scholarly journals A New Player in the Biorefineries Field: Phasin PhaP Enhances Tolerance to Solvents and Boosts Ethanol and 1,3-Propanediol Synthesis in Escherichia coli

2017 ◽  
Vol 83 (14) ◽  
Author(s):  
Mariela P. Mezzina ◽  
Daniela S. Álvarez ◽  
Diego E. Egoburo ◽  
Rocío Díaz Peña ◽  
Pablo I. Nikel ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Carbon Sources ◽  
Added Value ◽  
Heterologous Proteins ◽  
Bacterial Strains ◽  
Content Type ◽  
E Coli ◽  
The Road ◽  
Product Titer ◽  
Protein Group

ABSTRACT The microbial production of biofuels and other added-value chemicals is often limited by the intrinsic toxicity of these compounds. The phasin PhaP from the soil bacterium Azotobacter sp. strain FA8 is a polyhydroxyalkanoate granule-associated protein that protects recombinant Escherichia coli against several kinds of stress. PhaP enhances growth and poly(3-hydroxybutyrate) synthesis in polymer-producing recombinant strains and reduces the formation of inclusion bodies during overproduction of heterologous proteins. In this work, the heterologous expression of this phasin in E. coli was used as a strategy to increase tolerance to several biotechnologically relevant chemicals. PhaP was observed to enhance bacterial fitness in the presence of biofuels, such as ethanol and butanol, and other chemicals, such as 1,3-propanediol. The effect of PhaP was also studied in a groELS mutant strain, in which both GroELS and PhaP were observed to exert a beneficial effect that varied depending on the chemical tested. Lastly, the potential of PhaP and GroEL to enhance the accumulation of ethanol or 1,3-propanediol was analyzed in recombinant E. coli. Strains that overexpressed either groEL or phaP had increased growth, reflected in a higher final biomass and product titer than the control strain. Taken together, these results add a novel application to the already multifaceted phasin protein group, suggesting that expression of these proteins or other chaperones can be used to improve the production of biofuels and other chemicals. IMPORTANCE This work has both basic and applied aspects. Our results demonstrate that a phasin with chaperone-like properties can increase bacterial tolerance to several biochemicals, providing further evidence of the diverse properties of these proteins. Additionally, both the PhaP phasin and the well-known chaperone GroEL were used to increase the biosynthesis of the biotechnologically relevant compounds ethanol and 1,3-propanediol in recombinant E. coli. These findings open the road for the use of these proteins for the manipulation of bacterial strains to optimize the synthesis of diverse bioproducts from renewable carbon sources.

scholarly journals Production in Escherichia coli of Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) with Differing Monomer Compositions from Unrelated Carbon Sources

2011 ◽  
Vol 77 (14) ◽  
pp. 4886-4893 ◽  
Author(s):  
Quan Chen ◽  
Qian Wang ◽  
Guoqing Wei ◽  
Quanfeng Liang ◽  
Qingsheng Qi
Keyword(s):  
Escherichia Coli ◽  
Control Strategy ◽  
Biosynthetic Pathway ◽  
Feedback Inhibition ◽  
Carbon Sources ◽  
Threonine Deaminase ◽  
Content Type ◽  
E Coli ◽  
Key Factor ◽  
Threonine Biosynthesis

ABSTRACTThe industrial production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) has been hindered by high cost and a complex control strategy caused by the addition of propionate. In this study, based on analysis of the PHBV biosynthesis process, we developed a PHBV biosynthetic pathway from a single unrelated carbon source via threonine biosynthesis inEscherichia coli. To accomplish this, we (i) overexpressed threonine deaminase, which is the key factor for providing propionyl-coenzyme A (propionyl-CoA), from different host bacteria, (ii) removed the feedback inhibition of threonine by mutating and overexpressing thethrABCoperon inE. coli, and (iii) knocked out the competitive pathways of catalytic conversion of propionyl-CoA to 3-hydroxyvaleryl-CoA. Finally, we constructed a series of strains and mutants which were able to produce the PHBV copolymer with differing monomer compositions in a modified M9 medium supplemented with 20 g/liter xylose. The largest 3-hydroxyvalerate fraction obtained in the copolymer was 17.5 mol%.

scholarly journals Nanocapsular Dispersion of Thymol for Enhanced Dispersibility and Increased Antimicrobial Effectiveness against Escherichia coli O157:H7 and Listeria monocytogenes in Model Food Systems

