嗜乙酰乙酸棒杆菌Corynebacterium acetoacidophilum-Δldh缺氧条件下代谢葡萄糖途径的变化

缺氧条件下嗜乙酰乙酸棒杆菌Corynebacterium acetoacidophilum ATCC13870生长停滞,却能够代谢葡萄糖产生以乳酸和琥珀酸为主的有机酸。采用以sacB基因为反向筛选标记的同源重组染色体基因敲除系统,敲除嗜乙酰乙酸棒杆菌的乳酸脱氢酶基因,得到的Δldh菌株CCTCC NO.M20122041在缺氧条件下不产乳酸,葡萄糖消耗速率降低了29.3%,产琥珀酸和乙酸浓度分别提高45.6%和182%;NADH/NAD+值小于1(约0.7);磷酸烯醇式丙酮酸羧化酶和乙酸激酶的比酶活分别提高84%和12倍。说明嗜乙酰乙酸棒杆菌中乳酸合成途径的阻断驱使了琥珀酸和乙酸代谢途径加强,推测加强NADH供给和阻断乙酸产生支路可能是提高C.acetoacidophilum菌株产琥珀酸产量的有效途径。     

比较pta及ack敲除对钝齿棒杆菌产L-精氨酸生理代谢的影响

旁支代谢途径的截断有利于目的氨基酸合成途径的集流。基于基因组尺度代谢网络模型的预测,以钝齿棒杆菌(Corynebacterium crenatum)MT-M4为出发菌株,通过无痕敲除技术分别敲除了编码磷酸乙酰基转移酶的pta基因及编码乙酸激酶的ack基因,阻断了乙酸的合成。摇瓶发酵结果表明,pta缺失菌株精氨酸产量较出发菌株提高了25.60%,达15.46g/L。葡萄糖转化率提高了29.41%;ack缺失菌株精氨酸产量达13.82g/L,较出发菌株提高了12.81%,葡萄糖转化率提高了26.02%。同时,pta及ack敲除菌株的细胞生长较出发菌株均分别提高了9.19%及7.71%。因此,pta、ack的敲除不仅有利于精氨酸的合成,而且对菌体生长具有促进作用;但pta的敲除更有利于精氨酸的积累。     

乙酸合成途径阻断及NADH氧化酶表达对于谷氨酸棒杆菌生产乙偶姻的影响

【目的】研究乙酸合成途径阻断及NADH氧化酶表达对于谷氨酸棒杆菌生产乙偶姻的影响。【方法】在谷氨酸棒杆菌CGF2中异源表达als SD操纵子构建乙偶姻生产菌株CGT1,考察敲除乙酸生成途径cat和pqo对乙偶姻的影响。然后引入短乳杆菌的NADH氧化酶,在优化的溶氧条件下研究其对乙偶姻产量的影响。【结果】CGT1在摇瓶发酵中可积累6.27 g/L乙偶姻,敲除cat使乙偶姻产量显著提高30.94%,达到8.21 g/L;双敲除cat和pqo没有进一步提高产量。通过优化发酵的溶氧水平,乙偶姻产量达到10.06 g/L。在高溶氧水平下引入NADH氧化酶导致菌株的生长和糖代谢速率提高,但乙偶姻产量略有降低。在分批补料发酵中,重组菌株乙偶姻产量达到40.51 g/L,产率为0.51 g/(L?h)。【结论】在谷氨酸棒杆菌中阻断乙酸合成途径cat能够有效提高乙偶姻产量,NADH氧化酶在高溶氧水平下表达不利于乙偶姻的合成,需要进一步调节表达水平以确定其效果。     

产琥珀酸重组解脂酵母发酵副产物乙酸的代谢控制

琥珀酸是一种高附加值的有机酸,广泛用于食品、化工和农药领域。解脂酵母Yarrowia lipolytica作为新型强健的非传统酵母,近年来逐渐吸引了研究者的注意。前期通过基因敲除琥珀酸脱氢酶基因构建了一株产琥珀酸的重组解脂酵母PGC01003。由于糖酵解和TCA循环流量不协调,PGC01003分泌大量副产物乙酸,限制了琥珀酸产量的进一步提高。为降低乙酸的溢出,实现自然低pH值发酵生产琥珀酸,首先干扰旁路代谢,异源表达来自鼠沙门氏菌的乙酰辅酶A合酶,乙酸的产量下降至4.6 g/L,比对照降低了24.6%。而基因敲除乙酰辅酶A水解酶基因得到的重组菌PGC11505,发酵96 h乙酸分泌量只有0.4 g/L,琥珀酸产量提高到7.0 g/L,琥珀酸的转化率为0.30 g/g,为进一步构建高产琥珀酸的细胞工厂奠定基础。     

