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 fatty acyl-ACP reductase-dependent pathway to improve fatty alcohol production in Escherichia coli

Fatty alcohols are important components of surfactants and cosmetic products. The production of fatty alcohols from sustainable resources using microbial fermentation could reduce dependence on fossil fuels and greenhouse gas emission. However, the industrialization of this process has been hampered by the current low yield and productivity of this synthetic pathway. As a result of metabolic engineering strategies, an Escherichia coli mutant containing Synechococcus elongatus fatty acyl-ACP reductase showed improved yield and productivity. Proteomics analysis and in vitro enzymatic assays showed that endogenous E. coli AdhP is a major contributor to the reduction of fatty aldehydes to fatty alcohols. Both in vitro and in vivo results clearly demonstrated that the activity and expression level of fatty acyl-CoA/ACP reductase is the rate-limiting step in the current protocol. In 2.5-L fed-batch fermentation with glycerol as the only carbon source, the most productive E. coli mutant produced 0.75 g/L fatty alcohols (0.02 g fatty alcohol/g glycerol) with a productivity of up to 0.06 g/L/h. This investigation establishes a promising synthetic pathway for industrial microbial production of fatty alcohols.     

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.     

Development of an orthogonal fatty acid biosynthesis system in E. coli for oleochemical production

Here we report recombinant expression and activity of several type I fatty acid synthases that can function in parallel with the native Escherichia coli fatty acid synthase. Corynebacterium glutamicum FAS1A was the most active in E. coli and this fatty acid synthase was leveraged to produce oleochemicals including fatty alcohols and methyl ketones. Coexpression of FAS1A with the ACP/CoA-reductase Maqu2220 from Marinobacter aquaeolei shifted the chain length distribution of fatty alcohols produced. Coexpression of FAS1A with FadM, FadB, and an acyl-CoA-oxidase from Micrococcus luteus resulted in the production of methyl ketones, although at a lower level than cells using the native FAS. This work, to our knowledge, is the first example of in vivo function of a heterologous fatty acid synthase in E. coli. Using FAS1 enzymes for oleochemical production have several potential advantages, and further optimization of this system could lead to strains with more efficient conversion to desired products. Finally, functional expression of these large enzyme complexes in E. coli will enable their study without culturing the native organisms.     

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。     

Improving the tolerance of Escherichia coli to medium-chain fatty acid production

Microbial fatty acids are an attractive source of precursors for a variety of renewable commodity chemicals such as alkanes, alcohols, and biofuels. Rerouting lipid biosynthesis into free fatty acid production can be toxic, however, due to alterations of membrane lipid composition. Here we find that membrane lipid composition can be altered by the direct incorporation of medium-chain fatty acids into lipids via the Aas pathway in cells expressing the medium-chain thioesterase from Umbellularia californica (BTE). We find that deletion of the aas gene and sequestering exported fatty acids reduces medium-chain fatty acid toxicity, partially restores normal lipid composition, and improves medium-chain fatty acid yields.     

Purification of fatty acid ethyl esters by solid-phase extraction and high-performance liquid chromatography

We have developed a two-step method to purify fatty acid ethyl esters (FAEE) using solid-phase extraction (SPE), with a recovery of 70±3% (mean±S.E.M.) as assessed using ethyl oleate as a recovery marker from a standard lipid mixture in hexane. The first step of the SPE procedure involves application of a lipid mixture to an aminopropyl-silica column with simultaneous elution of FAEE and cholesteryl esters from the column with hexane. Gas chromatographic analysis of FAEE without interference from cholesteryl esters may be performed using the eluate from the aminopropyl-silica column, thus eliminating the need for an octadecylsily (ODS) column in this case. The FAEE can then be separated from the cholesteryl esters, if necessary, by chromatography on an ODS column and elution with isopropanol-water (5:1, v/v). Both the aminopropyl-silica and ODS columns were found to be effective for up to four uses. To permit isolation of specific FAEE species following isolation of total FAEE by the two-step SPE method, we have also developed a purification scheme for individaal FAEE by high-performance liquid chromatography (HPLC). Thus, this simple method allows for reproducible isolation of total FAEE by SPE and isolation of individual FAEE species by HPLC.     

