Metabolic engineering of Escherichia coli for efficient free fatty acid production from glycerol

Crude glycerol, generated as waste by-product in biodiesel production process, has been considered as an important carbon source for converting to value-added bioproducts recently. Free fatty acids (FFAs) can be used as precursors for the production of biofuels or biochemicals. Microbial biosynthesis of FFAs can be achieved by introducing an acyl–acyl carrier protein thioesterase into Escherichia coli. In this study, the effect of metabolic manipulation of FFAs synthesis cycle, host genetic background and cofactor engineering on FFAs production using glycerol as feed stocks was investigated. The highest concentration of FFAs produced by the engineered stain reached 4.82 g/L with the yield of 29.55% (g FFAs/g glycerol), about 83% of the maximum theoretical pathway value by the type II fatty acid synthesis pathway. In addition, crude glycerol from biodiesel plant was also used as feedstock in this study. The FFA production was 3.53 g/L with a yield of 24.13%. The yield dropped slightly when crude glycerol was used as a carbon source instead of pure glycerol, while it still can reach about 68% of the maximum theoretical pathway yield.     

Metabolic engineering of Escherichia coli W3110 for efficient production of homoserine from glucose

Efficient microbial cell factory for the production of homoserine from glucose has been developed by iterative and rational engineering of Escherichia coli W3110. The whole pathway from glucose to homoserine was divided into three groups, namely, glucose transport and glycolysis (‘up-stream’), TCA and glyoxylate cycles (‘mid-stream’), and homoserine module (conversion of aspartate to homoserine and its secretion; ‘down-stream’), and the carbon flux in each group as well as between the groups were accelerated and balanced. Altogether, ∼18 genes were modified for active and consistent production of homoserine during both the actively-growing and non-growing stages of cultivation. Finally, fed-batch, two-stage bioreactor experiments, separating the growth from the production stage, were conducted for 61 h, which gave the high titer of 110.8 g/L, yield of 0.64 g/g glucose and volumetric productivity of 1.82 g/L/h, with an insignificant amount of acetate (<0.5 g/L) as the only noticeable byproduct. The metabolic engineering strategy employed in this study should be applicable for the biosynthesis of other amino acids or chemicals derived from aspartic acid.     

Metabolic engineering of Escherichia coli for production of salvianic acid A via an artificial biosynthetic pathway

Salvianic acid A, a valuable derivative from L-tyrosine biosynthetic pathway of the herbal plant Salvia miltiorrhiza, is well known for its antioxidant activities and efficacious therapeutic potential on cardiovascular diseases. Salvianic acid A was traditionally isolated from plant root or synthesized by chemical methods, both of which had low efficiency. Herein, we developed an unprecedented artificial biosynthetic pathway of salvianic acid A in E. coli, enabling its production from glucose directly. In this pathway, 4-hydroxyphenylpyruvate was converted to salvianic acid A via D-lactate dehydrogenase (encoding by d-ldh from Lactobacillus pentosus) and hydroxylase complex (encoding by hpaBC from E. coli). Furthermore, we optimized the pathway by a modular engineering approach and deleting genes involved in the regulatory and competing pathways. The metabolically engineered E. coli strain achieved high productivity of salvianic acid A (7.1 g/L) with a yield of 0.47 mol/mol glucose.     

