L-Fucose production by engineered Escherichia coli

L -Fucose (6-deoxy-L -galactose) is a major constituent of glycans and glycolipids in mammals. Fucosylation of glycans can confer unique functional properties and may be an economical way to manufacture L -fucose. Research can extract L -fucose directly from brown algae, or by enzymatic hydrolysis of L -fucose-rich microbial exopolysaccharides. However, these L -fucose production methods are not economical or scalable for various applications. We engineered an Escherichia coli strain to produce L -fucose. Specifically, we modified the strain genome to eliminate endogenous L -fucose and lactose metabolism, produce 2′-fucosyllactose (2′-FL), and to liberate L -fucose from 2′-FL. This E. coli strain produced 16.7 g/L of L -fucose with productivity of 0.1 g·L⁻¹·h⁻¹ in a fed-batch fermentation. This study presents an efficient one-pot biosynthesis strategy to produce a monomeric form of L -fucose by microbial fermentation, making large-scale industrial production of L -fucose feasible.     

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.     

Butanol production from glycerol by recombinant Escherichia coli

Escherichia coli MG1655 (DE3) with the ability to synthesize butanol from glycerol was constructed by metabolic engineering. The genes thil, adhe2, bcs operon (crt, bcd, etfB, etfA, and hbd) were cloned into the plasmid vectors, pETDuet-1 and pACYCDuet-1, then the two resulting plasmids, pACYC-thl-bcs and pET-adhe2, were transferred to E. coli, and the recombinant strain was able to synthesize up to 18.5 mg/L butanol on a glycerol-containing medium. After the glycerol transport protein gene GlpF was expressed, the butanol production was improved to 22.7 mg/L. The competing pathway of byproducts, such as ethanol, succinate, and lactate, was subsequently deleted to improve the 1-butanol production to 97.9 mg/L. Moreover, a NADH regeneration system was introduced into the E. coli, and finally a 154.0 mg/L butanol titer was achieved in a laboratory-scale shake-flask experiment.     

Extracellular production of cloned alpha-amylase by Escherichia coli

Overexpression of Bacillus stearothermophilus gene coding for thermostable alpha-amylase in Escherichia coli was shown to cause outer-membrane damage leading to extracellular location of periplasmic proteins. Prolonged high expression of the alpha-amylase gene under lacZpo control eventually also lysed cells. Surprisingly, expression controlled by the pL promoter of phage lambda allowed specific release of periplasmic proteins into the growth medium without total cell lysis. Accumulation of alpha-amylase in the growth medium continued for at least 24 h under lambda pL control, whereas beta-lactamase activity ceased to increase beyond the exponential growth phase. The extent of outer membrane damage caused by alpha-amylase expression was monitored by following growth kinetics in the presence of lysozyme and by electron microscopy of the cells. Supplementing growth medium with Mg2+ restored the normal growth kinetics. It is suggested that periplasmic protein release caused by alpha-amylase overexpression is a stress response of the cell. A role for induced autolytic activity of the cell as a final effector of protein release is also proposed.     

Succinate production in Escherichia coli

Succinate has been recognized as an important platform chemical that can be produced from biomass. While a number of organisms are capable of succinate production naturally, this review focuses on the engineering of Escherichia coli for the production of four-carbon dicarboxylic acid. Important features of a succinate production system are to achieve an optimal balance of reducing equivalents generated by consumption of the feedstock, while maximizing the amount of carbon channeled into the product. Aerobic and anaerobic production strains have been developed and applied to production from glucose and other abundant carbon sources. Metabolic engineering methods and strain evolution have been used and supplemented by the recent application of systems biology and in silico modeling tools to construct optimal production strains. The metabolic capacity of the production strain, the requirement for efficient recovery of succinate, and the reliability of the performance under scaleup are important in the overall process. The costs of the overall biorefinery-compatible process will determine the economic commercialization of succinate and its impact in larger chemical markets.     

The production of D-acetoin by a transgenic Escherichia coli

A 6 kbp SphI fragment encoding genes for the enzymes α-acetolactate synthase (ALS), α-acetolactate decarboxylase (ALDC) and meso -2,3-butanediol dehydrogenase ( meso -BDH), involved in the formation of meso -2,3-butanediol ( meso -BD) from pyruvic acid, was cloned into plasmid pUC118. When derivatives of this plasmid were introduced into Escherichia coli JM109, the transformants were able to produce D -acetoin ( D -AC) without contamination with L -acetoin ( L -AC).     

