L-malate production by metabolically engineered Escherichia coli

Escherichia coli strains (KJ060 and KJ073) that were previously developed for succinate production have now been modified for malate production. Many unexpected changes were observed during this investigation. The initial strategy of deleting fumarase isoenzymes was ineffective, and succinate continued to accumulate. Surprisingly, a mutation in fumarate reductase alone was sufficient to redirect carbon flow into malate even in the presence of fumarase. Further deletions were needed to inactivate malic enzymes (typically gluconeogenic) and prevent conversion to pyruvate. However, deletion of these genes (sfcA and maeB) resulted in the unexpected accumulation of D-lactate despite the prior deletion of mgsA and ldhA and the absence of apparent lactate dehydrogenase activity. Although the metabolic source of this D-lactate was not identified, lactate accumulation was increased by supplementation with pyruvate and decreased by the deletion of either pyruvate kinase gene (pykA or pykF) to reduce the supply of pyruvate. Many of the gene deletions adversely affected growth and cell yield in minimal medium under anaerobic conditions, and volumetric rates of malate production remained low. The final strain (XZ658) produced 163 mM malate, with a yield of 1.0 mol (mol glucose(-1)), half of the theoretical maximum. Using a two-stage process (aerobic cell growth and anaerobic malate production), this engineered strain produced 253 mM malate (34 g liter(-1)) within 72 h, with a higher yield (1.42 mol mol(-1)) and productivity (0.47 g liter(-1) h(-1)). This malate yield and productivity are equal to or better than those of other known biocatalysts.     

利用代谢工程改善大肠杆菌的3-脱氢莽草酸生产

3-脱氢莽草酸是芳香族氨基酸合成代谢途径中的一种重要中间产物。除可作为一种高效的抗氧化剂,还可用于合成己二酸、香草醛等一些重要的化工产品,具有重要的应用价值。相关研究证明具有去酪氨酸反馈抑制的3-脱氧-D-阿拉伯庚酮糖-7-磷酸合成酶基因aroFFBR以及转酮醇酶基因tktA可以有效影响3-脱氢莽草酸的过量合成。通过增加aroFFBR和tktA串联过量表达的拷贝数,可使工程菌株在摇瓶发酵条件下3-脱氢莽草酸产量提高2.93倍。通过同源重组无痕基因敲除技术依次敲除出发菌大肠杆菌Escherichia coli AB2834的乳酸、乙酸、乙醇等副产物合成途径中的重要基因ldhA、ackA-pta和adhE,可使工程菌株的3-脱氢莽草酸产量进一步提高,达到了1.83 g/L,是初始出发菌株大肠杆菌E.coli AB2834产量的6.7倍。利用5 L发酵罐进行分批补料发酵,62 h后工程菌株3-脱氢莽草酸产量达到了25.48 g/L。本研究可为构建有应用前景的3-脱氢莽草酸生产菌株提供重要参考。     

代谢工程大肠杆菌利用甘油高效合成L-乳酸

以甘油为碳源高效合成L-乳酸有助于推进油脂水解产业和生物可降解材料制造业的共同发展。为此,首先分别从凝结芽胞杆菌Bacillus coagulans CICIM B1821和大肠杆菌Escherichia coli CICIM B0013中克隆了L-乳酸脱氢酶基因BcoaLDH和D-乳酸脱氢酶 (LdhA) 的启动子片段PldhA。将两条DNA片段连接组成了表达盒PldhA-BcoaLDH。然后将上述表达盒通过同源重组删除FMN为辅酶的L-乳酸脱氢酶编码基因lldD的同时克隆入ldhA基因缺失菌株E. coli CICIM B0013-080C (ack-pta pps pflB dld poxB adhE frdA ldhA)的染色体上,获得了L-乳酸高产菌株E. coli CICIM B0013-090B (B0013-080C,lldD::PldhA-BcoaLDH)。考察了菌株CICIM B0013-090B不同培养温度下代谢利用甘油和合成L-乳酸的特征后,建立并优化了一种新型L-乳酸变温发酵工艺。在7 L发酵罐上,发酵27 h,积累L-乳酸132.4 g/L,产酸强度4.90 g/(L·h),甘油到L-乳酸的得率为93.7％,L-乳酸的光学纯度达到99.95％。     

