Genetic engineering of Escherichia coli to enhance production of l-tryptophan

Reducing the accumulation of acetate in Escherichia coli cultures can decrease carbon efflux as by-products and reduce acetate toxicity, and therefore enable high cell density cultivation. The concentration of intracellular amino acids can be decreased by genetic modifications of the corresponding amino acid transport systems. This can increase the levels of amino acids in the fermentation broth by decreasing the feedback inhibition on the corresponding biosynthetic pathways. Here, the effects of genetic manipulation of phosphate acetyltransferase (pta), high affinity tryptophan transporter (mtr) and aromatic amino acid exporter (yddG) on l-tryptophan production were investigated. The pta mutants accumulated less acetate and showed higher capacity for producing l-tryptophan as compared with the parental strain. The strains lacking mtr, or overexpressed yddG, or with the both mtr knockout and yddG overexpression, accumulated lower concentrations of intracellular tryptophan but higher production of extracellular l-tryptophan. In the l-tryptohan fed-batch fermentation of an E. coli derived from TRTH0709/pMEL03 having deletion of pta-mtr and overexpression of yddG in a 30-L fermentor, the maximum concentration of l-tryptophan (48.68 g/L) was obtained, which represented a 15.96 % increase as compared with the parental strain. Acetate accumulated to a concentration of 0.95 g/L. The intracellular concentration of l-tryptophan was low, and the glucose conversion rate reached a high level of 21.87 %, which was increased by 15.53 % as compared with the parent strain.     

Metabolic engineering of Escherichia coli for biosynthesis of d-galactonate

d-galactose is an attractive substrate for bioconversion. Herein, Escherichia coli was metabolically engineered to convert d-galactose into d-galactonate, a valuable compound in the polymer and cosmetic industries. d-galactonate productions by engineered E. coli strains were observed in shake flask cultivations containing 2 g L^?1 d-galactose. Engineered E. coli expressing gld coding for galactose dehydrogenase from Pseudomonas syringae was able to produce 0.17 g L^?1 d-galactonate. Inherent metabolic pathways for assimilating both d-galactose and d-galactonate were blocked to enhance the production of d-galactonate. This approach finally led to a 7.3-fold increase with d-galactonate concentration of 1.24 g L^?1 and yield of 62.0 %. Batch fermentation in 20 g L^?1 d-galactose of E. coli ?galK?dgoK mutant expressing the gld resulted in 17.6 g L^?1 of d-galactonate accumulation and highest yield of 88.1 %. Metabolic engineering strategy developed in this study could be useful for industrial production of d-galactonate.     

Construction of an l-serine producing Escherichia coli via metabolic engineering

l-Serine is a nonessential amino acid, but plays a crucial role as a building block for cell growth. Currently, l-serine production is mainly dependent on enzymatic or cellular conversion. In this study, we constructed a recombinant Escherichia coli that can fermentatively produce l-serine from glucose. To accumulate l-serine, sdaA encoding the l-serine dehydratase, iclR encoding the isocitrate lyase regulator, and arcA encoding the aerobic respiration control protein were deleted in turn. In batch fermentation, the engineered E. coli strain YF-5 exhibited obvious l-serine accumulation but poor cell growth. To restore cell growth, aceB encoding the malate synthase was knocked out, and the engineered strain was then transformed with plasmid that overexpressed serA ^FR , serB, and serC genes. The resulting strain YF-7 produced 4.5 g/L l-serine in batch cultivation and 8.34 g/L l-serine in fed-batch cultivation.     

