高产L-精氨酸谷氨酸棒状杆菌的构建
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摘要
以产L-精氨酸诱变菌株谷氨酸棒状杆菌(Corynebacterium glutamicum)AJC为出发菌株,采用基因组编辑技术对其进行改造。首先,敲除阻遏蛋白Arg R和FarR,解除反馈阻遏作用;然后,敲除乳酸脱氢酶编码基因ldh和整合鸟氨酸乙酰转移酶编码基因argJ,阻断乳酸合成途径和增加前体物;最后,敲除谷氨酸分泌蛋白编码基因NCgl1221和整合乙酰谷氨酸激酶基因argB,减弱L-谷氨酸的胞外分泌,筛选一株L-精氨酸高产菌株。结果表明,获得一株高产L-精氨酸菌株AJC-4(C. glutamicum AJCΔargRΔfarRΔldh::PtufargJΔNCgl1221::PsodargB),该菌株在5 L发酵罐中发酵64 h后,L-精氨酸产量和糖酸转化率分别为78.0 g/L和0.38 g/g,较出发菌株AJC分别提高21.9%、18.8%;副产物乳酸和L-谷氨酸积累量分别为0.11 g/L、0.16 g/L,较出发菌株AJC分别降低96.8%、96.1%。
The L-arginine producing induced strain Corynebacterium glutamicum AJC was taken as the starting strain and modified by genome editing technology. Firstly, the repressor proteins Arg R and FarR were knocked out to relieve the feedback repressor effect. Secondly, the gene ldh encoding lactate dehydrogenase was knocked out, and the gene argJ encoding ornithine acetyltransferase was integrated to block the synthesis pathway of lactic acid and increase the accumulation of precursor. Finally, the gene NCgl1221 encoding glutamate secretory protein was knocked out, and the gene argB encoding acetylglutamate kinase was integrated to attenuate the extracellular secretion of L-glutamic acid. The high yield L-arginine strain was screened. The results showed that a high yield L-arginine strain AJC-4(C. glutamicum AJCΔargRΔfarRΔldh::PtufargJΔNCgl1221::PsodargB) was obtained. After the strain AJC-4 was cultured for 64 h in 5 L bioreactor, the yield of L-arginine and the conversion rate of glucose to acid were 78.0 g/L and 0.38 g/g, respectively, which were 21.9% and 18.8% higher than the starting strain AJC, respectively. The accumulation of by-product lactic acid and L-glutamate were 0.11 g/L and 0.16 g/L, respectively, which were 96.8% and 96.1% lower than the starting strain AJC, respectively.
The L-arginine producing induced strain Corynebacterium glutamicum AJC was taken as the starting strain and modified by genome editing technology. Firstly, the repressor proteins Arg R and FarR were knocked out to relieve the feedback repressor effect. Secondly, the gene ldh encoding lactate dehydrogenase was knocked out, and the gene argJ encoding ornithine acetyltransferase was integrated to block the synthesis pathway of lactic acid and increase the accumulation of precursor. Finally, the gene NCgl1221 encoding glutamate secretory protein was knocked out, and the gene argB encoding acetylglutamate kinase was integrated to attenuate the extracellular secretion of L-glutamic acid. The high yield L-arginine strain was screened. The results showed that a high yield L-arginine strain AJC-4(C. glutamicum AJCΔargRΔfarRΔldh::PtufargJΔNCgl1221::PsodargB) was obtained. After the strain AJC-4 was cultured for 64 h in 5 L bioreactor, the yield of L-arginine and the conversion rate of glucose to acid were 78.0 g/L and 0.38 g/g, respectively, which were 21.9% and 18.8% higher than the starting strain AJC, respectively. The accumulation of by-product lactic acid and L-glutamate were 0.11 g/L and 0.16 g/L, respectively, which were 96.8% and 96.1% lower than the starting strain AJC, respectively.
