灰褐链霉菌与黄色短杆菌属间原生质体融合提高ε-聚赖氨酸产量（英文）
|
摘要
为提高灰褐链霉菌(Streptomyces griseofuscus LS-6)的ε-聚赖氨酸(ε-poly-lysine,ε-PL)发酵水平,从增加内源性前体赖氨酸角度考虑,将S. griseofuscus LS-6与赖氨酸生产菌株黄色短杆菌(Brebvibacterium flavum S62)进行属间的原生质体融合。以S. griseofuscus LS-6产黑色素作为遗传标记挑选到1株融合子LS-32,其ε-PL摇瓶产量为2.82 g/L,是亲本菌株LS-6的1.73倍;LS-32在补料分批发酵中的ε-PL产量达到56.4 g/L,比亲本提高了38.9%。随机引物扩增多肽DNA技术显示融合子LS-32中确实有来自B. flavum S62的基因;融合子ε-PL代谢途径中关键酶的活力(己糖激酶、天冬氨酸激酶、ε-PL合成酶等)和胞内氨基酸含量(赖氨酸、谷氨酸、精氨酸等)均高于亲本,是LS-32高产ε-PL的重要原因。本研究明确了内源性赖氨酸对于提升ε-PL产量的重要性,且属间融合为ε-PL产生菌的育种研究提供了新的参考途径。
In order to improve the ε-poly-lysine(ε-PL) production of Streptomyces griseofuscus, intergeneric hybridization between S. griseofuscus and Brebvibacterium flavum for enhanced level of the endogenous precursor lysine in the cells was investigated for the first time in this study. One hybrid designated LS-32 was selected by using melanin as genetic marker, which produced 2.82 g/L of ε-PL in a shake flask, 1.73 folds as high as that of S. griseofuscus. In fed-batch fermentation, the ε-PL production of LS-32 was 56.4 g/L, which was increased by 38.9% as compared to that of S. griseofuscus. Randomly amplified polymorphic DNA(RAPD) analysis confirmed the intergeneric hybridization between S. griseofuscus and B. flavum. The activities of several key enzymes in LS-32 were greater than in S. griseofuscus. Furthermore, the concentrations of intracellular amino acids such as Asp, Glu and Arg in LS-32 were much higher than in S. griseofuscus. This research proves the importance of enhancing the endogenous lysine for improving ε-PL production and that intergeneric hybridization is a new breeding strategy for ε-PL production.
In order to improve the ε-poly-lysine(ε-PL) production of Streptomyces griseofuscus, intergeneric hybridization between S. griseofuscus and Brebvibacterium flavum for enhanced level of the endogenous precursor lysine in the cells was investigated for the first time in this study. One hybrid designated LS-32 was selected by using melanin as genetic marker, which produced 2.82 g/L of ε-PL in a shake flask, 1.73 folds as high as that of S. griseofuscus. In fed-batch fermentation, the ε-PL production of LS-32 was 56.4 g/L, which was increased by 38.9% as compared to that of S. griseofuscus. Randomly amplified polymorphic DNA(RAPD) analysis confirmed the intergeneric hybridization between S. griseofuscus and B. flavum. The activities of several key enzymes in LS-32 were greater than in S. griseofuscus. Furthermore, the concentrations of intracellular amino acids such as Asp, Glu and Arg in LS-32 were much higher than in S. griseofuscus. This research proves the importance of enhancing the endogenous lysine for improving ε-PL production and that intergeneric hybridization is a new breeding strategy for ε-PL production.
引文
[1]HIRAKI J,ICHIKAWA T,NINOMIYA S,et al.Use of ADMEstudies to confirm the safety of polylysine as a preservative in food[J].Regulatory Toxicology and Pharmacology,2003,37(2):328-340.
[2]SHIH I L,SHEN M H.Microbial synthesis of poly(ε-lysine)and its various applications[J].Bioresource Technology,2006,97(4):1148-1159.
[3]XU D L,YAO H Q,XU Z X,et al.Production ofε-poly-lysine by Streptomyces albulus PD-1 via solid-state fermentation[J].Bioresource Technology,2017,223(12):149-156.
[4]YAN P,SUN H B,LU P Q,et al.Enhancement ofε-poly-L-lysine synthesis in Streptomyces by exogenous glutathione[J].Bioprocess and Biosystems Engineering,2018,41(7):129-134.
[5]HIRAKI J,MASAKAZU H,HIROSHI M,et al.Improvedε-poly-L-lysine production of an S-(2-aminoethyl)-L-cysteine resistant mutant of Streptomyces albulus[J].Seibutsu Kogakkaishi,1998,76(5):487-493.
