for E. coli strains grown in the presence of 3-HP at levels as low as 20 g/L. To produce high titers of 3-HP the production host is required to balance production with overcoming inhibition. A known chemical exporter from E. coli that has been previously characterized for homoserine transport, rhtA, was evaluated for increased production of 3-HP. A mutant version of the exporter, rhtA(P2S) (SEQ ID NO. 30 nucleic acid, SEQ ID NO. 31 protein) was synthesized behind the PtpiA promoter and inserted into the pTRC-PyibD-MCR plasmid behind a terminator using the Gibson Assembly kit (NEB) according to manufacturer's instructions. The effects of overexpression of rhtA were evaluated in 1 L fermentation compared to the control plasmid without rhtA. As shown in FIG. 7, overexpression of rhtA resulted in a significant improvement in 3HP titer compared to the control production strain. Construction of plasmids expressing another putative transporter, ydcO (SEQ ID NO. 32 nucleic acid, SEQ ID NO. 33 protein) is carried out in the same manner.
Example 6
Bicarbonate Importer (Prophetic)
Increased import of bicarbonate to increase availability of bicarbonate for the ACCase reaction will increase production of malonyl-CoA and hence products derived metabolically from malonyl-CoA, such as 3-HP. The gene encoding the bicA bicarbonate transporter (SEQ ID NO. 13) of Synechococcus sp. was synthesized using codons optimized for expression in E. coli (SEQ ID NO. 14) and expressed using the E. coli tal promoter in a strain cotransformed with plasmids encoding ACCase and MCR functions. Production of 3-HP by this strain is compared to that achieved by a control strain without overexpressed bicA.








SEQUENCE LISTING



SEQ ID NO. 1: ACCA PROTEIN, HALOMONAS ELONGATA


MNPNYLDFEQPIAELQAKIEELRMVGNDSQVNLSDEIGRLEEKSRKLTES


 


IFKDLSAWQVSQLSRHPQRPYTLDYLEHVFTDFDELHGDRRFADDAAIVG


 


GVARLDDKPVMVIGHQKGRDVHEKVRRNFGMPRPEGYRKACRLMEMAERF


 


HMPVLTFIDTPGAYPGIDAEERGQSEAIAYNLGVMSRLKTPIISTVVGEG


 


GSGGALAIGVCDELAMLQYSTYSVISPEGCASILWKSADKASEAAQAMGI


 


TAERLKELGFVDTLIPEPLGGAHRQPSATAERIKTALLESLDRLETMETD


 


ALLERRYERLMSYGAPV


 


SEQ ID NO. 2: ACCA NUCLEIC ACID, HALOMONAS ELONGATA


(SYNTHETIC, CODON OPTIMIZE FOR E. COLI EXPRESSION)


ATGAATCCGAACTATCTGGACTTTGAACAACCGATCGCTGAACTGCAAGCCAAAATCGAA


 


GAACTGCGTATGGTGGGCAACGACTCACAGGTGAACCTGTCTGATGAAATTGGCCGTCTG


 


GAAGAAAAAAGTCGCAAACTGACCGAATCCATCTTTAAAGACCTGTCAGCGTGGCAAGTT


 


AGCCAACTGTCTCGTCATCCGCAACGCCCGTATACCCTGGATTACCTGGAACATGTCTTT


 


ACGGATTTCGACGAACTGCACGGTGACCGTCGCTTTGCAGATGACGCGGCCATTGTTGGC


 


GGTGTCGCTCGTCTGGATGACAAACCGGTCATGGTGATCGGCCATCAGAAAGGTCGTGAT


 


GTGCACGAAAAAGTTCGTCGCAACTTCGGCATGCCGCGCCCGGAAGGTTATCGTAAAGCG


 


TGCCGCCTGATGGAAATGGCCGAACGCTTTCACATGCCGGTGCTGACCTTCATTGATACG


 


CCGGGCGCATATCCGGGTATCGACGCTGAAGAACGTGGCCAAAGCGAAGCGATTGCCTAC


 


AATCTGGGTGTTATGTCGCGCCTGAAAACCCCGATTATCAGCACGGTGGTTGGCGAAGGC


 


GGTTCTGGCGGTGCACTGGCTATCGGTGTCTGCGATGAACTGGCGATGCTGCAATATAGT


 


ACCTACTCCGTGATTTCACCGGAAGGCTGTGCCTCGATCCTGTGGAAAAGCGCAGATAAA


 


GCTTCTGAAGCAGCTCAAGCGATGGGCATTACCGCCGAACGTCTGAAAGAACTGGGTTTC


 


GTTGACACGCTGATCCCGGAACCGCTGGGCGGTGCACATCGTCAGCCGAGTGCGACCGCC


 


GAACGCATTAAAACGGCCCTGCTGGAAAGCCTGGATCGCCTGGAAACGATGGAAACGGAT


 


GCCCTGCTGGAACGCCGCTATGAACGCCTGATGTCTTACGGTGCCCCGGTCTGA


 


SEQ ID NO. 3: ACCB PROTEIN, HALOMONAS ELONGATA


MDIRKVKKLIELLEESNISEIEIQEGEESVRISRHPNGTEHPQPAAPAWP


 


ATAAAPAPQPAAAPVESPAEVDEGPAYQGQPIVSPMVGTFYRAPAPGAKA


 


FVELGQSVKKGETVCIVEAMKMMNQIEADRDGVVEAILVEDGEPVEFEQP


 


MVVIS


 


SEQ ID NO. 4: ACCB NUCLEIC ACID HALOMONAS ELONGATA


(SYNTHETIC, CODON OPTIMIZE FOR E. COLI EXPRESSION)


ATGGACATCCGCAAAGTGAAAAAACTGATCGAACTGCTGGAAGAAAGTAACATCTCTGAA


 


ATTGAAATCCAAGAAGGCGAAGAAAGCGTGCGTATTAGTCGCCATCCGAACGGCACCGAA


 


CACCCGCAGCCGGCGGCACCGGCATGGCCGGCCACGGCAGCTGCGCCGGCGCCGCAACCG


 


GCCGCAGCTCCGGTGGAAAGCCCGGCAGAAGTTGATGAAGGCCCGGCTTATCAGGGTCAA


 


CCGATCGTTTCTCCGATGGTCGGCACCTTTTACCGTGCGCCGGCACCGGGTGCAAAAGCT


 


TTCGTCGAACTGGGCCAGAGCGTTAAAAAAGGTGAAACGGTCTGCATTGTGGAAGCCATG


 


AAAATGATGAATCAAATCGAAGCCGATCGCGACGGTGTGGTTGAAGCAATCCTGGTGGAA


 


GATGGTGAACCGGTGGAATTTGAACAGCCGATGGTGGTGATTAGTTAA


 


SEQ ID NO. 5: ACCC PROTEIN, HALOMONAS ELONGATA


MLDKVLIANRGEIALRILRACKELGIRTVAVHSKADRELMHVRLADEAVC


 


IGPASSAQSYLNIPALISAAEVTDTSAIHPGYGFLSENADFAEQVERSGF


 


TFIGPSAETIRLMGDKVSAINAMKEAGVPTVPGSNGPLGDDEGEILATAR


 


RIGYPVIIKAAAGGGGRGMRVVHAEGHLLSAVNVTRTEAHSSFGDGTVYM


 


EKFLENPRHVEVQVLADGQGNAIHLYDRDCSLQRRHQKVLEEAPAPGLDQ


 


QAREQVFKACRDACVKIGYRGAGTFEFLYENGEFFFIEMNTRVQVEHPVT


 


EMVTGVDIVREQLRIASGLPLSIRQEDVELSGHAFECRINAEDSRTFMPS


 


PGRVTLYHPPGGLGVRMDSHVYTGYTVPPHYDSLIGKLITWGDDRETALI


 


RMRNALDELLVEGIKTNTDLHKDLVRDGYFQQGGVNIHYLEKKLGL


 


SEQ ID NO. 6: ACCC NUCLEIC ACID, HALOMONAS ELONGATA


(SYNTHETIC, CODON OPTIMIZE FOR E. COLI EXPRESSION)


ATGCTGGACAAAGTGCTGATTGCGAATCGTGGCGAAATTGCGCTGCGTATCCTGCGTGCG


 


TGTAAAGAACTGGGTATCCGTACCGTCGCTGTTCATTCAAAAGCGGACCGTGAACTGATG


 


CACGTCCGCCTGGCCGATGAAGCAGTGTGCATTGGTCCGGCTAGCTCTGCGCAGTCGTAT


 


CTGAACATTCCGGCACTGATCAGTGCGGCCGAAGTGACCGACACGTCCGCGATCCATCCG


 


GGCTACGGTTTCCTGAGCGAAAATGCCGATTTTGCAGAACAAGTCGAACGTTCAGGTTTC


 


ACCTTTATTGGCCCGTCGGCCGAAACGATCCGCCTGATGGGTGATAAAGTTAGTGCTATT


 


AACGCGATGAAAGAAGCAGGCGTGCCGACCGTTCCGGGTTCCAATGGTCCGCTGGGTGAT


 


GACGAAGGTGAAATTCTGGCCACCGCACGTCGCATCGGCTATCCGGTTATTATCAAAGCA


 


GCTGCGGGCGGTGGCGGTCGTGGTATGCGTGTGGTTCATGCTGAAGGCCACCTGCTGAGC


 


GCGGTCAACGTGACCCGTACGGAAGCGCATAGTTCCTTCGGCGATGGCACCGTTTATATG


 


GAAAAATTTCTGGAAAACCCGCGTCACGTTGAAGTCCAGGTGCTGGCCGATGGCCAGGGT


 


AATGCAATTCATCTGTACGATCGCGACTGCTCTCTGCAACGTCGCCACCAAAAAGTGCTG


 


GAAGAAGCTCCGGCACCGGGTCTGGACCAGCAAGCACGTGAACAGGTTTTTAAAGCCTGC


 


CGCGATGCATGTGTCAAAATTGGTTATCGTGGCGCGGGCACCTTCGAATTTCTGTACGAA


 


AACGGCGAATTTTTCTTTATCGAAATGAATACGCGCGTTCAGGTCGAACATCCGGTGACC


 


GAAATGGTCACGGGTGTGGATATTGTTCGTGAACAGCTGCGTATCGCATCAGGTCTGCCG


 


CTGTCGATTCGCCAAGAAGACGTTGAACTGAGCGGTCATGCCTTCGAATGTCGTATCAAT


 


GCAGAAGATAGCCGCACCTTTATGCCGTCTCCGGGTCGTGTCACGCTGTATCACCCGCCG


 


GGCGGTCTGGGTGTCCGTATGGACAGCCATGTGTATACCGGCTACACGGTTCCGCCGCAC


 


TACGATTCTCTGATTGGTAAACTGATCACCTGGGGCGATGACCGTGAAACGGCTCTGATT


 


CGTATGCGCAACGCCCTGGATGAACTGCTGGTTGAAGGCATCAAAACCAATACGGATCTG


 


CACAAAGACCTGGTTCGCGATGGCTACTTTCAGCAAGGCGGTGTCAACATTCACTACCTG


 


GAAAAAAAACTGGGTCTGTAA


 


SEQ ID NO. 7: ACCD PROTEIN, HALOMONAS ELONGATA


MSWLDKIVPSVGRIQRKERRTSVPDGLWRKCPKCESVLYLPELEKHHNVC


 


PKCDHHLRLTARKRLDWFLDKEGREEIAADLEPVDRLKFRDSKKYKDRLS


 


AAQKATGEKDGLVAMRGTLEGLPVVAVAFEFTFMGGSMGAVVGEKFVRAA


 


TQALDEGVPLVCFSASGGARMQEALFSLMQMAKTSAALEKLKQAGVPYIS


 


VLTDPVFGGVSASLAMLGDLNIAEPNALIGFAGPRVIEQTVREQLPEGFQ


 


RSEFLLEHGAVDMIVHRQQIRERLGGVLRKLTHQPASGPAVVENDEPDLV


 


DAAEQAEPQPEAPEAVETSESEAPTEKGVEADSEETDESPRSGDNR


 


SEQ ID NO. 8: ACCD NUCLEIC ACID, HALOMONAS ELONGATA


(SYNTHETIC, CODON OPTIMIZE FOR E. COLI EXPRESSION)


ATGTCTTGGTTAGATAAAATCGTCCCGTCAGTGGGTCGCATTCAACGCAAAGAACGTCGC


 


ACCTCGGTCCCGGATGGTCTGTGGCGTAAATGCCCGAAATGTGAATCAGTTCTGTATCTG


 


CCGGAACTGGAAAAACATCACAACGTCTGCCCGAAATGTGATCATCACCTGCGTCTGACC


 


GCGCGTAAACGCCTGGACTGGTTCCTGGATAAAGAAGGCCGCGAAGAAATTGCGGCCGAC


 


CTGGAACCGGTGGATCGTCTGAAATTTCGCGACAGCAAAAAATACAAAGATCGTCTGAGC


 


GCGGCGCAGAAAGCAACCGGTGAAAAAGACGGTCTGGTGGCCATGCGTGGCACGCTGGAA


 


GGTCTGCCGGTGGTTGCAGTTGCTTTTGAATTTACCTTTATGGGCGGTAGCATGGGCGCA


 


GTCGTGGGTGAAAAATTCGTTCGTGCGGCCACGCAGGCTCTGGATGAAGGTGTGCCGCTG


 


GTTTGCTTCAGCGCATCTGGCGGTGCCCGCATGCAGGAAGCACTGTTTAGTCTGATGCAA


 


ATGGCTAAAACCTCCGCAGCTCTGGAAAAACTGAAACAGGCGGGCGTGCCGTATATTTCT


 


GTTCTGACGGACCCGGTCTTCGGCGGTGTGAGTGCGTCCCTGGCCATGCTGGGTGATCTG


 


AACATTGCAGAACCGAATGCTCTGATCGGCTTTGCGGGTCCGCGTGTCATCGAACAGACC


 


GTGCGCGAACAACTGCCGGAAGGCTTCCAGCGTTCAGAATTTCTGCTGGAACATGGTGCC


 


GTTGATATGATTGTCCACCGTCAGCAAATCCGTGAACGCCTGGGCGGTGTGCTGCGCAAA


 


CTGACGCATCAACCGGCATCGGGTCCGGCCGTTGTCGAAAATGATGAACCGGACCTGGTC


 


GATGCGGCCGAACAGGCAGAACCGCAACCGGAAGCACCGGAAGCTGTTGAAACCTCAGAA


 


TCGGAAGCACCGACGGAAAAAGGCGTGGAAGCAGACTCGGAAGAAACGGATGAATCACCG


 


CGCTCAGGCGACAACCGCTAA


 


SEQ ID NO. 9: FUSION ACCCB PROTEIN, E. COLI


MLDKIVIANRGEIALRILRACKELGIKTVAVHSSADRDLKHVLLADETVC


 


IGPAPSVKSYLNIPAIISAAEITGAVAIHPGYGFLSENANFAEQVERSGF


 


IFIGPKAETIRLMGDKVSAIAAMKKAGVPCVPGSDGPLGDDMDKNRAIAK


 


RIGYPVIIKASGGGGGRGMRVVRGDAELAQSISMTRAEAKAAFSNDMVYM


 


EKYLENPRHVEIQVLADGQGNAIYLAERDCSMQRRHQKVVEEAPAPGITP


 


ELRRYIGERCAKACVDIGYRGAGTFEFLFENGEFYFIEMNTRIQVEHPVT


 


EMITGVDLIKEQLRIAAGQPLSIKQEEVHVRGHAVECRINAEDPNTFLPS


 


PGKITRFHAPGGFGVRWESHIYAGYTVPPYYDSMIGKLICYGENRDVAIA


 


RMKNALQELIIDGIKTNVDLQIRIMNDENFQHGGTNIHYLEKKLGLQEKD


 


IRKIKKLIELVEESGISELEISEGEESVRISRAAPAASFPVMQQAYAAPM


 


MQQPAQSNAAAPATVPSMEAPAAAEISGHIVRSPMVGTFYRTPSPDAKAF


 


IEVGQKVNVGDTLCIVEAMKMMNQIEADKSGTVKAILVESGQPVEFDEPL


 


VVIE


 


SEQ ID NO. 10: FUSION ACCCB NUCLEIC ACID, E. COLI


ATGCTGGACAAGATCGTCATCGCCAACCGCGGCGAAATCGCCCTGCGCATCTTGCGCGCG


 


TGTAAAGAGCTGGGCATTAAGACTGTTGCCGTGCATTCCAGCGCAGACCGCGACCTGAAG


 


CATGTTCTGCTGGCCGACGAAACGGTTTGCATCGGTCCGGCACCGAGCGTGAAAAGCTAT


 


CTGAACATCCCGGCCATCATCTCTGCGGCAGAGATCACCGGTGCAGTGGCGATTCATCCG


 


GGCTACGGTTTCCTGAGCGAGAACGCTAACTTTGCTGAACAAGTGGAGCGTAGCGGTTTC


 


ATCTTCATTGGCCCTAAGGCGGAGACGATTCGCCTGATGGGCGACAAAGTGAGCGCCATT


 


GCAGCGATGAAAAAGGCCGGTGTGCCGTGTGTTCCGGGCAGCGATGGTCCGCTGGGTGAC


 


GATATGGACAAGAACCGTGCCATCGCTAAACGTATTGGCTACCCGGTCATTATCAAAGCC


 


TCTGGTGGTGGCGGTGGCCGTGGTATGCGTGTCGTCCGTGGTGATGCGGAACTGGCGCAA


 


AGCATCAGCATGACCCGTGCGGAAGCCAAAGCGGCGTTCTCTAACGATATGGTGTATATG


 


GAGAAGTATCTGGAGAATCCGCGCCACGTTGAAATCCAAGTTCTGGCGGATGGTCAGGGC


 


AATGCGATCTACTTGGCAGAACGTGATTGCTCCATGCAACGCCGTCATCAGAAGGTGGTG


 


GAAGAGGCACCGGCTCCGGGTATTACGCCGGAACTGCGTCGCTACATCGGTGAGCGCTGT


 


GCGAAAGCGTGTGTGGACATTGGTTACCGTGGTGCGGGTACGTTTGAGTTCCTGTTCGAA


 


AATGGTGAGTTTTACTTCATTGAAATGAATACCCGCATCCAGGTTGAGCACCCGGTGACC


 


GAGATGATTACTGGCGTTGATCTGATCAAAGAGCAACTGCGCATTGCGGCTGGTCAGCCG


 


CTGTCGATCAAGCAAGAAGAGGTGCACGTTCGTGGTCACGCGGTCGAGTGCCGTATCAAT


 


GCGGAGGACCCGAATACCTTTCTGCCGAGCCCTGGTAAGATCACTCGTTTTCACGCGCCA


 


GGTGGTTTTGGCGTTCGTTGGGAGTCTCACATCTACGCCGGTTACACCGTGCCGCCGTAC


 


TATGACAGCATGATTGGTAAACTGATCTGCTATGGCGAAAATCGTGATGTCGCGATCGCC


 


CGCATGAAAAACGCGCTGCAAGAGCTGATCATTGATGGCATTAAGACCAATGTGGATTTG


 


CAGATCCGCATTATGAACGACGAGAATTTCCAGCACGGCGGTACGAACATTCACTACCTG


 