Metabolic engineering of Escherichia coli for the production of fumaric acid

Fumaric acid is a naturally occurring organic acid that is an intermediate of the tricarboxylic acid cycle. Fungal species belonging to Rhizopus have traditionally been employed for the production of fumaric acid. In this study, Escherichia coli was metabolically engineered for the production of fumaric acid under aerobic condition. For the aerobic production of fumaric acid, the iclR gene was deleted to redirect the carbon flux through the glyoxylate shunt. In addition, the fumA, fumB, and fumC genes were also deleted to enhance fumaric acid formation. The resulting strain was able to produce 1.45 g/L of fumaric acid from 15 g/L of glucose in flask culture. Based on in silico flux response analysis, this base strain was further engineered by plasmid‐based overexpression of the native ppc gene, encoding phosphoenolpyruvate carboxylase (PPC), from the strong tac promoter, which resulted in the production of 4.09 g/L of fumaric acid. Additionally, the arcA and ptsG genes were deleted to reinforce the oxidative TCA cycle flux, and the aspA gene was deleted to block the conversion of fumaric acid into L ‐aspartic acid. Since it is desirable to avoid the use of inducer, the lacI gene was also deleted. To increase glucose uptake rate and fumaric acid productivity, the native promoter of the galP gene was replaced with the strong trc promoter. Fed‐batch culture of the final strain CWF812 allowed production of 28.2 g/L fumaric acid in 63 h with the overall yield and productivity of 0.389 g fumaric acid/g glucose and 0.448 g/L/h, respectively. This study demonstrates the possibility for the efficient production of fumaric acid by metabolically engineered E. coli. Biotechnol. Bioeng. 2013; 110: 2025–2034. © 2013 Wiley Periodicals, Inc.     

Metabolic engineering of Clostridium tyrobutyricum for enhanced butyric acid production from glucose and xylose

Clostridium tyrobutyricum is a promising microorganism for butyric acid production. However, its ability to utilize xylose, the second most abundant sugar found in lignocellulosic biomass, is severely impaired by glucose-mediated carbon catabolite repression (CCR). In this study, CCR in C. tyrobutyricum was eliminated by overexpressing three heterologous xylose catabolism genes (xylT, xylA and xlyB) cloned from C. acetobutylicum. Compared to the parental strain, the engineered strain Ct-pTBA produced more butyric acid (37.8 g/L vs. 19.4 g/L) from glucose and xylose simultaneously, at a higher xylose utilization rate (1.28 g/L·h vs. 0.16 g/L·h) and efficiency (94.3% vs. 13.8%), resulting in a higher butyrate productivity (0.53 g/L·h vs. 0.26 g/L·h) and yield (0.32 g/g vs. 0.28 g/g). When the initial total sugar concentration was ~120 g/L, both glucose and xylose utilization rates increased with increasing their respective concentration or ratio in the co-substrates but the total sugar utilization rate remained almost unchanged in the fermentation at pH 6.0. Decreasing the pH to 5.0 significantly decreased sugar utilization rates and butyrate productivity, but the effect was more pronounced for xylose than glucose. The addition of benzyl viologen (BV) as an artificial electron carrier facilitated the re-assimilation of acetate and increased butyrate production to a final titer of 46.4 g/L, yield of 0.43 g/g sugar consumed, productivity of 0.87 g/L·h, and acid purity of 98.3% in free-cell batch fermentation, which were the highest ever reported for butyric acid fermentation. The engineered strain with BV addition thus can provide an economical process for butyric acid production from lignocellulosic biomass.     

Metabolic engineering of Corynebacterium glutamicum for the production of 3-hydroxypropionic acid from glucose and xylose

3-Hydroxypropionic acid (3-HP) is a promising platform chemical which can be used for the production of various value-added chemicals. In this study,Corynebacterium glutamicum was metabolically engineered to efficiently produce 3-HP from glucose and xylose via the glycerol pathway. A functional 3-HP synthesis pathway was engineered through a combination of genes involved in glycerol synthesis (fusion of gpd and gpp from Saccharomyces cerevisiae) and 3-HP production (pduCDEGH from Klebsiella pneumoniae and aldehyde dehydrogenases from various resources). High 3-HP yield was achieved by screening of active aldehyde dehydrogenases and by minimizing byproduct synthesis (gapA^A1GΔldhAΔpta-ackAΔpoxBΔglpK). Substitution of phosphoenolpyruvate-dependent glucose uptake system (PTS) by inositol permeases (iolT1) and glucokinase (glk) further increased 3-HP production to 38.6 g/L, with the yield of 0.48 g/g glucose. To broaden its substrate spectrum, the engineered strain was modified to incorporate the pentose transport gene araE and xylose catabolic gene xylAB, allowing for the simultaneous utilization of glucose and xylose. Combination of these genetic manipulations resulted in an engineered C. glutamicum strain capable of producing 62.6 g/L 3-HP at a yield of 0.51 g/g glucose in fed-batch fermentation. To the best of our knowledge, this is the highest titer and yield of 3-HP from sugar. This is also the first report for the production of 3-HP from xylose, opening the way toward 3-HP production from abundant lignocellulosic feedstocks.     

