富马酸基因工程菌的构建
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摘要
富马酸是一种重要的化工原料和精细化工产品,在医药、化工、树脂等领域有着非常广泛的应用前景,其生物生产方法已经受到了广泛的重视,如微生物发酵法。本文以毕赤酵母作为出发菌株,研究考察米根霉富马酸酶基因、毕赤酵母丙酮酸羧化酶基因在毕赤酵母细胞中的过表达对富马酸胞质途径的影响,以及敲除副产物乳酸代谢过程中的关键酶乳酸脱氢酶基因,使得更多碳源流向富马酸胞质途径。
本实验克隆了毕赤酵母(Pichia pastoris GS115)中编码丙酮酸羧化酶的基因(PC),在毕赤酵母中进行了强化表达,以研究PC基因在毕赤酵母中的过量表达对TCA还原途径碳源分流及草酰乙酸、L-苹果酸生产的影响。为此,研究构建了表达载体pPIC3.5K-PC,并转化了毕赤酵母GS115。转化子经过表型鉴定,PCR分析和G418浓度梯度筛选获得了高拷贝的重组子(pas-01)。甲醇诱导表达后,经SDS-PAGE分析及PC活性检测,发现酶活提高了3倍,说明PC在毕赤酵母中获得了成功表达。以pPIC3.5K转化菌为对照,对该重组子进行了草酰乙酸、苹果酸的发酵研究,结果显示草酰乙酸产量(177.4 mg.L-1)提高了109.6%,L-苹果酸的产量(127.45 mg.L-1)提高33.7%,菌体生物量提高15.7%,表明PC的过量表达有助于L-苹果酸、草酰乙酸的积累,并且对菌体的生长有一定的促进作用。
为克隆米根霉细胞中的富马酸酶基因,本实验先后更换了二十对引物,尝试通过逆转录聚合酶链式反应(RT-PCR)扩增富马酸酶基因片段,未果。随后根据米根霉富马酸酶基因的保守序列设计引物,扩增得到250bp长度的保守序列,与模板相比同源性只有76%；说明目前所使用的这株米根霉3.2686细胞中的富马酸酶基因序列与1959年以色列学者报导的模板序列具有较大差异,该菌中的fumR是一个新的基因。
克隆了毕赤酵母中编码乳酸脱氢酶的基因LDH,同时也克隆了博来霉素抗性基因zeocin,构建了LDH敲除载体pMD18-T-LDHup-Zeocin-LDHdown,其中上游同源臂有900bp左右,下游同源臂包含500bp大小的序列,通过同源重组敲除LDH基因。
Fumaric acid is an important chemical raw materials and fine chemical products, has a very wide range of applications in pharmaceuticals, chemicals, resins and other fields, and its biological production methods have received wide attention, such as microbial fermentation. In this paper, we took Pichia pastoris as the starting strain and investigated influence of the expression of fumarase gene from Rhizopus oryzae CGMCC3.2686 and pyruvate carboxylase gene from Pichia pastoris on the cytoplasmic pathway of fumarate. We also knocked out lactate dehydrogenase gene, which is a key enzyme in the metabolism of lactic acid, to make way for more carbon flow of cytoplasmic fumarate.
In this study, we overexpressed PC gene in Pichia pastoris GS115, and investigated its influence on the diversion of carbon to the reductive TCA pathway and the yields of the oxaloacetate and L-malic acid. The expression vector pPIC3.5K-PC was constructed, and was introduced in Pichia pastoris GS115. A high-copy recombinant (pas-01) was screened out from 30 positive transformants by phenotypic identification, PCR analysis and G418 concentration gradient selection. After methanol induction, recombinant protein was analysed by SDS-PAGE and PC activity was detected. Results showed that PC was overexpressed in the Pichia pastoris, and the activity was increased 3-folds. Compared to the control (pPIC3.5K transformant), oxaloacetate production(177.4 mg.L-1) of the recombinant (pas-01) was increased by 109.6%, L-malic acid production (127.45 mg.L-1) increased by 33.7% and cell biomass increased by 10% through the fermentation. These results indicated that overexpression of PC is propitious to the accumulation of oxaloacetate and L-malic acid, which also play a significant role in promoting the growth of strain.
