大肠杆菌积累丙酮酸的研究
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摘要
丙酮酸是机体代谢的中间产物,在大肠杆菌中处于糖酵解途径的末端,它连接着EMP途径和TCA循环,一般用大肠杆菌发酵不会积累丙酮酸。但是在对大肠杆菌进行基因工程改造后,发现有丙酮酸出现,譬如lpdA基因敲除后的大肠杆菌。
lpdA基因编码的硫辛酰胺脱氢酶(lipoamide dehydrogenase,LPD)是丙酮酸脱氢酶复合体(pyruvate dehydrogenase complex,PDHc)的组成成分。有氧条件下,大肠杆菌中的PDHc是连接糖酵解途径和三羧酸循环的关键酶,在其催化作用下丙酮酸氧化脱羧生成乙酰辅酶A,该酶的缺陷会导致丙酮酸的积累。本文重点考察了lpdA基因敲除大肠杆菌(Escherichia coli lpdA~-)在不同碳源下积累丙酮酸的情况,并在此基因基础上,设想进一步敲除多个基因,构建出新的基因敲除的菌株,从分子水平考察新菌株对丙酮酸积累的影响。
第一,比较了敲除lpdA基因前后大肠杆菌的代谢情况,发现Escherichia colilpdA~-与野生型菌株相比,能够积累较高浓度的丙酮酸,而一般野生型菌株基本不积累丙酮酸。以葡萄糖为碳源对Escherichia coli lpdA~-进行游离培养,考察pH值、底物葡萄糖浓度等因素对丙酮酸积累情况的影响。发现lpdA基因敲除后,由于丙酮酸的积累,培养基pH值会迅速下降,影响菌体生长代谢,因此维持pH=7对Escherichia coli lpdA~-非常重要。通过对Escherichia coli lpdA~-的考察,最终获得了该菌株的一系列基本发酵参数:pH值恒定在7,有氧条件下培养20h后,30g/L葡萄糖基本耗尽,最高菌体浓度能达3.5g/L,丙酮酸的最终浓度为4.0g/L。
第二,分别以果糖、木糖及混合糖为碳源对Escherichia coli lpdA~-进行游离培养,通过比较不同单糖及混合糖培养结果,发现了混合糖培养条件下,20g/L葡萄糖及果糖的混合糖、20g/L木糖及果糖的混合糖培养的丙酮酸的最大浓度达到8g/L,是单独以20g/L葡萄糖、果糖或木糖培养的2倍。
第三,考察了Escherichia coli lpdA~-性能的基础上,对其进行多基因敲除操作的初探。采用PCR产物一步失活法敲除大肠杆菌的ldhA基因,首先,构建含有ldhA基因同源片段的卡那霉素抗性基因DNA;其次,将辅助质粒pkD46和卡那霉素抗性基因转化入细胞内;最后,通过抗性平板筛选和PCR确认,筛选到敲除ldhA基因的突变株。
Lipoamide dehydrogenase (LPD) coded by lpdA gene is an important composition of pyruvate dehydrogenase complex (PDHc). Under aerobic condition, PDHc of Escherichia coli is the key enzyme which connects EMP pathway with TCA cycle. Pyruvic acid converts into acetyl-CoA under the catalysis of PDHc whose disfigurement would lead to pyruvic acid accumulation. In this dissertation, lpdA gene knockout Escherichia coli (Escherichia coli lpdA~-) was studied for the purpose of investigating pyruvic acid accumulation. This fermentation aimed at enhancing pyruvic acid concentration, yield and prdouctivity. Farther, primary study on polygene knockout was carried out in order to construct a better genetic strain.
Firstly, comparison between wild type Escherichia coli and lpdA gene knockout Escherichia coli was carried out and the find was that Escherichia coli lpdA~- could accumulate pyruvic acid but wild type Escherichia coli could not. Free cell culture with glucose as carbon source was carried out and investigations about factor such as pH and concentration of glucose were done. pH=7 is a most important factors for lpdA gene knockout Escherichia coli, because the accumulation of pyruvic acid would lead to low pH which inhibited growth and metabolism of Escherichia coli. By the study on Escherichia coli lpdA~-, a series of parameters was obtained: pH=7, under aerobic condition with adding 30g/L glucose, the glucose was consumed in 20 hour and the final pyruvic acid concentration was 4.0g/L following the biomass raised to 3.5g/L ultimately.
