同源过表达fnr、pncB和fdhF对克雷伯菌产氢代谢的影响

Enhanced biohydrogen production by homologous over-expression of fnr, pncB, fdhF in Klebsiella sp. HQ-3

摘要:

利用代谢工程手段改造克雷伯菌Klebsiella sp. HQ-3产氢途径中相关代谢调控因子及辅酶因子，以构建高效产氢工程菌。利用简并引物，以Klebsiella sp. 总DNA为模板，克隆了甲酸-氢裂解酶系统的全局转录调控因子 (FNR) fnr基因、编码甲酸脱氢酶 (FDH-H) fdhF基因，以及NADH途径中编码烟酸转磷酸核糖激酶(NAPRTase)的pncB基因，构建了3种同源过表达重组菌株HQ-3-fnr、HQ-3-fdhF和HQ-3-pncB，以研究同源过表达产氢代谢调控因子及辅酶因子对克雷伯菌累积产氢、细胞生长、代谢终产物的影响。结果表明，过表达fnr、pncB和fdhF基因的克雷伯工程菌的产氢效率比携带空载体的克雷伯对照菌株分别提高12.26％、11.62％和7.28％；重组菌HQ-3-fnr、HQ-3-fdhF和HQ-3-pncB的葡萄糖利用率较克雷伯对照菌株HQ-3-C明显增加，过表达fnr、fdhF基因使代谢合成甲酸量增多；过表达pncB基因能促进NADH合成，使更多的NADH流入消耗NADH较多的乙醇与琥珀酸代谢路径，使得乙醇和琥珀酸含量增加，而乳酸含量降低。

Abstract:

To enhance biohydrogen production of Klebsiella sp. HQ-3, the global transcriptional factor (Fnr), formate dehydrogenase H (FDHH) and the pncB gene encoding the nicotinic acid phosphoribosyltransferase (NAPRTase) were for the first time over-expressed in Klebsiella sp. HQ-3. The fnr, fdhF, pncB genes were cloned from the genomic DNA of Klebsiella sp. HQ-3 by 3 pairs of universal primers, and introduced into the corresponding sites of the modified pET28a-Pkan, resulting in the plasmids pET28a-Pkan-fnr, pET28a-Pkan-fdhF and pET28a-Pkan-pncB. The 4 plasmids were then electroported into wild Klebsiella sp. HQ-3 to create HQ-3-fnr, HQ-3-fdhF, HQ-3-pncB and HQ-3-C, respectively. Hydrogen production was measured using a gas chromatograph and the metabolites were analyzed with a high-performance liquid chromatograph (HPLC). The results indicate that over-expression of fnr, fdhF and pncB significantly enhanced hydrogen production in the three recombinant strains. Hydrogen production per mol glucose for HQ-3 fnr, HQ-3 pncB, HQ-3 fdhF was 1.113, 1.106 and 1.063 mol of hydrogen/mol glucose, which was respectively increased by 12.26%, 11.62% and 7.28% compared with that of the control strain HQ-3-C (0.991 mol of hydrogen/mol glucose). Moreover, the analysis of HPLC showed that the concentrations of formate and lactate were markedly decreased, but succinate remained unchanged in culture media compared with those of the control strain HQ-3-C.