短小芽孢杆菌fadD基因的敲除及其对ω_(-1)-羟基脂肪酸产量的影响
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摘要
C18长碳链羟基脂肪酸中的ω_(-1)、ω_(-2)、ω_(-3)-羟基脂肪酸(ω_(-1)、ω_(-2)、ω_(-3)-HFA)可应用于机械工业、食品工业;而作为单体合成高分子聚合物时可提供最长的单体分子碳链,使得聚合物材料无单分子侧链,性能更优越,是当今绿色聚合物材料研究开发中最理想的单体成分之一。短小芽孢杆菌(Bacillus pumilus)M-F641是一株经过诱变后获得的ω_(-1)-羟基脂肪酸高产菌株,该菌株易于培养,且ω_(-1)-羟基脂肪酸转化率高(16.8%),为了能够进一步提高羟基脂肪酸的转化率(羟基脂肪酸与脂肪酸加入总量之比)和羟基脂肪酸产率(羟基脂肪酸与脂肪酸消耗量之比),本文对脂肪酸β-氧化代谢过程中参与长链脂肪酸活化、跨膜转运和编码脂酰CoA合成酶(Fatty acyl coenzyme A synthetases,FACS)的关键酶基因fadD进行了研究,主要研究内容如下:
首先,根据已发表的短小芽孢杆菌以及芽孢杆菌属的其它菌株的fadD基因设计了特异性引物,PCR扩增获得了B. pumilus M-F641的fadD基因序列,长度为1720bp,并利用MEGA 3.1、DNAStar软件对该基因序列进行了分析。
其次,构建重组表达质粒pET32M- fadD,转化到大肠杆菌BL21(DE3)中进行原核表达,IPTG诱导后表达产物经SDS-PAGE电泳分析表明重组质粒在约65 kDa处有明显的表达,并利用试剂盒测定了表达产物脂酰CoA合成酶的活性,酶活达到79.2U/mg。
最后,确定基因敲除策略,成功构建了自杀载体pMD18-T-fadD::Tetr,通过插入失活的方法对B. pumilus M-F641的fadD基因进行敲除,获得其fadD基因缺陷菌株B. pumilus M-F641A,经24h简单发酵验证,缺陷菌株B. pumilus M-F641A的ω-1羟基脂肪酸的产率为16.95%,但并不能证明产率有所提高,需经进一步发酵验证。
ω_(-1),ω_(-2),ω_(-3)-hydroxy fatty acids of C18 long-chain hydroxy fatty acids due to its hydroxyl group close to the first C-terminal and reaction nature live, can be applied to the surface-active agent, cosmetic ingredients, lubricants and anti-rust agent in machinery industry,and anti-bacterial agents in the food industry. They also work as a monomer in the synthesis of polymers to provide a maximum carbon chain which is no single-molecule side-chain and make the polymers’performance superior.ω-1-hydroxy fatty acid is one of the best monomers in research and development of green polymers. Bacillus pumilus M-F641 is a high-yielding strain forω-1-hydroxy fatty acids production obtained by mutagenesis, and it is easy to incubate. In order to improve the conversion rate of hydroxy fatty acids (the ratio of hydroxy fatty acids and total adding fatty acids) and the hydroxy fatty acid production rate (the ratio of hydroxy fatty acids and fatty acid consumption), in this paper, a key enzyme gene of fadD involved in long-chain fatty acid activation, transmembrane transport and encoding acyl-CoA synthetase in fatty acidβ-oxidation metabolic process was studied. The main research contents are as follows:
Firstly, B. pumilus M-F641's fadD gene was amplified by PCR method and the primer was designed according to published fadD genes of Bacillus pumilus and other Bacillus strains. Full-length sequences of fadD gene about 1720 bp were obtained. The sequences waere analyzed by using the MEGA 3.1 and DNAStar software;
Secondly, the recombinant expression plasmid pET32M-fadD was transformed into E. coli BL21 (DE3).after IPTG inducing , SDS-PAGE electrophoresis analysis showed a clear expression of pET32M-fadD. The recombinant expression acyl-CoA synthetase activity recached to 79.2U/mg;
Finally, the suicide vector pMD18-T-fadD::Tetr was succeessfully constructed,adopted deletion-insertion to disrupt fadD gene of B.pumilus M-F641 and got fadD knockout mutant B. pumilus M-F641A.After 24h simple fermentation validation, theω-1 hydroxy fatty acid production rate of B. pumilus M-F641A was 16.95%, but it can not prove that the yield has increased an subject to further fermentation to verify.