In order to clone fumarase gene from Rhizopus oryzae CGMCC3.2686, in this experiment we designed twenty pairs of primers and tried to clone fumarase gene by reverse transcription polymerase chain reaction (RT-PCR), but failed. Subsequently, according to the conserved sequence of fumarase gene from Rhizopus oryzae, we designed a pair of primers and amplified 250 bp length conserved sequence, compared with the template is only 76% homology; that this strain is currently used by the cells of Rhizopus oryzae CGMCC 3.2686 The gene sequence of fumarase 1959 template sequence Israeli scholars have reported large differences in the fumR bacteria is a new gene.
The LDH gene which encodes lactate dehydrogenase was cloned, and the bleomycin resistance gene zeocin was also cloned. At the same time, the knockout vector of LDH was constructed, named pMD18-T-LDHup-Zeocin-LDHdown, which has 900 bp upstream homologous arm and about 500 bp downstream homology arm in order to knockout the LDH genes from the genome of Pichia pastoris by homologous recombination.
本实验克隆了毕赤酵母(Pichia pastoris GS115)中编码丙酮酸羧化酶的基因(PC),在毕赤酵母中进行了强化表达,以研究PC基因在毕赤酵母中的过量表达对TCA还原途径碳源分流及草酰乙酸、L-苹果酸生产的影响。为此,研究构建了表达载体pPIC3.5K-PC,并转化了毕赤酵母GS115。转化子经过表型鉴定,PCR分析和G418浓度梯度筛选获得了高拷贝的重组子(pas-01)。甲醇诱导表达后,经SDS-PAGE分析及PC活性检测,发现酶活提高了3倍,说明PC在毕赤酵母中获得了成功表达。以pPIC3.5K转化菌为对照,对该重组子进行了草酰乙酸、苹果酸的发酵研究,结果显示草酰乙酸产量(177.4 mg.L-1)提高了109.6%,L-苹果酸的产量(127.45 mg.L-1)提高33.7%,菌体生物量提高15.7%,表明PC的过量表达有助于L-苹果酸、草酰乙酸的积累,并且对菌体的生长有一定的促进作用。
为克隆米根霉细胞中的富马酸酶基因,本实验先后更换了二十对引物,尝试通过逆转录聚合酶链式反应(RT-PCR)扩增富马酸酶基因片段,未果。随后根据米根霉富马酸酶基因的保守序列设计引物,扩增得到250bp长度的保守序列,与模板相比同源性只有76%；说明目前所使用的这株米根霉3.2686细胞中的富马酸酶基因序列与1959年以色列学者报导的模板序列具有较大差异,该菌中的fumR是一个新的基因。
克隆了毕赤酵母中编码乳酸脱氢酶的基因LDH,同时也克隆了博来霉素抗性基因zeocin,构建了LDH敲除载体pMD18-T-LDHup-Zeocin-LDHdown,其中上游同源臂有900bp左右,下游同源臂包含500bp大小的序列,通过同源重组敲除LDH基因。
Fumaric acid is an important chemical raw materials and fine chemical products, has a very wide range of applications in pharmaceuticals, chemicals, resins and other fields, and its biological production methods have received wide attention, such as microbial fermentation. In this paper, we took Pichia pastoris as the starting strain and investigated influence of the expression of fumarase gene from Rhizopus oryzae CGMCC3.2686 and pyruvate carboxylase gene from Pichia pastoris on the cytoplasmic pathway of fumarate. We also knocked out lactate dehydrogenase gene, which is a key enzyme in the metabolism of lactic acid, to make way for more carbon flow of cytoplasmic fumarate.