Metabolic engineering of Escherichia coli for the production of 1,2-propanediol from glycerol

Due to its availability, low‐price, and high degree of reduction, glycerol has become an attractive carbon source for the production of fuels and reduced chemicals. Using the platform we have established from the identification of key pathways mediating fermentative metabolism of glycerol, this work reports the engineering of Escherichia coli for the conversion of glycerol into 1,2‐propanediol (1,2‐PDO). A functional 1,2‐PDO pathway was engineered through a combination of overexpression of genes involved in its synthesis from the key intermediate dihydroxyacetone phosphate (DHAP) and the manipulation of the fermentative glycerol utilization pathway. The former included the overexpression of methylglyoxal synthase (mgsA), glycerol dehydrogenase (gldA), and aldehyde oxidoreductase (yqhD). Manipulation of the glycerol utilization pathway through the replacement of the native E. coli PEP‐dependent dihydroxyacetone kinase (DHAK) with an ATP‐dependent DHAK from C. freundii increased the availability of DHAP allowing for higher 1,2‐PDO production. Analysis of the major fermentative pathways indentified ethanol as a required co‐product while increases in 1,2‐PDO titer and yield were achieved through the disruption of the pathways for acetate and lactate production. Combination of these key metabolic manipulations resulted in an engineered E. coli strain capable of producing 5.6 g/L 1,2‐PDO, at a yield of 21.3% (w/w). This strain also performed well when crude glycerol, a by‐product of biodiesel production, was used as the substrate. The titer and yield achieved in this study were favorable to those obtained with the use of E. coli for the production of 1,2‐PDO from common sugars. Biotechnol. Bioeng. 2011; 108:867–879. © 2010 Wiley Periodicals, Inc.     

代谢工程改造大肠杆菌合成丙二酸

丙二酸是一种重要的有机二元羧酸,其应用价值遍及化工、医药、食品等领域。本文以大肠杆菌为底盘细胞,过表达了ppc、aspC、panD、pa0132、yneI和pyc基因,成功构建了丙二酸合成重组菌株大肠杆菌BL21(TPP)。该菌株在摇瓶发酵条件下,丙二酸产量达到0.61 g/L。在5 L发酵罐水平,采用间歇补料的方式丙二酸的积累量达3.32 g/L。本研究应用了融合蛋白技术,将ppc和aspC、pa0132和yneI分别进行融合表达,构建了工程菌BL21(SCR)。在摇瓶发酵水平,该菌株丙二酸的积累量达到了0.83 g/L,较出发菌株BL21(TPP)提高了36%。在5 L发酵罐中,工程菌BL21(SCR)的丙二酸产量最高达5.61 g/L,较出发菌株BL21(TPP)提高了69%。本研究实现了丙二酸在大肠杆菌中的生物合成,为构建丙二酸合成的细胞工厂提供了理论依据和技术基础,同时也对其他二元羧酸的生物合成具有启发和指导意义。     

Engineering Escherichia coli for efficient production of 5-aminolevulinic acid from glucose

5-Aminolevulinic acid (ALA) recently received much attention due to its potential applications in many fields. In this study, we developed a metabolic strategy to produce ALA directly from glucose in recombinant Escherichia coli via the C5 pathway. The expression of a mutated hemA gene, encoding a glutamyl-tRNA reductase from Salmonella arizona, significantly improved ALA production from 31.1 to 176 mg/L. Glutamate-1-semialdehyde aminotransferase from E. coli was found to have a synergistic effect with HemA^M from S. arizona on ALA production (2052 mg/L). In addition, we identified a threonine/homoserine exporter in E. coli, encoded by rhtA gene, which exported ALA due to its broad substrate specificity. The constructed E. coli DALA produced 4.13 g/L ALA in modified minimal medium from glucose without adding any other co-substrate or inhibitor. This strategy offered an attractive potential to metabolic production of ALA in E. coli.     

