乙酸胁迫下巴氏醋酸杆菌发酵过程中微环境水平的应答分析
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摘要
以Acetobacter pasteurianus CICIM B7003及其高产突变株A. pasteurianus CICIM B7003-02为研究对象,通过2株巴氏醋酸杆菌发酵过程中胞内微环境分析来揭示其耐酸机制。在7. 5 L发酵罐中比较分批和半连续发酵过程中其发酵动力学以及和胞内微环境变化。结果显示,分批发酵中其平均产酸速率达0. 836 g/(L·h),相对亲本菌株提高29. 6%。半连续发酵中酸启动时间缩短了31 h,微环境分析表明,突变株中与产酸直接相关的乙醇脱氢酶(alcohol dehydrogenase,ADH)和乙醛脱氢酶(acetaldehyde dehydrogenase,ALDH)的最高酶活分别提高27. 0%和15. 2%,辅酶Q9含量提高69. 5%。突变株拥有更高比例的十八碳烯酸,比亲本株高71. 4%。突变株最高胞内ATP含量是亲本株2. 33倍,胞内ATP与比生长速率呈正相关,胞内谷氨酸和天冬氨酸含量分别比亲本株提高10. 7%和18. 3%。突变株主要依靠加强乙醇呼吸链,增强ATP合成和关键氨基酸代谢等机制的协同作用提高酸胁迫抗性
This study was conducted to investigate changes in intracellular microenvironment of Acetobacter pasteurianus CICIM B7003 and its high-yield mutant A. pasteurianus CICIM B7003-02 to unravel the acid-tolerant mechanisms of A. pasteurianus. The fermentation kinetics of parent and mutant strain during batch and semi-continuous fermentation in a 7. 5 L fermenter under acidic stress,as well as changes in their intracellular microenvironment were compared. It was found that during batch fermentation,the average fermentation efficiency of the mutant strain reached 0. 836 g/( L·h),which increased 29. 6% compared against that of the parent strain. Moreover,the acid start-up time decreased 31 h during semi-continuous fermentation. Furthermore,enzymes that are directly related to acid production in the mutant strain,such as alcohol dehydrogenase and acetaldehyde dehydrogenase,increased their highest activities by 27. 0% and 15. 2%,respectively. Besides,the content of coenzyme Q9 increased by 69. 5%. In addition,the mutant strain had a higher proportion of octadecenoic acid,which was 71. 4% higher than that of the parent strain. The maximum intracellular ATP content of the mutant strain was 2. 33-fold higher,and intracellular ATP was positively correlated with the specific growth rate. Also,intracellular glutamate and aspartic acid increased by 10. 7% and 18. 3%,respectively. In conclusion,the mutant strain was mainly relied on the synergistic effects of strengthening ethanol respiratory chain,ATP synthesis,and critical amino acid metabolism to improve its acid resistance.
This study was conducted to investigate changes in intracellular microenvironment of Acetobacter pasteurianus CICIM B7003 and its high-yield mutant A. pasteurianus CICIM B7003-02 to unravel the acid-tolerant mechanisms of A. pasteurianus. The fermentation kinetics of parent and mutant strain during batch and semi-continuous fermentation in a 7. 5 L fermenter under acidic stress,as well as changes in their intracellular microenvironment were compared. It was found that during batch fermentation,the average fermentation efficiency of the mutant strain reached 0. 836 g/( L·h),which increased 29. 6% compared against that of the parent strain. Moreover,the acid start-up time decreased 31 h during semi-continuous fermentation. Furthermore,enzymes that are directly related to acid production in the mutant strain,such as alcohol dehydrogenase and acetaldehyde dehydrogenase,increased their highest activities by 27. 0% and 15. 2%,respectively. Besides,the content of coenzyme Q9 increased by 69. 5%. In addition,the mutant strain had a higher proportion of octadecenoic acid,which was 71. 4% higher than that of the parent strain. The maximum intracellular ATP content of the mutant strain was 2. 33-fold higher,and intracellular ATP was positively correlated with the specific growth rate. Also,intracellular glutamate and aspartic acid increased by 10. 7% and 18. 3%,respectively. In conclusion,the mutant strain was mainly relied on the synergistic effects of strengthening ethanol respiratory chain,ATP synthesis,and critical amino acid metabolism to improve its acid resistance.