Secondly, the fermentation with fructose, xylose and their mixture as carbons respectively was investigated. The results showed that the mixture of two kind of monosaccharide would raise the production of pyruvic acid. For instance, the maximum of pyruvic acid concentration with 20g/L glucose singlly is about 2g/L, but the maximum of pyruvic acid concentration with 10g/L gluocose and 10g/L fructose is 8g/L which is the quadruple of single glucose fermentation.
Third, according to capability of Escherichia coli lpdA~- to accumulate pyruvic acid, polygene knockout was investigated to construct a better genetic strain. One-step inactivation of chromosomal ldhA genes in Escherichia coli using PCR products were carried out. Construct linear kanamycin-resistant gene with ldhA gene homologous fragment first, and then transfer this DNA and vector pkD46 into the cell. After Screening by antibiotics and PCR, a mutant of ldhA knockout was obtained finally.
lpdA基因编码的硫辛酰胺脱氢酶(lipoamide dehydrogenase,LPD)是丙酮酸脱氢酶复合体(pyruvate dehydrogenase complex,PDHc)的组成成分。有氧条件下,大肠杆菌中的PDHc是连接糖酵解途径和三羧酸循环的关键酶,在其催化作用下丙酮酸氧化脱羧生成乙酰辅酶A,该酶的缺陷会导致丙酮酸的积累。本文重点考察了lpdA基因敲除大肠杆菌(Escherichia coli lpdA~-)在不同碳源下积累丙酮酸的情况,并在此基因基础上,设想进一步敲除多个基因,构建出新的基因敲除的菌株,从分子水平考察新菌株对丙酮酸积累的影响。
第一,比较了敲除lpdA基因前后大肠杆菌的代谢情况,发现Escherichia colilpdA~-与野生型菌株相比,能够积累较高浓度的丙酮酸,而一般野生型菌株基本不积累丙酮酸。以葡萄糖为碳源对Escherichia coli lpdA~-进行游离培养,考察pH值、底物葡萄糖浓度等因素对丙酮酸积累情况的影响。发现lpdA基因敲除后,由于丙酮酸的积累,培养基pH值会迅速下降,影响菌体生长代谢,因此维持pH=7对Escherichia coli lpdA~-非常重要。通过对Escherichia coli lpdA~-的考察,最终获得了该菌株的一系列基本发酵参数:pH值恒定在7,有氧条件下培养20h后,30g/L葡萄糖基本耗尽,最高菌体浓度能达3.5g/L,丙酮酸的最终浓度为4.0g/L。
第二,分别以果糖、木糖及混合糖为碳源对Escherichia coli lpdA~-进行游离培养,通过比较不同单糖及混合糖培养结果,发现了混合糖培养条件下,20g/L葡萄糖及果糖的混合糖、20g/L木糖及果糖的混合糖培养的丙酮酸的最大浓度达到8g/L,是单独以20g/L葡萄糖、果糖或木糖培养的2倍。
第三,考察了Escherichia coli lpdA~-性能的基础上,对其进行多基因敲除操作的初探。采用PCR产物一步失活法敲除大肠杆菌的ldhA基因,首先,构建含有ldhA基因同源片段的卡那霉素抗性基因DNA;其次,将辅助质粒pkD46和卡那霉素抗性基因转化入细胞内;最后,通过抗性平板筛选和PCR确认,筛选到敲除ldhA基因的突变株。
Lipoamide dehydrogenase (LPD) coded by lpdA gene is an important composition of pyruvate dehydrogenase complex (PDHc). Under aerobic condition, PDHc of Escherichia coli is the key enzyme which connects EMP pathway with TCA cycle. Pyruvic acid converts into acetyl-CoA under the catalysis of PDHc whose disfigurement would lead to pyruvic acid accumulation. In this dissertation, lpdA gene knockout Escherichia coli (Escherichia coli lpdA~-) was studied for the purpose of investigating pyruvic acid accumulation. This fermentation aimed at enhancing pyruvic acid concentration, yield and prdouctivity. Farther, primary study on polygene knockout was carried out in order to construct a better genetic strain.
