【作者】 李爽；

【导师】 闻建平；

【作者基本信息】 天津大学 ， 生物化工， 2010， 硕士

【摘要】 D-乳酸是众多手性物质的合成前体,广泛应用于化工、农业、食品、医药、环保等领域。目前,国际上主要采用乳酸菌发酵生产D-乳酸,发酵原料成本较高且产量较低,导致产品价格较高。与乳酸菌相比较,谷氨酸棒杆菌在厌氧条件下能够以相对简单的原料为底物,达到较高的乳酸生产效率,发酵优势明显,但目前国际上这方面的研究报道很少,本研究利用谷氨酸棒杆菌在厌氧条件下的这一发酵优势,首次在C. glutamicum Res167中进行了D-乳酸代谢途径的改造,敲除L-乳酸脱氢酶基因(L-lactate dehydrogenase , ldh)并完成优势外源D-乳酸脱氢酶表达载体的构建,为实现谷氨酸棒杆菌中高效率、低成本D-乳酸的生产奠定了基础。为了得到高纯度的D-乳酸,需要阻断谷氨酸棒杆菌中的L-乳酸代谢途径。因此,本文首先对D-乳酸及L-乳酸代谢途径进行分析,并对L-乳酸脱氢酶基因进行敲除研究。从C. glutamicum Res167基因组中克隆出ldh的上下游同源臂ldh1和ldh2,利用重叠PCR将其拼接成ldh1-ldh2,然后连接到C. glutamicum Res167中的自杀载体pK18mobsacB上,构建了谷氨酸棒杆菌ldh基因敲除载体pK18mobsacBΔldh。将该质粒转化至C. glutamicum Res167中,通过卡那霉素抗性正向筛选和蔗糖培养基反向筛选获得了双交换重组菌株C. gl utamicum Res167Δldh。进行固体平板培养基生长实验,结果显示,C. glutamicum Res167Δldh在以L-乳酸为唯一碳源的基本培养基上不能生长,初步证明了L-乳酸脱氢酶基因的缺失阻断了菌体中L-乳酸的代谢,完成了L-乳酸脱氢酶基因的敲除。为了进一步提高谷氨酸棒杆菌中D-乳酸的生产效率,本工作选择来源于保加利亚乳杆菌的D-乳酸脱氢酶(D-lactate dehydrogenase, ldhA)优势异源基因,构建了含有该基因的重组载体pXJM19-ldhA。实验首先对对谷氨酸棒杆菌-大肠杆菌中的穿梭表达载体pXJM 19在谷氨酸棒杆菌中的遗传稳定性进行了研究。经过100代传代培养,结果表明,在抗生素选择压力和无抗生素选择压力下其遗传稳定率分别为98%和94%,表明该表达载体具有较高的遗传稳定性。然后以pXJM19为载体,连入了保加利亚乳杆菌中的D-乳酸脱氢酶基因(D-lactate dehydrogenase, ldhA),完成了重组载体pXJM19-ldhA的构建工作。更多还原

【Abstract】 D-lactate is an important precursor of many chiral compounds, which have been applied in many fields, such as chemical engineering, agriculture, food industry, pharmacy, e nvironmental pr otection. At p resent, D -lactate is m ainly p roduced b y lactobacillus with t he s hortcoming of hi gh cost and low yield. Corynebacterium glutamate shows obvi ous a dvantages of s imple f eedstock a nd hi gh production i n lactate production by anaerobic fermentation, compared with Lactobacillus. Hence, C. glutamicum was co nsidered a s an ideal hos t f or l actate p roduction. I n t his w ork, D-lactate lactate biosynthesis pathway was reconstructed in C. glutamicum Res167 for th e f irst tim e. T he L-lactate de hydrogenase gene (ldh) w as knocked out successfully. Further, D-lactate d ehydrogenase (ldhA) overexpression v ector w as contrusted. The researches above lay a solid foundation for the high-efficiency and low-cost production of D-lactate.In order to obtain D-lactic acid of high purity, the L-lactic acid metabolic pathway needs to be blocked in Corynebacterium gl utamicum. Firstly, th e ldh gene w as knockout in C. glutamicum Res167. The upstream homologous arm of ldh (Ldh1) and downstream homologous arm of ldh (ldh2) were cloned from C. glutamicum Res167 genome and linked by overlap PCR resulted in ldh1-ldh2. Then it was inserted in pK18mobsacB to form the knockout vector. After transformed into C. glutamicum Res167, C. glutamicum Res167Δldh recombinants were screened by positive selection of Kmr, then by reversed selection of sucrose medium. The cultures in plats showed that C. glutamicum Res167Δldh recombinants could not grow in basal medium with the sole carbon source of L-lactate, indicating that the L-lactate metabolism of the microorganism was interrupted owing to the deletion of L-lactate dehydrogenase.The pXJM19 is a shuttle expressed plasmid between Corynebacterium glutamate and Escherichia coli. In order to confirm its genetic stability, 100 generations were subcultured. The results showed that stability rates reached to 98% and 94% with and without the antibiotics pressure respectively, which indicated good genetic stability of pXJM19 i n C. gl utamicum Res167. Then, t he e xpression pl asmid pXJM19-ldhA containing heterogeneous ldhA from Lactobacillus bulgaricus was constructed.更多还原