Bacillus amyloliquefaciens高效合成2,3-丁二醇及其发酵调控

Efficient Production of2,3-butanediol and its Regulation in Bacillus Amyloliquefaciens

作者: 专业:发酵工程 导师:许正宏 年度:2013 学位:博士 

关键词
解淀粉芽胞杆菌 2,3-丁二醇 乙偶姻 发酵调控 辅底物

Keywords
Bacillus amyloliquefaciens, 2,3-butanediol, acetoin, regulation, co-substrate
        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。
    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.
        

Bacillus amyloliquefaciens高效合成2,3-丁二醇及其发酵调控

摘要3-5
Abstract5-7
第一章 绪论12-22
    1.1 2,3-丁二醇的概述12
        1.1.1 2,3-丁二醇的性质12
        1.1.2 2,3-丁二醇的用途12
    1.2 2,3-丁二醇生产方法12-13
    1.3 微生物法合成 2,3-丁二醇的代谢途径13-14
    1.4 微生物合成 2,3-丁二醇的生理功能14
    1.5 2,3-丁二醇生产菌株14-15
    1.6 提高微生物合成 2,3-丁二醇效率的主要策略15-20
        1.6.1 菌种改良15-17
        1.6.2 培养基组分优化17-18
        1.6.3 发酵环境条件优化18-20
    1.7 本课题研究的意义与内容20-22
        1.7.1 本课题研究意义20
        1.7.2 本课题研究内容20-22
第二章 2,3-丁二醇安全高产菌株的分离、筛选及鉴定22-34
    2.1 引言22
    2.2 实验材料22-23
        2.2.1 土样22
        2.2.2 主要试剂22
        2.2.3 主要实验仪器22
        2.2.4 主要溶液及配制方法22-23
        2.2.5 培养基23
    2.3 实验方法23-27
        2.3.1 细菌的分离筛选23-24
        2.3.2 菌落及细胞形态观察24
        2.3.3 菌株的生理生化特征24
        2.3.4 16S rRNA 序列测定24
        2.3.5 基于 16S rRNA 序列分析的同源性与进化距离分析24-25
        2.3.6 分析方法25-27
    2.4 结果27-31
        2.4.1 菌株的分离与初筛27
        2.4.2 菌株复筛27-28
        2.4.3 菌株 B10-127 的菌体形态28
        2.4.4 菌株 B10-127 的生理生化特征28-29
        2.4.5 菌株 B10-127 的 16S rRNA 序列测定29-30
        2.4.6 B. amyloliquefaciens B10-127 生长曲线测定30
        2.4.7 B. amyloliquefaciens B10-127 发酵葡萄糖合成 2,3-丁二醇过程曲线30-31
    2.5 讨论31-32
    2.6 本章小结32-34
第三章 B.amyloliquefaciens生产 2,3-丁二醇摇瓶发酵条件研究34-50
    3.1 引言34
    3.2 材料与方法34-35
        3.2.1 菌种34
        3.2.2 培养基34
        3.2.3 培养方法34
        3.2.4 分析方法34-35
    3.3 结果35-48
        3.3.1 温度对菌株 B10-127 发酵生产 2,3-丁二醇的影响35
        3.3.2 摇床转速对菌株 B10-127 发酵生产 2,3-丁二醇的影响35-36
        3.3.3 初始 pH 对菌株 B10-127 发酵生产 2,3-丁二醇的影响36
        3.3.4 接种量对菌株 B10-127 发酵生产 2,3-丁二醇的影响36-37
        3.3.5 菌株 B10-127 对各类碳源利用情况37
        3.3.6 初始葡萄糖浓度对菌株 B10-127 发酵生产 2,3-丁二醇的影响37-38
        3.3.7 有机氮源对菌株 B10-127 发酵生产 2,3-丁二醇的影响38-39
        3.3.8 玉米浆对菌株 B10-127 发酵生产 2,3-丁二醇的影响39
        3.3.9 玉米浆和豆粕添加比例对菌株 B10-127 发酵生产 2,3-丁二醇的影响39-40
        3.3.10 无机氮源对菌株 B10-127 发酵生产 2,3-丁二醇的影响40
        3.3.11 K2HPO4 对菌株 B10-127 发酵生产 2,3-丁二醇的影响40-41
        3.3.12 有机酸对菌株 B10-127 发酵生产 2,3-丁二醇的影响41-42
        3.3.13 无机盐对菌株 B10-127 发酵生产 2,3-丁二醇的影响42
        3.3.14 Plackett-Burman Design 设计42-44
        3.3.15 最陡爬坡实验44
        3.3.16 Response Surface Methodology 分析实验44-46