2012 ◽  
Vol 78 (23) ◽  
pp. 8448-8453 ◽  
Author(s):  
Bhavini Shah ◽  
P. Michael Davidson ◽  
Qixin Zhong
Keyword(s):  
Escherichia Coli ◽  
Listeria Monocytogenes ◽  
Food Systems ◽  
Bacterial Strains ◽  
Content Type ◽  
Apple Cider ◽  
E Coli ◽  
Reduced Fat ◽  
Treatment Conditions ◽  
Food Applications

ABSTRACTEssential oils are marginally soluble in water, making it challenging to evenly disperse them in foods and resulting in an increased tendency to bind with food lipids and proteins, resulting in lowered antimicrobial efficacy. In the current study, free and nano-dispersed (ND) thymol were compared in terms of their antimicrobial efficacies againstEscherichia coliO157:H7 ATCC 43889 and 43894 andListeria monocytogenesstrains Scott A and 101 in apple cider and 2% reduced-fat milk. Apple cider was adjusted to pHs 5.5 and 3.5, and antimicrobial tests were performed at 0.3-, 0.5-, 0.75-, and 1.0-g/liter thymol concentrations at 35, 32, 25, and 4°C. Overall, 0.5 and 1.0 g/liter thymol in nano-dispersion and along with free thymol were inhibitory and bactericidal, respectively, against bacterial strains under all treatment conditions. At pH 5.5, 0.5 g/liter ND thymol was bacteriostatic againstL. monocytogenesandE. colifor up to 48 h. At pH 3.5,L. monocytogenescontrols did not survive beyond 12 h butE. colisurvived and was inhibited by 0.5 g/liter ND thymol after 12 and 48 h in apple cider.E. colistrains were significantly sensitive to 4°C and pH 3.5 (P< 0.05). When bacteria were tested in 2% reduced-fat milk at 35 or 32°C, ND and free thymol demonstrated inhibition at 4.5 g/liter. Thus, the current technology seems to be promising and novel, enabling thymol-containing nano-dispersions that are not only transparent but also effective against pathogens in food applications, especially in clear beverages.

scholarly journals Positive Effect of Carbon Sources on Natural Transformation in Escherichia coli: Role of Low-Level Cyclic AMP (cAMP)-cAMP Receptor Protein in the Derepression ofrpoS

2015 ◽  
Vol 197 (20) ◽  
pp. 3317-3328 ◽  
Author(s):  
Mengyue Guo ◽  
Huanyu Wang ◽  
Nengbin Xie ◽  
Zhixiong Xie
Keyword(s):  
Escherichia Coli ◽  
Natural Transformation ◽  
Carbon Sources ◽  
Transformation Frequency ◽  
Receptor Protein ◽  
Camp Receptor Protein ◽  
Content Type ◽  
E Coli ◽  
Camp Receptor

ABSTRACTNatural plasmid transformation ofEscherichia coliis a complex process that occurs strictly on agar plates and requires the global stress response factor σS. Here, we showed that additional carbon sources could significantly enhance the transformability ofE. coli. Inactivation of phosphotransferase system genes (ptsH,ptsG, andcrr) caused an increase in the transformation frequency, and the addition of cyclic AMP (cAMP) neutralized the promotional effect of carbon sources. This implies a negative role of cAMP in natural transformation. Further study showed thatcrpandcyaAmutations conferred a higher transformation frequency, suggesting that the cAMP-cAMP receptor protein (CRP) complex has an inhibitory effect on transformation. Moreover, we observed thatrpoSis negatively regulated by cAMP-CRP in early log phase and that bothcrpandcyaAmutants show no transformation superiority whenrpoSis knocked out. Therefore, it can be concluded that both thecrpandcyaAmutations derepressrpoSexpression in early log phase, whereby they aid in the promotion of natural transformation ability. We also showed that the accumulation of RpoS during early log phase can account for the enhanced transformation aroused by additional carbon sources. Our results thus demonstrated that the presence of additional carbon sources promotes competence development and natural transformation by reducing cAMP-CRP and, thus, derepressingrpoSexpression during log phase. This finding could contribute to a better understanding of the relationship between nutrition state and competence, as well as the mechanism of natural plasmid transformation inE. coli.IMPORTANCEEscherichia coli, which is not usually considered to be naturally transformable, was found to spontaneously take up plasmid DNA on agar plates. Researching the mechanism of natural transformation is important for understanding the role of transformation in evolution, as well as in the transfer of pathogenicity and antibiotic resistance genes. In this work, we found that carbon sources significantly improve transformation by decreasing cAMP. Then, the low level of cAMP-CRP derepresses the general stress response regulator RpoS via a biphasic regulatory pattern, thereby contributing to transformation. Thus, we demonstrate the mechanism by which carbon sources affect natural transformation, which is important for revealing information about the interplay between nutrition state and competence development inE. coli.