Phosphoenolpyruvate:glucose phosphotransferase system modification increases the conversion rate during <Emphasis Type="SmallCaps">l</Emphasis>-tryptophan production in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Escherichia coli FB-04(pta1), a recombinant l-tryptophan production strain, was constructed in our laboratory. However, the conversion rate (l-tryptophan yield per glucose) of this strain is somewhat low. In this study, additional genes have been deleted in an effort to increase the conversion rate of E. coli FB-04(pta1). Initially, the pykF gene, which encodes pyruvate kinase I (PYKI), was inactivated to increase the accumulation of phosphoenolpyruvate, a key l-tryptophan precursor. The resulting strain, E. coli FB-04(pta1)ΔpykF, showed a slightly higher l-tryptophan yield and a higher conversion rate in fermentation processes. To further improve the conversion rate, the phosphoenolpyruvate:glucose phosphotransferase system (PTS) was disrupted by deleting the ptsH gene, which encodes the phosphocarrier protein (HPr). The levels of biomass, l-tryptophan yield, and conversion rate of this strain, E. coli FB-04(pta1)ΔpykF/ptsH, were especially low during fed-batch fermentation process, even though it achieved a significant increase in conversion rate during shake-flask fermentation. To resolve this issue, four HPr mutations (N12S, N12A, S46A, and S46N) were introduced into the genomic background of E. coli FB-04(pta1)ΔpykF/ptsH, respectively. Among them, the strain harboring the N12S mutation (E. coli FB-04(pta1)ΔpykF-ptsHN12S) showed a prominently increased conversion rate of 0.178 g g^?1 during fed-batch fermentation; an increase of 38.0% compared with parent strain E. coli FB-04(pta1). Thus, mutation of the genomic of ptsH gene provided an alternative method to weaken the PTS and improve the efficiency of carbon source utilization.     

Effects of NADH kinase on NADPH-dependent biotransformation processes in Escherichia coli

Sufficient supply of NADPH is one of the most important factors affecting the productivity of biotransformation processes. In this study, construction of an efficient NADPH-regenerating system was attempted using direct phosphorylation of NADH by NADH kinase (Pos5p) from Saccharomyces cerevisiae for producing guanosine diphosphate (GDP)-l-fucose and ε-caprolactone in recombinant Escherichia coli. Expression of Pos5p in a fed-batch culture of recombinant E. coli producing GDP-l-fucose resulted in a maximum GDP-l-fucose concentration of 291.5 mg/l, which corresponded to a 51 % enhancement compared with the control strain. In a fed-batch Baeyer–Villiger (BV) oxidation of cyclohexanone using recombinant E. coli expressing Pos5p, a maximum ε-caprolactone concentration of 21.6 g/l was obtained, which corresponded to a 96 % enhancement compared with the control strain. Such an increase might be due to the enhanced availability of NADPH in recombinant E. coli expressing Pos5p. These results suggested that efficient regeneration of NADPH was possible by functional expression of Pos5p in recombinant E. coli, which can be applied to other NADPH-dependent biotransformation processes in E. coli.     

d-Lactic acid biosynthesis from biomass-derived sugars via Lactobacillus delbrueckii fermentation

Poly-lactic acid (PLA) derived from renewable resources is considered to be a good substitute for petroleum-based plastics. The number of poly l-lactic acid applications is increased by the introduction of a stereocomplex PLA, which consists of both poly-l and d-lactic acid and has a higher melting temperature. To date, several studies have explored the production of l-lactic acid, but information on biosynthesis of d-lactic acid is limited. Pulp and corn stover are abundant, renewable lignocellulosic materials that can be hydrolyzed to sugars and used in biosynthesis of d-lactic acid. In our study, saccharification of pulp and corn stover was done by cellulase CTec2 and sugars generated from hydrolysis were converted to d-lactic acid by a homofermentative strain, L. delbrueckii, through a sequential hydrolysis and fermentation process (SHF) and a simultaneous saccharification and fermentation process (SSF). 36.3 g L^?1 of d-lactic acid with 99.8 % optical purity was obtained in the batch fermentation of pulp and attained highest yield and productivity of 0.83 g g^?1 and 1.01 g L^?1 h^?1, respectively. Luedeking–Piret model described the mixed growth-associated production of d-lactic acid with a maximum specific growth rate 0.2 h^?1 and product formation rate 0.026 h^?1, obtained for this strain. The efficient synthesis of d-lactic acid having high optical purity and melting point will lead to unique stereocomplex PLA with innovative applications in polymer industry.     