Improved glutathione production by gene expression in Escherichia coli

AIMS: To improve glutathione (GSH) production in Escherichia coli by different genetic constructions containing GSH genes. METHODS AND RESULTS: GSH production was very low in E. coli by the expression of gshI gene. An increase of GSH production was achieved by the expression of both gshI and gshII genes in E. coli. A higher GSH production, namely 34.8 mg g(-1) wet cell weight, was obtained by simultaneous expression of two copies of gshI gene and one copy of gshII gene. CONCLUSIONS: The simultaneous expression of two copies of gshI gene and one copy of gshII gene resulted in a significant increase in GSH production. SIGNIFICANCE AND IMPACT OF THE STUDY: The expression strategy for GSH production described here can be used to increase gene expression and obtain high production rates in other multienzyme reaction systems.     

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。     

Reduction of aerobic acetate production by Escherichia coli.

Acetate excretion by Escherichia coli during aerobic growth on glucose is a major obstacle to enhanced recombinant protein production. We report here that the fraction of carbon flux through the anaplerotic pathways is one of the factors influencing acetate excretion. Flux analysis of E. coli central metabolic pathways predicts that increasing the fraction of carbon flux through the phosphoenolpyruvate carboxylase (PPC) pathway and the glyoxylate bypass reduces acetate production. We tested this prediction by overexpressing PPC and deregulating the glyoxylate bypass by using a fadR strain. Results show that the acetate yield by the fadR strain with PPC overexpression is decreased more than fourfold compared to the control, while the biomass yield is relatively unaffected. Apparently, the fraction of carbon flux through the anaplerotic pathways is one of the factors that influence acetate excretion. These results confirm the prediction of our flux analysis and further suggest that E. coli is not fully optimized for efficient utilization of glucose.     

A method of enhancing verocytotoxin production by Escherichia coli

The number of verocytotoxin producing Escherichia coli (VTEC) present in the faeces during an infection may be very low, making their detection difficult. We report a method for enhancing toxin production by VTEC using mitomycin C as an inducing agent with the aim of improving the detection of VTEC. In pure culture, mitomycin C enhanced toxin production up to 100-fold. When applied to mixed faecal culture, toxin could be detected in mitomycin C treated samples when standard cultures were negative and when substantially fewer verocytotoxin-producing bacteria were present. Use of this method may aid in the detection of VTEC and is appropriate for use in the routine diagnostic laboratory.     

Beta-lactamase production by Escherichia coli under different growth conditions

Facultative anaerobic bacteria, such as Escherichia coli, are more resistant to cephalosporin antibiotics during anaerobic growth. Strict anaerobic ambience reduces beta-lactamase production or the enzyme affinities for their substrates. A different balance between DNA gyrase and topoisomerase I activity, during aerobic and anaerobic growth condition, could be related to the bacteria behavior.     

One-step production of D-p-hydroxyphenylglycine by recombinant Escherichia coli strains

The gene encoding D-hydantoinase from Agrobacterium radiobacter NRRL B11291 was successfully cloned by use of polymerase chain reaction. A positive clone was scored, and its nucleotide sequence was further analyzed. The analysis by deleting various lengths of nucleotides from the amino terminus of the open reading frame revealed the putative regions for promoter and RBS site. By highly expressing both D-hydantoinase and carbamoylase, recombinant Escherichia coli strains were able to convert DL-hydroxyphenyl hydantoin (DL-HPH) to D-p-hydroxyphenylglycine (D-HPG) with a conversion yield of 97%, accounting for productivity 5 times higher than that obtained by A. radiobacter NRRL B11291. Immobilizing the recombinant cells with kappa-carrageenan could also achieve a conversion of 93%, while A. radiobacter NRRL B11291 attained 20% within the same period of reaction time. These results illustrate the feasibility in employing recombinant E. coli to accomplish one-step conversion of DL-HPH to D-HPG. In the process of improving D-HPG production, D-hydantoinase activity was increased 2.57-fold but carbamoylase activity remained constant, which resulted in only a 30% increase in the reaction rate. It suggests that carbamoylase is the step setting the pace of the reaction. Since the reaction substrate is highly insoluble, achieving sufficient agitation appears to be an important issue in this heterogeneous system. This view is further supported by the study on repeated use of cells, which shows that to reach a conversion of more than 90% free cells can be recycled six times, whereas immobilized cells can be used only twice. In conclusion, the poor reusability of immobilized cells is due to the fouling on the gel surface.     

