【作者】 杨套伟；

【导师】 许正宏；

【作者基本信息】 江南大学 ， 发酵工程， 2013， 博士

【摘要】 2,3-丁二醇是一种重要的生物基四碳平台化合物，被广泛应用于化工、食品、医药、燃料及航空航天等多个领域。随着石油资源日益短缺，微生物法合成2,3-丁二醇越来越受到人们的重视。目前，2,3-丁二醇高产菌株主要是克雷伯氏菌、产气肠杆菌和粘质沙雷氏菌，然而这些菌株都具有潜在致病性，不符合工业化安全生产的要求，虽然最近也有安全菌株（如枯草芽胞杆菌）的报道，但2,3-丁二醇产量偏低。本文通过传统育种的方法，筛选到一株具有工业化生产2,3-丁二醇潜力的安全菌株（解淀粉芽胞杆菌B10-127），并结合发酵调控与基因工程技术进行了以下研究工作：1.高产2,3-丁二醇安全菌株的筛选通过考察菌株在高浓度葡萄糖条件下生长及对葡萄糖利用效率与肌酸显色法相结合的方法，筛选到一株具有高产2,3-丁二醇潜力的菌株B10-127，通过细胞形态观察、生理生化特征鉴定和16Sr RNA序列分析，确定其为解淀粉芽胞杆菌（Bacillus amyloliquefaciens）。经初步发酵，2,3-丁二醇的产量为52.2g/L，生产强度为0.68g/(L h)。2.摇瓶水平发酵条件与培养基组分优化首先，在摇瓶水平上对解淀粉芽胞杆菌B10-127发酵葡萄糖合成2,3-丁二醇的培养条件进行优化，优化后的最适培养条件为：培养温度37℃，摇床转速150r/min，培养基初始pH6.5，接种量6%。随后，经过单因素和响应面综合优化，确定最佳培养基组分为：玉米浆31.9，豆粕22.0，柠檬酸铵5.6，K2HPO43H2O2.5，MgSO47H2O0.3， MnSO47H2O0.05，FeSO47H2O0.05，琥珀酸0.3g/L。在优化后的最适条件下培养，菌体量提高了14.6%，发酵周期从76h缩短至48h，2,3-丁二醇产量达到63.4g/L，提高了21.4%，生产强度提高了91.3%，副产物乙偶姻降低了34.4%。3.玉米浆对2,3-丁二醇发酵调控机理初探考察了玉米浆对2,3-丁二醇发酵的影响，结果发现：在低玉米浆浓度下，菌体生长速率较低，此时副产物乙偶姻大量积累；在高的玉米浆浓度下，菌体快速生长，菌体量较高，且此时菌株主要积累2,3-丁二醇，而其前体物质（乙偶姻）积累量很少；与不添加玉米浆相比，2,3-丁二醇产率提高了55.6%，生产强度提高了1.52倍，乙偶姻积累量降低了69.0%，2,3-丁二醇/乙偶姻的比值提高了3.99倍。随后，我们初步探讨了玉米浆对2,3-丁二醇发酵的调控机理。乙偶姻还原酶专一催化乙偶姻合成2,3-丁二醇，同时需要NADH的参与。在高的玉米浆浓度下，发现菌体长势良好，菌体量高，提高了胞内NADH水平，并提高了糖耗效率，缩短了发酵周期；同时，在此情况下，乙偶姻还原酶活力较高，乙偶姻被迅速转化为2,3-丁二醇，并导致胞内NADH/NAD+比值下降。4.3-磷酸甘油醛脱氢酶与乙偶姻还原酶共表达研究在EMP途径中，3-磷酸甘油醛脱氢酶（GAPDH）氧化3-磷酸甘油醛合成1,3-二磷酸甘油酸，需要等量的氧化型辅酶NAD+参与；在2,3-丁二醇支路中，乙偶姻还原酶（ACR）催化还原乙偶姻合成2,3-丁二醇，该步反应需要等量的还原型辅酶NADH参与，所以，在整个代谢途径中，GAPDH和ACR构成一个辅酶循环再生体系。我们首次尝试，将来源于解淀粉芽胞杆菌的依赖于NAD+的3-磷酸甘油醛脱氢酶和依赖于NADH的乙偶姻还原酶基因在解淀粉芽胞杆菌中过量表达，加强辅酶循环再生，成功的提高了发酵液中2,3-丁二醇的产量和生产强度。过量表达GAPD和ACR时，依赖于NADH的乙偶姻向2,3-丁二醇通量加强了16.7%，乙偶姻降低了60.9%，同样依赖于NADH的乳酸和琥珀酸支路的通量均呈现下降趋势，分别下降了25.9%和39.0%。5.发酵罐水平工艺参数的控制优化考察了溶氧对2,3-丁二醇合成的影响，结果发现：溶氧水平越高，菌体生长越快，发酵周期越短，但是2,3-丁二醇的产量越低，副产物乙偶姻的积累量越高。针对菌株在不同的发酵阶段对氧需求量的不同，我们采用分阶段控制转速的策略来调控发酵液中溶氧水平，具体方式如下：0-4h搅拌转速控制在较低水平350r/min，4-16h搅拌转速提高至400r/min，16h后搅拌转速降为350r/min。采用此三阶段搅拌转速调控策略进行2,3-丁二醇的分批发酵，结果发现：发酵28h，葡萄糖便消耗殆尽，此时葡萄糖消耗速度达到5.71g/(L h)，2,3-丁二醇最高产量达到72.8g/L，生产强度2.60g/(L h)（比恒定转速为300、350和400r/min的分批发酵相比，分别提高了85.7%、41.3%和23.8%）；乙偶姻产量下降至4.72g/L（比恒定转速为300、350和400r/min的分批发酵相比，分别降低了53.3%、47.1%和69.2%）。2,3-丁二醇发酵过程前期，pH会因为有机酸的合成而逐步下降，菌株会转而合成中性物质2,3-丁二醇以阻止生长环境过度酸化，而后pH逐渐升高；而2,3-丁二醇合成的最适pH偏酸性，所以中后期应控制pH在6.5以下。随后，采用pH分段控制-脉冲补料发酵策略，2,3-丁二醇的最高产量达到133.2g/L，此结果可与前期报道的2,3-丁二醇高产菌相媲美。6.粗甘油与糖蜜共底物发酵生产2,3-丁二醇研究为了降低2,3-丁二醇的生产成本，提升目的菌株利用前景。我们尝试利用生物柴油副产物粗甘油为底物进行2,3-丁二醇发酵实验。研究发现，菌株利用糖质原料的效率明显高于利用甘油的效率，为了提高甘油利用率，我们尝试将蔗糖作为甘油发酵的辅底物，结果发现：蔗糖作为辅底物时，显著提高了菌株利用甘油合成2,3-丁二醇的效率。甜菜糖蜜是制糖工业中的一种副产品，含有大量的蔗糖，将甜菜糖蜜（替代蔗糖）作为辅底物与甘油共发酵，结果发现，菌体生长和底物消耗速率得到显著提高，进而提高了菌株利用甘油合成2,3-丁二醇的效率，同时降低了生产成本。采用分阶段供氧策略和分段控制pH-脉冲流加发酵，2,3-丁二醇最大产量达到83.3g/L，生产强度达到0.85g/(L h)，此结果是目前报道的发酵粗甘油生产2,3-丁二醇的最高产量。更多还原

【Abstract】 Interest in this bioprocess has increased remarkably because2,3-butanediol (2,3-BD) has a large number of industrial applications, and microbial production will alleviate the dependence on oil supply for the production of platform chemicals. Additionally,2,3-BD has potential applications in the manufacture of printing inks, perfumes, fumigants, moistening and softening agents, explosives, plasticizers, foods, and pharmaceuticals. So far, absolutely unbeatable in efficient production of2,3-BD are Klebsiella pneumoniae, K. oxytoca, Enterobacter aerogenes and Serratia marcescens. It is important to note that mainly class2(pathogenic) microorganisms are employed in the2,3-BD fermentation. Industrial-scale fermentation requires obeying safety regulations, which implies that class2microorganisms are unwanted in such applications. Therefore, an urgent need for class1microorganisms (safe) is pronounced. Such microorganisms have been reported as2,3-BD producers, however, the ef ficiency of the production was much too low for an economic process. In the current study, a GRAS (Generally Recognized As Safe) strain of Bacillus amyloliquefaciens producing2,3-BD designated as B10-127was isolated in our lab. The strain B10-127produced2,3-BD effectively under the condition of20%glucose (quality concentration), showed a high-glucose tolerance. In this current study,2,3-BD production by was B. amyloliquefaciens was studied by using traditional fermentation regulatory methods and modern metabolic engineering technique. The detailed work was introduced as following:1. Isolation and identification of2,3-BD high production and safe microorganismsGenerally, only a strain with the abilities of high-glucose tolerance and effective glucose utilization can be an excellent candidate for use in the production of2,3-BD at an industrial scale. Based on this perspective, we designed a screening culture containing300g/L of glucose. The enrichment process was to select the interested strains, which grown quickly to increase its proportion in the mixed culture using glucose as the carbon source. And it was also combined with voges-proskavr test. When action (precursor of2,3-BD) was detected in the enrichment broth, it might imply that2,3-BD producing microorganisms existed in the culture. After purified several times, several strains which could tolerate glucose up to300g/L and produce2,3-BD effectively were isolated from a soil sample collected from grassland. One isolate was further identified as B. amyloliquefaciens B10-127by its16S rRNA gene sequence and physiological biochemical analysis. Under a unoptimized condition, the titer of2,3-BD were52.2g/L with a2,3-BD productivity of0.68g/(L h).2. Optimization of medium and process parameters for the production of2,3-BDThe optimization of flask fermentation conditions and cultural medium composition on2,3-BD production have been studied. The results showed that the optimal culture conditions included initial pH of6.5, cultivation at37oC, inocultun size of6%(v/v) and shaking speed of150r/min. Corn steep liquor, soybean meal and ammonium citrate were found to be the key factors in the fermentation according to the results obtained from the Plackett–Burman experimental design. The optimal con-centration range of the three factors was examined by the steepest ascent path, and their optimal concentration were further optimized via response surface methodological approach and determined to be31.9,22.0and5.58g/L, respectively. Under optimized conditions, biomass increased by14.6%, the fermentation time was shorten from76to48h, the titer of2,3-BD increased by21.4%, the productivity of2,3-BD increased by91.3%, and acetoin decreased by34.4%, when compared with the results obtained under unoptimized conditions.3. Effects of corn steep liquor on2,3-BD and acetoin productionIt was found that the initial concentration of corn steep liquor (CSL) have remarkable effects on not only2,3-butanediol (2,3-BD) and acetoin production, but also on the ratio of2,3-BD to acetoin. Acetoin reductase catalyzes the conversion of acetoin to2,3-BD. Results obtained from tests performed under low CSL levels were compared to that obtained under high CSL levels. When a high concentration of CSL was supplemented, cell growth was improved, acetoin reductase (ACR) was stimulated, the concentration of2,3-BD increased by55.6%, acetoin decreased by69.0%, and the