【作者】 张燎原；

【导师】 沈亚领；

【作者基本信息】 华东理工大学 ， 发酵工程， 2010， 博士

【摘要】 2,3-丁二醇(2,3-butanediol, BD)是一种重要的生物基化学品,广泛用于化工、食品、航空航天燃料等领域,可用来制备聚合物、油墨、香水、熏蒸剂、增湿剂、软化剂、增塑剂、药物手性载体等。本论文以粘质沙雷氏菌H30(Serratia marcescens H30)为出发菌株,通过传统的发酵调控方法结合现代代谢工程手段对生物法制备2,3-丁二醇开展了以下研究工作：1.发酵条件和发酵培养基的优化通过摇瓶实验,确定了粘质沙雷氏菌H30生长及2,3-丁二醇合成的最佳培养条件和最优发酵培养基组成。最佳培养条件为：初始pH值为7.0,过程pH值控制为6.0,培养温度为30℃,接种量为5%,250mL三角瓶最适装液量为50mL。在此基础上通过单因素实验、Plackett-Burman Design实验和Response Surface Methodology实验对粘质沙雷氏菌H30的发酵培养基组分进行了优化,获得了培养基的最优配方为：蔗糖90g／L,安琪酵母粉33.36g／L,柠檬酸钠10g／L,乙酸钠4g／L,硫酸锰0.1g／L,硫酸镁0.3g／L,磷酸二氢铵1g／L。2,3-丁二醇摇瓶发酵水平由15.93g／L提高到44.70g／L,发酵时间由48h缩短至15h。此外,在摇瓶中进行补料实验,发现维持残糖浓度在15-30g／L有利于2,3-丁二醇的合成。2.3.7L发酵罐实验及各类发酵罐逐级放大实验在发酵罐实验中,我们首先在3.7L发酵罐进行了细致的研究,通过比较几种不同的补料及调控策略：脉冲补料发酵、恒速补料发酵、恒底物浓度-RQ(呼吸熵)调控发酵和pH自控-恒底物浓度-RQ调控发酵,获得了2,3-丁二醇的产量分别为115.5g／L,117.14g／L,139.92g／L和130.65g／L。最终确立了恒底物浓度-RQ调控发酵策略,粘质沙雷氏菌细胞干重达16.05g／L,2,3-丁二醇的生产能力达到3.34g／L·h,产物得率为94.67%。在该发酵调控策略的基础上,我们在50L-5000L的发酵罐中进行了逐级放大,产量均超过130g／L,在5000L发酵罐中2,3-丁二醇的产量为130.2g／L。3.粘质沙雷氏菌生物表面活性剂缺失工程菌的构建粘质沙雷氏菌H30因合成生物表面活性剂导致发酵过程中产生大量泡沫,泡沫的产生势必需要加入大量的泡敌,否则将引起逃液、微生物污染和发酵过程无法控制等后果,然而,泡敌的大量加入又将导致发酵液的乳化和菌体活力的下降。为此,我们从粘质沙雷氏菌H30中钓取了编码生物表面活性剂合成酶的基因swrw,并对该基因进行了插入突变。生物表面活性剂基因的突变菌经摇瓶实验验证表明该基因的突变并不会影响2,3-丁二醇的合成,在3.7L发酵罐中对该突变菌进行了补料发酵实验,发酵57h,2,3-丁二醇最高产量可达152g／L。4.2.3-丁二醇合成途径相关基因的鉴定基于已测序的三株沙雷氏菌基因组数据,进行序列比对分析和引物设计,成功克隆了粘质沙雷氏菌H30中α-乙酰乳酸脱羧酶基因(budA)、α-乙酰乳酸合成酶基因(budB)和2,3-丁二醇脱氢酶基因(budC),结果表明budA基因、budB基因和一LysR型调控因子budR组成一操纵子,而budC基因独立于操纵子存在于基因组的其它位置,文献检索表明这些基因为首次克隆。利用生物信息学软件对2,3-丁二醇代谢途径中的三个基因(budA、budB和budC)进行分析,结果表明三个基因的开放阅读框长度分别为780bp、1686bp和756bp,分别编码259、561和251个氨基酸。利用在线蛋白分析软件计算可知,三个基因编码的蛋白分子量分别为28.96kD(budA)、60.70kD(budB)和27.43kD (budC),等电点分别为5.48、5.88和5.51,三个基因编码的蛋白均为酸性蛋白。同时利用pET28a载体表达体系,表达分析了这三个基因,所得蛋白的分子量大小与预测的分子量基本一致。5.2,3-丁二醇合成的调控机制为了确认LysR型转录调控因子budR的功能,对budR进行了插入突变。结果表明budR突变后无法合成2,3-丁二醇的前体物质乙偶姻,进而导致2,3-丁二醇无法形成,说明budR调控乙偶姻的合成。检测budR突变菌中2,3-丁二醇脱氢酶的活性依旧存在,说明粘质沙雷氏菌H30中2,3-丁二醇脱氢酶独立存在于基因组其他位置,不受budR的调控。针对发酵过程pH、溶氧和副产物有机酸对2,3-丁二醇的合成具有重要影响,我们构建了启动子：lacZ融合报告载体pPbud-lacZ,利用报告基因lacZ的表达水平分别考察了pH、溶氧和副产物有机酸对启动子转录水平的影响,结果表明pH对于启动子的转录水平影响不大,而溶氧和副产物有机酸对启动子转录水平具有重大影响,溶氧越低启动子转录水平越高,乙酸、乳酸、琥珀酸和柠檬酸均能在一定程度上促进启动子的转录,其中乙酸的效果最为明显。6.2,3-丁二醇高效重组菌的构建针对发酵前期乙偶姻积累量较高,我们构建了能够在粘质沙雷氏菌H30中组成表达2,3-丁二醇脱氢酶的载体pPbud-BDH,含有该载体的粘质沙雷氏菌H30经摇瓶实验表明2,3-丁二醇脱氢酶的过表达有利于降低发酵前期乙偶姻的积累,同时可以加速耗糖。将该工程菌在3.7L发酵罐中进行补料发酵实验,在42h 2,3-丁二醇的浓度可达151g／L,生产能力为3.59g／L·h,产物得率为94.97%。更多还原

【Abstract】 2,3-butanediol is an important biobased bulk chemical due to its extensive industrial application. It has been shown to have potential applications in the manufacture of printing inks, perfumes, fumigants, moistening and softening agents, explosives and plasticizers, and as a carrier for pharmaceuticals. In this current thesis,2,3-butanediol production by Serratia marcescens H30 was studied by using traditional fermentation regulatory methods and modern metabolic engineering technique. The detailed work was introduced as following:1. Optimization of fermentative conditions and medium compositionsThe optimization of flask fermentation conditions and cultural medium compositions for 2,3-butanediol production by Serratia marcescens H30 was investigated. The results showed that the optimal fermentation