Improvement of free fatty acid production in Escherichia coli using codon-optimized Streptococcus pyogenes acyl-ACP thioesterase

Fatty acyl–acyl carrier protein (ACP) thioesterase (acyl-ACP TE) from Streptococcus pyogenes (strain MGAS10270) was codon-optimized and expressed in Escherichia coli K-12 W3110 and Escherichia coli K-12 MG1655. By employing codon-optimized S. pyogenes acyl-ACP TE to improve the total free fatty acids (FFAs) and to tailor the composition of FFAs, high-specificity production of saturated fatty acids (C12, C14) and unsaturated fatty acids (C18:1 C18:2) was achieved in recombinants. E. coli SGJS41 and SGJS46 (codon-optimized acyl-ACP TE of S. pyogenes) demonstrated the highest intracellular total FFA content (339 mg/l vs 342 mg/l); in particular, the content of C12 and C14 FFAs was about 3–5 fold, and the content of C18:1 and C18:2 FFAs was about 8–42 fold higher than that in the control E. coli and E. coli JES1017 (original acyl-ACP TE of S. pyogenes).     

Enhancement of methane production from long chain fatty acid based effluents

Two anaerobic sludges previously loaded with oleate and palmitate accumulated 4570+/-257 and 5200+/-9 mgCOD-LCFAgVSS(-1), respectively. These sludges were incubated in batch assays and methane production was recorded after addition of 100-900 mg L(-1) of oleate and palmitate, respectively. The batch assays were conducted before and after allowing the depletion of the biomass-associated LCFA. The presence of biomass-associated LCFA decreased the capacity of both sludges to convert the added LCFA to methane. After the degradation of biomass-associated LCFA, the lag phases observed before the onset of methane production were significantly reduced, evidencing an increase in the tolerance of the acetotrophic methanogens to the presence of LCFA. In another experiment, three recurrent pulses were performed with a real dairy wastewater containing 3.6 gCOD L(-1), from which 53% was fat. Methane yields of 0.45+/-0.01, 0.88+/-0.02 and 1.29+/-0.08 gCOD-CH(4) gCOD(fed)(-1) were achieved in the first, second and third pulses, respectively, evidencing an increasing capacity of the sludge to convert substrate accumulated in previous additions.     

Identification of long chain specific aldehyde reductase and its use in enhanced fatty alcohol production in E. coli

Long chain fatty alcohols have wide application in chemical industries and transportation sector. There is no direct natural reservoir for long chain fatty alcohol production, thus many groups explored metabolic engineering approaches for its microbial production. Escherichia coli has been the major microbial platform for this effort, however, terminal endogenous enzyme responsible for converting fatty aldehydes of chain length C14-C18 to corresponding fatty alcohols is still been elusive. Through our in silico analysis we selected 35 endogenous enzymes of E. coli having potential of converting long chain fatty aldehydes to fatty alcohols and studied their role under in vivo condition. We found that deletion of ybbO gene, which encodes NADP⁺ dependent aldehyde reductase, led to >90% reduction in long chain fatty alcohol production. This feature was found to be strain transcending and reinstalling ybbO gene via plasmid retained the ability of mutant to produce long chain fatty alcohols. Enzyme kinetic study revealed that YbbO has wide substrate specificity ranging from C6 to C18 aldehyde, with maximum affinity and efficiency for C18 and C16 chain length aldehyde, respectively. Along with endogenous production of fatty aldehyde via optimized heterologous expression of cyanobaterial acyl-ACP reductase (AAR), YbbO overexpression resulted in 169 mg/L of long chain fatty alcohols. Further engineering involving modulation of fatty acid as well as of phospholipid biosynthesis pathway improved fatty alcohol production by 60%. Finally, the engineered strain produced 1989 mg/L of long chain fatty alcohol in bioreactor under fed-batch cultivation condition. Our study shows for the first time a predominant role of a single enzyme in production of long chain fatty alcohols from fatty aldehydes as well as of modulation of phospholipid pathway in increasing the fatty alcohol production.     

