【作者】 申佩弘；

【导师】 武波；

【作者基本信息】 广西大学 ， 微生物学， 2007， 博士

【摘要】 甲基营养菌是一类能利用甲基化合物为唯一碳源和能源的微生物,已有不少研究发现该类微生物能转化甲醇为多种有价值的化合物。前期的工作已从环境中筛选到一株能以甲醇为唯一碳源和能源的甲基营养杆菌菌株M.sp.MB200,该菌株利用丝氨酸循环途径来同化甲基化合物。为了提高其L-丝氨酸和乙醛酸的积累能力,通过研究和利用M.sp.MB200中与丝氨酸循环途径相关的关键基因glyA和hpr建重组菌株,以提高重组菌生物转化甲醇的能力。glyA基因是甲基营养型细菌中用于同化单碳化合物的丝氨酸循环中的第一个酶基因,它编码的丝氨酸羟甲基转移酶（SHMT）能可逆地催化甲叉四氢叶酸与甘氨酸结合生成L-丝氨酸。构建M.sp.MB200的部分DNA文库,从文库中的阳性克隆中得到一个带有启动子的glyA基因,该基因的开放阅读框编码434个氨基酸,计算分子量为48.23kDa,与已报道的M.extorquensAM1的glyA基因的核苷酸序列相似性为95%,它的氨基酸序列与M.extorquens AM1的SHMT氨基酸序列相似性为94%。通过pK18mob载体同源单交换构建了glyA基因的突变体MB200gTB,研究发现该突变体不能在以甲醇为唯一碳源的培养基上生长,但在多碳化合物上的生长并不受影响。突变体检测不到SHMT酶活性,也不能利用静息细胞体系产L-丝氨酸。该基因在大肠杆菌中能表达出SHMT酶活性。进一步将该基因连同其启动子一起克隆到广泛宿主载体pLAFR3上,并导入到出发菌株M.SP.MB200中,得到重组菌株M.sp.MB202。研究发现M.sp.MB202中的SHMT活性为野生菌株M.sp.MB200的3.5倍,利用静息细胞产L-丝氨酸的能力达到了11.4±0.6mg/mL,与出发菌株相比提高了约5倍。对重组菌的静息细胞产L-丝氨酸的条件进行进一步优化,结果发现,在50mg/mL湿菌体,甲醇浓度为70mg/mL,甘氨酸浓度为50 mg/mL,pH为8.8,温度为37℃,发酵48小时的条件下,MB202产L-丝氨酸的能力达到最佳,达到24.3±1.0 mg/mL。对固定化MB202细胞产L-丝氨酸也进行了研究,在0.3 g/mL菌体量、3%海藻酸钠、6%的CaCL₂的条件下,能发酵5批次,平均每批次产12.7±0.9mg的L-丝氨酸。实验结果为进一步利用甲醇利用菌和glyA基因生产L-丝氨酸提供了应用依据。羟基丙酮酸还原酶基因（hpr）编码的羟基丙酮酸还原酶能催化羟基丙酮酸盐还原成甘油酸,甘油酸进一步被代谢为乙醛酸。通过PCR扩增从M.sp.MB200菌株中得到一个hpr基因,该基因的大小与已报道的细菌中hpr基因大小相似。ORF大小为945bp,编码315个氨基酸的蛋白质,计算分子量约为37 KDa。经过初步的酶学特性分析发现,该HPR以NADPH作为辅酶,最适作用的pH值为5.5,酶的活性随温度的变化影响并不大。通过pK18mob载体同源单交换构建了hpr基因的突变体MB200hTB,研究发现该突变体不能在以甲醇为唯一碳源的培养基上生长,但在多碳化合物上的生长并不受影响,突变体检测不到HPR酶活性。利用广泛宿主载体pLAFR3带上hpr基因,导入到出发菌株MB200中,在lacZ启动子驱动下使得MB200中的hpr基因拷贝数增加,从而提高细胞内HPR的酶活性。实验结果表明重组菌株MB201中HPR的酶活性比野生型菌株MB200的高了2倍。菌株MB200和MB201均在多数生长后期时乙醛酸的含量达到最高,培养33小时后,MB201的乙醛酸含量为14.4±1.1 mg/mL,约为野生菌MB200的2倍。在利用微生物转化合成化合物的过程中,提高目标物产量的一个最重要的方法就是调节（提高或降低）基因的表达水平,尤其是通过基因工程的方法,增加菌株相应基因的拷贝数。本实验结果有利于推动进一步利用M.sp.MB200及其基因进行发酵生产。更多还原

