微生物发酵琥珀酸的研究进展

琥珀酸是一种重要的C_4平台化合物,生物基琥珀酸可作为合成大宗化学品的起始原料,在食品、医药、表面活性剂、洗涤剂、绿色溶剂、生物可降解塑料和动植物生长刺激物剂领域有广泛的应用前景。从可再生原料出发,发酵过程固定CO_2,使得微生物发酵生产琥珀酸具有良好的环境优势,成为近年研究的热点。围绕微生物发酵法生产琥珀酸迫切需要解决的主要问题,综述了国内外对琥珀酸生产菌的选育、其代谢机理与产酸条件、发酵过程工艺、产品提取等方面的研究现状。     

产琥珀酸放线杆菌发酵生产琥珀酸的研究进展

近年来,因瘤胃微生物产琥珀酸放线杆菌Actinobacillus succinogenes具有高的琥珀酸产量,并能够利用多种碳源进行发酵等优点,在利用发酵法生产琥珀酸领域具有广泛的应用前景和商业化价值,因而其代谢途径和发酵工艺等基础研究成为国内外研发的热点。近年来,人们在产琥珀酸放线杆菌的代谢途径、琥珀酸发酵动力学模型、新型经济培养基以及高产菌株选育等方面的研究取得了很大进展,对研发琥珀酸发酵工艺、降低生产成本和节能减耗等具有重要的理论意义。     

大肠杆菌生产琥珀酸研究进展

琥珀酸是一种重要的C4平台化合物,常作为合成通用化学品的起始原料,在化工、食品、医药、农药等领域有广泛的应用前景。与传统的化学合成法相比,微生物发酵生产琥珀酸的原料可再生,具有良好的环境优势,逐渐成为世界各国关注的热点。对近年来通过调控代谢途径提高大肠杆菌琥珀酸产率的研究进展进行了综述,分析了琥珀酸代谢网络及代谢途径中的关键酶基因进行的表达调控、NADH/NAD+的氧化还原态比例对琥珀酸发酵的影响等,并提出了利用RNAi技术改造大肠杆菌琥珀酸代谢途径的设想,以期为构建高产琥珀酸的工程菌株发酵生产琥珀酸提供新思路。     

维生素E琥珀酸酯的合成及表征

目的:合成维生素E琥珀酸酯并对其进行表征.方法:以d,1-α-生育酚和丁二酸酐为原料,吡啶为溶媒,合成了维生素E琥珀酸酯.采用紫外光谱法、红外光谱法、核磁共振氢谱和差示扫描量热分析对产物进行表征.结果:经验证,成功合成了维生素E琥珀酸酯.结论:成功合成了维生素E琥珀酸酯.     

鱼腥藻PCC7120中琥珀酸半醛脱氢酶的初步表征及结构分析

蓝藻琥珀酸半醛脱氢酶催化琥珀酸半醛到琥珀酸的转化,使得蓝藻的三羧酸循环变得完整。BLAST序列分析预测鱼腥藻PCC7120中all3556基因编码琥珀酸半醛脱氢酶。为了证实all3556基因编码蛋白的催化功能,构建了p ET28a-all3556表达质粒,并在大肠杆菌BL21(DE3)菌体中进行重组蛋白诱导表达;利用镍亲和层析方法对all3556蛋白进行了分离纯化。酶动力学测试表明all3556蛋白是一个NADP+-依赖型的琥珀酸半醛脱氢酶。生物信息学分析发现all3556蛋白和其他来源的琥珀酸半醛脱氢酶的氨基酸序列具有一定的同源性,在催化中心的氨基酸残基高度保守。     