Fatty alcohol production in engineered E. coli expressing Marinobacter fatty acyl-CoA reductases

Although successful production of fatty alcohols in metabolically engineered Escherichia coli with heterologous expression of fatty acyl-CoA reductase has been reported, low biosynthetic efficiency is still a hurdle to be overcome. In this study, we examined the characteristics of two fatty acyl-CoA reductases encoded by Maqu_2220 and Maqu_2507 genes from Marinobacter aquaeolei VT8 on fatty alcohol production in E. coli. Fatty alcohols with diversified carbon chain length were obtained by co-expressing Maqu_2220 with different carbon chain length-specific acyl-ACP thioesterases. Both fatty acyl-CoA reductases displayed broad substrate specificities for C12–C18 fatty acyl chains in vivo. The optimized mutant strain of E. coli carrying the modified tesA gene and fadD gene from E. coli and Maqu_2220 gene from Marinobacter aquaeolei VT8 produced fatty alcohols at a remarkable level of 1.725 g/L under the fermentation condition.     

Large-scale production and homogenous purification of long chain polysialic acids from E. coli K1

The study of new biomaterials is the objective of many current research projects in biotechnological medicine. A promising scaffold material for the application in tissue engineering or other biomedical applications is polysialic acid (polySia), a homopolymer of alpha2,8-linked sialic acid residues, which represents a posttranslational modification of the neural cell adhesion molecule and occurs in all vertebrate species. Some neuroinvasive bacteria like, e.g. Escherichia coli K1 (E. coli K1) use polySia as capsular polysaccharide. In this latter case long polySia chains with a degree of polymerization of >200 are linked to lipid anchors. Since in vertebrates no polySia degrading enzymes exist, the molecule has a long half-life in the organism, but degradation can be induced by the use of endosialidases, bacteriophage-derived enzymes with pronounced specificity for polySia. In this work a biotechnological process for the production of bacterial polysialic acid is presented. The process includes the development of a multiple fed-batch cultivation of the E. coli K1 strain and a complete downstream strategy of polySia. A controlled feed of substrate at low concentrations resulted in an increase of the carbon yield (C(product)/C(substrate)) from 2.2 to 6.6%. The downstream process was optimized towards purification of long polySia chains. Using a series of adjusted precipitation steps an almost complete depletion of contaminating proteins was achieved. The whole process yielded 1-2g polySia from a 10-l bacterial culture with a purity of 95-99%. Further product analysis demonstrated maximum chain length of >130 for the final product.     

Photoaffinity labeling of fatty acid-binding proteins involved in long chain fatty acid transport in Escherichia coli.

The photoreactive fatty acid 11-m-diazirinophenoxy-[11-3H]undecanoate was shown to be taken up specifically by the fatty acid transport system expressed in Escherichia coli grown on oleate. This photoreactive fatty acid analogue was therefore used to identify proteins involved in fatty acid uptake in E. coli. The fadL protein was labeled by the probe, confirmed to be exclusively in the outer membrane and to exhibit the heat modifiable behavior typical of outer membrane proteins. The apparent pI of the incompletely denatured form of the protein having the mobility of a 33-kDa protein was 4.6 while that of the fully denatured form was consistent with the calculated value of 5.2. The denaturation was reversible depending upon the protein to detergent ratios. The photoreactive fatty acid partitions into the outer membrane, resulting in extensive photolabeling of the lipid; a high affinity fatty acid-binding site is not apparent in total membranes labeled using free fatty acids due to this large binding capacity of the outer membrane. However, when the free fatty acid concentration was controlled by supplying it as a bovine serum albumin complex, the fadL protein exhibited saturable high affinity fatty acid binding, having an apparent Kd for the probe of 63 nM. The methods described very readily identify fatty acid-binding proteins: the fact that even when the sensitivity was increased 500-fold, no evidence was found for the presence of a fatty acid-binding protein in the inner membrane is consistent with the proposal that fatty acid permeation across the plasma membrane is not protein mediated but occurs by a simple diffusive mechanism.     