GAAAAGAAACTGGGCCTGCAAGAGAAAGACATCCGCAAGATCAAGAAGCTGATCGAACTG


 


GTGGAAGAGTCTGGCATCAGCGAGCTGGAGATCAGCGAAGGTGAAGAGAGCGTCCGTATT


 


TCCCGTGCGGCACCGGCAGCGAGCTTTCCGGTTATGCAGCAAGCATACGCCGCTCCGATG


 


ATGCAACAGCCGGCACAGAGCAACGCCGCTGCACCGGCGACCGTTCCAAGCATGGAGGCA


 


CCGGCAGCGGCCGAGATTTCGGGTCATATCGTGCGTAGCCCGATGGTGGGCACCTTCTAT


 


CGCACGCCGTCGCCGGACGCAAAAGCCTTCATCGAAGTCGGCCAGAAGGTCAATGTCGGC


 


GACACGCTGTGTATCGTTGAGGCAATGAAAATGATGAACCAGATTGAAGCGGATAAGAGC


 


GGTACTGTTAAAGCGATCCTGGTGGAATCCGGCCAGCCTGTTGAGTTCGATGAACCGCTG


 


GTTGTGATCGAGTAA


 


SEQ ID NO. 11: FUSION ACCDA PROTEIN, E. COLI


MSWIERIKSNITPTRKASIPEGVWTKCDSCGQVLYRAELERNLEVCPKCD


 


HHMRMTARNRLHSLLDEGSLVELGSELEPKDVLKFRDSKKYKDRLASAQK


 


ETGEKDALVVMKGTLYGMPVVAAAFEFAFMGGSMGSVVGARFVRAVEQAL


 


EDNCPLICFSASGGARMQEALMSLMQMAKTSAALAKMQERGLPYISVLTD


 


PTMGGVSASFAMLGDLNIAEPKALIGFAGPRVIEQTVREKLPPGFQRSEF


 


LIEKGAIDMIVRRPEMRLKLASILAKLMNLPAPNPEAPREGVVVPPVPDQ


 


EPEALSGGGGSGGGGSGGGGSGGGGSAAASLNFLDFEQPIAELEAKIDSL


 


TAVSRQDEKLDINIDEEVHRLREKSVELTRKIFADLGAWQIAQLARHPQR


 


PYTLDYVRLAFDEFDELAGDRAYADDKAIVGGIARLDGRPVMIIGHQKGR


 


ETKEKIRRNFGMPAPEGYRKALRLMQMAERFKMPIITFIDTPGAYPGVGA


 


EERGQSEAIARNLREMSRLGVPVVCTVIGEGGSGGALAIGVGDKVNMLQY


 


STYSVISPEGCASILWKSADKAPLAAEAMGIIAPRLKELKLIDSIIPEPL


 


GGAHRNPEAMAASLKAQLLADLADLDVLSTEDLKNRRYQRLMSYGYA


 


SEQ ID NO. 12: FUSION ACCDA NUCLEIC ACID; E. COLI


ATGAGCTGGATCGAGCGCATCAAGAGCAACATCACCCCGACCCGCAAGGCGAGCATCCCT


 


GAAGGCGTCTGGACCAAATGCGATAGCTGCGGTCAGGTTTTGTATCGTGCGGAGCTGGAG


 


CGTAACCTGGAAGTGTGCCCGAAATGCGACCATCACATGCGTATGACCGCTCGTAATCGT


 


CTGCATAGCCTGCTGGATGAGGGCAGCCTGGTCGAGCTGGGTAGCGAACTGGAACCGAAA


 


GATGTTCTGAAATTCCGTGATTCCAAGAAGTATAAGGATCGTTTGGCATCTGCACAAAAA


 


GAAACCGGTGAGAAGGACGCACTGGTTGTTATGAAAGGCACCCTGTATGGTATGCCGGTT


 


GTTGCTGCGGCGTTCGAGTTTGCGTTTATGGGTGGCAGCATGGGTTCCGTGGTGGGCGCA


 


CGCTTTGTGCGTGCCGTGGAGCAGGCGCTGGAGGATAACTGTCCTCTGATTTGTTTCAGC


 


GCGAGCGGTGGTGCGCGTATGCAAGAGGCCCTGATGAGCCTGATGCAGATGGCAAAAACC


 


TCGGCAGCCCTGGCGAAGATGCAAGAACGCGGCCTGCCGTACATTTCCGTCCTGACCGAC


 


CCTACGATGGGCGGTGTCAGCGCCAGCTTTGCGATGCTGGGTGATTTGAACATCGCAGAG


 


CCGAAGGCTCTGATTGGTTTTGCTGGTCCGCGTGTTATTGAACAGACGGTTCGCGAAAAG


 


TTGCCGCCTGGTTTCCAGCGCAGCGAGTTCCTGATTGAGAAAGGTGCCATCGACATGATC


 


GTTCGCCGTCCAGAAATGCGTCTGAAACTGGCGAGCATTCTGGCGAAATTGATGAATCTG


 


CCGGCTCCGAATCCTGAAGCACCGCGTGAGGGTGTCGTGGTTCCGCCGGTCCCGGACCAA


 


GAGCCGGAGGCTCTGAGCGGCGGAGGTGGCTCTGGTGGAGGCGGTTCAGGAGGCGGTGGC


 


AGTGGTGGCGGCGGATCTGCGGCAGCTTCTCTGAACTTCCTGGACTTCGAGCAGCCGATC


 


GCCGAACTGGAGGCGAAGATTGACAGCCTGACCGCGGTTAGCCGTCAAGATGAGAAACTG


 


GACATTAACATCGACGAAGAGGTCCACCGTTTGCGTGAGAAGTCTGTTGAACTGACTCGC


 


AAAATCTTTGCTGATTTGGGCGCATGGCAGATTGCCCAGTTGGCTCGCCACCCACAACGC


 


CCATATACCCTGGACTACGTGCGCCTGGCGTTTGACGAGTTCGACGAACTGGCAGGCGAC


 


CGCGCCTATGCGGACGATAAAGCAATTGTCGGCGGTATTGCTCGTTTGGATGGCCGTCCG


 


GTGATGATTATCGGCCATCAAAAAGGCCGCGAGACAAAGGAAAAGATTCGTCGTAATTTC


 


GGAATGCCGGCACCGGAGGGCTACCGCAAGGCCCTGCGTCTGATGCAAATGGCCGAACGC


 


TTTAAGATGCCGATTATCACGTTCATTGATACGCCTGGTGCGTACCCAGGCGTTGGTGCG


 


GAAGAGCGTGGTCAGAGCGAGGCCATCGCACGTAACCTGCGTGAGATGTCTCGTCTGGGT


 


GTGCCGGTCGTTTGCACCGTGATTGGCGAGGGCGGTAGCGGTGGTGCGTTGGCGATCGGT


 


GTCGGTGATAAGGTCAACATGCTGCAATACAGCACGTACAGCGTCATTAGCCCGGAAGGT


 


TGCGCTTCCATTCTGTGGAAGAGCGCGGATAAAGCACCATTGGCAGCGGAAGCGATGGGT


 


ATCATCGCACCGCGTCTGAAAGAACTGAAGTTGATTGATTCTATCATCCCGGAACCGCTG


 


GGCGGTGCTCACCGTAATCCGGAGGCGATGGCAGCCAGCCTGAAGGCCCAGCTGCTGGCG


 


GACCTGGCGGATCTGGACGTGCTGAGCACGGAGGATCTGAAAAACCGTCGCTATCAGCGC


 


TTGATGAGCTATGGCTACGCTTAA


 


SEQ ID NO. 13: BICA PROTEIN, SYNECHOCOCCUS SP.


MQITNKIHFRNIRGDIFGGLTAAVIALPMALAFGVASGAGAEAGLWGAVL


 


VGFFAALFGGTPTLISEPTGPMTVVMTAVIAHFTASAATPEEGLAIAFTV


 


VMMAGVFQIIFGSLKLGKYVTMMPYTVISGFMSGIGIILVILQLAPFLGQ


 


ASPGGGVIGTLQNLPTLLSNIQPGETALALGTVAIIWFMPEKFKKVIPPQ


 


LVALVLGTVIAFFVFPPEVSDLRRIGEIRAGFPELVRPSFSPVEFQRMIL


 


DAAVLGMLGCIDALLTSVVADSLTRTEHNSNKELIGQGLGNLFSGLFGGI


 


AGAGATMGTVVNIQSGGRTALSGLVRAFVLLVVILGAASLTATIPLAVLA


 


GIAFKVGVDIIDWSFLKRAHEISPKGALIMYGVILLTVLVDLIVAVGVGV


 


FVANVLTIERMSNLQSEKVQTVSDADDNIRLTTTEKRWLDEGQGRVLLFQ


 


LSGPMIFGVAKAIAREHNAMGDCDALVFDIGEVPHMGVTASLALENAIEE


 


ALDKERQVYIVGAAGQTRRRLEKLKLFKRVPPDKCLMSREEALKNAVLGI


 


YPHLADGVTAPSSEMG


 


SEQ ID NO. 14: BICA NUCLEIC ACID, SYNECHOCOCCUS SP.


ATGCAGATTACCAACAAGATCCATTTCCGTAACATTCGTGGCGACATTTTTGGTGGCCTG


 


ACCGCTGCTGTGATTGCGCTGCCGATGGCACTGGCTTTTGGTGTGGCAAGTGGTGCAGGT


 


GCAGAAGCAGGTCTGTGGGGTGCAGTTCTGGTGGGCTTTTTCGCAGCACTGTTCGGTGGT


 


ACGCCGACCCTGATTTCAGAACCGACGGGCCCGATGACCGTGGTTATGACGGCCGTGATC


 


GCACATTTTACCGCATCGGCAGCTACGCCGGAAGAAGGCCTGGCTATTGCGTTCACCGTC


 


GTGATGATGGCCGGTGTTTTTCAGATTATCTTCGGCAGCCTGAAACTGGGCAAGTATGTT


 


ACCATGATGCCGTACACGGTCATCAGTGGTTTTATGTCCGGTATTGGCATTATCCTGGTG


 


ATCCTGCAGCTGGCACCGTTCCTGGGTCAAGCCAGTCCGGGCGGTGGCGTTATTGGCACC


 


CTGCAGAACCTGCCGACGCTGCTGTCCAATATCCAACCGGGTGAAACCGCCCTGGCACTG


 


GGTACGGTCGCGATTATCTGGTTCATGCCGGAAAAGTTCAAGAAGGTTATCCCGCCGCAG


 


CTGGTTGCGCTGGTTCTGGGCACCGTCATCGCGTTTTTCGTGTTTCCGCCGGAAGTTAGC


 


GATCTGCGTCGCATTGGCGAAATCCGTGCAGGTTTCCCGGAACTGGTGCGTCCGAGCTTT


 


TCTCCGGTTGAATTTCAGCGCATGATTCTGGATGCGGCCGTGCTGGGCATGCTGGGTTGC


 


ATCGATGCGCTGCTGACCAGCGTTGTCGCCGACTCTCTGACGCGTACCGAACATAACAGC


 


AATAAAGAACTGATTGGTCAGGGCCTGGGTAACCTGTTTTCTGGCCTGTTCGGTGGTATT


 


GCTGGTGCAGGTGCAACGATGGGCACCGTGGTTAATATCCAAAGTGGTGGCCGTACCGCA


 


CTGTCCGGTCTGGTGCGTGCTTTTGTTCTGCTGGTCGTGATTCTGGGTGCAGCTTCTCTG


 


ACGGCAACCATTCCGCTGGCTGTGCTGGCAGGCATCGCCTTTAAAGTGGGTGTTGATATT


 


ATCGACTGGTCATTCCTGAAACGCGCCCACGAAATCTCGCCGAAGGGCGCACTGATTATG


 


TATGGTGTGATCCTGCTGACCGTCCTGGTGGATCTGATTGTTGCGGTCGGCGTGGGTGTT


 


TTTGTCGCCAACGTTCTGACCATCGAACGTATGTCAAATCTGCAGTCGGAAAAAGTCCAA


 


ACCGTGAGCGATGCGGATGACAACATTCGCCTGACCACGACCGAAAAGCGTTGGCTGGAC


 


GAGGGTCAGGGTCGTGTGCTGCTGTTTCAACTGTCTGGCCCGATGATTTTCGGTGTTGCA


 


AAAGCTATCGCGCGTGAACATAACGCAATGGGTGATTGCGACGCTCTGGTGTTTGATATT


 


GGCGAAGTCCCGCACATGGGTGTGACCGCAAGTCTGGCTCTGGAAAATGCGATTGAAGAA


 


GCCCTGGACAAAGAACGCCAGGTTTACATCGTCGGTGCAGCAGGTCAAACCCGTCGCCGT


 


CTGGAAAAACTGAAGCTGTTTAAACGCGTGCCGCCGGATAAGTGTCTGATGTCCCGTGAA


 


GAAGCACTGAAGAATGCTGTTCTGGGTATCTATCCGCATCTGGCTGACGGTGTTACGGCT


 


CCGAGTTCCGAAATGGGCTAA


 


SEQ ID NO. 15: MCR DNA ORGANISM NAME: SULFOLOBUS TOKODAII









ATGTCTCGTC GCACCCTGAA AGCGGCTATC CTGGGCGCCA CCGGCCTGGT TGGTATCGAA
  60



 


TATGTCCGTA TGCTGTCAAA TCATCCGTAT ATCAAACCGG CGTATCTGGC CGGCAAAGGT
 120


 


TCAGTTGGCA AACCGTACGG TGAAGTGGTT CGTTGGCAGA CCGTTGGCCA AGTCCCGAAA
 180


 


GAAATCGCCG ATATGGAAAT TAAACCGACG GACCCGAAAC TGATGGATGA CGTGGATATT
 240


 


ATCTTTTCGC CGCTGCCGCA GGGTGCGGCC GGTCCGGTTG AAGAACAATT TGCAAAAGAA
 300


 


GGCTTCCCGG TCATCAGCAA CTCTCCGGAT CATCGTTTCG ATCCGGACGT CCCGCTGCTG
 360


 


GTGCCGGAAC TGAATCCGCA CACCATTAGT CTGATCGATG AACAGCGCAA ACGTCGCGAA
 420


 


TGGAAAGGTT TTATTGTTAC CACGCCGCTG TGCACGGCAC AAGGTGCAGC TATCCCGCTG
 480


 


GGTGCTATCT TCAAAGATTA CAAAATGGAC GGCGCGTTCA TTACCACGAT CCAGAGTCTG
 540


 


TCCGGTGCAG GTTACCCGGG TATCCCGTCT CTGGATGTCG TGGACAACAT TCTGCCGCTG
 600


 


GGCGATGGTT ATGACGCGAA AACCATTAAA GAAATCTTCC GTATTCTGTC AGAAGTTAAA
 660


 


CGCAATGTCG ATGAACCGAA ACTGGAAGAC GTTTCGCTGG CGGCCACCAC GCATCGTATC
 720


 


GCCACCATTC ATGGCCACTA TGAAGTGCTG TACGTTAGTT TTAAAGAAGA AACCGCAGCT
 780


 


GAAAAAGTGA AAGAAACGCT GGAAAACTTC CGCGGTGAAC CGCAGGATCT GAAACTGCCG
 840


 


ACCGCACCGT CCAAACCGAT TATCGTCATG AATGAAGATA CGCGTCCGCA AGTGTACTTT
 900


 


GATCGCTGGG CTGGCGACAT TCCGGGTATG AGCGTTGTCG TGGGCCGTCT GAAACAGGTG
 960


 


AACAAACGTA TGATCCGCCT GGTGTCTCTG ATTCACAATA CCGTTCGCGG TGCGGCGGGC
1020


 


GGTGGCATCC TGGCTGCTGA ACTGCTGGTT GAAAAAGGTT ACATTGAAAA A
1071


 








<212> TYPE: DNA



 


<211> LENGTH: 1071


SEQUENCENAME: SULFOLOBUS TOKODAII MCR DNA


 


SEQ ID NO. 16: MCR PROTEIN ORGANISM NAME: SULFOLOBUS TOKODAII









MSRRTLKAAI LGATGLVGIE YVRMLSNHPY IKPAYLAGKG SVGKPYGEVV RWQTVGQVPK
  60



 


EIADMEIKPT DPKLMDDVDI IFSPLPQGAA GPVEEQFAKE GFPVISNSPD HRFDPDVPLL
 120


 


VPELNPHTIS LIDEQRKRRE WKGFIVTTPL CTAQGAAIPL GAIFKDYKMD GAFITTIQSL
 180


 


SGAGYPGIPS LDVVDNILPL GDGYDAKTIK EIFRILSEVK RNVDEPKLED VSLAATTHRI
 240


 


ATIHGHYEVL YVSFKEETAA EKVKETLENF RGEPQDLKLP TAPSKPIIVM NEDTRPQVYF
 300


 


DRWAGDIPGM SVVVGRLKQV NKRMIRLVSL IHNTVRGAAG GGILAAELLV EKGYIEK
 357


 








<212> TYPE: PRT



 


<211> LENGTH: 357


SEQUENCENAME: SULFOLOBUS TOKODAII MCR PROTEIN


 


SEQ ID NO. 17: NEMA DNA ORGANISM NAME: ESCHERICHIA COLI









ATGTCATCTG AAAAACTGTA TTCCCCACTG AAAGTGGGCG CGATCACGGC GGCAAACCGT
  60



 


ATTTTTATGG CACCGCTGAC GCGTCTGCGC AGTATTGAAC CGGGTGACAT TCCTACCCCG
 120


 


TTGATGGCGG AATACTATCG CCAACGTGCC AGTGCCGGTT TGATTATTAG TGAAGCCACG
 180


 


CAAATTTCTG CCCAGGCAAA AGGATATGCA GGTGCGCCTG GCATCCATAG TCCGGAGCAA
 240


 


ATTGCCGCAT GGAAAAAAAT CACCGCTGGC GTTCATGCTG AAAATGGTCA TATGGCCGTG
 300


 


CAGCTGTGGC ACACCGGACG CATTTCTCAC GCCAGCCTGC AACCTGGCGG TCAGGCACCG
 360


 


GTAGCGCCTT CAGCACTTAG CGCGGGAACA CGTACTTCTC TGCGCGATGA AAATGGTCAG
 420


 


GCGATCCGTG TTGAAACATC CATGCCGCGT GCGCTTGAAC TGGAAGAGAT TCCAGGTATC
 480


 


GTCAATGATT TCCGTCAGGC CATTGCTAAC GCGCGTGAAG CCGGTTTTGA TCTGGTAGAG
 540


 


CTCCACTCTG CTCACGGTTA TTTGCTGCAT CAGTTCCTTT CTCCTTCTTC AAACCATCGT
 600


 


ACCGATCAGT ACGGCGGCAG CGTGGAAAAT CGCGCACGTT TGGTACTGGA AGTGGTCGAT
 660


 


GCCGGGATTG AAGAATGGGG TGCCGATCGC ATTGGCATTC GCGTTTCACC AATCGGTACT
 720


 


TTCCAGAACA CAGATAACGG CCCGAATGAA GAAGCCGATG CACTGTATCT GATTGAACAA
 780


 


