重组大肠杆菌利用木糖发酵产高纯度L-乳酸

对重组大肠杆菌JH16利用木糖产高纯度的三一乳酸进行研究。通过无氧管驯化EscherwhiacdiJH12菌株得到E．coliJH16,驯化后的菌株茵体浓度提高了31％,乙酸积累减少了43％;在摇瓶中考察不同Mg2＋浓度对EcoliJHl6产三一乳酸的影响,确定最适Mg2＋质量浓度为0．25g/L;EcoEJH16以60g/L木糖为C源,在7L全自动发酵罐中添加0．25g／LMg2＋,乳酸积累量提高了18％,达38．18g/L,乳酸纯度高达95％;E．coliJH16在30g／L木糖和30g／L葡萄糖混合C源中,优先利用葡萄糖,当葡萄糖质量浓度低于1．56g/L后,菌体开始利用木糖进行乳酸发酵,最终得到39g／L乳酸。     

Role of xylose transporters in xylitol production from engineered Escherichia coli

Escherichia coli W3110 was previously engineered to co-utilize glucose and xylose by replacing the wild-type crp gene with a crp* mutant encoding a cAMP-independent CRP variant (Cirino et al., 2006 [Cirino, P.C., Chin, J.W., Ingram, L.O., 2006. Engineering Escherichia coli for xylitol production from glucose-xylose mixtures. Biotechnol. Bioeng. 95, 1167-1176.]). Subsequent deletion of the xylB gene (encoding xylulokinase) and expression of xylose reductase from Candida boidinii (CbXR) resulted in a strain which produces xylitol from glucose-xylose mixtures. In this study we examine the contributions of the native E. coli xylose transporters (the d-xylose/proton symporter XylE and the d-xylose ABC transporter XylFGH) and CRP* to xylitol production in the presence of glucose and xylose. The final batch xylitol titer with strain PC09 (Delta xylB and crp*) is reduced by 40% upon deletion of xylG and by 60% upon deletion of both xyl transporters. Xylitol production by the wild-type strain (W3110) expressing CbXR is not reduced when xylE and xylG are deleted, demonstrating tight regulation of the xylose transporters by CRP and revealing significant secondary xylose transport. Finally, plasmid expression of XylE or XylFGH with CbXR in PC07 (Delta xylB and wild-type crp) growing on glucose results in xylitol titers similar to that achieved with PC09 and provides an alternative strategy to the use of CRP*.     

Metabolic pathway optimization for biosynthesis of 1,2,4-butanetriol from xylose by engineered Escherichia coli

1,2,4-Butanetriol (BT) and related derivatives have been widely used in many fields, especially in the military and in medicine. In this paper, we systematically optimized the BT biosynthetic pathway. We first investigated the activities of various NADH dependent aldehyde reductases (ALRs), which catalyze the fourth reaction in the four-step pathway for BT production from xylose in E. coli, and found that a combination of multiple endogenous enzymes catalyzed aldehyde reduction in the BT production bioprocess and that YqhD in E. coli was a main ALR for BT production. In addition, ADH2 from Saccharomyces cerevisiae can effectively catalyze 3,4-dihydroxybutanal to BT. Also, YjhG was identified as the major xylonate dehydratase and was co-overexpressed with YqhD, resulting in an improvement of BT production by 30%. Moreover, we identified and eliminated the competing branch pathway by inactivating 2-keto acid reductases (yiaE). Finally, the combination of these approaches led to BT production of 5.1 g/L. In summary, our study provides insights into the biosynthetic pathway for BT production, demonstrates an effective strategy to enhance BT production, and paves the way toward in-depth research on BT biosynthesis.     

Improved conversion of fumarate to succinate by Escherichia coli strains amplified for fumarate reductase

Two recombinant plasmid Escherichia coli strains containing amplified fumarate reductase activity converted fumarate to succinate at significantly higher rates and yields than a wild-type E. coli strain. Glucose was required for the conversion of fumarate to succinate, and in the absence of glucose or in cultures with a low cell density, malate accumulated. Two-dimensional gel electrophoretic analysis of proteins from the recombinant DNA and wild-type strains showed that increased quantities of both large and small fumarate reductase subunits were expressed in the recombinant DNA strains.     

Improved conversion of fumarate to succinate by Escherichia coli strains amplified for fumarate reductase.

Two recombinant plasmid Escherichia coli strains containing amplified fumarate reductase activity converted fumarate to succinate at significantly higher rates and yields than a wild-type E. coli strain. Glucose was required for the conversion of fumarate to succinate, and in the absence of glucose or in cultures with a low cell density, malate accumulated. Two-dimensional gel electrophoretic analysis of proteins from the recombinant DNA and wild-type strains showed that increased quantities of both large and small fumarate reductase subunits were expressed in the recombinant DNA strains.     