环境因素对琥珀酸放线杆菌Actinobacillus succinogenes CGMCC 1593发酵生产丁二酸的影响

琥珀酸放线杆菌是发酵生产有应用前景的生物基原料-丁二酸的微生物。本研究室从牛瘤胃中筛选获得一株琥珀酸放线杆菌Actinobacillus succinogenes CGMCC 1593, 分析了环境气体、pH、氧化还原电位(ORP)环境因素对琥珀酸放线杆菌A. succinogenes CGMCC 1593发酵生产丁二酸的影响。结果表明: CO2不仅提供了A. succinogenes CGMCC 1593发酵生产丁二酸的最佳气体环境, 也是发酵生产丁二酸的底物之一; MgCO3是A. succinogenes CGMCC 1593发酵过程较好的pH调节剂, 发酵过程维持pH7.1~6.2, 可满足菌体生长与产酸的要求; 发酵液初始ORP过低, 不利于菌体生长, ORP在-270 mV时对丁二酸产生有利。在菌体对数生长期结束时, 通过Na2S·9H2O降低发酵液ORP到-270 mV, 发酵48 h时可产丁二酸37 g/L, 摩尔产率达到129%。这对深入研究A. succinogenes CGMCC 1593发酵生产丁二酸具有参考价值。     

环境因素对琥珀酸放线杆菌Actinobacillus succinogenes CGMCC 1593发酵生产丁二酸的影响

琥珀酸放线杆菌是发酵生产有应用前景的生物基原料-丁二酸的微生物。本研究室从牛瘤胃中筛选获得一株琥珀酸放线杆菌Actinobacillus succinogenes CGMCC 1593, 分析了环境气体、pH、氧化还原电位(ORP)环境因素对琥珀酸放线杆菌A. succinogenes CGMCC 1593发酵生产丁二酸的影响。结果表明: CO2不仅提供了A. succinogenes CGMCC 1593发酵生产丁二酸的最佳气体环境, 也是发酵生产丁二酸的底物之一; MgCO3是A. succinogenes CGMCC 1593发酵过程较好的pH调节剂, 发酵过程维持pH7.1~6.2, 可满足菌体生长与产酸的要求; 发酵液初始ORP过低, 不利于菌体生长, ORP在-270 mV时对丁二酸产生有利。在菌体对数生长期结束时, 通过Na2S·9H2O降低发酵液ORP到-270 mV, 发酵48 h时可产丁二酸37 g/L, 摩尔产率达到129%。这对深入研究A. succinogenes CGMCC 1593发酵生产丁二酸具有参考价值。     

大肠杆菌产琥珀酸基因工程研究进展

生物合成琥珀酸摆脱了对不可再生战略资源石油的依赖,以其社会、经济和环境效益展现出良好的发展前景。野生型大肠杆菌的琥珀酸生产强度难以满足生物合成琥珀酸工业化的要求,但遗传背景清楚,容易改造。近年来,人们深入研究了大肠杆菌的琥珀酸代谢途径,通过强化大肠杆菌琥珀酸合成途径、抑制琥珀酸旁路代谢途径、构建产琥珀酸乙醛酸循环和有氧生产体系等多种基因工程策略,对大肠杆菌进行菌株改造和代谢进化筛选,提高了琥珀酸产量。综述了大肠杆菌产琥珀酸的基因工程研究进展。     

产琥珀酸工程菌株的发酵工艺条件优化

对实验室构建的产琥珀酸大肠杆菌工程菌株(E.coliQZ1111)进行发酵工艺条件研究。以AM1低盐培养基为基础,研究不同C、N源及其质量浓度,培养基初始pH和发酵温度等因素对琥珀酸的影响,并在5L发酵罐中进行了补料-分批发酵实验。优化后的发酵条件为葡萄糖20g/L,玉米浆10g/L,pH6.4,发酵温度37℃。在5L发酵罐中培养,琥珀酸产量达到47.9g/L。     