CTGGGTAAAC GCGGCATTGC TTATCTGCAT ATGTCAGAAC CAGATTGGGC GGGGGGTGAA
 840


 


CCGTATACTG ATGCGTTCCG CGAAAAAGTA CGCGCCCGTT TCCACGGTCC GATTATCGGC
 900


 


GCAGGTGCAT ACACAGTAGA AAAAGCTGAA ACGCTGATCG GCAAAGGGTT AATTGATGCG
 960


 


GTGGCATTTG GTCGTGACTG GATTGCGAAC CCGGATCTGG TCGCCCGCTT GCAGCGCAAA
1020


 


GCTGAGCTTA ACCCACAGCG TGCCGAAAGT TTCTACGGTG GCGGCGCGGA AGGCTATACC
1080


 


GATTACCCGA CGTTGTAA
1098


 








<212> TYPE: DNA



 


<211> LENGTH: 1098


SEQUENCENAME: ESCHERICHIA COLI NEMA DNA


 


SEQ ID NO. 18: NEMA PROTEIN ORGANISM NAME: ESCHERICHIA COLI









MSSEKLYSPL KVGAITAANR IFMAPLTRLR SIEPGDIPTP LMAEYYRQRA SAGLIISEAT
  60



 


QISAQAKGYA GAPGIHSPEQ IAAWKKITAG VHAENGHMAV QLWHTGRISH ASLQPGGQAP
 120


 


VAPSALSAGT RTSLRDENGQ AIRVETSMPR ALELEEIPGI VNDFRQAIAN AREAGFDLVE
 180


 


LHSAHGYLLH QFLSPSSNHR TDQYGGSVEN RARLVLEVVD AGIEEWGADR IGIRVSPIGT
 240


 


FQNTDNGPNE EADALYLIEQ LGKRGIAYLH MSEPDWAGGE PYTDAFREKV RARFHGPIIG
 300


 


AGAYTVEKAE TLIGKGLIDA VAFGRDWIAN PDLVARLQRK AELNPQRAES FYGGGAEGYT
 360


 


DYPTL
 365


 








<212> TYPE: PRT



 


<211> LENGTH: 365


SEQUENCENAME: ESCHERICHIA COLI NEMA PROTEIN


 


SEQ ID NO. 19: RUTE DNA ORGANISM NAME: ESCHERICHIA COLI









ATGAACGAAG CCGTTAGCCC AGGTGCGCTT AGCACCCTGT TCACCGATGC CCGCACTCAC
  60



 


AACGGCTGGC GGGAGACACC CGTCAGCGAT GAGACGTTAC GGGAGATTTA TGCCCTGATG
 120


 


AAATGGGGGC CGACATCAGC TAACTGTTCT CCGGCACGGA TCGTGTTTAC CCGCACGGCA
 180


 


GAAGGAAAAG AACGTCTGCG CCCGGCACTT TCCAGCGGCA ATCTGCAAAA AACCCTGACC
 240


 


GCGCCCGTCA CCGCTATCGT CGCCTGGGAC AGTGAATTTT ATGAACGGTT ACCACTACTG
 300


 


TTTCCCCACG GTGATGCCCG CAGTTGGTTT ACCTCCAGCC CACAACTTGC CGAAGAAACA
 360


 


GCGTTTCGCA ACAGTTCCAT GCAGGCGGCC TATCTGATCG TCGCCTGCCG GGCGCTGGGA
 420


 


CTGGATACCG GCCCGATGTC GGGCTTTGAC CGTCAACACG TGGACGACGC CTTTTTTACG
 480


 


GGCAGCACGC TGAAGAGCAA TCTGCTGATT AATATCGGCT ATGGCGATAG CAGCAAGCTT
 540


 


TATGCGCGCC TGCCACGTCT GTCCTTTGAA GAAGCCTGCG GGCTGTTGTA A
 591


 








<212> TYPE: DNA



 


<211> LENGTH: 591


SEQUENCENAME: ESCHERICHIA COLI RUTE DNA


 


SEQ ID NO. 20 RUTE PROTEIN ORGANISM NAME: ESCHERICHIA COLI









MNEAVSPGAL STLFTDARTH NGWRETPVSD ETLREIYALM KWGPTSANCS PARIVFTRTA
  60



 


EGKERLRPAL SSGNLQKTLT APVTAIVAWD SEFYERLPLL FPHGDARSWF TSSPQLAEET
 120


 


AFRNSSMQAA YLIVACRALG LDTGPMSGFD RQHVDDAFFT GSTLKSNLLI NIGYGDSSKL
 180


 


YARLPRLSFE EACGLL
 196


 








<212> TYPE: PRT



 


<211> LENGTH: 196


SEQUENCENAME: ESCHERICHIA COLI RUTE PROTEIN


 


SEQ ID NO. 21: YDFG DNA ORGANISM NAME: ESCHERICHIA COLI









ATGGTCGTTT TAGTAACTGG AGCAACGGCA GGTTTTGGTG AATGCATTAC TCGTCGTTTT
  60



 


ATTCAACAAG GGCATAAAGT TATCGCCACT GGCCGTCGCC AGGAACGGTT GCAGGAGTTA
 120


 


AAAGACGAAC TGGGAGATAA TCTGTATATC GCCCAACTGG ACGTTCGCAA CCGCGCCGCT
 180


 


ATTGAAGAGA TGCTGGCATC GCTTCCTGCC GAGTGGTGCA ATATTGATAT CCTGGTAAAT
 240


 


AATGCCGGCC TGGCGTTGGG CATGGAGCCT GCGCATAAAG CCAGCGTTGA AGACTGGGAA
 300


 


ACGATGATTG ATACCAACAA CAAAGGCCTG GTATATATGA CGCGCGCCGT CTTACCGGGT
 360


 


ATGGTTGAAC GTAATCATGG TCATATTATT AACATTGGCT CAACGGCAGG TAGCTGGCCG
 420


 


TATGCCGGTG GTAACGTTTA CGGTGCGACG AAAGCGTTTG TTCGTCAGTT TAGCCTGAAT
 480


 


CTGCGTACGG ATCTGCATGG TACGGCGGTG CGCGTCACCG ACATCGAACC GGGTCTGGTG
 540


 


GGTGGTACCG AGTTTTCCAA TGTCCGCTTT AAAGGCGATG ACGGTAAAGC AGAAAAAACC
 600


 


TATCAAAATA CCGTTGCATT GACGCCAGAA GATGTCAGCG AAGCCGTCTG GTGGGTGTCA
 660


 


ACGCTGCCTG CTCACGTCAA TATCAATACC CTGGAAATGA TGCCGGTTAC CCAAAGCTAT
 720


 


GCCGGACTGA ATGTCCACCG TCAGTAA
 747


 








<212> TYPE: DNA



 


<211> LENGTH: 747


SEQUENCENAME: ESCHERICHIA COLI YDFG DNA


 


SEQ ID NO. 22: YDFG PROTEIN ORGANISM NAME: ESCHERICHIA COLI









MVVLVTGATA GFGECITRRF IQQGHKVIAT GRRQERLQEL KDELGDNLYI AQLDVRNRAA
  60



 


IEEMLASLPA EWCNIDILVN NAGLALGMEP AHKASVEDWE TMIDTNNKGL VYMTRAVLPG
 120


 


MVERNHGHII NIGSTAGSWP YAGGNVYGAT KAFVRQFSLN LRTDLHGTAV RVTDIEPGLV
 180


 


GGTEFSNVRF KGDDGKAEKT YQNTVALTPE DVSEAVWWVS TLPAHVNINT LEMMPVTQSY
 240


 


AGLNVHRQ
 248


 








<212> TYPE: PRT



 


<211> LENGTH: 248


SEQUENCENAME: ESCHERICHIA COLI YDFG PROTEIN


 


SEQ ID NO. 23: MMSB DNA ORGANISM NAME: P. AERUGINOSA









ATGGCCGACA TTGCGTTTCT GGGTCTGGGC AATATGGGCG GTCCGATGGC CGCGAACCTG
  60



 


CTGAAAGCCG GCCACCGTGT GAATGTGTTC GACCTGCAAC CAAAAGCGGT CCTGGGCTTG
 120


 


GTTGAGCAAG GCGCGCAGGG CGCAGACTCT GCTCTGCAAT GTTGTGAGGG TGCGGAGGTC
 180


 


GTGATTTCTA TGCTGCCAGC AGGCCAGCAT GTGGAAAGCC TGTACCTGGG CGATGATGGT
 240


 


CTGCTGGCAC GCGTGGCGGG CAAGCCTTTG CTGATTGACT GTAGCACCAT CGCACCGGAA
 300


 


ACGGCGCGTA AGGTGGCGGA GGCAGCCGCA GCAAAGGGCC TGACGCTGCT GGATGCCCCG
 360


 


GTTTCGGGCG GTGTCGGTGG TGCCCGTGCA GGTACGCTGT CGTTTATCGT GGGTGGTCCG
 420


 


GCGGAGGGTT TTGCGCGTGC GCGTCCGGTT CTGGAGAATA TGGGTCGCAA CATTTTCCAC
 480


 


GCGGGTGATC ACGGCGCTGG TCAGGTGGCG AAAATCTGTA ACAACATGCT GCTGGGTATC
 540


 


TTGATGGCGG GCACCGCCGA AGCCTTGGCG CTGGGCGTCA AAAACGGTCT GGACCCGGCA
 600


 


GTGCTGTCCG AAGTGATGAA ACAGAGCAGC GGTGGTAACT GGGCGCTGAA TCTGTACAAT
 660


 


CCGTGGCCGG GTGTGATGCC GCAGGCCCCA GCCTCTAATG GCTACGCAGG CGGCTTCCAA
 720


 


GTGCGCCTGA TGAACAAAGA CCTGGGCCTG GCGCTGGCGA ATGCGCAAGC GGTCCAAGCG
 780


 


AGCACCCCGC TGGGCGCACT GGCCCGTAAC CTGTTTAGCC TGCACGCTCA AGCCGACGCC
 840


 


GAGCACGAAG GTCTGGACTT CAGCTCTATT CAAAAACTGT ATCGCGGTAA GGATTAG
 897


 








<212> TYPE: DNA



 


<211> LENGTH: 897


SEQUENCE NAME: P. AERUGINOSA MMSB DNA


 


SEQ ID NO. 24: MMSB PROTEIN ORGANISM NAME: P. AERUGINOSA









MADIAFLGLG NMGGPMAANL LKAGHRVNVF DLQPKAVLGL VEQGAQGADS ALQCCEGAEV
  60



 


VISMLPAGQH VESLYLGDDG LLARVAGKPL LIDCSTIAPE TARKVAEAAA AKGLTLLDAP
 120


 


VSGGVGGARA GTLSFIVGGP AEGFARARPV LENMGRNIFH AGDHGAGQVA KICNNMLLGI
 180


 


LMAGTAEALA LGVKNGLDPA VLSEVMKQSS GGNWALNLYN PWPGVMPQAP ASNGYAGGFQ
 240


 


VRLMNKDLGL ALANAQAVQA STPLGALARN LFSLHAQADA EHEGLDFSSI QKLYRGKD
 298


 








<212> TYPE: PRT



 


<211> LENGTH: 298


SEQUENCENAME: P. AERUGINOSA MMSB PROTEIN


 


SEQ ID NO. 25: NDSD DNA ORGANISMNAME: PSEUDOMONAS AERUGINOSA









ATGGGCAAAC AGATCGCCTT CATCGGCCTG GGCCATATGG GCGCACCTAT GGCCACCAAC
  60



 


CTGCTGAAGG CCGGCTACCT GCTGAATGTG TTCGACCTGG TGCAGAGCGC CGTGGATGGT
 120


 


TTAGTGGCCG CAGGTGCAAG TGCAGCACGC AGTGCACGCG ATGCCGTTCA GGGTGCCGAC
 180


 


GTGGTGATCA GCATGCTGCC TGCCAGCCAA CACGTGGAGG GTCTGTACCT GGACGACGAT
 240


 


GGTCTGCTGG CCCACATTGC CCCTGGCACC TTAGTGCTGG AGTGCAGCAC AATCGCCCCG
 300


 


ACCAGTGCAC GCAAGATTCA TGCAGCAGCC CGCGAGCGTG GTCTGGCAAT GCTGGACGCA
 360


 


CCGGTTAGCG GTGGTACAGC AGGTGCCGCA GCAGGCACCC TGACCTTCAT GGTGGGCGGT
 420


 


GACGCCGAAG CCCTGGAAAA AGCACGCCCG CTGTTTGAGG CAATGGGCCG TAACATCTTC
 480


 


CATGCCGGCC CTGATGGCGC AGGTCAGGTG GCCAAAGTGT GCAATAACCA GCTGCTGGCA
 540


 


GTGCTGATGA TCGGTACCGC CGAGGCAATG GCACTGGGCG TGGCAAACGG CTTAGAGGCC
 600


 


AAGGTGCTGG CAGAAATCAT GCGCCGTAGT AGCGGCGGTA ACTGGGCCCT GGAGGTGTAC
 660


 


AACCCGTGGC CTGGCGTGAT GGAGAATGCA CCGGCCAGTC GTGACTACAG CGGCGGTTTC
 720


 


ATGGCACAGC TGATGGCCAA GGACCTGGGC TTAGCCCAAG AGGCAGCCCA AGCCAGCGCC
 780


 


AGTAGTACCC CGATGGGCAG CTTAGCCCTG AGTCTGTACC GCTTACTGCT GAAGCAGGGC
 840


 


TACGCCGAAC GCGACTTCAG CGTGGTGCAG AAGCTGTTCG ACCCGACCCA AGGCCAGTAA
 900


 








<212> TYPE: DNA



 


<211> LENGTH: 900


SEQUENCENAME: P. AERUGINOSA NDSD DNA


 


SEQ ID NO. 26: NDSD PROTEIN PSEUDOMONAS AERUGINOSA









MGKQIAFIGL GHMGAPMATN LLKAGYLLNV FDLVQSAVDG LVAAGASAAR SARDAVQGAD
  60



 


VVISMLPASQ HVEGLYLDDD GLLAHIAPGT LVLECSTIAP TSARKIHAAA RERGLAMLDA
 120


 


PVSGGTAGAA AGTLTFMVGG DAEALEKARP LFEAMGRNIF HAGPDGAGQV AKVCNNQLLA
 180


 


VLMIGTAEAM ALGVANGLEA KVLAEIMRRS SGGNWALEVY NPWPGVMENA PASRDYSGGF
 240


 


MAQLMAKDLG LAQEAAQASA SSTPMGSLAL SLYRLLLKQG YAERDFSVVQ KLFDPTQGQ
 299


 








<212> TYPE: PRT



 


<211> LENGTH: 299


SEQUENCENAME: P. AERUGINOSA NDSD PROTEIN


 


SEQ ID NO. 27: FABI(TS) PROTEIN ORGANISM NAME: ESCHERICHIA COLI









MGFLSGKRIL VTGVASKLSI AYGIAQAMHR EGAELAFTYQ NDKLKGRVEE FAAQLGSDIV
  60



 


LQCDVAEDAS IDTMFAELGK VWPKFDGFVH SIGFAPGDQL DGDYVNAVTR EGFKIAHDIS
 120


 


SYSFVAMAKA CRSMLNPGSA LLTLSYLGAE RAIPNYNVMG LAKASLEANV RYMANAMGPE
 180


 


GVRVNAISAG PIRTLAASGI KDFRKMLAHC EAVTPIRRTV TIEDVGNSAA FLCSDLSAGI
 240


 


FGEVVHVDGG FSIAAMNELE LK
 262


 








<212> TYPE: PRT



 


<211> LENGTH: 262


SEQUENCENAME: ESCHERICHIA COLI FABI(TS) PROTEIN


 


SEQ ID NO. 28: FABB(TS) PROTEIN ORGANISM NAME: ESCHERICHIA COLI









MKRAVITGLG IVSSIGNNQQ EVLASLREGR SGITFSQELK DSGMRSHVWG NVKLDTTGLI
  60



 


DRKVVRFMSD ASIYAFLSME QAIADAGLSP EAYQNNPRVG LIAGSGGGSP RFQVFGADAM
 120


 


RGPRGLKAVG PYVVTKAMAS GVSACLATPF KIHGVNYSIS SACATSAHCI GNAVEQIQLG
 180


 


KQDIVFAGGG EELCWEMACE FDAMGALSTK YNDTPEKASR TYDAHRDGFV IAGGGGMVVV
 240


 


EELEHALARG AHIYAEIVGY GATSDGADMV APSGEGAVRC MKMAMHGVDT PIDYLNSHGT
 300


 


STPVGDVKEL AAIREVFGDK SPAISATKVM TGHSLGAAGV QEAIYSLLML EHGFIAPSIN
 360


 


IEELDEQAAG LNIVTETTDR ELTTVMSNSF GFGGTNATLV MRKLKD
 406


 


<212> TYPE: PRT



 


<211> LENGTH: 406



SEQUENCENAME: ESCHERICHIA COLI FABB(TS) PROTEIN



 


SEQ ID NO. 29: FABD(TS) PROTEIN ORGANISM NAME: ESCHERICHIA COLI



MTQFAFVFPG QGSQTVGMLA DMAASYPIVE ETFAEASAAL GYDLWALTQQ GPAEELNKTW
  60


 


QTQPALLTAS VALYRVWQQQ GGKAPAMMAG HSLGEYSALV CAGVIDFADA VRLVEMRGKF
 120


 


MQEAVPEGTG AMAAIIGLDD ASIAKACEEA AEGQVVSPVN FNSPGQVVIA GHKEAVERAG
 180


 


AACKAAGAKR ALPLPVSVPS HCALMKPAAD KLAVELAKIT FNAPTVPVVN NVDVKCETNG
 240


 


DAIRDALVRQ LYNPVQQTKS VEYMAAQGVE HLYEVGPGKV LTGLTKRIVD TLTASALNEP
 300


 


SAMAAALEL
 309


 








<212> TYPE: PRT



 


<211> LENGTH: 309


SEQUENCENAME: ESCHERICHIA COLI FABD(TS) PROTEIN


 


SEQ ID NO. 30: RHTA DNA ORGANISM NAME: ESCHERICHIA COLI









ATGAGCGGTT CATTACGTAA AATGCCGGTC TGGTTACCAA TAGTCATATT GCTCGTTGCC
  60



 


ATGGCGTCTA TTCAGGGTGG AGCCTCGTTA GCTAAGTCAC TTTTTCCTCT GGTGGGCGCA
 120


 


CCGGGTGTCA CTGCGCTGCG TCTGGCATTA GGAACGCTGA TCCTCATCGC GTTCTTTAAG
 180


 


CCATGGCGAC TGCGCTTTGC CAAAGAGCAA CGGTTACCGC TGTTGTTTTA CGGCGTTTCG
 240


 


CTGGGTGGGA TGAATTATCT TTTTTATCTT TCTATTCAGA CAGTACCGCT GGGTATTGCG
 300


 


GTGGCGCTGG AGTTCACCGG ACCACTGGCG GTGGCGCTGT TCTCTTCTCG TCGCCCGGTA
 360


 


GATTTCGTCT GGGTTGTGCT GGCGGTTCTT GGTCTGTGGT TCCTGCTACC GCTGGGGCAA
 420


 