Enhancement of γ-aminobutyric acid production in recombinant Corynebacterium glutamicum by co-expressing two glutamate decarboxylase genes from Lactobacillus brevis

γ-Aminobutyric acid (GABA), a non-protein amino acid, is a bioactive component in the food, feed and pharmaceutical fields. To establish an effective single-step production system for GABA, a recombinant Corynebacterium glutamicum strain co-expressing two glutamate decarboxylase (GAD) genes (gadB1 and gadB2) derived from Lactobacillus brevis Lb85 was constructed. Compared with the GABA production of the gadB1 or gadB2 single-expressing strains, GABA production by the gadB1–gadB2 co-expressing strain increased more than twofold. By optimising urea supplementation, the total production of l-glutamate and GABA increased from 22.57 ± 1.24 to 30.18 ± 1.33 g L^?1, and GABA production increased from 4.02 ± 0.95 to 18.66 ± 2.11 g L^?1 after 84-h cultivation. Under optimal urea supplementation, l-glutamate continued to be consumed, GABA continued to accumulate after 36 h of fermentation, and the pH level fluctuated. GABA production increased to a maximum level of 27.13 ± 0.54 g L^?1 after 120-h flask cultivation and 26.32 g L^?1 after 60-h fed-batch fermentation. The conversion ratio of l-glutamate to GABA reached 0.60–0.74 mol mol^?1. By co-expressing gadB1 and gadB2 and optimising the urea addition method, C. glutamicum was genetically improved for de novo biosynthesis of GABA from its own accumulated l-glutamate.     

Biosynthesis of ethylene glycol in Escherichia coli

Ethylene glycol (EG) is an important platform chemical with steadily expanding global demand. Its commercial production is currently limited to fossil resources; no biosynthesis route has been delineated. Herein, a biosynthesis route for EG production from d-xylose is reported. This route consists of four steps: d-xylose?→?d-xylonate?→?2-dehydro-3-deoxy-d-pentonate?→?glycoaldehyde?→?EG. Respective enzymes, d-xylose dehydrogenase, d-xylonate dehydratase, 2-dehydro-3-deoxy-d-pentonate aldolase, and glycoaldehyde reductase, were assembled. The route was implemented in a metabolically engineered Escherichia coli, in which the d-xylose?→?d-xylulose reaction was prevented by disrupting the d-xylose isomerase gene. The most efficient construct produced 11.7 g?L^?1 of EG from 40.0 g?L^?1 of d-xylose. Glycolate is a carbon-competing by-product during EG production in E. coli; blockage of glycoaldehyde?→?glycolate reaction was also performed by disrupting the gene encoding aldehyde dehydrogenase, but from this approach, EG productivity was not improved but rather led to d-xylonate accumulation. To channel more carbon flux towards EG than the glycolate pathway, further systematic metabolic engineering and fermentation optimization studies are still required to improve EG productivity.     

Poly(l-diaminopropionic acid), a novel non-proteinic amino acid oligomer co-produced with poly(ε-l-lysine) by Streptomyces albulus PD-1

Poly(ε-l-lysine) (ε-PL) producer strain Streptomyces albulus PD-1 secreted a novel polymeric substance into its culture broth along with ε-PL. The polymeric substance was purified to homogeneity and identified. Matrix-assisted laser desorption ionization-time of flight mass spectrometry and nuclear magnetic resonance spectroscopy as well as other analytical techniques revealed that the substance was poly(l-diaminopropionic acid) (PDAP). PDAP is an l-α,β-diaminopropionic acid oligomer linking between amino and carboxylic acid functional groups. The molecular weight of PDAP ranged from 500 to 1500 Da, and no co-polymers composed of l-diaminopropionic acid and l-lysine were present in the culture broth. Compared with ε-PL, PDAP exhibited stronger inhibitory activities against yeasts but weaker activities against bacteria. ε-PL and PDAP co-production was also investigated. Both ε-PL and PDAP were synthesized during the stationary phase of growth, and the final ε-PL and PDAP concentration reached 21.7 and 4.8 g L^-1, respectively, in fed-batch fermentation. Citric acid feeding resulted in a maximum ε-PL concentration of 26.1 g L^-1 and a decrease in the final concentration of PDAP to 3.8 g L^-1. No studies on ε-PL and PDAP co-production in Streptomyces albulus have been reported previously, and inhibition of by-products such as PDAP is potentially useful in ε-PL production.     

