鈍齒棒桿菌SYPH5-8合成L-精氨酸的代謝工程改造
發(fā)布時(shí)間：2019-05-05 05:47
【摘要】：L-精氨酸(L-Arginine，簡稱L-Arg)作為具有多種生理功能的半必需氨基酸，廣泛應(yīng)用于醫(yī)藥、食品和化工等領(lǐng)域。鈍齒棒桿菌(Corynebacterium crenatum) SYPH5-8是經(jīng)過多級(jí)誘變篩選獲得的能夠利用糖質(zhì)原料發(fā)酵生產(chǎn)L-精氨酸的菌株，本文以SYPH5-8為出發(fā)菌株，運(yùn)用代謝工程手段，對(duì)其L-精氨酸合成途徑的限速步驟實(shí)施改造，探討改造后菌株發(fā)生代謝變化的機(jī)制，強(qiáng)化L-精氨酸的合成途徑。主要研究結(jié)果如下： (1)考察L-精氨酸對(duì)SYPH5-8中N-乙酰谷氨酸激酶(N-acetylglutamate kinase,CcNAGK, argB)的反饋抑制情況，測得其半數(shù)反饋抑制常數(shù)(I0.5)為1.36mM，當(dāng)L-精氨酸添加量達(dá)5mM時(shí)，CcNAGK的酶活力僅保持3.0%，以上結(jié)果表明：SYPH5-8雖然經(jīng)過多級(jí)誘變及L-精氨酸結(jié)構(gòu)類似物抗性篩選，但仍未解除L-精氨酸對(duì)CcNAGK的反饋抑制作用。將CcNAGK與已知蛋白質(zhì)晶體結(jié)構(gòu)的N-乙酰谷氨酸激酶(L-精氨酸敏感型)進(jìn)行氨基酸序列的同源性比對(duì)，選擇14個(gè)可能與L-精氨酸結(jié)合相關(guān)的位點(diǎn)進(jìn)行體外突變，，發(fā)現(xiàn)209位精氨酸突變?yōu)楸彼?R209A)或268位組氨酸突變?yōu)樘於０?H268N)不改變酶的催化活性，同時(shí)突變體的I0.5提高約40倍，當(dāng)添加15mM L-精氨酸時(shí)，突變體的酶活力可保持99%以上，以上結(jié)果表明：CcNAGK中R209A或H268N位點(diǎn)突變可解除L-精氨酸對(duì)其反饋抑制作用。 (2)在SYPH5-8基因組的argB中分別精確引入R209A及H268N突變點(diǎn)，對(duì)獲得的重組菌SYPH5-8-NAGKR209A(簡稱：R-8)及SYPH5-8-NAGKH268N(簡稱：H-7)進(jìn)行發(fā)酵特性研究，發(fā)現(xiàn)其生長緩慢，L-精氨酸的積累速率降低，但L-精氨酸合成途徑的中間代謝產(chǎn)物(L-鳥氨酸及L-瓜氨酸)大量積累。對(duì)重組菌的arg基因簇進(jìn)行轉(zhuǎn)錄分析，發(fā)現(xiàn)argG及argH的轉(zhuǎn)錄水平顯著降低，同時(shí)酶活力也相應(yīng)降低，推斷其是L-瓜氨酸及L-鳥氨酸積累的關(guān)鍵因素。 (3)為加強(qiáng)表達(dá)argGH基因，構(gòu)建了重組質(zhì)粒pDXW-10-argGH，轉(zhuǎn)化R-8及H-7獲得重組菌R-8-argGH (簡稱：R-8-GH)及H-7-argGH (簡稱：H-7-GH)。發(fā)酵64h后，H-7-GH較H-7的L-精氨酸產(chǎn)量提高了1.4倍，L-鳥氨酸及L-瓜氨酸含量分別降低了79.3%及82.8%，R-8-GH表現(xiàn)為相同的趨勢。H-7-GH與SYPH5-8相比，發(fā)酵時(shí)間縮短16h，L-精氨酸生產(chǎn)能力提高84.6%，糖酸轉(zhuǎn)化效率提高1倍，主要副產(chǎn)物L(fēng)-賴氨酸及L-異亮氨酸分別降低了51.4%及53.7%。優(yōu)化了重組菌發(fā)酵培養(yǎng)基中的主要營養(yǎng)組分，當(dāng)添加14g/L酵母粉及8g/L牛肉膏時(shí)，菌體生物量為19.9g/L，較優(yōu)化前(14.1g/L)提高41.1%，L-精氨酸產(chǎn)量為45.1g/L，較優(yōu)化前(30.1g/L)提高49.8%。在此基礎(chǔ)上優(yōu)化了碳氮源的補(bǔ)加方式，發(fā)酵80h，菌體生物量為22.7g/L，L-精氨酸產(chǎn)量為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.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R914.5
本文編號(hào)：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.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R914.5
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