scholarly journals Gene Dispensability in Escherichia coli Grown in Thirty Different Carbon Environments

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Madeline Tong ◽  
Shawn French ◽  
Sara S. El Zahed ◽  
Wai kit Ong ◽  
Peter D. Karp ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Carbon Source ◽  
High Throughput ◽  
Carbon Metabolism ◽  
Gene Deletion ◽  
Carbon Sources ◽  
Central Metabolism ◽  
Content Type ◽  
E Coli ◽  
Growth Phenotype

ABSTRACT Central metabolism is a topic that has been studied for decades, and yet, this process is still not fully understood in Escherichia coli, perhaps the most amenable and well-studied model organism in biology. To further our understanding, we used a high-throughput method to measure the growth kinetics of each of 3,796 E. coli single-gene deletion mutants in 30 different carbon sources. In total, there were 342 genes (9.01%) encompassing a breadth of biological functions that showed a growth phenotype on at least 1 carbon source, demonstrating that carbon metabolism is closely linked to a large number of processes in the cell. We identified 74 genes that showed low growth in 90% of conditions, defining a set of genes which are essential in nutrient-limited media, regardless of the carbon source. The data are compiled into a Web application, Carbon Phenotype Explorer (CarPE), to facilitate easy visualization of growth curves for each mutant strain in each carbon source. Our experimental data matched closely with the predictions from the EcoCyc metabolic model which uses flux balance analysis to predict growth phenotypes. From our comparisons to the model, we found that, unexpectedly, phosphoenolpyruvate carboxylase (ppc) was required for robust growth in most carbon sources other than most trichloroacetic acid (TCA) cycle intermediates. We also identified 51 poorly annotated genes that showed a low growth phenotype in at least 1 carbon source, which allowed us to form hypotheses about the functions of these genes. From this list, we further characterized the ydhC gene and demonstrated its role in adenosine efflux. IMPORTANCE While there has been much study of bacterial gene dispensability, there is a lack of comprehensive genome-scale examinations of the impact of gene deletion on growth in different carbon sources. In this context, a lot can be learned from such experiments in the model microbe Escherichia coli where much is already understood and there are existing tools for the investigation of carbon metabolism and physiology (1). Gene deletion studies have practical potential in the field of antibiotic drug discovery where there is emerging interest in bacterial central metabolism as a target for new antibiotics (2). Furthermore, some carbon utilization pathways have been shown to be critical for initiating and maintaining infection for certain pathogens and sites of infection (3–5). Here, with the use of high-throughput solid medium phenotyping methods, we have generated kinetic growth measurements for 3,796 genes under 30 different carbon source conditions. This data set provides a foundation for research that will improve our understanding of genes with unknown function, aid in predicting potential antibiotic targets, validate and advance metabolic models, and help to develop our understanding of E. coli metabolism.

scholarly journals A Modular, Tn7-Based System for Making Bioluminescent or Fluorescent Salmonella and Escherichia coli Strains

2016 ◽  
Vol 82 (16) ◽  
pp. 4931-4943 ◽  
Author(s):  
Dylan J. Shivak ◽  
Keith D. MacKenzie ◽  
Nikole L. Watson ◽  
J. Alex Pasternak ◽  
Brian D. Jones ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
High Throughput ◽  
Constitutive Expression ◽  
Bacterial Strains ◽  
Efficient System ◽  
Transposase Gene ◽  
Content Type ◽  
E Coli

