过量表达异柠檬酸裂解酶对ldh-1谷氨酸棒状杆菌产丁二酸的影响

谷氨酸棒状杆菌SA001是缺失了乳酸脱氢酶基因 (ldhA) 的菌株。为了增加厌氧条件下经异柠檬酸到丁二酸的代谢通量,以提高丁二酸的产量。将来自大肠杆菌Escherichia coli K12的异柠檬酸裂解酶基因导入谷氨酸棒状杆菌SA001 (SA001/pXMJ19-aceA) 中。该菌经0.8 mmol/L的IPTG有氧诱导12 h后,转入厌氧发酵16 h,丁二酸的产量为10.38 g/L,丁二酸的生产强度为0.83 g/(L·h)。与出发菌株比较,异柠檬酸裂解酶的酶活提高了5.8倍,丁二酸的产量提高了48%。结果表明过量表达异柠檬酸裂解酶可以增加由乙醛酸途径流向丁二酸的代谢流。     

木薯粉同步糖化发酵(SSF)产丁二酸

【目的】通过优化产琥珀酸放线杆菌GXAS137同步糖化发酵木薯粉产丁二酸的发酵培养基,提高丁二酸产量,降低生产成本。【方法】在单因素试验的基础上,先利用Plackett-Burman试验设计筛选出影响丁二酸发酵的重要参数,再采用正交试验确定重要参数的最佳水平。【结果】价格低廉玉米浆可用作氮源,影响丁二酸产量的重要参数是木薯粉、玉米浆、碱式碳酸镁和糖化酶浓度。最佳条件为(g/L):木薯粉100,玉米浆14,糖化酶2.0 AGU/g底物,碱式碳酸镁75。优化后丁二酸产量达到69.31 g/L,丁二酸得率为90.01%,生产强度为1.44 g/(L·h)。与初始条件(52.34 g/L)相比,丁二酸浓度提高了32.42%。并利用1.3 L发酵罐对SSF与SHF两种发酵工艺进行了比较,SSF丁二酸产量(72.21 g/L)远高于SHF(56.86 g/L)。【结论】产琥珀酸放线杆菌同步糖化发酵木薯粉丁二酸产量高,生产成本低,具有较好的工业化应用前景。     

乳糖诱导丙酮酸羧化酶基因在大肠杆菌中的表达及对丁二酸产量的影响

大肠杆菌DC1515是敲除葡萄糖磷酸转移酶(ptsG)、乳酸脱氢酶(ldhA)、丙酮酸甲酸裂解酶(pflA)基因的菌株,具有发酵生产丁二酸的潜力。为进一步提高菌株DC1515的丁二酸生产能力,将枯草芽孢杆菌丙酮酸羧化酶(pyc)基因转入其中。用乳糖代替IPTG诱导pyc表达,确定了最佳乳糖加入时间、乳糖浓度及诱导温度。在此基础上,考察了补加乳糖对丁二酸产量的影响。结果表明:由于ptsG基因缺失,当培养基中葡萄糖浓度达到15g/L时,乳糖诱导作用并不受葡萄糖抑制。优化诱导条件后,pyc过表达菌株的丁二酸产量达15.17g/L,为对照菌株的1.78倍。间歇补加乳糖2次至浓度为1g/L,丁二酸产量可进一步增至17.54g/L。研究结果为以葡萄糖为底物生产丁二酸的过程中乳糖诱导外源基因在大肠杆菌中的表达奠定了基础。乳糖诱导降低了成本,有利于实现丁二酸发酵生产的工业化。     

发酵产丁二酸过程中废弃细胞的循环利用

对厌氧发酵产丁二酸后的废弃细胞进行破壁处理,考察了以细胞水解液作为有机氮源重新用于丁二酸发酵的可行性。比较了超声破碎、盐溶、酶解3种方法破碎细胞获得的水解液作为氮源发酵产丁二酸的效果,结果表明酶解制得的细胞水解液效果最佳。以总氮含量为1.11g/L的酶解液(相当于10g/L酵母膏)作为氮源发酵,丁二酸产量可达42.0g/L,继续增大酶解液用量对耗糖、产酸能力没有显著提高。将细胞酶解液与5g/L酵母膏联用发酵36h后,丁二酸产量达75.5g/L,且丁二酸生产强度为2.10g/(L·h),比使用10g/L酵母膏时提高了66.7%。因此,厌氧发酵产丁二酸结束后的废弃细胞酶解液可以替代原培养基中50%的酵母膏用于发酵。     

