# Patent

## Title
Microbial production of muconic acid and salicylic acid

## Abstract

The invention provides a recombinant microorganism that has been genetically engineered to contain metabolic pathway for the production of muconic acid from a salicylic acid intermediate. The genetically engineered metabolic pathway comprises both biosynthetic and biodegradative elements.

## Background
CONTINUING APPLICATION DATA
This application is a continuation application of U.S. patent application Ser. No. 14/622,155, filed Feb. 13, 2015, which claims the benefit of U.S. Provisional Application Ser. No. 61/939,593, filed Feb. 13, 2014, each of which is incorporated by reference herein.


SEQUENCE LISTING
This application contains a Sequence Listing electronically submitted to the United States Patent and Trademark Office via EFS-Web as an ASCII text file entitled “235-02380101_SequenceListing_ST25.txt” having a size of 31 kilobytes and created on Feb. 12, 2015. The information contained in the Sequence Listing is incorporated by reference herein.
BACKGROUND
Fossil fuels supply the world with not only energy but also important feedstocks for chemical industry. However, the shrinking availability of fossil reserves and the deteriorating environment compel people to explore renewable alternatives for the production of fuels, chemicals, and pharmaceuticals. Fortunately, the metabolic diversity of biological systems provides us with an extremely rich chemical repertoire. In recent years, the development of metabolic engineering has enabled the establishment of microbial chemical factories by constituting heterologous or non-natural biosynthetic pathways into genetically advantageous microbial hosts (Ajikumar et al., 2010 Science 330:70-4; Anthony et al., 2009 Metab. Eng. 11:13-9; Atsumi et al., 2008 Metab. Eng. 10:305-11; Huang et al., 2013 Biotechnol. Bioeng. 110:3188-96; Lin et al., 2013b Metab. Eng. 18:69-77; Lin et al., 2012 Microb. Cell Fact. 11:42; Shen et al., 2008 Metab. Eng. 10:312-20; Shen et al., 2012 J. Ind. Microbiol. Biotechnol. 39:1725-9; Zhang et al., 2008 Proc. Natl. Acad. Sci. U. S. A. 105:20653-8).
Muconic acid (MA) is a platform chemical that serves as the precursor to several bio-plastics. It is also a naturally-occurring metabolite. Muconic acid is present in biological systems as an intermediate in the microbial degradation of aromatic hydrocarbons (Fuchs et al., 2011 Nat. Rev. Microbiol. 9:803-16). In past 20 years, many efforts have been made for the microbial production of muconic acid. Draths and Frost reported the earliest study on the artificial biosynthesis of muconic acid in Escherichia coli from renewable carbon source glucose (Draths et al., 1994 1 Am. Chem. Soc. 116:399-400). By introducing three heterologous enzymes 3-dehydroshikimate dehydratase, protocatechuic acid decarboxylase and catechol 1,2-dioxygenase (CDO), the carbon flux was redirected from the E. coli native shikimate pathway to the biosynthesis of muconic acid. Metabolically optimized strains carrying this artificial pathway were able to produce up to 2.4 g/L of muconic acid via two-stage bioconversion in shake flasks (Draths et al., 1994 J. Am. Chem. Soc. 116:399-400) and 38.6 g/L via fed-batch fermentation (Niu et al., 2002 Biotechnol. Prog. 18:201-11). Afterwards, the same pathway was reconstituted in Saccharomyces cerevisiae (Weber et al., 2012 Appl. Environ. Microbiol. 78:8421-30), and the highest titer reported was nearly 141 mg/L (Curran et al., 2013 Metab. Eng. 15:55-66).
Muconic acid is easily converted into adipic acid by chemical hydrogenation, and adipic acid is a direct building block for nylon-6,6 and polyurethane (Sun et al., 2013 Appl. Environ. Microbiol. 79:4024-30). In addition, muconic acid is a synthetic precursor to terephthalic acid, a chemical used for manufacturing polyethylene terephthalate (PET) and polyester (Curran et al., 2013 Metab. Eng. 15:55-66). The global production of adipic acid and terephthalic acid is 2.8 and 71 million metric tons, respectively (Curran et al., 2013 Metab. Eng. 15:55-66).
Salicylic acid (SA) is an important drug precursor mainly used for producing pharmaceuticals such as aspirin and lamivudine (an anti-HIV drug). Like muconic acid, it is a naturally-occurring metabolite. In biological systems, salicylic acid serves not only as a plant hormone (Chen et al., 2009 Plant Signal Behav. 4:493-6) but also as a biosynthetic precursor of bacterial siderophore (Gaille et al., 2002 J. Biol. Chem. 277:21768-75). Salicylic acid esters and salts used in sunscreens and medicaments account for another large portion of salicylic acid consumption. The global market for salicylic acid products was estimated to be $292.5 million in 2012 and is expected to reach $521.2 million in 2019, growing at an annual increase of 8.6% (“Salicylic Acid Market for Pharmaceutical, Skin care, Hair care and Other Applications-Global Industry Analysis, Size, Share, Growth, Trends, and Forecast 2013-2019,” 2013 Transparency Market Research).
Muconic acid and salicylic acid are thus naturally-occurring organic acids having great commercial value. Muconic acid is a potential platform chemical for the manufacture of several widely-used consumer plastics; while salicylic acid is mainly used for producing pharmaceuticals, skincare and haircare products. At present, commercial production of muconic acid, salicylic acid, adipic acid, and terephthalic acid predominantly relies on organic chemical synthesis using petroleum-derived chemicals, such as benzene, as starting materials. These chemical synthesis processes are considered nonrenewable and environmentally unfriendly. Therefore, it is of great importance to develop “green” synthetic approaches that can utilize renewable feedstocks.
SUMMARY OF THE INVENTION
The present invention involves the construction of a biosynthetic pathway for muconic acid and salicylic acid production using recombinant microorganisms such as E. coli. Muconic acid and salicylic acid are high-value commodity chemicals. Producing high-value chemicals through microbial conversion is an attractive alternative to the current petroleum-based chemical industry.
A novel pathway for muconic acid and salicylic acid production is described and validated. The novel biosynthetic pathway involves a plurality of enzymatic modules and is exemplified in the figures and examples included herewith. The biosynthetic pathway can be introduced into any commercially useful microorganism, such as E. coli. Advantageously, the resulting metabolically engineered microorganism can be employed in large scale fermentations to produce muconic acid, salicylic acid, or other metabolites in the engineered pathway that are of interest. Muconic acid, salicylic acid, or their metabolites can be isolated and further derivatized chemically or enzymatically to yield a large array of additional commercially important products such as adipic acid and terephthalic acid, which in turn can be incorporated into other materials including bio-based polymers such as polyethylene terephthalate (PET) and polyester.
The present invention provides cells, for example microbial cells, also referred to herein as microorganisms, which are genetically engineered for the production of muconic acid from a salicylic acid intermediate, as well as methods for making the genetically engineered cells and methods for producing and isolating salicylic acid, muconic acid, and/or their derivatives and downstream metabolites, from the cells or cell culture.
In one aspect, the invention provides a genetically engineered cell having a genetically engineered metabolic pathway for the production of muconic acid from a salicylic acid intermediate. The genetically engineered cell is preferably a microbial cell, i.e., a microorganism. The genetically engineered metabolic pathway can include at least one enzyme associated with the biosynthesis of salicylic acid, and at least one enzyme associated with the conversion of salicylic acid to muconic acid. In some embodiments, the enzyme associated with the biosynthesis of salicylic acid includes one or both of an isochorismate synthase (ICS) and an isochorismate pyruvate lyase (IPL). The enzymes associated with the biosynthesis of salicylic acid can be introduced into the host cell using a “synthetic” enzymatic module. In some embodiments, the enzyme associated with the conversion of salicylic acid to muconic acid includes one or both of a salicylate 1-monoxygenase (SMO) and a catechol 1,2-dioxygenase (CDO). The enzymes associated with the conversion of salicylic acid to muconic acid can be introduced into the host cell using a “degradative” enzymatic module. In an exemplary embodiment, a genetically engineered microorganism includes a first module (i.e., a synthetic enzymatic module) expressing an isochorismate synthase (ICS) and an isochorismate pyruvate lyase (IPL), and a second module (i.e., a degradative enzymatic module) expressing a salicylate 1-monoxygenase (SMO) and a catechol 1,2-dioxygenase (CDO). The first and second modules may be present on different plasmids, or they can be present on the same plasmid.
The genetically engineered metabolic pathway optionally further includes at least one enzyme associated with enhanced chorismate availability. In some embodiments, the enzyme or enzymes associated with enhanced chorismate availability include enzymes that increase carbon flow toward chorismate. An enzyme or enzymes associated with enhanced chorismate availability can be introduced into the host cell using a “precursor enhancing” enzymatic module. In an exemplary embodiment, a genetically engineered microorganism optionally includes a third module (i.e., a precursor enhancing enzymatic module) expressing one or more enzymes encoded by aroL, ppsA, tktA, and/or aroGfbr. The third module may be present on a plasmid. In exemplary embodiments, the first and third modules are present on the same or different high copy number plasmid(s), and the second module is present on a low copy number plasmid.
The genetically engineered biosynthetic pathway can include one or more enzymes that are heterologous to the host cell, and/or it can include one or more enzymes that are naturally occurring in the host cell. In the case of an enzyme naturally occurring in the host cell, the enzyme can be expressed at naturally occurring levels, or it can be overexpressed as a component of the genetically engineered pathway.
In some aspects, the genetically engineered microorganism of the invention has been further engineered to overproduce at least one aromatic amino acid, such as phenylalanine or tyrosine. The genetically engineered microorganism of the invention may be further engineered to knock out one or more enzymes involved in biosynthesis of an aromatic amino acid. Exemplary enzymes encoding an aromatic amino acid include pheA, tyrA and trpD. In some embodiments, pheA and tyrA are knocked out, and trpD, if present, is not disrupted.
The genetically engineered microorganism of the invention is preferably a bacterium or a yeast. In some embodiments, the genetically engineered microorganism is selected from Escherichia coli, Bacillus subtilis Bacillus licheniformis, Alcaligenes eutrophus, Rhodococcus erythropolis, Paenibacillus macerans, Pseudomonas putida, Enterococcus faecium, Saccharomyces cerevisiae, Lactobacillus plantarum, Enterococcus gallinarium and Enterococcus faecalis. An exemplary genetically engineered microorganism is E. coli. 
In exemplary embodiments, the genetically engineered microorganism includes at least one plasmid selected from the group consisting of pCS-EP, pZE-EP, pSA-EP, pCS-NC, pZE-NC, pSA-NC, pCS-APTA, pZE-EP-APTA.
The invention also provides a method for using the genetically engineered host cell, for example the genetically engineered microorganism, to produce a downstream metabolite of chorismate, preferably an organic acid such as salicylic acid or muconic acid. The genetically engineered cell is cultured under conditions to produce the organic acid, and the organic is optionally separated from the genetically engineered cell. Optionally, the organic acid is isolated and/or purified.
The words “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.
The terms “comprises” and variations thereof do not have a limiting meaning where these terms appear in the description and claims.
Unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one.
Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.
The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows a novel artificial pathway for the biosynthesis of MA. (A) Solid arrows indicate native metabolic pathways in E. coli; dashed arrows indicate the introduced artificial pathway. ICS, isochorismate synthase; IPL, isochorismate pyruvate lyase; SMO, salicylate 1-monoxygenase; CDO, catechol 1,2-dioxygenase. (B) Includes a delineation of an EP module (an exemplary “synthetic” enzymatic module), an NC module (an exemplary “degradative” enzymatic module), and an APTA module (an exemplary “precursor enhancing” enzymatic module) of a novel artificial pathway for the biosynthesis of MA.
FIG. 2 shows transformation of a phenylalanine producing strain into an SA overproducer. (A) Schematic representation of the aromatic amino acid biosynthetic pathways in E. coli. Grey-colored arrows refer to the native carbon flow of E. coli strain ATCC31884, while the blue-colored arrow indicates the desired carbon flow after metabolic engineering. CHO, chorismate; PPA, prephenate; PPY, phenylpyruvate; HPP, 4-hydroxyphenylpyruvate; ANT, anthranilate; PRA, 5-phosphoribosyl-anthranilate. (B) Production of SA by metabolically engineered strains. pZE-EP, pCS-EP, and pSA-EP indicate that EntC and PchB are co-expressed using high-, medium-, and low-copy plasmids, respectively. All data points are reported as mean±s.d. from three independent experiments. (C) Growth inhibition assay to evaluate the toxicity of SA towards QH4 strain. All data points are reported as mean±s.d. from two independent experiments.
FIG. 3 shows activity of the salicylate 1-monoxygase (SMO) encoded by nahGopt. (A) Kinetic parameters of the SMO. The Km and Vmax values were estimated with OriginPro8 through non-linear regression of the Michaelis-Menten equation. The protein concentration [E] of the SMO in the reaction systems was 0.97 nM. The kcat value was calculated according to the formula kcat=Vmax/[E]. All data points are reported as mean±s.d. from two independent experiments. (B) Conversion of SA to MA using the wild type E. coli strain carrying pZE-NC. Data points are reported as mean±s.d. from three independent experiments. Error bars are defined as s.d.
FIG. 4 shows modular optimization of the MA biosynthetic pathway. (A) Gene organization of the three modules: EP, NC, and APTA. entC, pchB, nahGopt, and catA encode ICS, IPL, SMO, and CDO, respectively. (B) Optimization of MA production by adjusting the copy number of each module. All data points are reported as mean from three independent experiments. Asterisk mark indicates an untested construct.
FIG. 5 shows exemplary polynucleotides sequences and exemplary amino acid sequences for use in the invention. (A) Amino acid sequence of EntC from E. coli (SEQ ID NO:1). (B) Amino acid sequence of PchB from P. fluorescence (SEQ ID NO:2). (C) Amino acid sequence of NahG from E. coli (SEQ ID NO:3). (D) Amino acid sequence of CatA from P. putida (SEQ ID NO:4). (E) Polynucleotide sequence of codon-optimized nahG from E. coli (SEQ ID NO:5). (F) Polynucleotide sequence of catA from E. coli (SEQ ID NO:6). (G) Amino acid sequence of AroL from E. coli (SEQ ID NO:7). (H) Amino acid sequence of PpsA from E. coli (SEQ ID NO:8). (I) Amino acid sequence TktA of from E. coli (SEQ ID NO:9). (J) Amino acid sequence of feedback-inhibition-resistant AroG from E. coli (SEQ ID NO:10).



DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
The present invention provides a genetically engineered microorganism (also referred to herein as a genetically engineered microbe) containing a biosynthetic pathway resulting in production of muconic acid (MA) from chorismate, via a salicylic acid (SA) intermediate. Also provided by the invention are methods of making the genetically engineered organism, and methods of using the genetically engineered organism, for example to produce muconic acid, salicylic acid, or other metabolites.
This novel biosynthetic pathway is a de novo muconic acid biosynthetic pathway that extends the shikimate pathway by combining a salicylic acid biosynthetic pathway (via a first “synthetic” enzymatic module), with a partial salicylic acid degradation pathway (via a second “degradative” enzymatic module), thereby creatively bridging SA biosynthesis with its partial degradation pathway. Efficient production of MA was surprising because biosynthesis and degradation of a specific molecule (in this case, salicylic acid) typically do not occur simultaneously in natural settings. The resulting biosynthetic pathway thus quite unexpectedly yielded an efficient microbial platform for the production of muconic and other compounds for which muconic is a precursor.
The biosynthetic pathway optionally includes a third “precursor enhancing” enzymatic module designed to increase the availability of chorismate, which is the precursor for salicylic acid synthesis via the synthetic module.
An illustrative biosynthetic pathway is shown in FIG. 1.
Biosynthetic Pathway
The biosynthetic pathway that is engineered into the host microorganism includes at least two, and preferably three, enzymatic modules. In one embodiment, the genetically engineered organism includes, or is engineered to include, a first “synthetic” module, which supplies enzymatic activity to produce salicylic acid from chorismate, and a second “degradative” module, which supplies enzymatic activity to produce muconic acid from salicylic acid. In another embodiment, the genetically engineered organism further includes, or is engineered to include, a third “precursor enhancing” enzymatic molecule which increases availability of chorismate.
“Synthetic” module. A first module is associated with salicylic acid (SA) biosynthesis. Chorismate, for example, an intermediate of the shikimate pathway, can serve as starting material for the enzymatic synthesis of salicylic acid. Chorismate can be used as a substrate by synthase, such as an isochorismate synthase (ICS) to produce isochorismate. The isochorismate produced can be converted to salicylic acid by the action of a lyase, such as an isochorismate pyruvate lyase (IPL). An exemplary synthetic module can thus include one or more polynucleotides that operably encode isochorismate synthase (ICS) activity and one or more polynucleotides that operably encode isochorismate pyruvate lyase (IPL) activity. A preferred synthetic module expresses an isochorismate synthase (ICS) and an isochorismate pyruvate lyase (IPL) to achieve SA biosynthesis, but other enzymes capable of catalyzing the conversion of chorismate to salicylic acid can be used.
The isochorismate synthase (ICS) that converts chorismate to isochorismate may be any enzyme having ICS activity. Enzymes having ICS activity are known to the skilled worker and are readily obtained. Examples include, but are not limited to, enzymes encoded by pchA from P. aeruginosa, entC from E. coli, and menF from E. coli. In one embodiment, the sequence of the ICS can be SEQ ID NO:1; however, the ICS of the invention is not limited to any particular ICS enzyme. Enzymes having ICS activity may also be obtained fromMycobacterium species and other Pseudomonas species, as well as other genera.
The isochorismate pyruvate lyase (IPL) that uses isochorismate as a substrate and converts it to salicylic acid may be any enzyme having IPL activity. Such enzymes are known to the skilled worker and are readily obtained. Examples include, but are not limited to, enzymes encoded by pchB from P. aeruginosa and/or P. fluorescence. In one embodiment, the sequence of the IPL can be SEQ ID NO:2; however, the IPL of the invention is not limited to any particular IPL enzyme. Enzymes having isochorismate pyruvate lyase activity may also be obtained from Mycobacterium species and other Pseudomonas species, as well as other genera.
Preferred enzymes are EntC from E. coli (SEQ ID NO:1) and the PchB from P. fluorescence (SEQ ID NO:2), which represent highly efficient ICS and IPL, respectively. Exemplary polynucleotides which encode the preferred enzymes are entC and pchB, respectively. In E. coli, the synthetic module preferably expresses entC and pchB and is for that reason termed the “EP module” in Example I. Exemplary components of the synthetic module are described in more detail in Example I and also, for example, in Lin et al., 2013 Nat. Commun. 4:2603; and US Patent Application Publication No. 2014/0370557, published Dec. 18, 2014; each of which is incorporated by reference herein.
“Degradative” module. A second module is associated with converting salicylic acid (SA) into muconic acid (MA). Salicylic acid can be used as a substrate for an oxygenase, such as a monoxygenase (MO) or a dioxygenase (DO), such as a salicylate 1-monoxygenase (SMO), to produce catechol. The catechol produced can be converted to muconic acid by the action of an oxygenase, such as a monoxygenase (MO) or a dioxygenase (DO), such as catechol 1,2-dioxygenase (CDO). An exemplary degradative module can thus include one or more polynucleotides that operably encode salicylate 1-monoxygenase (SMO) activity and one or more polynucleotides that operably encode catechol 1,2-dioxygenase (CDO) activity. A preferred degradative module expresses a salicylate 1-monoxygenase (SMO) and a catechol 1,2-dioxygenase (CDO) to convert SA to MA, but other enzymes capable of catalyzing the conversion of SA to MA can be used.
The salicylate 1-monoxygenase (SMO) that converts salicylic acid to catechol may be any enzyme having SMO activity. Enzymes having SMO activity are known to the skilled worker and are readily obtained. In one embodiment, the sequence of the SMO can be SEQ ID NO:3; however, the SMO of the invention is not limited to any particular SMO enzyme. Examples include, but are not limited to, enzymes encoded by nahG from P. putida DOT-T1E, paantABC from P. aeruginosa PAO1, pfantABC from P. fluorescens Migula, and ppbenABCD from P. putida KT2440. Enzymes having SMO activity may also be obtained from other Pseudomonas species, as well as other genera.
Two anthranilate 1,2-dioxygenases (ADOs) from P. aeruginosa PAO1 (encoded by paantABC) and P. fluorescens Migula (encoded by pfantABC) and a benzoate 1,2-dioxygenase (BDO) from P. putida KT2440 (encoded by ppbenABCD) can catalyze the conversion of anthranilate to catechol (Sun et al., 2013 Appl. Environ. Microbiol. 79:4024-30). Given the structure similarity of the substrates anthranilate (2-aminobenzoate) and SA (2-hydroxybenzoate), these enzymes are also considered as having SMO activity. Thus, enzymes having SMO activity can include dioxygenases such as anthranilate dioxygenase (ADO) and benzoate dioxygenase (BDO). Enzymes having SMO activity may also be obtained from other Pseudomonas species, as well as other genera.
The catechol 1,2-dioxygenase (CDO) that converts catechol to muconic acid may be any enzyme having CDO activity. Enzymes having CDO activity are known to the skilled worker and are readily obtained. In one embodiment, the sequence of the CDO can be SEQ ID NO:4; however, the CDO of the invention is not limited to any particular CDO enzyme. Examples include, but are not limited to, enzymes encoded by catA from P. putida KT-2440 and/or P. aeruginosa. Enzymes having CDO activity may also be obtained from other Pseudomonas species, as well as other genera.
Preferred enzymes are NahG from E. coli (SEQ ID NO:3) and the CatA from P. putida (SEQ ID NO:4), which represent highly efficient SMO and CDO, respectively. In one embodiment, the SMO can be encoded by SEQ ID NO:5; however, the invention is not limited to any particular sequence encoding the SMO enzyme. In one embodiment, the CDO can be encoded by SEQ ID NO:6; however, the invention is not limited to any particular sequence encoding the CDO enzyme. Exemplary polynucleotides which encode the preferred enzymes are nahG (preferably a codon-optimized version thereof; SEQ ID NO:5) and catA (SEQ ID NO:6), respectively. In E. coli, the degradative module preferably expresses nahG and catA, and is for that reason termed the “NC module” in Example I. Exemplary components of the degradative module are described in more detail in Example 1.
“Precursor Enhancing” Module. An optional third module is associated with enhanced chorismate availability. The optional “precursor enhancing” module operably encodes one or more enzymes that increase chorismate availability. Pathways such as the glycolysis pathway and the pentose phosphate pathway can be extended to feed into the shikimate pathway which can be used to enhance chorismate production (FIG. 1). Several rate limiting steps in the production of chorismate are known and can be manipulated to result in a microbe that produces more chorismate. Preferably, the precursor enhancing module expresses one or more enzymes that increase carbon flow toward chorismate. Enzymes that increase carbon flow toward chorismate are known to the skilled worker and are readily obtained. Exemplary enzymes include, but are not limited to, shikimate kinase, phosphoenolpyruvate synthase, transketolase, and 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, preferably feedback-inhibition-resistant 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, 3-dehydroquinate synthase, 3-dehydroquinate dehydratase, shikimate dehydrogenase, 3-phosphoshikimate-1-carboxyvinyltransferase, and chorismate synthase. Examples include, but are not limited to, AroL, AroF, AroH, AroG, PpsA, and TktA. In some embodiments, one or more of the AroL, AroF, AroH, AroG enzymes may be in the form of feedback-inhibition-resistant (fbr) enzymes. In one embodiment, the sequence of the AroL can be SEQ ID NO:7; however, the AroL of the invention is not limited to any particular AroL enzyme. In one embodiment, the sequence of the PpsA can be SEQ ID NO:8; however, the PpsA of the invention is not limited to any particular PpsA enzyme. In one embodiment, the sequence of the TktA can be SEQ ID NO:9; however, the TktA of the invention is not limited to any particular TktA enzyme. In one embodiment, the sequence of the AroGfbr can be SEQ ID NO:10; however, the AroGfbr of the invention is not limited to any particular AroGfbr enzyme. Preferred enzymes operably encoded by a precursor enhancing module are AroL (SEQ ID NO:7), PpsA (SEQ ID NO:8), TktA (SEQ ID NO:9), and a feedback-inhibition-resistant AroG (SEQ ID NO:10) from E. coli. Exemplary polynucleotides which encode preferred enzymes are aroL, ppsA, tktA, and aroGfbr, respectively. In E. coli, the precursor enhancing module preferably expresses aroL, ppsA, tktA, and aroGfbr to increase chorismate availability, and for that reason is termed the “APTA module” in Example I. Exemplary components of the precursor enhancing module are described in more detail in Example I and also, for example, in Lin et al., 2013 Nat. Commun. 4:2603; and US Patent Application Publication No. 2014/0370557, published Dec. 18, 2014; each of which is incorporated by reference herein.
