钝齿棒杆菌SYPH5-8合成L-精氨酸的代谢工程改造
发布时间：2019-05-05 05:47
【摘要】：L-精氨酸(L-Arginine，简称L-Arg)作为具有多种生理功能的半必需氨基酸，广泛应用于医药、食品和化工等领域。钝齿棒杆菌(Corynebacterium crenatum) SYPH5-8是经过多级诱变筛选获得的能够利用糖质原料发酵生产L-精氨酸的菌株，本文以SYPH5-8为出发菌株，运用代谢工程手段，对其L-精氨酸合成途径的限速步骤实施改造，探讨改造后菌株发生代谢变化的机制，强化L-精氨酸的合成途径。主要研究结果如下： (1)考察L-精氨酸对SYPH5-8中N-乙酰谷氨酸激酶(N-acetylglutamate kinase,CcNAGK, argB)的反馈抑制情况，测得其半数反馈抑制常数(I0.5)为1.36mM，当L-精氨酸添加量达5mM时，CcNAGK的酶活力仅保持3.0%，以上结果表明：SYPH5-8虽然经过多级诱变及L-精氨酸结构类似物抗性筛选，但仍未解除L-精氨酸对CcNAGK的反馈抑制作用。将CcNAGK与已知蛋白质晶体结构的N-乙酰谷氨酸激酶(L-精氨酸敏感型)进行氨基酸序列的同源性比对，选择14个可能与L-精氨酸结合相关的位点进行体外突变，，发现209位精氨酸突变为丙氨酸(R209A)或268位组氨酸突变为天冬酰胺(H268N)不改变酶的催化活性，同时突变体的I0.5提高约40倍，当添加15mM L-精氨酸时，突变体的酶活力可保持99%以上，以上结果表明：CcNAGK中R209A或H268N位点突变可解除L-精氨酸对其反馈抑制作用。 (2)在SYPH5-8基因组的argB中分别精确引入R209A及H268N突变点，对获得的重组菌SYPH5-8-NAGKR209A(简称：R-8)及SYPH5-8-NAGKH268N(简称：H-7)进行发酵特性研究，发现其生长缓慢，L-精氨酸的积累速率降低，但L-精氨酸合成途径的中间代谢产物(L-鸟氨酸及L-瓜氨酸)大量积累。对重组菌的arg基因簇进行转录分析，发现argG及argH的转录水平显著降低，同时酶活力也相应降低，推断其是L-瓜氨酸及L-鸟氨酸积累的关键因素。 (3)为加强表达argGH基因，构建了重组质粒pDXW-10-argGH，转化R-8及H-7获得重组菌R-8-argGH (简称：R-8-GH)及H-7-argGH (简称：H-7-GH)。发酵64h后，H-7-GH较H-7的L-精氨酸产量提高了1.4倍，L-鸟氨酸及L-瓜氨酸含量分别降低了79.3%及82.8%，R-8-GH表现为相同的趋势。H-7-GH与SYPH5-8相比，发酵时间缩短16h，L-精氨酸生产能力提高84.6%，糖酸转化效率提高1倍，主要副产物L-赖氨酸及L-异亮氨酸分别降低了51.4%及53.7%。优化了重组菌发酵培养基中的主要营养组分，当添加14g/L酵母粉及8g/L牛肉膏时，菌体生物量为19.9g/L，较优化前(14.1g/L)提高41.1%，L-精氨酸产量为45.1g/L，较优化前(30.1g/L)提高49.8%。在此基础上优化了碳氮源的补加方式，发酵80h，菌体生物量为22.7g/L，L-精氨酸产量为51.7g/L。
[Abstract]:As a semi-essential amino acid with many physiological functions, L-arginine (L-Arg) is widely used in many fields such as medicine, food and chemical industry. Actinobacillus obtuse (Corynebacterium crenatum) SYPH5-8 is a strain which can produce L-arginine by using sugar raw material after multi-stage mutagenesis screening. In this paper, SYPH5-8 was used as the starting strain and metabolic engineering method was used to produce L-arginine. The rate-limiting steps of L-arginine synthesis pathway were modified to explore the mechanism of metabolic changes and strengthen the synthesis pathway of L-arginine. The main results are as follows: (1) the feedback inhibition of L-arginine on N-acetylglutamic kinase (N-acetylglutamate kinase,CcNAGK, argB) in SYPH5-8 was investigated, and the half feedback inhibition constant (I0.5) of L-arginine was 1.36 mm. When L-arginine was added to 5mM, the enzyme activity of CcNAGK remained only 3.0%. The above results showed that although SYPH5-8 was screened by multi-level mutagenesis and screening of L-arginine structure analogues, the enzyme activity of LArg was only 3.0%. However, the feedback inhibitory effect of L-arginine on CcNAGK was not relieved. The amino acid sequence of CcNAGK was compared with that of N-acetylglutamic kinase (L-arginine sensitive type) with known protein crystal structure. Fourteen sites which might be associated with L-arginine binding were selected for in vitro mutation. It was found that the mutation of arginine at position 209 to alanine (R209A) or histidine at position 268 to asparagine (H268N) did not change the catalytic activity of the enzyme, and the I0.5 of the mutant increased by about 40 times. When 15mM L-arginine was added, the activity of the mutant was increased by 40 times. The enzyme activity of the mutant was over 99%. The above results indicated that the mutation of R209A or H268N in CcNAGK could relieve the feedback inhibitory effect of L-arginine on the mutant. (2) R209A and H268N mutation sites were introduced into the argB of SYPH5-8 genome, respectively. The fermentation characteristics of the recombinant strains SYPH5-8-NAGKR209A and SYPH5-8-NAGKH268N were studied, and the results showed that R209A and H268N mutants grew slowly, and the results showed that R209A and H268N mutation sites were introduced into the R209A and H268N mutation sites respectively. The accumulation rate of L-arginine decreased, but the intermediate metabolites of L-arginine synthesis pathway (L-ornithine and L-citrulline) accumulated a lot. The transcription analysis of the arg gene cluster of the recombinant bacteria showed that the transcription level of argG and argH decreased significantly, and the enzyme activity also decreased accordingly. It was inferred that it was the key factor for the accumulation of L-citrulline and L-ornithine. (3) in order to enhance the expression of argGH gene, recombinant plasmid pDXW-10-argGH, was constructed and transformed into R-8-argGH (R-8-GH) and H-7-argGH (H-7-GH). After 64 h fermentation, the yield of L-arginine in H-7-GH was increased by 1.4 times, and the contents of L-ornithine and L-citrulline were reduced by 79.3% and 82.8%, respectively. R-8-GH showed the same trend. Compared with SYPH5-8, the fermentation time of H-7-GH was shortened by 16 hours, the production capacity of L-arginine was increased by 84.6%, and the conversion efficiency of sugar and acid was doubled. The main by-products L-lysine and L-isoleucine decreased by 51.4% and 53.7%, respectively. The main nutrient components in the fermentation medium were optimized. When 14g/L yeast powder and 8g/L beef extract were added, the biomass of bacteria was 19.9 g / L, which was 41.1% higher than that before optimization (14.1g/L). The L-arginine yield was 45.1 g / L, which was 49.8% higher than that of pre-optimization (30.1g/L). After 80 h fermentation, the biomass and L-arginine yield were 22.7 g / L and 51.7 g / L, respectively, and the yield of L-arginine was 51.7 g 路L-1 路L-1, and the yield of L-arginine was 51.7 g / L.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R914.5