Fed-batch two-phase production of alanine by a metabolically engineered Escherichia coli

dl-Alanine was produced from glucose in an Escherichia coli pfl pps poxB ldhA aceEF pTrc99A-alaD strain which lacked pyruvate-formate lyase, phosphoenolpyruvate (PEP) synthase, pyruvate oxidase, lactate dehydogenase, components of the pyruvate dehydogenase complex and over-produced alanine dehydrogenase (ALD). A two-phase process was developed with cell growth under aerobic conditions followed by alanine production under anaerobic conditions. Using the batch mode, cells grew to 5.3 g/l in 9 h with the accumulation of 6–10 g acetate/l, and under subsequent anaerobic conditions achieved 34 g alanine/l in 13 h with a yield of 0.86 g/g glucose. Using the fed-batch mode at μ = 0.15 h⁻¹, only about 1 g acetate/l formed in the 25 h required for the cells to reach 5.6 g/l, and 88 g alanine/l accumulated during the subsequent 23 h. This fed-batch process attained an alanine volumetric productivity of 4 g/lh during the production phase, and a yield that was essentially 1 g/g.     

Improving 1,3-propanediol production from glycerol in a metabolically engineered Escherichia coli by reducing accumulation of sn-glycerol-3-phosphate

High levels of glycerol significantly inhibit cell growth and 1,3-propanediol (1,3-PD) production in anaerobic glycerol fermentation by genetically engineered Escherichia coli (E. coli) strains expressing genes from the Klebsiella pneumoniae dha (K.pneumoniae) regulon. We have previously demonstrated that 1,3-PD production by the engineered E. coli can be improved by reducing the accumulation of methylglyoxal. This study focuses on investigation of another lesser-known metabolite in the pathways related to 1,3-PD production-glycerol-3-phosphate (G3P). When grown anaerobically on glycerol in the absence of an exogenous acceptor, the engineered E. coli strains have intracellular G3P levels that are significantly higher than those in K. pneumoniae, a natural 1,3-PD producer. Furthermore, in the engineered E. coli strains, the G3P levels increase with increasing glycerol concentrations, whereas, in K. pneumoniae, the concentrations of G3P remain relatively constant. Addition of fumarate, which can stimulate activity of anaerobic G3P dehydrogenase, into the fermentation medium led to a greater than 30-fold increase in the specific activity of anaerobic G3P dehydrogenase and a significant decrease in concentrations of intracellular G3P and resulted in better cell growth and an improved production of 1,3-PD. This indicates that the low activity of G3P dehydrogenase in the absence of an exogenous electron acceptor is one of the reasons for G3P accumulation. In addition, spent media from E.coli Lin61, a glycerol kinase (responsible for conversion of glycerol to G3P) mutant, contains greatly decreased concentrations of G3P and shows improved production of 1,3-PD (by 2.5-fold), when compared to media from its parent strain E. coli K10. This further suggests that G3P accumulation is one of the reasons for the inhibition of 1,3-PD production during anaerobic fermentation.     

Directed evolution of metabolically engineered Escherichia coli for carotenoid production

We have previously introduced a reconstructed isoprenoid pathway into Escherichia coli that exhibits amplified biosynthetic flux to geranylgeranyl diphosphate (GGPP), a common isoprenoid precursor. It was shown that GGPP synthase is an important rate-controlling enzyme in this reconstructed isoprenoid pathway. In this investigation, we applied directed evolution to GGPP synthase from Archaeoglobus fulgidus to enable the enhanced production of carotenoids in metabolically engineered E. coli. Eight mutants were isolated, and the best one increased lycopene production by 100%. Among the mutants that were isolated, mutation points were clustered in four "hot regions". The "hottest" region is located in the sequence upstream of the coding region, which presumably improves the expression level of the enzyme. The other three are within the coding sequence and are believed to improve the enzyme-specific activity in E coli. These results demonstrate that modulating both enzymatic expression and specific activity are important for optimizing the metabolic flux distribution.     