The enzymes expressed from the modules may be heterologous with respect to the host organism, or they may be naturally found in the host organism. For example, when E. coli is a host organism, one or more on the enzymes that are expressed from the one or more modules can be native E. coli enzymes, and one or more of the expressed enzymes can be from other (non-E. coli) organisms (i.e., heterologous). When an enzyme that is native to the host organism is expressed from a module, that enzyme is overexpressed in the host organism relative to a host organism without the module. Overexpression of naturally occurring enzymes, and expression of enzymes from other hosts, via the multiple modules, allows optimization and fine-tuning of the various enzymes that make up the overall genetically engineered biosynthetic pathway.
Host Cells
The novel metabolic pathway described herein is introduced into a host cell using genetic engineering techniques. The term “cell” is meant to include any type of biological cell. The host cell can be a eukaryotic cell or a prokaryotic cell. Preferably, the host cell is a prokaryotic cell such as a bacterial cell; however single cell eukaryotes such as protists or yeasts are also useful as host cells. Preferred host cells are microbial cells, preferably the cells of single-celled microbes such as bacterial cells or yeast cells, and are also referred to herein as “microorganisms.” Exemplary host microorganisms that can be metabolically engineered to incorporate the biosynthetic pathway described herein include Escherichia, Salmonella, Clostridium, Zymomonas, Pseudomonas, Bacillus, Rhodococcus, Alcaligenes, Klebsiella, Paenibacillus, Lactobacillus, Enterococcus, Arthrobacter, Brevibacterium, Corynebacterium Candida, Hansenula, Pichia and Saccharomyces. Preferred hosts include Escherichia coli, Bacillus subtilis Bacillus licheniformis, Alcaligenes eutrophus, Rhodococcus erythropolis, Paenibacillus macerans, Pseudomonas putida, Enterococcus faecium, Saccharomyces cerevisiae, Lactobacillus plantarum, Enterococcus gallinarium and Enterococcus faecalis. 
A cell that has been genetically engineered as described herein for muconic acid biosynthesis may be referred to as a “host” cell, a “recombinant” cell, a “metabolically engineered” cell, a “genetically engineered” cell or simply an “engineered” cell. These and similar terms are used interchangeably. As used herein, “genetically engineered” refers to a cell into which has been introduced at least one exogenous polynucleotide and has been altered “by the hand of man.” For example, “genetically engineered” refers to a cell that contains one or more artificial sequences of nucleotides which have been created through standard molecular cloning techniques to bring together genetic material that is not natively found together. DNA sequences used in the construction of recombinant DNA molecules can originate from any species. Alternatively, DNA sequences that do not occur anywhere in nature may be created by the chemical synthesis of DNA, and incorporated into recombinant molecules. “Genetically engineered” also refers to a cell that has been genetically manipulated such that one or more endogenous nucleotides have been altered. For example, a cell is a genetically engineered cell by virtue of introduction of an alteration of endogenous nucleotides into a suitable cell. For instance, a regulatory region, such as a promoter, could be altered to result in increased or decreased expression of an operably linked endogenous coding region.
Genetically engineered cells are also referred to as “metabolically engineered” cells when the genetic engineering modifies or alters one or more particular metabolic pathways so as to cause a change in metabolism. The goal of metabolic engineering is to improve the rate and conversion of a substrate into a desired product. General laboratory methods for introducing and expressing or overexpressing native and nonnative proteins such as enzymes in many different cell types (including bacteria, plants, and animals) are routine and well known in the art; see, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual., Cold Spring Harbor Laboratory Press (1989), and Methods for General and Molecular Bacteriology, (eds. Gerhardt et al.) American Society for Microbiology, chapters 13-14 and 16-18 (1994).
While certain embodiments of the method are described herein using E. coli as the microbe, the method is not limited to E. coli and there are a number of other options for microbes suitable for use in the method. Other suitable microbial hosts for the production of MA and SA include, but are not limited to, prokaryotes such as members of the genera Escherichia, Salmonella, Bacillus, Corynebacterium, and cyanobacteria, and eukaryotes such as fungi, including Saccharomyces cerevisiae. 
Introduction of the Biosynthetic Pathway into a Cell
In one embodiment, the genetically engineered organism includes, or is engineered to include, a first “synthetic” module, which supplies enzymatic activity to produce salicylic acid from chorismate, and a second “degradative” module, which supplies enzymatic activity to produce muconic acid from salicylic acid. In another embodiment, the genetically engineered organism further includes, or is engineered to include, a third “precursor enhancing” enzymatic molecule which increases availability of chorismate.
The introduction of the novel biosynthetic pathway of the invention into a cell involves expression or overexpression of one or more enzymes included in the biosynthetic pathway described herein. An enzyme is “overexpressed” in a recombinant cell when the enzyme is expressed at a level higher than the level at which it is expressed in a comparable wild-type cell. In cells that do not express a particular endogenous enzyme, or in cells in which the enzyme is not endogenous (i.e., the enzyme is not native to the cell), any level of expression of that enzyme in the cell is deemed an “overexpression” of that enzyme for purposes of the present invention.
As will be appreciated by a person of skill in the art, overexpression of an enzyme can be achieved through a number of molecular biology techniques. For example, overexpression can be achieved by introducing into the host cell one or more copies of a polynucleotide encoding a desired enzyme. A polynucleotide encoding a desired enzyme may be integrated in the genetically engineered microorganism's chromosome, or present in the genetically engineered microorganism as an extrachromosomal element, such as a plasmid or episome. Each coding region may be on a separate plasmid, all coding regions may be on one plasmid, or different coding regions may be grouped together on some combination thereof.
In one embodiment of the method of making the genetically engineered microorganism and resultant engineered microorganism, the polynucleotides that operably encode the selected enzymes, which are engineered into the host organism, are present on one or more plasmids. A separate plasmid can be used for each module, or two or more modules can be combined on the same plasmid. As a result, the genetically engineered host organism may include one, two or three plasmids to form the complete biosynthetic pathway for synthesis of muconic acid.
In one embodiment, the genetically engineered microorganism contains a biosynthetic pathway for the production of muconic acid that has been optimized to produce enhanced carbon flux into the pathway. Contrary to common wisdom, since high copy number plasmids typically yield better results in biosynthetic pathways, it was surprisingly found that expression of the degradative module on a low copy number plasmid resulted in dramatically improved MA production. A preferred genetically engineered microorganism contains a synthetic module on a high copy number plasmid and a degradative module on a relatively low copy number plasmid. An example of a high copy number plasmid is a plasmid having a copy number of over 20, or over 40. An example of a low copy number plasmid is a plasmid having a copy number under 20, or under 10. A precursor enhancer module, such as an APTA module, is optionally included on either high copy number plasmid, a medium copy number plasmid, or a low copy number plasmid.
In one embodiment, a genetically engineered microorganism of the invention contains three plasmids: a first plasmid expressing enzymes associated with the biosynthesis of salicylic acid, preferably an isochorismate synthase (ICS) and an isochorismate pyruvate lyase (IPL); a second plasmid expressing enzymes associated with the degradation of salicylic acid and its conversion to muconic acid, preferably a salicylate 1-monoxygenase (SMO) and a catechol 1,2-dioxygenase (CDO); and optionally third plasmid expressing enzymes associated with enhanced chorismate availability, preferably expressing one or more of aroL, ppsA, tktA, and aroGfbr, encoding shikimate kinase, phosphoenolpyruvate synthase, transketolase and feedback-inhibition-resistant 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, respectively.
In another embodiment, a genetically engineered microorganism of the invention contains two plasmids: a first plasmid expressing enzymes associated with the biosynthesis of salicylic acid, preferably an isochorismate synthase (ICS) and an isochorismate pyruvate lyase (IPL), and also expressing one or more enzymes associated with enhanced chorismate availability, preferably at least one of aroL, ppsA, tktA, and aroGfbr; and a second plasmid expressing enzymes associated with the degradation of salicylic acid and its conversion to muconic acid, preferably a salicylate 1-monoxygenase (SMO) and a catechol 1,2-dioxygenase (CDO).
The use of modules for different sections of the overall biosynthetic pathway provides significant advantages. Initially, the modules can be incorporated into separate plasmids. Expression levels for the various modules (each module operably encoding one or more enzymes associated with a portion of the overall metabolic pathway) can be conveniently controlled and tested by means of plasmid copy number. When it is found that low copy number is effective or even preferred, as described below for the salicylic acid “degradation” portion of the novel muconic acid pathway (the degradation module), copy number in the host can be reduced, thereby minimizing the metabolic burden to the host cell and conserving resources. When it is found that two (or more) modules are preferably expressed at the same level, they can be combined on a single plasmid. For example, as described below, both the synthetic module involved in biosynthesis of salicylic acid and the precursor enhancing module associated with increased availability of chorismate can optionally be included on the same plasmid, as higher expression of the enzymes encoded in those modules is beneficial. Overall, the use of multiple modules allows production of the desired end product, in this case for example, muconic acid, to be optimized by testing different expression levels.
In some embodiments, the host organism may produce, or may have been engineered to overproduce one or more aromatic amino acids, preferably phenylalanine and tyrosine. Such a host organism is then optionally further engineered to interrupt one or more metabolic pathways that utilize chorismate in the production of aromatic acids, for example by eliminating or knocking out one or more genes responsible for the first committing catalytic steps in the biosynthesis of phenylalanine, tyrosine, and tryptophan, such as pheA, tyrA, and trpD, respectively. Preferably, only pheA and tyrA of the competing pathways are eliminated or knocked out; trpD is not disrupted.
Production of Salicylic Acid, Muconic Acid, Derivatives, and Downstream Metabolites