3个植物源苯甲醇酰基转移酶合成乙酸肉桂酯的研究

[背景]乙酸肉桂酯是一种重要的香料化合物,在化妆品和食品工业上具有广泛的应用,传统的生产方法主要依靠植物提取和化学合成。[目的]通过筛选不同植物源的酰基转移酶,利用大肠杆菌从头合成乙酸肉桂酯。[方法]首先,通过在苯丙氨酸高产菌BPHE中表达异源基因苯丙氨酸解氨酶(Phenylalanine Ammonia-Lyase from Arabidopsis thaliana,AtPAL)、对羟基肉桂酰辅酶A连接酶(Hydroxycinnamate:CoA Ligase from Petroselinum crispum,Pc4CL)和肉桂酰辅酶 A 还原酶(Cinnamyl-CoA Reductase from Arabidopsis thaliana,AtCCR),并结合大肠杆菌自身的内源性醇脱氢酶(Alcohol Dehydrogenases,ADHs)或醛酮还原酶(Aldo-Keto Reductases,AKRs)的催化作用构建了从苯丙氨酸到肉桂醇的生物合成途径。然后,苯甲醇苯甲酰转移酶(Benzyl Alcohol O-Benzoyltransferase from Nicotiana tabacum,ANN09798;Benzyl Alcohol O-Benzoyltransferase from Clarkia breweri,ANN09796)或苯甲醇乙酰转移酶(Benzyl Alcohol Acetyltransferase from Clarkia breweri,BEAT)被引入到上述重组大肠杆菌中发酵培养生产乙酸肉桂酯。最后,在大肠杆菌中过表达乙酰辅酶A合成酶(Acetyl Coenzyme A Synthetase,ACS)来提高底物乙酰辅酶A的量。[结果]探讨了 3个植物源苯甲醇酰基转移酶生物合成乙酸肉桂酯的能力,并应用于合成乙酸肉桂酯的细胞工厂,最终使乙酸肉桂酯最高产量达到166.9±6.6mg/L。[结论]植物源苯甲醇酰基转移酶具有一定的底物宽泛性,能以肉桂醇为底物催化合成乙酸肉桂酯。首次利用植物源的苯甲醇酰基转移酶合成乙酸肉桂酯,为微生物细胞工厂以葡萄糖作为碳源生产乙酸肉桂酯提供参考。     

代谢工程改造枯草芽孢杆菌发酵生产乙偶姻

乙偶姻是枯草芽孢杆菌的主要代谢产物,它作为一种食用香精,广泛应用于食品、烟草、化妆品、清洁剂、酒类等行业。本研究首先在不产芽孢的枯草芽孢杆菌(BSD1,阻断了芽孢的合成途径)中敲除了2,3-丁二醇脱氢酶(BDH)的编码基因bdh A、乳酸脱氢酶(LDH)的编码基因ldh和乙酸激酶的编码基因(ACK)ack A,随后克隆了来自菌株B.subtilis168的α-乙酰乳酸合成酶(ALS)和α-乙酰乳酸脱羧酶(ALDC)基因als S和als D,并将其在上述敲除菌中过量表达,结果表明阻断副产物合成途径和加强乙偶姻合成途径关键酶的表达,会显著提高乙偶姻的产量,最终乙偶姻产量达到38.08 g/L,产率为0.45 g·L~(-1)·h~(-1),产率提高了约87.5%。     

