甲基营养菌M.sp.MB200中serA基因功能初探

serA基因编码3-磷酸甘油酸脱氢酶（PGDH）,此酶在合成L-丝氨酸的第一步起催化作用。利用质粒pCM80分别与野生型基因serA、缺失了C末端ACT功能域的突变基因serA△77构建了重组体表达菌MB200/pCM80serA和MB200/pCM80serA△77。通过分析发现野生型基因重组表达菌的PGDH酶活受L-丝氨酸浓度影响较大,当反应体系中的L-丝氨酸浓度为40μmol/L时,酶活减少了约1/3;缺失了ACT功能域的基因表达菌的PGDH酶活基本不变,不受L-丝氨酸浓度的影响。进一步在静息细胞反应条件下,对重组菌和野生菌株MB200进行发酵产L-丝氨酸的研究,实验发现MB200/pCM80serA产L-丝氨酸的量为13.6mg/mL,MB200/pCM80serA△77产L-丝氨酸的量为16.8 mg/mL,而野生型菌株M.sp MB200的产量为6.4 mg/mL,重组菌的L-丝氨酸产量都有所提高,且MB200/pCM80serA△77的产量提高更多。结果证实了甲基营养菌MB200中serA基因与产L-丝氨酸具有反馈抑制关系,为进一步解除反馈抑制并发酵产L-丝氨酸奠定了基础。

Function of serA Gene in M. sp. MB200

3-phosphoglycerate dehydrogenase（PGDH）, encoded by serA gene, catalyzes the first step in the synthesis of l-serine in some microorganisms. Two recombinants of MB200/pCM80serA（based on the wild-type gene）and MB200/pCM80serA△77（based on the mutant gene missing 77 amino acid in the C-terminal） were constructed using pCM80 as vector. Enzyme activity analysis showed that the PGDH activity in strain MB200/pCM80serA was affected largely by the concentration of L-serine, and was reduced by about one-third when the concentration of L-serine was 40 μmol/L, while the PGDH activity in strain MB200/pCM80serA△77 was not influenced by the concentration of L-serine. L-serine productivity of strains through the resting cell reaction systems, and the result found that the L-serine production of MB200/pCM80serA and MB200/pCM80serA△77 was 13.6 mg/mL and 16.8 mg/mL respectively, and MB200 of 6.4 mg/mL. L-serine production in recombinant bacteria has risen and yields of MB200/pCM80serA△77 is more higher. Results confirmed that serA gene in M. sp MB200 inhibited by L-serine,and laid the foundation for further relieving feedback inhibition and produce L-serine using M. sp MB200.