Conversion of glucose‐xylose mixtures to pyruvate using a consortium of metabolically engineered Escherichia coli

Two strains of Escherichia coli were engineered to accumulate pyruvic acid from two sugars found in lignocellulosic hydrolysates by knockouts in the aceE, ppsA, poxB, and ldhA genes. Additionally, since glucose and xylose are typically consumed sequentially due to carbon catabolite repression in E. coli, one strain (MEC590) was engineered to grow only on glucose while a second strain (MEC589) grew only on xylose. On a single substrate, each strain generated pyruvate at a yield of about 0.60 g/g in both continuous culture and batch culture. In a glucose‐xylose mixture under continuous culture, a consortium of both strains maintained a pyruvate yield greater than 0.60 g/g when three different concentrations of glucose and xylose were sequentially fed into the system. In a fed‐batch process, both sugars in a glucose‐xylose mixture were consumed simultaneously to accumulate 39 g/L pyruvate in less than 24 h at a yield of 0.59 g/g.     

Identification of bottlenecks in Escherichia coli engineered for the production of CoQ(10)

In this work, Escherichia coli was engineered to produce a medically valuable cofactor, coenzyme Q₁₀ (CoQ₁₀), by removing the endogenous octaprenyl diphosphate synthase gene and functionally replacing it with a decaprenyl diphosphate synthase gene from Sphingomonas baekryungensis. In addition, by over-expressing genes coding for rate-limiting enzymes of the aromatic pathway, biosynthesis of the CoQ₁₀ precursor para-hydroxybenzoate (PHB) was increased. The production of isoprenoid precursors of CoQ₁₀ was also improved by the heterologous expression of a synthetic mevalonate operon, which permits the conversion of exogenously supplied mevalonate to farnesyl diphosphate. The over-expression of these precursors in the CoQ₁₀-producing E. coli strain resulted in an increase in CoQ₁₀ content, as well as in the accumulation of an intermediate of the ubiquinone pathway, decaprenylphenol (10P-Ph). In addition, the over-expression of a PHB decaprenyl transferase (UbiA) encoded by a gene from Erythrobacter sp. NAP1 was introduced to direct the flux of DPP and PHB towards the ubiquinone pathway. This further increased CoQ₁₀ content in engineered E. coli, but decreased the accumulation of 10P-Ph. Finally, we report that the combined over-production of isoprenoid precursors and over-expression of UbiA results in the decaprenylation of para-aminobenzoate, a biosynthetic precursor of folate, which is structurally similar to PHB.     

Active site complementation in engineered heterodimers of Escherichia coli glutathione reductase created in vivo

By directed mutagenesis of the cloned Escherichia coli gor gene encoding the dimeric flavoprotein glutathione reductase, Cys-47 (a cysteine residue forming an essential charge-transfer complex with enzyme-bound FAD) was converted to serine (C47S) and His-439 (required to facilitate protonation of the reduced glutathione) was converted to glutamine (H439Q). Both mutant genes were placed in the same plasmid, pHD, where each of them came under the control of a strong tac promoter. This was designed to achieve equal over-expression of both genes in the same E. coli cell. The parental homo-dimers show no (C47S) or very little (H439Q) activity as glutathione reductases. The formation in vivo of heterodimers, carrying one crippled and one fully functional active site, was detected by absorbance spectroscopy and fluorescence emission spectrometry of enzyme-bound FAD and by active site complementation. The fractional distribution of homo- and hetero-dimers was in accord with that expected for a random association of enzyme subunits. In a homo-dimer, the H439Q mutation leads to a big fall in the value of Km for NADPH which binds some 1.8 nm from the point of mutation (Berry, A., Scrutton, N.S. & Perham, R. N. Biochemistry 28, 1264-1269 (1989)). However, the one active site in the H439Q/C47S hetero-dimer exhibited kinetic parameters similar to those of the wild-type enzyme. Thus, the effect of the H439Q mutation must be retained within the active site that accommodates it and is not transmitted through the protein to the second active site across the subunit interface.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of Sphingomonas aldehyde dehydrogenase catalyzing the conversion of various aromatic aldehydes to their carboxylic acids

Natural products represent an important source of drugs in a number of therapeutic fields, e.g. antiinfectives and cancer therapy. Natural products are considered as biologically validated lead structures, and evolution of compounds with novel or enhanced biological properties is expected from the generation of structural diversity in natural product libraries. However, natural products are often structurally complex, thus precluding reasonable synthetic access for further structure-activity relationship studies. As a consequence, natural product research involves semisynthetic or biotechnological approaches. Among the latter are mutasynthesis (also known as mutational biosynthesis) and precursor-directed biosynthesis, which are based on the cellular uptake and incorporation into complex antibiotics of relatively simple biosynthetic building blocks. This appealing idea, which has been applied almost exclusively to bacteria and fungi as producing organisms, elegantly circumvents labourious total chemical synthesis approaches and exploits the biosynthetic machinery of the microorganism. The recent revitalization of mutasynthesis is based on advancements in both chemical syntheses and molecular biology, which have provided a broader available substrate range combined with the generation of directed biosynthesis mutants. As an important tool in supporting combinatorial biosynthesis, mutasynthesis will further impact the future development of novel secondary metabolite structures.     