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

Benzoic acid (BA) is an important platform aromatic compound in chemical industry and is widely used as food preservatives in its salt forms. Yet, current manufacture of BA is dependent on petrochemical processes under harsh conditions. Here we report the de novo production of BA from glucose using metabolically engineered Escherichia coli strains harboring a plant-like β-oxidation pathway or a newly designed synthetic pathway. First, three different natural BA biosynthetic pathways originated from plants and one synthetically designed pathway were systemically assessed for BA production from glucose by in silico flux response analyses. The selected plant-like β-oxidation pathway and the synthetic pathway were separately established in E. coli by expressing the genes encoding the necessary enzymes and screened heterologous enzymes under optimal plasmid configurations. BA production was further optimized by applying several metabolic engineering strategies to the engineered E. coli strains harboring each metabolic pathway, which included enhancement of the precursor availability, removal of competitive reactions, transporter engineering, and reduction of byproduct formation. Lastly, fed-batch fermentations of the final engineered strain harboring the β-oxidation pathway and the strain harboring the synthetic pathway were conducted, which resulted in the production of 2.37 ± 0.02 g/L and 181.0 ± 5.8 mg/L of BA from glucose, respectively; the former being the highest titer reported by microbial fermentation. The metabolic engineering strategies developed here will be useful for the production of related aromatics of high industrial interest.     

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.     

Application of a lipase-immobilized silica monolith bioreactor to the production of fatty acid methyl esters

We developed a highly efficient bioreactor loaded with a lipase-immobilized non-shrinkable silica monolith by adopting a two-step sol–gel method, i.e., preparing a methyltrimethoxysilane (MTMS)-based silica monolith followed by coating of the latter with more hydrophobic alkyl-substituted silicates that entrapped lipase. We applied this type of bioreactor to the production of fatty acid methyl esters through methanolysis of rapeseed oil in n-hexane by Rhizopus oryzae lipase. As the result of screening alkyltrimethoxysilanes mixed with tetramethoxysilane (TMOS) during sol–gel coating, propyltrimethoxysilane (PTMS) gave the best performance, and the lipase immobilized therein exhibited ca. 10 times higher activity than non-immobilized lipase powder. The amount of the PTMS-based silicates with which the MTMS-based silica monolith was coated was optimized by adjusting the molar ratio of silanes (mixture of PTMS and TMOS at 4:1) to 100 mol of water. Conversion rate was highest at the molar ratio of 0.4 mol silanes to 100 mol of water, which was ca. 10 times higher than that with lipase deposited on the MTMS-based silica monolith. Conversion rate was approximately 1.5 times higher in the flow-through monolith bioreactor than in the batch slurry reactor.     

内消旋-二氨基庚二酸脱氢酶关键氨基酸位点的改造提高对烷基取代2-酮酸的催化活力

【背景】高效实现D-氨基酸的生物合成一直是人们关注的热点。内消旋-二氨基庚二酸脱氢酶(meso-diaminopimelate dehydrogenase,DAPDH)能够直接催化2-酮酸和氨合成D-氨基酸。【目的】提高DAPDH对烷基取代2-酮酸的催化活力,并解释其催化机制。【方法】以来源于嗜热共生杆菌(Symbiobacteriumthermophilum)的内消旋-二氨基庚二酸脱氢酶(StDAPDH)为模板,在前期结构分析结合被选择位点突变结果的基础上,确定对H227位进行定点饱和突变,并以D-丙氨酸、D-2-氨基丁酸、D-正缬氨酸、D-谷氨酸为底物进行筛选。【结果】获得突变体H227Q和H227N。突变体H227Q对丙酮酸、2-氧代丁酸、2-氧代戊酸、2-酮戊二酸的比活力比野生型分别提高了10.9、11.5、8.6和7.6倍。动力学参数表明,突变体H227Q同时提高了酶对底物的亲和力及催化常数,使其对丙酮酸的催化效率(k_(cat)/K_m)相较于野生型提高了9.4倍。利用分子模拟技术分析突变体H227Q与产物氨基酸之间的相互作用表明,227位的谷氨酰胺通过与氨基酸的羧酸形成氢键,使得氨基酸产物Cα上的氢和辅酶烟酰胺环C4原子之间的距离缩短。【结论】利用定向进化技术提高DAPDH对烷基取代2-酮酸的催化活力,有助于开发新型的高效生物催化剂,这些工作也为下一步继续进行更具挑战性的D-氨基酸研究提供了基础。     

