基于木质纤维产丁醇高产菌株选育及关键酶基因表达分析

发布时间：2018-02-23 19:40

本文关键词： 糖丁基梭状芽孢杆菌生物丁醇基因组改组原生质体融合进化工程实时荧光定量PCR 响应面　出处：《中南林业科技大学》2017年博士论文　论文类型：学位论文

【摘要】：随着全球化石能源的日渐枯竭和快速增长的能源需求,能源资源约束的日益加剧和生态环境问题的不断突出,保障能源安全和发展绿色可再生能源已引起各国政府的高度重视。因此,利用可再生的木质纤维素生物质来生产替代燃料受到了广泛的关注。而生物丁醇作为一种能与汽油以任意比例混和使用甚至替代汽油的可再生清洁能源也迎来了全新的发展机遇。但利用木质纤维生产生物丁醇目前尚存在产量低,原料利用率不高等难题。鉴于此,本研究从菌种的选育出发,以具有广谱碳水化合物发酵能力的糖丁基梭状芽孢杆菌(Clostridium saccharobutylicum)ATCC BAA-117为出发菌株,将基因组改组技术与进化工程育种相结合,进行了适应高浓度杨木水解液的生物丁醇高产菌株的选育,成功获得了一株能高效利用杨木水解液发酵高产丁醇的重组进化菌株gsGD-1。同时,对选育菌株gsGD-1利用杨木水解液产丁醇的发酵条件进行了优化,并进一步采用实时荧光定量PCR的方法对出发菌株和改组后高产重组进化菌株gsGD-1之间的丁醇合成酶关键基因表达差异进行了分析。研究内容及取得的主要结果如下:1.建立了用于基因组改组的遗传多样性亲本菌株库。出发菌株Clostridium saccharobutylicum ATCC BAA-117经紫外诱变(UV)、核糖体工程育种技术和丁醇耐受性筛选三种方法获得9株能稳定遗传的正向性状菌株,包括丁醇产量高于出发菌株的突变菌6株,分别为UV-1、UV-2、UV-3、RE-1、RE-2和RE-3,其丁醇产量均比原始菌株提高20%以上;丁醇耐受性高于出发菌株的突变株3株,分别为HBT-1、HBT-2和HBT-3,其丁醇耐受性均较原始菌株提高25%以上。这9株菌株组成基因组改组的亲本菌株库,用于后续的递推式原生质体融合实验。2.获得了Clostridium saccharobutylicum原生质体制备、再生和融合的最佳条件。通过单因素实验、中心组合实验设计与响应面分析得到亲本库菌株原生质体制备的最优条件为:菌培养时间为12h,采用溶菌酶的酶浓度为7.11mg/mL,在酶解温度为39℃条件下,酶解处理1h,糖丁基梭状芽孢杆菌原生质体的制备率与再生率之积达到了50.8%;原生质体融合的最佳条件为:PEG4000的浓度为30%、融合温度为37℃、融合时间为20min和Ca2+浓度为45mM,融合率最高为4.9×10-5。3.利用基因组改组技术选育了 2株高产丁醇重组菌株GS3-11和GS3-255。以亲本菌株库的9株菌株进行递推式原生质体融合,经过三轮有效的基因组改组,对获得的融合子进行了抗性平板初筛和摇瓶发酵复筛,获得了 2株能稳定遗传的高产和高丁醇耐受性重组菌株GS3-11和GS3-255,在3L发酵罐中进行发酵培养,丁醇的产量分别达到12.51 g/L和12.20g/L,均比出发菌株提高60%以上。4.用进化工程的方法筛选出一株能适应高浓度杨木水解液的进化菌株gsGD-1。首先确定了各菌株在不同杨木水解液浓度下的代时和筛选压力,得出GS3-11的抗高浓度杨木水解液的能力要优于GS3-255。进一步通过在恒定杨木水解液浓度和梯度杨木水解液浓度下对GS3-11进行持续进化培养,发现梯度杨木水解液浓度进化筛选高浓度杨木水解液耐受性菌株效果优于恒定杨木水解液浓度下的进化筛选。筛选得到的进化菌株gsGD-1在杨木水解液中还原糖浓度为60g/L条件下进行摇瓶发酵和3L发酵罐发酵培养,其最大OD值为2.05,生物丁醇的最大产量为10.21g/L,比进化筛选前菌株GS3-11的丁醇最大产量(3.45 g/L)提高196%。5.对进化菌株gsGD-1利用杨木水解液发酵产丁醇的发酵条件进行了优化。通过P-B实验、中心组合实验设计和响应面分析得到gsGD-1发酵产生物丁醇的最优发酵条件为:杨木水解液中还原糖浓度为52.79 g/L,(NH4)2SO43.0g/L,酵母粉2.54 g/L,CaCO35.43g/L,KH2PO40.75g/L,K2HPO40.81 g/L,MgSO4·7H2O0.2g/L,FeSO4·7H2O 0.3g/L,MnSO40.01g/L,初始 pH7.0,接种量 9.7%,装液量 76.4%和发酵温度 36.5℃。在此优化条件下发酵培养,菌株gsGD-1最大生物丁醇产量为15.64g/L,较优化前(12.57g/L)提高了 24.4%,较原始菌株(7.12 g/L)提高了 120%。6.对进化菌株gsGD-1和原始菌株丁醇生物合成中关键酶基因的差异表达进行了分析。以管家基因16srRNA为内参,利用实时荧光定量PCR法检测了原始菌株Clostridium saccharobutylicum ATCC BAA-117 与进化菌株 gsGD-1 在丁醇生物合成过程中产酸中期与产溶剂中期关键酶基因的表达差异。2-△△Ct相对定量分析结果表明:(1)进化菌株的产酸基因ack和buk在产酸阶段的表达量均低于原始菌株,约为原始菌株ack和buk基因表达量的65%,而在产溶剂阶段的表达量比原始菌株要高,约为原始菌株的1.5倍。(2)进化菌株的产溶剂基因adhE、aadc、ctfAB和bdhB的表达量在产酸阶段,除aadc变化不明显外,其他三个基因adhE、ctfAB和bdhB的表达量均高于原始菌株,分别为原始菌株的2.67、1.8和1.6倍;在产溶剂阶段的表达量均高于原始菌株,分别为原始菌株的2.51、2.82、10.5和1.34倍。(3)进化菌株与丁醇耐受性相关的基因gldA在产酸阶段的表达量约为原始菌株的40%,在产溶剂阶段gldA的表达量仍然低于原始菌株,约为原始菌株的60%;进化菌株的丁醇耐受性基因hsp90在产酸阶段的表达量略高于原始菌株,在产溶剂阶段hsp90的表达量则为原始菌株1.5倍。可见,进化菌株丁醇产量比原始菌株高,与产溶剂基因的上调表达和与丁醇耐受性相关基因的差异表达(gldA基因的表达下调和hsp90基因的上调表达)有紧密联系。
[Abstract]:As the fossil energy depletion and the rapid growth of energy demand, increasing energy and environmental problems of resource constraints continue to highlight the energy security and the development of green renewable energy has aroused the attention of governments. Therefore, the production of alternative fuels has been widely concerned about the use of renewable lignocellulosic biomass and biomass. As a kind of butanol with gasoline in any proportion and mixed use of renewable clean energy alternative to gasoline also ushered in a new development opportunity. But the use of bio butanol production of wood fiber is low yield, the utilization rate of raw materials is not high. In view of this problem, this study from the strains of the broad-spectrum carbohydrate fermentation ability the sugar butyl Clostridium (Clostridium saccharobutylicum) ATCC BAA-117 strain genome will change Group technology and evolutionary engineering breeding combination, to adapt to the selection of high yield strain of bio butanol high concentration poplar hydrolyzate, successfully obtained a strain of efficient utilization of poplar wood hydrolysate fermentation yield of recombinant strain gsGD-1. butanol evolution and fermentation conditions on the breeding of strain gsGD-1 by poplar hydrolyzate of butanol production and further optimization, using real time quantitative PCR method for butanol synthetase gene between strain and reconstituted high recombinant strain gsGD-1 evolution differential expression was analyzed. The main research contents and results are as follows: 1. the genetic diversity for genome shuffling of parental strains. The strain Clostridium saccharobutylicum ATCC library BAA-117 through ultraviolet mutagenesis (UV), ribosome engineering breeding techniques and butanol tolerance three methods screened 9 strains positive genetic stability To the characters of strains, including butanol yield higher than that of the original strain mutation of 6 strains, respectively UV-1, UV-2, UV-3, RE-1, RE-2 and RE-3, the butanol yield were 20% higher than that of original strain above; butanol tolerance higher than that of the original strain of the 3 mutant strains were HBT-1, HBT-2 and HBT-3, improve the butanol tolerance than the original strain. More than 25% of the 9 strains were composed of genome shuffling startingstrain library for recursive protoplast fusion experiment of.2. obtained Clostridium saccharobutylicum subsequent protoplast preparation, regeneration and fusion. The optimum conditions by single factor experiment and response surface analysis of central composite design to obtain the optimal conditions of parents Library of protoplast preparation for bacteria culture time was 12h, the enzyme concentration of lysozyme was 7.11mg/mL, the enzymolysis temperature is 39 DEG C, enzyme treatment 1H, sugar Ding Ji Clostridium native Plasmid preparation rate and regeneration rate of the product reached 50.8%; the optimum conditions of protoplast fusion: the concentration of PEG4000 is 30%, the fusion temperature of 37 DEG C, the fusion time was 20min and the concentration of Ca2+ was 45mM, the fusion rate was up to 4.9 * 10-5.3. by genome shuffling 2 high-yield butanol recombinant strain GS3-11 and GS3-255. to the parent strain Library of 9 strains of recursive protoplast fusion, genome shuffling through effective three, to obtain the fusion of resistance screening plate and shaking flask screening, the 2 strains can be stably inherited high yield and high butanol tolerance of recombinant strains GS3-11 and GS3-255. Fermentation in 3L fermentor, butanol production reached 12.51 g/L and 12.20g/L, were more than 60% higher than the original strain.4. by evolutionary engineering method was screened to adapt to the evolution of bacteria with high concentration of poplar hydrolyzate Line gsGD-1. first identified the strains in different poplar hydrolysate concentration under generation and screening pressure, obtained GS3-11 resistance to a high concentration of poplar hydrolyzate is superior to GS3-255. further by a constant poplar hydrolysate and poplar hydrolysate concentration gradient of GS3-11 continued evolution of culture, found that the gradient of Yang wood hydrolysate concentration evolution of high concentration screening poplar