基因改造Rhodobacter sphaeroides提高

Genetic manipulation of Rhodobacter sphaeroides for improved production of coenzyme Q10

摘要:

【目的】通过敲除类球红细菌2.4.1基因组中八氢番茄素合成酶基因crtB, 让异戊二烯前体更多流向辅酶Q10的合成。引入大肠杆菌编码的分支酸裂解酶基因ubiC和4-羟苯甲酸转移酶基因ubiA, 提高4-羟苯甲酸的合成和与聚异戊二烯的连接, 从而提高类球红细菌的辅酶Q10产量。【方法】以自杀型质粒pSUP202为载体, 构建包含crtB基因上游2.5 kb片段, 壮观霉素抗性基因, ubiC、ubiA基因和crtB基因下游2.5 kb片段的基因置换质粒, 利用结合转移方法转入类球红细菌2.4.1中, 利用抗性机制筛选双交换突变株, RT-PCR方法检测引入的ubiC和ubiA基因转录。用HPLC方法测定出发菌株和基因改造菌株的辅酶Q10产量。【结果】成功构建出基因置换质粒, 筛选出发生基因置换的突变株, RT-PCR证实了外源基因的转录, 并且突变株辅酶Q10的产量比出发菌株提高40%。【结论】大肠杆菌的ubiC和ubiA基因能够利用自身启动子在类球红细菌中表达, 利用基因改造的方法能成功提高类球红细菌的辅酶Q10产量。

Abstract:

[Objective] The objective of this study was to improve the production of coenzyme Q10 of Rhodobacter sphaeroides by replacing crtB (encoding phytoene synthase) with ubiC and ubiA (encoding chorismate pyruvate-lyase and 4-hydroxybenzoate: polyprenyldiphosphate 3-polyprenyltransferase, respectively) from Escherochia coli DH5α. [Methods] The plasmid for gene replacement was constructed with a suicide pSUP202 as vector, and its insertions, including the up and down streams of crtB in R. sphaerodies 2.4.1, spectinomycin resistance gene, ubiC and ubiA from E. coli DH5α were obtained by PCR. RT-PCR was used to detect the transcription of genes. HPLC was used to quantify the production of coenzyme Q10. [Results] The gene replacement mutant of R. sphaerodies was constructed, in which the crtB was replaced by ubiC and ubiA. The transcription of heterologous genes was confirmed by RT-PCR. The productivity of gene engineered strain was 1.6 fold of wide type. [Conclusion] The strain of R. sphaerodies with improved production of coenzyme Q10 was obtained successfully, and the ubiC and ubiA from E. coli could transcript with its native promoter in R. sphaerodies.