conditions included initial pH of 7.0, process controlled to pH 6.0, cultivation at 30℃, inoculum size of 5%(v/v) and 50mL medium in 250mL flask. On the basis of the above fermentation conditions, the concentrations of medium components were optimized in shake flask fermentations by using single factor experiment, Plackett-Burman design and Response Surface methodology. And the optimal medium (g/L) (sucrose 90; yeast extract 33.36; sodium citrate 10; sodium acetate 4; MnSO4 0.1; MgSO4 0.3; NH4H2PO4 3) was obtained. It could improve 2,3-butanediol production from 15.93 g/L to 44.7g/L and shorten fermentation period from 48h to 15h. Fed-batch experiments in flask showed residual sucrose concentration of 15-30g/L favored 2,3-butanediol production.2. The experiments in 3.7L bioreactor and scale up in 50L-5000L bioreactorsThe fermentation experiments were firstly carried out in 3.7L bioreactor. Several feeding and regulatory strategies, including pulse fed batch, constant feed rate fed batch, constant residual sucrose concentration fed batch with respiratory quotient (RQ) control and pH self-control with constant residual sucrose concentration fed batch and RQ control, were compared for improving the production of 2,3-butanediol. The obtained 2,3-butanediol concentrations were 115.5g/L,117.14g/L,139.92g/L and 130.65g/L, respectively. Ultimately, a suitable control strategy which combined the RQ control with the constant residual sucrose concentration fed batch was developed. Using this strategy, the DCW (Dry Cell Weight) of 16.05g/L with the 2,3-butanediol productivity of 3.34g/L-h and the yield of 94.67%was obtained. Then we performed fermentation scale up experiments in 50L-5000L bioreactors using the above strategy. The 2,3-butanediol concentrations obtained in 50L-5000L bioreactors were over 130g/L, and in 5000L bioreactor the 2,3-butanediol concentrations of 130.2g/L was achieved.3. Construction of the swrw mutant encoding a biosurfactant synthase in S. marcescens H30Biosurfactant synthesis by S. marcescens H30 during the fermentation process for 2,3-butanediol producton results in a lot foam formation, which is harmful to the fermentation process due to microbial pollution. In addition, excessive anti-foam agent added would influence the microbial activity and emulsify fermentation broth. So we amplified and cloned the swrw gene encoding the biosurfactant synthase in S. marcescens H30. A swrw mutant by mutagenesis with the suicide vector was constructed successfully. The flask experiment results of the swrw mutant on the basis of optimized medium showed that the swrw inactivation had no effect on the growth and 2,3-butanediol production. In 3.7L bioreactor, the swrw mutant was performed fermentation experiments using constant residual sucrose concentration fed batch-RQ control strategy. The maximum 2,3-butanediol concentration of 152g/L was obtained at 57h.4. Cloning, characterization and expression of the genes involved in 2,3-butanediol pathway from S. marcescens H30Based on the genome sequence of Serratia genus, we successfully cloned the genes involved in 2,3-butanediol pathway from S. marcescens