Re-engineering Escherichia coli for ethanol production

A lactate producing derivative of Escherichia coli KO11, strain SZ110, was re-engineered for ethanol production by deleting genes encoding all fermentative routes for NADH and randomly inserting a promoterless mini-Tn5 cassette (transpososome) containing the complete Zymomonas mobilis ethanol pathway (pdc, adhA, and adhB) into the chromosome. By selecting for fermentative growth in mineral salts medium containing xylose, a highly productive strain was isolated in which the ethanol cassette had been integrated behind the rrlE promoter, designated strain LY160 (KO11, Δfrd::celY _Ec ΔadhE ΔldhA, ΔackA lacA::casAB _Ko rrlE::(pdc _Zm -adhA _Zm -adhB _Zm -FRT-rrlE) pflB ⁺ ). This strain fermented 9% (w/v) xylose to 4% (w/v) ethanol in 48 h in mineral salts medium, nearly equal to the performance of KO11 with Luria broth.     

Recombinant protein production in Escherichia coli

Growing needs for efficient recombinant production pose new challenges; starting from cell growth optimization under overexpression conditions, improving vectors, gene and protein sequence to suit them to protein biosynthesis machinery of the host, through extending the knowledge of protein folding, fusion protein construction, and coexpression systems, to improvements in protein purification and renaturation technologies. Hitherto Escherichia coli is the most defined and the cheapest protein biosynthesis system. With its wealth of available mutants tested is the best suited to economically test new gene constructs and to scale up the recombinant protein production.     

Biochemical production capabilities of Escherichia coli

Microbial metabolism provides at mechanism for the conversion of substrates into useful biochemicals. Utilization of microbes in industrial processes requires a modification of their natural metabolism in order to increase the efficiency of the desired conversion. Redirection of metabolic fluxes forms the basis of the newly defined field of metabolic engineering. In this study we use a flux balance based approach to study the biosynthesis of the 20 amino acids and 4 nucleotides as biochemical products. These amino acids and nucleotides are primary products of biosynthesis as well as important industrial products and precursors for the production of other biochemicals. The biosynthetic reactions of the bacterium Escherichia coli have been formulated into a metabolic network, and growth has been defined as a balanced drain on the metabolite pools corresponding to the cellular composition. Theoretical limits on the conversion of glucose, glycerol, and acetate substrates to biomass as well as the biochemical products have been computed. The substrate that results in the maximal carbon conversion to a particular product is identified. Criteria have been developed to identify metabolic constraints in the optimal solutions. The constraints of stoichiometry, energy, and redox have been determined in the conversions of glucose, glycerol, and acetate substrates into the biochemicals. Flux distributions corresponding to the maximal production of the biochemicals are presented. The goals of metabolic engineering are the optimal redirection of fluxes from generating biomass toward producing the desired biochemical. Optimal biomass generation is shown to decrease in a piecewise linear manner with increasing product formation. In some cases, synergy is observed between biochemical production and growth, leading to an increased overall carbon conversion. Balanced growth and product formation are important in a bioprocess, particularly for nonsecreted products. (c) 1993 John Wiley & Sons, Inc.     

Polyhydroxyalkanoate production in recombinant Escherichia coli

Abstract The bacterial species Escherichia coli has proven to be a powerful tool in the molecular analysis of polyhydroxyalkanoate (PHA) biosynthesis. In addition, E. coli holds promise as a source for economical PHA production. Using this microorganism, clones have been developed in our laboratory which direct the synthesis of poly-β-hydroxybutyrate (PHB) to levels as high as 95% of the cell dry weight. These clones have been further enhanced by the addition of a genetically mediated lysis system that allows the PHB granules to be released gently and efficiently. This paper describes these developments, as well as the use of an E. coli strain to produce the copolymer poly-(3-hydroxybutyrate- co -3-hydroxyvalerate (PHB- co -3-).