Enhancing the supply of oxaloacetate for l-glutamate production by pyc overexpression in different Corynebacterium glutamicum

During l-glutamate production, phosphoenolpyruvate carboxylase and pyruvate carboxylase (PCx) play important roles in supplying oxaloacetate to the tricarboxylic acid cycle. To explore the significance of PCx for l-glutamate overproduction, the pyc gene encoding PCx was amplified in Corynebacterium glutamicum GDK-9 triggered by biotin limitation and CN1021 triggered by a temperature shock, respectively. In the fed-batch cultures, GDK-9pXMJ19pyc exhibited 7.4 % lower l-alanine excretion and no improved l-glutamate production. In contrast, CN1021pXMJ19pyc finally exhibited 13 % lower l-alanine excretion and identical l-glutamate production, however, 8.5 % higher l-glutamate production was detected during a short period of the fermentation. It was indicated that pyc overexpression in l-glutamate producer strains, especially CN1021, increased the supply of oxaloacetate for l-glutamate synthesis and decreased byproduct excretion at the pyruvate node.     

Modification of glycolysis and its effect on the production of l-threonine in Escherichia coli

High concentrations of acetate, the main by-product of Escherichia coli (E. coli) high cell density culture, inhibit bacterial growth and l-threonine production. Since metabolic overflux causes acetate accumulation, we attempted to reduce acetate production by redirecting glycolysis flux to the pentose phosphate pathway by deleting the genes encoding phosphofructokinase (pfk) and/or pyruvate kinase (pyk) in an l-threonine-producing strain of E. coli, THRD. pykF, pykA, pfkA, and pfkB deletion mutants produced less acetate (9.44 ± 0.83, 3.86 ± 0.88, 0.30 ± 0.25, and 6.99 ± 0.85 g/l, respectively) than wild-type THRD cultures (19.75 ± 0.93 g/l). THRDΔpykF and THRDΔpykA produced 11.05 and 5.35 % more l-threonine, and achieved a 10.91 and 5.60 % higher yield on glucose, respectively. While THRDΔpfkA grew more slowly and produced less l-threonine than THRD, THRDΔpfkB produced levels of l-threonine (102.28 ± 2.80 g/l) and a yield on glucose (0.34 g/g) similar to that of THRD. The dual deletion mutant THRDΔpfkBΔpykF also achieved low acetate (7.42 ± 0.81 g/l) and high l-threonine yields (111.37 ± 2.71 g/l). The level of NADPH in THRDΔpfkA cultures was depressed, whereas all other mutants produced more NADPH than THRD did. These results demonstrated that modification of glycolysis in E. coli THRD reduced acetate production and increased accumulation of l-threonine.     

Enhancement of ε-poly-l-lysine production coupled with precursor l-lysine feeding in glucose–glycerol co-fermentation by Streptomyces sp. M-Z18

ε-Poly-l-lysine (ε-PL), one of the only two homo-poly amino acids known in nature, is used as a preservative. In this study, strategies of feeding precursor l-lysine into 5 L laboratory scale fermenters, including optimization of l-lysine concentration and time, was investigated to optimize the production of ε-PL by Streptomyces sp. M-Z18. The optimized strategy was then used in ε-PL fed-batch fermentation in which glucose and glycerol served as mixed carbon sources. In this way, a novel ε-PL production strategy involving precursor l-lysine coupled with glucose–glycerol co-fermentation was developed. Under optimal conditions, ε-PL production reached 37.6 g/l, which was 6.2 % greater than in a previous study in which glucose and glycerol co-fermentation was performed without added l-lysine (35.14 g/l). To the best of our knowledge, this is the first report of the enhancement of ε-PL production through l-lysine feeding to evaluate the use of fermenters. Meanwhile, the role of l-lysine in the promotion of ε-PL production, participating ε-PL synthesis as a whole, was first determined using the l-[U–¹³C] lysine labeling method. It has been suggested that the bottleneck of ε-PL synthesis in Streptomyces sp. M-Z18 is in the biosynthesis of precursor l-lysine. The information obtained in the present work may facilitate strain improvement and efficient large-scale ε-PL production.     

d-Tagatose production in the presence of borate by resting Lactococcus lactis cells harboring Bifidobacterium longum l-arabinose isomerase