With respect to methods, the invention includes not only methods for making the genetically engineered microbe, but also methods for using the genetically engineered microbe, for example to produce a desired biochemical. In one method, the genetically engineered microbe is cultured under conditions to produce a downstream metabolite of chorismate, preferably an organic acid, such as salicylic acid and/or muconic acid. Culturing can be small scale or large scale; it can be aerobic or anaerobic. Preferably, the genetically engineered organism is cultured in a large scale fermentation system. The salicylic acid, muconic acid or other metabolite is separated from the microorganism and optionally isolated and purified. The salicylic acid, muconic acid, or other metabolite can be isolated from the host cell, or it can be isolated from the cell supernatant. The salicylic acid, muconic acid, or other metabolite can be secreted by the host cell and then isolated or purified, or it can be removed or separated from the host cell by solubilization, permeabilization, enzymatic action, mechanical crushing, or any other method to separate the biochemical from the host cell or cellular components.
Muconic acid, salicylic acid or other metabolite produced by and optionally isolated from the host cell can be further chemically or enzymatically derivatized. Muconic acid is a potential platform chemical for the manufacture of several widely-used consumer plastics, and is a synthetic precursor of a number of commercially relevant compounds, including adipic acid, terephthalic acid, caprolactam, hexamethylenediamine, and adiponitrile. Salicylic acid is mainly used for producing pharmaceuticals, skincare and haircare products. Muconic acid, salicylic acid, and their derivatives or downstream metabolites can be incorporated into many different types of materials such as polymeric compounds, including heteropolymers, copolymers and block copolymers, for example polyethylene terephthalate (PET) and polyester, as well as pharmaceutical, cosmetic, detergent, and industrial compositions. Presently, commercial production of muconic acid, salicylic acid, and their various derivatives such as adipic acid and terephthalic acid predominantly relies on organic chemical synthesis using petroleum-derived chemicals as starting materials. A “green” synthesis of salicylic acid, muconic acid and other compounds using the microbial biosynthetic pathway described herein represents a novel, environmentally sound advance in the art.
The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.
EXAMPLES
Example 1
Extending Shikimate Pathway for the Production of Muconic Acid and Its Precursor Salicylic Acid in Escherichia coli 
cis,cis-Muconic acid (MA) and salicylic acid (SA) are naturally-occurring organic acids having great commercial value. MA is a potential platform chemical for the manufacture of several widely-used consumer plastics; while SA is mainly used for producing pharmaceuticals (for example, aspirin and lamivudine) and skincare and haircare products. At present, MA and SA are commercially produced by organic chemical synthesis using petro-derived aromatic chemicals, such as benzene, as starting materials, which is not environmentally friendly. Here, we report a novel approach for efficient microbial production of MA via extending shikimate pathway by introducing the hybrid of an SA biosynthetic pathway with its partial degradation pathway. First, we engineered a well-developed phenylalanine producing Escherichia coli strain into an SA overproducer by introducing isochorismate synthase and isochorismate pyruvate lyase. The engineered stain is able to produce 1.2 g/L of SA from simple carbon sources, which is the highest titer reported so far. Further, the partial SA degradation pathway involving salicylate 1-monoxygenase and catechol 1,2-dioxygenase is established to achieve the conversion of SA to MA. Finally, a de novo MA biosynthetic pathway is assembled by integrating the established SA biosynthesis and degradation modules. Modular optimization enables the production of 1.5 g/L MA within 48 h in shake flasks. This study not only establishes an efficient microbial platform for the production of SA and MA, but also demonstrates a generalizable pathway design strategy for the de novo biosynthesis of valuable degradation metabolites.
Fossil fuels supply the world with not only energy but also important feedstocks for chemical industry. However, the shrinking availability of fossil reserves and the deteriorating environment compel people to explore renewable alternatives for the production of fuels, chemicals, and pharmaceuticals. Fortunately, the metabolic diversity of biological systems provides us with an extremely rich chemical repertoire. In recent years, the development of metabolic engineering has enabled the establishment of microbial chemical factories by constituting heterologous or non-natural biosynthetic pathways into genetically advantageous microbial hosts (Ajikumar et al., 2010 Science 330:70-4; Anthony et al., 2009 Metab. Eng. 11:13-9; Atsumi et al., 2008 Metab. Eng. 10:305-11; Huang et al., 2013 Biotechnol. Bioeng. 110:3188-96; Lin et al., 2013 Metab. Eng. 18:69-77; Lin et al., 2012 Microb. Cell Fact. 11:42; Shen et al., 2008 Metab. Eng. 10:312-20; Shen et al., 2012 J. Ind. Microbiol. Biotechnol. 39:1725-9; Zhang et al., 2008 Proc. Natl. Acad. Sci. U. S. A. 105:20653-8). In this study, we report our work on extending shikimate pathway for the production of two industrially important chemicals, cis,cis-muconic acid (MA) and its biosynthetic precursor salicylic acid (SA) in Escherichia coli. 
MA is a platform chemical that serves as the precursor to several bio-plastics. It can be easily converted into adipic acid by chemical hydrogenation, and the latter one is a direct building block for nylon-6,6 and polyurethane (Sun et al., 2013 Appl. Environ. Microbiol. 79:4024-30). In addition, MA is a synthetic precursor to terephthalic acid, a chemical used for manufacturing polyethylene terephthalate (PET) and polyester (Curran et al., 2013 Metab. Eng. 15:55-66). The global production of adipic acid and terephthalic acid is 2.8 and 71 million metric tons, respectively (Curran et al., 2013 Metab. Eng. 15:55-66). SA is an important drug precursor mainly used for producing pharmaceuticals such as aspirin and lamivudine (an anti-HIV drug). SA esters and salts used in sunscreens and medicaments account for another large portion of SA consumption. The global market for SA products was estimated to be $292.5 million in 2012 and is expected to reach $521.2 million in 2019, growing at an annual increase of 8.6% (“Salicylic Acid Market for Pharmaceutical, Skin care, Hair care and Other Applications-Global Industry Analysis, Size, Share, Growth, Trends, and Forecast 2013-2019,” 2013 Transparency Market Research). Currently, commercial production of adipic acid, terephthalic acid, and SA predominantly relies on organic chemical synthesis using petroleum-derived chemicals such as benzene as starting materials. These chemical synthesis processes are considered nonrenewable and environmentally unfriendly. Therefore, it is of great importance to develop “green” synthetic approaches that can utilize renewable feedstocks.
In fact, MA and SA are both naturally-occurring metabolites. MA is an intermediate in the microbial degradation of aromatic hydrocarbons (Fuchs et al., 2011 Nat. Rev. Microbiol. 9:803-16); while SA serves not only as a plant hormone (Chen et al., 2009 Plant Signal Behav. 4:493-6) but also as a biosynthetic precursor of bacterial siderophore (Gaille et al., 2002 J. Biol. Chem. 277:21768-75). In past 20 years, many efforts have been made for the microbial production of MA. Draths and Frost reported the earliest study on the artificial biosynthesis of MA in Escherichia coli from renewable carbon source glucose (Draths et al., 1994 J. Am. Chem. Soc. 116:399-400). By introducing three heterologous enzymes 3-dehydroshikimate dehydratase, protocatechuic acid decarboxylase and catechol 1,2-dioxygenase (CDO), the carbon flux was redirected from the E. coli native shikimate pathway to the biosynthesis of MA. Metabolically optimized strains carrying this artificial pathway were able to produce up to 2.4 g/L of MA via two-stage bioconversion in shake flasks (Draths et al., 1994 J. Am. Chem. Soc. 116:399-400) and 38.6 g/L via fed-batch fermentation (Niu et al., 2002 Biotechnol. Prog. 18:201-11). Afterwards, the same pathway was reconstituted in Saccharomyces cerevisiae (Weber et al., 2012 Appl. Environ. Microbiol. 78:8421-30), and the highest titer reported was nearly 141 mg/L (Curran et al., 2013 Metab. Eng. 15:55-66). Very recently, our group reported the construction of a different artificial pathway in E. coli by shunting tryptophan biosynthesis from anthranilate, which led to the production of 389 mg/L MA in shake flasks (Sun et al., 2013 Appl. Environ. Microbiol. 79:4024-30). By contrast, much less attention has been paid on engineering microbes for the production of SA, except for our recent work, in which SA was produced as a biosynthetic precursor to 4-hydroxycoumarin. Over-expression of heterologous enzymes isochorismate synthase (ICS) and isochorismate pyruvate lyase (IPL) in a wild-type E. coli strain resulted in the accumulation of 158.5 mg/L of SA in the culture (Lin et al., 2013 Nat. Commun. 4:2603).
In this example, we first reconstitute and optimize the biosynthesis of SA in E. coli by engineering a well-developed phenylalanine overproducing strain, yielding 1.2 g/L of SA, which is the highest titer reported so far. Further, the partial SA degradation pathway involving salicylate 1-monoxygenase (SMO) and catechol 1,2-dioxygenase (CDO) is established to achieve the conversion of SA to MA. On these bases, a novel MA biosynthetic approach is established by introducing the hybrid of the SA biosynthetic pathway and its partial degradation pathway (FIG. 1). Through modular optimization, the generated optimal strain produces about 1.5 g/L of MA in shake flasks. See also Lin et al. “Extending shikimate pathway for the production of muconic acid and its precursor salicylic acid in Escherichia coli,” 2014 Metab. Eng. 23:62-69.
Materials and Methods
Media, Strains, and Plasmids
Luria-Bertani (LB) medium was used for inoculants preparation, cell propagation, and protein expression; while modified M9 medium was used for de novo microbial production of SA and MA. LB medium contains 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl. The modified M9 (M9Y) medium contains 10 g/L glycerol, 2.5 g/L glucose, 6 g/L Na2HPO4, 0.5 g/L NaCl, 3 g/L KH2PO4, 1 g/L NH4Cl, 246.5 mg/L MgSO4, 14.7 mg/L CaCl2, 1 g/L yeast extract, 2 g/L MOPS, vitamin B1 (2.0 mg), H3BO3 (1.25 mg), NaMoO4.2H2O (0.15 mg), CoCl2.6H2O (0.7 mg), CuSO4.5H2O (0.25 mg), MnCl2.4H2O (1.6 mg), and ZnSO4.7H2O (0.3 mg). When needed, ampicillin, kanamycin, and chloramphenicol were added to the medium to the final concentrations of 100, 50, and 34 μg/ml, respectively. E. coli XL1-Blue was used as the host strain for plasmid construction and propagation. E. coli BW25113 was used as a wild-type (wt) strain for in vivo enzyme assays and feeding experiments. E. coli BL21 Star (DE3) was used for protein expression and purification. E. coli ATCC31884 is a phenylalanine overproducing strain purchased from American Type Culture Collection (ATCC) and was used for constructing the derivative strains SXX1 and QH4 (Huang et al., 2013 Biotechnol. Bioeng. 110:3188-96). pZE12-luc, pCS27, and pSA74 are high-, medium- and low-copy plasmids, respectively, used for expressing pathway enzymes. Plasmid pETDuet-1 was employed for protein expression and purification. The details of the strains and plasmids used in this study are depicted in Table 1.