引文
[1] SAICHANAN,MATSUSHITA K,ADACHI O,et al. Acetic acid bacteria:A group of bacteria with versatile biotechnological applications[J]. Biotechnology Advances,2015,33(6):1 260-1 271.
[2] TRCEK J,MIRA N P,JARBOE L R. Adaptation and tolerance of bacteria against acetic acid[J]. Applied Microbiology and Biotechnology,2015,99(15):6 215-6 229.
[3] XIA K,ZANG N,ZHANG J,et al. New insights into the mechanisms of acetic acid resistance in Acetobacter pasteurianus using i TRAQ-dependent quantitative proteomic analysis[J]. International Journal of Food Microbiology,2016,238:241-251.
[4] TRCEK J,TOYAMA H,CZUBA J,et al. Correlation between acetic acid resistance and characteristics of PQQ-dependent ADH in acetic acid bacteria[J]. Applied Microbiology and Biotechnology,2006,70(3):366-373.
[5] FUKAYA M,TAKEMURA H,TAYAMA K,et al. The aar C gene responsible for acetic acid assimilation confers acetic acid resistance on acetobacter aceti[J]. Journal of Fermentation and Bioengineering,1993,76(4):270-275.
[6] NAKANO S,FUKAYA M,HORINOUCHI S. Putative ABC transporter responsible for acetic acid resistance in Acetobacter aceti[J]. Applied and Environmental Microbiology,2006,72(1):497-505.
[7] TRCEK J,JERNEJC K,MATSUSHITA K. The highly tolerant acetic acid bacterium Gluconacetobacter europaeus adapts to the presence of acetic acid by changes in lipid composition,morphological properties and PQQ-dependent ADH expression[J]. Extremophiles, 2007, 11(4):627-635.
[8] HANADA T,KASHIMA Y,KOSUGI A,et al. A gene encoding phosphatidylethanolamine N-methyltransferase from Acetobacter aceti and some properties of its disruptant[J].Bioscience Biotechnology and Biochemistry, 2001, 65(12):2 741-2 748.
[9] LIU Y,TANG H,LIN Z,et al. Mechanisms of acid tolerance in bacteria and prospects in biotechnology and bioremediation[J]. Biotechnology Advances,2015,33(7):1 484-1 492.
[10] COTTERP D,COLIN H. Surviving the acid test:responses of gram-positive bacteria to low p H[J]. Microbiology and Molecular Biology Reviews:MMBR,2003,67(3):429-453.
[11] MATSUSHITA K,INOUE T,ADACHI O,et al. Acetobacter aceti possesses a proton motive force-dependent efflux system for acetic acid[J]. Journal of Bacteriology,2005,187(13):4 346-4 352.
[12] SAINZ F,MAS A,TORIJA M J. Effect of ammonium and amino acids on the growth of selected strains of Gluconobacter and Acetobacter[J]. International Journal of Food Microbiology,2017,242:45-52.
[13] QI Z,DONG D,YANG H,et al. Improving fermented quality of cider vinegar via rational nutrient feeding strategy[J]. Food Chemistry,2017,224:312-319.
[14] YIN H,ZHANG R,XIA M,et al. Effect of aspartic acid and glutamate on metabolism and acid stress resistance of Acetobacter pasteurianus[J]. Microbial Cell Factories,2017,16(1):109.
[15] ZHENG Y,CHANG Y,XIE S,et al. Impacts of bioprocess engineering on product formation by Acetobacter pasteurianus[J]. Applied Microbiology and Biotechnology,2018,102(6):2 535-2 541.
[16] QI Z,WANG W,YANG H,et al. Mutation of Acetobacter pasteurianus by UV irradiation under acidic stress for high-acidity vinegar fermentation[J]. International Journal of Food Science and Technology,2014,49(2):468-476.