Bifidobacterium longum NRRL B-41409 l-arabinose isomerase (l-AI) was overexpressed in Lactococcus lactis using a phosphate depletion inducible expression system. The resting L. lactis cells harboring the B. longum l-AI were used for production of d-tagatose from d-galactose in the presence of borate buffer. Multivariable analysis suggested that high pH, temperature and borate concentration favoured the conversion of d-galactose to d-tagatose. Almost quantitative conversion (92 %) was achieved at 20 g L^?1 substrate and at 37.5 °C after 5 days. The d-tagatose production rate of 185 g L^?1 day^?1 was obtained at 300 g L^?1 galactose, at 1.15 M borate, and at 41 °C during 10 days when the production medium was changed every 24 h. There was no significant loss in productivity during ten sequential 24 h batches. The initial d-tagatose production rate was 290 g L^?1 day^?1 under these conditions.     

Biotransformation of l-ornithine from l-arginine using whole-cell recombinant arginase

A recombinant arginase was generated for a whole-cell biotransformation system to convert l-arginine to l-ornithine in Escherichia coli. The gene ARG1 coding arginase from Bos taurus liver was synthesized and expressed in E. coli BL21 (DE3) via pETDuet-1. The recombinant arginase was used to catalyze l-arginine to l-ornithine and urea. The reaction was optimal at pH 9.5 and 37 °C. Manganese (10^?5 M) and Emulsifier OP-10 [0.033 % (v/v)] could promote arginase activity. In a scale up study, l-arginine conversion rate reached 98 % with a final concentration of 111.52 g l-ornithine/l.     

Strategy for pH control and pH feedback-controlled substrate feeding for high-level production of l-tryptophan by Escherichia coli

Optimum production of l-tryptophan by Escherichia coli depends on pH. Here, we established conditions for optimizing the production of l-tryptophan. The optimum pH range was 6.5–7.2, and pH was controlled using a three-stage strategy [pH 6.5 (0–12 h), pH 6.8 (12–24 h), and pH 7.2 (24–38 h)]. Specifically, ammonium hydroxide was used to adjust pH during the initial 24 h, and potassium hydroxide and ammonium hydroxide (1:2, v/v) were used to adjust pH during 24–38 h. Under these conditions, NH₄ ⁺ and K⁺ concentrations were kept below the threshold for inhibiting l-tryptophan production. Optimization was also accomplished using ratios (v/v) of glucose to alkali solutions equal to 4:1 (5–24 h) and 6:1 (24–38 h). The concentration of glucose and the pH were controlled by adjusting the pH automatically. Applying a pH-feedback feeding method, the steady-state concentration of glucose was maintained at approximately 0.2 ± 0.02 g/l, and acetic acid accumulated to a concentration of 1.15 ± 0.03 g/l, and the plasmid stability was 98 ± 0.5 %. The final, optimized concentration of l-tryptophan was 43.65 ± 0.29 g/l from 52.43 ± 0.38 g/l dry cell weight.     

Improvement of l-lactic acid production by osmotic-tolerant mutant of Lactobacillus casei at high temperature

l-Lactic acid production by Lactobacillus casei was used as a model to study the mechanism of substrate inhibition and the strategy for enhancing l-lactic acid production. It was found that the concentration of cell growth and l-lactate decreased with the increase of glucose concentration and fermentation temperature. To enhance the osmotic stress resistance of the strain at high temperature, a mutant G-03 was screened and selected with 360?g/L glucose at 45°C as the selective criterion. To further increase the cell growth for lactic acid production, 3?g/L of biotin was supplemented to the medium. As a result, l-lactate concentration by the mutant G-03 reached 198.2?g/L (productivity of 5.5?g?L^?1?h^?1) at 41°C in a 7-L fermentor with 210?g/L glucose as carbon source. l-Lactate concentration and productivity of mutant G-03 were 115.2% and 97.8% higher than those of the parent strain, respectively. The strategy for enhancing l-lactic acid production by increasing osmotic stress resistance at high temperature may provide an alternative approach to enhance organic acid production with other strains.     