[6]LI S,CHEN X S,REN X D,et al.Combining genome shuffling and interspecific hybridization among Streptomyces improvedε-poly-L-lysine production[J].Applied Biochemistry and Biotechnology,2013,169(3):338-350.
[7]LI S,LI F,CHEN X S,et al.Genome shuffling enhancedε-poly-L-lysine production by improving glucose tolerance of Streptomyces graminearus[J].Applied Biochemistry and Biotechnology,2012,166(6):414-423.
[8]WANG L,CHEN X S,WU G Y,et al.Genome shuffling and gentamicin resistance to improveε-poly-L-lysine productivity of Streptomyces albulus W-156[J].Applied Biochemistry and Biotechnology,2016,5(4):416-426.DOI:10.1007/s12010-016-2190-9.
[9]WANG L,CHEN X S,WU G Y,et al.Enhancedε-poly-L-lysine production by inducing double antibiotic-resistant mutations in Streptomyces albulus[J].Bioprocess and Biosystems Engineering,2016,12(5):325-338.DOI:10.1007/s00449-016-1695-5.
[10]SHIMA S,OSHIMA S,SAKAI H.Biosynthesis ofε-poly-L-lysine by washed mycelium of Streptomyces albulus No.346[J].Nippon Nogeikagaku Kaishi,1983,57(8):221-226.
[11]ZENG X,ZHAO J J,CHEN X S,et al.Insights into the simultaneous utilization of glucose and glycerol by Streptomyces albulus M-Z18for highε-poly-L-lysine productivity[J].Bioprocess and Biosystems Engineering,2017,40(5):1775-1785.
[12]CHEN X S,REN X D,ZENG X,et al.Enhancement ofε-poly-L-lysine production coupled with precursor L-lysine feeding in glucoseglycerol co-fermentation by Streptomyces sp.M-Z18[J].Bioprocess and Biosystems Engineering,2013,36(7):1843-1849.
[13]HIROHARA H,TAKEHARA M,SAIMURA M.Biosynthesis of poly(ε-L-lysine)s in two newly isolated strains of Streptomyces sp.[J].Applied Microbiology and Biotechnology,2006,73(6):321-331.
[14]LI S,TANG L,CHEN X S,et al.Isolation and characterization of a novel epsilon-poly-L-lysine producing strain:Streptomyces griseofuscus[J].Journal of Industrial Microbiology and Biotechnology,2011,38(6):557-563.
[15]LIU Y J,CHEN X S,ZHAO J J,et al.Development of microtiter plate culture method for rapid screening ofε-poly-L-lysine-producing strains[J].Applied Microbiology and Biotechnology,2017,183(6):1209-1223.
[16]PATNAIK R,LOUIE S,GAVRILOVIC V,et al.Genome shuffling of Lactobacillus for improved acid tolerance[J].Nature Biotechnology,2002,20(11):707-712.
[17]KAHAR P,IWATA T,HIRAKI J,et al.Enhancement ofε-poly-lysine production by Streptomyces albulus strain 410 using pH control[J].Journal of Bioscience and Bioengineering,2001,91(3):190-194.
[18]HAMANO Y,NICCHU I.ε-Poly-L-lysine producer,Streptomyces albulus,has feedback inhibition resistant aspartokinase[J].Applied Microbiology and Biotechnology,2007,76(7):873-882.
[19]MURMU J,WILLIAM C.Phosphoenolpyruvate carboxylase protein kinase from developing castor oil seeds:partial purification,characterization,and reversible control by photosynthate supply[J].Planta,2007,226(14):1299-1310.
[20]KIYOHARA H,TOSHIRO W,JUNKO I.Intergeneric hybridization between Monascus anka and Asperyillus oryzae by protoplast fusion[J].Applied Microbiology and Biotechnology,1990,33(8):671-676.
[21]ROJAN P,GANGADHARAN D,MADHAVAN K.Genome shuffling of Lactobacillus delbrueckii mutant and Bacillus amyloliquefaciens through protoplasmic fusion for L-lactic acid production from starchy wastes[J].Bioresource Technology,2008,99(12):8008-8015.
[22]NALIN R,DAVID H.Spheroplast fusion as a mode of genetic recombination in mycobacteria[J].Journal of General Microbiology,1983,129(14):227-237.
[23]JOHN F,HENDRIK E.Interspecific hybridization between Penicillium chlysogenum and Penicillium cyaneofulvum following protoplast fusion[J].Molecular Genetics and Genomics,1977,157(6):281-284.