In this study, we overexpressed PC gene in Pichia pastoris GS115, and investigated its influence on the diversion of carbon to the reductive TCA pathway and the yields of the oxaloacetate and L-malic acid. The expression vector pPIC3.5K-PC was constructed, and was introduced in Pichia pastoris GS115. A high-copy recombinant (pas-01) was screened out from 30 positive transformants by phenotypic identification, PCR analysis and G418 concentration gradient selection. After methanol induction, recombinant protein was analysed by SDS-PAGE and PC activity was detected. Results showed that PC was overexpressed in the Pichia pastoris, and the activity was increased 3-folds. Compared to the control (pPIC3.5K transformant), oxaloacetate production(177.4 mg.L-1) of the recombinant (pas-01) was increased by 109.6%, L-malic acid production (127.45 mg.L-1) increased by 33.7% and cell biomass increased by 10% through the fermentation. These results indicated that overexpression of PC is propitious to the accumulation of oxaloacetate and L-malic acid, which also play a significant role in promoting the growth of strain.
In order to clone fumarase gene from Rhizopus oryzae CGMCC3.2686, in this experiment we designed twenty pairs of primers and tried to clone fumarase gene by reverse transcription polymerase chain reaction (RT-PCR), but failed. Subsequently, according to the conserved sequence of fumarase gene from Rhizopus oryzae, we designed a pair of primers and amplified 250 bp length conserved sequence, compared with the template is only 76% homology; that this strain is currently used by the cells of Rhizopus oryzae CGMCC 3.2686 The gene sequence of fumarase 1959 template sequence Israeli scholars have reported large differences in the fumR bacteria is a new gene.
The LDH gene which encodes lactate dehydrogenase was cloned, and the bleomycin resistance gene zeocin was also cloned. At the same time, the knockout vector of LDH was constructed, named pMD18-T-LDHup-Zeocin-LDHdown, which has 900 bp upstream homologous arm and about 500 bp downstream homology arm in order to knockout the LDH genes from the genome of Pichia pastoris by homologous recombination.
引文
[1]高翠英,李彦威,贾浩旭.富马酸及衍生物的应用研究进展[J].广东化工,2007,34(171)：78-79
[2]Robinson, W.D and Mount, et al. Maleic anhydride, maleic acid and fumaric acid[J]. Encyclopedia of Chemical technology,1978,14:770-793
[3]Walter J.Stefaniak, Cheektowaga, et al. Manufacture of fumaric acid[P]. US patent NO2914559,1959
[4]TR Felthouse, JC Burnett, et al. Maleic Anhydride, Maleic acid, and Fumaric acid[J]. Encyclopedia of Chemical technology,1995,15:893-928
[5]黄肾琦.富马酸氯马斯汀片[J].中国新药杂志,1998,7(3)：194-195
[6]于波涛,尧剑虹,舒明锡,曾仁杰.富马酸比索洛尔片制备工艺与质量控制研究[J].药学服务与研究,2005,5(3)：251-253
[7]Carol A.Roa Engel, Adrie J.J.Straathof, Tiemen W.Zijlmans, Walter M.van Gulik,Luuk A.M.van der Wielen.Fumaric acid production by fermentation[J]. Applied Microbiology and Biotechnology,2008,78:379-389
[8]关公凑,黄耀威等.由糖醛合成富马酸[J].广州化工,2001,29(2)：20-21
[9]Y.Zhou, J.Du, GT.Tsao, et al. Comparision of fumaric acid production by Rhizopus oryzae using different neutralizing agents[J]. Bioprocess Engineering Joural,2002,25:179-181
