本文關(guān)鍵詞： 糖丁基梭狀芽孢桿菌 生物丁醇 基因組改組 原生質(zhì)體融合 進(jìn)化工程 實(shí)時(shí)熒光定量PCR 響應(yīng)面　出處：《中南林業(yè)科技大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：隨著全球化石能源的日漸枯竭和快速增長(zhǎng)的能源需求,能源資源約束的日益加劇和生態(tài)環(huán)境問(wèn)題的不斷突出,保障能源安全和發(fā)展綠色可再生能源已引起各國(guó)政府的高度重視。因此,利用可再生的木質(zhì)纖維素生物質(zhì)來(lái)生產(chǎn)替代燃料受到了廣泛的關(guān)注。而生物丁醇作為一種能與汽油以任意比例混和使用甚至替代汽油的可再生清潔能源也迎來(lái)了全新的發(fā)展機(jī)遇。但利用木質(zhì)纖維生產(chǎn)生物丁醇目前尚存在產(chǎn)量低,原料利用率不高等難題。鑒于此,本研究從菌種的選育出發(fā),以具有廣譜碳水化合物發(fā)酵能力的糖丁基梭狀芽孢桿菌(Clostridium saccharobutylicum)ATCC BAA-117為出發(fā)菌株,將基因組改組技術(shù)與進(jìn)化工程育種相結(jié)合,進(jìn)行了適應(yīng)高濃度楊木水解液的生物丁醇高產(chǎn)菌株的選育,成功獲得了一株能高效利用楊木水解液發(fā)酵高產(chǎn)丁醇的重組進(jìn)化菌株gsGD-1。同時(shí),對(duì)選育菌株gsGD-1利用楊木水解液產(chǎn)丁醇的發(fā)酵條件進(jìn)行了優(yōu)化,并進(jìn)一步采用實(shí)時(shí)熒光定量PCR的方法對(duì)出發(fā)菌株和改組后高產(chǎn)重組進(jìn)化菌株gsGD-1之間的丁醇合成酶關(guān)鍵基因表達(dá)差異進(jìn)行了分析。研究?jī)?nèi)容及取得的主要結(jié)果如下:1.建立了用于基因組改組的遺傳多樣性親本菌株庫(kù)。出發(fā)菌株Clostridium saccharobutylicum ATCC BAA-117經(jīng)紫外誘變(UV)、核糖體工程育種技術(shù)和丁醇耐受性篩選三種方法獲得9株能穩(wěn)定遺傳的正向性狀菌株,包括丁醇產(chǎn)量高于出發(fā)菌株的突變菌6株,分別為UV-1、UV-2、UV-3、RE-1、RE-2和RE-3,其丁醇產(chǎn)量均比原始菌株提高20%以上;丁醇耐受性高于出發(fā)菌株的突變株3株,分別為HBT-1、HBT-2和HBT-3,其丁醇耐受性均較原始菌株提高25%以上。這9株菌株組成基因組改組的親本菌株庫(kù),用于后續(xù)的遞推式原生質(zhì)體融合實(shí)驗(yàn)。2.獲得了Clostridium saccharobutylicum原生質(zhì)體制備、再生和融合的最佳條件。通過(guò)單因素實(shí)驗(yàn)、中心組合實(shí)驗(yàn)設(shè)計(jì)與響應(yīng)面分析得到親本庫(kù)菌株原生質(zhì)體制備的最優(yōu)條件為:菌培養(yǎng)時(shí)間為12h,采用溶菌酶的酶濃度為7.11mg/mL,在酶解溫度為39℃條件下,酶解處理1h,糖丁基梭狀芽孢桿菌原生質(zhì)體的制備率與再生率之積達(dá)到了50.8%;原生質(zhì)體融合的最佳條件為:PEG4000的濃度為30%、融合溫度為37℃、融合時(shí)間為20min和Ca2+濃度為45mM,融合率最高為4.9×10-5。3.利用基因組改組技術(shù)選育了 2株高產(chǎn)丁醇重組菌株GS3-11和GS3-255。以親本菌株庫(kù)的9株菌株進(jìn)行遞推式原生質(zhì)體融合,經(jīng)過(guò)三輪有效的基因組改組,對(duì)獲得的融合子進(jìn)行了抗性平板初篩和搖瓶發(fā)酵復(fù)篩,獲得了 2株能穩(wěn)定遺傳的高產(chǎn)和高丁醇耐受性重組菌株GS3-11和GS3-255,在3L發(fā)酵罐中進(jìn)行發(fā)酵培養(yǎng),丁醇的產(chǎn)量分別達(dá)到12.51 g/L和12.20g/L,均比出發(fā)菌株提高60%以上。4.用進(jìn)化工程的方法篩選出一株能適應(yīng)高濃度楊木水解液的進(jìn)化菌株gsGD-1。