H30. The sequencing result showed the budA, budB and budR genes, encoded a-acetolactate decarboxylase (a-ALDC), a-acetolactate synthase (a-ALS) and a LysR type regulatory factor, located in one operon designated acetoin operon. While the budC gene encoded 2,3-butanediol dehydrogenase (acetoin reductase) exsited in another position of the genome. Search of the knowledge database comfirmed that this was the first report of the genes involved in 2,3-butanediol pathway from S. marcescens H30. Bioinformatics analysis showed that the strengths of the budA, budB and budC genes were 780bp,1686bp and 756bp respectively, and encoded proteins of 259,561 and 251 residues. Their molecular weights and isoelectric points were 28.96kD and 5.48,60.7kD and 5.88,27.43kD and 5.51. They were acidic proteins judged from the calculated pI values. Expression products of the genes with pET28a system exhibited comparable molecular weights using SDS-PAGE analysis.5. Regulatory mechanism of 2,3-butanediol biosynthesis by S. marcescens H30To identify the function of budR, a budR mutant by mutagensis with the suicide vector was constructed successfully. The fermentation experiments showed that the budR gene regulated the precursor (acetoin) biosynthesis of 2,3-butanediol as a positive regulatory factor. While assay of 2,3-butanediol dehydrogenase from the budR mutant showed that the budC gene encoding 2,3-butanediol dehydrogenase from S. marcescens H30 remained its catalytic activity. The results indicated that the transcription and expression of the budC gene was not regulated by the budR gene.A lacZ reporter vector pPbud-lacZ fusing the promoter of the acetoin operon and the lacZ gene from PSV-beta-gal was constructed and used to analyze the effects of pH, dissolve oxygen and organic acid on the transcriptional level of the promoter. The results showed that pH had little effect on the transcriptional level of the promoter, while dissolve oxygen and organic acid had significant effect on the transcriptional level of the promoter. The expression of the lacZ gene was raised with the dissolve oxygen lowering. For organic acids, acetic acid, lactic acid, succinic acid and citric acid could increase partially the expression of the lacZ gene, it seemed acetic aicd was the best additive of the fermentation medium.6. Construction of engineering strain for 2,3-butanediol productionIn order to decrease the accumulation of acetoin in the fermentation process for 2,3-production by S. marcescens H30, a constitutive expression vector pPbud-BDH was constructed for over-expression of 2,3-butanediol dehydrogenase (BDH) from Klebsiella pneumoniae in S. marcescens H30. Flask experiments using the engineering strain showed that over-expression of BDH in S. marcescens H30 could lower acetoin accumulation and accelerate the sucrose consumption. In 3.7L bioreactor, the engineering strain was performed fermentation experiments using constant residual sucrose concentration fed batch-RQ control strategy. The maximum 2,3-butanediol concentration of 151g/L was obtained at 42h with the 2,3-butanediol productivity of 3.59g/L-h and the yield of 94.97%.更多还原