Metabolic engineering Corynebacterium glutamicum for the l-lysine production by increasing the flux into l-lysine biosynthetic pathway

The experiments presented here were based on the conclusions of our previous results. In order to avoid introduction of expression plasmid and to balance the NADH/NAD ratio, the NADH biosynthetic enzyme, i.e., NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GADPH), was replaced by NADP-dependent GADPH, which was used to biosynthesize NADPH rather than NADH. The results indicated that the NADH/NAD ratio significantly decreased, and glucose consumption and l-lysine production drastically improved. Moreover, increasing the flux through l-lysine biosynthetic pathway and disruption of ilvN and hom, which involve in the branched amino acid and l-methionine biosynthesis, further improved l-lysine production by Corynebacterium glutamicum. Compared to the original strain C. glutamicum Lys5, the l-lysine production and glucose conversion efficiency (α) were enhanced to 81.0 ± 6.59 mM and 36.45 % by the resulting strain C. glutamicum Lys5-8 in shake flask. In addition, the by-products (i.e., l-threonine, l-methionine and l-valine) were significantly decreased as results of genetic modification in homoserine dehydrogenase (HSD) and acetohydroxyacid synthase (AHAS). In fed-batch fermentation, C. glutamicum Lys5-8 began to produce l-lysine at post-exponential growth phase and continuously increased over 36 h to a final titer of 896 ± 33.41 mM. The l-lysine productivity was 2.73 g l^?1 h^?1 and the α was 47.06 % after 48 h. However, the attenuation of MurE was not beneficial to increase the l-lysine production because of decreasing the cell growth. Based on the above-mentioned results, we get the following conclusions: cofactor NADPH, precursor, the flux through l-lysine biosynthetic pathway and DCW are beneficial to improve l-lysine production in C. glutamicum.     

Microbial monomers custom-synthesized to build true bio-derived aromatic polymers

Aromatic polymers include novel and extant functional materials although none has been produced from biotic building blocks derived from primary biomass glucose. Here we screened microbial aromatic metabolites, engineered bacterial metabolism and fermented the aromatic lactic acid derivative β-phenyllactic acid (PhLA). We expressed the Wickerhamia fluorescens gene (pprA) encoding a phenylpyruvate reductase in Escherichia coli strains producing high levels of phenylalanine, and fermented optically pure (>99.9 %) D-PhLA. Replacing pprA with bacterial ldhA encoding lactate dehydrogenase generated L-PhLA, indicating that the produced enzymes converted phenylpyruvate, which is an intermediate of phenylalanine synthesis, to these chiral PhLAs. Glucose was converted under optimized fermentation conditions to yield 29 g/l d-PhLA, which was purified from fermentation broth. The product satisfied the laboratory-scale chemical synthesis of poly(d-PhLA) with M _w 28,000 and allowed initial physiochemical characterization. Poly(d-PhLA) absorbed near ultraviolet light, and has the same potential as all other biomass-derived aromatic bioplastics of phenylated derivatives of poly(lactic acid). This approach to screening and fermenting aromatic monomers from glucose exploits a new era of bio-based aromatic polymer design and will contribute to petroleum conservation and carbon dioxide fixation.     

Engineered Bacillus subtilis 168 produces l-malate by heterologous biosynthesis pathway construction and lactate dehydrogenase deletion

In the present work, Bacillus subtilis was engineered to produce l-malate. Initially, the study revealed that the slight fumarase activity under anaerobic conditions is extremely favourable for l-malate one-step fermentation accumulation. Subsequently, an efficient heterologous biosynthesis pathway formed by Escherichia coli phosphoenolpyruvate carboxylase and Saccharomyces cerevisiae malate dehydrogenase was introduced into B. subtilis, which led to 6.04?±?0.19?mM l-malate production. Finally, the l-malate production was increased 1.5-fold to 9.18?±?0.22?mM by the deletion of lactate dehydrogenase. Under two-stage fermentation conditions, the engineered B. subtilis produced up to 15.65?±?0.13?mM l-malate, which was 86.3?% higher than that under anaerobic fermentation conditions. Though the l-malate production by the recombinant was low, this is the first attempt to produce l-malate in engineered B. subtilis and paves the way for further improving l-malate production in B. subtilis.