[17]朱小明,夏小乐,杨海麟,等.巴氏醋酸杆菌沪酿1. 01乙醇氧化产醋酸关键酶的研究[J].食品工业科技,2013,34(2):167-170.
[18]亓正良,杨海麟,夏小乐,等.巴氏醋杆菌高酸度醋发酵过程的能量代谢分析[J].微生物学通报,2013,40(12):2 171-2 181.
[19]亓正良,杨海麟,夏小乐,等.巴氏醋酸杆菌对发酵中醋酸胁迫的生理应答[J].微生物学报,2014,54(3):299-308.
[20] FOUNTOULAKIS M,LAHM H W. Hydrolysis and amino acid composition of proteins[J]. Journal of Chromatography A,1998,826(2):109-134.
[21] HARRISON C J,HAYER-HARTL M,LIBERTO M Di,et al. Crystal structure of the nucleotide exchange factor Grp E bound to the ATPase domain of the molecular chaperone Dna K[J]. Science,1997,276(5 311):431-435.
[22] ZHENG Y,ZHANG R,YIN H,et al. Acetobacter pasteurianus metabolic change induced by initial acetic acid to adapt to acetic acid fermentation conditions[J]. Applied Microbiology and Biotechnology,2017,101(18):7 007-7 016.
[23] ISHIKAWA M,OKAMOTO-KAINUMA A,MATSUI K,et al. Cloning and characterization of clp B in Acetobacter pasteurianus NBRC 3283[J]. Journal of Bioscience and Bioengineering,2010,110(1):69-71.
[24] DENICH T J,BEAUDETTE L A,LEE H,et al. Effect of selected environmental and physico-chemical factors on bacterial cytoplasmic membranes[J]. Journal of Microbiological Methods,2003,52(2):149-182.
[25] WANG B,SHAO Y,CHENG F. Overview on mechanisms of acetic acid resistance in acetic acid bacteria[J].World Journal of Microbiology and Biotechnology,2015,31(2):255-263.
[26]马新凤,陈义伦,周波,等.巴氏醋酸杆菌沪酿1. 01对液体保藏中醋酸胁迫的生理应答[J].食品与发酵工业,2016,42(1):42-47.
[2] TRCEK J,MIRA N P,JARBOE L R. Adaptation and tolerance of bacteria against acetic acid[J]. Applied Microbiology and Biotechnology,2015,99(15):6 215-6 229.
[3] XIA K,ZANG N,ZHANG J,et al. New insights into the mechanisms of acetic acid resistance in Acetobacter pasteurianus using i TRAQ-dependent quantitative proteomic analysis[J]. International Journal of Food Microbiology,2016,238:241-251.
[4] TRCEK J,TOYAMA H,CZUBA J,et al. Correlation between acetic acid resistance and characteristics of PQQ-dependent ADH in acetic acid bacteria[J]. Applied Microbiology and Biotechnology,2006,70(3):366-373.
[5] FUKAYA M,TAKEMURA H,TAYAMA K,et al. The aar C gene responsible for acetic acid assimilation confers acetic acid resistance on acetobacter aceti[J]. Journal of Fermentation and Bioengineering,1993,76(4):270-275.
[6] NAKANO S,FUKAYA M,HORINOUCHI S. Putative ABC transporter responsible for acetic acid resistance in Acetobacter aceti[J]. Applied and Environmental Microbiology,2006,72(1):497-505.
[7] TRCEK J,JERNEJC K,MATSUSHITA K. The highly tolerant acetic acid bacterium Gluconacetobacter europaeus adapts to the presence of acetic acid by changes in lipid composition,morphological properties and PQQ-dependent ADH expression[J]. Extremophiles, 2007, 11(4):627-635.
[8] HANADA T,KASHIMA Y,KOSUGI A,et al. A gene encoding phosphatidylethanolamine N-methyltransferase from Acetobacter aceti and some properties of its disruptant[J].Bioscience Biotechnology and Biochemistry, 2001, 65(12):2 741-2 748.