产L-苹果酸重组大肠杆菌的构建

基于产琥珀酸重组大肠杆菌E.coli B0013-1050的琥珀酸合成途径,利用Red同源重组技术结合Xer/dif重组系统敲除富马酸酶基因fumB、fumC,苹果酸酶基因maeB,构建L-苹果酸合成途径,最终得到重组大肠杆菌E.coli2030,该菌株在15 L发酵罐中,产L-苹果酸12.5 g/L,葡萄糖-苹果酸转化率为52.1%,同时对发酵产物中主要杂酸丙酮酸和琥珀酸的生产原因进行了初步的探讨与分析。为进一步提高L-苹果酸的转化率,整合表达来源于黄曲霉的苹果酸脱氢酶基因,构建重组菌E.coli 2040,在15 L发酵罐中产L-苹果酸14 g/L,葡萄糖-苹果酸转化率提高到60.3%。     

枯草芽孢杆菌氧化还原感应全局调控因子Rex对乙偶姻合成的影响

【背景】枯草芽孢杆菌体内含有一种可响应胞内氧化还原水平的因子,称之为氧化还原感应全局调控因子Rex (由基因ydiH编码)。Rex可通过感知辅酶NADH/NAD+水平的变化来调节胞内氧化还原平衡。【目的】研究Rex对枯草芽孢杆菌乙偶姻合成和辅因子代谢的相关性。【方法】利用比较转录组挖掘乙偶姻和2,3-丁二醇可逆转化过程中显著差异的基因,并通过Cre/lox基因敲除技术敲除ydiH、acuA (乙酰AcsA)和acoC (二氢脂酰胺乙酰转移酶)。随后,利用实时荧光定量PCR (RT-qPCR)技术分析敲除菌株中乙偶姻相关基因的转录水平。【结果】通过发酵实验发现,敲除ydiH会在一定程度上抑制菌体的生长速率,但发酵前期乙偶姻单位细胞产量和底物转化率都得到了显著提高;敲除acuA和acoC后,对乙偶姻合成、菌体生长和糖耗速率均影响不大;敲除ydiH后,与乙偶姻合成相关基因alsR (alsSD的正转录调控因子)、alsS (α-乙酰乳酸合成酶)、alsD (α-乙酰乳酸脱羧酶)和bdhA (2,3-丁二醇脱氢酶)的转录水平显著上调。【结论】枯草芽孢杆菌氧化还原感应全局调控因子Rex通过抑制与乙偶姻相关基因的转录水平影响乙偶姻合成。本研究首次报道了枯草芽孢杆菌中Rex和乙偶姻合成的相关性,为探索Rex如何通过调控相关基因的转录来影响胞内氧化还原稳态奠定了基础,也为提高枯草芽孢杆菌工业化生产强度和底物转化率提供了借鉴。     

proC及putP基因的敲除对钝齿棒杆菌产L-精氨酸生理代谢的影响

为了选育精氨酸高产菌株,基于谷氨酸棒杆菌的基因组尺度代谢网络模型的指导,以钝齿棒杆菌(Corynebacterium crenatum)MT-M4为出发菌株,通过基因敲除技术构建了pro C和put P敲除菌株。摇瓶发酵结果表明,pro C敲除菌株精氨酸产量达到9.94g/L,较出发菌株提高了15.90%,葡萄糖转化率提高了26.02%。由于其生长受到明显抑制,因此在发酵液中外源添加24mmol/L的脯氨酸,结果发现其精氨酸产量达到12.22g/L,且菌株恢复生长。put P敲除菌株精氨酸产量达到12.23g/L,较出发菌株提高了42.70%,葡萄糖转化率提高了49.31%。以上结果显示,put P的敲除比pro C的敲除更有利于精氨酸的合成,put P的敲除对菌株的生理代谢基本无影响且无需外添加脯氨酸。     

肉毒碱乙酰转移酶基因敲除对长链二元酸生产代谢过程的影响

在利用热带假丝酵母发酵生产长链二元酸的过程中 ,脂肪酸将进入 β 氧化途径 ,代谢产生能量 ,从而降低产物收率。首次以负责运输的肉毒碱乙酰转移酶为改造目标 ,在肉毒碱乙酰转移酶基因中插入潮霉素B抗性基因 ,构建DNA转化质粒 ,并进行一次同源重组 ,得到肉毒碱乙酰转移酶基因单拷贝敲除的基因工程菌。根据摇瓶实验结果 ,该基因工程菌与原始菌株相比 ,十三碳二元酸的产量与摩尔转化率分别提高了 13 0 %和 11 8%。     

