Extracellular recombinant protein production from <Emphasis Type="Italic">Escherichia coli</Emphasis>

Escherichia coli is the most commonly used host for recombinant protein production and metabolic engineering. Extracellular production of enzymes and proteins is advantageous as it could greatly reduce the complexity of a bioprocess and improve product quality. Extracellular production of proteins is necessary for metabolic engineering applications in which substrates are polymers such as lignocelluloses or xenobiotics since adequate uptake of these substrates is often an issue. The dogma that E. coli secretes no protein has been challenged by the recognition of both its natural ability to secrete protein in common laboratory strains and increased ability to secrete proteins in engineered cells. The very existence of this review dedicated to extracellular production is a testimony for outstanding achievements made collectively by the community in this regard. Four strategies have emerged to engineer E. coli cells to secrete recombinant proteins. In some cases, impressive secretion levels, several grams per liter, were reached. This secretion level is on par with other eukaryotic expression systems. Amid the optimism, it is important to recognize that significant challenges remain, especially when considering the success cannot be predicted a priori and involves much trials and errors. This review provides an overview of recent developments in engineering E. coli for extracellular production of recombinant proteins and an analysis of pros and cons of each strategy.     

Synthesis of citramalic acid from glycerol by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Citramalic acid (citramalate) serves as a five-carbon precursor for the chemical synthesis of methacrylic acid. We compared citramalate and acetate accumulation from glycerol using Escherichia coli strains expressing a modified citramalate synthase gene cimA from Methanococcus jannaschii. These studies revealed that gltA coding citrate synthase, leuC coding 3-isopropylmalate dehydratase, and acetate pathway genes play important roles in elevating citramalate and minimizing acetate formation. Controlled 1.0 L batch experiments confirmed that deletions in all three acetate-production genes (poxB, ackA, and pta) were necessary to reduce acetate formation to less than 1 g/L during citramalate production from 30 g/L glycerol. Fed-batch processes using MEC568/pZE12-cimA (gltA leuC ackA-pta poxB) generated over 31 g/L citramalate and less than 2 g/L acetate from either purified or crude glycerol at yields exceeding 0.50 g citramalate/g glycerol in 132 h. These results hold promise for the viable formation of citramalate from unrefined glycerol.     

Improvement of <Emphasis Type="SmallCaps">d</Emphasis>-lactate productivity in recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> by coupling production with growth

Coupling lactate fermentation with cell growth was investigated in shake-flask and bioreactor cultivation systems by increasing aeration to improve lactate productivity in Escherichia coli CICIM B0013-070 (ackA pta pps pflB dld poxB adhE frdA). In shake-flasks, cells reached 1 g dry wt/l then, cultivated at 100 rpm and 42°C, achieved a twofold higher productivity of lactic acid compared to aerobic and O₂-limited two-phase fermentation. The cells in the bioreactor yielded an overall volumetric productivity of 5.5 g/l h and a yield of 86 g lactic acid/100 g glucose which were 66% higher and the same level compared to that of the aerobic and O₂-limited two-phase fermentation, respectively, using scaled-up conditions optimized from shake-flask experiments. These results have revealed an approach for improving production of fermentative products in E. coli.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-phenylalanine from glycerol by a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

The production of l-phenylalanine is conventionally carried out by fermentations that use glucose or sucrose as the carbon source. This work reports on the use of glycerol as an inexpensive and abundant sole carbon source for producing l-phenylalanine using the genetically modified bacterium Escherichia coli BL21(DE3). Fermentations were carried out at 37°C, pH 7.4, using a defined medium in a stirred tank bioreactor at various intensities of impeller agitation speeds (300–500 rpm corresponding to 0.97–1.62 m s⁻¹ impeller tip speed) and aeration rates (2–8 L min⁻¹, or 1–4 vvm). This highly aerobic fermentation required a good supply of oxygen, but intense agitation (impeller tip speed ~1.62 m s⁻¹) reduced the biomass and l-phenylalanine productivity, possibly because of shear sensitivity of the recombinant bacterium. Production of l-phenylalanine was apparently strongly associated with growth. Under the best operating conditions (1.30 m s⁻¹ impeller tip speed, 4 vvm aeration rate), the yield of l-phenylalanine on glycerol was 0.58 g g⁻¹, or more than twice the best yield attainable on sucrose (0.25 g g⁻¹). In the best case, the peak concentration of l-phenylalanine was 5.6 g L⁻¹, or comparable to values attained in batch fermentations that use glucose or sucrose. The use of glycerol for the commercial production of l-phenylalanine with E. coli BL21(DE3) has the potential to substantially reduce the cost of production compared to sucrose- and glucose-based fermentations.     

