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食品与发酵工业  2021, Vol. 47 Issue (16): 22-30    DOI: 10.13995/j.cnki.11-1802/ts.027019
  研究报告 本期目录 | 过刊浏览 | 高级检索 |
酿酒酵母PDC5基因的缺失对2-苯乙醇合成的影响及相关代谢改造
朱灵桓1,2,3, 徐沙1,2, 李由然1,2, 张梁1,2, 石贵阳1,2*
1(江南大学 生物工程学院,江苏 无锡,214122)
2(粮食发酵工艺与技术国家工程实验室(江南大学),江苏 无锡,214122)
3(河北科技大学 食品与生物学院,河北 石家庄,050018)
Improvement of 2-phenylethanol production by deleting gene PDC5 and related metabolic strategies in Saccharomyces cerevisiae
ZHU Linghuan1,2,3, XU Sha1,2, LI Youran1,2, ZHANG Liang1,2, SHI Guiyang1,2*
1(School of Biotechnology, Jiangnan University, Wuxi 214122, China)
2(National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China)
3(College of Food Science and Biology, Hebei University of Science and Technology, Shijiazhuang 050018, China)
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摘要 该文研究了酿酒酵母中编码丙酮酸脱羧酶的基因PDC5的缺失对2-苯乙醇合成的影响,并将其应用于2-苯乙醇高产菌株的代谢改造策略。首先利用CRISPR/Cas9双质粒敲除体系构建pdc5△突变株,通过优化过的培养条件进行摇瓶发酵,发现基因PDC5的缺失能够促进酵母合成2-苯乙醇。分别表达芳香族转氨酶Aro8p和Aro9p,发现在缺失Aro9p的突变株中过表达Aro8p能够促进2-苯乙醇的合成。由此构建了重组菌株RM59-810(pdc5aro9△ PTPI-ARO8-TTT PTPI-ARO10-TTT),在6.7 g/L L-苯丙氨酸培养基中培养120 h后2-苯乙醇的产量为3.85 g/L,摩尔转化率为0.67 mol/mol,是对照菌株的1.33倍,得率为0.5 g/g L-苯丙氨酸。该文为加强酿酒酵母合成2-苯乙醇的能力提供了一种新的代谢工程改造策略,为研究丙酮酸脱羧酶Pdc5p可能存在的调控作用提供了依据。
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朱灵桓
徐沙
李由然
张梁
石贵阳
关键词:  酿酒酵母  PDC5  ARO8  2-苯乙醇  代谢改造    
Abstract: As an important aromatic alcohols with favorable flavor and superior property, 2-phenylethanol has been widely used in food, medicine, and chemical industry. In this study, the deletion of phenylpyruvate decarboxylase gene PDC5 was found to improve the synthesis of 2-phenylethanol, and related metabolic strategy of the Ehrlich pathway was rational reconstructed in Saccharomyces cerevisiae for further product-enhancement. Strain RM22 (pdc5△) was obtained using CRISPR/Cas9, and improved 2-phenylethanol production was observed in the optimized culture conditions. Additionally, by expressing aromatic transaminase Aro8p and Aro9p respectively, we found that overexpressing of Aro8p in aro9-deleted strain was an efficient strategy to increase 2-phenylethanol production. To further improve the production of 2-phenylethanol, metabolic engineered strain RM-59810 (pdc5aro9△PTPI-ARO8-TTT PTPI-ARO10-TTT) was constructed, resulting in 3.85 g/L of 2-phenylethanol from 6.7 g/L phenylalanine after 120 h, and the molar conversion rate was 0.67 mol/mol, which was 1.33-fold than that of the control strain, with a yield of 0.5 g/g L-phenylalanine. In this study, a new efficient strategy was provided to improve 2-phenylethanol production in S. cerevisiae, and also provided a primary foundation for studying the possible regulatory effects of pyruvate decarboxylase Pdc5p.
Key words:  Saccharomyces cerevisiae    PDC5    ARO8    2-phenylethanol    metabolic modification
收稿日期:  2021-02-08      修回日期:  2021-03-09                发布日期:  2021-09-10      期的出版日期:  2021-08-25
基金资助: 国家自然科学基金项目(31571817)
作者简介:  博士,讲师(石贵阳教授为通讯作者,E-mail:gyshi@jiangnan.edu.cn)
引用本文:    
朱灵桓,徐沙,李由然,等. 酿酒酵母PDC5基因的缺失对2-苯乙醇合成的影响及相关代谢改造[J]. 食品与发酵工业, 2021, 47(16): 22-30.
ZHU Linghuan,XU Sha,LI Youran,et al. Improvement of 2-phenylethanol production by deleting gene PDC5 and related metabolic strategies in Saccharomyces cerevisiae[J]. Food and Fermentation Industries, 2021, 47(16): 22-30.
链接本文:  
http://sf1970.cnif.cn/CN/10.13995/j.cnki.11-1802/ts.027019  或          http://sf1970.cnif.cn/CN/Y2021/V47/I16/22
[1] HUA D L, XU P.Recent advances in biotechnological production of 2-phenylethanol[J].Biotechnology Advances, 2011, 29(6):654-660.
[2] WANG Y Q, ZHANG H, LU X Y, et al.Advances in 2-phenylethanol production from engineered microorganisms[J].Biotechnology Advances, 2019, 37(3):403-409.
[3] GU Y, MA J, ZHU Y, et al.Refactoring Ehrlich pathway for high-yield 2-phenylethanol production in Yarrowia lipolytica[J].ACS Synthetic Biology, 2020, 9(3):623-633.