ABSTRACTOur goal was to develop a robust tagging method that can be used to track bacterial strainsin vivo. To address this challenge, we adapted two existing systems: a modular plasmid-based reporter system (pCS26) that has been used for high-throughput gene expression studies inSalmonellaandEscherichia coliand Tn7transposition. We generated kanamycin- and chloramphenicol-resistant versions of pCS26 with bacterial luciferase, green fluorescent protein (GFP), and mCherry reporters under the control of σ70-dependent promoters to provide three different levels of constitutive expression. We improved upon the existing Tn7system by modifying the delivery vector to accept pCS26 constructs and moving the transposase genes from a nonreplicating helper plasmid into a temperature-sensitive plasmid that can be conditionally maintained. This resulted in a 10- to 30-fold boost in transposase gene expression and transposition efficiencies of 10−8to 10−10inSalmonella entericaserovar Typhimurium andE. coliAPEC O1, whereas the existing Tn7system yielded no successful transposition events. The new reporter strains displayed reproducible signaling in microwell plate assays, confocal microscopy, andin vivoanimal infections. We have combined two flexible and complementary tools that can be used for a multitude of molecular biology applications within theEnterobacteriaceae. This system can accommodate new promoter-reporter combinations as they become available and can help to bridge the gap between modern, high-throughput technologies and classical molecular genetics.IMPORTANCEThis article describes a flexible and efficient system for tagging bacterial strains. Using our modular plasmid system, a researcher can easily change the reporter type or the promoter driving expression and test the parameters of these new constructsin vitro. Selected constructs can then be stably integrated into the chromosomes of desired strains in two simple steps. We demonstrate the use of this system inSalmonellaandE. coli, and we predict that it will be widely applicable to other bacterial strains within theEnterobacteriaceae. This technology will allow for improvedin vivoanalysis of bacterial pathogens.

scholarly journals Improved production of the non-native cofactor F420 in Escherichia coli

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mihir V. Shah ◽  
Hadi Nazem-Bokaee ◽  
James Antoney ◽  
Suk Woo Kang ◽  
Colin J. Jackson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Expression System ◽  
Carbon Sources ◽  
Fold Increase ◽  
High Yield ◽  
Antibiotic Biosynthesis ◽  
Bacterial Strains ◽  
E Coli ◽  
A Genome

AbstractThe deazaflavin cofactor F420 is a low-potential, two-electron redox cofactor produced by some Archaea and Eubacteria that is involved in methanogenesis and methanotrophy, antibiotic biosynthesis, and xenobiotic metabolism. However, it is not produced by bacterial strains commonly used for industrial biocatalysis or recombinant protein production, such as Escherichia coli, limiting our ability to exploit it as an enzymatic cofactor and produce it in high yield. Here we have utilized a genome-scale metabolic model of E. coli and constraint-based metabolic modelling of cofactor F420 biosynthesis to optimize F420 production in E. coli. This analysis identified phospho-enol pyruvate (PEP) as a limiting precursor for F420 biosynthesis, explaining carbon source-dependent differences in productivity. PEP availability was improved by using gluconeogenic carbon sources and overexpression of PEP synthase. By improving PEP availability, we were able to achieve a ~ 40-fold increase in the space–time yield of F420 compared with the widely used recombinant Mycobacterium smegmatis expression system. This study establishes E. coli as an industrial F420-production system and will allow the recombinant in vivo use of F420-dependent enzymes for biocatalysis and protein engineering applications.

scholarly journals MhpA Is a Hydroxylase Catalyzing the Initial Reaction of 3-(3-Hydroxyphenyl)Propionate Catabolism in Escherichia coli K-12

2019 ◽  
Vol 86 (4) ◽  
Author(s):  
Ying Xu ◽  
Ning-Yi Zhou
Keyword(s):  
Escherichia Coli ◽  
Liquid Chromatography ◽  
High Performance ◽  
Initial Reaction ◽  
Functional Identification ◽  
Bacterial Strains ◽  
Content Type ◽  
E Coli ◽  
C Terminus ◽  
K 12

