Improvement of free fatty acid production in Escherichia coli using codon-optimized Streptococcus pyogenes acyl-ACP thioesterase

Fatty acyl–acyl carrier protein (ACP) thioesterase (acyl-ACP TE) from Streptococcus pyogenes (strain MGAS10270) was codon-optimized and expressed in Escherichia coli K-12 W3110 and Escherichia coli K-12 MG1655. By employing codon-optimized S. pyogenes acyl-ACP TE to improve the total free fatty acids (FFAs) and to tailor the composition of FFAs, high-specificity production of saturated fatty acids (C12, C14) and unsaturated fatty acids (C18:1 C18:2) was achieved in recombinants. E. coli SGJS41 and SGJS46 (codon-optimized acyl-ACP TE of S. pyogenes) demonstrated the highest intracellular total FFA content (339 mg/l vs 342 mg/l); in particular, the content of C12 and C14 FFAs was about 3–5 fold, and the content of C18:1 and C18:2 FFAs was about 8–42 fold higher than that in the control E. coli and E. coli JES1017 (original acyl-ACP TE of S. pyogenes).     

Development of Escherichia coli MG1655 strains to produce long chain fatty acids by engineering fatty acid synthesis (FAS) metabolism

The goal of this research was to develop recombinant Escherichia coli to improve fatty acid synthesis (FAS). Genes encoding acetyl-CoA carboxylase (accA, accB, accC), malonyl-CoA-[acyl-carrier-protein] transacylase (fabD), and acyl-acyl carrier protein thioesterase (EC 3.1.2.14 gene), which are all enzymes that catalyze key steps in the synthesis of fatty acids, were cloned and over-expressed in E. coli MG1655. The acetyl-CoA carboxylase (ACC) enzyme catalyzes the addition of CO₂ to acetyl-CoA to generate malonyl-CoA. The enzyme encoded by the fabD gene converts malonyl-CoA to malonyl-[acp], and the EC 3.1.2.14 gene converts fatty acyl-ACP chains to long chain fatty acids. All the genes except for the EC 3.1.2.14 gene were homologous to E. coli genes and were used to improve the enzymatic activities to over-express components of the FAS pathway through metabolic engineering. All recombinant E. coli MG1655 strains containing various gene combinations were developed using the pTrc99A expression vector. To observe changes in metabolism, the in vitro metabolites and fatty acids produced by the recombinants were analyzed. The fatty acids (C16) from recombinant strains were produced 1.23-2.41 times higher than that from the wild type.     

Alteration of the fatty acid profile of Streptomyces coelicolor by replacement of the initiation enzyme 3-ketoacyl acyl carrier protein synthase III (FabH)

The first elongation step of fatty acid biosynthesis by a type II dissociated fatty acid synthases is catalyzed by 3-ketoacyl-acyl carrier protein (ACP) synthase III (KASIII, FabH). This enzyme, encoded by the fabH gene, catalyzes a decarboxylative condensation between an acyl coenzyme A (CoA) primer and malonyl-ACP. In organisms such as Escherichia coli, which generate only straight-chain fatty acids (SCFAs), FabH has a substrate preference for acetyl-CoA. In streptomycetes and other organisms which produce a mixture of both SCFAs and branched-chain fatty acids (BCFAs), FabH has been shown to utilize straight- and branched-chain acyl-CoA substrates. We report herein the generation of a Streptomyces coelicolor mutant (YL/ecFabH) in which the chromosomal copy of the fabH gene has been replaced and the essential process of fatty acid biosynthesis is initiated by plasmid-based expression of the E. coli FabH (bearing only 35% amino acid identity to the Streptomyces enzyme). The YL/ecFabH mutant produces predominantly SCFAs (86%). In contrast, BCFAs predominate (~70%) in both the S. coelicolor parental strain and S. coelicolor YL/sgFabH (a ΔfabH mutant carrying a plasmid expressing the Streptomyces glaucescens FabH). These results provide the first unequivocal evidence that the substrate specificity of FabH observed in vitro is a determinant of the fatty acid made in an organism. The YL/ecFabH strain grows significantly slower on both solid and liquid media. The levels of FabH activity in cell extracts of YL/ecFabH were also significantly lower than those in cell extracts of YL/sgFabH, suggesting that a decreased rate of fatty acid synthesis may account for the observed decreased growth rate. The production of low levels of BCFAs in YL/ecFabH suggests either that the E. coli FabH is more tolerant of different acyl-CoAs substrates than previously thought or that there is an additional pathway for initiation of BCFA biosynthesis in Streptomyces coelicolor.     