大肠杆菌木糖生物合成琥珀酸的代谢工程研究

目的:对大肠杆菌进行代谢网络改造,考察木糖好氧发酵生产琥珀酸的可行性。方法:以有氧条件下大肠杆菌木糖生物合成琥珀酸的代谢途径分析为基础,以大肠杆菌BL21为出发菌株,通过P1噬菌体一步敲除法敲除琥珀酸脱氢酶基因(sdhA)、磷酸转乙酰基酶基因(pta)、丙酮酸脱氢酶基因(poxB)及异柠檬酸裂解酶阻遏物基因(iclR),构建木糖好氧发酵生产琥珀酸的大肠杆菌工程菌JLS400(△poxB△pta△iclR△sdhA)。将携带磷酸烯醇式丙酮酸羧化酶基因的质粒pJW225转化到JLS400中。结果:摇瓶发酵结果表明,构建的工程菌能以木糖为碳源,在好氧发酵条件下琥珀酸产率较高,副产物仅有少量乙酸和丙酮酸。结论:基因工程大肠杆菌JLS400pJW225的构建,为有氧条件下以木糖为原料生产琥珀酸的进一步研究奠定了基础。     

Recent advances in production of succinic acid from lignocellulosic biomass

Production of succinic acid via separate enzymatic hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) are alternatives and are environmentally friendly processes. These processes have attained considerable positions in the industry with their own share of challenges and problems. The high-value succinic acid is extensively used in chemical, food, pharmaceutical, leather and textile industries and can be efficiently produced via several methods. Previously, succinic acid production via chemical synthesis from petrochemical or refined sugar has been the focus of interest of most reviewers. However, these expensive substrates have been recently replaced by alternative sustainable raw materials such as lignocellulosic biomass, which is cheap and abundantly available. Thus, this review focuses on succinic acid production utilizing lignocellulosic material as a potential substrate for SSF and SHF. SSF is an economical single-step process which can be a substitute for SHF — a two-step process where biomass is hydrolyzed in the first step and fermented in the second step. SSF of lignocellulosic biomass under optimum temperature and pH conditions results in the controlled release of sugar and simultaneous conversion into succinic acid by specific microorganisms, reducing reaction time and costs and increasing productivity. In addition, main process parameters which influence SHF and SSF processes such as batch and fed-batch fermentation conditions using different microbial strains are discussed in detail.     

糠醛和5-羟甲基糠醛对大肠杆菌产丁二酸的影响

利用可再生生物质特别是木质纤维素水解液来生产平台化合物丁二酸,是目前研究的热点。虽然许多研究者相继报道了木质纤维素水解液对菌株生长和丁二酸生产存在一定抑制作用,但并没有水解液中各种抑制物对菌株影响的相关动力学研究及机理研究。我们选择了两种代表性木质纤维素水解液抑制物,即糠醛和5-羟甲基糠醛,系统研究了它们对大肠杆菌的生长和丁二酸生产的影响。结果表明：糠醛和5-羟甲基糠醛的初始抑制浓度均为0.8 g/L。当糠醛浓度大于6.4 g/L,5-羟甲基糠醛浓度大于12.8 g/L时,菌株生长完全受到抑制。在最高耐受浓度下,糠醛的存在使菌株生物量比对照菌株下降77.8%,丁二酸产量下降36.1%。5-羟甲基糠醛的存在使菌株生物量比对照菌株降低13.6%,丁二酸产量降低18.3%。糠醛和5-羟甲基糠醛具有明显的协同作用。体外酶活测定表明丁二酸生产途径中关键酶磷酸烯醇式丙酮酸羧化酶、苹果酸脱氢酶、富马酸还原酶均受糠醛和5-羟甲基糠醛抑制。研究结果对丁二酸生产用纤维素水解液的预处理和脱毒工艺开发具有指导作用,有利于实现丁二酸发酵生产的工业化。     

New reactive extraction systems for separation of bio-succinic acid

Biotechnologically produced succinic acid has the potential to displace maleic acid and its uses and to become an important feedstock for the chemical industry. In addition to optimized production strains and fermentation processes, an efficient separation of succinic acid from the aqueous fermentation broth is indispensable to compete with the current petrochemical production processes. In this context, high molecular weight amines are known to be effective extractants for organic acids. For this reason, as a first step of isolation and purification, the reactive extraction of succinic acid was studied by mixing aqueous succinic acid solutions with 448 different amine–solvent mixtures as extraction agents (mixer-settler studies). The extraction agents consist either of one amine and one solvent (208 reactive extraction systems) or two amines and two solvents (240 reactive extraction systems). Maximum extraction yields of succinic acid from an aqueous solution with 423 mM succinic acid at pH 2.0 were obtained with more than 95% yield with trihexylamine solved in 1-octanol or with dihexylamine and diisooctylamine solved in 1-octanol and 1-hexanol. Applying these optimized reactive extraction systems with Escherichia coli fermentation broth resulted in extraction yields of 78–85% due to the increased ionic strength of the fermentation supernatant and the co-extraction of other organic acids (e.g., lactic acid and acetic acid), which represent typical fermentation byproducts.     