[10]李学坤,张昆等.富马酸的合成及应用[J].现代化工,2005,25(增刊)：81-83
[11]Ulrich Hartel, wolfgang Buckel. Fermentation of trans-aconitate via citrate, oxaloacetate, and pyruvate by Acidaminococcus fermentans[J]. Archives of Microbiology,1996,166: 342-349
[12]王普,虞炳钧,张福明,王雷.生物转化法生产L-苹果酸的研究(I)——富马酸酶产生菌株的筛选[J].食品与发酵工业,1996,1：18-19
[13]K.Belafi-Bako, N.Nemestothy, L.Gubicza. A study on applications of membrane techniques in bioconversion of fumaric acid to L-malic acid[J]. Desalination,2004, 162:301-306
[14]景晓辉,王之婉.诱变温特曲霉转化富马酸为L-苹果酸的研究[J].化学世界,2005：486-488
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[16]Toshiaki Nakajima-kambe, Takehiro Nozue, et al. Bioconversion of Maleic acid to Fumaric acid to by Pseudomonas alcaligenes Strain XD-01[J]. Journal of fermentation and Bioengineering,1997,84:165-168
[17]PAUL V.ATTWOOD. The Structure and the Mechanism of Action of Pyruvate Carboxylase[J]. International Journal of Biochemistry and Cell Biology,1995, 27(3):231-234
[18]Carol A, Roa Engel Adrie J, et al. Fumaric acid production by fermentation[J]. Applied Microbiology and Biotechnology,2008,78:379-385
[19]Osmani SA, Scrutton MC. The sub-cellular localization and regulatory properties of pyruvate carboxylase from Rhizopus arrhizus. European Journal of Biochemistry,1985, 147:119-128
[20]PAUL V.ATTWOOD. The Structure and the Mechanism of Action of Pyruvate Carboxylase[J]. International Journal of Biochemistry and Cell Biology,1995,27(3):231-234
[21]Suzuki T, Sato M, Yoshida T and Tuboi S. Rat liver mito-chondrial and cytosolic fumarases with identical amino acid sequences are encoded from a single gene. Journal, of Biochemistry,1989,264:2581-2586
[22]Woods SA, Schwartzbach SD, Guest JR. Two biochemically different distinct classes offumarase in Escherichia coli [J]. Biochimical and Biophysical Research Communications,1988,954:14-26
[23]姜绍通,郑志,潘丽军,李兴江,张志英.L-乳酸米根霉发酵体系LDH活力及代谢调控研究[J].食品科学,2005,26(1)：41-44
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[26]王清路,李俏俏,薛金艳,窦烨,王红艳,张玉军,张杰.巴斯德毕赤酵母表达系统的特点及应用[J].生物技术通讯,2006,17(4)：640-641
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[30]Werten M M, Vanden B T J, Wind R D, et al. High-yield secretion of recombinant gelatins by Pichia pastoris[J]. Yeast,1999,15:1087-1096
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[45]T.FERAIN, J.N.HOBBS, JR, J.RICHARDSON, N.BERNARD, D.GARMYN, P.HOLS, N.E.ALLEN, J.DELCOUR. Knockout of the Two 1dh Genes Has a Major Impact on Peptidoglycan Precursor Synthesis in Lactobacillus plantarum[J]. JOURNAL OF BACTERIOLOGY,1996:5431-5437
[2]Robinson, W.D and Mount, et al. Maleic anhydride, maleic acid and fumaric acid[J]. Encyclopedia of Chemical technology,1978,14:770-793
[3]Walter J.Stefaniak, Cheektowaga, et al. Manufacture of fumaric acid[P]. US patent NO2914559,1959
[4]TR Felthouse, JC Burnett, et al. Maleic Anhydride, Maleic acid, and Fumaric acid[J]. Encyclopedia of Chemical technology,1995,15:893-928
[5]黄肾琦.富马酸氯马斯汀片[J].中国新药杂志,1998,7(3)：194-195
[6]于波涛,尧剑虹,舒明锡,曾仁杰.富马酸比索洛尔片制备工艺与质量控制研究[J].药学服务与研究,2005,5(3)：251-253
