大肠杆菌高效合成L-苯甘氨酸的研究进展

doi:10.13560/j.cnki.biotech.bull.1985.2020-0779

摘要/Abstract

摘要：

自然界存在着多种氨基酸,除用于蛋白质合成的20种外,大量用于合成具有生物活性的物质,广泛应用于食品、医药等多个领域。其中,非天然芳香族氨基酸L-苯甘氨酸作为一种重要的组成单元广泛的应用于盘尼西林、维吉霉素S、原始霉素I等β-内酰胺类抗生素的生物合成当中。目前L-苯甘氨酸主要通过化学法合成,但该方法合成收率低、污染大,且不易得到单一手性的化合物。由于生物合成L-苯甘氨酸具有反应条件温和、产物立体选择性好的优势,因此受到了广泛的关注。通过对L-苯甘氨酸两条生物合成途径的解析,合成所需相关酶的筛选及辅因子平衡再生等,逐步形成了以苯乙酮酸、扁桃酸和L-苯丙氨酸为底物的合成线路。主要对L-苯甘氨酸的生物合成途径及生物合成策略展开综述,为研究者提供优化方向,以期为高效工业化生物合成L-苯甘氨酸提供理论参考。

关键词: L-苯甘氨酸, 生物合成, 合成策略

Abstract:

There are many kinds of amino acids in nature. In addition to the 20 kinds used in protein synthesis,a large number of amino acids are used to synthesize biologically active substances,which are widely used in food,medicine and other fields. Among them,the unnatural aromatic amino acid L-phenylglycine as an important component is widely used in the biosynthesis of β-lactam antibiotics such as penicillin,virginomycin S,and protomycin I. At present,L-phenylglycine is mainly synthesized by chemical methods,but this method has low synthesis yield,high pollution,and it is not easy to obtain monochiral compounds. Biosynthesis of L-phenylglycine has the advantages of mild reaction conditions and good product stereoselectivity,so it has received extensive attention. Through the analysis of the two biosynthetic pathways of L-phenylglycine,the screening of relevant enzymes required for synthesis and the balance regeneration of cofactors,etc.,A synthetic route with acetophenone acid,mandelic acid and L-phenylalanine as substrates was gradually formed.This article mainly reviews the biosynthetic pathways and biosynthetic strategies of L-phenylglycine,and provides researchers with optimization directions,with a view to providing a theoretical reference for efficient industrial biosynthesis of L-phenylglycine.

Key words: L-phenylglycine, Biosynthesis, Synthetic strategy

陶宇丞, 吕旭冰, 程圣杰, 王彦雯, 王文峰, 焦朕, 王鹏超. 大肠杆菌高效合成L-苯甘氨酸的研究进展[J]. 生物技术通报, 2021, 37(3): 175-184.

TAO Yu-cheng, LV Xu-bing, CHENG Sheng-jie, WANG Yan-wen, WANG Wen-feng, JIAO Zhen, WANG Peng-chao. Research Progress on the Efficient Synthesis of Phenylglycine by Escherichia coli[J]. Biotechnology Bulletin, 2021, 37(3): 175-184.

图/表 5

图1 由葡萄糖生成L-苯甘氨酸的途径 E4P：四磷酸赤藓糖;PEP：磷酸烯醇丙酮酸;DAHP：3-脱氧-D-阿拉伯糖基庚酸-7-磷酸酯;CHOR：分支酸;Hmas：羟基扁桃酸合成酶;Hmo：羟基扁桃酸氧化酶;HpgAT：羟基苯甘氨酸氨基转移酶;PglB/C：丙酮酸脱羧酶E1;PglA：羟烷基脱氢酶;PglD：II型硫酯酶;PglE：苯甘氨酸氨基转移酶

图2 芳香族氨基酸的生物合成途径 E4P：四磷酸赤藓糖;PEP：磷酸烯醇丙酮酸;NAHP：2-酮-3-脱氧-D-阿拉伯庚酮糖酸-7-磷酸;EPSP：5-烯醇式丙酮酰莽草酸-3-磷酸;CA：分支酸;Prephenate：预苯酸;4-HPP：对羟基苯丙酮酸;PP：苯丙酮酸;L-TYR：L-酪氨酸;L-PHE：L-苯丙氨酸;TYR1：prephenate dehydrogenase;PHA2：prephenate dehydratase