Identification of membrane anchor polypeptides of Escherichia coli fumarate reductase

Fumarate reductase of Escherichia coli has been shown to be a membrane-bound enzyme composed of a 69,000-dalton catalytic-flavin-containing subunit and a 27,000-dalton nonheme-iron-containing subunit. Using gene cloning and amplification techniques, we have observed two additional polypeptides encoded by the frd operon, with apparent molecular weights of 15,000 and 14,000, which are expressed when E. coli is grown anaerobically on glycerol plus fumarate. Expression of these two small polypeptides is necessary for the two large subunits to associate with the membrane. The four subunits remain associated in Triton X-100 extracts of the membrane, and a holoenzyme form of fumarate reductase containing one copy of each of the four polypeptides has been isolated. Unlike the well-characterized two-subunit form, the holoenzyme is not dependent on anions for activity and is not labile at alkaline pH. In these respects, it more closely resembles the membrane-bound activity.     

Reduction of methylglyoxal in Escherichia coli K12 by an aldehyde reductase and alcohol dehydrogenase

Two enzymes, one NADPH-dependent and another NADH-dependent which catalyze the reduction of methylglyoxal to acetol have been isolated and substantially purified from crude extracts of Escherichia coli K₁₂ cells. Substrate specificity and formation of acetol as the reaction product by both the enzymes, reversibility of NADH-dependent enzyme with alcohols as substrates and inhibitor study with NADPH-dependent enzyme indicate that NADPH-dependent and NADH-dependent enzymes are identical with an aldehyde reductase (EC 1.1.1.2) and alcohol dehydrogenase (EC 1.1.1.1) respectively. The K_m for methylglyoxal have been determined to be 0.77 mM for NADPH-dependent and 3.8 mM for NADH-dependent enzyme. Stoichiometrically equimolar amount of acetol is formed from methylglyoxal by both NADPH- and NADH-dependent enzymes. In phosphate buffer, both the enzymes are active in the pH range of 5.8–6.6 with no sharp pH optimum. Molecular weight of both the enzymes were found to be 100,000 ± 3,000 by gel filtration on a Sephacryl S-200 column. Both NADPH- and NADH-dependent enzymes are sensitive to sulfhydryl group reagents.     

Purification and properties of beef liver aldehyde reductase catalyzing the reduction of D-erythrose 4-phosphate

An aldehyde reductase catalyzing the NADPH-dependent reduction of D-erythrose 4-phosphate to D-erythritol 4-phosphate was purified from beef liver. It was proved to be homogeneous by polyacrylamide gel electrophoresis, sodium dodecyl sulfate polyacrylamide gel electrophoresis and ultracentrifugation analysis. The enzyme was proved to be a monomeric enzyme and its molecular weight was about 40,000. The enzyme was able to reduce not only tetroses but also trioses, aromatic aldehydes, D-glucuronate and succinic semialdehyde. Apparent Km-values for aromatic aldehydes were lower than those for tetroses, trioses, D-glucuronate and succinic semi-aldehyde. Barbiturates and valproate were potent inhibitors of the enzyme and their apparent K1-values were in the range of 80-180 microM. Quercitrin was the most potent inhibitor and its K1-value was about 7 microM. From the viewpoint of substrate specificity and inhibitor sensitivity, it seems that the enzyme belongs to the high-Km type aldehyde reductases.     

Engineering the isobutanol biosynthetic pathway in Escherichia coli by comparison of three aldehyde reductase/alcohol dehydrogenase genes

Biofuels synthesized from renewable resources are of increasing interest because of global energy and environmental problems. We have previously demonstrated production of higher alcohols from Escherichia coli using a 2-keto acid-based pathway. Here, we have compared the effect of various alcohol dehydrogenases (ADH) for the last step of the isobutanol production. E. coli has the yqhD gene which encodes a broad-range ADH. Isobutanol production significantly decreased with the deletion of yqhD, suggesting that the yqhD gene on the genome contributed to isobutanol production. The adh genes of two bacteria and one yeast were also compared in E. coli harboring the isobutanol synthesis pathway. Overexpression of yqhD or adhA in E. coli showed better production than ADH2, a result confirmed by activity measurements with isobutyraldehyde.     