Over-expression of 3-ketoacyl-ACP synthase III or malonyl-CoA-ACP transacylase gene induces monomer supply for polyhydroxybutyrate production in Escherichia coli HB101

A Pseudomonas sp. 61-3 malonyl-CoA-ACP transacylase gene (fabD _Ps) was cloned by Southern analysis using an equivalent gene of Escherichia coli (fabD _Ec) as a probe. Some recombinant E. coli HB101 strains harboring fabD _Ps, fabD _Ec, or E. coli 3-ketoacyl-ACP synthase III gene (fabH _Ec) with Aeromonas caviae polyhydroxyalkanoate synthase gene (phaC _Ac) were constructed and grown on one-stage cultivation in Luria-Bertani broth containing glucose as carbon source. These strains accumulated 5 to 11 wt% of poly (3-hydroxybutyrate) (PHB) within cells. Over-expression of fabH _Ec, fabD _Ec, or fabD _Ps has been suggested to lead the monomer-supply of (R)-3-hydroxybutyryl-CoA for PHB synthesis in E. coli cells.     

Effect of acetate formation pathway and long chain fatty acid CoA-ligase on the free fatty acid production in E. coli expressing acy-ACP thioesterase from Ricinus communis

Microbial biosynthesis of fatty acid like chemicals from renewable carbon sources has attracted significant attention in recent years. Free fatty acids can be used as precursors for the production of fuels or chemicals. Wild type E. coli strains produce fatty acids mainly for the biosynthesis of lipids and cell membranes and do not accumulate free fatty acids as intermediates in lipid biosynthesis. However, free fatty acids can be produced by breaking the fatty acid elongation through the overexpression of an acyl-ACP thioesterase. Since acetyl-CoA might be an important factor for fatty acid synthesis (acetate formation pathways are the main competitive pathways in consuming acetyl-CoA or pyruvate, a precursor of acetyl-CoA), and the long chain fatty acid CoA-ligase (FadD) plays a pivotal role in the transport and activation of exogenous fatty acids prior to their subsequent degradation, we examined the composition and the secretion of the free fatty acids in four different strains including the wild type MG1655, a mutant strain with inactivation of the fatty acid beta-oxidation pathway (fadD mutant (ML103)), and mutant strains with inactivation of the two major acetate production pathways (an ack-pta (acetate kinase/phosphotransacetylase), poxB (pyruvate oxidase) double mutant (ML112)) and a fadD, ack-pta, poxB triple mutant (ML115). The engineered E. coli cells expressing acyl-ACP thioesterase with glucose yield is higher than 40% of theoretical yield. Compared to MG1655(pXZ18) and ML103(pXZ18), acetate forming pathway deletion strains such as ML112(pXZ18) and ML115(pXZ18) produced similar quantity of total free fatty acids, which indicated that acetyl-CoA availability does not appear to be limiting factor for fatty acid production in these strains. However, these strains did show significant differences in the composition of free fatty acids. Different from MG1655(pXZ18) and ML103(pXZ18), acetate formation pathway deletion strains such as ML112(pXZ18) and ML115(pXZ18) produced similar level of C14, C16:1 and C16 free fatty acids, and the free fatty acid compositions of both strains did not change significantly with time. In addition, the strains bearing the fadD mutation showed significant differences in the quantities of free fatty acids found in the broth. Finally, we examined two potential screening methods for selecting and isolating high free fatty acids producing cells.     

Improving poly-3-hydroxybutyrate production in Escherichia coli by combining the increase in the NADPH pool and acetyl-CoA availability

The biosynthesis of poly-3-hydroxybutyrate (P3HB), a biodegradable bio-plastic, requires acetyl-CoA as precursor and NADPH as cofactor. Escherichia coli has been used as a heterologous production model for P3HB, but metabolic pathway analysis shows a deficiency in maintaining high levels of NADPH and that the acetyl-CoA is mainly converted to acetic acid by native pathways. In this work the pool of NADPH was increased 1.7-fold in E. coli MG1655 through plasmid overexpression of the NADP⁺-dependent glyceraldehyde 3-phosphate dehydrogenase gene (gapN) from Streptococcus mutans (pTrcgapN). Additionally, by deleting the main acetate production pathway (ackA-pta), the acetic acid production was abolished, thus increasing the acetyl-CoA pool. The P3HB biosynthetic pathway was heterologously expressed in strain MG1655 Δack-pta/pTrcgapN, using an IPTG inducible vector with the P3HB operon from Azotobacter vinelandii (pPHB _Av ). Cultures were performed in controlled fermentors using mineral medium with glucose as the carbon source. Accordingly, the mass yield of P3HB on glucose increased to 73 % of the maximum theoretical and was 30 % higher when compared to the progenitor strain (MG1655/pPHB _Av ). In comparison with the wild type strain expressing pPHB _Av , the specific accumulation of PHB (g_PHB/g_DCW) in MG1655 Δack-pta/pTrcgapN/pPHB _Av increased twofold, indicating that as the availability of NADPH is raised and the production of acetate abolished, a P3HB intracellular accumulation of up to 84 % of the E. coli dry weight is attainable.     

