关键基因的修饰对枯草芽孢杆菌尿苷合成的影响

Effect of key-gene modification on uridine biosynthesis in Bacillus subtilis

摘要:

【目的】探究磷酸核糖焦磷酸(PRPP)合成酶(prs)和氨甲酰磷酸合成酶(pyrAA/pyrAB)的点突变,以及异源5'-核苷酸酶(sdt1)的过表达,对枯草芽孢杆菌尿苷生物合成的影响。【方法】依据推断的变构位点,分别在prs基因和pyrAB基因编码序列中引入点突变;将点突变的prs基因在染色体xylR位点整合表达,pyrAB基因则在染色体原位被修饰; sdt1基因在染色体sacB位点整合过表达。通过对重组菌摇瓶发酵液中尿苷、胞苷和尿嘧啶的分析,表征相关基因修饰对尿苷合成的影响。【结果】在PRPP合成酶中引入Asn120Ser、Leu135Ile和Glu52Gly或Val312Ala点突变,分别导致尿苷积累量提高67%和96%。进一步在氨甲酰磷酸合成酶中引入Ser948Phe、Thr977Ala和Lys993Ile点突变,导致尿苷积累量又增加了182%,达到6.97 g/L。在此基础上,过表达异源5'-核苷酸酶,导致尿苷产量增加17%,达到8.16 g/L。【结论】PRPP合成酶和氨甲酰磷酸合成酶的酶活或反馈抑制调节机制,是限制尿苷过量合成的重要因素。PRPP合成酶的Asn120Ser和Leu135Ile点突变,以及氨甲酰磷酸合成酶的Ser948Phe、Thr977Ala和Lys993Ile点突变,能够显著促进尿苷合成。PRPP合成酶附加的Glu52Gly或Val312Ala点突变,有利于尿苷合成。异源的嘧啶专一性5'-核苷酸酶的引入,也对尿苷的合成有明显的促进作用。

Abstract:

[Objective] We studied several crucial factors influencing the uridine biosynthesis in Bacillus subtilis, including mutations of phosphoribosylpyrophosphate synthetase(PRPP synthetase)(prs) and carbamyl phosphate synthetase(pyrAA/pyrAB), and overexpression of heterologous 5'-nucleotidase(sdt1).[Methods] According to the inferred allosteric sites, we introduced point mutation into coding sequences of prs and pyrAB. The mutated prs gene was integratedly expressed in the xylR locus of the chromosome and the pyrAB gene was modified in-situ. The sdt1 gene was overexpressed in the saB locus of the chromosome. The effect of the genetic modification on uridine biosynthesis was characterized by the analysis of uridine, cytidine and uracil in the fermentation broth.[Results] The mutations of Asn120Ser, Leu135Ile, Glu52Gly or Val312Ala on PRPP synthase resulted in an increase of uridine production by 67% and 96%, respectively. The mutations of Ser948Phe, Thr977Ala and Lys993Ile on carbamyl phosphate synthase resulted in a 182% increase of uridine yield to 6.97 g/L. The overexpression of heterologous 5'-nucleotidase resulted in a 17% increase of uridine yield to 8.16 g/L.[Conclusion] The activity and regulation mechanism of PRPP synthase and carbamyl phosphate synthase was an important factor to limit the excessive synthesis of uridine. Asn120Ser and Leu135Ile mutations of PRPP synthase and Ser948Phe, Thr977Ala and Lys993Ile mutations of carbamyl phosphate synthase will facilitate the biosynthesis of uridine. The additional Glu52Gly and Val312Ala mutations of PRPP synthase were beneficial for uridine biosynthesis. The reaction from UMP to uridine also limited the biosynthesis of uridine in B. subtilis.