利用代谢工程构建D-甘露醇生产菌株

D-甘露醇广泛应用于食品、制药、化学品工业等领域。从野生型大肠杆菌出发,将来自假肠膜明串珠菌Leuconostoc pseudomesenteroides ATCC 12291菌株的甘露醇脱氢酶与果糖转运蛋白编码基因整合到大肠杆菌ATCC 8739的染色体中,并失活其他的发酵途径 (丙酮酸甲酸裂解酶、乳酸脱氢酶、富马酸还原酶、乙醇脱氢酶、甲基乙二醛合成酶和丙酮酸氧化酶) ,构建了一株遗传稳定的D-甘露醇生产菌株。使用无机盐培养基和葡萄糖果糖作为混合碳源,厌氧发酵6 d,D-甘露醇产量达1.2 mmol/L。基于细胞生长和D-甘露醇合成的偶联,进一步通过代谢进化技术提高细胞合成D-甘露醇的生产能力。经过80代的驯化,D-甘露醇产量提高了2.6倍,甘露醇脱氢酶的活性提高了2.8倍。构建获得的遗传稳定的工程菌能直接发酵糖生产D-甘露醇,不需添加抗生素、诱导剂和甲酸,在工业化生产时有一定优势。     

Production of d-ribose by metabolically engineered Escherichia coli

Escherichia coli was metabolically engineered for the production of d-ribose, a functional five-carbon sugar, from xylose. For the accumulation of d-ribose, two genes of transketolase catalyzing the conversion of d-ribose-5-phosphate to sedoheptulose-7-phosphate in pentose phosphate pathway were disrupted to create a transketolase-deficient E. coli SGK013. In batch fermentation, E. coli SGK013 grew by utilizing glucose and then started to produce d-ribose from xylose after glucose depletion. E. coli SGK013 produced 0.75 g/L of d-ribose, which was identical to the standard d-ribose as confirmed by HPLC and LC/MS analyses. To improve D-ribose production, the ptsG gene encoding the glucose-specific IICB component was disrupted additionally, resulting in the construction of E. coli SGK015. The carbon catabolite repression-negative E. coli SGK015 utilized xylose and glucose simultaneously and produced up to 3.75 g/L of d-ribose, which is a 5-fold improvement compared to that of E. coli SGK013.     

Homolactate fermentation by metabolically engineered Escherichia coli strains

We report the homofermentative production of lactate in Escherichia coli strains containing mutations in the aceEF, pfl, poxB, and pps genes, which encode the pyruvate dehydrogenase complex, pyruvate formate lyase, pyruvate oxidase, and phosphoenolpyruvate synthase, respectively. The process uses a defined medium and two distinct fermentation phases: aerobic growth to an optical density of about 30, followed by nongrowth, anaerobic production. Strain YYC202 (aceEF pfl poxB pps) generated 90 g/liter lactate in 16 h during the anaerobic phase (with a yield of 0.95 g/g and a productivity of 5.6 g/liter . h). Ca(OH)(2) was found to be superior to NaOH for pH control, and interestingly, significant succinate also accumulated (over 7 g/liter) despite the use of N(2) for maintaining anaerobic conditions. Strain ALS961 (YYC202 ppc) prevented succinate accumulation, but growth was very poor. Strain ALS974 (YYC202 frdABCD) reduced succinate formation by 70% to less than 3 g/liter. (13)C nuclear magnetic resonance analysis using uniformly labeled acetate demonstrated that succinate formation by ALS974 was biochemically derived from acetate in the medium. The absence of uniformly labeled succinate, however, demonstrated that glyoxylate did not reenter the tricarboxylic acid cycle via oxaloacetate. By minimizing the residual acetate at the time that the production phase commenced, the process with ALS974 achieved 138 g/liter lactate (1.55 M, 97% of the carbon products), with a yield of 0.99 g/g and a productivity of 6.3 g/liter . h during the anaerobic phase.     

Enhanced production of plasmid DNA by engineered Escherichia coli strains

Escherichia coli strains VH33 (PTS? GalP? strain displaying a strongly reduced overflow metabolism) and VH34 (additionally lacking the pyruvate kinase A) were evaluated for the production of a plasmid DNA (pDNA) vaccine. The parent (W3110) and mutant strains were cultured using 10 g of glucose/L. While the specific growth rates of the three strains were similar, they presented differences in the accumulation of acetate. W3110 accumulated up to 4 g/L of acetate, VH33 produced 1.4 g/L, and VH34 only 0.78 g/L. VH33 and VH34 produced 76% and 300% more pDNA than W3110. Moreover, VH34 demanded 33% less oxygen than VH33 and W3110, which can be advantageous for large-scale applications.     