        3.3.17 优化前后 2,3-丁二醇发酵过程曲线46-48
    3.4 讨论48-49
    3.5 本章小结49-50
第四章 玉米浆对 2,3-丁二醇发酵调控的机理初探50-60
    4.1 引言50
    4.2 材料与方法50-51
        4.2.1 菌种与质粒50
        4.2.2 培养基50
        4.2.3 培养方法50
        4.2.4 基因组 DNA 提取50
        4.2.5 PCR 产物克隆50-51
        4.2.6 细胞粗提取液的制备及 ACR 酶活测定方法51
        4.2.7 细胞内 NADH/NAD+ 提取及测定方法51
        4.2.8 分析方法51
    4.3 结果51-57
        4.3.1 玉米浆添加量对菌株 B10-127 生长的影响51-52
        4.3.2 玉米浆添加量对菌株 B10-127 利用葡萄糖消耗速率的影响52-53
        4.3.3 玉米浆添加量对 2,3-丁二醇发酵的影响53
        4.3.4 玉米浆添加量对胞内乙偶姻还原酶酶活的影响53-54
        4.3.5 玉米浆添加量对胞内 NADH 和 NAD+ 浓度的影响54-55
        4.3.6 acr 基因敲除菌株的构建及酶活测定55-56
        4.3.7 玉米浆浓度对突变菌株 acr ::cat 发酵合成乙偶姻的影响56-57
    4.4 讨论57-59
    4.5 本章小结59-60
第五章 通过加强辅酶循环再生促进 2,3-丁二醇合成60-73
    5.1 引言60
    5.2 材料与方法60-62
        5.2.1 菌种与质粒60
        5.2.2 培养基60
        5.2.3 培养方法60-61
        5.2.4 解淀粉芽胞杆菌基因组 DNA 提取61
        5.2.5 PCR 产物克隆61
        5.2.6 枯草芽胞杆菌转化方法61
        5.2.7 重组质粒稳定性分析61
        5.2.8 细胞粗提取液的制备及酶活测定方法61-62
        5.2.9 分析方法62
    5.3 结果62-70
        5.3.1 gapA 和 acr 表达体系的构建和鉴定62-65
        5.3.2 gapA 和 acr 基因在解淀粉芽胞杆菌中的表达分析65
        5.3.3 过量表达 gapA 基因对解淀粉芽胞杆菌生长及 2,3-丁二醇发酵的影响65-68
        5.3.4 过量表达 acr 基因对解淀粉芽胞杆菌生长及 2,3-丁二醇发酵的影响68
        5.3.5 过量共表达 gapA 和 acr 基因对解淀粉芽胞杆菌生长及 2,3-丁二醇发酵的影响68-69
        5.3.6 重组菌 AG 代谢分析69-70
        5.3.7 玉米浆浓度对重组菌株 AG 生长和 2,3-丁二醇发酵的影响70
    5.4 讨论70-72
    5.5 本章小结72-73
第六章 发酵罐水平 2,3-丁二醇发酵工艺控制研究73-85
    6.1 引言73
    6.2 材料与方法73-74
        6.2.1 菌种73
        6.2.2 培养基73
        6.2.3 培养方法73
        6.2.4 分析方法73-74
    6.3 结果74-81
        6.3.1 pH 对 2,3-丁二醇发酵的影响74-75
        6.3.2 不同溶氧水平菌株发酵生产 2,3-丁二醇的过程曲线75-77
        6.3.3 不同溶氧水平下菌株发酵生产 2,3-丁二醇的动力学特征77-78
        6.3.4 多阶段供氧控制模式的提出和实验验证78
        6.3.5 分批补料发酵78-81
    6.4 讨论81-84
    6.5 本章小结84-85
第七章 廉价底物粗甘油与糖蜜共底物发酵高效生产 2,3-丁二醇85-96
    7.1 引言85
    7.2 材料与方法85
        7.2.1 菌株85
        7.2.2 培养基85
        7.2.3 培养方法85
        7.2.4 分析方法85
    7.3 结果85-93
        7.3.1 解淀粉芽胞杆菌对生物柴油副产物粗甘油的利用情况85-86
        7.3.2 糖作为辅底物对解淀粉芽胞杆菌利用甘油合成 2,3-丁二醇的影响86-88
        7.3.3 利用糖蜜作为辅底物提高菌株利用甘油合成 2,3-丁二醇的效率88-89
        7.3.4 糖蜜与粗甘油共底物发酵合成 2,3-丁二醇供氧工艺研究89-91
        7.3.5 糖蜜与粗甘油共底物流加发酵合成 2,3-丁二醇91-93
    7.4 讨论93-94
    7.5 本章小结94-96
主要结论与展望96-98
    主要结论96-97
    展望97-98
论文创新点98-99
致谢99-101
参考文献101-108
附录Ⅰ:作者在攻读博士学位期间发表的论文及申请的专利108-109
附录Ⅱ:菌株 B10-127的 16Sr RNA部分序列109-110
附录Ⅲ:来源于解淀粉芽胞杆菌 B10-127的乙偶姻还原酶基因序列110-111
附录Ⅳ:来源于解淀粉芽胞杆菌 B10-127的 3-磷酸甘油醛脱氢酶基因序列111


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