TABLE 1





Strains and Plasmids used in this study.

















Strain
Genotype
Source





XL1-Blue
recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB
Stratagene



lacIqZΔM15 Tn10 (Tetr)]


BW25113
rrnBT14 ΔlacZWJ16 hsdR514 ΔaraBADAH33 ΔrhaBADLD78
CGSC


QH4
E. coli ATCC31884 with pheA and tyrA disrupted
A


SXX1
QH4 with trpD disrupted
This study





Plasmids
Description
Reference





pZE12-luc
PLlacO1, colE ori, luc, Ampr
B, D


pCS27
PLlacO1, P15A ori, Kanr
B, D


pSA74
PLlacO1, pSC101 ori, Cmr
B, D


pETDuet-1
pT7, PBR322 ori, Ampr
Novagen


pZE-NahG
pZE12-luc containing nahGopt
This study


pZE-paantABC
pZE12-luc containing antABC from P. aeruginosa PAO1
C


pZE-pfantABC
pZE12-luc containing antABC from P. fluorescens Migula
C


pZE-ppbenABCD
pZE12-luc containing benABCD from P. putida KT2440
C


pET-NahG
pETDuet-1 containing nahGopt
This study


pZE-EP
pZE12-luc containing entC from E. coli and pchB from
B, D



P. fluorescens Migula


pCS-EP
pCS27 containing entC from E. coli and pchB from P. fluorescens
This study



Migula


pSA-EP
pSA74 containing entC from E. coli and pchB from P. fluorescens
This study



Migula


pZE-NC
pZE12-luc containing nahGopt and catA from P. putida KT2440
This study


pCS-NC
pCS27 containing nahGopt and catA from P. putida KT2440
This study


pSA-NC
pSA74 containing nahGopt and catA from P. putida KT2440
This study


pCS-APTA
pCS27 containing aroL, ppsA, tktA, aroGfbr from E. coli
B, D


pZE-EP-APTA
pZE12-luc containing PLlacO1-EP and PLlacO1-APTA
B, D


pZE-EP-NC
pZE12-luc PLlacO1-EP and PLlacO1-NC
This study


pCS-NC-APTA
pCS27 containing PLlacO1-NC and PLlacO1-APTA
This study





A: Huang et al., 2013 Biotechnol. Bioeng. 110:3188-96


B: Lin et al., 2013 Nat. Commun. 4:2603


C: Sun et al., 2013 Appl. Environ. Microbiol. 79:4024-30


D: U.S. patent application Pub. No. 2014/0370557, published Dec. 18, 2014






DNA Manipulation

Plasmids pZE-ppbenABCD, pZE-paantABC, pZE-pfantABC were constructed in our previous study (Sun et al., 2013 Appl. Environ. Microbiol. 79:4024-30). The codon-optimized gene nahGopt was synthesized and then subcloned into pZE12-luc vector using KpnI and XbaI, and into pETDuet-1 using NcoI and XhoI, yielding plasmids pZE-NahG and pET-NahG, respectively. The coding sequence of nahGopt (SEQ ID NO:5) is provided below:







Sequence of nahGopt (SEQ ID NO: 5):


ATGCAGAACAGTACCAGCGCCCTGAACGTTAGCATCATTGGCGGCGGTAT


 


CGCAGGCGTTGCACTGGCCCTGGACTTATGTCGCCACGCCCACCTGAACG


 


TGCAGCTGTTCGAGGCAGCCCCGGCCTTTGGCGAAGTTGGTGCCGGTGTT


 


AGCTTCGGCGCCAATGCAGTGCGTGCAATCGCCGGTCTGGGTATCGCAGA


 


GCCGTACGGCAAAATTGCCGACAGTAATCCGGCCCCGTGGCAGGACATCT


 


GGTTCGAATGGCGCAATGGCCGTGATGCCAAATACCTGGGTTGCAGCGTT


 


GCCGAAGGCGTTGGTCAAAGCAGTGTGCACCGTGCCGATTTCCTGGACGC


 


TCTGGCTTCTCAGCTGCCGGACGGTATCGCTCAGTTCGGTAAACGTGCTC


 


AGCGTGTTGAACAGGACGGTGAGCAAGTGCGTGTGACATTCACAGACGGC


 


AGCGAGCACCGCTGCGATCTGCTGATTGGTGCCGACGGTATCAAGAGTAG


 


CATCCGTGACCACGTGTTACAGGGCCTGAATCAACCGCTGGCAAGCCCGC


 


GTTTTAGCGGCACCTGCGCCTATCGCGGTCTGATCGATAGCCAGCAGCTG


 


CGTGAGGCCTATCGTGCCCGTGGCGTGGACGAGCATCTGATTGACGTGCC


 


GCAGATGTACCTGGGCCTGGACGGCCACATCCTGACCTTCCCGGTTAAAC


 


AAGGCCGCCTGATCAACGTGGTGGCCTTCATCAGTGACCGCAGCCAACCG


 


AACCCGGTTTGGCCGAGCGATACACCGTGGGTTCGTAATGCCACCCAAGC


 


CGAGATGCTGGCCGCATTCGAGGGCTGGGATGATGCAGCCCAAGTGCTGC


 