首先,根据已发表的短小芽孢杆菌以及芽孢杆菌属的其它菌株的fadD基因设计了特异性引物,PCR扩增获得了B. pumilus M-F641的fadD基因序列,长度为1720bp,并利用MEGA 3.1、DNAStar软件对该基因序列进行了分析。
其次,构建重组表达质粒pET32M- fadD,转化到大肠杆菌BL21(DE3)中进行原核表达,IPTG诱导后表达产物经SDS-PAGE电泳分析表明重组质粒在约65 kDa处有明显的表达,并利用试剂盒测定了表达产物脂酰CoA合成酶的活性,酶活达到79.2U/mg。
最后,确定基因敲除策略,成功构建了自杀载体pMD18-T-fadD::Tetr,通过插入失活的方法对B. pumilus M-F641的fadD基因进行敲除,获得其fadD基因缺陷菌株B. pumilus M-F641A,经24h简单发酵验证,缺陷菌株B. pumilus M-F641A的ω-1羟基脂肪酸的产率为16.95%,但并不能证明产率有所提高,需经进一步发酵验证。
ω_(-1),ω_(-2),ω_(-3)-hydroxy fatty acids of C18 long-chain hydroxy fatty acids due to its hydroxyl group close to the first C-terminal and reaction nature live, can be applied to the surface-active agent, cosmetic ingredients, lubricants and anti-rust agent in machinery industry,and anti-bacterial agents in the food industry. They also work as a monomer in the synthesis of polymers to provide a maximum carbon chain which is no single-molecule side-chain and make the polymers’performance superior.ω-1-hydroxy fatty acid is one of the best monomers in research and development of green polymers. Bacillus pumilus M-F641 is a high-yielding strain forω-1-hydroxy fatty acids production obtained by mutagenesis, and it is easy to incubate. In order to improve the conversion rate of hydroxy fatty acids (the ratio of hydroxy fatty acids and total adding fatty acids) and the hydroxy fatty acid production rate (the ratio of hydroxy fatty acids and fatty acid consumption), in this paper, a key enzyme gene of fadD involved in long-chain fatty acid activation, transmembrane transport and encoding acyl-CoA synthetase in fatty acidβ-oxidation metabolic process was studied. The main research contents are as follows:
Firstly, B. pumilus M-F641's fadD gene was amplified by PCR method and the primer was designed according to published fadD genes of Bacillus pumilus and other Bacillus strains. Full-length sequences of fadD gene about 1720 bp were obtained. The sequences waere analyzed by using the MEGA 3.1 and DNAStar software;
Secondly, the recombinant expression plasmid pET32M-fadD was transformed into E. coli BL21 (DE3).after IPTG inducing , SDS-PAGE electrophoresis analysis showed a clear expression of pET32M-fadD. The recombinant expression acyl-CoA synthetase activity recached to 79.2U/mg;
Finally, the suicide vector pMD18-T-fadD::Tetr was succeessfully constructed,adopted deletion-insertion to disrupt fadD gene of B.pumilus M-F641 and got fadD knockout mutant B. pumilus M-F641A.After 24h simple fermentation validation, theω-1 hydroxy fatty acid production rate of B. pumilus M-F641A was 16.95%, but it can not prove that the yield has increased an subject to further fermentation to verify.
引文
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