The <Emphasis Type="Italic">Lactococcus lactis</Emphasis> FabF fatty acid synthetic enzyme can functionally replace both the FabB and FabF proteins of <Emphasis Type="Italic">Escherichia coli</Emphasis> and the FabH protein of <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

The genome of Lactococcus lactis encodes a single long chain 3-ketoacyl-acyl carrier protein synthase. This is in contrast to its close relative, Enterococcus faecalis, and to Escherichia coli, both of which have two such enzymes. In E. faecalis and E. coli, one of the two long chain synthases (FabO and FabB, respectively) has a role in unsaturated fatty acid synthesis that cannot be satisfied by FabF, the other long chain synthase. Since L. lactis has only a single long chain 3-ketoacyl-acyl carrier protein synthase (annotated as FabF), it seemed likely that this enzyme must function both in unsaturated fatty acid synthesis and in elongation of short chain acyl carrier protein substrates to the C18 fatty acids found in the cellular phospholipids. We report that this is the case. Expression of L. lactis FabF can functionally replace both FabB and FabF in E. coli, although it does not restore thermal regulation of phospholipid fatty acid composition to E. coli fabF mutant strains. The lack of thermal regulation was predictable because wild-type L. lactis was found not to show any significant change in fatty acid composition with growth temperature. We also report that overproduction of L. lactis FabF allows growth of an L. lactis mutant strain that lacks the FabH short chain 3-ketoacyl-acyl carrier protein synthase. The strain tested was a derivative (called the ∆fabH bypass strain) of the original fabH deletion strain that had acquired the ability to grow when supplemented with octanoate. Upon introduction of a FabF overexpression plasmid into this strain, growth proceeded normally in the absence of fatty acid supplementation. Moreover, this strain had a normal rate of fatty acid synthesis and a normal fatty acid composition. Both the ∆fabH bypass strain that overproduced FabF and the wild type strain incorporated much less exogenous octanoate into long chain phospholipid fatty acids than did the ∆fabH bypass strain. Incorporation of octanoate and decanoate labeled with deuterium showed that these acids were incorporated intact as the distal methyl and methylene groups of the long chain fatty acids.     

Complete Genome Sequence and Comparative Analysis of the Wild-type Commensal Escherichia coli Strain SE11 Isolated from a Healthy Adult

We sequenced and analyzed the genome of a commensal Escherichia coli (E. coli) strain SE11 (O152:H28) recently isolated from feces of a healthy adult and classified into E. coli phylogenetic group B1. SE11 harbored a 4.8 Mb chromosome encoding 4679 protein-coding genes and six plasmids encoding 323 protein-coding genes. None of the SE11 genes had sequence similarity to known genes encoding phage- and plasmid-borne virulence factors found in pathogenic E. coli strains. The comparative genome analysis with the laboratory strain K-12 MG1655 identified 62 poorly conserved genes between these two non-pathogenic strains and 1186 genes absent in MG1655. These genes in SE11 were mostly encoded in large insertion regions on the chromosome or in the plasmids, and were notably abundant in genes of fimbriae and autotransporters, which are cell surface appendages that largely contribute to the adherence ability of bacteria to host cells and bacterial conjugation. These data suggest that SE11 may have evolved to acquire and accumulate the functions advantageous for stable colonization of intestinal cells, and that the adhesion-associated functions are important for the commensality of E. coli in human gut habitat.Key words: Escherichia coli, commensal, human gut, genome sequencing     

De novo creation of MG1655-derived E. coli strains specifically designed for plasmid DNA production