Metabolic engineering of Escherichia coli for the production of succinic acid from glucose

Bio-based succinate production from renewable resources has prospective economic and environmental benefits that caused heightened interest towards the study of succinate-producing microorganisms. The pathways of succinate formation have been well studied, and microorganisms that are capable of biomass convertion into the target substance (bacteria of the genera Actinobacillus, Anaerobiospirillum, and Mannheimia) have been isolated and characterized; however, the realization of economically feasible industrial processes using native producers still remains a challenge. Traditionally, the Escherichia coli bacterium has been used as a workhouse to develop new processes for the biosynthesis of many valuable chemicals due to the extensive knowledge of its metabolism, available genetic tools, and good growth characteristics, combined with low nutrient requirements. This review is focused on modern rational approaches to the construction of recombinant E. coli strains that efficiently produce succinic acid from glucose.     

Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production

Limonene is a valuable monoterpene used in the production of several commodity chemicals and medicinal compounds. Among them, perillyl alcohol (POH) is a promising anti-cancer agent that can be produced by hydroxylation of limonene. We engineered E. coli with a heterologous mevalonate pathway and limonene synthase for production of limonene followed by coupling with a cytochrome P450, which specifically hydroxylates limonene to produce POH. A strain containing all mevalonate pathway genes in a single plasmid produced limonene at titers over 400 mg/L from glucose, substantially higher than has been achieved in the past. Incorporation of a cytochrome P450 to hydroxylate limonene yielded approximately 100 mg/L of POH. Further metabolic engineering of the pathway and in situ product recovery using anion exchange resins would make this engineered E. coli a potential production platform for any valuable limonene derivative.     

Metabolic engineering of Escherichia coli for high production of 1,5-pentanediol via a cadaverine-derived pathway

1,5-Pentanediol (1,5-PDO) is a high value-added chemical which is widely used as a monomer in the polymer industry. There are no natural organisms that could directly produce 1,5-PDO from renewable carbon sources. In this study, we report metabolic engineering of Escherichia coli for high-level production of 1,5-PDO from glucose via a cadaverine-derived pathway. In the newly proposed pathway, cadaverine can be converted to 1,5-PDO via 5-hydroxyvalerate (5-HV) by introducing only one heterologous enzyme in E. coli. Different endogenous genes of E. coli were screened and heterologous carboxylic acid reductase genes were tested to build a functional pathway. Compared to the previously reported pathways, the engineered cadaverine-based pathway has a higher theoretical yield (0.70 mol/mol glucose) and higher catalytic efficiency. By further combining strategies of pathway engineering and process engineering, we constructed an engineered E. coli strain that could produce 2.62 g/L 1,5-PDO in shake-flask and 9.25 g/L 1,5-PDO with a yield of 0.28 mol/mol glucose in fed-batch fermentation. The proposed new pathway and engineering strategies reported here should be useful for developing biological routes to produce 1,5-PDO for real application.     