Eliminating side products and increasing succinate yields in engineered strains of Escherichia coli C

Derivatives of Escherichia coli C were previously described for succinate production by combining the deletion of genes that disrupt fermentation pathways for alternative products (ldhA::FRT, adhE::FRT, ackA::FRT, focA-pflB::FRT, mgsA, poxB) with growth-based selection for increased ATP production. The resulting strain, KJ073, produced 1.2 mol of succinate per mol glucose in mineral salts medium with acetate, malate, and pyruvate as significant co-products. KJ073 has been further improved by removing residual recombinase sites (FRT sites) from the chromosomal regions of gene deletion to create a strain devoid of foreign DNA, strain KJ091(DeltaldhA DeltaadhE DeltaackA DeltafocA-pflB DeltamgsA DeltapoxB). KJ091 was further engineered for improvements in succinate production. Deletion of the threonine decarboxylase (tdcD; acetate kinase homologue) and 2-ketobutyrate formate-lyase (tdcE; pyruvate formate-lyase homologue) reduced the acetate level by 50% and increased succinate yield (1.3 mol mol(-1) glucose) by almost 10% as compared to KJ091 and KJ073. Deletion of two genes involved in oxaloacetate metabolism, aspartate aminotransferase (aspC) and the NAD(+)-linked malic enzyme (sfcA) (KJ122) significantly increased succinate yield (1.5 mol mol(-1) glucose), succinate titer (700 mM), and average volumetric productivity (0.9 g L(-1) h(-1)). Residual pyruvate and acetate were substantially reduced by further deletion of pta encoding phosphotransacetylase to produce KJ134 (DeltaldhA DeltaadhE DeltafocA-pflB DeltamgsA DeltapoxB DeltatdcDE DeltacitF DeltaaspC DeltasfcA Deltapta-ackA). Strains KJ122 and KJ134 produced near theoretical yields of succinate during simple, anaerobic, batch fermentations using mineral salts medium. Both may be useful as biocatalysts for the commercial production of succinate.     

Catabolite regulation of the pta gene as part of carbon flow pathways in Bacillus subtilis

In Bacillus subtilis, the products of the pta and ackA genes, phosphotransacetylase and acetate kinase, play a crucial role in the production of acetate, one of the most abundant by-products of carbon metabolism in this gram-positive bacterium. Although these two enzymes are part of the same pathway, only mutants with inactivated ackA did not grow in the presence of glucose. Inactivation of pta had only a weak inhibitory effect on growth. In contrast to pta and ackA in Escherichia coli, the corresponding B. subtilis genes are not cotranscribed. Expression of the pta gene was increased in the presence of glucose, as has been reported for ackA. The effects of the predicted cis-acting catabolite response element (CRE) located upstream from the promoter and of the trans-acting proteins CcpA, HPr, Crh, and HPr kinase on the catabolite regulation of pta were investigated. As for ackA, glucose activation was abolished in ccpA and hprK mutants and in the ptsH1 crh double mutant. Footprinting experiments demonstrated an interaction between CcpA and the pta CRE sequence, which is almost identical to the proposed CRE consensus sequence. This interaction occurs only in the presence of Ser-46-phosphorylated HPr (HPrSer-P) or Ser-46-phosphorylated Crh (CrhSer-P) and fructose-1,6-bisphosphate (FBP). In addition to CcpA, carbon catabolite activation of the pta gene therefore requires at least two other cofactors, FBP and either HPr or Crh, phosphorylated at Ser-46 by the ATP-dependent Hpr kinase.     

