【作者】 段云霞；

【导师】 赵学明；

【作者基本信息】 天津大学 ， 生物化工， 2009， 博士

【摘要】 本文围绕核黄素生物合成反应,研究了异源核黄素操纵子的扩增对工程菌核黄素合成的影响。在此基础上,对产核黄素枯草芽孢杆菌进行了代谢工程改造:通过在宿主菌株中分别扩增zwf基因,缺失ptsG基因,表达异源vgb基因等手段,构建了一系列产核黄素枯草芽孢杆菌基因工程菌。本文得到的主要结果如下:利用生物信息学原理从已经完成全基因测序的三种微生物（B. cereus ATCC 10987, B. cereus ATCC 14579和Geobacillus stearother mophilu）中寻找核黄素操纵子序列,对其进行核黄素操纵子基因的注释。通过核黄素缺陷型菌株的营养互补实验,证实了三种异源核黄素操纵子能够在枯草芽孢杆菌中表达。通过在B. subtilis RH13中表达三种不同的P43-rib（异源操纵子）,发现蜡样芽孢杆菌（B. cereus ATCC 14579）核黄素操纵子对枯草芽孢杆菌核黄素产量的影响最大。将含有P43-rib_{B.cereus ATCC 14579}的片段整合到B.subtilis RH33染色体上得到的工程菌B. subtilis PY,在含8 %葡萄糖的发酵培养基中核黄素产量达到4.3 g/l,与出发菌相比提高了近27 %。在此基础上,研究了在工程菌B. subtilis PY中过量表达zwf基因（编码6-磷酸葡萄糖脱氢酶）对菌体核黄素合成的影响。实验结果表明:zwf基因的过量表达增加了PP途径的通量,提高了核黄素合成前体物5-磷酸核酮糖（Ru5P）的胞内浓度,在含8 %葡萄糖的发酵培养基中核黄素产量达到5.4 g/l与出发菌相比提高了约25 %。其次,考察了缺失工程菌B. subtilis PY的ptsG基因对其生理和核黄素产量的影响。实验结果表明:该基因的缺失降低了菌株对葡萄糖的吸收速率,EMP和TCA耦联性加强,在一定程度上减少了发酵副产物的大量积累,最大限度地将反应底物转化为菌体和目标产物。在含8 %葡萄糖的发酵培养基中最高菌体浓度增加22%,核黄素产量提高达到5.1 g/l,与出发菌相比提高了19 %,但与此同时发酵周期由48 h延长至72 h。对工程菌及其出发菌通量分布的计算结果表明:ptsG基因缺失的工程菌减少了EMP途径的通量,增强了TCA途径的通量,弱化了溢流代谢,底物转化率由出发菌的0.01 mol核黄素/mol葡萄糖提高到0.016 mol核黄素/mol葡萄糖。最后,本文研究了在B. subtilis PY中表达vgb基因对工程菌生理和核黄素产量的影响。实验结果表明:vgb基因的表达提高宿主菌氧化磷酸化效率,ATP/ADP由出发菌的1.3增加到工程菌的3.2。含vgb基因的工程菌在5 l发酵罐中流加发酵48 h,发酵结果显示菌体浓度增加28 %,核黄素产量达到13.3 g/l,比出发菌核黄素产量提高了约20 %。工程菌及其出发菌通量分布的计算结果表明:vgb基因的表达使工程菌PP途径通量增加,有效调节EMP途径和TCA循环途径的耦合性,在一定程度上消弱了溢流代谢。更多还原

【Abstract】 Based on the riboflavin （VB₂） biosynthetic reactions, B. subtilis PY strain was constructed to study the effect of heterologous rib operon on riboflavin biosynthesis. Then metabolic engineering of B. subtilis PY for riboflavin production were studied by over-expression of zwf gene, expression of heterologous vgb （Vitreoscilla hemoglobin） gene and deletion of ptsG gene in B. subtilis PY, respectively. The main results presented in this work are as follows:Fragments containing the whole rib operons of B. cereus ATCC 10987, B. cereus ATCC 14579 and Geobacillus stearother mophilu were detected and annotated respectively from GenBank. Three heterologous rib operons were operative in B. subtilis that was identified by riboflavin auxotroph complementary experiments.Three fragments of P43-rib, which were expressed in B. subtilis RH13, were respectively constructed by replacing native prompters of the three heteroglous rib operons with constitutively expressed strong promoter P43. It indicated that expression of P43-rib_{B.cereus ATCC 14579B} enhances riboflavin production most efficiently. Subsequently, P43-rib_{B.cereus ATCC 14579B} was integrated into the chromosome of B. subtilis RH33, yielding transformant B. subtilis PY. 4.3 g/l of riboflavin was achieved in batch culture of B. subtilis PY with 8 % glucose as carbon source, which was 27 % enhancement compared to the host strain.The effect of zwf gene （encoding glucose 6-phosphate dehydrogenase） amplification on riboflavin production of strain B. subtilis PY was studied in this thesis. Metbolites analysis indicated that the flux of PP pathway increased. With the increasing of the intracellular concentration of precursor ribulose 5-phosphate （Ru5P）, the riboflavin production of B. subtilis achieved 5.4 g/l in batch fermentation medium containing 8 % glucose, which was 25 % enhancement compared to the host strain.ptsG gene of B. subtilis PY, which encoding EII in glucose phosphotransferase system,was inactivated. Compared to the host strain, ptsG-disrupted stain accumulated less acid, and directed more carbon source to biomass and riboflavin biosynthetic pathway, while the glucose uptake rate was declined. 22% more biomass and 19 % higher riboflavin were achieved, compared to the host strain in batch culture with 8 % glucose. However, the fermentation cycle was prolonged from 48 h to 72 h. A metabolic flux distribution analysis indicated that both the fluxes in EMP pathway and also in the fluxes of overflow metabolism decreased in ptsG-disrupted strain while the riboflavin yield was increased from 0.01 mol riboflavin/mol glucose to 0.016 mol riboflavin/mol glucose.Vitreoscilla hemoglobin （VHb） gene was integrated into the chromosome of B. subtilis PY, and its influence on physiological characters and riboflavin production has been investigated. The presence of VHb not only enhanced efficiency of oxidative phosphorylation, but increased the ratio of ATP/ADP from 1.3 to 3.2, which as a result improved the biomass and riboflavin production. Compared with the control strain under fed-batch fermentation in 5 l fermentor, 28 % higher cell growth density and about 20 % improvement of the yield of riboflavin were obtained. A metabolic flux distribution analysis indicated that VHb had significantly enhanced the fluxes through PPP and pathways for biomass biosynthesis. The overflow metabolism was also decreased.更多还原