含苏氨酸操纵子重组质粒的构建及其对大肠杆菌L-苏氨酸积累的影响

Construction of recombinant plasmids containing threonine operon and their effects on L-threonine accumulation

摘要:

摘要：【目的】通过分子生物学手段构建重组质粒，将其转入野生型大肠杆菌W3110，分析含苏氨酸操纵子基因的质粒及质粒定点突变解除反馈抑制时，对L-苏氨酸积累的影响。【方法】以W3110染色体DNA为模板，PCR扩增苏氨酸操纵子基因，即启动子THrLp、编码前导肽基因thrL以及thrA、thrB、thrC基因，通过重叠延伸PCR的方法对thrA基因定点突变，解除苏氨酸对它的反馈抑制，构建出重组表达质粒WYE112和WYE134，5 L发酵实验测定L-苏氨酸的产量。【结果】经5 L发酵罐发酵产酸实验，W3110的L-苏氨酸产量为0.036 ± 0.004 g/L，携带含苏氨酸操纵子质粒的W3110菌株L-苏氨酸产量为2.590 ± 0.115 g/L，质粒上thrA解除反馈抑制后，L-苏氨酸的产量增加到9.223 ± 1.279 g/L。【结论】过表达苏氨酸操纵子基因可以使L-苏氨酸积累，进一步解除thrA基因的反馈抑制，可以增强L-苏氨酸积累的效果，为L-苏氨酸工程菌改造的进一步研究奠定了基础。

Abstract:

Abstract: [Objective] We reconstructed two recombinant plasmids and studied their effects on L-threonine accumulation of Escherichia coli W3110. [Methods] We amplified the threonine operon containing ThrLp promoter, lead peptide thrL, thrA thrB and thrC genes by PCR from E. coli W3110 chromosome and ligated it into the pMD19 T-vector. Site-directed mutation were carried out by gene splicing by overlap extension PCR to release the feedback inhibition of aspartokinase I (thrA). Two recombinant plasmids WYE112 and WYE134 were transformed into E. coli W3110 by electroporation. Fed-batch cultures of E. coli W3110 were carried out in 5-Liter fermentors and the L-threonine concentration was measured by HPLC. [Results] Fed-batch fermentation results showed that E. coli W3110 could accumulate little L-threonine (0.036 ± 0.004 g/L) but recombinant E. coli W3110 harboring the plasmid WYE112 containing a threonine operon exhibited a L-threonine production of 2.590 ± 0.115 g/L. Furthermore, L-threonine production reached 9.223 ± 1.279 g/L when the feedback inhibition of thrA was released. [Conclusion] Overexpression of threonine operon can lead to the accumulation of L-threonine. Further release of feedback inhibition of aspartokinase I can enhance its accumulation.