Engineering of Acetate Recycling and Citrate Synthase to Improve Aerobic Succinate Production in Corynebacterium glutamicum

Corynebacterium glutamicum lacking the succinate dehydrogenase complex can produce succinate aerobically with acetate representing the major byproduct. Efforts to increase succinate production involved deletion of acetate formation pathways and overexpression of anaplerotic pathways, but acetate formation could not be completely eliminated. To address this issue, we constructed a pathway for recycling wasted carbon in succinate-producing C. glutamicum. The acetyl-CoA synthetase from Bacillus subtilis was heterologously introduced into C. glutamicum for the first time. The engineered strain ZX1 (pEacsA) did not secrete acetate and produced succinate with a yield of 0.50 mol (mol glucose)⁻¹. Moreover, in order to drive more carbon towards succinate biosynthesis, the native citrate synthase encoded by gltA was overexpressed, leading to strain ZX1 (pEacsAgltA), which showed a 22% increase in succinate yield and a 62% decrease in pyruvate yield compared to strain ZX1 (pEacsA). In fed-batch cultivations, strain ZX1 (pEacsAgltA) produced 241 mM succinate with an average volumetric productivity of 3.55 mM h⁻¹ and an average yield of 0.63 mol (mol glucose) ⁻¹, making it a promising platform for the aerobic production of succinate at large scale.     

Evaluation of Genetic Manipulation Strategies on d-Lactate Production by Escherichia coli

In order to rationally manipulate the cellular metabolism of Escherichia coli for D: -lactate production, single-gene and multiple-gene deletions with mutations in acetate kinase (ackA), phosphotransacetylase (pta), phosphoenolpyruvate synthase (pps), pyruvate formate lyase (pflB), FAD-binding D-lactate dehydrogenase (dld), pyruvate oxidase (poxB), alcohol dehydrogenase (adhE), and fumarate reductase (frdA) were tested for their effects in two-phase fermentations (aerobic growth and oxygen-limited production). Lactate yield and productivity could be improved by single-gene deletions of ackA, pta, pflB, dld, poxB, and frdA in the wild type E. coli strain but were unfavorably affected by deletions of pps and adhE. However, fermentation experiments with multiple-gene mutant strains showed that deletion of pps in addition to ackA-pta deletions had no effect on lactate production, whereas the additional deletion of adhE in E. coli B0013-050 (ackA-pta pps pflB dld poxB) increased lactate yield. Deletion of all eight genes in E. coli B0013 to produce B0013-070 (ackA-pta pps pflB dld poxB adhE frdA) increased lactate yield and productivity by twofold and reduced yields of acetate, succinate, formate, and ethanol by 95, 89, 100, and 93%, respectively. When tested in a bioreactor, E. coli B0013-070 produced 125 g/l D-lactate with an increased oxygen-limited lactate productivity of 0.61 g/g h (2.1-fold greater than E. coli B0013). These kinetic properties of D-lactate production are among the highest reported and the results have revealed which genetic manipulations improved D-lactate production by E. coli.     

Genetic changes to optimize carbon partitioning between ethanol and biosynthesis in ethanologenic Escherichia coli

The production of ethanol from xylose by ethanologenic Escherichia coli strain KO11 was improved by adding various medium supplements (acetate, pyruvate, and acetaldehyde) that prolonged the growth phase by increasing cell yield and volumetric productivity (approximately twofold). Although added pyruvate and acetaldehyde were rapidly metabolized, the benefit of these additives continued throughout fermentation. Both additives increased the levels of extracellular acetate through different mechanisms. Since acetate can be reversibly converted to acetyl coenzyme A (acetyl-CoA) by acetate kinase and phosphotransacetylase, the increase in cell yield caused by each of the three supplements is proposed to result from an increase in the pool of acetyl-CoA. A similar benefit was obtained by inactivation of acetate kinase (ackA), reducing the production of acetate (and ATP) and sparing acetyl-CoA for biosynthetic needs. Inactivation of native E. coli alcohol-aldehyde dehydrogenase (adhE), which uses acetyl-CoA as an electron acceptor, had no beneficial effect on growth, which was consistent with a minor role for this enzyme during ethanol production. Growth of KO11 on xylose appears to be limited by the partitioning of carbon skeletons into biosynthesis rather than the level of ATP. Changes in acetyl-CoA production and consumption provide a useful approach to modulate carbon partitioning. Together, these results demonstrate that xylose fermentation to ethanol can be improved in KO11 by redirecting small amounts of pyruvate away from fermentation products and into biosynthesis. Though negligible with respect to ethanol yield, these small changes in carbon partitioning reduced the time required to complete the fermentation of 9.1% xylose in 1% corn steep liquor medium from over 96 h to less than 72 h.     