图3 全细胞催化生成苯甘氨酸步骤图 ArMR：扁桃酸消旋酶;LhDMDH：D-扁桃酸脱氢酶;EsLeuDH：亮氨酸脱氢酶

图4 以L-苯丙酮酸为前体的L-苯甘氨酸生物合成途径

表1 L-苯甘氨酸合成

底物	优化策略	L-苯甘氨酸	参考文献
苯乙酮酸60 g/L	LeuDH、FDH	60.2 g/L	[35]
苯乙酮酸60 g/L	BcAADH	60 g/L	[38]
扁桃酸30.4g/L	LhDMDH	30 g/L	[39]
D,L-扁桃酸 7.6g/L	ArMR、LhDMDH、 EsLeuDH	5.85 g/L	[40]
扁桃酸5 g/L	d-MDH、AADH	4.67 g/L	[8]
苯丙氨酸33 g/L	Hmas、Hmo、HpgT	51.6 mg/g DCW	[28]
葡萄糖10g/L	Hmo、HpgT	16 mg/L	[46]

参考文献 46

[1]	Langen LMV, Rantwijk FV, Švedas VK, et al. Penicillin acylase-catalyzed peptide synjournal:a chemo-enzymatic route to stereoisomers of 3, 6-diphenylpiperazine-2, 5-dione[J]. Tetrahedron Asymmetry, 2000,11(5):1077-1083. doi: 10.1016/S0957-4166(00)00027-6 URL
[2]	Toma R, Brieke C, Cryle M, et al. Structural aspects of phenylglycines, their biosynjournal and occurrence in peptide natural products[J]. Nat Prod Rep, 2015,32(8):1207-1235. doi: 10.1039/c5np00025d URL pmid: 25940955
[3]	Ningsih F, Kitani S, Fukushima E, et al. VisG is essential for biosynjournal of virginiamycin S, a streptogramin type B antibiotic, as a provider of the nonproteinogenic amino acid phenylglycine[J]. Microbiology, 2011,157(11):3213-3220. doi: 10.1099/mic.0.050203-0 URL
[4]	Teshima T, Nishikawa M, Kubota I, et al. The structure of an antibiotic, dityromycin[J]. Tetrahedron Letters, 1988,29(16):1963-1966. doi: 10.1016/S0040-4039(00)82090-0 URL
[5]	陆军民. 国内苯甘氨酸类产品的生产与市场前景[J]. 浙江化工, 2002,33(1):46-48.
	Lu JM. Production and market prospects of domestic phenylglycine products[J]. Zhejiang Chemical Industry, 2002,33(1):46-48.
[6]	Croteau R, Ketchum REB, Long RM, et al. Taxol biosynjournal and molecular genetics[J]. Phytochem Rev, 2006,5:75-97. pmid: 20622989
[7]	Tang C, Ding P, Shi H, et al. One-pot synjournal of phenylglyoxylic acid from racemic mandelic acids via cascade biocatalysis[J]. J Agricu Food Chem, 2019,67(10):2946-2953. doi: 10.1021/acs.jafc.8b07295 URL
[8]	Resch V, Fabian W, Kroutil W, Deracemisation of mandelic acid to optically pure non-natural L-phenylglycine via a redox-neutral biocatalytic cascade[J]. Advanced Synjournal & Catalysis, 2010,352(6):993-997.
[9]	李华冲, 晏菊芳, 苏小燕, 等. L-苯甘氨酸衍生物的合成及其抗糖尿病活性[J]. 合成化学, 2011(4):441-445.
	Li HC, Yan JF, Su XY, et al. Synjournal and antidiabetic activity of L-phenylglycine derivates[J]. Chinese Journal of Synthetic Chemistry, 2011(4):441-445.
[10]	崔哲峰. 化学合成联酶法生产D-对羟基苯甘氨酸的研究[D]. 天津:天津大学, 2003.
	Cui ZF. Study on the production of D-phydroxyphenylglycine by chemical synthesis linked enzyme[D]. Tianjin:Tianjin University, 2003.
[11]	Zhang NF, Li JT, Li HZ, et al. Synpatent of DL-phenylglycine by halogenated ammonation of phenylacetic acid：China, 95119414.3[P]. 1996-09-11.
[12]	王素青, 郭建权. 芳基甘氨酸的不对称合成[J]. 北京师范大学学报:自然科学版, 1994,30(3):387-390.
	Wang SQ, Guo JQ. Asymmetric synjournal of arylglycine[J]. Journal of Beijing Normal University:Natural Science, 1994,30(3):387-390.
[13]	徐亚荣. 苯甘氨酸、对氯苯甘氨酸合成工艺的研究[D]. 南京:南京工业大学, 2005.
	Xu YR. Study on the synthesis process of phenylglycine and p-chlorophenylglycine[D]. Nanjing:Nanjing University of Technology, 2005.
[14]	Wang JB, Reetz MT. Biocatalysis:chiral cascades[J]. Nature Chemistry, 2015,7(12):948-949. doi: 10.1038/nchem.2408 URL pmid: 26587707
