【作者】 郑辉杰；

【导师】 赵学明；

【作者基本信息】 天津大学 ， 生物化工， 2009， 博士

【摘要】 本文以菊糖芽孢乳杆菌SP-BME126为出发菌株,将基因组重排技术和进化工程相结合,进行了具有耐酸性的菊糖芽孢乳杆菌D-乳酸高产株的选育。首先通过UV、DES诱变和pH梯度筛选三种方法,筛选出8株遗传稳定性较高的菌株,构建了基因组重排的亲本菌株库。利用响应面实验设计方法对菊糖芽孢乳杆菌原生质体的制备及再生条件进行了研究,其优化条件为:菌龄12h,酶浓度7.75mg/mL,酶解时间1.59h,酶解温度38℃。在此条件下菊糖芽孢乳杆菌原生质体制备率和再生率的乘积可达到59.6%,在RSM优化工艺条件的基础上,得到8种原生质体,分别研究了PEG浓度、融合温度、融合时间以及Ca2+浓度对原生质体融合的影响。经过三轮有效的基因组重排,得到高产D-乳酸的基因组重排菌F3-3,通过在MRS发酵培养基(pH5.0)中发酵培养,其菌体OD值达到3.84,D-乳酸最大积累浓度为66.5g/L。用进化工程的方法对基因组重排菌F3-3进行了耐酸性的进化选择。首先确定了菌株在不同pH下的代时和筛选压力,通过在恒定pH和梯度pH下的持续进化选择,发现梯度pH进化筛选耐酸性菌株效果优于恒定pH下的筛选。将进化工程菌EV-1在pH5.0的MRS发酵培养基中进行发酵罐培养,其最大OD值为3.89,D-乳酸的最大产量为85.2g/L。通过将96孔板和生物传感器分析有机结合,确定了在高通量筛选中菌株培养时间为3天,D-乳酸提取溶剂为无菌水,以0.015%溴甲酚紫作为微孔板培养中的筛选试剂,在此基础上确定了一种用于胞外代谢物的菌株高通量筛选方法。利用GC/MS和HPLC等分析手段对D-乳酸发酵过程中胞内外的代谢产物进行了分析,推测出14种胞内代谢物;建立了S. inulinus菌的代谢通量模型;通过代谢通量分析和关键酶活分析,对比了进化工程菌EV-1和出发菌株SP-BME126在不同发酵时期的代谢通量分布情况和关键酶活的变化。通过单因素实验、P-B实验和中心组合实验设计对进化工程菌EV-1的发酵工艺条件进行了优化,其优化条件为:葡萄糖95g/L、蛋白胨22g/L、种龄24h、接种量5%、碳酸钙3g/100mL、装液量112mL/500mL、温度35℃。在pH5.0的MRS优化培养基中发酵培养,菌株最大D-乳酸产量为91.7g/L,葡萄糖转化率达到96.5%,较优化前的进化工程菌株EV-1(85.2g/L)提高了7.63%。更多还原

【Abstract】 In this paper, evolutionary engineering was integrated with genome shuffling technology to generate the evolved Sporolactobacillus inulinus (SP-BME126) with acid tolerance and high lactic acid production in laboratory-scale fermentation.Eight mutants with high genetic stabilities were obtained by using ultraviolet radiation, DES mutations and pH gradient screenings were used as the start population for genome shuffling. The conditions for protoplast preparation and regeneration were studied by response surface methodology (RSM), and the obtained optimal condition was: seed age 12 h, lysozyme concentration 7.75 mg/l, treating time 1.59 h and 38℃, which resulted that the product of protoplast preparation and regeneration was above 59.6 %. Then, eitht protoplasts were obtained based on the optimized condition from RSM. The effects of PEG concentration, Ca2+ concentration, fusion time and temperature on protoplast fusion were studied. A D-lactic acid producing mutant F3-3 was obtained by three rounds of genome shuffling. Under the fermentation condition (pH5.0) in MRS culture broth, the strain F3-3 could produce 66.5 g/L D-lactic acid.At a 5 L fermentor, F3-3 acquired high acid tolerance by evolution engineering. The generation time and selection pressure of F3-3 were determined and it was found that the efficiency of pH-gradient selection for obtaining strains with higher acid tolerance was much better than that of immobilized pH selection. Under the fermentation condition, the evolved strain EV-1 could produce D-lactic acid 85.2 g/L at pH5.0.The 96-well plate was integrated with biosensor to determine the content of lactic acid in the colonies. The culture time of strains in 96-well plate was 3 days. Sterile water was employed as solvent for the abstract of lactic acid from the colonies. The solution of 0.015 % bromcresol purple was applied to strain screening in 96-well plate. So a platform was established for high-throughput screening of strains producing extracellular metabolite.A scheme of metabolomics analysis by GC/MS was developed for detecting the metabolites in the broth. Fourteen intracellular metabolites were confirmed according to the GC/MS data. A quantitative metabolic flux model was developed and tested for Sporolactobacillus inulinus. Accordingly, the metabolic flux distribution was determined between the evolved strain EV-1 and the parental strain SP-BME126 at different fermentation phase.The optimal fermentation condition for D-lactic acid production was investigated by single factor experiment, Plackett-Burman design, central composite design and response surface methodology (RSM). The obtained optimal condition was: glucose 95 g/L, peptone 22 g/L, seed age 24 h, inoculum size 5 %, calcium carbonate 3 g/100mL, medium volume 112 mL/500mL and temperature 35℃. Under this condition, the evolved strain EV-1 showed 96.5 % yield efficiency, led to 7.63 % increase in D-lactic acid production from 85.2 g/L to 91.7 g/L.更多还原