GACGTTTCCC ATGTCGATTT AACCGGCTGT GCGCTGGCAC TGGGGGCCGG GGCTTGTTGG
 480


 


GCTATTTACA TTTTAAGTGG GCAACGCGCA GGAGCGGAAC ATGGCCCTGC GACGGTGGCA
 540


 


ATTGGTTCGT TGATTGCAGC GTTAATTTTC GTGCCAATTG GAGCGCTTCA GGCTGGTGAA
 600


 


GCACTCTGGC ACTGGTCGGT TATTCCATTG GGTCTGGCTG TCGCTATTCT CTCGACCGCT
 660


 


CTGCCTTATT CGCTGGAAAT GATTGCCCTC ACCCGTTTGC CAACACGGAC ATTTGGTACG
 720


 


CTGATGAGCA TGGAACCGGC GCTGGCTGCC GTTTCCGGGA TGATTTTCCT CGGAGAAACA
 780


 


CTGACACCCA TACAGCTACT GGCGCTCGGC GCTATCATCG CCGCTTCAAT GGGGTCTACG
 840


 


CTGACAGTAC GCAAAGAGAG CAAAATAAAA GAATTAGACA TTAATTAA
 888


 








<212> TYPE: DNA



 


<211> LENGTH: 888


SEQUENCENAME: ESCHERICHIA COLI RHTA DNA


 


SEQ ID NO. 31: RHTA PROTEIN ORGANISM NAME: ESCHERICHIA COLI









MSGSLRKMPV WLPIVILLVA MASIQGGASL AKSLFPLVGA PGVTALRLAL GTLILIAFFK
  60



 


PWRLRFAKEQ RLPLLFYGVS LGGMNYLFYL SIQTVPLGIA VALEFTGPLA VALFSSRRPV
 120


 


DFVWVVLAVL GLWFLLPLGQ DVSHVDLTGC ALALGAGACW AIYILSGQRA GAEHGPATVA
 180


 


IGSLIAALIF VPIGALQAGE ALWHWSVIPL GLAVAILSTA LPYSLEMIAL TRLPTRTFGT
 240


 


LMSMEPALAA VSGMIFLGET LTPIQLLALG AIIAASMGST LTVRKESKIK ELDIN
 295


 








<212> TYPE: PRT



 


<211> LENGTH: 295


SEQUENCENAME: ESCHERICHIA COLI RHTA PROTEIN


 


SEQ ID NO. 32: YDCO DNA ORGANISM NAME: ESCHERICHIA COLI









ATGCGACTCC TCCGGCAAAA CGGAAGTTTA TCACTTGTGC GTTATAACGG ACAAATGCTA
  60



 


CGGTGCCTGT ACGCTATAAC GCACGAGGTG ACTATGCGTC TGTTTTCTAT TCCTCCACCC
 120


 


ACGCTACTGG CGGGGTTTCT GGCGGTATTA ATTGGCTACG CCAGTTCAGC GGCAATAATC
 180


 


TGGCAAGCAG CGATTGTCGC CGGAGCCACC ACTGCACAAA TCTCTGGCTG GATGACGGCG
 240


 


CTGGGGCTGG CAATGGGCGT CAGTACGCTG ACTCTGACAT TATGGTATCG CGTACCTGTT
 300


 


CTCACCGCAT GGTCAACGCC TGGCGCGGCT TTGTTGGTCA CCGGATTGCA GGGACTAACA
 360


 


CTTAACGAAG CCATCGGCGT TTTTATTGTC ACCAACGCGC TAATAGTCCT CTGCGGCATA
 420


 


ACGGGACTCT TTGCTCGTCT GATGCGCATT ATTCCGCACT CGCTTGCGGC GGCAATGCTT
 480


 


GCCGGGATTT TATTACGCTT TGGTTTACAG GCGTTTGCCA GTCTGGACGG TCAATTTACG
 540


 


TTGTGTGGAA GTATGTTGCT GGTATGGCTG GCAACCAAGG CCGTTGCGCC GCGCTATGCG
 600


 


GTAATTGCCG CGATGATTAT TGGGATCGTG ATCGTCATCG CGCAAGGTGA CGTTGTCACA
 660


 


ACTGATGTTG TCTTTAAACC CGTTCTCCCC ACTTATATTA CCCCTGATTT TTCGTTTGCT
 720


 


CACAGCCTGA GCGTTGCACT CCCCCTTTTT CTGGTGACGA TGGCATCGCA AAACGCACCG
 780


 


GGTATCGCAG CAATGAAAGC AGCTGGATAT TCGGCTCCTG TTTCGCCATT AATTGTATTT
 840


 


ACTGGATTGC TGGCACTGGT TTTTTCCCCT TTCGGCGTTT ATTCCGTCGG TATTGCGGCA
 900


 


ATCACCGCGG CTATTTGCCA AAGCCCGGAA GCGCATCCGG ATAAAGATCA ACGTTGGCTG
 960


 


GCCGCTGCCG TTGCAGGCAT TTTCTATTTG CTCGCAGGTC TGTTTGGTAG TGCCATTACC
1020


 


GGGATGATGG CTGCCCTGCC CGTAAGTTGG ATCCAGATGC TGGCAGGTCT GGCGCTGTTA
1080


 


AGTACCATCG GCGGCAGTTT GTATCAGGCG CTGCATAATG AGCGTGAGCG AGACGCGGCG
1140


 


GTGGTGGCAT TTCTGGTAAC GGCAAGTGGA TTGACGCTGG TCGGGATTGG TTCTGCGTTT
1200


 


TGGGGATTAA TTGCCGGAGG CGTTTGTTAC GTGGTGTTGA ATTTAATCGC TGACAGAAAC
1260


 


CGATATTGA
1269


 








<212> TYPE: DNA



 


<211> LENGTH: 1269


SEQUENCENAME: ESCHERICHIA COLI YDCO DNA


 


SEQ ID NO. 33: YDCO PROTEIN ORGANISM NAME: ESCHERICHIA COLI









MRLLRQNGSL SLVRYNGQML RCLYAITHEV TMRLFSIPPP TLLAGFLAVL IGYASSAAII
  60



 


WQAAIVAGAT TAQISGWMTA LGLAMGVSTL TLTLWYRVPV LTAWSTPGAA LLVTGLQGLT
 120


 


LNEAIGVFIV TNALIVLCGI TGLFARLMRI IPHSLAAAML AGILLRFGLQ AFASLDGQFT
 180


 


LCGSMLLVWL ATKAVAPRYA VIAAMIIGIV IVIAQGDVVT TDVVFKPVLP TYITPDFSFA
 240


 


HSLSVALPLF LVTMASQNAP GIAAMKAAGY SAPVSPLIVF TGLLALVFSP FGVYSVGIAA
 300


 


ITAAICQSPE AHPDKDQRWL AAAVAGIFYL LAGLFGSAIT GMMAALPVSW IQMLAGLALL
 360


 


STIGGSLYQA LHNERERDAA VVAFLVTASG LTLVGIGSAF WGLIAGGVCY VVLNLIADRN
 420


 


RY
 422


 








<212> TYPE: PRT



 


<211> LENGTH: 422


SEQUENCENAME: ESCHERICHIA COLI YDCO PROTEIN


 


SEQ ID NO. 34: ACRD ORGANISM NAME: ESCHERICHIA COLI


MANFFIDRPI FAWVLAILLC LTGTLAIFSL PVEQYPDLAP PNVRVTANYP GASAQTLENT


 


VTQVIEQNMT GLDNLMYMSS QSSGTGQASV TLSFKAGTDP DEAVQQVQNQ LQSAMRKLPQ


 


AVQNQGVTVR KTGDTNILTI AFVSTDGSMD KQDIADYVAS NIQDPLSRVN GVGDIDAYGS


 


QYSMRIWLDP AKLNSFQMTA KDVTDAIESQ NAQIAVGQLG GTPSVDKQAL NATINAQSLL


 


QTPEQFRDIT LRVNQDGSEV RLGDVATVEM GAEKYDYLSR FNGKPASGLG VKLASGANEM


 


ATAELVLNRL DELAQYFPHG LEYKVAYETT SFVKASIEDV VKTLLEAIAL VFLVMYLFLQ


 


NFRATLIPTI AVPVVLMGTF SVLYAFGYSV NTLTMFAMVL AIGLLVDDAI VVVENVERIM


 


SEEGLTPREA TRKSMGQIQG ALVGIAMVLS AVFVPMAFFG GTTGAIYRQF SITIVAAMVL


 


SVLVAMILTP ALCATLLKPL KKGEHHGQKG FFAWFNQMFN RNAERYEKGV AKILHRSLRW


 


IVIYVLLLGG MVFLFLRLPT SFLPLEDRGM FTTSVQLPSG STQQQTLKVV EQIEKYYFTH


 


EKDNIMSVFA TVGSGPGGNG QNVARMFIRL KDWSERDSKT GTSFAIIERA TKAFNQIKEA


 


RVIASSPPAI SGLGSSAGFD MELQDHAGAG HDALMAARNQ LLALAAENPE LTRVRHNGLD


 


DSPQLQIDID QRKAQALGVA IDDINDTLQT AWGSSYVNDF MDRGRVKKVY VQAAAPYRML


 


PDDINLWYVR NKDGGMVPFS AFATSRWETG SPRLERYNGY SAVEIVGEAA PGVSTGTAMD


 


IMESLVKQLP NGFGLEWTAM SYQERLSGAQ APALYAISLL VVFLCLAALY ESWSVPFSVM


 


LVVPLGVIGA LLATWMRGLE NDVYFQVGLL TVIGLSAKNA ILIVEFANEM NQKGHDLFEA


 


TLHACRQRLR PILMTSLAFI FGVLPMATST GAGSGGQHAV GTGVMGGMIS ATILAIYFVP


 


LFFVLVRRRF PLKPRPE


 


SEQ ID NO. 35: ATTA ORGANISM NAME: ESCHERICHIA COLI


MAHPPLLHLQ DITLSLGGNP LLDGAGFAVG RGERLCLVGR NGSGKSTLLK IAAGVIQPDS


 


GSVFVQPGAS LRYLPQEPDL SAYATTADYV VGQIGDPDMA WRATPLLDAL GLTGRESTQN


 


LSGGEGRRCA IAGVLAAAPD VLLLDEPTNH LDMPTIEWLE RELLSLGAMV IISHDRRLLS


 


TLSRSVVWLD RGVTRRLDEG FGRFEAWREE VLEQEERDAH KLDRKIAREE DWMRYGVTAR


 


RKRNVRRVRE LADLRTARKE AIRAPGTLTL NTQLRPHRKL VAVAEDISKA WGEKQVVRHL


 


DLRILRGDRL GIVGANGAGK TTLLRMLTGL DQPDSGTISL GPSLNMVTLD QQRRTLNPER


 


TLADTLTEGG GDMVQVGTEK RHVVGYMKDF LFRPEQARTP VSALSGGERG RLMLACALAK


 


PSNLLVLDEP TNDLDLETLD ILQDMLASCE GTVLLVSHDR DFLDRVATSV LATEGDGNWI


 


EYAGGYSDML AQRHQKPLTT ASVVENEPTK PKETTAARGP TKKLSYKDQF ALDNLPKEME


 


KLEAQAANCV KNWQIQIYME KTPRSLRNFR LIYRSSKQSW QNLKNAGWNW K


 


SEQ ID NO. 36: BCR ORGANISM NAME: ESCHERICHIA COLI


MTTRQHSSFA IVFILGLLAM LMPLSIDMYL PALPVISAQF GVPAGSTQMT LSTYILGFAL


 


GQLIYGPMAD SFGRKPVVLG GTLVFAAAAV ACALANTIDQ LIVMRFFHGL AAAAASVVIN


 


ALMRDIYPKE EFSRMMSFVM LVTTIAPLMA PIVGGWVLVW LSWHYIFWIL ALAAILASAM


 


IFFLIKETLP PERRQPFHIR TTIGNFAALF RHKRVLSYML ASGFSFAGMF SFLSAGPFVY


 


IEINHVAPEN FGYYFALNIV FLFVMTIFNS RFVRRIGALN MFRSGLWIQF IMAAWMVISA


 


LLGLGFWSLV VGVAAFVGCV SMVSSNAMAV ILDEFPHMAG TASSLAGTFR FGIGAIVGAL


 


LSLATFNSAW PMIWSIAFCA TSSILFCLYA SRPKKR


 


SEQ ID NO. 37: CUSA ORGANISM NAME: ESCHERICHIA COLI


MIEWIIRRSV ANRFLVLMGA LFLSIWGTWT IINTPVDALP DLSDVQVIIK TSYPGQAPQI


 


VENQVTYPLT TTMLSVPGAK TVRGFSQFGD SYVYVIFEDG TDPYWARSRV LEYLNQVQGK


 


LPAGVSAELG PDATGVGWIY EYALVDRSGK HDLADLRSLQ DWFLKYELKT IPDVAEVASV


 


GGVVKEYQVV IDPQRLAQYG ISLAEVKSAL DASNQEAGGS SIELAEAEYM VRASGYLQTL


 


DDFNHIVLKA SENGVPVYLR DVAKVQIGPE MRRGIAELNG EGEVAGGVVI LRSGKNAREV


 


IAAVKDKLET LKSSLPEGVE IVTTYDRSQL IDRAIDNLSG KLLEEFIVVA VVCALFLWHV


 


RSALVAIISL PLGLCIAFIV MHFQGLNANI MSLGGIAIAV GAMVDAAIVM IENAHKRLEE


 


WQHQHPDATL DNKTRWQVIT DASVEVGPAL FISLLIITLS FIPIFTLEGQ EGRLFGPLAF


 


TKTYAMAGAA LLAIVVIPIL MGYWIRGKIP PESSNPLNRF LIRVYHPLLL KVLHWPKTTL


 


LVAALSVLTV LWPLNKVGGE FLPQINEGDL LYMPSTLPGI SAAEAASMLQ KTDKLIMSVP


 


EVARVFGKTG KAETATDSAP LEMVETTIQL KPQEQWRPGM TMDKIIEELD NTVRLPGLAN


 


LWVPPIRNRI DMLSTGIKSP IGIKVSGTVL ADIDAMAEQI EEVARTVPGV ASALAERLEG


 


GRYINVEINR EKAARYGMTV ADVQLFVTSA VGGAMVGETV EGIARYPINL RYPQSWRDSP


 


QALRQLPILT PMKQQITLAD VADIKVSTGP SMLKTENARP TSWIYIDARD RDMVSVVHDL


 


QKAIAEKVQL KPGTSVAFSG QFELLERANH KLKLMVPMTL MIIFVLLYLA FRRVGEALLI


 


ISSVPFALVG GIWLLWWMGF HLSVATGTGF IALAGVAAEF GVVMLMYLRH AIEAVPSLNN


 


PQTFSEQKLD EALYHGAVLR VRPKAMTVAV IIAGLLPILW GTGAGSEVMS RIAAPMIGGM


 


ITAPLLSLFI IPAAYKLMWL HRHRVRK


 


SEQ ID NO. 38: CYNX ORGANISM NAME: ESCHERICHIA COLI


MLLVLVLIGL NMRPLLTSVG PLLPQLRQAS GMSFSVAALL TALPVVTMGG LALAGSWLHQ


 


HVSERRSVAI SLLLIAVGAL MRELYPQSAL LLSSALLGGV GIGIIQAVMP SVIKRRFQQR


 


TPLVMGLWSA ALMGGGGLGA AITPWLVQHS ETWYQTLAWW ALPAVVALFA WWWQSAREVA


 


SSHKTTTTPV RVVFTPRAWT LGVYFGLING GYASLIAWLP AFYIEIGASA QYSGSLLALM


 


TLGQAAGALL MPAMARHQDR RKLLMLALVL QLVGFCGFIW LPMQLPVLWA MVCGLGLGGA


 


FPLCLLLALD HSVQPAIAGK LVAFMQGIGF IIAGLAPWFS GVLRSISGNY LMDWAFHALC


 


VVGLMIITLR FAPVRFPQLW VKEA


 


SEQ ID NO. 39: DEDA ORGANISM NAME: ESCHERICHIA COLI


MDLIYFLIDF ILHIDVHLAE LVAEYGVWVY AILFLILFCE TGLVVTPFLP GDSLLFVAGA


 


LASLETNDLN VHMMVVLMLI AAIVGDAVNY TIGRLFGEKL FSNPNSKIFR RSYLDKTHQF


 


YEKHGGKTII LARFVPIVRT FAPFVAGMGH MSYRHFAAYN VIGALLWVLL FTYAGYFFGT


 


IPMVQDNLKL LIVGIIVVSI LPGVIEIIRH KRAAARAAK


 


SEQ ID NO. 40: EAMA ORGANISM NAME: ESCHERICHIA COLI


MSRKDGVLAL LVVVVWGLNF VVIKVGLHNM PPLMLAGLRF MLVAFPAIFF VARPKVPLNL


 


LLGYGLTISF AQFAFLFCAI NFGMPAGLAS LVLQAQAFFT IMLGAFTFGE RLHGKQLAGI


 


ALAIFGVLVL IEDSLNGQHV AMLGFMLTLA AAFSWACGNI FNKKIMSHST RPAVMSLVIW


 


SALIPIIPFF VASLILDGSA TMIHSLVTID MTTILSLMYL AFVATIVGYG IWGTLLGRYE


 


TWRVAPLSLL VPVVGLASAA LLLDERLTGL QFLGAVLIMT GLYINVFGLR WRKAVKVGS


 


SEQ ID NO. 41: EAMB ORGANISM NAME: ESCHERICHIA COLI


MTPTLLSAFW TYTLITAMTP GPNNILALSS ATSHGFRQST RVLAGMSLGF LIVMLLCAGI


 


SFSLAVIDPA AVHLLSWAGA AYIVWLAWKI ATSPTKEDGL QAKPISFWAS FALQFVNVKI


 


ILYGVTALST FVLPQTQALS WVVGVSVLLA MIGTFGNVCW ALAGHLFQRL FRQYGRQLNI


 


VLALLLVYCA VRIFY


 


SEQ ID NO. 42: EMRB ORGANISM NAME: ESCHERICHIA COLI


MQQQKPLEGA QLVIMTIALS LATFMQVLDS TIANVAIPTI AGNLGSSLSQ GTWVITSFGV


 


ANAISIPLTG WLAKRVGEVK LFLWSTIAFA IASWACGVSS SLNMLIFFRV IQGIVAGPLI


 


PLSQSLLLNN YPPAKRSIAL ALWSMTVIVA PICGPILGGY ISDNYHWGWI FFINVPIGVA


 


VVLMTLQTLR GRETRTERRR IDAVGLALLV IGIGSLQIML DRGKELDWFS SQEIIILTVV


 


AVVAICFLIV WELTDDNPIV DLSLFKSRNF TIGCLCISLA YMLYFGAIVL LPQLLQEVYG


 


YTATWAGLAS APVGIIPVIL SPIIGRFAHK LDMRRLVTFS FIMYAVCFYW RAYTFEPGMD


 


FGASAWPQFI QGFAVACFFM PLTTITLSGL PPERLAAASS LSNFTRTLAG SIGTSITTTM


 


WTNRESMHHA QLTESVNPFN PNAQAMYSQL EGLGMTQQQA SGWIAQQITN QGLIISANEI


 