TGGAGTGCATCCCGACCCCTAGTCTGTGGGCCCTGCACGACCTGGCAGAA


 


TTACCGGGCTACGTTCACGGCCGTGTTGGCTTAATCGGCGACGCCGCCCA


 


CGCAATGTTACCGCATCAGGGTGCCGGTGCAGGTCAGGGTCTGGAGGATG


 


CCTGGTTACTGGCCCGCCTGCTGGAAGACCCGAAGGTGCTGGACAAACGC


 


CCGCAGGCAGTGCTGGATGCCTATGACGCCGTTCGTCGTCCTCGTGCCTG


 


CCGTGTGCAGCGTACCAGCTTCGAGGCCGGCGAACTGTATGAGTTCCGTG


 


ACCCGGCCGTGTTAGCCGACGAGGAGCGTCTGGGCAAAGTTCTGGCCGAA


 


CGCTTTGACTGGCTGTGGAACCACGACATGCAGGAAGACTTATTACAGGC


 


CCGTGAGCTGCTGGGTTTACGTGCCCAAGCCGCCTAA






The plasmids pZE-EP, pCS-APTA, pZE-EP-APTA were constructed in our previous study (Lin et al., 2013 Nat. Commun. 4:2603). The expression cassette PLlacO1-EP was amplified by PCR from pZE-EP and inserted into plasmids pCS27 and pSA74 between SacI and SpeI, yielding plasmids pCS-EP and pSA-EP, respectively. The gene catA was amplified from Pseudomonas putida KT2440 genomic DNA. Plasmid pZE-NC was created by subcloning nahGopt and catA into plasmid pZE12-luc using KpnI, XhoI, and XbaI. The expression cassette PLlacO1-NC was amplified by PCR from pZE-NC and inserted into plasmid pCS27, pSA74, pZE-EP, and pCS-APTA between SacI and SpeI, yielding plasmids pCS-NC, pSA-NC, pZE-EP-NC (two operons) and pCS-NC-APTA (two operons).

Screening SMOs In Vivo

To evaluate the activity of enzymes converting SA to catechol, E. coli BW25113 was transformed with the expression vectors pZE-ppbenABCD, pZE-paantABC, pZE-pfantABC and pZE-NahG, separately. The resultant transformants were inoculated in 3 ml LB liquid medium containing 100 μg/ml of ampicillin and grown aerobically at 37° C. The overnight cultures were inoculated into 20 ml fresh LB medium and left to grow at 37° C. till OD600 reached 0.6 and then induced with 0.25 mM of IPTG at 37° C. for additional 3 h. Then the cells were harvested, and re-suspended in M9Y medium (OD600=2.1-2.7). SA was added into the cell suspension to a final concentration of 2 mM. The flasks were incubated with shaking at 37° C. for 10 min for the SMO encoded by nahGopt and 1 h for ppBenABCD, paAntABC, and pfAntABC. Samples were taken by removing cell pellets and the product (catechol) concentrations were measured with HPLC. The in vivo activity was expressed as μM/min/OD.
In Vitro SMO Enzyme Assay
To express and purify the enzyme, E. coli BL21 Star (DE3) was transformed with the expression plasmid pET-NahG. A fresh transformant was inoculated in 3 ml LB medium containing 100 μg/ml of ampicillin and grown aerobically at 37° C. Overnight cultures were inoculated into 50 ml fresh LB medium and left to grow at 37° C. till OD600 reached 0.6 and then induced at 30° C. with 0.5 mM IPTG for another 3 h. Cells were then harvested and lysed by French Press. The recombinant protein with an N-terminal multi-histidine tag was purified using His-Spin protein miniprep kit (ZYMO RESEARCH). The enzyme concentration was measured using BCA kit (Pierce Chemicals). The standard enzyme assay was performed by making an assay mixture containing 500 μM NADH, 10 μM FAD, 0.97 nM purified enzyme, and SA as the substrate. The final volume was adjusted to 1 ml with Kpi buffer (20 mM, pH=7.0). The substrate concentrations varied from 0 to 100 μM. The reaction system was kept at 37° C. for 1 min and stopped by adding 50 μL HCl (20%). The reaction rates were calculated by measuring the formation of catechol via HPLC. The kinetic parameters were estimated with OriginPro8 through non-linear regression of the Michaelis-Menten equation.
Feeding Experiments
Feeding experiments were conducted to examine the production of MA from SA. E. coli BW25113 was transformed with the plasmid pZE-NC. Single colonies were inoculated into 3 ml LB medium containing 100 μg/ml ampicillin and grown aerobically at 37° C. 200 μl of overnight cultures were inoculated into 20 ml LB medium containing 100 μg/ml ampicillin. The cultures were left to grow at 37° C. till OD600 reached 0.6 and then induced with 0.25 mM IPTG. After 3 h induction, cells were harvested, re-suspended in 20 ml of M9Y medium containing 3 mM of SA. Then SA was continuously fed into the cultures at 3 mM/h. Samples were taken at 2 h, 5 h and 10 h and the product concentrations were analyzed by HPLC.
De Novo Production of SA and MA
Overnight LB cultures of the producing strains were inoculated at 3% into the M9Y medium containing appropriated antibiotics and cultivated at 37° C. with shaking at 300 rpm. IPTG was added to the cultures to a final concentration of 0.25 mM at 0 h. Samples were taken every 24 hours. OD600 values were measured and the concentrations of the products and intermediates were analyzed by HPLC.
HPLC Analysis
SA (from SIGMA ALDRICH), catechol (from Alfa Aesar), MA (from ACROS ORGANICS) were used as standards. Both the standards and samples were analyzed and quantified by HPLC (Dionex Ultimate 3000) equipped with a reverse phase ZORBAX SB-C18 column and an Ultimate 3000 Photodiode Array Detector. Solvent A was water with 0.1% formic acid, and solvent B was methanol. The column temperature was set to 28° C. The following gradient was used at a flow rate of 1 ml/min: 5 to 50% solvent B for 15 min, 50 to 5% solvent B for 1 min, and 5% solvent B for an additional 4 min. Quantification of SA, catechol, and MA was based on the peak areas at absorbance of specific wavelengths (329 nm for SA, 274 nm for catechol, and 260 nm for MA). Glucose, glycerol, and acetate were quantified using a previously described method (Shen et al., 2012 J. Ind. Microbiol. Biotechnol. 39:1725-9).
Results
A novel Artificial Biosynthetic Pathway Towards MA Production
SA is a widely occurring aromatic metabolite which is produced not only by plants as a phytohormone but also by some bacteria as an intermediate in the biosynthesis of siderophore (Chen et al., 2009 Plant Signal Behav. 4:493-6; Gaille et al., 2002 Biol. Chem. 277:21768-75). For its biosynthesis in bacteria, only two enzymes isochorismate synthase (ICS) and isochorismate pyruvate lyase (IPL) are required to synthesize SA from chorismate, a pivotal metabolite in shikimate pathway (Gaille et al., 2002 J. Biol. Chem. 277:21768-75; Gaille et al., 2003 J. Biol. Chem. 278:16893-8). In contrast, some bacterial species such as Pseudomonas were reported to be capable of utilizing SA as a carbon and energy source, during which SA is degraded via catechol and MA (Fuchs et al., 2011 Nat. Rev. Microbiol. 9:803-16; Seo et al., 2009 Int. I Environ. Res. Public. Health. 6:278-309). In nature, however, the catabolism and anabolism of SA usually do not occur simultaneously in time and space. In this work, we reconstitute and synchronize the SA biosynthesis pathway catalyzed by ICS and IPL and its partial degradation pathway catalyzed by SMO and CDO, leading to a novel biosynthetic approach towards MA production from renewable carbon sources (FIG. 1).
Transformation of a Phenylalanine Overproducer to an SA Overproducer
Re-directing carbon flux from the native shikimate pathway towards salicylate biosynthesis is the first step towards establishing the artificial MA biosynthetic pathway. Previously, we have reported that the EntC from E. coli and the PchB from P. fluorescence are the most efficient ICS and IPL, respectively, among all the screened enzymes. When EntC and PchB were co-expressed in wild type E. coli host, the resulting strain produced 158.5 mg/L of SA after 32-hour cultivation (Lin et al., 2013 Nat. Commun. 4:2603). To further elevate the production of SA, we focused on engineering a well-developed phenylalanine overproducing strain E. coli ATCC31884, since this strain has been successfully modified to produce tyrosine and caffeic acid efficiently (Huang et al., 2013 Biotechnol. Bioeng. 110:3188-96; Patnaik et al., 2008 Biotechnol. Bioeng. 99:741-52). To eliminate undesired consumption of chorismate, we disrupted the competing pathway genes pheA, tyrA, and trpD from ATCC31884 which encode the enzymes responsible for the first committing catalytic steps in the biosynthesis of phenylalanine, tyrosine, and tryptophan, respectively, generating the strain SXX1 (FIG. 2A). However, when EntC and PchB were over-expressed in SXX1 with the high-copy plasmid (pZE-EP), the resulting strain only produced 177.95 mg/L of SA after 48 hour cultivation (FIG. 2B), which is not significantly improved compared with the wild type host strain carrying pZE-EP. Meanwhile, we observed that the final cell density of this mutant strain was quite low (OD600=2.5), suggesting that the simultaneous disruption of pheA, tyrA and trpD impaired the cell growth.
Then we turned to another ATCC31884 derived strain QH4 with pheA and tyrA disrupted. QH4 carrying pZE-EP produced 778.16 mg/L SA by the end of 48 h. As we expected, the dramatic increase in SA production was accompanied by the improvement of cell growth (final OD600=4.6). Further, to test the impact of plasmid copy number on SA production, we constructed another two plasmids pCS-EP (medium copy number) and pSA-EP (low copy number). As shown in FIG. 2B, QH4 carrying pCS-EP and pSA-EP produced 621.05 and 207.20 mg/L SA, respectively, indicating that the reduced copy number of plasmids used for expressing EntC and PchB resulted in lower production of SA. Therefore, high-level expression of the pathway enzymes is preferred to redirect more carbon flux into the artificial pathway. On this basis, we further boosted the availability of chorismate by eliminating the bottlenecks associated with the shikimate pathway. For this purpose, we employed a previously constructed chorismate-boosting plasmid expressing aroL, ppsA, tktA, and the feedback inhibition resistant mutant of aroG (aroGfbr) (FIG. 1), namely pCS-APTA (medium copy number) (Lin et al., 2013 Nat. Commun. 4:2603). When pCS-APTA was co-transferred with pZE-EP into QH4, the resulting strain produced 1179.92 mg/L of SA at 48 h, a 51.6% increase in titer compared with its parent strain (QH4 containing pZE-EP). To our knowledge, this is the highest titer for SA production via microbial production approaches. However, when the APTA expression cassette was moved into the high-copy-number plasmid pZE-EP resulting in pZE-EP-APTA, the SA production of QH4 carrying pZE-EP-APTA (813.97 mg/L) was not improved significantly (FIG. 2B) compared with that of QH4 carrying pZE-EP alone. SA production for all the strains followed the growth-dependent pattern.
In addition, to evaluate the toxicity of SA against the host strain, a growth inhibition assay was conducted. We added SA into the cell cultures at different final concentrations ranging from 0-1000 mg/L and detected its impact on cell growth. As shown in FIG. 3C, SA exhibited toxicity against QH4 cells especially when its concentration exceeded 500 mg/L. 1000 mg/L SA completely inhibited the cell proliferation. Notably, strain QH4 carrying pZE-EP and pCS-APTA produced 1179.92 mg/L SA. This result suggested that on one hand QH4 can develop some degree of tolerance towards SA when the cells were initially exposed to low concentration of the produced SA; on the other hand, this strain has probably reached its maximum SA production capacity, which was constrained by its SA tolerance.
Screening for an Efficient SMO
Following the reconstitution and optimization of an SA biosynthetic pathway, our subsequent effort was focused on constructing the SA degradation pathway. SMO catalyzes the first enzymatic step of SA degradation, and thus it is of great importance to have an efficient SMO available for the pathway assembly. Pseudomonas species (for example, P. putida) are known to demonstrate diverse metabolism, especially their capability of degrading a variety of aromatic hydrocarbon pollutants (Seo et al., 2009 Int. J. Environ. Res. Public. Health. 6:278-309). A putative SMO (encoded by nahG) was identified from the genome of P. putida DOT-T1E. For the screening purpose, we synthesized a codon-optimized gene of nahGopt. In addition, we have previously reported two anthranilate 1,2-dioxygenases (ADOs) from P. aeruginosa PAO1 (encoded by paantABC) and P. fluorescens Migula (encoded by pfantABC) and a benzoate 1,2-dioxygenase (BDO) from P. putida KT2440 (encoded by ppbenABCD) that can catalyzed the conversion of anthranilate to catechol (Sun et al., 2013 Appl. Environ. Microbiol. 79:4024-30). Given the structure similarity of the substrates anthranilate (2-aminobenzoate) and SA (2-hydroxybenzoate), these enzymes were also considered as candidates for SMO screening. To test their catalytic activities, the genes paantABC, pfantABC, ppBenABCD, and nahGopt were cloned into a high-copy-number plasmid pZE12-luc separately, resulting in the corresponding expression vectors used for in vivo assays. The wild type E. coli cells carrying these expression vectors were cultivated and then fed with 2 mM SA. Their activities were estimated by measuring the catechol formation rates. As indicated in Table 2, the SMO encoded by nahGopt showed the highest in vivo activity (54.78 μM/min/OD) among all the tested enzymes, while the ADOs and BDO also exhibited activity towards SA to generate catechol, although their catalytic activities were much lower.
Furthermore, to measure the kinetic parameters of the SMO encoded by nahGopt, a multi-histidine tag was fused its N-terminus, and the recombinant protein was purified. The result of in vitro enzyme assay showed that the Km and kcat of the SMO were 4.37 μM and 96.56 s−1, respectively, indicating its high substrate affinity and catalytic efficiency (FIG. 3A).