Overproduction of free fatty acids in E. coli: implications for biodiesel production

Whereas microbial fermentation processes for producing ethanol and related alcohol biofuels are well established, biodiesel (methyl esters of fatty acids) is exclusively derived from plant oils. Slow cycle times for engineering oilseed metabolism and the excessive accumulation of glycerol as a byproduct are two major drawbacks of deriving biodiesel from plants. Although most bacteria produce fatty acids as cell envelope precursors, the biosynthesis of fatty acids is tightly regulated at multiple levels. By introducing four distinct genetic changes into the E. coli genome, we have engineered an efficient producer of fatty acids. Under fed-batch, defined media fermentation conditions, 2.5 g/L fatty acids were produced by this metabolically engineered E. coli strain, with a specific productivity of 0.024 g/h/g dry cell mass and a peak conversion efficiency of 4.8% of the carbon source into fatty acid products. At least 50% of the fatty acids produced were present in the free acid form.     

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.     

Synthesis of long chain acyl-enzyme thioesters by modified fatty acid synthetases and their hydrolysis by a mammary gland thioesterase.

The fatty acid synthetase multienzyme from lactating rat mammary gland was modified either by removal of the two thioesterase I domains with trypsin or by inhibiting the thioesterase I activity with phenylmethanesulfonyl fluoride. The modified multienzymes are able to convert acetyl-CoA, malonyl-CoA, and NADPH to long chain acyl moieties (C₁₆C₂₂), which are covalently bound to the enzyme through thioester linkage, but they are unable to release the acyl groups as free fatty acids. A single enzyme-bound, long chain acyl thioester is formed by each molecule of modified multienzyme. Kinetic studies showed that the modified multienzymes rapidly elongate the acetyl primer moiety to a C₁₆ thioester and that further elongation to C₁₈, C₂₀, and C₂₂ is progressively slower. Thioesterase II, a mammary gland enzyme which is not part of the fatty acid synthetase multienzyme, can release the acyl moiety from its thioester linkage to either modified multienzyme. Kinetic data are consistent with the formation of an enzyme—substrate complex between thioesterase II and the acylated modified multienzymes. The present study demonstrates that the ability of thioesterase II to modify the product specificity of normal fatty acid synthetase is most likely attributable to the capacity of thioesterase II for hydrolysis of acyl moieties from thioester linkage to the multienzyme.     

Metabolic engineering of omega3-very long chain polyunsaturated fatty acid production by an exclusively acyl-CoA-dependent pathway

omega3-Very long chain polyunsaturated fatty acids (VLCPUFA) are essential for human development and brain function and, thus, are indispensable components of the human diet. The current main source of VLCPUFAs is represented by ocean fish stocks, which are in severe decline, and the development of alternative, sustainable sources of VLCPUFAs is urgently required. Our research aims at exploiting the powerful infrastructure available for the large scale culture of oilseed crops, such as rapeseed, to produce VLCPUFAs such as eicosapentaenoic acid in transgenic plants. VLCPUFA biosynthesis requires repeated desaturation and repeated elongation of long chain fatty acid substrates. In previous experiments the production of eicosapentaenoic acid in transgenic plants was found to be limited by an unexpected bottleneck represented by the acyl exchange between the site of desaturation, endoplasmic reticulum-associated phospholipids, and the site of elongation, the cytosolic acyl-CoA pool. Here we report on the establishment of a coordinated, exclusively acyl-CoA-dependent pathway, which avoids the rate-limiting transesterification steps between the acyl lipids and the acyl-CoA pool during VLCPUFA biosynthesis. The pathway is defined by previously uncharacterized enzymes, encoded by cDNAs isolated from the microalga Mantoniella squamata. The conceptual enzymatic pathway was established and characterized first in yeast to provide proof-of-concept data for its feasibility and subsequently in seeds of Arabidopsis thaliana. The comparison of the acyl-CoA-dependent pathway with the known lipid-linked pathway for VLCPUFA biosynthesis showed that the acyl-CoA-dependent pathway circumvents the bottleneck of switching the Delta6-desaturated fatty acids between lipids and acyl-CoA in Arabidopsis seeds.     