引文
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[16]文瑶.大肠杆菌工程菌HBUT-L的耐乳酸盐驯化及其发酵产L-乳酸的研究[D].武汉:湖北工业大学,2016.
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[21]蔡冬梅.精氨酸高产菌株钝齿棒杆菌中arg J基因的功能研究[D].无锡:江南大学,2009.
[2]代张超,李吕木,卫爱莲,等.畜禽饲用精氨酸的研究进展[J].中国饲料,2018(13):5-9.
[3]WU G,BAZER F W,DAVIS T A,et al.Arginine metabolism and nutrition in growth,health and disease[J].Amino Acids,2009,37(1):153-168.
[4]程功,徐建中,张伟国.L-精氨酸生物合成机制及其代谢工程育种研究进展[J].微生物学通报,2016,43(6):1379-1387.
[5]赵鑫,肖玉平,廖延智,等.L-精氨酸高产菌株的亚硝基胍诱变选育和种子培养基的优化研究[J].中国酿造,2014,33(9):81-85.
[6]孙安然,宋伟,刘佳,等.生物酶法合成L-精氨酸衍生物的研究进展[J].生物工程学报(英文版),2018,34(2):165-176.
[7]占米林.辅因子代谢工程改造谷氨酸棒杆菌合成L-精氨酸[D].无锡:江南大学,2018.
[8]PARK S H,KIM H U,KIM T Y,et al.Metabolic engineering of Corynebacterium glutamicum for L-arginine production[J].Nat Commun,2014,5:4618.
[9]DOU W,XU M,CAI D,et al.Improvement of L-arginine production by overexpression of a bifunctional ornithine acetyltransferase in Corynebacterium crenatum[J].Appl Biochem Biotech,2011,165(3-4):845-855.
[10]IKEDA M,MITSUHASHI S,TANAKA K,et al.Reengineering of a Corynebacterium glutamicum L-arginine and L-citrulline producer[J].Appl Environ Microbiol,2009,75(6):1635-1641.
[11]谭延振.谷氨酸棒状杆菌基因敲除系统的构建[J].无锡:江南大学,2012.
[12]李刚,程度,李宝健,等.利用高效Ca Cl2转化法实现质粒的共转化[J].生物技术,2003,13(6):31-33.
[13]刘子强,张震,蔡萌萌,等.稀糖分罐发酵对L-色氨酸发酵的影响[J].中国酿造,2018,37(4):132-136.
[14]黎永贤,陈梓芬,覃亮.Acc Q.Tag内标法测定精氨酸发酵液中精氨酸含量[J].化工管理,2015(20):134-135.
[15]王楠楠.谷氨酸棒杆菌γ-氨基丁酸及其前体物质L-谷氨酸合成的代谢途径改造[D].无锡:江南大学,2015.
[16]文瑶.大肠杆菌工程菌HBUT-L的耐乳酸盐驯化及其发酵产L-乳酸的研究[D].武汉:湖北工业大学,2016.
[17]LARSEN R,BUIST G,KUIPERS O P,et al.Arg R and Ahr C are both required for regulation of arginine metabolism in Lactococcus lactis[J].J Bacteriol,2004,186(4):1147-1157.
[18]LEE S Y,PARK J M,LEE J H,et al.Interaction of transcriptional repressor Arg R with transcriptional regulator Far R at the arg B promoter region in Corynebacterium glutamicum[J].Appl Environ Microbiol,2011,77(3):711-718.
[19]朱进伟,陈青山,张伟国.L-精氨酸生物合成的代谢流量分析[J].微生物学通报,2008,35(3):395-401.
[20]SIEBERT D,WENDISCH V F.Metabolic pathway engineering for production of 1,2-propanediol and 1-propanol by Corynebacterium glutamicum[J].Biotechnol Biofuels,2015,8(1):91.
[21]蔡冬梅.精氨酸高产菌株钝齿棒杆菌中arg J基因的功能研究[D].无锡:江南大学,2009.