首先確定了各菌株在不同楊木水解液濃度下的代時(shí)和篩選壓力,得出GS3-11的抗高濃度楊木水解液的能力要優(yōu)于GS3-255。進(jìn)一步通過(guò)在恒定楊木水解液濃度和梯度楊木水解液濃度下對(duì)GS3-11進(jìn)行持續(xù)進(jìn)化培養(yǎng),發(fā)現(xiàn)梯度楊木水解液濃度進(jìn)化篩選高濃度楊木水解液耐受性菌株效果優(yōu)于恒定楊木水解液濃度下的進(jìn)化篩選。篩選得到的進(jìn)化菌株gsGD-1在楊木水解液中還原糖濃度為60g/L條件下進(jìn)行搖瓶發(fā)酵和3L發(fā)酵罐發(fā)酵培養(yǎng),其最大OD值為2.05,生物丁醇的最大產(chǎn)量為10.21g/L,比進(jìn)化篩選前菌株GS3-11的丁醇最大產(chǎn)量(3.45 g/L)提高196%。5.對(duì)進(jìn)化菌株gsGD-1利用楊木水解液發(fā)酵產(chǎn)丁醇的發(fā)酵條件進(jìn)行了優(yōu)化。通過(guò)P-B實(shí)驗(yàn)、中心組合實(shí)驗(yàn)設(shè)計(jì)和響應(yīng)面分析得到gsGD-1發(fā)酵產(chǎn)生物丁醇的最優(yōu)發(fā)酵條件為:楊木水解液中還原糖濃度為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,接種量 9.7%,裝液量 76.4%和發(fā)酵溫度 36.5℃。在此優(yōu)化條件下發(fā)酵培養(yǎng),菌株gsGD-1最大生物丁醇產(chǎn)量為15.64g/L,較優(yōu)化前(12.57g/L)提高了 24.4%,較原始菌株(7.12 g/L)提高了 120%。6.對(duì)進(jìn)化菌株gsGD-1和原始菌株丁醇生物合成中關(guān)鍵酶基因的差異表達(dá)進(jìn)行了分析。以管家基因16srRNA為內(nèi)參,利用實(shí)時(shí)熒光定量PCR法檢測(cè)了原始菌株Clostridium saccharobutylicum ATCC BAA-117 與進(jìn)化菌株 gsGD-1 在丁醇生物合成過(guò)程中產(chǎn)酸中期與產(chǎn)溶劑中期關(guān)鍵酶基因的表達(dá)差異。2-△△Ct相對(duì)定量分析結(jié)果表明:(1)進(jìn)化菌株的產(chǎn)酸基因ack和buk在產(chǎn)酸階段的表達(dá)量均低于原始菌株,約為原始菌株ack和buk基因表達(dá)量的65%,而在產(chǎn)溶劑階段的表達(dá)量比原始菌株要高,約為原始菌株的1.5倍。(2)進(jìn)化菌株的產(chǎn)溶劑基因adhE、aadc、ctfAB和bdhB的表達(dá)量在產(chǎn)酸階段,除aadc變化不明顯外,其他三個(gè)基因adhE、ctfAB和bdhB的表達(dá)量均高于原始菌株,分別為原始菌株的2.67、1.8和1.6倍;在產(chǎn)溶劑階段的表達(dá)量均高于原始菌株,分別為原始菌株的2.51、2.82、10.5和1.34倍。(3)進(jìn)化菌株與丁醇耐受性相關(guān)的基因gldA在產(chǎn)酸階段的表達(dá)量約為原始菌株的40%,在產(chǎn)溶劑階段gldA的表達(dá)量仍然低于原始菌株,約為原始菌株的60%;進(jìn)化菌株的丁醇耐受性基因hsp90在產(chǎn)酸階段的表達(dá)量略高于原始菌株,在產(chǎn)溶劑階段hsp90的表達(dá)量則為原始菌株1.5倍�？梢�(jiàn),進(jìn)化菌株丁醇產(chǎn)量比原始菌株高,與產(chǎn)溶劑基因的上調(diào)表達(dá)和與丁醇耐受性相關(guān)基因的差異表達(dá)(gldA基因的表達(dá)下調(diào)和hsp90基因的上調(diào)表達(dá))有緊密聯(lián)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.

【學(xué)位授予單位】：中南林業(yè)科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q93

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