FWMSAGIFLV LLGLVWFAKP PFGAGGGGGG AH


 


SEQ ID NO. 43: EMRD ORGANISM NAME: ESCHERICHIA COLI


MKRQRNVNLL LMLVLLVAVG QMAQTIYIPA IADMARDLNV REGAVQSVMG AYLLTYGVSQ


 


LFYGPISDRV GRRPVILVGM SIFMLATLVA VTTSSLTVLI AASAMQGMGT GVGGVMARTL


 


PRDLYERTQL RHANSLLNMG ILVSPLLAPL IGGLLDTMWN WRACYLFLLV LCAGVTFSMA


 


RWMPETRPVD APRTRLLTSY KTLFGNSGFN CYLLMLIGGL AGIAAFEACS GVLMGAVLGL


 


SSMTVSILFI LPIPAAFFGA WFAGRPNKRF STLMWQSVIC CLLAGLLMWI PDWFGVMNVW


 


TLLVPAALFF FGAGMLFPLA TSGAMEPFPF LAGTAGALVG GLQNIGSGVL ASLSAMLPQT


 


GQGSLGLLMT LMGLLIVLCW LPLATRMSHQ GQPV


 


SEQ ID NO. 44: EMRKY ORGANISM NAME: ESCHERICHIA COLI


MAITKSTPAP LTGGTLWCVT IALSLATFMQ MLDSTISNVA IPTISGFLGA STDEGTWVIT


 


SFGVANAIAI PVTGRLAQRI GELRLFLLSV TFFSLSSLMC SLSTNLDVLI FFRVVQGLMA


 


GPLIPLSQSL LLRNYPPEKR TFALALWSMT VIIAPICGPI LGGYICDNFS WGWIFLINVP


 


MGIIVLTLCL TLLKGRETET SPVKMNLPGL TLLVLGVGGL QIMLDKGRDL DWFNSSTIII


 


LTVVSVISLI SLVIWESTSE NPILDLSLFK SRNFTIGIVS ITCAYLFYSG AIVLMPQLLQ


 


ETMGYNAIWA GLAYAPIGIM PLLISPLIGR YGNKIDMRLL VTFSFLMYAV CYYWRSVTFM


 


PTIDFTGIIL PQFFQGFAVA CFFLPLTTIS FSGLPDNKFA NASSMSNFFR TLSGSVGTSL


 


TMTLWGRRES LHHSQLTATI DQFNPVFNSS SQIMDKYYGS LSGVLNEINN EITQQSLSIS


 


ANEIFRMAAI AFILLTVLVW FAKPPFTAKG VG


 


SEQ ID NO. 45: EMRY ORGANISM NAME: ESCHERICHIA COLI


MAITKSTPAP LTGGTLWCVT IALSLATFMQ MLDSTISNVA IPTISGFLGA STDEGTWVIT


 


SFGVANAIAI PVTGRLAQRI GELRLFLLSV TFFSLSSLMC SLSTNLDVLI FFRVVQGLMA


 


GPLIPLSQSL LLRNYPPEKR TFALALWSMT VIIAPICGPI LGGYICDNFS WGWIFLINVP


 


MGIIVLTLCL TLLKGRETET SPVKMNLPGL TLLVLGVGGL QIMLDKGRDL DWFNSSTIII


 


LTVVSVISLI SLVIWESTSE NPILDLSLFK SRNFTIGIVS ITCAYLFYSG AIVLMPQLLQ


 


ETMGYNAIWA GLAYAPIGIM PLLISPLIGR YGNKIDMRLL VTFSFLMYAV CYYWRSVTFM


 


PTIDFTGIIL PQFFQGFAVA CFFLPLTTIS FSGLPDNKFA NASSMSNFFR TLSGSVGTSL


 


TMTLWGRRES LHHSQLTATI DQFNPVFNSS SQIMDKYYGS LSGVLNEINN EITQQSLSIS


 


ANEIFRMAAI AFILLTVLVW FAKPPFTAKG VG


 


SEQ ID NO. 46: GARP ORGANISM NAME: ESCHERICHIA COLI


MILDTVDEKK KGVHTRYLIL LIIFIVTAVN YADRATLSIA GTEVAKELQL SAVSMGYIFS


 


AFGWAYLLMQ IPGGWLLDKF GSKKVYTYSL FFWSLFTFLQ GFVDMFPLAW AGISMFFMRF


 


MLGFSEAPSF PANARIVAAW FPTKERGTAS AIFNSAQYFS LALFSPLLGW LTFAWGWEHV


 


FTVMGVIGFV LTALWIKLIH NPTDHPRMSA EELKFISENG AVVDMDHKKP GSAAASGPKL


 


HYIKQLLSNR MMLGVFFGQY FINTITWFFL TWFPIYLVQE KGMSILKVGL VASIPALCGF


 


AGGVLGGVFS DYLIKRGLSL TLARKLPIVL GMLLASTIIL CNYTNNTTLV VMLMALAFFG


 


KGFGALGWPV ISDTAPKEIV GLCGGVFNVF GNVASIVTPL VIGYLVSELH SFNAALVFVG


 


CSALMAMVCY LFVVGDIKRM ELQK


 


SEQ ID NO. 47: GUDP ORGANISM NAME: ESCHERICHIA COLI


MSSLSQAASS VEKRTNARYW IVVMLFIVTS FNYGDRATLS IAGSEMAKDI GLDPVGMGYV


 


FSAFSWAYVI GQIPGGWLLD RFGSKRVYFW SIFIWSMFTL LQGFVDIFSG FGIIVALFTL


 


RFLVGLAEAP SFPGNSRIVA AWFPAQERGT AVSIFNSAQY FATVIFAPIM GWLTHEVGWS


 


HVFFFMGGLG IVISFIWLKV IHEPNQHPGV NKKELEYIAA GGALINMDQQ NTKVKVPFSV


 


KWGQIKQLLG SRMMIGVYIG QYCINALTYF FITWFPVYLV QARGMSILKA GFVASVPAVC


 


GFIGGVLGGI ISDWLMRRTG SLNIARKTPI VMGMLLSMVM VFCNYVNVEW MIIGFMALAF


 


FGKGIGALGW AVMADTAPKE ISGLSGGLFN MFGNISGIVT PIAIGYIVGT TGSFNGALIY


 


VGVHALIAVL SYLVLVGDIK RIELKPVAGQ


 


SEQ ID NO. 48: HSRA ORGANISM NAME: ESCHERICHIA COLI


MSDKKKRSMA GLPWIAAMAF FMQALDATIL NTALPAIAHS LNRSPLAMQS AIISYTLTVA


 


MLIPVSGWLA DRFGTRRIFT LAVSLFTLGS LACALSNSLP QLVVFRVIQG IGGAMMMPVA


 


RLALLRAYPR NELLPVLNFV AMPGLVGPIL GPVLGGVLVT WATWHWIFLI NIPIGIAGLL


 


YARKHMPNFT TARRRFDITG FLLFGLSLVL FSSGIELFGE KIVASWIALT VIVTSIGLLL


 


LYILHARRTP NPLISLDLFK TRTFSIGIVG NIATRLGTGC VPFLMPLMLQ VGFGYQAFIA


 


GCMMAPTALG SIIAKSMVTQ VLRRLGYRHT LVGITVIIGL MIAQFSLQSP AMAIWMLILP


 


LFILGMAMST QFTAMNTITL ADLTDDNASS GNSVLAVTQQ LSISLGVAVS AAVLRVYEGM


 


EGTTTVEQFH YTFITMGIIT VASAAMFMLL KTTDGNNLIK RQRKSKPNRV PSESE


 


SEQ ID NO. 49: LEUE ORGANISM NAME: ESCHERICHIA COLI


MFAEYGVLNY WTYLVGAIFI VLVPGPNTLF VLKNSVSSGM KGGYLAACGV FIGDAVLMFL


 


AWAGVATLIK TTPILFNIVR YLGAFYLLYL GSKILYATLK GKNSEAKSDE PQYGAIFKRA


 


LILSLTNPKA ILFYVSFFVQ FIDVNAPHTG ISFFILAATL ELVSFCYLSF LIISGAFVTQ


 


YIRTKKKLAK VGNSLIGLMF VGFAARLATL QS


 


SEQ ID NO. 50: MDLB ORGANISM NAME: ESCHERICHIA COLI


MRSFSQLWPT LKRLLAYGSP WRKPLGIAVL MMWVAAAAEV SGPLLISYFI DNMVAKNNLP


 


LKVVAGLAAA YVGLQLFAAG LHYAQSLLFN RAAVGVVQQL RTDVMDAALR QPLSEFDTQP


 


VGQVISRVTN DTEVIRDLYV TVVATVLRSA ALVGAMLVAM FSLDWRMALV AIMIFPVVLV


 


VMVIYQRYST PIVRRVRAYL ADINDGFNEI INGMSVIQQF RQQARFGERM GEASRSHYMA


 


RMQTLRLDGF LLRPLLSLFS SLILCGLLML FGFSASGTIE VGVLYAFISY LGRLNEPLIE


 


LTTQQAMLQQ AVVAGERVFE LMDGPRQQYG NDDRPLQSGT IEVDNVSFAY RDDNLVLKNI


 


NLSVPSRNFV ALVGHTGSGK STLASLLMGY YPLTEGEIRL DGRPLSSLSH SALRQGVAMV


 


QQDPVVLADT FLANVTLGRD ISEERVWQAL ETVQLAELAR SMSDGIYTPL GEQGNNLSVG


 


QKQLLALARV LVETPQILIL DEATASIDSG TEQAIQHALA AVREHTTLVV IAHRLSTIVD


 


ADTILVLHRG QAVEQGTHQQ LLAAQGRYWQ MYQLQLAGEE LAASVREEES LSA


 


SEQ ID NO. 51: MDTD ORGANISM NAME: ESCHERICHIA COLI


MTDLPDSTRW QLWIVAFGFF MQSLDTTIVN TALPSMAQSL GESPLHMHMV IVSYVLTVAV


 


MLPASGWLAD KVGVRNIFFT AIVLFTLGSL FCALSGTLNE LLLARALQGV GGAMMVPVGR


 


LTVMKIVPRE QYMAAMTFVT LPGQVGPLLG PALGGLLVEY ASWHWIFLIN IPVGIIGAIA


 


TLLLMPNYTM QTRRFDLSGF LLLAVGMAVL TLALDGSKGT GLSPLTIAGL VAVGVVALVL


 


YLLHARNNNR ALFSLKLFRT RTFSLGLAGS FAGRIGSGML PFMTPVFLQI GLGFSPFHAG


 


LMMIPMVLGS MGMKRIVVQV VNRFGYRRVL VATTLGLSLV TLLFMTTALL GWYYVLPFVL


 


FLQGMVNSTR FSSMNTLTLK DLPDNLASSG NSLLSMIMQL SMSIGVTIAG LLLGLFGSQH


 


VSVDSGTTQT VFMYTWLSMA LIIALPAFIF ARVPNDTHQN VAISRRKRSA Q


 


SEQ ID NO. 52: MDTG ORGANISM NAME: ESCHERICHIA COLI


MSPCENDTPI NWKRNLIVAW LGCFLTGAAF SLVMPFLPLY VEQLGVTGHS ALNMWSGIVF


 


SITFLFSAIA SPFWGGLADR KGRKLMLLRS ALGMGIVMVL MGLAQNIWQF LILRALLGLL


 


GGFVPNANAL IATQVPRNKS GWALGTLSTG GVSGALLGPM AGGLLADSYG LRPVFFITAS


 


VLILCFFVTL FCIREKFQPV SKKEMLHMRE VVTSLKNPKL VLSLFVTTLI IQVATGSIAP


 


ILTLYVRELA GNVSNVAFIS GMIASVPGVA ALLSAPRLGK LGDRIGPEKI LITALIFSVL


 


LLIPMSYVQT PLQLGILRFL LGAADGALLP AVQTLLVYNS SNQIAGRIFS YNQSFRDIGN


 


VTGPLMGAAI SANYGFRAVF LVTAGVVLFN AVYSWNSLRR RRIPQVSN


 


SEQ ID NO. 53: MDTL ORGANISM NAME: ESCHERICHIA COLI


MSRFLICSFA LVLLYPAGID MYLVGLPRIA ADLNASEAQL HIAFSVYLAG MAAAMLFAGK


 


VADRSGRKPV AIPGAALFII ASVFCSLAET STLFLAGRFL QGLGAGCCYV VAFAILRDTL


 


DDRRRAKVLS LLNGITCIIP VLAPVLGHLI MLKFPWQSLF WAMAMMGIAV LMLSLFILKE


 


TRPAAPAASD KPRENSESLL NRFFLSRVVI TTLSVSVILT FVNTSPVLLM EIMGFERGEY


 


ATIMALTAGV SMTVSFSTPF ALGIFKPRTL MITSQVLFLA AGITLAVSPS HAVSLFGITL


 


ICAGFSVGFG VAMSQALGPF SLRAGVASST LGIAQVCGSS LWIWLAAVVG IGAWNMLIGI


 


LIACSIVSLL LIMFVAPGRP VAAHEEIHHH A


 


SEQ ID NO. 54: MDTM ORGANISM NAME: ESCHERICHIA COLI


MPRFFTRHAA TLFFPMALIL YDFAAYLSTD LIQPGIINVV RDFNADVSLA PAAVSLYLAG


 


GMALQWLLGP LSDRIGRRPV LITGALIFTL ACAATMFTTS MTQFLIARAI QGTSICFIAT


 


VGYVTVQEAF GQTKGIKLMA IITSIVLIAP IIGPLSGAAL MHFMHWKVLF AIIAVMGFIS


 


FVGLLLAMPE TVKRGAVPFS AKSVLRDFRN VFCNRLFLFG AATISLSYIP MMSWVAVSPV


 


ILIDAGSLTT SQFAWTQVPV FGAVIVANAI VARFVKDPTE PRFIWRAVPI QLVGLSLLIV


 


GNLLSPHVWL WSVLGTSLYA FGIGLIFPTL FRFTLFSNKL PKGTVSASLN MVILMVMSVS


 


VEIGRWLWFN GGRLPFHLLA VVAGVIVVFT LAGLLNRVRQ HQAAELVEEQ


 


SEQ ID NO. 55: MHPT ORGANISM NAME: ESCHERICHIA COLI


MSTRTPSSSS SRLMLTIGLC FLVALMEGLD LQAAGIAAGG IAQAFALDKM QMGWIFSAGI


 


LGLLPGALVG GMLADRYGRK RILIGSVALF GLFSLATAIA WDFPSLVFAR LMTGVGLGAA


 


LPNLIALTSE AAGPRFRGTA VSLMYCGVPI GAALAATLGF AGANLAWQTV FWVGGVVPLI


 


LVPLLMRWLP ESAVFAGEKQ SAPPLRALFA PETATATLLL WLCYFFTLLV VYMLINWLPL


 


LLVEQGFQPS QAAGVMFALQ MGAASGTLML GALMDKLRPV TMSLLIYSGM LASLLALGTV


 


SSFNGMLLAG FVAGLFATGG QSVLYALAPL FYSSQIRATG VGTAVAVGRL GAMSGPLLAG


 


KMLALGTGTV GVMAASAPGI LVAGLAVFIL MSRRSRIQPC ADA


 


SEQ ID NO. 56: RHTA ORGANISM NAME: ESCHERICHIA COLI


MPGSLRKMPV WLPIVILLVA MASIQGGASL AKSLFPLVGA PGVTALRLAL GTLILIAFFK


 


PWRLRFAKEQ RLPLLFYGVS LGGMNYLFYL SIQTVPLGIA VALEFTGPLA VALFSSRRPV


 


DFVWVVLAVL GLWFLLPLGQ DVSHVDLTGC ALALGAGACW AIYILSGQRA GAEHGPATVA


 


IGSLIAALIF VPIGALQAGE ALWHWSVIPL GLAVAILSTA LPYSLEMIAL TRLPTRTFGT


 


LMSMEPALAA VSGMIFLGET LTPIQLLALG AIIAASMGST LTVRKESKIK ELDIN


 


SEQ ID NO. 57: RHTB ORGANISM NAME: ESCHERICHIA COLI


MTLEWWFAYL LTSIILSLSP GSGAINTMTT SLNHGYRGAV ASIAGLQTGL AIHIVLVGVG


 


LGTLFSRSVI AFEVLKWAGA AYLIWLGIQQ WRAAGAIDLK SLASTQSRRH LFQRAVFVNL


 


TNPKSIVFLA ALFPQFIMPQ QPQLMQYIVL GVTTIVVDII VMIGYATLAQ RIALWIKGPK


 


QMKALNKIFG SLFMLVGALL ASARHA


 


SEQ ID NO. 58: RHTC ORGANISM NAME: ESCHERICHIA COLI


MLMLFLTVAM VHIVALMSPG PDFFFVSQTA VSRSRKEAMM GVLGITCGVM VWAGIALLGL


 


HLIIEKMAWL HTLIMVGGGL YLCWMGYQML RGALKKEAVS APAPQVELAK SGRSFLKGLL


 


TNLANPKAII YFGSVFSLFV GDNVGTTARW GIFALIIVET LAWFTVVASL FALPQMRRGY


 


QRLAKWIDGF AGALFAGFGI HLIISR


 


SEQ ID NO. 59: YAHN ORGANISM NAME: ESCHERICHIA COLI


MMQLVHLFMD EITMDPLHAV YLTVGLFVIT FFNPGANLFV VVQTSLASGR RAGVLTGLGV


 


ALGDAFYSGL GLFGLATLIT QCEEIFSLIR IVGGAYLLWF AWCSMRRQST PQMSTLQQPI


 


SAPWYVFFRR GLITDLSNPQ TVLFFISIFS VTLNAETPTW ARLMAWAGIV LASIIWRVFL


 


SQAFSLPAVR RAYGRMQRVA SRVIGAIIGV FALRLIYEGV TQR


 


SEQ ID NO. 60: YAJR ORGANISM NAME: ESCHERICHIA COLI


MNDYKMTPGE RRATWGLGTV FSLRMLGMFM VLPVLTTYGM ALQGASEALI GIAIGIYGLT


 


QAVFQIPFGL LSDRIGRKPL IVGGLAVFAA GSVIAALSDS IWGIILGRAL QGSGAIAAAV


 


MALLSDLTRE QNRTKAMAFI GVSFGITFAI AMVLGPIITH KLGLHALFWM IAILATTGIA


 


LTIWVVPNSS THVLNRESGM VKGSFSKVLA EPRLLKLNFG IMCLHILLMS TFVALPGQLA


 


DAGFPAAEHW KVYLATMLIA FGSVVPFIIY AEVKRKMKQV FVFCVGLIVV AEIVLWNAQT


 


QFWQLVVGVQ LFFVAFNLME ALLPSLISKE SPAGYKGTAM GVYSTSQFLG VAIGGSLGGW


 


INGMFDGQGV FLAGAMLAAV WLTVASTMKE PPYVSSLRIE IPANIAANEA LKVRLLETEG


 


IKEVLIAEEE HSAYVKIDSK VTNRFEIEQA IRQA


 


SEQ ID NO. 61: YBBP ORGANISM NAME: ESCHERICHIA COLI


MIARWFWREW RSPSLLIVWL ALSLAVACVL ALGNISDRME KGLSQQSREF MAGDRALRSS


 