Efficient production of indigoidine in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Indigoidine is a bacterial natural product with antioxidant and antimicrobial activities. Its bright blue color resembles the industrial dye indigo, thus representing a new natural blue dye that may find uses in industry. In our previous study, an indigoidine synthetase Sc-IndC and an associated helper protein Sc-IndB were identified from Streptomyces chromofuscus ATCC 49982 and successfully expressed in Escherichia coli BAP1 to produce the blue pigment at 3.93 g/l. To further improve the production of indigoidine, in this work, the direct biosynthetic precursor l-glutamine was fed into the fermentation broth of the engineered E. coli strain harboring Sc-IndC and Sc-IndB. The highest titer of indigoidine reached 8.81 ± 0.21 g/l at 1.46 g/l l-glutamine. Given the relatively high price of l-glutamine, a metabolic engineering technique was used to directly enhance the in situ supply of this precursor. A glutamine synthetase gene (glnA) was amplified from E. coli and co-expressed with Sc-indC and Sc-indB in E. coli BAP1, leading to the production of indigoidine at 5.75 ± 0.09 g/l. Because a nitrogen source is required for amino acid biosynthesis, we then tested the effect of different nitrogen-containing salts on the supply of l-glutamine and subsequent indigoidine production. Among the four tested salts including (NH₄)₂SO₄, NH₄Cl, (NH₄)₂HPO₄ and KNO₃, (NH₄)₂HPO₄ showed the best effect on improving the titer of indigoidine. Different concentrations of (NH₄)₂HPO₄ were added to the fermentation broths of E. coli BAP1/Sc-IndC+Sc-IndB+GlnA, and the titer reached the highest (7.08 ± 0.11 g/l) at 2.5 mM (NH₄)₂HPO₄. This work provides two efficient methods for the production of this promising blue pigment in E. coli.     

Peroxidase activity of cytochrome <Emphasis Type="Italic">bd</Emphasis> from <Emphasis Type="Italic">Escherichia coli</Emphasis>

Cytochrome bd from Escherichia coli is able to oxidize such substrates as guaiacol, ferrocene, benzohydroquinone, and potassium ferrocyanide through the peroxidase mechanism, while none of these donors is oxidized in the oxidase reaction (i.e. in the reaction that involves molecular oxygen as the electron acceptor). Peroxidation of guaiacol has been studied in detail. The dependence of the rate of the reaction on the concentration of the enzyme and substrates as well as the effect of various inhibitors of the oxidase reaction on the peroxidase activity have been tested. The dependence of the guaiacol-peroxidase activity on the H₂O₂ concentration is linear up to the concentration of 8 mM. At higher concentrations of H₂O₂, inactivation of the enzyme is observed. Guaiacol markedly protects the enzyme from inactivation induced by peroxide. The peroxidase activity of cytochrome bd increases with increasing guaiacol concentration, reaching saturation in the range from 0.5 to 2.5 mM, but then starts falling. Such inhibitors of the ubiquinol-oxidase activity of cytochrome bd as cyanide, pentachlorophenol, and 2-n-heptyl 4-hydroxyquinoline-N-oxide also suppress its guaiacol-peroxidase activity; in contrast, zinc ions have no influence on the enzyme-catalyzed peroxidation of guaiacol. These data suggest that guaiacol interacts with the enzyme in the center of ubiquinol binding and donates electrons into the di-heme center of oxygen reduction via heme b ₅₅₈, and H₂O₂ is reduced by heme d. Although the peroxidase activity of cytochrome bd from E. coli is low compared to peroxidases, it might be of physiological significance for the bacterium itself and plays a pathophysiological role for humans and animals.     