常压室温等离子体诱变高效利用木糖产丁二酸菌株

大肠杆菌Escherichia coli AFP111是E. coli NZN111 (△pflAB△ldhA) 的ptsG自发突变株,其转化1 mol的木糖合成丁二酸的过程中净产生1.67 mol ATP,但是转化1 mol的木糖合成丁二酸的过程中实际需要2.67 mol ATP,因此在厌氧条件下,ATP的供给不足导致E. coli AFP111不能代谢木糖。采用常压室温等离子体射流诱变产丁二酸大肠杆菌菌株,在厌氧条件下,利用以木糖为碳源的M9培养基,筛选得到一株可以代谢木糖并积累丁二酸的突变株DC111。该突变菌株在发酵培养基中,72 h内可以消耗10.52 g/L木糖产6.46 g/L的丁二酸,丁二酸的得率达到了0.78 mol/mol。而且突变株中伴有ATP产生的磷酸烯醇式丙酮酸羧激酶 (PCK) 途径得到加强,PCK的比酶活相对于出发菌株提高了19.33倍,使得其在厌氧条件下能够有足够的ATP供给来代谢木糖发酵产丁二酸。     

响应面优化产琥珀酸放线杆菌生产丁二酸

丁二酸是一种重要的C4化合物平台,可以合成一系列重要化合物。文中对产琥珀酸放线杆菌Actinobacillus succinogenes GXAS137发酵生产丁二酸培养基成分进行优化。通过单因素和Plackett-Burman试验设计筛选出影响丁二酸发酵的重要参数,采用最陡爬坡实验逼近最大丁二酸生产区域后,利用Box-Behnken设计确定重要参数的最佳水平。筛选结果表明,影响丁二酸产量的重要参数是葡萄糖、酵母提取物和碱式碳酸镁浓度。最佳条件为(g/L)：葡萄糖70.00,酵母提取物9.20,碱式碳酸镁58.10。优化后丁二酸产量达到47.64 g/L。与初始条件 (36.89 g/L) 相比,丁二酸浓度提高了30 %。在最佳工艺条件下得到的试验结果与模型预测值很吻合,说明建立的模型是有效的。     

高效液相色谱法在测定丁二酸厌氧发酵体系中有机酸的应用

建立了一种利用高效液相色谱法定量分析丁二酸厌氧发酵体系中多种有机酸的方法。利用Alltech反相Prevail有机酸色谱柱,以25 mmol.L-1KH2PO4(pH2.5)作为流动相,流速1 mL.min-1,采用紫外检测器,于215 nm处检测,能将丁二酸厌氧发酵体系中多种有机酸完全分离并准确定量。有机酸的回收率均在99%~103%之间。本方法能够快速、精确测定丁二酸厌氧发酵体系中多种有机酸含量,并初步应用于该发酵体系培养基成分优化方面,对于指导厌氧代谢调控生产丁二酸具有重要意义。     

Actinobacillus succinogenes 130Z厌氧发酵产丁二酸条件初步研究

对Actinobacillus succinogenes130Z厌氧发酵产丁二酸的培养条件进行了初步研究。研究了不同有机氮源,不同碳、氮源浓度配比、CO2供体、培养温度,培养基起始pH值对菌株生长和产酸的影响,并在5 L发酵罐中进行了放大试验。结果表明最佳培养基配方为(g/L):葡萄糖10,酵母膏5,NaHCO310,Na2HPO40.3,NaH2PO4.2H2O 9.6,K2HPO43,MgCl20.2,MnCl20.2,NaCl 0.1;pH7.0。在最佳条件下,血清瓶37℃培养24 h,丁二酸产量达到8.3 g/L,在5 L发酵罐中培养,葡萄糖质量浓度分别为10和100 g/L时,丁二酸产量分别达到8.2和45.6 g/L,收率分别为80%和65%。     

最新专利文摘

CN1814747:一种微生物发酵生产丁二酸的菌种和方法本专利具体涉及一种发酵糖质原料产生丁二酸的微生物琥珀酸放线杆菌(Actinobacillus succinogenes)SW 0580,保藏号CGMCC No·1593,以及利用该微生物发酵生产丁二酸的方法。该微生物是从瘤胃中分离并鉴定的琥珀酸放线杆菌(Actino     

碳源对重组大肠杆菌两阶段发酵产丁二酸的影响

研究了在好氧培养基中分别添加不同碳源对两阶段发酵菌体生长、酶活及代谢产物分布的影响,结果表明添加4mmol/L葡萄糖和12,54,80mmol/L乙酸钠均可以提高好氧阶段的菌体密度和相关酶活。将不同条件下培养的菌体转接厌氧发酵后,厌氧阶段的酶活和代谢产物分布也发生改变。进一步对酶活及代谢产物分析表明：Escherichia coli NZN111(sfcA)厌氧发酵过程中,磷酸烯醇式丙酮酸羧化激酶(PCK)是产丁二酸的关键酶,丙酮酸激酶(PYK)主要和副产物丙酮酸的积累有关,异柠檬酸裂解酶(ICL)对丁二酸产量也有一定影响。好氧培养基中添加80mmol/L乙酸钠,厌氧发酵结束时丁二酸的质量收率可达89.0%,相比对照提高了16.6%。     