[7]Carol A.Roa Engel, Adrie J.J.Straathof, Tiemen W.Zijlmans, Walter M.van Gulik,Luuk A.M.van der Wielen.Fumaric acid production by fermentation[J]. Applied Microbiology and Biotechnology,2008,78:379-389
[8]关公凑,黄耀威等.由糖醛合成富马酸[J].广州化工,2001,29(2)：20-21
[9]Y.Zhou, J.Du, GT.Tsao, et al. Comparision of fumaric acid production by Rhizopus oryzae using different neutralizing agents[J]. Bioprocess Engineering Joural,2002,25:179-181
[10]李学坤,张昆等.富马酸的合成及应用[J].现代化工,2005,25(增刊)：81-83
[11]Ulrich Hartel, wolfgang Buckel. Fermentation of trans-aconitate via citrate, oxaloacetate, and pyruvate by Acidaminococcus fermentans[J]. Archives of Microbiology,1996,166: 342-349
[12]王普,虞炳钧,张福明,王雷.生物转化法生产L-苹果酸的研究(I)——富马酸酶产生菌株的筛选[J].食品与发酵工业,1996,1：18-19
[13]K.Belafi-Bako, N.Nemestothy, L.Gubicza. A study on applications of membrane techniques in bioconversion of fumaric acid to L-malic acid[J]. Desalination,2004, 162:301-306
[14]景晓辉,王之婉.诱变温特曲霉转化富马酸为L-苹果酸的研究[J].化学世界,2005：486-488
[15]BARBARA E.WRIGHT, Angelika Longacre, et al. Models of Metabolism in Rhizopus oryzae[J]. Journal of Theor Biology,1996,182(3):453-457
[16]Toshiaki Nakajima-kambe, Takehiro Nozue, et al. Bioconversion of Maleic acid to Fumaric acid to by Pseudomonas alcaligenes Strain XD-01[J]. Journal of fermentation and Bioengineering,1997,84:165-168
[17]PAUL V.ATTWOOD. The Structure and the Mechanism of Action of Pyruvate Carboxylase[J]. International Journal of Biochemistry and Cell Biology,1995, 27(3):231-234
[18]Carol A, Roa Engel Adrie J, et al. Fumaric acid production by fermentation[J]. Applied Microbiology and Biotechnology,2008,78:379-385
[19]Osmani SA, Scrutton MC. The sub-cellular localization and regulatory properties of pyruvate carboxylase from Rhizopus arrhizus. European Journal of Biochemistry,1985, 147:119-128
[20]PAUL V.ATTWOOD. The Structure and the Mechanism of Action of Pyruvate Carboxylase[J]. International Journal of Biochemistry and Cell Biology,1995,27(3):231-234
[21]Suzuki T, Sato M, Yoshida T and Tuboi S. Rat liver mito-chondrial and cytosolic fumarases with identical amino acid sequences are encoded from a single gene. Journal, of Biochemistry,1989,264:2581-2586
[22]Woods SA, Schwartzbach SD, Guest JR. Two biochemically different distinct classes offumarase in Escherichia coli [J]. Biochimical and Biophysical Research Communications,1988,954:14-26
[23]姜绍通,郑志,潘丽军,李兴江,张志英.L-乳酸米根霉发酵体系LDH活力及代谢调控研究[J].食品科学,2005,26(1)：41-44
[24]T.FERAIN, J.N.HOBBS, J.RICHARDSON, N.BERNARD, D.GARMYN, P.HOLS, N.E.ALLEN, J.DELCOUR. Knockout of the Two ldh Genes Has a Major Impact on Peptidoglycan Precursor Synthesis in Lactobacillus plantarum[J]. JOURNAL OF BACTERIOLOGY,1996:5431-5437
[25]吴万贵,袁俐,王仙.毕赤酵母高效表达外源基因的研究进展[J].农垦医学,2008-02,30(1)：56-58.
[26]王清路,李俏俏,薛金艳,窦烨,王红艳,张玉军,张杰.巴斯德毕赤酵母表达系统的特点及应用[J].生物技术通讯,2006,17(4)：640-641
[27]王勇.毕赤酵母表达外源基因研究进展[J].微生物学免疫学进展,2004,32(1)：62-66
[28]Cereghino J L, Cregg J M. Heterologous protein expression in the methylotrophic yeast Pichia pastoris[J]. FEMA Microbiology Review,2000,24(1):45-66
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