ABSTRACT Escherichia coli K-12 and some other strains have been reported to be capable of utilizing 3-(3-hydroxyphenyl)propionate (3HPP), one of the phenylpropanoids from lignin. Although other enzymes involved in 3HPP catabolism and their corresponding genes from its degraders have been identified, 3HPP 2-hydroxylase, catalyzing the first step of its catabolism, has yet to be functionally identified at biochemical and genetic levels. In this study, we investigated the function and characteristics of MhpA from E. coli strain K-12 (MhpAK-12). Gene deletion and complementation showed that mhpA was vital for its growth on 3HPP, but the mhpA deletion strain was still able to grow on 3-(2,3-dihydroxyphenyl)propionate (DHPP), the hydroxylation product transformed from 3HPP by MhpAK-12. MhpAK-12 was overexpressed and purified, and it was likely a polymer and tightly bound with an approximately equal number of moles of FAD. Using NADH or NADPH as a cofactor, purified MhpAK-12 catalyzed the conversion of 3HPP to DHPP at a similar efficiency. The conversion from 3HPP to DHPP by purified MhpAK-12 was confirmed using high-performance liquid chromatography and liquid chromatography-mass spectrometry. Bioinformatics analysis indicated that MhpAK-12 and its putative homologues belonged to taxa that were phylogenetically distant from functionally identified FAD-containing monooxygenases (hydroxylases). Interestingly, MhpAK-12 has approximately an extra 150 residues at its C terminus in comparison to its close homologues, but its truncated versions MhpAK-12400 and MhpAK-12480 (with 154 and 74 residues deleted from the C terminus, respectively) both lost their activities. Thus, MhpAK-12 has been confirmed to be a 3HPP 2-hydroxylase catalyzing the conversion of 3HPP to DHPP, the initial reaction of 3HPP degradation. IMPORTANCE Phenylpropionate and its hydroxylated derivatives resulted from lignin degradation ubiquitously exist on the Earth. A number of bacterial strains have the ability to grow on 3HPP, one of the above derivatives. The hydroxylation was thought to be the initial and vital step for its aerobic catabolism via the meta pathway. The significance of our research is the functional identification and characterization of the purified 3HPP 2-hydroxylase MhpA from Escherichia coli K-12 at biochemical and genetic levels, since this enzyme has not previously been expressed from its encoding gene, purified, and characterized in any bacteria. It will not only fill a gap in our understanding of 3HPP 2-hydroxylase and its corresponding gene for the critical step in microbial 3HPP catabolism but also provide another example of the diversity of microbial degradation of plant-derived phenylpropionate and its hydroxylated derivatives.

scholarly journals Phenotypic Diversity Caused by Differential RpoS Activity among Environmental Escherichia coli Isolates

2011 ◽  
Vol 77 (22) ◽  
pp. 7915-7923 ◽  
Author(s):  
Sarah M. Chiang ◽  
Tao Dong ◽  
Thomas A. Edge ◽  
Herb E. Schellhorn
Keyword(s):  
Escherichia Coli ◽  
Phenotypic Diversity ◽  
Carbon Sources ◽  
Enteric Bacteria ◽  
Environmental Isolates ◽  
Bacterial Populations ◽  
Content Type ◽  
Phenotypic Differences ◽  
Adaptive Mutations ◽  
E Coli

ABSTRACTEnteric bacteria deposited into the environment by animal hosts are subject to diverse selective pressures. These pressures may act on phenotypic differences in bacterial populations and select adaptive mutations for survival in stress. As a model to study phenotypic diversity in environmental bacteria, we examined mutations of the stress response sigma factor, RpoS, in environmentalEscherichia coliisolates. A total of 2,040 isolates from urban beaches and nearby fecal pollution sources on Lake Ontario (Canada) were screened for RpoS function by examining growth on succinate and catalase activity, two RpoS-dependent phenotypes. TherpoSsequence was determined for 45 isolates, including all candidate RpoS mutants, and of these, six isolates were confirmed as mutants with the complete loss of RpoS function. Similarly to laboratory strains, the RpoS expression of these environmental isolates was stationary phase dependent. However, the expression of RpoS regulon members KatE and AppA had differing levels of expression in several environmental isolates compared to those in laboratory strains. Furthermore, after platingrpoS+isolates on succinate, RpoS mutants could be readily selected from environmentalE. coli. Naturally isolated and succinate-selected RpoS mutants had lower generation times on poor carbon sources and lower stress resistance than theirrpoS+isogenic parental strains. These results show that RpoS mutants are present in the environment (with a frequency of 0.003 among isolates) and that, similarly to laboratory and pathogenic strains, growth on poor carbon sources selects forrpoSmutations in environmentalE. coli. RpoS selection may be an important determinant of phenotypic diversification and, hence, the survival ofE. coliin the environment.

scholarly journals The General Phosphotransferase System Proteins Localize to Sites of Strong Negative Curvature in Bacterial Cells