[24]XIA J,XU Z,FENG X H,et al.The regulatory effect of citric acid on the co-production of poly(epsilon-lysine)and poly(L-diaminopropionic acid)in Streptomyces albulus PD-1[J].Bioprocess and Biosystems Engineering,2014,37(4):2095-2103.
[25]ZENG X,CHEN X S,REN X D,et al.Insights into the role of glucose and glycerol as a mixed carbon source in the improvement of epsilon-poly-L-lysine productivity[J].Applied Biochemistry and Biotechnology,2014,173(10):2211-2224.
[26]XU Z,XIA J,SUN Z,et al.Effects of oxygen-vectors on the synthesis of epsilon-poly-lysine and the metabolic characterization of Streptomyces albulus PD-1[J].Biochemical Engineering Journal,2015,94(9):58-64.
[27]HELENA B,HUGH G.Phosphoenolpyruvate carboxylase from Streptomyces coelicolor A3(2):purification of the enzyme,cloning of the ppc gene and over-expression of the protein in a streptomycete[J].Biochemical Journal,1993,293(8):131-136.
[28]GU Y Y,WANG X M,YANG C,et al.Effects of chromosomal integration of the vitreoscilla hemoglobin gene(vgb)and S-adenosylmethionine synthetase gene(metK)onε-poly-L-lysine synthesis in Streptomyces albulus NK660[J].Applied Microbiology and Biotechnology,2016,178(15):1445-1457.
[29]VRANCHEN G,RIMAUX T,WECKX S.Environmental pHdetermines citrulline and ornithine release through the arginine deiminase pathway in Lactobacillus fermentum IMDO 13010[J].International Journal of Food Microbiology,2009,135(13):216-222.
[30]ZHANG Y,XU J,YUAN J.Artificial neural network-genetic algorithm based optimization for the immobilization of cellulase on the smart polymer eudragit L-100[J].Bioresource Technology,2010,101(9):3153-3158.
[2]SHIH I L,SHEN M H.Microbial synthesis of poly(ε-lysine)and its various applications[J].Bioresource Technology,2006,97(4):1148-1159.
[3]XU D L,YAO H Q,XU Z X,et al.Production ofε-poly-lysine by Streptomyces albulus PD-1 via solid-state fermentation[J].Bioresource Technology,2017,223(12):149-156.
[4]YAN P,SUN H B,LU P Q,et al.Enhancement ofε-poly-L-lysine synthesis in Streptomyces by exogenous glutathione[J].Bioprocess and Biosystems Engineering,2018,41(7):129-134.
[5]HIRAKI J,MASAKAZU H,HIROSHI M,et al.Improvedε-poly-L-lysine production of an S-(2-aminoethyl)-L-cysteine resistant mutant of Streptomyces albulus[J].Seibutsu Kogakkaishi,1998,76(5):487-493.
[6]LI S,CHEN X S,REN X D,et al.Combining genome shuffling and interspecific hybridization among Streptomyces improvedε-poly-L-lysine production[J].Applied Biochemistry and Biotechnology,2013,169(3):338-350.
[7]LI S,LI F,CHEN X S,et al.Genome shuffling enhancedε-poly-L-lysine production by improving glucose tolerance of Streptomyces graminearus[J].Applied Biochemistry and Biotechnology,2012,166(6):414-423.
[8]WANG L,CHEN X S,WU G Y,et al.Genome shuffling and gentamicin resistance to improveε-poly-L-lysine productivity of Streptomyces albulus W-156[J].Applied Biochemistry and Biotechnology,2016,5(4):416-426.DOI:10.1007/s12010-016-2190-9.
[9]WANG L,CHEN X S,WU G Y,et al.Enhancedε-poly-L-lysine production by inducing double antibiotic-resistant mutations in Streptomyces albulus[J].Bioprocess and Biosystems Engineering,2016,12(5):325-338.DOI:10.1007/s00449-016-1695-5.
[10]SHIMA S,OSHIMA S,SAKAI H.Biosynthesis ofε-poly-L-lysine by washed mycelium of Streptomyces albulus No.346[J].Nippon Nogeikagaku Kaishi,1983,57(8):221-226.
[11]ZENG X,ZHAO J J,CHEN X S,et al.Insights into the simultaneous utilization of glucose and glycerol by Streptomyces albulus M-Z18for highε-poly-L-lysine productivity[J].Bioprocess and Biosystems Engineering,2017,40(5):1775-1785.
[12]CHEN X S,REN X D,ZENG X,et al.Enhancement ofε-poly-L-lysine production coupled with precursor L-lysine feeding in glucoseglycerol co-fermentation by Streptomyces sp.M-Z18[J].Bioprocess and Biosystems Engineering,2013,36(7):1843-1849.
