该实验考察和比较增强回补途径对谷氨酸棒状杆菌合成L-异亮氨酸的影响。通过以L-异亮氨酸生产菌Corynebacterium glutamicum YI为出发菌株,分别采用基因组整合和质粒的方式过表达磷酸烯醇式丙酮酸羧化酶编码基因ppc及丙酮酸羧化酶编码基因pyc。结果表明,获得pyc基因组整合和质粒过表达菌株ILE01和ILE02,摇瓶条件下菌株L-异亮氨酸产量分别提高17.3%(6.1 g/L)和9.6% (5.7 g/L)、发酵罐条件下分别提高11.7% (24.8 g/L)和8.1%(24.0 g/L)。获得ppc基因组整合和质粒过表达菌株ILE03和 ILE04,摇瓶条件下菌株L-异亮氨酸产量分别提高30.8% (6.8 g/L)和13.5%(5.9 g/L)、发酵罐条件下分别提高15.8% (25.7 g/L)和9.5% (24.3 g/L)。此外,过表达pyc和ppc还可不同程度地提高L-异亮氨酸转化率。然而采用质粒过表达pyc和ppc均使得菌株生物量下降。因此,过表达pyc和ppc均能显著提高L-异亮氨酸产量和转化率,基因组整合的过表达方式效果优于质粒过表达。该研究首次比较并报道了增强谷氨酸棒杆菌回补途径对谷氨酸棒杆菌生产L-异亮氨酸的影响,可为其代谢工程改造提供参考。
To clarify and compare the effects of enhancing the anaplerotic pathways on L-isoleucine production performance by Corynebacterium glutamicum. The genes of pyc and ppc were overexpressed by replacing the native promoter with Ptuf or by inserting the genes to plasmid in a L-isoleucine producer, C. glutamicum YI.The pyc overexpressed via genome-integration in ILE01 and via plasmid in ILE02 resulted in 17.3% (6.1 g/L) and 9.6% (5.7 g/L) increase in production of L-isoleucine by batch fermentation; as well as 11.7% (24.8 g/L) and 8.1% (24.0 g/L) by fed-batch fermentation. By comparison, the ppc overexpressed via the two strategies lead to 30.8% (6.8 g/L) and 13.5% (5.9 g/L), 15.8% (25.7 g/L) and 9.5% (24.3 g/L) increase by the two fermentation processes. Meanwhile, overexpression of pyc and ppc resulted in improvement of L-isoleucine yield. However, overexpression of pyc and ppc in ILE02 and ILE04 resulted in cell growth decreased. Overexpression of pyc and ppc both resulted in remarkably promotion of L-isoleucine production performance and the genome-integration overexpressing strategy was more profitable. This study first reports the effects of enhancing anaplerotic pathways on L-isoleucine production by C. glutamicum. The results would supply the reference for metabolic engineering of C. glutamicum.
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