TABLE 2







In vivo activity of salicylate 1-monoxygenases


from Pseudomonas species.













In vivo activity



Genes
Source
(μM/min/OD)







paantABC
P. aeruginosa
0.17 ± 0.01



pfantABC
P. fluorescence
0.16 ± 0.01



ppbenABCD
P. putida
1.89 ± 0.02



nahGopt
P. putida
54.78 ± 1.65 










Bioconversion of SA to MA

Conversion of SA to MA is a part of the SA degradation pathway, which involves the action of SMO and CDO. Given that an efficient CDO from P. putida KT-2440 (encoded by catA) has been identified previously in our lab (Sun et al., 2013 Appl. Environ. Microbiol. 79:4024-30), we aimed to assemble this pathway by co-expressing the SMO and the CDO in E. coli. For this purpose, the genes nahGopt and catA were cloned into a high-copy-number plasmid as an operon, yielding an expression vector pZE-NC. To explore its potential in the bioconversion of SA to MA, the wild type E. coli strain carrying pZE-NC was pre-cultivated in LB medium till the OD600 values reached 4-4.5 and then transferred to M9Y medium containing 3 mM SA. Considering the toxicity issue, the concentration of SA was maintained below 500 mg/L (3.62 mM) by continuous feeding at 3 mM/h. By the end of 2 and 5 h, the titers of MA reached 938.4 and 2313.0 mg/L, respectively (FIG. 3B), indicating the high conversion rate from SA to MA with this plasmid construct.
Efficient De Novo MA Production Via Modular Optimization
With the well-constructed SA biosynthesis and partial degradation pathways, we assembled the complete MA producing pathway by introducing the two modules simultaneously into E. coli using plasmid pZE-EP-NC, a high-copy expression vector with two operons. To our surprise, E. coli QH4 carrying pZE-EP-NC (Table 3, strain LS-1) only produced 5.27 mg/L MA after 48 h cultivation; while intermediates (SA and catechol) were not accumulated in the cultures. Besides, we observed that this strain underwent a longer lag phase before entering the exponential phase. We reasoned that the two modules were co-expressed in an unharmonious manner, which apparently exerted negative influence on the cell viability and productivity. To address this issue, we performed modular optimization to adjust the relative expression levels of each module. In this case, we decomposed the whole pathway into three modules (FIG. 4A), namely EP module (expressing entC and pchB responsible for SA biosynthesis), NC module (expressing nahGopt and catA responsible for converting SA to MA) and APTA module (expressing aroL, ppsA, tktA, and aroGfbr to increase chorismate availability). To ensure the maximum carbon flux towards the artificial pathway, the EP module was always fixed on high-copy plasmids. The expression level of NC module was optimized using high-, medium-, and low-copy plasmids. For APTA module, we attempted the high- and medium-copy expression in addition to the native expression (single copy). The plasmid combinations used for the modular optimization were listed in Table 3. As the result shown in Table 3 and FIG. 4B, compared with APTA module, NC module had much more impact on the performance of MA production. Expression of the NC module on a low-copy plasmid resulted in dramatically improved MA production, suggesting that relatively lower expression level of the NC module was more beneficial for this artificial pathway. Furthermore, over-expression of APTA module further enhanced the titer by about 20% (from LS6 to LS7 and LS8). Placing APTA module on the high-copy plasmid resulted in slightly higher MA production (1453.64 mg/L) than that on the medium-copy plasmid (1425.71 mg/L). Through modular optimization, the MA titer was improved by 275 folds compared with that of the initial test with strain LS-1. For all the MA producing strains, no accumulation of SA and catechol was observed, indicating the high robustness of NC module. We also analyzed the by-product accumulation and carbon source consumption for the best two strains LS-7 and LS-8. By the end of 48 h, Strain LS-8 consumed all the glucose (2.5 g/L) and glycerol (10 g/L), while strain LS-7 consumed all the glucose but left around 3 g/L glycerol unconsumed. Acetate was the major by-product for both strains (1.5-2.5 g/L). The OD600 values and MA titers for the engineered E. coli strains at 24 h and 48 h are listed in Table 4.







TABLE 3







Combinations of plasmids for modular optimization.










Strain
Module Copy No.

Titers












name
EP
NC
APTA
Plasmid combinations
(mg/L)





LS-1
H
H
S
pZE-EP-NC
 5.27 ± 1.14


LS-2
H
H
M
pZE-EP-NC, pCS-APTA
 23.13 ± 23.06


LS-3
H
M
S
pZE-EP, pCS-NC
 119.18 ± 23.15


LS-4
H
M
M
pZE-EP, pCS-NC-APTA
 177.64 ± 33.58


LS-5
H
M
H
pZE-EP-APTA, pCS-NC
 382.05 ± 30.62


LS-6
H
L
S
pZE-EP, pSA-NC
1179.26 ± 61.58


LS-7
H
L
M
pZE-EP, pSA-NC,
1425.71 ± 41.08






pCS-APTA


LS-8
H
L
H
pZE-EP-APTA, pSA-NC
1453.64 ± 98.88





* H, M and L indicate high-, medium-, and low-copy number plasmids; S refers to native expression with a single copy in the E. coli genome.













TABLE 4







The OD600 values and MA titers for the engineered E. coli strains.









Strain
24 h
48 h











name
OD600
Titer (mg/L)
OD600
Titer (mg/L)





LS-1
4.38 ± 1.50
3.35 ± 1.29
7.24 ± 0.50
 5.27 ± 1.14


LS-2
4.42 ± 2.51
29.52 ± 27.10
6.53 ± 0.49
 23.13 ± 23.06


LS-3
3.45 ± 0.19
83.38 ± 16.32
3.61 ± 0.28
 119.18 ± 23.15


LS-4
3.67 ± 0.32
60.20 ± 21.28
6.74 ± 0.07
 177.64 ± 33.58


LS-5
3.97 ± 0.46
100.09 ± 23.95 
6.69 ± 0.54
 382.05 ± 30.62


LS-6
4.08 ± 0.19
418.84 ± 22.70 
6.24 ± 0.26
1179.26 ± 61.58


LS-7*
3.16 ± 0.11
528.42 ± 9.03 
4.82 ± 0.22
1425.71 ± 41.08


LS-8*
3.72 ± 0.84
638.40 ± 31.23 
6.30 ± 0.23
1453.64 ± 98.88





*For LS-7 and LS-8, the OD600 values and MA titers at 72 h were slightly lower than those at 48 h.