REVPQAWLEE AQKRGLKVGK QLTFATMTFA GDTPQLANVK AVDDIYPMYG DLQTNPPGLK


 


PQAGSVLLAP RLMALLNLKT GDTIDVGDAT LRIAGEVIQE PDSGFNPFQM APRLMMNLAD


 


VDKTGAVQPG SRVTWRYKFG GNENQLDGYE KWLLPQLKPE QRWYGLEQDE GALGRSMERS


 


QQFLLLSALL TLLLAVAAVA VAMNHYCRSR YDLVAILKTL GAGRAQLRKL IVGQWLMVLT


 


LSAVTGGAIG LLFENVLMVL LKPVLPAALP PASLWPWLWA LGTMTVISLL VGLRPYRLLL


 


ATQPLRVLRN DVVANVWPLK FYLPIVSVVV VLLLAGLMGG SMLLWAVLAG AVVLALLCGV


 


LGWMLLNVLR RMTLKSLPLR LAVSRLLRQP WSTLSQLSAF SLSFMLLALL LVLRGDLLDR


 


WQQQLPPESP NYFLINIATE QVAPLKAFLA EHQIVPESFY PVVRARLTAI NDKPTEGNED


 


EALNRELNLT WQNTRPDHNP IVAGNWPPKA DEVSMEEGLA KRLNVALGDT VTFMGDTQEF


 


RAKVTSLRKV DWESLRPNFY FIFPEGALDG QPQSWLTSFR WENGNGMLTQ LNRQFPTISL


 


LDIGAILKQV GQVLEQVSRA LEVMVVLVTA CGMLLLLAQV QVGMRQRHQE LVVWRTLGAG


 


KKLLRTTLWC EFAMLGFVSG LVAAIGAETA LAVLQAKVFD FPWEPDWRLW IVLPCSGALL


 


LSLFGGWLGA RLVKGKALFR QFAG


 


SEQ ID NO. 62: YBIF (RHTA) ORGANISM NAME: ESCHERICHIA COLI


MPGSLRKMPV WLPIVILLVA MASIQGGASL AKSLFPLVGA PGVTALRLAL GTLILIAFFK


 


PWRLRFAKEQ RLPLLFYGVS LGGMNYLFYL SIQTVPLGIA VALEFTGPLA VALFSSRRPV


 


DFVWVVLAVL GLWFLLPLGQ DVSHVDLTGC ALALGAGACW AIYILSGQRA GAEHGPATVA


 


IGSLIAALIF VPIGALQAGE ALWHWSVIPL GLAVAILSTA LPYSLEMIAL TRLPTRTFGT


 


LMSMEPALAA VSGMIFLGET LTPIQLLALG AIIAASMGST LTVRKESKIK ELDIN


 


SEQ ID NO. 63: YBJJ ORGANISM NAME: ESCHERICHIA COLI


MTVNSSRNAL KRRTWALFMF FFLPGLLMAS WATRTPAIRD ILSVSIAEMG GVLFGLSIGS


 


MSGILCSAWL VKRFGTRNVI LVTMSCALIG MMILSLALWL TSPLLFAVGL GVFGASFGSA


 


EVAINVEGAA VEREMNKTVL PMMHGFYSLG TLAGAGVGMA LTAFGVPATV HILLAALVGI


 


APIYIAIQAI PDGTGKNAAD GTQHGEKGVP FYRDIQLLLI GVVVLAMAFA EGSANDWLPL


 


LMVDGHGFSP TSGSLIYAGF TLGMTVGRFT GGWFIDRYSR VAVVRASALM GALGIGLIIF


 


VDSAWVAGVS VVLWGLGASL GFPLTISAAS DTGPDAPTRV SVVATTGYLA FLVGPPLLGY


 


LGEHYGLRSA MLVVLALVIL AAIVAKAVAK PDTKTQTAME NS


 


SEQ ID NO. 64: YCAP ORGANISM NAME: ESCHERICHIA COLI


MKAFDLHRMA FDKVPFDFLG EVALRSLYTF VLVFLFLKMT GRRGVRQMSL FEVLIILTLG


 


SAAGDVAFYD DVPMVPVLIV FITLALLYRL VMWLMAHSEK LEDLLEGKPV VIIEDGELAW


 


SKLNNSNMTE FEFFMELRLR GVEQLGQVRL AILETNGQIS VYFFEDDKVK PGLLILPSDC


 


TQRYKVVPES ADYACIRCSE IIHMKAGEKQ LCPRCANPEW TKASRAKRVT


 


SEQ ID NO. 65: YDCO ORGANISM NAME: ESCHERICHIA COLI


MRLFSIPPPT LLAGFLAVLI GYASSAAIIW QAAIVAGATT AQISGWMTAL GLAMGVSTLT


 


LTLWYRVPVL TAWSTPGAAL LVTGLQGLTL NEAIGVFIVT NALIVLCGIT GLFARLMRII


 


PHSLAAAMLA GILLRFGLQA FASLDGQFTL CGSMLLVWLA TKAVAPRYAV IAAMIIGIVI


 


VIAQGDVVTT DVVFKPVLPT YITPDFSFAH SLSVALPLFL VTMASQNAPG IAAMKAAGYS


 


APVSPLIVFT GLLALVFSPF GVYSVGIAAI TAAICQSPEA HPDKDQRWLA AAVAGIFYLL


 


AGLFGSAITG MMAALPVSWI QMLAGLALLS TIGGSLYQAL HNERERDAAV VAFLVTASGL


 


TLVGIGSAFW GLIAGGVCYV VLNLIADRNRY


 


SEQ ID NO. 66: YDDG ORGANISM NAME: ESCHERICHIA COLI


MTRQKATLIG LIAIVLWSTM VGLIRGVSEG LGPVGGAAAI YSLSGLLLIF TVGFPRIRQI


 


PKGYLLAGSL LFVSYEICLA LSLGYAATHH QAIEVGMVNY LWPSLTILFA ILFNGQKTNW


 


LIVPGLLLAL VGVCWVLGGD NGLHYDEIIN NITTSPLSYF LAFIGAFIWA AYCTVTNKYA


 


RGFNGITVFV LLTGASLWVY YFLTPQPEMI FSTPVMIKLI SAAFTLGFAY AAWNVGILHG


 


NVTIMAVGSY FTPVLSSALA AVLLSAPLSF SFWQGALMVC GGSLLCWLAT RRG


 


SEQ ID NO. 67: YDED (EAMA) ORGANISM NAME: ESCHERICHIA COLI


MSRKDGVLAL LVVVVWGLNF VVIKVGLHNM PPLMLAGLRF MLVAFPAIFF VARPKVPLNL


 


LLGYGLTISF AQFAFLFCAI NFGMPAGLAS LVLQAQAFFT IMLGAFTFGE RLHGKQLAGI


 


ALAIFGVLVL IEDSLNGQHV AMLGFMLTLA AAFSWACGNI FNKKIMSHST RPAVMSLVIW


 


SALIPIIPFF VASLILDGSA TMIHSLVTID MTTILSLMYL AFVATIVGYG IWGTLLGRYE


 


TWRVAPLSLL VPVVGLASAA LLLDERLTGL QFLGAVLIMT GLYINVFGLR WRKAVKVGS


 


SEQ ID NO. 68: YDGE (MDTI) ORGANISM NAME: ESCHERICHIA COLI


MAQFEWVHAA WLALAIVLEI VANVFLKFSD GFRRKIFGLL SLAAVLAAFS ALSQAVKGID


 


LSVAYALWGG FGIAATLAAG WILFGQRLNR KGWIGLVLLL AGMIMVKLA


 


SEQ ID NO. 69: YDHC ORGANISM NAME: ESCHERICHIA COLI


MQPGKRFLVW LAGLSVLGFL ATDMYLPAFA AIQADLQTPA SAVSASLSLF LAGFAAAQLL


 


WGPLSDRYGR KPVLLIGLTI FALGSLGMLW VENAATLLVL RFVQAVGVCA AAVIWQALVT


 


DYYPSQKVNR IFAAIMPLVG LSPALAPLLG SWLLVHFSWQ AIFATLFAIT VVLILPIFWL


 


KPTTKARNNS QDGLTFTDLL RSKTYRGNVL IYAACSASFF AWLTGSPFIL SEMGYSPAVI


 


GLSYVPQTIA FLIGGYGCRA ALQKWQGKQL LPWLLVLFAV SVIATWAAGF ISHVSLVEIL


 


IPFCVMAIAN GAIYPIVVAQ ALRPFPHATG RAAALQNTLQ LGLCFLASLV VSWLISISTP


 


LLTTTSVMLS TVVLVALGYM MQRCEEVGCQ NHGNAEVAHS ESH


 


SEQ ID NO. 70: YDHP ORGANISM NAME: ESCHERICHIA COLI


MKINYPLLAL AIGAFGIGTT EFSPMGLLPV IARGVDVSIP AAGMLISAYA VGVMVGAPLM


 


TLLLSHRARR SALIFLMAIF TLGNVLSAIA PDYMTLMLSR ILTSLNHGAF FGLGSVVAAS


 


VVPKHKQASA VATMFMGLTL ANIGGVPAAT WLGETIGWRM SFLATAGLGV ISMVSLFFSL


 


PKGGAGARPE VKKELAVLMR PQVLSALLTT VLGAGAMFTL YTYISPVLQS ITHATPVFVT


 


AMLVLIGVGF SIGNYLGGKL ADRSVNGTLK GFLLLLMVIM LAIPFLARNE FGAAISMVVW


 


GAATFAVVPP LQMRVMRVAS EAPGLSSSVN IGAFNLGNAL GAAAGGAVIS AGLGYSFVPV


 


MGAIVAGLAL LLVFMSARKQ PETVCVANS


 


SEQ ID NO. 71: YDIN ORGANISM NAME: ESCHERICHIA COLI


MSQNKAFSTP FILAVLCIYF SYFLHGISVI TLAQNMSSLA EKFSTDNAGI AYLISGIGLG


 


RLISILFFGV ISDKFGRRAV ILMAVIMYLL FFFGIPACPN LTLAYGLAVC VGIANSALDT


 


GGYPALMECF PKASGSAVIL VKAMVSFGQM FYPMLVSYML LNNIWYGYGL IIPGILFVLI


 


TLMLLKSKFP SQLVDASVTN ELPQMNSKPL VWLEGVSSVL FGVAAFSTFY VIVVWMPKYA


 


MAFAGMSEAE ALKTISYYSM GSLVCVFIFA ALLKKMVRPI WANVFNSALA TITAAIIYLY


 


PSPLVCNAGA FVIGFSAAGG ILQLGVSVMS EFFPKSKAKV TSIYMMMGGL ANFVIPLITG


 


YLSNIGLQYI IVLDFTFALL ALITAIIVFI RYYRVFIIPE NDVRFGERKF CTRLNTIKHR


 


G


 


SEQ ID NO. 72: YDIM ORGANISM NAME: ESCHERICHIA COLI


MKNPYFPTAL GLYFNYLVHG MGVLLMSLNM ASLETLWQTN AAGVSIVISS LGIGRLSVLL


 


FAGLLSDRFG RRPFIMLGMC CYMAFFFGIL QTNNIIIAYV FGFLAGMANS FLDAGTYPSL


 


MEAFPRSPGT ANILIKAFVS SGQFLLPLII SLLVWAELWF GWSFMIAAGI MFINALFLYR


 


CTFPPHPGRR LPVIKKTTSS TEHRCSIIDL ASYTLYGYIS MATFYLVSQW LAQYGQFVAG


 


MSYTMSIKLL SIYTVGSLLC VFITAPLIRN TVRPTTLLML YTFISFIALF TVCLHPTFYV


 


VIIFAFVIGF TSAGGVVQIG LTLMAERFPY AKGKATGIYY SAGSIATFTI PLITAHLSQR


 


SIADIMWFDT AIAAIGFLLA LFIGLRSRKK TRHHSLKENV APGG


 


SEQ ID NO. 73: YDJE ORGANISM NAME: ESCHERICHIA COLI


MEQYDQIGAR LDRLPLARFH YRIFGIISFS LLLTGFLSYS GNVVLAKLVS NGWSNNFLNA


 


AFTSALMFGY FIGSLTGGFI GDYFGRRRAF RINLLIVGIA ATGAAFVPDM YWLIFFRFLM


 


GTGMGALIMV GYASFTEFIP ATVRGKWSAR LSFVGNWSPM LSAAIGVVVI AFFSWRIMFL


 


LGGIGILLAW FLSGKYFIES PRWLAGKGQI AGAECQLREV EQQIEREKSI RLPPLTSYQS


 


NSKVKVIKGT FWLLFKGEML RRTLVAITVL IAMNISLYTI TVWIPTIFVN SGIDVDKSIL


 


MTAVIMIGAP VGIFIAALII DHFPRRLFGS TLLIIIAVLG YIYSIQTTEW AILIYGLVMI


 


FFLYMYVCFA SAVYIPELWP THLRLRGSGF VNAVGRIVAV FTPYGVAALL THYGSITVFM


 


VLGVMLLLCA LVLSIFGIET RKVSLEEISE VN


 


SEQ ID NO. 74: YDJF ORGANISM NAME: ESCHERICHIA COLI


MAAKDRIQAI KQMVANDKKV TVSNLSGIFQ VTEETIRRDL EKLEDEGFLT RTYGGAVLNT


 


AMLTENIHFY KRASSFYEEK QLIARKALPF IDNKTTMAAD SSSTVMELLK LLQDRSGLTL


 


LTNSAEAIHV LAQSEIKVVS TGGELNKNTL SLQGRITKEI IRRYHVDIMV MSCKGLDINS


 


GALDSNEAEA EIKKTMIRQA TEVALLVDHS KFDRKAFVQL ADFSHINYII TDKSPGAEWI


 


AFCKDNNIQL VW


 


SEQ ID NO. 75: YDJI ORGANISM NAME: ESCHERICHIA COLI


MLADIRYWEN DATNKHYAIA HFNVWNAEML MGVIDAAEEA KSPVIISFGT GFVGNTSFED


 


FSHMMVSMAQ KATVPVITHW DHGRSMEIIH NAWTHGMNSL MRDASAFDFE ENIRLTKEAV


 


DFFHPLGIPV EAELGHVGNE TVYEEALAGY HYTDPDQAAE FVERTGCDSL AVAIGNQHGV


 


YTSEPQLNFE VVKRVRDAVS VPLVLHGASG ISDADIKTAI SLGIAKINIH TELCQAAMVA


 


VKENQDQPFL HLEREVRKAV KERALEKIKL FGSDGKAE


 


SEQ ID NO. 76: YDJK ORGANISM NAME: ESCHERICHIA COLI


MEQITKPHCG ARLDRLPDCR WHSSMFAIVA FGLLVCWSNA VGGLILAQLK ALGWTDNSTT


 


ATFSAITTAG MFLGALVGGI IGDKTGRRNA FILYEAIHIA SMVVGAFSPN MDFLIACRFV


 


MGVGLGALLV TLFAGFTEYM PGRNRGTWSS RVSFIGNWSY PLCSLIAMGL TPLISAEWNW


 


RVQLLIPAIL SLIATALAWR YFPESPRWLE SRGRYQEAEK VMRSIEEGVI RQTGKPLPPV


 


VIADDGKAPQ AVPYSALLTG VLLKRVILGS CVLIAMNVVQ YTLINWLPTI FMTQGINLKD


 


SIVLNTMSMF GAPFGIFIAM LVMDKIPRKT MGVGLLILIA VLGYIYSLQT SMLLITLIGF


 


FLITFVYMYV CYASAVYVPE IWPTEAKLRG SGLANAVGRI SGIAAPYAVA VLLSSYGVTG


 


VFILLGAVSI IVAIAIATIG IETKGVSVES LSIDAVANK


 


SEQ ID NO. 77: YEAN ORGANISM NAME: ESCHERICHIA COLI


MTCSTSLSGK NRIVLIAGIL MIATTLRVTF TGAAPLLDTI RSAYSLTTAQ TGLLTTLPLL


 


AFALISPLAA PVARRFGMER SLFAALLLIC AGIAIRSLPS PYLLFGGTAV IGGGIALGNV


 


LLPGLIKRDF PHSVARLTGA YSLTMGAAAA LGSAMVVPLA LNGFGWQGAL LMLMCFPLLA


 


LFLWLPQWRS QQHANLSTSR ALHTRGIWRS PLAWQVTLFL GINSLVYYVI IGWLPAILIS


 


HGYSEAQAGS LHGLLQLATA APGLLIPLFL HHVKDQRGIA AFVALMCAVG AVGLCFMPAH


 


AITWTLLFGF GSGATMILGL TFIGLRASSA HQAAALSGMA QSVGYLLAAC GPPLMGKIHD


 


ANGNWSVPLM GVAILSLLMA IFGLCAGRDK EIR


 


SEQ ID NO. 78: YEAS (LEUE) ORGANISM NAME: ESCHERICHIA COLI


MFAEYGVLNY WTYLVGAIFI VLVPGPNTLF VLKNSVSSGM KGGYLAACGV FIGDAVLMFL


 


AWAGVATLIK TTPILFNIVR YLGAFYLLYL GSKILYATLK GKNSEAKSDE PQYGAIFKRA


 


LILSLTNPKA ILFYVSFFVQ FIDVNAPHTG ISFFILAATL ELVSFCYLSF LIISGAFVTQ


 


YIRTKKKLAK VGNSLIGLMF VGFAARLATL QS


 


SEQ ID NO. 79: YEBS ORGANISM NAME: ESCHERICHIA COLI


MALNTPQITP TKKITVRAIG EELPRGDYQR CPQCDMLFSL PEINSHQSAY CPRCQAKIRD


 


GRDWSLTRLA AMAFTMLLLM PFAWGEPLLH IWLLGIRIDA NVMQGIWQMT KQGDAITGSM


 


VFFCVIGAPL ILVTSIAYLW FGNRLGMNLR PVLLMLERLK EWVMLDIYLV GIGVASIKVQ


 


DYAHIQAGVG LFSFVALVIL TTVTLSHLNV EELWERFYPQ RPATRRDEKL RVCLGCHFTG


 


YPDQRGRCPR CHIPLRLRRR HSLQKCWAAL LASIVLLLPA NLLPISIIYL NGGRQEDTIL


 


SGIMSLASSN IAVAGIVFIA SILVPFTKVI VMFTLLLSIH FKCQQGLRTR ILLLRMVTWI


 


GRWSMLDLFV ISLTMSLINR DQILAFTMGP AAFYFGAAVI LTILAVEWLD SRLLWDAHES


 


GNARFDD


 


SEQ ID NO. 80: YEDA ORGANISM NAME: ESCHERICHIA COLI


MRFRQLLPLF GALFALYIIW GSTYFVIRIG VESWPPLMMA GVRFLAAGIL LLAFLLLRGH


 


KLPPLRPLLN AALIGLLLLA VGNGMVTVAE HQNVPSGIAA VVVATVPLFT LCFSRLFGIK


 


TRKLEWVGIA IGLAGIIMLN SGGNLSGNPW GAILILIGSI SWAFGSVYGS RITLPVGMMA


 