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Sutharsan Govindarajan ◽  
Yair Elisha ◽  
Keren Nevo-Dinur ◽  
Orna Amster-Choder
Keyword(s):  
Escherichia Coli ◽  
Negative Curvature ◽  
Carbon Sources ◽  
Bacterial Cells ◽  
Phosphotransferase System ◽  
Content Type ◽  
E Coli ◽  
Negatively Curved ◽  
Subcellular Domains ◽  
Geometric Cues

ABSTRACTThe bacterial cell poles are emerging as subdomains where many cellular activities take place, but the mechanisms for polar localization are just beginning to unravel. The general phosphotransferase system (PTS) proteins, enzyme I (EI) and HPr, which control preferential use of carbon sources in bacteria, were recently shown to localize near theEscherichia colicell poles. Here, we show that EI localization does not depend on known polar constituents, such as anionic lipids or the chemotaxis receptors, and on the cell division machinery, nor can it be explained by nucleoid occlusion or localized translation. Detection of the general PTS proteins at the budding sites of endocytotic-like membrane invaginations in spherical cells and their colocalization with the negative curvature sensor protein DivIVA suggest that geometric cues underlie localization of the PTS system. Notably, the kinetics of glucose uptake by spherical and rod-shapedE. colicells are comparable, implying that negatively curved “pole-like” sites support not only the localization but also the proper functioning of the PTS system in cells with different shapes. Consistent with the curvature-mediated localization model, we observed the EI protein fromBacillus subtilisat strongly curved sites in bothB. subtilisandE. coli. Taken together, we propose that changes in cell architecture correlate with dynamic survival strategies that localize central metabolic systems like the PTS to subcellular domains where they remain active, thus maintaining cell viability and metabolic alertness.IMPORTANCEDespite their tiny size and the scarcity of membrane-bounded organelles, bacteria are capable of sorting macromolecules to distinct subcellular domains, thus optimizing functionality of vital processes. Understanding the cues that organize bacterial cells should provide novel insights into the complex organization of higher organisms. Previously, we have shown that the general proteins of the phosphotransferase system (PTS) signaling system, which governs utilization of carbon sources in bacteria, localize to the poles ofEscherichia colicells. Here, we show that geometric cues, i.e., strong negative membrane curvature, mediate positioning of the PTS proteins. Furthermore, localization to negatively curved regions seems to support the PTS functionality.

scholarly journals Metabolic Engineering of a Novel Muconic Acid Biosynthesis Pathway via 4-Hydroxybenzoic Acid in Escherichia coli

2015 ◽  
Vol 81 (23) ◽  
pp. 8037-8043 ◽  
Author(s):  
Sudeshna Sengupta ◽  
Sudhakar Jonnalagadda ◽  
Lakshani Goonewardena ◽  
Veeresh Juturu
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
Carbon Sources ◽  
Raw Material ◽  
Hydroxybenzoic Acid ◽  
Muconic Acid ◽  
Content Type ◽  
E Coli ◽  
Catechol 1,2 Dioxygenase ◽  
K 12

ABSTRACTcis,cis-Muconic acid (MA) is a commercially important raw material used in pharmaceuticals, functional resins, and agrochemicals. MA is also a potential platform chemical for the production of adipic acid (AA), terephthalic acid, caprolactam, and 1,6-hexanediol. A strain ofEscherichia coliK-12, BW25113, was genetically modified, and a novel nonnative metabolic pathway was introduced for the synthesis of MA from glucose. The proposed pathway converted chorismate from the aromatic amino acid pathway to MA via 4-hydroxybenzoic acid (PHB). Three nonnative genes,pobA,aroY, andcatA, coding for 4-hydroxybenzoate hydrolyase, protocatechuate decarboxylase, and catechol 1,2-dioxygenase, respectively, were functionally expressed inE. colito establish the MA biosynthetic pathway.E. colinative genesubiC,aroFFBR,aroE, andaroLwere overexpressed and the genesptsH,ptsI,crr, andpykFwere deleted from theE. coligenome in order to increase the precursors of the proposed MA pathway. The final engineeredE. colistrain produced nearly 170 mg/liter of MA from simple carbon sources in shake flask experiments. The proposed pathway was proved to be functionally active, and the strategy can be used for future metabolic engineering efforts for production of MA from renewable sugars.

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