ratio of2,3-BD to acetoin increased by3.99-fold. Compared to the BD/AC ratio obtained in low CSL levels, the BD/AC ratio was much higher when CSL levels were high. This indicates that the NADH-oxidizing branch of acetoin to2,3-BD was enhanced, resulting in the decrease of the NADH/NAD+ratio.4. Enhanced2,3-BD production by overexpressing a NADH/NAD+regeneration systemIn the2,3-BD metabolic pathway, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of3-phosphate glyceraldehydes to1,3-bisphosphoglycerate with concomitant reduction of NAD+to NADH while acetoin reductase (ACR) catalyzes the conversion of acetoin to2,3-butanediol with concomitant oxidation of NADH to NAD+. So GAPDH and ACR are bound up with regeneration of cofactors. In this study, we firstly introduced extra copies of GAPDH/ACR enzymes into the GRAS strain B. amyloliquefaciens and studied their effect on2,3-BD fermentation. It was found that no difference of NADH and NAD+levels was observed between mutant and parent strain. While for mutant strain glucose fluxes were redistributed, in the NADH-dependent pathways, yield of2,3-BD of was16.7%higher and yields of by-products acetoin, lactate and succinate were separately60.9%,39.0%and25.9%lower than that of parent strain.5. The experiments in a5-L biorcaetorThe pH and oxygen supply are the very important variables in the2,3-BD fermentation. Batch fermentative production of2,3-BD by B. amyloliquefaciens was investigated using various oxygen supply methods though varying agitation speed. Based on the analysis of three kinetic parameters including specific cell growth rate (μ), specific glucose consumption rate (qs) and specific2,3-BD formation rate (qp), a multi-stage agitation speed control strategy, aimed at achieving high concentration, high yield and high productivity of2,3-BD, was proposed. At the first4h, agitation speed was controlled at350r/min, subsequently agitation speed was raised to400r/min until16h, and then, agitation speed was reduced to350r/min. Finally, the maximum titer of2,3-butanediol reached72.8g/L with the productivity of2.60g/(L h), which were8.2%and23.8%over the best results controlled by constant agitation speeds. In a pulse fed-batch fermentation, combining with a two-stage pH control strategy, the2,3-BD concentration and productivity were significantly improved to133.2g/L and2.78g/(L h), respectively.6. Enhancement of waste glycerol consumption with beet molasses co-fermentationThe production of2,3-BD from glycerol was inhibited when compared with the equivalent cultures performed on sugars. In the current study, Bacillus amyloliquefaciens was first reported to exhibit a remarkable producing potency of2,3-BD from biodiesel-derived glycerol, and the beet molasses was employed as co-substrate. Surprisedly, the molasses addition stimulated significant increases of2,3-BD production, and simultaneously reduced the duration of fermentation. At the beginning of fermentation the molasses addition enhanced the productivity of2,3-BD, and molasses fed during the fermentation increased the conversion rate of2,3-BD. In fed-batch fermentation,15g/L of molasses were added to the bioreactor before inoculation, and after6h, a solution containing80%glycerol and15%molasses was then fed into the bioreactor. The2,3-BD concentration, conversion, and productivity were improved significantly to83.3g/L,0.42g/g, and0.87g/(L h), respectively. To our knowledge, these results might hit a new record on2,3-BD fermentation from biodiesel-derived glycerol.更多还原