GAIEMLAAGV VLMIASMIAG EKLTALPSLS GFLAVGYLAL FGSIIAINAY MYLIRNVSPA


 


LATSYAYVNP VVAVLLGTGL GGETLSKIEW LALGVIVFAV VLVTLGKYLF PAKPVVAPVI


 


QDASSE


 


SEQ ID NO. 90: YEEO ORGANISM NAME: ESCHERICHIA COLI


MLRHILTAKN LLSNPIFKFP NCLPFLSTVC CICRQFVGEN LCSFADSPSL FEMWFHFLQL


 


RSALNISSAL RQVVHGTRWH AKRKSYKVLF WREITPLAVP IFMENACVLL MGVLSTFLVS


 


WLGKDAMAGV GLADSFNMVI MAFFAAIDLG TTVVVAFSLG KRDRRRARVA TRQSLVIMTL


 


FAVLLATLIH HFGEQIIDFV AGDATTEVKA LALTYLELTV LSYPAAAITL IGSGALRGAG


 


NTKIPLLING SLNILNIIIS GILIYGLFSW PGLGFVGAGL GLTISRYIGA VAILWVLAIG


 


FNPALRISLK SYFKPLNFSI IWEVMGIGIP ASVESVLFTS GRLLTQMFVA GMGTSVIAGN


 


FIAFSIAALI NLPGSALGSA STIITGRRLG VGQIAQAEIQ LRHVFWLSTL GLTAIAWLTA


 


PFAGVMASFY TQDPQVKHVV VILIWLNALF MPIWSASWVL PAGFKGARDA RYAMWVSMLS


 


MWGCRVVVGY VLGIMLGWGV VGVWMGMFAD WAVRAVLFYW RMVTGRWLWK YPRPEPQKCE


 


KKPVVSE


 


SEQ ID NO. 91: YEGH ORGANISM NAME: ESCHERICHIA COLI


MEWIADPSIW AGLITLIVIE LVLGIDNLVF IAILAEKLPP KQRDRARVTG LLLAMLMRLL


 


LLASISWLVT LTQPLFSFRS FTFSARDLIM LFGGFFLLFK ATMELNERLE GKDSNNPTQR


 


KGAKFWGVVT QIVVLDAIFS LDSVITAVGM VDHLLVMMAA VVIAISLMLM ASKPLTQFVN


 


SHPTIVILCL SFLLMIGFSL VAEGFGFVIP KGYLYAAIGF SVMIEALNQL AIFNRRRFLS


 


ANQTLRQRTT EAVMRLLSGQ KEDAELDAET ASMLVDHGNQ QIFNPQERRM IERVLNLNQR


 


TVSSIMTSRH DIEHIDLNAP EEEIRQLLER NQHTRLVVTD GDDAEDLLGV VHVIDLLQQS


 


LRGEPLNLRV LIRQPLVFPE TLPLLPALEQ FRNARTHFAF VVDEFGSVEG IVTLSDVTET


 


IAGNLPNEVE EIDARHDIQK NADGSWTANG HMPLEDLVQY VPLPLDEKRE YHTIAGLLME


 


YLQRIPKPGE EVQVGDYLLK TLQVESHRVQ KVQIIPLRKD GEMEYEV


 


SEQ ID NO. 92: YGGA (ARGO) ORGANISM NAME: ESCHERICHIA COLI


MFSYYFQGLA LGAAMILPLG PQNAFVMNQG IRRQYHIMIA LLCAISDLVL ICAGIFGGSA


 


LLMQSPWLLA LVTWGGVAFL LWYGFGAFKT AMSSNIELAS AEVMKQGRWK IIATMLAVTW


 


LNPHVYLDTF VVLGSLGGQL DVEPKRWFAL GTISASFLWF FGLALLAAWL APRLRTAKAQ


 


RIINLVVGCV MWFIALQLAR DGIAHAQALF S


 


SEQ ID NO. 93: YFCJ ORGANISM NAME: ESCHERICHIA COLI


MTAVSQTETR SSANFSLFRI AFAVFLTYMT VGLPLPVIPL FVHHELGYGN TMVGIAVGIQ


 


FLATVLTRGY AGRLADQYGA KRSALQGMLA CGLAGGALLL AAILPVSAPF KFALLVVGRL


 


ILGFGESQLL TGALTWGLGI VGPKHSGKVM SWNGMAIYGA LAVGAPLGLL IHSHYGFAAL


 


AITTMVLPVL AWACNGTVRK VPALAGERPS LWSVVGLIWK PGLGLALQGV GFAVIGTFVS


 


LYFASKGWAM AGFTLTAFGG AFVVMRVMFG WMPDRFGGVK VAIVSLLVET VGLLLLWQAP


 


GAWVALAGAA LTGAGCSLIF PALGVEVVKR VPSQVRGTAL GGYAAFQDIA LGVSGPLAGM


 


LATTFGYSSV FLAGAISAVL GIIVTILSFR RG


 


SEQ ID NO. 94: YFIK (EAMB) ORGANISM NAME: ESCHERICHIA COLI


MTPTLLSAFW TYTLITAMTP GPNNILALSS ATSHGFRQST RVLAGMSLGF LIVMLLCAGI


 


SFSLAVIDPA AVHLLSWAGA AYIVWLAWKI ATSPTKEDGL QAKPISFWAS FALQFVNVKI


 


ILYGVTALST FVLPQTQALS WVVGVSVLLA MIGTFGNVCW ALAGHLFQRL FRQYGRQLNI


 


VLALLLVYCA VRIFY


 


SEQ ID NO. 95: YHJE ORGANISM NAME: ESCHERICHIA COLI


MQATATTLDH EQEYTPINSR NKVLVASLIG TAIEFFDFYI YATAAVIVFP HIFFPQGDPT


 


AATLQSLATF AIAFVARPIG SAVFGHFGDR VGRKATLVAS LLTMGISTVV IGLLPGYATI


 


GIFAPLLLAL ARFGQGLGLG GEWGGAALLA TENAPPRKRA LYGSFPQLGA PIGFFFANGT


 


FLLLSWLLTD EQFMSWGWRV PFIFSAVLVI IGLYVRVSLH ESPVFEKVAK AKKQVKIPLG


 


TLLTKHVRVT VLGTFIMLAT YTLFYIMTVY SMTFSTAAAP VGLGLPRNEV LWMLMMAVIG


 


FGVMVPVAGL LADAFGRRKS MVIITTLIIL FALFAFNPLL GSGNPILVFA FLLLGLSLMG


 


LTFGPMGALL PELFPTEVRY TGASFSYNVA SILGASVAPY IAAWLQTNYG LGAVGLYLAA


 


MAGLTLIALL LTHETRHQSL


 


SEQ ID NO. 96: YIDE ORGANISM NAME: ESCHERICHIA COLI


MSDIALTVSI LALVAVVGLF IGNVKFRGIG LGIGGVLFGG IIVGHFVSQA GMTLSSDMLH


 


VIQEFGLILF VYTIGIQVGP GFFASLRVSG LRLNLFAVLI VIIGGLVTAI LHKLFDIPLP


 


VVLGIFSGAV TNTPALGAGQ QILRDLGTPM EMVDQMGMSY AMAYPFGICG ILFTMWMLRV


 


IFRVNVETEA QQHESSRTNG GALIKTINIR VENPNLHDLA IKDVPILNGD KIICSRLKRE


 


ETLKVPSPDT IIQLGDLLHL VGQPADLHNA QLVIGQEVDT SLSTKGTDLR VERVVVTNEN


 


VLGKRIRDLH FKERYDVVIS RLNRAGVELV ASGDISLQFG DILNLVGRPS AIDAVANVLG


 


NAQQKLQQVQ MLPVFIGIGL GVLLGSIPVF VPGFPAALKL GLAGGPLIMA LILGRIGSIG


 


KLYWFMPPSA NLALRELGIV LFLSVVGLKS GGDFVNTLVN GEGLSWIGYG ALITAVPLIT


 


VGILARMLAK MNYLTMCGML AGSMTDPPAL AFANNLHPTS GAAALSYATV YPLVMFLRII


 


TPQLLAVLFW SIG


 


SEQ ID NO. 97: YIGK (RHTB) ORGANISM NAME: ESCHERICHIA COLI


MTLEWWFAYL LTSIILSLSP GSGAINTMTT SLNHGYRGAV ASIAGLQTGL AIHIVLVGVG


 


LGTLFSRSVI AFEVLKWAGA AYLIWLGIQQ WRAAGAIDLK SLASTQSRRH LFQRAVFVNL


 


TNPKSIVFLA ALFPQFIMPQ QPQLMQYIVL GVTTIVVDII VMIGYATLAQ RIALWIKGPK


 


QMKALNKIFG SLFMLVGALL ASARHA


 


SEQ ID NO. 98: YIGJ (RHTC) ORGANISM NAME: ESCHERICHIA COLI


MLMLFLTVAM VHIVALMSPG PDFFFVSQTA VSRSRKEAMM GVLGITCGVM VWAGIALLGL


 


HLIIEKMAWL HTLIMVGGGL YLCWMGYQML RGALKKEAVS APAPQVELAK SGRSFLKGLL


 


TNLANPKAII YFGSVFSLFV GDNVGTTARW GIFALIIVET LAWFTVVASL FALPQMRRGY


 


QRLAKWIDGF AGALFAGFGI HLIISR


 


SEQ ID NO. 99: YIJE ORGANISM NAME: ESCHERICHIA COLI


MSAAGKSNPL AISGLVVLTL IWSYSWIFMK QVTSYIGAFD FTALRCIFGA LVLFIVLLLR


 


GRGMRPTPFK YTLAIALLQT CGMVGLAQWA LVSGGAGKVA ILSYTMPFWV VIFAALFLGE


 


RLRRGQYFAI LIAAFGLFLV LQPWQLDFSS MKSAMLAILS GVSWGASAIV AKRLYARHPR


 


VDLLSLTSWQ MLYAALVMSV VALLVPQREI DWQPTVFWAL AYSAILATAL AWSLWLFVLK


 


NLPASIASLS TLAVPVCGVL FSWWLLGENP GAVEGSGIVL IVLALALVSR KKKEAVSVKRI


 


SEQ ID NO. 100: YJII (KPTA) ORGANISM NAME: ESCHERICHIA COLI


MAKYNEKELA DTSKFLSFVL RHKPEAIGIV LDREGWADID KLILCAQKAG KRLTRALLDT


 


VVATSDKKRF SYSSDGRCIR AVQGHSTSQV AISFAEKTPP QFLYHGTASR FLDEIKKQGL


 


IAGERHYVHL SADEATARKV GARHGSPVIL TVKAQEMAKR GLPFWQAENG VWLTSTVAVE


 


FLEW


 


SEQ ID NO. 101: YJIJ ORGANISM NAME: ESCHERICHIA COLI


MPSSTHPVER FSFSTALFGM LVLTLGMGLG RFLYTPMLPV MMAEGSFSFS QLSWIASGNY


 


AGYLAGSLLF SFGAFHQPSR LRPFLLASAL ASGLLILAMA WLPPFILVLL IRVLAGVASA


 


GMLIFGSTLI MQHTRHPFVL AALFSGVGIG IALGNEYVLA GLHFDLSSQT LWQGAGALSG


 


MMLIALTLLM PSKKHAITPM PLAKTEQQIM SWWLLAILYG LAGFGYIIVA TYLPLMAKDA


 


GSPLLTAHLW TLVGLSIVPG CFGWLWAAKR WGALPCLTAN LLVQAICVLL TLASDSPLLL


 


IISSLGFGGT FMGTTSLVMT IARQLSVPGN LNLLGFVTLI YGIGQILGPA LTSMLSNGTS


 


ALASATLCGA AALFIAALIS TVQLFKLQVV TS


 


SEQ ID NO. 102: YJIO (MDTM) ORGANISM NAME: ESCHERICHIA COLI


MPRFFTRHAA TLFFPMALIL YDFAAYLSTD LIQPGIINVV RDFNADVSLA PAAVSLYLAG


 


GMALQWLLGP LSDRIGRRPV LITGALIFTL ACAATMFTTS MTQFLIARAI QGTSICFIAT


 


VGYVTVQEAF GQTKGIKLMA IITSIVLIAP IIGPLSGAAL MHFMHWKVLF AIIAVMGFIS


 


FVGLLLAMPE TVKRGAVPFS AKSVLRDFRN VFCNRLFLFG AATISLSYIP MMSWVAVSPV


 


ILIDAGSLTT SQFAWTQVPV FGAVIVANAI VARFVKDPTE PRFIWRAVPI QLVGLSLLIV


 


GNLLSPHVWL WSVLGTSLYA FGIGLIFPTL FRFTLFSNKL PKGTVSASLN MVILMVMSVS


 


VEIGRWLWFN GGRLPFHLLA VVAGVIVVFT LAGLLNRVRQ HQAAELVEEQ


 


SEQ ID NO. 103: YKGH ORGANISM NAME: ESCHERICHIA COLI


MREQIKQDID LIEILFYLKK KIRVILFIMA ICMAMVLLFL YINKDNIKVI YSLKINQTTP


 


GILVSCDSNN NFACQTTMTE DVIQRITTFF HTSPDVKNRE IRLEWSGDKR ALPTAEEEIS


 


RVQASIIKWY ASEYHNGRQV LDEIQTPSAI NSELYTKMIY LTRNWSLYPN GDGCVTISSP


 


EIKNKYPAAI CLALGFFLSI VISVMFCLVK KMVDEYQQNS GQ


 


SEQ ID NO. 104: YPJD ORGANISM NAME: ESCHERICHIA COLI


MQRLEQRSPD AILLLFLIAQ TVDITMPVFA LLALVAYSVS LALIVPGLLQ KNGGWRRMAI


 


ISAVIALVCH AIALEARILP DGDSGQNLSL LNVGSLVSLM ICTVMTIVAS RNRGWLLLPI


 


VYAFALINLA LATFMPNEYI THLEATPGML VHIGLSLFSY ATLIIAALYA LQLAWIDYQL


 


KNKKLAFNQE MPPLMSIERK MFHITQIGVV LLTLTLCTGL FYMHNLFSME NIDKAVLSIV


 


AWFVYIVLLW GHYHEGWRGR RVVWFNVAGA VILTLAYFGS RIVQQLIS


 


SEQ ID NO. 105: YTFF ORGANISM NAME: ESCHERICHIA COLI


MPVMISGVLY ALLAGLMWGL IFVGPLIVPE YPAMLQSMGR YLALGLIALP IAWLGRVRLR


 


QLARRDWLTA LMLTMMGNLI YYFCLASAIQ RTGAPVSTMI IGTLPVVIPV FANLLYSQRD


 


GKLAWGKLAP ALICIGIGLA CVNIAELNHG LPDFDWARYT SGIVLALVSV VCWAWYALRN


 


ARWLRENPDK HPMMWATAQA LVTLPVSLIG YLVACYWLNT QTPDFSLPFG PRPLVFISLM


 


VAIAVLCSWV GALCWNVASQ LLPTVILGPL IVFETLAGLL YTFLLRQQMP PLMTLSGIAL


 


LVIGVVIAVR AKPEKPLTES VSES


 


SEQ ID NO. 106: YTFL ORGANISM NAME: ESCHERICHIA COLI


MLNSILVILC LIAVSAFFSM SEISLAASRK IKLKLLADEG NINAQRVLNM QENPGMFFTV


 


VQIGLNAVAI LGGIVGDAAF SPAFHSLFSR YMSAELSEQL SFILSFSLVT GMFILFADLT


 


PKRIGMIAPE AVALRIINPM RFCLYVCTPL VWFFNGLANI IFRIFKLPMV RKDDITSDDI


 


YAVVEAGALA GVLRKQEHEL IENVFELESR TVPSSMTPRE NVIWFDLHED EQSLKNKVAE


 


HPHSKFLVCN EDIDHIIGYV DSKDLLNRVL ANQSLALNSG VQIRNTLIVP DTLTLSEALE


 


SFKTAGEDFA VIMNEYALVV GIITLNDVMT TLMGDLVGQG LEEQIVARDE NSWLIDGGTP


 


IDDVMRVLDI DEFPQSGNYE TIGGFMMFML RKIPKRTDSV KFAGYKFEVV DIDNYRIDQL


 


LVTRIDSKAT ALSPKLPDAK DKEESVA


 


SEQ ID NO. 107: CHLOROFLEXUS AGGREGANS (CGMCR)


<400> 785


MET SER VAL THR GLY ARG LEU ALA GLY LYS ILE ALA LEU ILE THR GLY


1       5          10         15


 


GLY ALA GLY ASN ILE GLY SER GLU MET THR ARG ARG PHE LEU ALA GLU


      20         25         30


 


GLY ALA THR VAL ILE ILE SER GLY ARG ASN SER ALA LYS LEU ALA ALA


    35         40         45


 


LEU ALA GLU ARG LEU ARG SER GLU ALA GLY VAL PRO ALA LYS ARG ILE


  50         55         60


 


ASP LEU GLU VAL MET ASP GLY SER ASP PRO ALA ALA VAL ARG ALA GLY


65         70         75         80


 


VAL ALA ALA ILE ILE GLY ARG HIS GLY HIS ILE ASP ILE LEU VAL ASN


       85          90          95


 


ASN ALA GLY SER THR GLY ALA GLN ARG ARG LEU ALA GLU ILE PRO LEU


     100         105         110


 


ASN GLU THR ASP ARG ASP LEU ASP ASP GLU GLU ALA LEU SER THR SER


   115         120         125


 


VAL ALA ASN LEU LEU GLY MET ALA TRP HIS LEU MET ARG ILE LEU SER


 130         135         140


 


PRO HIS MET PRO PRO GLY SER ALA ILE ILE ASN ILE SER THR ILE PHE


145         150         155         160


 


SER ARG ALA GLU TYR TYR GLY ARG ILE PRO TYR VAL VAL PRO LYS ALA


        165         170         175


 


ALA LEU ASN THR LEU THR GLN ILE ALA ALA ARG GLU LEU GLY ILE ARG


    180         185         190


 


GLY ILE ARG VAL ASN THR ILE PHE PRO GLY PRO ILE GLU SER GLU ARG


     195         200         205


 


ILE GLN THR VAL PHE GLN ARG MET ASP GLN LEU LYS GLY ARG PRO GLU


  210         215         220


 


GLY ASP THR ALA SER GLN PHE LEU ALA THR MET ARG LEU TYR ARG ALA


225         230         235         240


 


ASN ASP GLN GLY GLN LEU GLU ARG ARG PHE PRO THR ILE CYS ASP VAL


        245         250         255


 


ALA ASP ALA ALA VAL PHE LEU ALA SER ASP GLU ALA ALA ALA LEU THR


      260         265         270


 


GLY GLU THR ILE GLU VAL THR HIS GLY MET GLU LEU PRO THR SER SER


    275         280         285


 


GLU THR SER LEU LEU ALA ARG THR ASP LEU ARG THR ILE ASP ALA ASN


  290         295         300


 


GLY ARG THR THR LEU ILE CYS ALA GLY ASP GLN ILE GLU GLU VAL MET


305         310         315         320


 


ALA LEU THR GLY MET LEU ARG THR CYS GLY SER GLU VAL ILE ILE GLY


        325         330         335


 