本文编号：2469295
[Abstract]:As a semi-essential amino acid with many physiological functions, L-arginine (L-Arg) is widely used in many fields such as medicine, food and chemical industry. Actinobacillus obtuse (Corynebacterium crenatum) SYPH5-8 is a strain which can produce L-arginine by using sugar raw material after multi-stage mutagenesis screening. In this paper, SYPH5-8 was used as the starting strain and metabolic engineering method was used to produce L-arginine. The rate-limiting steps of L-arginine synthesis pathway were modified to explore the mechanism of metabolic changes and strengthen the synthesis pathway of L-arginine. The main results are as follows: (1) the feedback inhibition of L-arginine on N-acetylglutamic kinase (N-acetylglutamate kinase,CcNAGK, argB) in SYPH5-8 was investigated, and the half feedback inhibition constant (I0.5) of L-arginine was 1.36 mm. When L-arginine was added to 5mM, the enzyme activity of CcNAGK remained only 3.0%. The above results showed that although SYPH5-8 was screened by multi-level mutagenesis and screening of L-arginine structure analogues, the enzyme activity of LArg was only 3.0%. However, the feedback inhibitory effect of L-arginine on CcNAGK was not relieved. The amino acid sequence of CcNAGK was compared with that of N-acetylglutamic kinase (L-arginine sensitive type) with known protein crystal structure. Fourteen sites which might be associated with L-arginine binding were selected for in vitro mutation. It was found that the mutation of arginine at position 209 to alanine (R209A) or histidine at position 268 to asparagine (H268N) did not change the catalytic activity of the enzyme, and the I0.5 of the mutant increased by about 40 times. When 15mM L-arginine was added, the activity of the mutant was increased by 40 times. The enzyme activity of the mutant was over 99%. The above results indicated that the mutation of R209A or H268N in CcNAGK could relieve the feedback inhibitory effect of L-arginine on the mutant. (2) R209A and H268N mutation sites were introduced into the argB of SYPH5-8 genome, respectively. The fermentation characteristics of the recombinant strains SYPH5-8-NAGKR209A and SYPH5-8-NAGKH268N were studied, and the results showed that R209A and H268N mutants grew slowly, and the results showed that R209A and H268N mutation sites were introduced into the R209A and H268N mutation sites respectively. The accumulation rate of L-arginine decreased, but the intermediate metabolites of L-arginine synthesis pathway (L-ornithine and L-citrulline) accumulated a lot. The transcription analysis of the arg gene cluster of the recombinant bacteria showed that the transcription level of argG and argH decreased significantly, and the enzyme activity also decreased accordingly. It was inferred that it was the key factor for the accumulation of L-citrulline and L-ornithine. (3) in order to enhance the expression of argGH gene, recombinant plasmid pDXW-10-argGH, was constructed and transformed into R-8-argGH (R-8-GH) and H-7-argGH (H-7-GH). After 64 h fermentation, the yield of L-arginine in H-7-GH was increased by 1.4 times, and the contents of L-ornithine and L-citrulline were reduced by 79.3% and 82.8%, respectively. R-8-GH showed the same trend. Compared with SYPH5-8, the fermentation time of H-7-GH was shortened by 16 hours, the production capacity of L-arginine was increased by 84.6%, and the conversion efficiency of sugar and acid was doubled. The main by-products L-lysine and L-isoleucine decreased by 51.4% and 53.7%, respectively. The main nutrient components in the fermentation medium were optimized. When 14g/L yeast powder and 8g/L beef extract were added, the biomass of bacteria was 19.9 g / L, which was 41.1% higher than that before optimization (14.1g/L). The L-arginine yield was 45.1 g / L, which was 49.8% higher than that of pre-optimization (30.1g/L). After 80 h fermentation, the biomass and L-arginine yield were 22.7 g / L and 51.7 g / L, respectively, and the yield of L-arginine was 51.7 g 路L-1 路L-1, and the yield of L-arginine was 51.7 g / L.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R914.5
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