[13]HIROHARA H,TAKEHARA M,SAIMURA M.Biosynthesis of poly(ε-L-lysine)s in two newly isolated strains of Streptomyces sp.[J].Applied Microbiology and Biotechnology,2006,73(6):321-331.
[14]LI S,TANG L,CHEN X S,et al.Isolation and characterization of a novel epsilon-poly-L-lysine producing strain:Streptomyces griseofuscus[J].Journal of Industrial Microbiology and Biotechnology,2011,38(6):557-563.
[15]LIU Y J,CHEN X S,ZHAO J J,et al.Development of microtiter plate culture method for rapid screening ofε-poly-L-lysine-producing strains[J].Applied Microbiology and Biotechnology,2017,183(6):1209-1223.
[16]PATNAIK R,LOUIE S,GAVRILOVIC V,et al.Genome shuffling of Lactobacillus for improved acid tolerance[J].Nature Biotechnology,2002,20(11):707-712.
[17]KAHAR P,IWATA T,HIRAKI J,et al.Enhancement ofε-poly-lysine production by Streptomyces albulus strain 410 using pH control[J].Journal of Bioscience and Bioengineering,2001,91(3):190-194.
[18]HAMANO Y,NICCHU I.ε-Poly-L-lysine producer,Streptomyces albulus,has feedback inhibition resistant aspartokinase[J].Applied Microbiology and Biotechnology,2007,76(7):873-882.
[19]MURMU J,WILLIAM C.Phosphoenolpyruvate carboxylase protein kinase from developing castor oil seeds:partial purification,characterization,and reversible control by photosynthate supply[J].Planta,2007,226(14):1299-1310.
[20]KIYOHARA H,TOSHIRO W,JUNKO I.Intergeneric hybridization between Monascus anka and Asperyillus oryzae by protoplast fusion[J].Applied Microbiology and Biotechnology,1990,33(8):671-676.
[21]ROJAN P,GANGADHARAN D,MADHAVAN K.Genome shuffling of Lactobacillus delbrueckii mutant and Bacillus amyloliquefaciens through protoplasmic fusion for L-lactic acid production from starchy wastes[J].Bioresource Technology,2008,99(12):8008-8015.
[22]NALIN R,DAVID H.Spheroplast fusion as a mode of genetic recombination in mycobacteria[J].Journal of General Microbiology,1983,129(14):227-237.
[23]JOHN F,HENDRIK E.Interspecific hybridization between Penicillium chlysogenum and Penicillium cyaneofulvum following protoplast fusion[J].Molecular Genetics and Genomics,1977,157(6):281-284.
[24]XIA J,XU Z,FENG X H,et al.The regulatory effect of citric acid on the co-production of poly(epsilon-lysine)and poly(L-diaminopropionic acid)in Streptomyces albulus PD-1[J].Bioprocess and Biosystems Engineering,2014,37(4):2095-2103.
[25]ZENG X,CHEN X S,REN X D,et al.Insights into the role of glucose and glycerol as a mixed carbon source in the improvement of epsilon-poly-L-lysine productivity[J].Applied Biochemistry and Biotechnology,2014,173(10):2211-2224.
[26]XU Z,XIA J,SUN Z,et al.Effects of oxygen-vectors on the synthesis of epsilon-poly-lysine and the metabolic characterization of Streptomyces albulus PD-1[J].Biochemical Engineering Journal,2015,94(9):58-64.
[27]HELENA B,HUGH G.Phosphoenolpyruvate carboxylase from Streptomyces coelicolor A3(2):purification of the enzyme,cloning of the ppc gene and over-expression of the protein in a streptomycete[J].Biochemical Journal,1993,293(8):131-136.
[28]GU Y Y,WANG X M,YANG C,et al.Effects of chromosomal integration of the vitreoscilla hemoglobin gene(vgb)and S-adenosylmethionine synthetase gene(metK)onε-poly-L-lysine synthesis in Streptomyces albulus NK660[J].Applied Microbiology and Biotechnology,2016,178(15):1445-1457.
[29]VRANCHEN G,RIMAUX T,WECKX S.Environmental pHdetermines citrulline and ornithine release through the arginine deiminase pathway in Lactobacillus fermentum IMDO 13010[J].International Journal of Food Microbiology,2009,135(13):216-222.
[30]ZHANG Y,XU J,YUAN J.Artificial neural network-genetic algorithm based optimization for the immobilization of cellulase on the smart polymer eudragit L-100[J].Bioresource Technology,2010,101(9):3153-3158.