Firstly, comparison between wild type Escherichia coli and lpdA gene knockout Escherichia coli was carried out and the find was that Escherichia coli lpdA~- could accumulate pyruvic acid but wild type Escherichia coli could not. Free cell culture with glucose as carbon source was carried out and investigations about factor such as pH and concentration of glucose were done. pH=7 is a most important factors for lpdA gene knockout Escherichia coli, because the accumulation of pyruvic acid would lead to low pH which inhibited growth and metabolism of Escherichia coli. By the study on Escherichia coli lpdA~-, a series of parameters was obtained: pH=7, under aerobic condition with adding 30g/L glucose, the glucose was consumed in 20 hour and the final pyruvic acid concentration was 4.0g/L following the biomass raised to 3.5g/L ultimately.
Secondly, the fermentation with fructose, xylose and their mixture as carbons respectively was investigated. The results showed that the mixture of two kind of monosaccharide would raise the production of pyruvic acid. For instance, the maximum of pyruvic acid concentration with 20g/L glucose singlly is about 2g/L, but the maximum of pyruvic acid concentration with 10g/L gluocose and 10g/L fructose is 8g/L which is the quadruple of single glucose fermentation.
Third, according to capability of Escherichia coli lpdA~- to accumulate pyruvic acid, polygene knockout was investigated to construct a better genetic strain. One-step inactivation of chromosomal ldhA genes in Escherichia coli using PCR products were carried out. Construct linear kanamycin-resistant gene with ldhA gene homologous fragment first, and then transfer this DNA and vector pkD46 into the cell. After Screening by antibiotics and PCR, a mutant of ldhA knockout was obtained finally.
引文
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2.Slavinska V.,Sile D.,Korchagovae E.,Katkevich M.,Lukevics E.,New way for preparation of 4-phenyl-2-oxobutyric acid ethyl ester.Synthetic communication,1996,26(11):2229-2233
3.马翠卿,生物催化法生产丙酮酸(盐)的研究(博士学位论文),山东大学,济南,2003
4.韩广甸,赵树纬,李述文等编译.有机制备化学手册(中册),北京,石油工业出版社,1997:235-236
5.杨辉琼,易翔,郭贤烙,乳酸氧气氧化法制备丙酮酸,化学世界,2002,06:307-308
6.姜胜斌,喻宗沅.丙酮酸(酯)的合成方法评述.湖北化.1999,16(5):1-3
7.Pinxt H.H.C.M.,Kuster B.F.M.,Matin G.B.,Promoter effects in the Pt-catalysed oxidation of propylene glycol.Applied catalysis A:General,2000,191:45-54
8.Yokota A.,Iota S.,Takao S.,Tryptophan production by a lipoic acid auxotroph Enterobacter aerogenes having both pyruvie acid productivity and high truytophanase activity.Agric,Biol,Chem.,1989,53:2037-2044
9.Yanse H.,Mori N.,Masuda M.,Pyruvate production by Enterococcus casseliflavus A212 from gluconate in an alkaline medium.J,Ferment,Bioeng.,1992,73:287-291
10.Yokota A.,Terasawa Y.,Takaota N.,Pyruvate productiom by an F1-ATPase-defective mutant of Escherichia coli.Biosci.Biotechnol.Biochem.,1994,58:2164-2169
11.Besnainou B.,Giani D.,Sahut C.,Process for the production of pyruvia acid by fermentation.European Patent.NO.EP 312453
12.Takao S.,Tanida M.,Pyruvic acid production by Schizophyllum commune.J,,Ferment,Technol.,1982,60:277-280
13.Uchio R.,Kikuchi K.,Hirose Y.,Process for produeting pyruvic acid by fermentation.US Patent.No.39993543
14.Yonehara T.,Miyata R.,Fermentative production of pyruvate from glucose by Torulopsis glabrata.J,Ferment,Bioeng.,1994,78(2):155-159
15.Miyata R.,Yonehara T.,Improvement of fermentative production of pyruvate from glucose by Torulopsisi glabrata IFO0005.J,,Ferment,Bioeng.,1996,82(5):475-479
16.Miyata R.,Yonehara T.,Fermentative manufacture of pyruvic acid andi its salts.JP Patent.,1997,09047292
17.袁辉,华子春,丙酮酸发酵能力的提高,微生物学杂志,2000,19(6):23-24
18.Miyata R.,Yonehara T.Breeding of high-pyruvate-production Totuglopsis glabrata with acquired reduced pyruvate decarboxylase.J,Biosci,Bioeng.,1999,88(2):173-177
19.Izumi Y.,Matsumura Y.,Tani Y.,Yamada H.,Pyruvic acid production from 1,2-propanediol by thiamin-requiring Acinetobacter sp.80-M.Agric,Biol,Chem.,1982,46:2673-2679
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