聚丁二酸丁二醇酯(PBS)的生物降解的研究进展

聚丁二酸丁二醇酯(poly(butylene succinate), PBS)是一种人工合成的脂肪族聚酯化合物。PBS的生产成本低、热稳定性好,具有良好加工性能、机械性能以及力学性能等优点。本文就近年来PBS在生物降解方面的研究进展进行了综述,具体包括PBS的生物堆肥降解、PBS的微生物降解以及PBS降解酶的相关研究。最后对PBS生物降解研究进展做出了总结。     

Thermophilic microbial degradation of polyethylene succinate

This paper examined the biodegradability of a new aliphatic polyester, polyethylene succinate (PES), at a high incubation temperature of 50°C. The distribution and population of total colonies and of PES degrading micro organisms on polymer-emulsified agar plates were determined using the plate count and clear zone methods. The PES-decomposers were present in six of 10 soil samples and the total number ranged from 2.0×104 to 2.2×106 c.f.u./g of samples. Degrading microorganisms constituted between 20 and 80% of the total colonies on PES–agar plates. A single PES-degrading strain, TT96, was isolated and tested for its biodegrading capacity on PES powder and on other aliphatic polyesters: poly(beta-hydroxybutyrate) (PHB), polycaprolactone (PCL), poly(butylene succinate) (PBS), and poly(L-lactide) (PLA). Degraded films of PES and PBS were presented and compared using scanning electron microscopy. Strain TT96 was able to create clear zones on all the polymers used, except on PHB-agar plates. Liquid culture test after 2 weeks showed that TT96 completely degraded PCL powder but had very little activity on other samples. Scanning electron micrograph confirmed the microbial attack of TT96 on PES and PBS films. PES film surfaces were degraded more uniformly compared to PBS films which were decomposed only in some parts.     

Properties of a bacterium which degrades solid poly(tetramethylene succinate)-co-adipate, a biodegradable plastic

Various microorganisms were screened for their ability to degrade poly(tetramethylene succinate)-co-(tetramethylene adipate) (PBSA). Strain BS-3, which was newly isolated from a soil sample, was selected as the best strain. From taxonomical studies, the strain was tentatively ascribed to belong to the genus Acidovorax, most probably to the species A. delafieldii. Strain BS-3 could degrade both solid and emulsified PBSA, and also emulsified poly(tetramethylene succinate). During the degradation, a lipase activity was observed in the culture broth. This lipase activity was induced more strongly by PBSA than by tributyrin or triolein which are typical substrates of lipase. These observations strongly suggest that this lipase was involved in the PBSA biodegradation in strain BS-3.     

Isolation and characterization of a bacterium that degrades various polyester-based biodegradable plastics

Microorganisms isolated from soil samples were screened for their ability to degrade various biodegradable polyester-based plastics. The most active strain, designated as strain TB-13, was selected as the best strain for degrading these plastics. From its phenotypic and genetic characteristics, strain TB-13 was closely related to Paenibacillus amylolyticus. It could degrade poly(lactic acid), poly(butylene succinate), poly(butylene succinate-co-adipate), poly(caprolactone) and poly(ethylene succinate) but not poly(hydroxybutylate-co-valerate). However, it could not utilize these plastics as sole carbon sources. Both protease and esterase activities, which may be involved in the degradation of plastic, were constitutively detected in the culture broth.     

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.     

Micelle-supported electroenzymology: Succinate dehydrogenation by escherichia coli fumarate reductase in decylubiquinone and octyl glucoside micelles

The concept of micelle-supported electroenzymology is demonstrated using a system consisting of the membrane enzyme Escherichia coli fumarate reductase (FRD), the amphiphilic coenzyme analogue decylubiquinone (DU), the micelle-forming surfactant n-octyl glucoside (OG), and a gold electrode. The OG micelles provide a hydrophobic, membrane mimetic medium for FRD and DU to exchange electrons while the gold electrode serves to regenerate DU. When succinate is presented to the FRD/DU/OG micelle system, electroenzymatic oxidation of succinate to fumarate occurs as evidenced using cyclic voltammetry. DU is shown to be the only electroactive species in the system; and as increasing amounts of succinate are added, the expected increase in the peak anodic (oxidative) current and decrease in the peak cathodic (reductive) current are observed. The peak anodic current approaches a limiting value with succinate concentration in qualitative agreement with simple Michaelis-Menten enzyme kinetics. When the strong competitive inhibitor oxaloacetate is added, enzymatic oxidation of succinate is inhibited as indicated by no change in the peak anodic and cathodic currents with increasing succinate concentration. (c) 1993 John Wiley & Sons, Inc.     