Regulation of NAD(H) pool and NADH/NAD(+) ratio by overexpression of nicotinic acid phosphoribosyltransferase for succinic acid production in Escherichia coli NZN111

Succinic acid is not the dominant fermentation product from glucose in wild-type Escherichia coli W1485. To reduce byproduct formation and increase succinic acid accumulation, pyruvate formate-lyase and lactate dehydrogenase, encoded by pflB and ldhA genes, were inactivated. However, E. coli NZN111, the ldhA and pflB deletion strain, could not utilize glucose anaerobically due to the block of NAD(+) regeneration. To restore glucose utilization, overexpression of nicotinic acid phosphoribosyltransferase, a rate limiting enzyme of NAD(H) synthesis encoded by the pncB gene, resulted in a significant increase in cell mass and succinic acid production. Furthermore, the results indicated a significant increase in NAD(H) pool size, and decrease in the NADH/NAD(+) ratio from 0.64 to 0.13, in particular, the concentration of NAD(+) increased 6.2-fold during anaerobic fermentation. In other words, the supply of enough NAD(+) for NADH oxidation by regulation of NAD(H) salvage synthesis mechanism could improve the cell growth and glucose utilization anaerobically. In addition, the low NADH/NAD(+) ratio also change the metabolite distribution during the dual-phase fermentation. As a result, there was a significant increase in succinic acid production, and it is provided further evidence that regulation of NAD(H) pool and NADH/NAD(+) ratio was very important for succinic acid production.     

大肠杆菌AFP111菌体回收连续转化生产丁二酸

在利用大肠杆菌AFP111厌氧发酵生产丁二酸过程中,随着产物丁二酸的不断积累,菌体活力和产酸能力逐渐降低,而通过回收菌体在新鲜培养基中重复发酵,可延长厌氧发酵时间,但是丁二酸生产效率较低。为了提高菌体回收丁二酸的转化效率,通过在回收菌体时有氧诱导 3 h,以纯水为培养基,进行丁二酸转化发酵。在连续进行 3 批次的发酵后,丁二酸的总产量和最终收率分别为 56.50 g/L和90%,生产速率达到了 0.81 g/(L·h),比未诱导情况下的生产速率提高了13%。     

Succinic acid production from wheat using a biorefining strategy

The biosynthesis of succinic acid from wheat flour was investigated in a two-stage bio-process. In the first stage, wheat flour was converted into a generic microbial feedstock either by fungal fermentation alone or by combining fungal fermentation for enzyme and fungal bio-mass production with subsequent flour hydrolysis and fungal autolysis. In the second stage, the generic feedstock was converted into succinic acid by bacterial fermentation by Actinobacillus succinogenes. Direct fermentation of the generic feedstock produced by fungal fermentation alone resulted in a lower succinic acid production, probably due to the low glucose and nitrogen concentrations in the fungal broth filtrate. In the second feedstock production strategy, flour hydrolysis conducted by mixing fungal broth filtrate with wheat flour generated a glucose-rich stream, while the fungal bio-mass was subjected to autolysis for the production of a nutrient-rich stream. The possibility of replacing a commercial semi-defined medium by these two streams was investigated sequentially. A. succinogenes fermentation using only the wheat-derived feedstock resulted in a succinic acid concentration of almost 16 g l^–1 with an overall yield of 0.19 g succinic acid per g wheat flour. These results show that a wheat-based bio-refinery employing coupled fungal fermentation and subsequent flour hydrolysis and fungal autolysis can lead to a bacterial feedstock for the efficient production of succinic acid.     