PHE ARG SER GLU ALA ALA LEU ALA GLN PHE GLU GLN ALA ILE GLY GLU


     340         345         350


 


SER ARG ARG LEU ALA GLY GLU SER PHE ILE PRO PRO ILE ALA LEU PRO


   355         360         365


 


ILE ASP LEU ARG ASN PRO SER THR ILE ASP ALA LEU PHE ASP TRP ALA


  370         375         380


 


GLY GLU ASN THR GLY GLY ILE HIS ALA ALA VAL ILE LEU PRO ALA SER


385         390         395         400


 


GLY ARG GLU PRO ALA THR GLN VAL ILE ASP ILE ASP ASP ALA HIS VAL


        405         410         415


 


GLN ALA PHE LEU ASN ASP GLU ILE VAL GLY SER ILE ILE ILE ALA SER


      420         425         430


 


ARG LEU ALA ARG TYR TRP GLN ALA GLN ARG ILE ALA PRO GLY ALA ARG


    435         440         445


 


ALA ARG GLU PRO ARG VAL ILE PHE LEU SER ASN GLY ALA SER THR ALA


  450         455         460


 


GLY ASN PRO TYR GLY ARG ILE GLN SER ALA ALA ILE GLU GLN LEU ILE


465         470         475         480


 


ARG VAL TRP ARG HIS GLU ALA ALA LEU ASP TYR GLU ARG ALA THR ALA


        485         490         495


 


ALA GLY GLU ARG VAL LEU PRO ALA VAL TRP ALA SER GLN ILE VAL ARG


      500         505         510


 


PHE ALA ASN ARG SER LEU GLU GLY LEU GLU PHE ALA CYS ALA TRP THR


    515         520         525


 


ALA GLN LEU LEU HIS SER GLN ARG ARG ILE ASN GLU ILE THR LEU THR


  530         535         540


 


ILE PRO ALA ASP ILE SER ALA THR THR GLY ALA ARG SER ALA SER VAL


545         550         555         560


 


GLY TRP ALA GLU SER LEU ILE GLY LEU HIS LEU GLY LYS VAL ALA LEU


        565         570         575


 


ILE THR GLY GLY SER ALA GLY ILE GLY GLY GLN ILE GLY ARG LEU LEU


      580         585         590


 


ALA LEU SER GLY ALA ARG VAL MET LEU ALA ALA ARG ASP PRO HIS LYS


    595         600         605


 


LEU GLU GLN ILE GLN ALA THR ILE ARG ALA GLU LEU ALA GLU VAL GLY


  610         615         620


 


TYR THR ASP VAL GLU GLU ARG VAL GLN ILE ALA PRO GLY CYS ASP VAL


625         630         635         640


 


SER SER GLU GLU GLN LEU VAL ASP LEU VAL GLU ARG THR LEU ALA ALA


        645         650         655


 


PHE GLY THR VAL ASP TYR LEU ILE ASN ASN ALA GLY ILE ALA GLY VAL


      660         665         670


 


GLU GLU MET VAL ILE ASP MET PRO VAL GLU GLY TRP ARG ASN THR LEU


    675         680         685


 


TYR ALA ASN LEU ILE SER ASN TYR SER LEU MET ARG LYS LEU ALA PRO


  690         695         700


 


LEU MET LYS LYS GLN GLY SER GLY TYR VAL LEU ASN VAL SER SER TYR


705         710         715         720


 


PHE GLY GLY GLU LYS ASP ALA ALA ILE PRO TYR PRO ASN ARG ALA ASP


        725         730         735


 


TYR ALA VAL SER LYS ALA GLY GLN ARG ALA MET ALA GLU VAL PHE ALA


      740         745         750


 


ARG PHE LEU GLY PRO GLU ILE GLN ILE ASN ALA ILE ALA PRO GLY PRO


    755         760         765


 


VAL GLU GLY ASP ARG LEU ARG GLY THR GLY GLU ARG PRO GLY LEU PHE


  770         775         780


 


ALA ARG ARG ALA ARG LEU ILE LEU GLU ASN LYS ARG LEU ASN GLU LEU


785         790         795         800


 


HIS ALA ALA LEU ILE THR ALA ALA ARG THR ASP ASN ARG PRO MET ARG


        805         810         815


 


GLU LEU VAL GLU LEU LEU LEU PRO ASN ASP VAL ALA ALA LEU ALA GLN


      820         825         830


 


HIS PRO ALA ALA PRO ASP VAL LEU ARG THR LEU ALA LYS ARG PHE GLN


    835         840         845


 


SER GLU GLY ASP PRO ALA ALA SER SER SER SER PHE LEU LEU ASN ARG


  850         855         860


 


SER ILE ALA ALA LYS LEU LEU ALA ARG LEU ILE ASN GLY GLY TYR ASP


865         870         875         880


 


LEU PRO ALA ASP ILE PHE ALA ASN LEU ALA VAL PRO PRO ASP PRO PHE


        885         890         895


 


PHE THR ARG ALA GLN ILE ASP ARG GLU ALA ARG LYS VAL ARG ASP GLY


      900         905         910


 


ILE MET GLY MET LEU TYR LEU GLN ARG MET PRO THR GLU PHE ASP VAL


    915         920         925


 


ALA MET ALA THR VAL TYR TYR LEU ALA ASP ARG ASN VAL SER GLY GLU


  930         935         940


 


THR PHE HIS PRO SER GLY GLY LEU ARG TYR GLU ARG THR PRO THR GLY


945         950         955         960


 


GLY GLU LEU PHE GLY LEU PRO ALA PRO GLU ARG LEU ALA GLU LEU VAL


        965         970         975


 


GLY SER THR VAL TYR LEU ILE GLY GLU HIS LEU THR GLU HIS LEU ASN


      980         985         990


 


LEU LEU ALA ARG ALA TYR LEU GLU ARG TYR GLY ALA ARG GLN VAL VAL


    995         1000         1005


 


MET ILE VAL GLU THR GLU ALA GLY ALA GLU LYS MET ARG HIS LEU


  1010        1015         1020


 


LEU HIS ASP HIS VAL GLU ALA GLY ARG LEU PRO ILE ILE VAL ALA


  1025        1030         1035


 


GLY ASP GLN ILE GLU ALA ALA ILE ASP GLN ALA ILE ALA ASN TYR


  1040        1045         1050


 


GLY ARG PRO GLY PRO VAL VAL CYS THR PRO PHE ARG PRO LEU PRO


  1055        1060         1065


 


SER ALA PRO LEU VAL GLY ARG LYS ASP SER ASP TRP SER THR VAL


  1070        1075         1080


 


LEU SER GLU ALA GLU PHE ALA GLU LEU CYS GLU HIS GLN LEU THR


  1085        1090         1095


 


HIS HIS PHE ARG VAL ALA ARG LYS ILE ALA LEU SER ASP GLY ALA


  1100        1105         1110


 


SER LEU ALA LEU VAL THR PRO GLU THR THR ALA THR SER SER THR


  1115        1120         1125


 


GLU GLN PHE ALA LEU ALA ASN PHE VAL LYS THR THR LEU HIS ALA


  1130        1135         1140


 


PHE THR ALA THR ILE GLY VAL GLU SER GLU ARG THR ALA GLN ARG


  1145        1150         1155


 


ILE LEU ILE ASN GLN VAL ASP LEU THR ARG ARG ALA ARG ALA GLU


  1160        1165         1170


 


GLU PRO ARG ASP PRO ARG GLU ARG GLN GLN GLU LEU GLU ARG PHE


  1175        1180         1185


 


ILE GLU ALA VAL LEU LEU VAL THR ALA PRO LEU PRO PRO GLU ALA


  1190        1195         1200


 


ASP THR ARG TYR ALA GLY ARG ILE HIS ARG GLY ARG ALA ILE THR VAL


  1205        1210         1215


 


SEQ ID NO. 108: MCR OSCILLOCHLORIS TRICHOIDES (OTMCR)


MFMTRLNDKIALITGGAGTIGEVITRRYLEEGATVVMAGRNRDKLDRYRERLITEFHALP


 


ERVMVVRMDGSSNAEVRMGIAAVVAHFGRIDILVNNAGSAGARQRLPAIPLLRSELQADE


 


TETLADSIGNLIGITWNLIRAAAPFMPAGSSVINISTIFARTDYYGRIPYVVPKAALHAL


 


TLAAATELGERGIRVNQINPGPIDSDRIRTVFRRMDELKGVPEQSTADGFFQMMRLRRPN


 


AEGDLVKGFPKTLDVANVAVFLGSAESAALSGETLDVTHGMAVPTESRTTLTSRPGLRAV


 


DGSGHTTLICVGDQIEEAAALTGVLRACGAEVVIGFRSRAAIARFDHLIERGRHLPSQEH


 


VAPVLLYLNPTEPESIDQALRWMATNLDLPTSVIILPAQRQPLPPSVVRASDEEVAYFLR


 


DELSGMIVLASRLARFWQQATLAPGNAPIQPRVLFMTNPDDGQGNLYAEILRAGVEQLCR


 


VWRHESQLDYTRLAQMDAHPPHIRPVWANQLVRFANNEQENLEYCCAWVAKILLSERTIE


 


ELNLYLPRQIGSTTGSRQPSFGWAENLIGLHLGKTALITGGSAGIGSQIARLLALSGARV


 


MLCARDERKLIQMRDMIIAELTEVGYNQVESRVQICAGCDVGEEEQLEIAVQRTLDLFGH


 


VDYLINNAGIAGAEEMVLDLPLEAWQRTLRTNLISNYSLIRKLAPQMKSRGSGYILNVSS


 


YFGGEKYAAIPYPNRADYAVSKAGQRALGEALARLLGPEVQINAMAPGPVEGERLRGSGD


 


RPGLFLRRGRLILENKRLNDLHATLIAAERETQVGMRDLLARLLHNDVCALIDDPAAPTH


 


LRALAERIWEQSDPNSYARAFFMNANIATKLLARLFNADQIDAQTFHTSQPNLPPEPFFA


 


RTQIDREARRVRDGVMSMLYLQRMPTEFDVALATVYYLNDRSVSGETFHPSGGLRHERTP


 


TGAELYGSPAPQRLASLAGSTVYLIGESMAAHLEALARAYIERYAATRVVLICATPAGVE


 


RFSHHLADHLASGALAILSAEEGIEAALSEALRRFGPPGPVVSTPFQPLPSQPLIGRNDS


 


DWSTVLDVAGFSAMCEQQLTHHFRVTRKLSLVAGVSLVLVTPETDSHSSTEQFALANFVK


 


TTLHAFTATVGVECERTAHRILVNQVDLGRQARAEEPRSPAEQAQEMERFIDAIMLTTAP


 


IPAEEDNRYTGRIYRGRAITV


 


SEQ ID NO. 109: CHLOROFLEXUS AURANTIACUS (CAMCR)


MSGTGRLAGKIALITGGAGNIGSELTRRFLAEGATVIISGRNRAKLTALAERMQAEAGVP


 


AKRIDLEVMDGSDPVAVRAGIEAIVARHGQIDILVNNAGSAGAQRRLAEIPLTEAELGPG


 


AEETLHASIANLLGMGWHLMRIAAPHMPVGSAVINVSTIFSRAEYYGRIPYVTPKAALNA


 


LSQLAARELGARGIRVNTIFPGPIESDRIRTVFQRMDQLKGRPEGDTAHHFLNTMRLCRA


 


NDQGALERRFPSVGDVADAAVFLASAESAALSGETIEVTHGMELPACSETSLLARTDLRT


 


IDASGRTTLICAGDQIEEVMALTGMLRTCGSEVIIGFRSAAALAQFEQAVNESRRLAGAD


 


FTPPIALPLDPRDPATIDAVFDWGAGENTGGIHAAVILPATSHEPAPCVIEVDDERVLNF


 


LADEITGTIVIASRLARYWQSQRLTPGARARGPRVIFLSNGADQNGNVYGRIQSAAIGQL


 


IRVWRHEAELDYQRASAAGDHVLPPVWANQIVRFANRSLEGLEFACAWTAQLLHSQRHIN


 


EITLNIPANISATTGARSASVGWAESLIGLHLGKVALITGGSAGIGGQIGRLLALSGARV


 


MLAARDRHKLEQMQAMIQSELAEVGYTDVEDRVHIAPGCDVSSEAQLADLVERTLSAFGT


 


VDYLINNAGIAGVEEMVIDMPVEGWRHTLFANLISNYSLMRKLAPLMKKQGSGYILNVSS


 


YFGGEKDAAIPYPNRADYAVSKAGQRAMAEVFARFLGPEIQINAIAPGPVEGDRLRGTGE


 


RPGLFARRARLILENKRLNELHAALIAAARTDERSMHELVELLLPNDVAALEQNPAAPTA


 


LRELARRFRSEGDPAASSSSALLNRSIAAKLLARLHNGGYVLPADIFANLPNPPDPFFTR


 


AQIDREARKVRDGIMGMLYLQRMPTEFDVAMATVYYLADRNVSGETFHPSGGLRYERTPT


 


GGELFGLPSPERLAELVGSTVYLIGEHLTEHLNLLARAYLERYGARQVVMIVETETGAET


 


MRRLLHDHVEAGRLMTIVAGDQIEAAIDQAITRYGRPGPVVCTPFRPLPTVPLVGRKDSD


 


WSTVLSEAEFAELCEHQLTHHFRVARKIALSDGASLALVTPETTATSTTEQFALANFIKT


 


TLHAFTATIGVESERTAQRILINQVDLTRRARAEEPRDPHERQQELERFIEAVLLVTAPL


 


PPEADTRYAGRIHRGRAITV

## Claims

1. A genetically modified microorganism comprising a heterologous gene encoding an acetyl-CoA carboxylase (ACCase) polypeptide fusion wherein the polypeptide fusion comprises at least two functional subunits of an ACCase enzyme selected from the group consisting of accA-accB, accA-accC, accA-accD, accB-accC, accB-accD, accC-accD, accA-accB-accC, accA-accB-accD, accA-accC-accD, accB-accC-accD and accA-accB-accC-accD.

2. The genetically modified microorganism of claim 1, wherein said microorganism is capable of converting a renewable carbon source to at least 60 grams/liter 3-hydroxypropionic acid (3-HP).

3. The genetically modified microorganism of claim 1, wherein the microorganism encodes an active ACCase enzyme comprising two or more polypeptide fusions wherein at least two of the polypeptide fusions are encoded by genes expressed on a same plasmid.

4. The genetically modified microorganism of claim 1, wherein the microorganism encodes an active ACCase enzyme comprising two or more polypeptide fusions wherein at least two of the polypeptide fusions are encoded for by genes expressed on different plasmids.

5. The genetically modified microorganism of claim 1, wherein the microorganism encodes an active ACCase enzyme and wherein at least one subunit of the enzyme is not fused to another subunit.

6. The genetically modified microorganism of claim 5, wherein the subunit that is not fused to another subunit is expressed by a gene on the same plasmid as the gene encoding the polypeptide fusion.

7. The genetically modified microorganism of claim 5, wherein the gene that encodes the subunit that is not fused to another subunit is expressed on a different plasmid than the gene that encodes the fusion.

8. The genetically modified microorganism of claim 1, wherein the gene encoding an acetyl-CoA carboxylase polypeptide encodes an accA-accD polypeptide fusion, and further comprises nucleotide sequence encoding for an accB polypeptide subunit, and a nucleotide sequence encoding for an accC subunit.

9. The genetically modified microorganism of claim 1, wherein the microorganism encodes an active ACCase enzyme comprising two or more polypeptide fusions wherein the stoichiometric ratio of the polypeptide fusions relative to one another is between 1 to 10.

10. The genetically modified microorganism of claim 9, wherein the stoichiometric ratio of the polypeptide fusions relative to one another is between about 1 to about 2 or between about 7 to about 9.

11. The genetically modified microorganism of claim 1, wherein the microorganism encodes an active ACCase enzyme and wherein the ACCase enzyme comprises an accA-accD polypeptide fusion, an accB polypeptide subunit that is not fused to another polypeptide subunit, and an accC polypeptide subunit that is not fused to another polypeptide subunit.

12. The genetically modified microorganism of claim 1, wherein the polypeptide fusion comprises two, three, or four different ACCase subunits in a single polypeptide.

13. The genetically modified microorganism of claim 1, wherein the microorganism comprises a stoichiometric ratio of
a) accB and accC fusion to accA and accD fusion;
b) accA and accD fusion to accB and accC fusion;
c) accC and accD fusion to accA and accB fusion;
d) accA and accB fusionto accC and accD fusion;
e) accA subunit to accB subunit;
f) accB subunit to accA subunit;
g) accA subunit to accC subunit;
h) accC subunit to accA subunit;
i) accA subunit to accD subunit;
j) accD subunit to accA subunit;
k) accB subunit to accC subunit;
l) accC subunit to accB subunit;
m) accB subunit to accD subunit;
n) accD subunit to accB subunit;
o) accC subunit to accD subunit; or
p) acct) subunit to accC subunit;

of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 2:1, 2:3, 2:4, 2:5, 2:6, 2:7, 2:8, 3:1, 3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 3:1, 3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 4:1, 4:3, 4:4, 4:5, 4:6, 4:7, 4:8, 5:1, 5:3, 5:4, 5:5, 5:6, 5:7, 5:8, 6:1, 6:3, 6:4, 6:5, 6:6, 6:7, 6:8, 7:1, 7:3, 7:4, 7:5, 7:6, 7:7 7:8, 8:1, 8:3, 8:4, 8:5, 8:6, 8:7, or 8:8 in low, medium, high or inducible expression.

14. The genetically modified microorganism of claim 1, wherein the microorganism comprises a polypeptide fusion of accA-aaD, a subunit of accB that is not fused to another subunit, and an accC subunit that is not fused to another subunit wherein the molar ratios for accA:accB:accC:accD are about 1:2:1:1.

15. The genetically modified microorganism of claim 1, wherein the microorganism comprises an active ACCase enzyme that comprises a polypeptide fusion of accA-aaD, a subunit of accB that is not fused to another subunit, and an accC subunit that is not fused to another subunit wherein the molar ratios for accDAfusion:accB:accC are about 1:2:1.

16. A method of producing chemical products comprising culturing the genetically modified microorganism of claim 1 in a suitable medium and under suitable conditions to produce a desired chemical product selected from the group consisting of 3-hydroxypropionic acid (3-HP) or a derivative of, 1,4-butanediol, butanol, isobutanol, polyketide chemical products and C4-C18 fatty acid chains.

17. The method of claim 16, wherein said chemical is converted to acrylic acid, acrylates, 1,3-propanediol, malonic acid, ethyl-3-hydroxypropionate, ethyl ethoxy propionate, propiolactone, acrylamide, or acrylonitrile to make consumer products.

18. The method of claim 16, wherein said chemical is oligomerized or polymerized to form polyacrylic acid, methyl acrylate, acrylamide, acrylonitrile, propiolactone, ethyl 3-HP, ethyl acrylate, n-butyl acrylate, hydroxypropyl acrylate, hydroxyethyl acrylate, isobutyl acrylate, 2-ethylhexyl acrylate, and acrylic acid ester to which an alkyl or aryl addition may be made, and/or to which halogens, aromatic amines or amides, and aromatic hydrocarbons may be added to make consumer products.