Efficient production of polylactic acid and its copolymers by metabolically engineered Escherichia coli

Polylactic acid (PLA) is one of the promising biodegradable polymers, which has been produced in a rather complicated two-step process by first producing lactic acid by fermentation followed by ring opening polymerization of lactide, a cyclic dimer of lactic acid. Recently, we reported the production of PLA and its copolymers by direct fermentation of metabolically engineered Escherichia coli equipped with the evolved propionate CoA-transferase and polyhydroxyalkanoate (PHA) synthase using glucose as a carbon source. When employing these initially constructed E. coli strains, however, it was necessary to use an inducer for the expression of the engineered genes and to feed succinate for proper cell growth. Here we report further metabolic engineering of E. coli strain to overcome these problems for more efficient production of PLA and its copolymers. This allowed efficient production of PLA and its copolymers without adding inducer and succinate. The finally constructed recombinant E. coli JLXF5 strain was able to produce P(3HB-co-39.6 mol% LA) having the molecular weight of 141,000 Da to 20 g l⁻¹ with a polymer content of 43 wt% in a chemically defined medium by the pH-stat fed-batch culture.     

Homofermentative production of d-lactic acid from sucrose by a metabolically engineered Escherichia coli

Escherichia coli W, a sucrose-positive strain, was engineered for the homofermentative production of d-lactic acid through chromosomal deletion of the competing fermentative pathway genes (adhE, frdABCD, pta, pflB, aldA) and the repressor gene (cscR) of the sucrose operon, and metabolic evolution for improved anaerobic cell growth. The resulting strain, HBUT-D, efficiently fermented 100?g?sucrose?l^?1 into 85?g?d-lactic acid?l^?1 in 72–84?h in mineral salts medium with a volumetric productivity of ~1?g?l^?1?h^?1, a product yield of 85?% and d-lactic acid optical purity of 98.3?%, and with a minor by-product of 4?g?acetate?l^?1. HBUT-D thus has great potential for production of d-lactic acid using an inexpensive substrate, such as sugar cane and/or beet molasses, which are primarily composed of sucrose.     

Efficient production of 2-deoxy-scyllo-inosose from d-glucose by metabolically engineered recombinant Escherichia coli

2-Deoxy-scyllo-inosose (DOI) is a six-membered carbocycle formed from d-glucose-6-phosphate catalyzed by 2-deoxy-scyllo-inosose synthase (DOIS), a key enzyme in the biosynthesis of 2-deoxystreptamine-containing aminocyclitol antibiotics. DOI is valuable as a starting material for the benzene-free synthesis of catechol and other benzenoids. We constructed a series of metabolically engineered Escherichia coli strains by introducing a DOIS gene (btrC) from Bacillus circulans and disrupting genes for phosphoglucose isomerase, d-glucose-6-phosphate dehydrogenase, and phosphoglucomutase (pgi, zwf and pgm, respectively). It was found that deletion of the pgi gene, pgi and zwf genes, pgi and pgm genes, or all pgi, zwf and pgm genes significantly improved DOI production by recombinant E. coli in 2YTG medium (3% glucose) up to 7.4, 6.1, 11.6, and 8.4 g l(-1), respectively, compared with that achieved by wild-type recombinant E. coli (1.5 g l(-1)). Moreover, E. coli mutants with disrupted pgi, zwf and pgm genes showed strongly enhanced DOI productivity of up to 29.5 g l(-1) (99% yield) in the presence of mannitol as a supplemental carbon source. These results demonstrated that DOI production by metabolically engineered recombinant E. coli may provide a novel, efficient approach to the production of benzenoids from renewable d-glucose.     

Enhanced N-acetyl-d-neuraminic production from glycerol and N-acetyl-d-glucosamine by metabolically engineered Escherichia coli with a two-stage pH-shift control strategy

Journal of Industrial Microbiology & Biotechnology - Typical N-acetyl-d-neuraminic acid (Neu5Ac) production uses N-acetyl-d-glucosamine (GlcNAc) and excess pyruvate as substrates in the...     