A synthetic pathway for the production of 2-hydroxyisovaleric acid in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Synthetic biology, encompassing the design and construction of novel artificial biological pathways and organisms and the redesign of existing natural biological systems, is rapidly expanding the number of applications for which biological systems can play an integral role. In the context of chemical production, the combination of synthetic biology and metabolic engineering approaches continues to unlock the ability to biologically produce novel and complex molecules from a variety of feedstocks. Here, we utilize a synthetic approach to design and build a pathway to produce 2-hydroxyisovaleric acid in Escherichia coli and demonstrate how pathway design can be supplemented with metabolic engineering approaches to improve pathway performance from various carbon sources. Drawing inspiration from the native pathway for the synthesis of the 5-carbon amino acid l-valine, we exploit the decarboxylative condensation of two molecules of pyruvate, with subsequent reduction and dehydration reactions enabling the synthesis of 2-hydroxyisovaleric acid. Key to our approach was the utilization of an acetolactate synthase which minimized kinetic and regulatory constraints to ensure sufficient flux entering the pathway. Critical host modifications enabling maximum product synthesis from either glycerol or glucose were then examined, with the varying degree of reduction of these carbons sources playing a major role in the required host background. Through these engineering efforts, the designed pathway produced 6.2 g/L 2-hydroxyisovaleric acid from glycerol at 58% of maximum theoretical yield and 7.8 g/L 2-hydroxyisovaleric acid from glucose at 73% of maximum theoretical yield. These results demonstrate how the combination of synthetic biology and metabolic engineering approaches can facilitate bio-based chemical production.     

Lipoprotein trafficking in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Bacterial lipoproteins comprise a subset of membrane proteins that are covalently modified with lipids at the amino-terminal Cys. Lipoproteins are involved in a wide variety of functions in bacterial envelopes. Escherichia coli has more than 90 species of lipoproteins, most of which are located on the periplasmic surface of the outer membrane, while others are located on that of the inner membrane. In order to elucidate the mechanisms by which outer-membrane-specific lipoproteins are sorted to the outer membrane, biochemical, molecular biological and crystallographic approaches have been taken. Localization of lipoproteins on the outer membrane was found to require a lipoprotein-specific sorting machinery, the Lol system, which is composed of five proteins (LolABCDE). The crystal structures of LolA and LolB, the periplasmic chaperone and outer-membrane receptor for lipoproteins, respectively, were determined. On the basis of the data, we discuss here the mechanism underlying lipoprotein transfer from the inner to the outer membrane through Lol proteins. We also discuss why inner membrane-specific lipoproteins remain on the inner membrane.     

Expression of <Emphasis Type="Italic">recA</Emphasis> gene of <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

Plasmids pKS5 and pKSrec30 carrying normal and mutant alleles of the Deinococcus recA gene controlled by the lactose promoter slightly increase radioresistance of Escherichia coli cells with mutations in genes recA and ssb. The RecA protein of D. radiodurans is expressed in E. coli cells, and its synthesis can be supplementary induced. The radioprotective effect of the xenologic protein does not exceed 1.5 fold and yields essentially to the contribution of plasmid pUC19-recA1.1 harboring the E. coli recA ⁺ gene in the recovery of resistance of the ΔrecA deletion mutant. These data suggest that the expression of D. radiodurans recA gene in E. coli cells does not complement mutations at gene recA in the chromosome possibly due to structural and functional peculiarities of the D. radiodurans RecA protein.     