Lipase-Catalyzed Synthesis of Poly(1,4-Butanediol Succinate) in Organic Solvent

During the last decade, lipase has gained interest as a biocatalyst for synthesis in organic solvent systems. The paper describes the lipase catalyzed oligocondensation of bis(2-chloroethyl) succinate and 1,4-butanediol to obtain poly (1,4-butanediol succinate). The reaction was carried out at 37°C in organic solvents without any addition of water. Various lipases and solvents were screened to obtain a maximum degree of polymerization. Based on gel permeation chromatography, the highest average molecular weight of the oligomer obtained was 1570 g/mol with a polydispersity of 1.2 when a mixture of 70% diisopropyl ether and 30% chloroform was used as a solvent. The degree of polymerization was 8 in this case. According to thin-layer chromatography, a trimer (HO(CH₂)₄OCO(CH₂)₂COO(CH₂)₄OH) was formed at an early stage, with a subsequent condensation with bis(2-chloroethyl) succinate to give higher oligomers. The structure of the oligomers was confirmed by ¹³C NMR and IR spectra.     

Culture conditions' impact on succinate production by a high succinate producing Escherichia coli strain

This work aimed to identify the key operational factors that significantly affect succinate production by the high succinate producing Escherichia coli strain SBS550MG (pHL413), which bears mutations inactivating genes adhE ldhA iclR ackpta::Cm(R) and overexpresses the pyruvate carboxylase from Lactococcus lactis. The considered factors included glucose concentration, cell density, CO(2) concentration in the gas stream, pH, and temperature. The results showed that high glucose concentrations inhibited succinate production and that there is a compromise between the total succinate productivity and succinate specific productivity, where the total productivity increased with the increase in cell density and the specific productivity decreased with cell density, probably due to mass transfer limitation. On the other hand, a CO(2) concentration of 100% in the gas stream showed the highest specific succinate productivity, probably by favoring pyruvate carboxylation, increasing the OAA pool that later is converted into succinate. A full factorial design of experiments was applied to analyze the pH and temperature effects on succinate production in batch bioreactors, where succinate yield was not significantly affected by either temperature (37 to 43°C) or pH (6.5 to 7.5). Additionally, the temperature effect on succinate productivity and titer was not significant, in the range tested. On the other hand, a pH of 6.5 showed very low productivity, whereas pH values of 7.0 and 7.5 resulted in significantly higher specific productivities and higher titers. The increase on pH value from 7.0 to 7.5 did not show significant improvement. Then, pH 7.0 should be chosen because it involves a lower cost in base addition.     

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.     

Microbial degradation of aliphatic and aliphatic-aromatic co-polyesters

Biodegradable plastics (BPs) have attracted much attention since more than a decade because they can easily be degraded by microorganisms in the environment. The development of aliphatic-aromatic co-polyesters has combined excellent mechanical properties with biodegradability and an ideal replacement for the conventional nondegradable thermoplastics. The microorganisms degrading these polyesters are widely distributed in various environments. Although various aliphatic, aromatic, and aliphatic-aromatic co-polyester-degrading microorganisms and their enzymes have been studied and characterized, there are still many groups of microorganisms and enzymes with varying properties awaiting various applications. In this review, we have reported some new microorganisms and their enzymes which could degrade various aliphatic, aromatic, as well as aliphatic-aromatic co-polyesters like poly(butylene succinate) (PBS), poly(butylene succinate)-co-(butylene adipate) (PBSA), poly(ε-caprolactone) (PCL), poly(ethylene succinate) (PES), poly(l-lactic acid) (PLA), poly(3-hydroxybutyrate) and poly(3-hydoxybutyrate-co-3-hydroxyvalterate) (PHB/PHBV), poly(ethylene terephthalate) (PET), poly(butylene terephthalate) (PBT), poly(butylene adipate-co-terephthalate (PBAT), poly(butylene succinate-co-terephthalate) (PBST), and poly(butylene succinate/terephthalate/isophthalate)-co-(lactate) (PBSTIL). The mechanism of degradation of aliphatic as well as aliphatic-aromatic co-polyesters has also been discussed. The degradation ability of microorganisms against various polyesters might be useful for the treatment and recycling of biodegradable wastes or bioremediation of the polyester-contaminated environments.