Discussion

The extreme diversity of metabolism in living organisms generates an enormous number of natural products and metabolic intermediates, a substantial portion of which are of industrial and/or pharmaceutical importance. Over the past decades, metabolic engineering promoted the microbial production of a variety of valuable molecules, including bio-fuels, bulk chemicals, and pharmaceuticals (Oliver et al., 2013 Proc. Natl. Acad. Sci. U. S. A. 110:1249-54; Rabinovitch-Deere et al., 2013 Chem. Rev. 113:4611-32; Xu et al., 2013 Curr. Opin. Biotechnol. 24:291-299; Zhang et al., 2011 ChemSusChem. 4:1068-70). In this study, we devised a novel artificial pathway for the efficient production of MA by bridging the SA biosynthesis with its partial degradation pathway. In fact, biosynthesis and degradation of a specific molecule usually do not occur simultaneously in nature, since from the perspective of energy, it is not metabolically economic for organisms to survive nutrient-poor environment. But from the perspective of microbial production, it is a feasible strategy to link a degradation pathway to certain biosynthetic pathway and further to the microbial host's native metabolism, which can lead to the expansion of native metabolism and de novo production of a valuable degradation intermediate from inexpensive and renewable carbon sources. In addition to MA, we think this design strategy can also be generalized to establish the artificial biosynthesis of other degradation intermediates.
The shikimate pathway is the only route in bacteria leading to the biosynthesis of aromatic compounds. Before this study, two artificial pathways derived from the shikimate pathway have been reported for the microbial production of MA. Draths and Frost reported the first pathway that shunts the shikimate pathway via 3-dehydroshikimate by introducing three heterologous enzymes. To ensure high-level production, the competing pathway that consumes 3-dehydroshikimate was deleted. However, the knockout of shikimate dehydrogenase (aroE) disrupted not only the biosynthesis of aromatic amino acids but also the formation of other important aromatic molecules, such as 4-hydroxybenzoate, the precursor of the respiratory chain component, ubiquinol. Therefore, six supplements (L-phenylalanine, L-tyrosine, L-tryptophan, p-aminobenzoic acid, p-hydroxybenzoic acid, and 2,3-dihydroxybenzoic acid) were added in the fermentation medium to maintain the cell growth and high productivity, which increased the production cost (Draths et al., 1994 J. Am. Chem. Soc. 116:399-400; Niu et al., 2002 Biotechnol. Prog. 18:201-11). Recently, our group reported another MA producing pathway that shunts the tryptophan biosynthesis from anthranilate. This pathway keeps the integrity of the shikimate pathway, but involves a rate-limiting transamination step converting chorismate into anthranilate. This reaction requires the participation of glutamine, a less abundant amino acid, which limited the efficiency of the whole pathway (Sun et al., 2013 Appl. Environ. Microbiol. 79:4024-30). Comparatively, the pathway developed in this study does not disrupt the shikimate pathway either; meanwhile, all the catalytic steps in this pathway are very efficient. Although phenylalanine and tyrosine biosynthesis was deleted for high-level MA production, cell growth could be easily restored by supplementing a small amount of yeast extract (1 g/L).
In general, current metabolic engineering approaches towards the reconstitution of artificial pathways in heterologous hosts involve very limited regulatory mechanism, which frequently brings undesired effects. On one hand, unregulated expression of heterologous enzymes can cause cell toxicity and metabolic imbalance because of either the enzymes themselves or the toxic intermediates and products the enzymes generate. On the other hand, excessive expression of pathway enzymes can result in the waste of cellular resources and bring metabolic burden to the hosts, which may limit yield and productivity. Therefore, it is desirable to control the expression of pathway enzymes at a proper and balanced level. In recent years, the development of synthetic biology tools enabled the fine-tuning of protein expression level, including the adjustment of gene copy number (Lin et al., 2013 Nat. Commun. 4:2603), promoter strength (Hammer et al., 2006 Trends Biotechnol. 24:53-5), mRNA stability (Smolke et al., 2001 Metab. Eng. 3:313-21) and RBS binding efficiency (Salis et al., 2009 Nat. Biotechnol. 27:946-50). In addition, dynamic regulatory circuits were also developed to control pathway enzyme expression in response to the accumulation of certain intermediates (Dahl et al., 2013 Nat. Biotechnol. 31:1039-46; Zhang et al., 2012 Nat. Biotechnol. 30:354-9). These strategies were proved to be very effective for those relatively simple metabolic pathways. However, as artificial biosynthetic pathways become more complex, an increasing number of enzymes are involved in the pathway assembly. It becomes much more laborious and time-consuming to exhaustively explore the optimal expression level for each of the pathway enzymes simultaneously. In this study, we employed a simplified method of modular optimization to balance pathway enzyme expression. Using this approach, a multi-step pathway can be decomposed into several modules which can be initially optimized individually. Then the relative expression level of each module can be further adjusted in the context of the whole pathway. This strategy greatly reduces the number of engineering targets and combinations and has been used to achieve the efficient production of a variety of molecules (Ajikumar et al., 2010 Science 330:70-4; Juminaga et al., 2012 Appl. Environ. Microbiol. 78:89-98; Lin et al., 2013 Nat. Commun. 4:2603; Xu et al., 2013 Nat. Commun. 4:1409). In this work, the modular optimization enabled the improvement of MA production by 275 folds, which demonstrated its generalizable potential to the optimization of other complex pathways.
In conclusion, we established a novel MA biosynthetic pathway by connecting the SA biosynthesis with its partial degradation pathway and achieved the efficient production of MA and its precursor SA. However, cellular toxicity remains to be a challenge that limits the production of these chemicals, especially for SA. To address this issue, it is necessary to seek and develop more resistant E. coli strains. A recent work reported the engineering of efflux pumps which successfully improved the tolerance of E. coli toward non-native product n-butanol (Fisher et al., 2014 ACS Synth. Biol. 3:30-40). Besides, some microbial species other than E. coli exhibit high tolerance towards toxic chemicals, such as Pseudomonas. Given the increasing availability of genetic manipulation tools, it becomes more feasible to transfer and engineer the demonstrated pathways into these microorganisms.
The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.
All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

## Claims

1. A genetically engineered microorganism comprising a genetically engineered metabolic pathway for the production of muconic acid from a salicylic acid intermediate, said pathways comprising:
a plurality of enzymes associated with the biosynthesis of salicylic acid, said plurality of enzymes comprising an isochorismate synthase (ICS) and an isochorismate pyruvate lyase (IPL); and
a plurality of enzymes associated with the conversion of salicylic acid to muconic acid, said plurality of enzymes comprising a salicylate 1-monoxygenase (SMO) and a catechol 1,2-dioxygenase (CDO).

2. The genetically engineered microorganism of claim 1 wherein the genetically engineered metabolic pathway further comprises at least one enzyme associated with enhanced chorismate availability.

3. The genetically engineered microorganism of claim 2 wherein the enzyme associated with enhanced chorismate availability is selected from the group consisting of a shikimate kinase, a phosphoenolpyruvate synthase, a transketolase, a 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, a 3-dehydroquinate synthase, a 3-dehydroquinate dehydratase, a shikimate dehydrogenase, a 3-phosphoshikimate-1-carboxyvinyltransferase, and a chorismate synthase.

4. The genetically engineered microorganism of claim 1 comprising:
a first module expressing an isochorismate synthesis (ICS) and an isochorismate pyruvate lyase (IPL); and
a second module expressing a salicylate 1-monoxygenase (SMO) and a catechol 1,2-dioxygenase (CDO).

5. The genetically engineered microorganism of claim 4 wherein the first and second modules are present on different plasmids.

6. The genetically engineered microorganism of claim 4 further comprising a third module expressing at least one enzyme that increases carbon flow toward chorismate.

7. The genetically engineered microorganism of claim 6 wherein the third module is present on a plasmid.

8. The genetically engineered microorganism of claim 7 wherein the first and third modules are present on a high copy number plasmid, and wherein the second module is present on a low copy number plasmid.

9. The genetically engineered microorganism of claim 1 which has been further engineered to overproduce at least one aromatic amino acid.

10. The genetically engineered microorganism of claim 9 wherein the aromatic amino acid comprises at least one of phenylalanine and tyrosine.

11. The genetically engineered microorganism of claim 9 which has been further engineered to knock out one or more enzyme involved in biosynthesis of an aromatic amino acid, wherein the enzyme is encoded by pheA, tyrA or trpD.

12. The genetically engineered microorganism of claim 11 wherein pheA and tyrA are knocked out, and wherein trpD, if present, is not disrupted.

13. The genetically engineered microorganism of claim 1 comprising a bacterium or a yeast.

14. The genetically engineered microorganism of claim 13 selected from the group consisting of Escherichia coli, Bacillus subtilis Bacillus licheniformis, Alcaligenes eutrophus, Rhodococcus erythropolis, Paenibacillus macerans, Pseudomonas putida, Enterococcus faecium, Saccharomyces cerevisiae, Lactobacillus plantarum, Enterococcus gallinarium and Enterococcus faecalis.

15. The genetically engineered microorganism of claim 14 which comprises E. coli.

16. The genetically engineered microorganism of claim 15 comprising at least one plasmid selected from the group consisting of pCS-EP, pZE-EP, pSA-EP, pCS-NC, pZE-NC, pSA-NC, pCS-APTA, pZE-EP-APTA.

17. The genetically engineered microorganism of claim 1 wherein the genetically engineered biosynthetic pathway comprises at least one enzyme that is heterologous to the host organism.

18. The genetically engineered microorganism of claim 1 wherein the genetically engineered metabolic pathway comprises at least one enzyme that is naturally occurring in the host organism.

19. A method for producing organic acid, comprising:
culturing the genetically engineered microorganism of claim 1 under conditions to produce an downstream metabolite of chorismate comprising an organic acid.

20. The method of claim 19 wherein the organic acid comprises salicylic acid.

21. The method of claim 19 wherein the organic acid comprises muconic acid.

22. The method of claim 19 further comprising separating the organic acid from the genetically engineered microorganism.

23. The method of claim 19 further comprising isolating and purifying the organic acid.

24. The genetically engineered microorganism of claim 3 wherein the enzyme associated with enhanced chorismate availability is encoded by at least one of aroL, ppsA, tktA, and aroGfbr.