Expression of the pfl gene and resulting metabolite flux distribution in nuo and ackA-pta E. coli mutant strains

Our laboratory previously studied the interaction between nuo and the acetate-producing pathway encoded by ackA-pta in Escherichia coli. We examined metabolic patterns, particularly the ethanol and acetate production rates, of several mutant strains grown under anaerobic growth conditions. Since the pyruvate formate-lyase (PFL) pathway is the major route for acetyl-CoA and formate production under anaerobic conditions, we examined the effects of nuo and ackA/pta mutations on the expression of pyruvate formate-lyase (pfl) under anaerobic conditions. The ackA-pta mutant has a pfl::lacZ expression level much higher than that of the wild-type strain, and cultures also exhibit the highest ethanol production. Real-time PCR demonstrated that the adhE gene expression in the ack-pta mutant strain was approximately 100 fold that of the same gene in the ackA-pta nuo mutant strain. This result correlates with the observed ethanol production rates in cultures of the strain. However, the lack of exact correlation between the ethanol production rates and the RT-PCR data suggests additional regulation actions at the posttranslation level. In addition, the activity of the pfl gene as indicated by mRNA levels was also considerably greater in theack-pta mutant. We can conclude that deletions of nuo and ack/pta can partially affect the expression of the genes encoding adhE and pfl under anaerobic conditions.     

Combining metabolic engineering and metabolic evolution to develop nonrecombinant strains of Escherichia coli C that produce succinate and malate

Derivatives of Escherichia coli C were engineered to produce primarily succinate or malate in mineral salts media using simple fermentations (anaerobic stirred batch with pH control) without the addition of plasmids or foreign genes. This was done by a combination of gene deletions (genetic engineering) and metabolic evolution with over 2,000 generations of growth-based selection. After deletion of the central anaerobic fermentation genes (ldhA, adhE, ackA), the pathway for malate and succinate production remained as the primary route for the regeneration of NAD+. Under anaerobic conditions, ATP production for growth was obligately coupled to malate dehydrogenase and fumarate reductase by the requirement for NADH oxidation. Selecting strains for improved growth co-selected increased production of these dicarboxylic acids. Additional deletions were introduced as further improvements (focA, pflB, poxB, mgsA). The best succinate biocatalysts, strains KJ060(ldhA, adhE, ackA, focA, pflB) and KJ073(ldhA, adhE, ackA, focA, pflB, mgsA, poxB), produce 622-733 mM of succinate with molar yields of 1.2-1.6 per mole of metabolized glucose. The best malate biocatalyst, strain KJ071(ldhA, adhE, ackA, focA, pflB, mgsA), produced 516 mM malate with molar yields of 1.4 per mole of glucose metabolized.     

Redistribution of metabolic fluxes in Escherichia coli with fermentative lactate dehydrogenase overexpression and deletion

Under anaerobic conditions, competition for pyruvate between the branch point enzymes pyruvate formate lyase (PFL, Km = 2 mM) and fermentative lactate dehydrogenase (LDH, Km = 7.2 mM) determines the partition of carbon flux. Two Escherichia coli mutant strains, one deficient in ackA, pta, and ldhA and the other overexpressing LDH, were constructed to systematically analyze the effects of these perturbations in the existing pathways on the redistribution of carbon fluxes. Deletion of the lactate and acetate synthesis pathways was detrimental to cell growth. Carbon flux is forced through ethanol and formate production pathways, resulting in a concomitant increase in those fluxes. In addition, overexpression of LDH simultaneously increases the common flux as well as the flux to the competing acetyl-CoA branch. Overexpression of lactate dehydrogenase (ldhA) in the parent strain increases the lactate synthesis rate from 0.19 to 0.40 mmol/g-biomass-h when the LDH activities increases from 1.3 to 15.3 units. Even an increase of more than 10 times in the LDH activity fails to divert a large fraction of the carbon flux to lactate; the majority of the flux still channels through the acetyl-CoA branch. Overexpression of LDH in the parent strain simultaneously increases the common flux as well as the flux through the acetyl-CoA branch. Subsequently, the flux amplification factors (or deviation indices which can be related to the flux control coefficients) are positive for all three fluxes occurring at the pyruvate node.