Metabolic engineering of Escherichia coli for the production of polylactic acid and its copolymers

Polylactic acid (PLA) is a promising biomass‐derived polymer, but is currently synthesized by a two‐step process: fermentative production of lactic acid followed by chemical polymerization. Here we report production of PLA homopolymer and its copolymer, poly(3‐hydroxybutyrate‐co‐lactate), P(3HB‐co‐LA), by direct fermentation of metabolically engineered Escherichia coli. As shown in an accompanying paper, introduction of the heterologous metabolic pathways involving engineered propionate CoA‐transferase and polyhydroxyalkanoate (PHA) synthase for the efficient generation of lactyl‐CoA and incorporation of lactyl‐CoA into the polymer, respectively, allowed synthesis of PLA and P(3HB‐co‐LA) in E. coli, but at relatively low efficiency. In this study, the metabolic pathways of E. coli were further engineered by knocking out the ackA, ppc, and adhE genes and by replacing the promoters of the ldhA and acs genes with the trc promoter based on in silico genome‐scale metabolic flux analysis in addition to rational approach. Using this engineered strain, PLA homopolymer could be produced up to 11 wt% from glucose. Also, P(3HB‐co‐LA) copolymers containing 55–86 mol% lactate could be produced up to 56 wt% from glucose and 3HB. P(3HB‐co‐LA) copolymers containing up to 70 mol% lactate could be produced to 46 wt% from glucose alone by introducing the Cupriavidus necator β‐ketothiolase and acetoacetyl‐CoA reductase genes. Thus, the strategy of combined metabolic engineering and enzyme engineering allowed efficient bio‐based one‐step production of PLA and its copolymers. This strategy should be generally useful for developing other engineered organisms capable of producing various unnatural polymers by direct fermentation from renewable resources. Biotechnol. Bioeng. 2010; 105: 161–171. © 2009 Wiley Periodicals, Inc.     

代谢工程改造大肠杆菌提高乙醇酸产率

乙醇酸(Glycolate)是一种在工业上有多种用途的重要化合物。本研究首先在大肠杆菌MG1655(DE3)中敲除了ldh A(乳酸脱氢酶),获得菌株Mgly1,作为出发菌株。然后通过调节乙醇酸合成途径的关键酶——异柠檬酸裂解酶(ace A)、乙醛酸还原酶(ycd W)、异柠檬酸脱氢酶激酶/磷酸化酶(ace K)的表达水平,得到乙醇酸产率为0.24 g/g葡萄糖(占理论产率的28.2%)。过量表达柠檬酸合成酶(glt A),乙醇酸产率提高到0.326 g/g葡萄糖(占理论产率的38.3%)。然后在Mgly1中敲除了glc B和ace B(苹果酸合成酶),减少了乙醇酸合成的前体乙醛酸的消耗。最终获得的工程菌株Mgly335乙醇酸产率达到0.522 g/g葡萄糖(占理论产率的61.4%)。     

Metabolic engineering of Escherichia coli for quinolinic acid production by assembling L-aspartate oxidase and quinolinate synthase as an enzyme complex

Quinolinic acid (QA) is a key intermediate of nicotinic acid (Niacin) which is an essential human nutrient and widely used in food and pharmaceutical industries. In this study, a quinolinic acid producer was constructed by employing comprehensive engineering strategies. Firstly, the quinolinic acid production was improved by deactivation of NadC (to block the consumption pathway), NadR (to eliminate the repression of L-aspartate oxidase and quinolinate synthase), and PtsG (to slow the glucose utilization rate and achieve a more balanced metabolism, and also to increase the availability of the precursor phosphoenolpyruvate). Further modifications to enhance quinolinic acid production were investigated by increasing the oxaloacetate pool through overproduction of phosphoenolpyruvate carboxylase and deactivation of acetate-producing pathway enzymes. Moreover, quinolinic acid production was accelerated by assembling NadB and NadA as an enzyme complex with the help of peptide-peptide interaction peptides RIAD and RIDD, which resulted in up to 3.7 g/L quinolinic acid being produced from 40 g/L glucose in shake-flask cultures. A quinolinic acid producer was constructed in this study, and these results lay a foundation for further engineering of microbial cell factories to efficiently produce quinolinic acid and subsequently convert this product to nicotinic acid for industrial applications.     