[9] LIU Y,TANG H,LIN Z,et al. Mechanisms of acid tolerance in bacteria and prospects in biotechnology and bioremediation[J]. Biotechnology Advances,2015,33(7):1 484-1 492.
[10] COTTERP D,COLIN H. Surviving the acid test:responses of gram-positive bacteria to low p H[J]. Microbiology and Molecular Biology Reviews:MMBR,2003,67(3):429-453.
[11] MATSUSHITA K,INOUE T,ADACHI O,et al. Acetobacter aceti possesses a proton motive force-dependent efflux system for acetic acid[J]. Journal of Bacteriology,2005,187(13):4 346-4 352.
[12] SAINZ F,MAS A,TORIJA M J. Effect of ammonium and amino acids on the growth of selected strains of Gluconobacter and Acetobacter[J]. International Journal of Food Microbiology,2017,242:45-52.
[13] QI Z,DONG D,YANG H,et al. Improving fermented quality of cider vinegar via rational nutrient feeding strategy[J]. Food Chemistry,2017,224:312-319.
[14] YIN H,ZHANG R,XIA M,et al. Effect of aspartic acid and glutamate on metabolism and acid stress resistance of Acetobacter pasteurianus[J]. Microbial Cell Factories,2017,16(1):109.
[15] ZHENG Y,CHANG Y,XIE S,et al. Impacts of bioprocess engineering on product formation by Acetobacter pasteurianus[J]. Applied Microbiology and Biotechnology,2018,102(6):2 535-2 541.
[16] QI Z,WANG W,YANG H,et al. Mutation of Acetobacter pasteurianus by UV irradiation under acidic stress for high-acidity vinegar fermentation[J]. International Journal of Food Science and Technology,2014,49(2):468-476.
[17]朱小明,夏小乐,杨海麟,等.巴氏醋酸杆菌沪酿1. 01乙醇氧化产醋酸关键酶的研究[J].食品工业科技,2013,34(2):167-170.
[18]亓正良,杨海麟,夏小乐,等.巴氏醋杆菌高酸度醋发酵过程的能量代谢分析[J].微生物学通报,2013,40(12):2 171-2 181.
[19]亓正良,杨海麟,夏小乐,等.巴氏醋酸杆菌对发酵中醋酸胁迫的生理应答[J].微生物学报,2014,54(3):299-308.
[20] FOUNTOULAKIS M,LAHM H W. Hydrolysis and amino acid composition of proteins[J]. Journal of Chromatography A,1998,826(2):109-134.
[21] HARRISON C J,HAYER-HARTL M,LIBERTO M Di,et al. Crystal structure of the nucleotide exchange factor Grp E bound to the ATPase domain of the molecular chaperone Dna K[J]. Science,1997,276(5 311):431-435.
[22] ZHENG Y,ZHANG R,YIN H,et al. Acetobacter pasteurianus metabolic change induced by initial acetic acid to adapt to acetic acid fermentation conditions[J]. Applied Microbiology and Biotechnology,2017,101(18):7 007-7 016.
[23] ISHIKAWA M,OKAMOTO-KAINUMA A,MATSUI K,et al. Cloning and characterization of clp B in Acetobacter pasteurianus NBRC 3283[J]. Journal of Bioscience and Bioengineering,2010,110(1):69-71.
[24] DENICH T J,BEAUDETTE L A,LEE H,et al. Effect of selected environmental and physico-chemical factors on bacterial cytoplasmic membranes[J]. Journal of Microbiological Methods,2003,52(2):149-182.
[25] WANG B,SHAO Y,CHENG F. Overview on mechanisms of acetic acid resistance in acetic acid bacteria[J].World Journal of Microbiology and Biotechnology,2015,31(2):255-263.
[26]马新凤,陈义伦,周波,等.巴氏醋酸杆菌沪酿1. 01对液体保藏中醋酸胁迫的生理应答[J].食品与发酵工业,2016,42(1):42-47.
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