Long chain fatty acid synthesis in developing castor bean seeds I. The operation of the path from acetate to long chain fatty acids in a subcellular particulate system

The path of LFA synthesis from acetate in developing castorbean seeds was associated with subcellular 10,000 g particles.Further fractionation of these particles by a stepwise densitygradient method showed the high possibility that the site ofLFA synthesis is the proplastid. A study on cofactor requirementswhen [1-¹⁴C]acetate predominantly incorporated into LFAs indicatedthat synthesis would be achieved by acetyl-CoA carboxylation,malonyl-ACP condensation. ATP, CoA, HCO₃^– and Mg⁺⁺ orMn⁺⁺ were essential for synthesis from acetate by the 10,000gparticulate system. Results of inhibhitor experiment suggestedthat the supply of ATP to the LFA synthesizing system is broughtabout by mitochondrial oxidative phosphorylation, when acetateis the sole precursor for LFA synthesis in this system. TheNADPH generating system was contained in the paticles, althoughthe addition of NADP⁺ and G-6-P increased synthesis. NADH markedlystimulated LFA synthesis from acetate. The primary role of NADHseems to be as a direct reductant in both steps involving thereduction and oxidative desaturation of fatty acid chains; particularly,in the former step, although NADH partially contributes to thesupply of ATP as a respiratory substrate. It is unlikely thatNADH works as a hydrogen donor to NADP⁺. LFA synthesis by thecastor bean particulate system was not stimulated by light,thus differing from that by leaf chloroplasts. (Received July 23, 1973; )     

Enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) via manipulating the fatty acid beta-oxidation pathway in E. coli

Acyl-CoA dehydrogenase gene (yafH) of Escherichia coli was expressed together with polyhydroxyalkanoate synthase gene (phaC(Ac)) and (R)-enoyl-CoA hydratase gene (phaJ(Ac)) from Aeromonas caviae. The expression plasmids were introduced into E. coli JM109, DH5 alpha and XL1-blue, respectively. Compared with the strains harboring only phaC(Ac) and phaJ(Ac), all recombinant E. coli strains harboring yafH, phaC(Ac) and phaJ(Ac) accumulated at least four times more poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx). Cell dry weights produced by all recombinants containing yafH were also considerably higher than that without yafH. The addition of acrylic acid which serves as inhibitor for beta-oxidation and may lead to more precursor supply for PHA synthesis did not result in improved PHBHHx production compared with that of the overexpression of yafH. It appeared that the overexpression of acyl-CoA dehydrogenase gene (yafH) enhanced the supply of enoyl-CoA which is the substrate of (R)-enoyl-CoA hydratase. With the enhanced precursor supply, the recombinants accumulated more PHBHHx.     

Metabolic flux analysis of Escherichia coli deficient in the acetate production pathway and expressing the Bacillus subtilis acetolactate synthase

Several approaches to reduce acetate accumulation in Escherichia coli cultures have recently been reported. This reduction subsequently led to a significant enhancement in recombinant protein production. In those studies, metabolically engineered E. coli strains with reduced acetate synthesis rates were constructed through the modification of glucose uptake rate, the elimination of critical enzymes that are involved in the acetate formation pathways, and the redirection of carbon flux toward less inhibitory byproducts. In particular, it has been shown that strains carrying the Bacillus subtilis acetolactate synthase (ALS) gene not only produce less acetate but also have a higher ATP yield. Metabolic flux analysis of carbon flux distribution of the central metabolic pathways and at the pyruvate branch point revealed that this strain has the ability to channel excess pyruvate to the much less toxic compound, acetoin. The main focus of this study is the systematic analysis of the effects of small perturbations in the host's existing pathways on the redistribution of carbon fluxes. Specifically, a mutant with deleted acetate kinase (ACK) and acetyl phosphotransferase (PTA) was constructed and studied. Results from the metabolic analysis of carbon redistribution show the ackA-pta mutation will reduce acetate level at the expense of the growth rate. In addition, in the ackA-pta deficient strain a much higher lactate formation rate with simultaneously lower formate and ethanol synthesis rates was found. Expression of the B. subtilis ALS in ackA-pta mutants further reduces acetate levels while cell density similar to that of the parent strain is attained.     

游离整体细胞法工业化生产L-天冬氨酸

利用游离整体细胞催化技术在工业规模上进行Ｌ－天冬氨酸的生产,发现游离整体细胞催化技术不仅缩短了工艺流程,减少了设备投资,而且反应效率（生产效率）、生产能力、生产成本等方面均优于传统的固定化细胞法。