Engineering a <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> biosynthesis pathway into <Emphasis Type="Italic">Escherichia coli</Emphasis> for high yield triglyceride production

Background

Microbial lipid production represents a potential alternative feedstock for the biofuel and oleochemical industries. Since Escherichia coli exhibits many genetic, technical, and biotechnological advantages over native oleaginous bacteria, we aimed to construct a metabolically engineered E. coli strain capable of accumulating high levels of triacylglycerol (TAG) and evaluate its neutral lipid productivity during high cell density fed-batch fermentations.

Results

The Streptomyces coelicolor TAG biosynthesis pathway, defined by the acyl-CoA:diacylglycerol acyltransferase (DGAT) Sco0958 and the phosphatidic acid phosphatase (PAP) Lppβ, was successfully reconstructed in an E. coli diacylglycerol kinase (dgkA) mutant strain. TAG production in this genetic background was optimized by increasing the levels of the TAG precursors, diacylglycerol and long-chain acyl-CoAs. For this we carried out a series of stepwise optimizations of the chassis by 1) fine-tuning the expression of the heterologous SCO0958 and lpp β genes, 2) overexpression of the S. coelicolor acetyl-CoA carboxylase complex, and 3) mutation of fadE, the gene encoding for the acyl-CoA dehydrogenase that catalyzes the first step of the β-oxidation cycle in E. coli. The best producing strain, MPS13/pET28-0958-ACC/pBAD-LPPβ rendered a cellular content of 4.85% cell dry weight (CDW) TAG in batch cultivation. Process optimization of fed-batch fermentation in a 1-L stirred-tank bioreactor resulted in cultures with an OD_600nm of 80 and a product titer of 722.1 mg TAG L^-1 at the end of the process.

Conclusions

This study represents the highest reported fed-batch productivity of TAG reached by a model non-oleaginous bacterium. The organism used as a platform was an E. coli BL21 derivative strain containing a deletion in the dgkA gene and containing the TAG biosynthesis genes from S. coelicolor. The genetic studies carried out with this strain indicate that diacylglycerol (DAG) availability appears to be one of the main limiting factors to achieve higher yields of the storage compound. Therefore, in order to develop a competitive process for neutral lipid production in E. coli, it is still necessary to better understand the native regulation of the carbon flow metabolism of this organism, and in particular, to improve the levels of DAG biosynthesis.

     

Cloning and expression of the sucrose phosphorylase gene from <Emphasis Type="Italic">Leuconostoc </Emphasis><Emphasis Type="Italic">mesenteroides</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The gene encoding sucrose phosphorylase (742sp) in Leuconostoc mesenteroides NRRL B-742 was cloned and expressed in Escherichia coli. The nucleotide sequence of the transformed 742sp comprised an ORF of 1,458 bp giving a protein with calculated molecular mass of 55.3 kDa. 742SPase contains a C-terminal amino acid sequence that is significantly different from those of other Leu. mesenteroides SPases. The purified 742SPase had a specific activity of 1.8 U/mg with a K _m of 3 mM with sucrose as a substrate; optimum activity was at 37°C and pH 6.7. The purified 742SPase transferred the glucosyl moiety of sucrose to cytosine monophosphate (CMP). Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="SmallCaps">L</Emphasis>-Tyrosine production by deregulated strains of <Emphasis Type="Italic">Escherichia coli</Emphasis>

The excretion of the aromatic amino acid l-tyrosine was achieved by manipulating three gene targets in the wild-type Escherichia coli K12: The feedback-inhibition-resistant (fbr) derivatives of aroG and tyrA were expressed on a low-copy-number vector, and the TyrR-mediated regulation of the aromatic amino acid biosynthesis was eliminated by deleting the tyrR gene. The generation of this l-tyrosine producer, strain T1, was based only on the deregulation of the aromatic amino acid biosynthesis pathway, but no structural genes in the genome were affected. A second tyrosine over-producing strain, E. coli T2, was generated considering the possible limitation of precursor substrates. To enhance the availability of the two precursor substrates phosphoenolpyruvate and erythrose-4-phosphate, the ppsA and the tktA genes were over-expressed in the strain T1 background, increasing l-tyrosine production by 80% in 50-ml batch cultures. Fed-batch fermentations revealed that l-tyrosine production was tightly correlated with cell growth, exhibiting the maximum productivity at the end of the exponential growth phase. The final l-tyrosine concentrations were 3.8 g/l for E. coli T1 and 9.7 g/l for E. coli T2 with a yield of l-tyrosine per glucose of 0.037 g/g (T1) and 0.102 g/g (T2), respectively.     