hydrolyzate superior tolerance strain constant poplar hydrolysate concentration evolution. The evolution of screening screened strain gsGD-1 in poplar hydrolyzate in reducing sugar concentration is 60g/L under the condition of shaking flask and 3L fermentor fermentation, the maximum OD was 2.05. The maximum yield of bio butanol is 10.21g/L, the maximum yield of butanol than evolution before screening of strain GS3-11 (3.45 g/L) to improve the 196%.5. fermentation conditions on the use of butanol fermentation evolution strain gsGD-1 poplar hydrolyzate Parts are optimized. Through the P-B experiment, central composite experiment design and response surface analysis to obtain the optimal fermentation conditions of gsGD-1 fermentation bio butanol: Poplar hydrolyzate in reducing sugar concentration was 52.79 g/L (NH4) 2SO43.0g/L, yeast powder 2.54 g/L, CaCO35.43g/L, KH2PO40.75g, /L, K2HPO40.81, g/L, MgSO4, 7H2O0.2g/L, FeSO4 7H2O, 0.3g/L, MnSO40.01g/L, initial pH7.0, inoculation volume 9.7%, volume 76.4% and fermentation temperature 36.5. Under the optimized fermentation conditions, the strain gsGD-1 maximum butanol yield was 15.64g/L, compared with before optimization (12.57g/L) increased by 24.4%, compared with the original strain (7.12 g/L) increased the difference of 120%.6. on key enzymes of evolution gsGD-1 strain and original strain of butanol biosynthesis gene expression was analyzed. The housekeeping gene 16srRNA was used to detect the original strain Clostridium, saccharobutylic using real-time fluorescence quantitative PCR method Um ATCC and BAA-117 gsGD-1 analysis of expression difference in strain evolution process of middle acid medium and the biosynthesis of butanol producing solvent medium key enzyme gene.2- Delta Ct relative quantitative results showed that: (1) the evolution of strains producing acid gene ACK and buk expression in the acid production phase were significantly lower than that of the original strain is about ACK and the original strain expression of buk 65%, and in the expression of solventogenic phase than the original strain to be high, about 1.5 times of the original strain. (2) the solventogenic genes adhE, evolutionary strains of AADC, expression of ctfAB and bdhB in the acid production phase, in addition to AADC did not change significantly, the other three genes adhE, the expression of ctfAB and bdhB were higher than that of the original strain, respectively. The original strain 2.67,1.8 and 1.6 times; the expression level in solventogenic phase were higher than that of the original strain, respectively. The original strain 2.51,2.82,10.5 and 1.34 times. (3) the evolution of strain and butanol tolerance. The expression of gldA in the acid production phase was about 40% of the original strain, the expression of solventogenic phase gldA was still lower than that of the original strain, the original strain is about 60%; butanol tolerance of Hsp90 gene expression in the acid producing strains evolution stage was slightly higher than the original strain, the expression of Hsp90 in solventogenic phase the amount is 1.5 times as the original strain. The strain evolution of butanol production than the original strain, expression and solventogenic genes were up-regulated and correlated with alcohol tolerance gene (downregulation of the expression of the gldA gene and HSP90 gene up-regulated) are closely linked.

【学位授予单位】：中南林业科技大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q93

【相似文献】