Increasing reducing power output (NADH) of glucose catabolism for reduction of xylose to xylitol by genetically engineered Escherichia coli AI05

Anaerobic homofermentative production of reduced products requires additional reducing power (NADH and/or NADPH) output from glucose catabolism. Previously, with an anaerobically expressed pyruvate dehydrogenase operon (aceEF-lpd), we doubled the reducing power output to four NADH per glucose (or 1.2 xylose) catabolized anaerobically, which satisfied the NADH requirement to establish a non-transgenic homoethanol pathway (1 glucose or 1.2 xylose ? 2 acetyl-CoA + 4 NADH ? 2 ethanol) in the engineered strain, Escherichia coli SZ420 (?frdBC ?ldhA ?ackA ?focA-pflB ?pdhR::pflBp6-pflBrbs-aceEF-lpd). In this study, E. coli SZ420 was further engineered for reduction of xylose to xylitol by (1) deleting the alcohol dehydrogenase gene (adhE) to divert NADH from the ethanol pathway; (2) deleting the glucose-specific PTS permease gene (ptsG) to eliminate catabolite repression and allow simultaneous uptake of glucose and xylose; (3) cloning the aldose reductase gene (xylI) of Candida boidinii to reduce xylose to xylitol. The resulting strain, E. coli AI05 (pAGI02), could in theory simultaneously uptake glucose and xylose, and utilize glucose as a source of reducing power for the reduction of xylose to xylitol, with an expected yield of four xylitol for each glucose consumed (Y_RPG = 4) under anaerobic conditions. In resting cell fermentation tests using glucose and xylose mixtures, E. coli AI05 (pAGI02) achieved an actual Y_RPG value of ~3.6, with xylitol as the major fermentation product and acetate as the by-product.     

High-yield anthocyanin biosynthesis in engineered Escherichia coli

Anthocyanins are red, purple, or blue plant water-soluble pigments. In the past two decades, anthocyanins have received extensive studies for their anti-oxidative, anti-inflammatory, anti-cancer, anti-obesity, anti-diabetic, and cardioprotective properties. In the present study, anthocyanin biosynthetic enzymes from different plant species were characterized and employed for pathway construction leading from inexpensive precursors such as flavanones and flavan-3-ols to anthocyanins in Escherichia coli. The recombinant E. coli cells successfully achieved milligram level production of two anthocyanins, pelargonidin 3-O-glucoside (0.98 mg/L) and cyanidin 3-O-gluside (2.07 mg/L) from their respective flavanone precursors naringenin and eriodictyol. Cyanidin 3-O-glucoside was produced at even higher yields (16.1 mg/L) from its flavan-3-ol, (+)-catechin precursor. Further studies demonstrated that availability of the glucosyl donor, UDP-glucose, was the key metabolic limitation, while product instability at normal pH was also identified as a barrier for production improvement. Therefore, various optimization strategies were employed for enhancing the homogenous synthesis of UDP-glucose in the host cells while at the same time stabilizing the final anthocyanin product. Such optimizations included culture medium pH adjustment, the creation of fusion proteins and the rational manipulation of E. coli metabolic network for improving the intracellular UDP-glucose metabolic pool. As a result, production of pelargonidin 3-O-glucoside at 78.9 mg/L and cyanidin 3-O-glucoside at 70.7 mg/L was achieved from their precursor flavan-3-ols without supplementation with extracellular UDP-glucose. These results demonstrate the efficient production of the core anthocyanins for the first time and open the possibility for their commercialization for pharmaceutical and nutraceutical applications.     

High-yield resveratrol production in engineered Escherichia coli

Plant polyphenols have been the subject of several recent scientific investigations since many of the molecules in this class have been found to be highly active in the human body, with a plethora of health-promoting activities against a variety of diseases, including heart disease, diabetes, and cancer, and with even the potential to slow aging. Further development of these potent natural therapeutics hinges on the formation of robust industrial production platforms designed using specifically selected as well as engineered protein sources along with the construction of optimal expression platforms. In this work, we first report the investigation of various stilbene synthases from an array of plant species considering structure-activity relationships, their expression efficiency in microorganisms, and their ability to synthesize resveratrol. Second, we looked into the construct environment of recombinantly expressed stilbene synthases, including different promoters, construct designs, and host strains, to create an Escherichia coli strain capable of producing superior resveratrol titers sufficient for commercial usage. Further improvement of metabolic capabilities of the recombinant strain aimed at improving the intracellular malonyl-coenzyme A pool, a resveratrol precursor, resulted in a final improved titer of 2.3 g/liter resveratrol.