【Abstract】 Methylotrophic bacteria are a group of microorganisms that are able to use compounds containing one-carbon as well as multi-carbons as energy and carbon sources.These organisms can convert methanol to chemicals and materials biologically.A strain named MB200,can utilize methanol as the sole carbon and energy soure,was obtained from environment in our former work.This bacteria pocesses L-serine cycle to assimilate methanol.In order to enhance accumulating L-serine amd glyoxylate by this strain,the improved strains were constructed through increase the expressing level of two key genes （hpr,glyA）involved in L-serine cycle.glyA,a gene which encodes serinehydroxy methyltransferase（SHMT）in organisms,SHMT plays an improtant role as the first enzyme in the assimilation of C1 compounds through the transfer of formaldehyde to glycine,thus giving the main intermediate in the pathway,serine.A glyA gene was cloned from the genomic DNA of M.sp MB200,this gene encdes for the 434-amino-acid protein with a calculated molecular mass of 48230 Da.The amino-acid sequence of the enzyme showed identity to the sequence of the enzyme from M.extorquens AM1 （94%）.The transfermed E.coli cells could express SHMT which has enzyme activity.With the suicide plasmid pK18mob,a mutant MB200gTB inactivated in glyA gene has been constructed through homologous recombination caused by a single crossover event.The experiments showed that this mutant can’t utilize methanol as the sole carbon source and can’t accumulate L-serine any more,no SHMT enzyme activity can be detected in the cells,but could grow on the medium with multi-carbon coumpounds.This gene was ligated into the vector pLAFR3 to obtained recombinant plasmid pLAFRg,which was transfered into M.sp.MB200 to generate MB202.The improved strain MB202 was found to exhibit the higher L-serine productivity than the wild strain MB200,11.4±0.6 mg/mL L-serine was produced from 20mg/mL glycine and 70mg/mL methanol in 2d when used a resting cell system of MB202,the yield was about 5-fold of that produced by MB200.After optmizing the fermentation conditions,an optimun conditions for resting cell system were:methanol 70 mg/mL;glycine 50 mg/mL;pH8.8;and 37 degrees C,under the under above conditions,the improved strain MB202 can produce 24.3±1.0 mg/mL L-serine.When use the conditions of 0.3 g/mL of cells（as wet matter）,3%sodium alginate and 6% CaCL₂,the average output of L-serine produced by immobilized cells is 12.7±0.9 mg/mL per bacth and can be fermented for 5 bacthes.The results will facilitate enhancing the conversion rate of methanol to L-serine using M.sp strains.hpr encodes hydroxypyruvate reductase（HPR）,an enzyme catalyzes the reversible conversion of hydroxypyruvate to D（-）glycerate,and D（-）glycerate is further metabolized to glyoxylate in the serine cycle in M.sp bacteria.A hpr gene was amplified from the total DNA of M.sp.MB200,sequenceing analysis showed that an ORF of 945bp was included in this fragment coding 315 amino acids with a molecular weight of approximately 37,000 Da,The purified HPR enzyme was performed to use NADPH as cofactor,the optimal PH for the reduction of hydroxypyruvate is about 5.5,and temprature has litter effect on it’s activity.The mutant MB200hTB was construct by the suicide plasmid pK18mob ligated a part of glyA sequence.The result showed that MB200hTB had no HPR activity and lost its ability to grow on C-1 compounds but retained the ability to grow on C-2 compounds and multi-carbon compounds.The gene was ligated into the vector pLAFR3 to obtained the recombinant plasmid pLAFRh,which was transferred into M.sp.MB200 to generate an recombinant strain MB201. Homologous expression of hpr under the control of the lacZ promoter led to the enhanced glyoxylate accumulation in cultures of Methylobacterium sp MB201. The yield of glyoxylate reached 14.4±1.1 mg/mL,representing nearly a two-fold increase when compared with the wild-type strain.An important strategy to develop a synthesis method of commercial chemicals and materials by bacteria is to deregulate and increase the levels of gene expression,especially to increase the copy number of genes,by genetic engineering.In this report,a glyA gene with it’s promoter was over-expressed in M.sp.MB200 lead to ehance the yield of L-serine,and a hpr gene was over-expressed under the control of lacZ promoter in the pLAFR₃ vector in M. sp.MB200 to produce glyoxylate.The results will facilitate researching and applicating M.sp.MB200 and it’s genes to biosynthesize more products.更多还原