Expression of <Emphasis Type="Italic">Treponema denticola</Emphasis> Oligopeptidase B in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Treponema denticola is a small anaerobic spirochete often isolated from periodontal lesions and closely associated with periodontal diseases. This bacterium possesses a particular arginine peptidase activity (previously called BANA-peptidase or trypsin-like enzyme) that is common to the three cultivable bacterial species most highly associated with severe periodontal disease. We recently reported the identification of the opdB locus that encodes the BANA-peptidase activity of T. denticola through DNA sequencing and mutagenesis studies. In the present study, we report expression of T. denticola OpdB peptidase in Escherichia coli. The opdB PCR product was cloned into pET30b and then transformed into the E. coli BL21 (DE3)/pLysS expression strain. Assays of enzymatic activities in E. coli containing T. denticola opdB showed BANA-peptidase activity similar to that of T. denticola. Availability of this recombinant expression system producing active peptidase will facilitate characterization of the potential role of this peptidase in periodontal disease etiology.     

Regioselective glucosidation of <Emphasis Type="Italic">trans</Emphasis>-resveratrol in <Emphasis Type="Italic">Escherichia coli</Emphasis> expressing glucosyltransferase from <Emphasis Type="Italic">Phytolacca americana</Emphasis>

A glucosyltransferase (GT) of Phytolacca americana (PaGT3) was expressed in Escherichia coli and purified for the synthesis of two O-β-glucoside products of trans-resveratrol. The reaction was moderately regioselective with a ratio of 4′-O-β-glucoside: 3-O-β-glucoside at 10:3. We used not only the purified enzyme but also the E. coli cells containing the PaGT3 gene for the synthesis of glycoconjugates. E. coli cell cultures also have other advantages, such as a shorter incubation time compared with cultured plant cells, no need for the addition of exogenous glucosyl donor compounds such as UDP-glucose, and almost complete conversion of the aglycone to the glucoside products. Furthermore, a homology model of PaGT3 and mutagenesis studies suggested that His-20 would be a catalytically important residue.     

Cloning and expression of <Emphasis Type="Italic">mel</Emphasis> gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain.     

An engineered non-oxidative glycolysis pathway for acetone production in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To find new metabolic engineering strategies to improve the yield of acetone in Escherichia coli.

Results

Results of flux balance analysis from a modified Escherichia coli genome-scale metabolic network suggested that the introduction of a non-oxidative glycolysis (NOG) pathway would improve the theoretical acetone yield from 1 to 1.5 mol acetone/mol glucose. By inserting the fxpk gene encoding phosphoketolase from Bifidobacterium adolescentis into the genome, we constructed a NOG pathway in E.coli. The resulting strain produced 47 mM acetone from glucose under aerobic conditions in shake-flasks. The yield of acetone was improved from 0.38 to 0.47 mol acetone/mol glucose which is a significant over the parent strain.

Conclusions

Guided by computational analysis of metabolic networks, we introduced a NOG pathway into E. coli and increased the yield of acetone, which demonstrates the importance of modeling analysis for the novel metabolic engineering strategies.

     

Function of the <Emphasis Type="Italic">rel</Emphasis> Gene in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Sokawa et al. suggest that rel^- strains of Escherichia coli possess abnormal protein synthesizing machinery, which cannot carry out normal protein synthesis when the supply of amino-acids is limited.     