The interest in plasmid DNA (pDNA) as a biopharmaceutical has been increasing over the last several years, especially after the approval of the first DNA vaccines. New pDNA production strains have been created by rationally mutating genes selected on the basis of Escherichia coli central metabolism and plasmid properties. Nevertheless, the highly mutagenized genetic background of the strains used makes it difficult to ascertain the exact impact of those mutations. To explore the effect of strain genetic background, we investigated single and double knockouts of two genes, pykF and pykA, which were known to enhance pDNA synthesis in two different E. coli strains: MG1655 (wild-type genetic background) and DH5α (highly mutagenized genetic background). The knockouts were only effective in the wild-type strain MG1655, demonstrating the relevance of strain genetic background and the importance of designing new strains specifically for pDNA production. Based on the obtained results, we created a new pDNA production strain starting from MG1655 by knocking out the pgi gene in order to redirect carbon flux to the pentose phosphate pathway, enhance nucleotide synthesis, and, consequently, increase pDNA production. GALG20 (MG1655ΔendAΔrecAΔpgi) produced 25-fold more pDNA (19.1 mg/g dry cell weight, DCW) than its parental strain, MG1655ΔendAΔrecA (0.8 mg/g DCW), in glucose. For the first time, pgi was identified as an important target for constructing a high-yielding pDNA production strain.     

2D [<Superscript>1</Superscript>H,<Superscript>13</Superscript>C] NMR study of carbon fluxes during glucose utilization by <Emphasis Type="Italic">Escherichia coli</Emphasis> MG1655

Carbon fluxes through main pathways of glucose utilization in Escherichia coli cells-glycolysis, pentose phosphate pathway (PPP), and Enther-Doudoroff pathway (EDP)—were studied. Their ratios were analyzed in E. coli strains MG1655, MG1655Δ(edd-eda), MG1655Δ(zwf, edd-eda), and MG1655Δ(pgi, edd-eda). It was shown that the carbon flux through glycolysis was the main route of glucose utilization, averaging ca. 80%. Inactivation of EDP did not affect growth parameters. Nevertheless, it altered carbon fluxes through the tricarboxylic acid cycles and energy metabolism in the cell. Inactivation of PPP decreased growth rate to a lesser degree than glycolysis inactivation.     

Induction of Oxidative Stress by High Hydrostatic Pressure in Escherichia coli

Using leaderless alkaline phosphatase as a probe, it was demonstrated that pressure treatment induces endogenous intracellular oxidative stress in Escherichia coli MG1655. In stationary-phase cells, this oxidative stress increased with the applied pressure at least up to 400 MPa, which is well beyond the pressure at which the cells started to become inactivated (200 MPa). In exponential-phase cells, in contrast, oxidative stress increased with pressure treatment up to 150 MPa and then decreased again, together with the cell counts. Anaerobic incubation after pressure treatment significantly supported the recovery of MG1655, while mutants with increased intrinsic sensitivity toward oxidative stress (katE, katF, oxyR, sodAB, and soxS) were found to be more pressure sensitive than wild-type MG1655. Furthermore, mild pressure treatment strongly sensitized E. coli toward t-butylhydroperoxide and the superoxide generator plumbagin. Finally, previously described pressure-resistant mutants of E. coli MG1655 displayed enhanced resistance toward plumbagin. In one of these mutants, the induction of endogenous oxidative stress upon high hydrostatic pressure treatment was also investigated and found to be much lower than in MG1655. These results suggest that, at least under some conditions, the inactivation of E. coli by high hydrostatic pressure treatment is the consequence of a suicide mechanism involving the induction of an endogenous oxidative burst.     

Adaptation of Escherichia coli to Elevated Sodium Concentrations Increases Cation Tolerance and Enables Greater Lactic Acid Production

Adaptive evolution was employed to generate sodium (Na⁺)-tolerant mutants of Escherichia coli MG1655. Four mutants with elevated sodium tolerance, designated ALS1184, ALS1185, ALS1186, and ALS1187, were independently isolated after 73 days of serial transfer in medium containing progressively greater Na⁺ concentrations. The isolates also showed increased tolerance of K⁺, although this cation was not used for selective pressure. None of the adapted mutants showed increased tolerance to the nonionic osmolyte sucrose. Several physiological parameters of E. coli MG1655 and ALS1187, the isolate with the greatest Na⁺ tolerance, were calculated and compared using glucose-limited chemostats. Genome sequencing showed that the ALS1187 isolate contained mutations in five genes, emrR, hfq, kil, rpsG, and sspA, all of which could potentially affect the ability of E. coli to tolerate Na⁺. Two of these genes, hfq and sspA, are known to be involved in global regulatory processes that help cells endure a variety of cellular stresses. Pyruvate formate lyase knockouts were constructed in strains MG1655 and ALS1187 to determine whether increased Na⁺ tolerance afforded increased anaerobic generation of lactate. In fed-batch fermentations, E. coli ALS1187 pflB generated 76.2 g/liter lactate compared to MG1655 pflB, which generated only 56.3 g/liter lactate.     