Fatty acids, alcohol and fatty acid ethyl esters: Toxic Ca signal generation and pancreatitis

Pancreatitis, a potentially fatal disease in which the pancreas digests itself as well as its surroundings, is a well recognized complication of hyperlipidemia. Fatty acids have toxic effects on pancreatic acinar cells and these are mediated by large sustained elevations of the cytosolic Ca²⁺ concentration. An important component of the effect of fatty acids is due to inhibition of mitochondrial function and subsequent ATP depletion, which reduces the operation of Ca²⁺-activated ATPases in both the endoplasmic reticulum and the plasma membrane. One of the main causes of pancreatitis is alcohol abuse. Whereas the effects of even high alcohol concentrations on isolated pancreatic acinar cells are variable and often small, fatty acid ethyl esters – synthesized by combination of alcohol and fatty acids – consistently evoke major Ca²⁺ release from intracellular stores, subsequently opening Ca²⁺ entry channels in the plasma membrane. The crucial trigger for pancreatic autodigestion is intracellular trypsin activation. Although there is still uncertainty about the exact molecular mechanism by which this Ca²⁺-dependent process occurs, progress has been made in identifying a subcellular compartment – namely acid post-exocytotic endocytic vacuoles – in which this activation takes place.     

代谢工程改造野生耐酸酵母生产L-乳酸

以选育低pH条件下高产L-乳酸的酵母菌为目的,从自然样品中筛选分离得到一株能在pH 2.5 (乳酸调节) 的培养基中生长且不利用乳酸的酵母 (初步鉴定为木兰假丝酵母Candida magnolia);进一步将来源于米根霉As3.819的乳酸脱氢酶编码基因 (ldhA) 插入含有G418抗性基因的酵母穿梭载体,构建了重组质粒pYX212-kanMX-ldhA,电转化入野生型C. magnolia中,筛选获得了一株具有产L-乳酸能力的重组菌株C. magnolia-2;通过发酵实验表明,该重组菌产L-乳酸的最     

Metabolic evolution of two reducing equivalent-conserving pathways for high-yield succinate production in Escherichia coli

Reducing equivalents are an important cofactor for efficient synthesis of target products. During metabolic evolution to improve succinate production in Escherichia coli strains, two reducing equivalent-conserving pathways were activated to increase succinate yield. The sensitivity of pyruvate dehydrogenase to NADH inhibition was eliminated by three nucleotide mutations in the lpdA gene. Pyruvate dehydrogenase activity increased under anaerobic conditions, which provided additional NADH. The pentose phosphate pathway and transhydrogenase were activated by increased activities of transketolase and soluble transhydrogenase SthA. These data suggest that more carbon flux went through the pentose phosphate pathway, thus leading to production of more reducing equivalent in the form of NADPH, which was then converted to NADH through soluble transhydrogenase for succinate production. Reverse metabolic engineering was further performed in a parent strain, which was not metabolically evolved, to verify the effects of activating these two reducing equivalent-conserving pathways for improving succinate yield. Activating pyruvate dehydrogenase increased succinate yield from 1.12 to 1.31 mol/mol, whereas activating the pentose phosphate pathway and transhydrogenase increased succinate yield from 1.12 to 1.33 mol/mol. Activating these two pathways in combination led to a succinate yield of 1.5 mol/mol (88% of theoretical maximum), suggesting that they exhibited a synergistic effect for improving succinate yield.     

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.