High-level expression of the xylanase from <Emphasis Type="Italic">Thermomyces lanuginosus</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

According to the amino acid sequence, a codon-optimized xylanase gene (xynA1) from Thermomyces lanuginosus DSM 5826 was synthesized to construct the expression vector pHsh-xynA1. After optimization of the mRNA secondary structure in the translational initiation region of pHsh-xynA1, free energy of the 70 nt was changed from −6.56 to −4.96 cal/mol, and the spacing between AUG and the Shine-Dalgarno sequence was decreased from 15 to 8 nt. The expression level was increased from 1.3 to 13% of total cell protein. A maximum xylanase activity of 47.1 U/mL was obtained from cellular extract. The recombinant enzyme was purified 21.5-fold from the cellular extract of Escherichia coli by heat treatment, DEAE-Sepharose FF column and t-Butyl-HIC column. The optimal temperature and pH were 65 °C and pH 6.0, respectively. The purified enzyme was stable for 30 min over the pH range of 5.0–8.0 at 60 °C, and had a half-life of 3 h at 65 °C.     

Systematic engineering of pentose phosphate pathway improves <Emphasis Type="Italic">Escherichia coli</Emphasis> succinate production

Background

Succinate biosynthesis of Escherichia coli is reducing equivalent-dependent and the EMP pathway serves as the primary reducing equivalent source under anaerobic condition. Compared with EMP, pentose phosphate pathway (PPP) is reducing equivalent-conserving but suffers from low efficacy. In this study, the ribosome binding site library and modified multivariate modular metabolic engineering (MMME) approaches are employed to overcome the low efficacy of PPP and thus increase succinate production.

Results

Altering expression levels of different PPP enzymes have distinct effects on succinate production. Specifically, increased expression of five enzymes, i.e., Zwf, Pgl, Gnd, Tkt, and Tal, contributes to increased succinate production, while the increased expression of two enzymes, i.e., Rpe and Rpi, significantly decreases succinate production. Modular engineering strategy is employed to decompose PPP into three modules according to position and function. Engineering of Zwf/Pgl/Gnd and Tkt/Tal modules effectively increases succinate yield and production, while engineering of Rpe/Rpi module decreases. Imbalance of enzymatic reactions in PPP is alleviated using MMME approach. Finally, combinational utilization of engineered PPP and SthA transhydrogenase enables succinate yield up to 1.61 mol/mol glucose, which is 94% of theoretical maximum yield (1.71 mol/mol) and also the highest succinate yield in minimal medium to our knowledge.

Conclusions

In summary, we systematically engineered the PPP for improving the supply of reducing equivalents and thus succinate production. Besides succinate, these PPP engineering strategies and conclusions can also be applicable to the production of other reducing equivalent-dependent biorenewables.

     

Phosphoenolpyruvate-supply module in <Emphasis Type="Italic">Escherichia coli</Emphasis> improves <Emphasis Type="Italic">N</Emphasis>-acetyl-<Emphasis Type="SmallCaps">d</Emphasis>-neuraminic acid biocatalysis

Objectives

N-Acetyl-d-neuraminic acid (Neu5Ac) is often synthesized from exogenous N-acetylglucosamine (GlcNAc) and excess pyruvate. We have previously constructed a recombinant Escherichia coli strain for Neu5Ac production using GlcNAc and intracellular phosphoenolpyruvate (PEP) as substrates (Zhu et al. Biotechnol Lett 38:1–9, 2016).

Results

PEP synthesis-related genes, pck and ppsA, were overexpressed within different modes to construct PEP-supply modules, and their effects on Neu5Ac production were investigated. All the PEP-supply modules enhanced Neu5Ac production. For the best module, pCDF-pck-ppsA increased Neu5Ac production to 8.6 ± 0.15 g l^?1, compared with 3.6 ± 0.15 g l^?1 of the original strain. Neu5Ac production was further increased to 15 ± 0.33 g l^?1 in a 1 l fermenter.

Conclusions

The PEP-supply module can improve the intracellular PEP supply and enhance Neu5Ac production, which benefited industrial Neu5Ac production.