L-精氨酸是一种人体必需氨基酸,在医疗保健、食品添加等制造行业中商业价值显著。针对1株产L-精氨酸的钝齿棒杆菌(Corynebacterium crenatum)SYPA5-5进行代谢改造,通过比较异源乙酰辅酶A合成酶(acetyl-CoA synthetases,ACS)的酶学性质,构建了过表达ACS的重组钝齿棒杆菌SYPA-ACS,能有效利用乙酸提高菌株产L-精氨酸能力。首先在大肠杆菌中分别克隆表达及纯化4种不同细菌来源的ACS,发现巴氏醋酸杆菌(Acetobacter pasteurianus) ATCC 33445来源的ApACS1酶活力最高,为784.59 U/mL,最适反应的pH为7.0,最适反应温度37 ℃,与其他3种ACS来源相比,ApACS1具有更好的pH和温度稳定性。然后通过pXMJ19载体,将A.pasteurianus来源的编码基因acs1在C.crenatum中表达,有效提高了乙酰辅酶A含量。利用5 L发酵罐,将SYPA-ACS菌株培养96 h后,L-精氨酸的产量达53.43 g/L,产率为0.577 g/(L·h),相较于出发菌株SYPA5-5,产量提高了27.48%。该策略既可以利用乙酸,降低乙酸对菌株的危害作用,又能提高乙酰辅酶A的含量,促进L-精氨酸的积累。研究结果为L-精氨酸的制备提供了绿色、高效的合成策略,具有工业化应用潜力。
L-Arginine is essential amino acid that is widely used in medicine, food addition, and other industries. The metabolic pathway engineering of Corynebacterium crenatum SYPA5-5 was performed to further enhance the flux of L-arginine biosynthesis. Comparing the enzymatic properties of acetyl-CoA synthase (ACS) from different bacterial sources, and heterologous expression in C. crenatum SYPA-ACS to effectively utilize acetic acid and increase L-arginine yield. Firstly, the four genes encoding ACS from different bacterial sources were cloned and expressed in Escherichia coli, and the highest activity of ApACS1 from Acetobacter pasteurianus ATCC 33445 was 784.59 U/mL, the optimum pH was 7.0 and temperature was 37 ℃. Compared with the other ACSs, ApACS1 has better pH and temperature stability. The acs1 from A. pasteurianus was then cloned into plasmid pXMJ19 and expressed in C. crenatum, which was effectively improving the acetyl-CoA content. The recombinant strain SYPA-ACS was cultured in 5 L fermenter for 96 h, L-arginine accumulation increased to 53.43 g/L with the productivity of 0.577 g/(L·h) in the recombinant SYPA-ACS, which was approximately 27.48% higher than that of SYPA5-5. The ACS from A. pasteurianus has been screened and overexpressed in C. crenatum, which could use acetic acid to improve the content of acetyl-CoA and achieve L-arginine accumulation. Thus, this strategy provides a safe and efficient synthesis of L-arginine and has important industrial application potential.
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