Malonyl-acyl carrier protein decarboxylase activity promotes fatty acid and cell envelope biosynthesis in Proteobacteria

Bacterial fatty acid synthesis in Escherichia coli is initiated by the condensation of an acetyl-CoA with a malonyl-acyl carrier protein (ACP) by the β-ketoacyl-ACP synthase III enzyme, FabH. E. coli ΔfabH knockout strains are viable because of the yiiD gene that allows FabH-independent fatty acid synthesis initiation. However, the molecular function of the yiiD gene product is not known. Here, we show the yiiD gene product is a malonyl-ACP decarboxylase (MadA). MadA has two independently folded domains: an amino-terminal N-acetyl transferase (GNAT) domain (MadA^N) and a carboxy-terminal hot dog dimerization domain (MadA^C) that encodes the malonyl-ACP decarboxylase function. Members of the proteobacterial Mad protein family are either two domain MadA (GNAT-hot dog) or standalone MadB (hot dog) decarboxylases. Using structure-guided, site-directed mutagenesis of MadB from Shewanella oneidensis, we identified Asn45 on a conserved catalytic loop as critical for decarboxylase activity. We also found that MadA, MadA^C, or MadB expression all restored normal cell size and growth rates to an E. coli ΔfabH strain, whereas the expression of MadA^N did not. Finally, we verified that GlmU, a bifunctional glucosamine-1-phosphate N-acetyl transferase/N-acetyl-glucosamine-1-phosphate uridylyltransferase that synthesizes the key intermediate UDP-GlcNAc, is an ACP binding protein. Acetyl-ACP is the preferred glucosamine-1-phosphate N-acetyl transferase/N-acetyl-glucosamine-1-phosphate uridylyltransferase substrate, in addition to being the substrate for the elongation-condensing enzymes FabB and FabF. Thus, we conclude that the Mad family of malonyl-ACP decarboxylases supplies acetyl-ACP to support the initiation of fatty acid, lipopolysaccharide, peptidoglycan, and enterobacterial common antigen biosynthesis in Proteobacteria.     

Engineering of a robust Escherichia coli chassis and exploitation for large-scale production processes

In large-scale bioprocesses microbes are exposed to heterogeneous substrate availability reducing the overall process performance. A series of deletion strains was constructed from E. coli MG1655 aiming for a robust phenotype in heterogeneous fermentations with transient starvation. Deletion targets were hand-picked based on a list of genes derived from previous large-scale simulation runs. Each gene deletion was conducted on the premise of strict neutrality towards growth parameters in glucose minimal medium. The final strain of the series, named E. coli RM214, was cultivated continuously in an STR-PFR (stirred tank reactor – plug flow reactor) scale-down reactor. The scale-down reactor system simulated repeated passages through a glucose starvation zone. When exposed to nutrient gradients, E. coli RM214 had a significantly lower maintenance coefficient than E. coli MG1655 (Δm_s = 0.038 g_Glucose/g_CDW/h, p < 0.05). In an exemplary protein production scenario E. coli RM214 remained significantly more productive than E. coli MG1655 reaching 44% higher eGFP yield after 28 h of STR-PFR cultivation. This study developed E. coli RM214 as a robust chassis strain and demonstrated the feasibility of engineering microbial hosts for large-scale applications.     

Development of a Biofilm Production-Deficient Escherichia coli Strain as a Host for Biotechnological Applications

Bacteria form biofilms by adhering to biotic or abiotic surfaces. This phenomenon causes several problems, including a reduction in the transport of mass and heat, an increase in resistance to antibiotics, and a shortening of the lifetimes of modules in bioindustrial fermentors. To overcome these difficulties, we created a biofilm production-deficient Escherichia coli strain, BD123, by deleting genes involved in curli biosynthesis and assembly, Δ(csgG-csgC); colanic acid biosynthesis and assembly, Δ(wcaL-wza); and type I pilus biosynthesis, Δ(fimB-fimH). E. coli BD123 remained mostly in the form of planktonic cells under the conditions tested and became more sensitive to the antibiotics streptomycin and rifampin than the wild-type E. coli MG1655: the growth of BD123 was inhibited by one-fourth of the concentrations needed to inhibit MG1655. In addition, the transformation efficiency of BD123 was about 20 times higher than that of MG1655, and the production and secretion of recombinant proteins were ~16% and ~25% greater, respectively, with BD123 than with MG1655. These results indicate that the newly created biofilm production-deficient strain of E. coli displays several key properties that substantially enhance its utility in the biotechnology arena.