Co‐culture engineering for microbial biosynthesis of 3‐amino‐benzoic acid in Escherichia coli

3‐amino‐benzoic acid (3AB) is an important building block molecule for production of a wide range of important compounds such as natural products with various biological activities. In the present study, we established a microbial biosynthetic system for de novo 3AB production from the simple substrate glucose. First, the active 3AB biosynthetic pathway was reconstituted in the bacterium Escherichia coli, which resulted in the production of 1.5 mg/L 3AB. In an effort to improve the production, an E. coli‐E. coli co‐culture system was engineered to modularize the biosynthetic pathway between an upstream strain and an downstream strain. Specifically, the upstream biosynthetic module was contained in a fixed E. coli strain, whereas a series of E. coli strains were engineered to accommodate the downstream biosynthetic module and screened for optimal production performance. The best co‐culture system was found to improve 3AB production by 15 fold, compared to the mono‐culture approach. Further engineering of the co‐culture system resulted in biosynthesis of 48 mg/L 3AB. Our results demonstrate co‐culture engineering can be a powerful new approach in the broad field of metabolic engineering.     

Systems engineering of Escherichia coli for n-butane production

Rising concerns about climate change and sustainable energy have attracted efforts towards developing environmentally friendly alternatives to fossil fuels. Biosynthesis of n-butane, a highly desirable petro-chemical, fuel additive and diluent in the oil industry, remains a challenge. In this work, we first engineered enzymes Tes, Car and AD in the termination module to improve the selectivity of n-butane biosynthesis, and ancestral reconstruction and a synthetic RBS significantly improved the AD abundance. Next, we did ribosome binding site (RBS) calculation to identify potential metabolic bottlenecks, and then mitigated the bottleneck with RBS engineering and precursor propionyl-CoA addition. Furthermore, we employed a model-assisted strain design and a nonrepetitive extra-long sgRNA arrays (ELSAs) and quorum sensing assisted CRISPRi to facilitate a dynamic two-stage fermentation. Through systems engineering, n-butane production was increased by 168-fold from 0.04 to 6.74 mg/L. Finally, the maximum n-butane production from acetate was predicted using parsimonious flux balance analysis (pFBA), and we achieved n-butane production from acetate produced by electrocatalytic CO reduction. Our findings pave the way for selectively producing n-butane from renewable carbon source.     

代谢工程方法改造大肠杆菌生产胸苷

胸苷是抗艾滋病药物司他夫定(3′-脱氧-2′,3′-双脱氢胸苷)和叠氮胸苷的重要前体物质。应用代谢工程方法对大肠杆菌Escherichia coli BL21(DE3)生物合成胸苷进行了研究。通过敲除E.coli BL21嘧啶回补途径的deo A、tdk和udp三个基因,BS03工程菌株能够积累21.6 mg/L胸苷。为了增加合成胸苷前体物核糖-5-磷酸和NADPH的供给,进一步敲除pgi和pyr L使工程菌BS05胸苷的产量提高到90.5 mg/L。而通过过表达胸苷合成途径的ush A、thy A、dut、ndk、nrd A和nrd B六个基因,菌株BS08胸苷的产量能达到272 mg/L。通过分批补料发酵,BS08最终可以积累1 248.8 mg/L的胸苷。本研究结果表明经过代谢工程改造的E.coli BL21具有良好的胸苷合成能力和应用潜力。     

Metabolic engineering of plants for polyunsaturated fatty acid production

Very-long-chain polyunsaturated fatty acids (VLCPUFAs) have demonstrated health benefits. Currently, the main sources for these fatty acids are oils from fish and microbes. However, shrinking fish populations and the high cost of microbial oil extraction are making the economic sustainability of these sources questionable. Metabolic engineering of oilseed crops could provide a novel and sustainable source of VLCPUFAs. Recently, genes encoding desaturases and elongases from microbes have been identified and successfully expressed in oilseed plants. However, the levels of VLCPUFAs produced in transgenic plants expressing these genes are still much lower than those found in native microbes. This review assesses the recent progress and future perspectives in the metabolic engineering of PUFAs in plants.