[4] 牛明福,李亚恒,陈金帅.马克斯克鲁维酵母生物转化2-PE工艺优化及耐高温特性分析[J].食品与发酵工业, 2018, 44(2):15-20.
NIU M F,LI Y H,CHEN J S, et al.Optimization and characterization of 2-phenylethanol bioconversion by thermotolerant yeast Kluyveromyces marxianus[J].Food and Fermentation Industries, 2018, 44(2):15-20.
[5] HUANG C J, LEE S L, CHOU C C.Production of 2-phenylethanol, a flavor ingredient, by Pichia fermentans L-5 under various culture conditions[J].Food Research International, 2001, 34:277-282.
[6] KIM B, CHO B R, HAHN J S.Metabolic engineering of Saccharomyces cerevisiae for the production of 2-phenylethanol via Ehrlich pathway[J].Biotechnology and Bioengineering, 2014, 111(1):115-124.
[7] JIN D, GU B, XIONG D, et al.A Transcriptomic analysis of Saccharomyces cerevisiae under the stress of 2-phenylethanol[J].Current Microbiology, 2018, 75(8):1 068-1 076.
[8] ETSCHMANN M M, BLUEMKE W, SELL D, et al.Biotechnological production of 2-phenylethanol[J].Applied Microbiology and Biotechnology, 2002, 59(1):1-8.
[9] IRAQUI I, VISSERS S, CARTIAUX M, et al.Characterisation of Saccharomyces cerevisiae ARO8 and ARO9 genes encoding aromatic aminotransferases I and II reveals a new aminotransferase subfamily[J].Molecular and General Genetics, 1998, 257:238-248.
[10] BULFER S L, BRUNZELLE J S, TRIEVEL R C.Crystal structure of Saccharomyces cerevisiae Aro8, a putative alpha-aminoadipate aminotransferase[J].Protein Science, 2013, 22(10):1 417-1 424.
[11] KIM S, LEE K, BAE S J, et al.Promoters inducible by aromatic amino acids and gamma-aminobutyrate (GABA) for metabolic engineering applications in Saccharomyces cerevisiae[J].Applied Microbiology and Biotechnology, 2015, 99(6):2 705-2 714.
[12] LEE K, SUNG C, KIM B G, et al.Activation of Aro80 transcription factor by heat-induced aromatic amino acid influx in Saccharomyces cerevisiae[J].Biochemical and Biophysical Research Communications, 2013, 438(1):43-47.
[13] LEE K, HAHN J S.Interplay of Aro80 and GATA activators in regulation of genes for catabolism of aromatic amino acids in Saccharomyces cerevisiae[J].Molecular Microbiology, 2013, 88(6):1 120-1 134.
[14] CHOO J H, HAN C, LEE D W, et al.Molecular and functional characterization of two pyruvate decarboxylase genes, PDC1 and PDC5, in the thermotolerant yeast Kluyveromyces marxianus[J].Applied Microbiology and Biotechnology, 2018, 102(8):3 723-3 737.
[15] BRION C, AMBROSET C, DELOBEL P.Deciphering regulatory variation of THI genes in alcoholic fermentation indicate an impact of Thi3p on PDC1 expression[J].BMC Genomics, 2014, 15(1 085):1-11.
[16] VURALHAN Z, LUTTIK M A, TAI S L, et al.Physiological characterization of the ARO10-dependent, broad-substrate-specificity 2-oxo acid decarboxylase activity of Saccharomyces cerevisiae[J].Applied and Environmental Microbiology, 2005, 71(6):3 276-3 284.
[17] ROMAGNOLI G, LUTTIK M A, KOTTER P, et al.Substrate specificity of thiamine pyrophosphate-dependent 2-oxo-acid decarboxylases in Saccharomyces cerevisiae[J].Applied and Environmental Microbiology, 2012, 78(21):7 538-7 548.
[18] MULLER E H, RICHARDS E J, NORBECK J.Thiamine repression and pyruvate decarboxylase autoregulation independently control the expression of the Saccharomyces cerevisiae PDC5 gene[J].FEBS Letters, 1999, 449:245-250.
[19] YIN S, ZHOU H, XIAO X, et al.Improving 2-phenylethanol production via Ehrlich pathway using genetic engineered Saccharomyces cerevisiae strains[J].Current Microbiology, 2015, 70(5):762-767.
[20] WANG Z Y, JIANG M Y, GUO X N, et al.Reconstruction of metabolic module with improved promoter strength increases the productivity of 2-phenylethanol in Saccharomyces cerevisiae[J].Microbial Cell Factories, 2018, 17(1):60.
[21] STARK D, KORNMANN H, MUNCH T, et al.Novel type of in situ extraction:Use of solvent containing microcapsules for the bioconversion of 2-phenylethanol from L-phenylalanine by Saccharomyces cerevisiae[J].Biotechnology and Bioengineering, 2003, 83(4):376-385.
[22] MEI J F, MIN H, LIU Z M.Enhanced biotransformation of L-phenylalanine to 2-phenylethanol using an in situ product adsorption technique[J].Process Biochemistry, 2009, 44(8):886-890.
[23] VURALHAN Z, MORAIS M, TAI S, et al.Identification and characterization of phenylpyruvate decarboxylase genes in Saccharomyces cerevisiae[J].Applied and Environmental Microbiology, 2403, 69(8):4 534-4 541.
[24] NOSAKA K, ESAKI H, ONOZUKA M, et al.Facilitated recruitment of Pdc2p, a yeast transcriptional activator, in response to thiamin starvation[J].FEMS Microbiology Letters, 2012, 330(2):140-147.
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