Integrated separation process for isolation and purification of biosuccinic acid

Biotechnologically produced succinic acid has the potential to displace maleic acid and its uses. Therefore, it is of high interest for the chemical, pharmaceutical, and food industry.In addition to optimized production strains and fermentation processes, an efficient separation of succinic acid from the aqueous fermentation broth is indispensable to compete with the current petrochemical production of succinic acid. Isolation and purification of succinic acid from an Escherichia coli fermentation broth were studied with two amine-based reactive extraction systems: (i) trihexylamine in 1-octanol and (ii) diisooctylamine and dihexylamine in a mixture of 1-octanol and 1-hexanol. Back extraction of succinic acid from the organic phase was carried out using an aqueous trimethylamine solution. The trimethylammonium succinate generated after back extraction was split with an evaporation-based crystallization.The focus was on process integration, for example, reuse of the applied amines for extraction and back extraction. It was shown that the maximum trimethylamine concentration for back extraction should not exceed the stoichiometric amount (2 mol trimethylamine/mol the succinic acid in the organic phase) to ensure maximal extraction yields with the reused organic phase in subsequent extractions. Moreover, mixer-settler extraction and back extraction of succinic acid were scaled up from the milliliter- to the liter-scale making use of liquid–liquid centrifuges. The overall yield was 83.5% of the succinic acid from thefermentation supernatant. The final purity of the succinic acid crystals was 99.5%. Organic phase and amines can easily be recycled and reused.     

Recovery of succinic acid produced by fermentation of a metabolically engineered Mannheimia succiniciproducens strain

There have recently been much advances in the production of succinic acid, an important four-carbon dicarboxylic acid for many industrial applications, by fermentation of several natural and engineered bacterial strains. Mannheimia succiniciproducens MBEL55E isolated from bovine rumen is able to produce succinic acid with high efficiency, but also produces acetic, formic and lactic acids just like other anaerobic succinic acid producers. We recently reported the development of an engineered M. succiniciproducens LPK7 strain which produces succinic acid as a major fermentation product while producing much reduced by-products. Having an improved succinic acid producer developed, it is equally important to develop a cost-effective downstream process for the recovery of succinic acid. In this paper, we report the development of a simpler and more efficient method for the recovery of succinic acid. For the recovery of succinic acid from the fermentation broth of LPK7 strain, a simple process composed of a single reactive extraction, vacuum distillation, and crystallization yielded highly purified succinic acid (greater than 99.5% purity, wt%) with a high yield of 67.05wt%. When the same recovery process or even multiple reactive extraction steps were applied to the fermentation broth of MBEL55E, lower purity and yield of succinic acid were obtained. These results suggest that succinic acid can be purified in a cost-effective manner by using the fermentation broth of engineered LPK7 strain, showing the importance of integrating the strain development, fermentation and downstream process for optimizing the whole processes for succinic acid production.     

From genome sequence to integrated bioprocess for succinic acid production by <Emphasis Type="Italic">Mannheimia succiniciproducens</Emphasis>

Mannheimia succiniciproducens is a capnophilic gram-negative bacterium isolated from bovine rumen. Wild-type M. succiniciproducens can produce succinic acid as a major fermentation product with acetic, formic, and lactic acids as byproducts during the anaerobic cultivation using several different carbon sources. Succinic acid is an important C4 building block chemical for many applications. Here, we review the progress made with M. succiniciproducens for efficient succinic acid production; the approaches taken towards the development of an integrated process for succinic acid production are described, which include strain isolation and characterization, complete genome sequencing and annotation, development of genetic tools for metabolic engineering, strain development by systems approach of integrating omics and in silico metabolic analysis, and development of fermentation and recovery processes. We also describe our current effort on further improving the performance of M. succiniciproducens and optimizing the mid- and downstream processes. Finally, we finish this mini-review by discussing the issues that need to be addressed to make this process of fermentative succinic acid production employing M. succiniciproducens to reach the industrial-scale process.