Biosynthesis of enantiopure (S)-3-hydroxybutyric acid in metabolically engineered Escherichia coli

A biosynthetic pathway for the production of (S)-3-hydroxybutyric acid (S3HB) from glucose was established in recombinant Escherichia coli by introducing the beta-ketothiolase gene from Ralstonia eutropha H16, the (S)-3-hydroxybutyryl-CoA dehydrogenase gene from R. eutropha H16, or Clostridium acetobutylicum ATCC824, and the 3-hydroxyisobutyryl-CoA hydrolase gene from Bacillus cereus ATCC14579. Artificial operon consisting of these genes was constructed and was expressed in E. coli BL21 (DE3) codon plus under T7 promoter by isopropyl beta-D: -thiogalactoside (IPTG) induction. Recombinant E. coli BL21 (DE3) codon plus expressing the beta-ketothiolase gene, the (S)-3-hydroxybutyryl-CoA dehydrogenase gene, and the 3-hydroxyisobutyryl-CoA hydrolase gene could synthesize enantiomerically pure S3HB to the concentration of 0.61 g l(-1) from 20 g l(-1) of glucose in Luria-Bertani medium. Fed-batch cultures of recombinant E. coli BL21 (DE3) codon plus were carried out to achieve higher titer of S3HB with varying induction time and glucose concentration during fermentation. Protein expression was induced by addition of 1 mM IPTG when cell concentration reached 10 and 20 g l(-1) (OD(600) = 30 and 60), respectively. When protein expression was induced at 60 of OD(600) and glucose was fed to the concentration of 15 g l(-1), 10.3 g l(-1) of S3HB was obtained in 38 h with the S3HB productivity of 0.21 g l(-1)h(-1). Lowering glucose concentration to 5 g l(-1) and induction of protein expression at 30 of OD(600) significantly reduced final S3HB concentration to 3.7 g l(-1), which also resulted in the decrease of the S3HB productivity to 0.05 g l(-1)h(-1).     

Production of p-Aminobenzoic acid by metabolically engineered Escherichia coli

The production of chemical compounds from renewable resources is an important issue in building a sustainable society. In this study, Escherichia coli was metabolically engineered by introducing T7lac promoter-controlled aroF^fbr, pabA, pabB, and pabC genes into the chromosome to overproduce para-aminobenzoic acid (PABA) from glucose. Elevating the copy number of chromosomal P_T7lac-pabA-pabB distinctly increased the PABA titer, indicating that elevation of 4-amino-4-deoxychorismic acid synthesis is a significant factor in PABA production. The introduction of a counterpart derived from Corynebacterium efficiens, pabAB (ce), encoding a fused PabA and PabB protein, resulted in a considerable increase in the PABA titer. The introduction of more than two copies of P_T7lac-pabAB (ce-mod), a codon-optimized pabAB (ce), into the chromosome of a strain that simultaneously overexpressed aroF^fbr and pabC resulted in 5.1?mM PABA from 55.6?mM glucose (yield 9.2%). The generated strain produced 35?mM (4.8?g?L^?1) PABA from 167?mM glucose (yield 21.0%) in fed-batch culture.     

Synthesis of methyl ketones by metabolically engineered Escherichia coli

Methyl ketones are a group of highly reduced platform chemicals with widespread applications in the fragrance, flavor and pharmacological industries. Current methods for the industrial production of methyl ketones include oxidation of hydrocarbons, but recent advances in the characterization of methyl ketone synthases from wild tomato have sparked interest towards the development of microbial platforms for the industrial production of methyl ketones. A functional methyl ketone biosynthetic pathway was constructed in Escherichia coli by over-expressing two genes from Solanum habrochaites: shmks2, encoding a 3-ketoacyl-ACP thioesterase, and shmks1, encoding a beta-decarboxylase. These enzymes enabled methyl ketone synthesis from 3-ketoacyl-ACP, an intermediate in the fatty acid biosynthetic cycle. The production of 2-nonanone, 2-undecanone, and 2-tridecanone by MG1655 pTH-shmks2-shmks1 was initially detected by nuclear magnetic resonance and gas chromatography–mass spectrometry analyses at levels close to 6?mg/L. The deletion of major fermentative pathways leading to ethanol (adhE), lactate (ldhA), and acetate (pta, poxB) production allowed for the carbon flux to be redirected towards methyl ketone production, doubling total methyl ketone concentration. Variations in methyl ketone production observed under different working volumes in flask experiments led to a more detailed analysis of the effects of oxygen availability on methyl ketone concentration in order to determine optimal levels of oxygen. The methyl ketone concentration achieved with MG1655 ?adhE ?ldhA ?poxB ?pta pTrcHis2A-shmks2-shmks1, the best performer strain in this study, was approximately 500?mg/L, the highest reported for an engineered microorganism. Through the establishment of optimal operating conditions and by executing rational metabolic engineering strategies, we were able to increase methyl ketone concentrations by almost 75-fold from the initial confirmatory levels.