[15]	Mast YJ, Wohlleben W, Schinko E. Identification and functional characterization of phenylglycine biosynthetic genes involved in pristinamycin biosynjournal in Streptomyces pristinaespiralis[J]. Journal of Biotechnology, 2010,155(1):63-67. doi: 10.1016/j.jbiotec.2010.12.001 URL pmid: 21146568
[16]	Miguel S, Guo W, Feng X, et al. Investigating strain dependency in the production of aromatic compounds in Saccharomyces cerevisiae[J]. Biotechnology & Bioengineering, 2016,113(12):2676-2685. doi: 10.1002/bit.26037 URL pmid: 27317047
[17]	Curran KA, Leavitt JM, Karim AS, et al. Metabolic engineering of muconic acid production in Saccharomyces cerevisiae[J]. Metab Eng, 2012,15:55-66. URL pmid: 23164574
[18]	Tyagi N, Saini D, Guleria R, et al. Designing an Escherichia coli strain for phenylalanine overproduction by metabolic engineering[J]. Mol Biotechnol, 2017,59(4-5):168-178. doi: 10.1007/s12033-017-9999-5 URL pmid: 28374116
[19]	Braus GH. Aromatic amino acid biosynjournal in the yeast Saccharomyces cerevisiae:A model system for the regulation of a eukaryotic biosynthetic pathway[J]. Microbiological Reviews, 1991,55(3):349-370. pmid: 1943992
[20]	Suástegui M, Shao Z. Yeast factories for the production of aromatic compounds:from building blocks to plant secondary metabolites[J]. Journal of Industrial Microbiology & Biotechnology, 2016,43(11):1611-1624. URL pmid: 27581441
[21]	Sun ZT, Ning YY, Liu LX, et al. Metabolic engineering of the L-phenylalanine pathway in Escherichia coli for the production of S- or R-mandelic acid[J]. Microb Cell Fac, 2011,10(1):71.
[22]	雷呈祥, 范长胜, 等. 大肠杆菌pheA基因在黄色短杆菌中的克隆与表达[J]. 生物工程学报, 1996(S1):184-189.
	Lei CX, Fan CS, et al. Cloning and expression of Escherichia coli pheA gene in Brevibacterium flavum[J]. Chinese Journal of Biotechnology, 1996(S1):184-189.
[23]	Müller U, Assema FV, Gunsior M, et al. Metabolic engineering of the E. coli L-phenylalanine pathway for the production of D-phenylglycine(D-Phg)[J]. Metab Eng, 2006,8(3):196-208. doi: 10.1016/j.ymben.2005.12.001 URL pmid: 16466681
[24]	叶玉成, 刘双平, 张梁, 等. 对羟基扁桃酸合酶基因的克隆表达及催化特性[J]. 微生物学通报, 2014,20(2):80-90.
	Ye YC, Liu SP, Zhang L, et al. Cloning, expression and catalytic characterization of hydroxymandelate synthase[J]. Microbiology China, 2014,20(2):80-90.
[25]	Li FF, Zhao Y, Li BZ, et al. Engineering Escherichia coli for production of 4-hydroxymandelic acid using glucose-xylose mixture[J]. Microbial Cell Factories, 2016,15(1):90. doi: 10.1186/s12934-016-0489-4 URL
[26]	Gelfand DH, Rudo N. Mapping of the aspartate and aromatic amino acid aminotransferase genes tyrB and aspC[J]. Journal of Bacteriology, 1977,130(1):441-444. doi: 10.1128/JB.130.1.441-444.1977 URL pmid: 323238
[27]	Liu SP, Xiao MR, Zhang L, et al. Production of L-phenylalanine from glucose by metabolic engineering of wild type Escherichia coli W3110[J]. Process Biochemistry, 2013,48(3):413-419. doi: 10.1016/j.procbio.2013.02.016 URL
[28]	谢雪原. 双酶偶联制备L-苯丙氨酸[D]. 南京:南京工业大学, 2004.
	Xie XY. Preparation of L-phenylalanine by double enzyme coupling[D]Nanjing:Nanjing University of Technology, 2004.
[29]	Osipenkov N, Kulik A, Mast Y. Characterization of the phenylglycine aminotransferase PglE from Streptomyces pristinaespiralis[J]. J Biotechnol, 2018,278:34-38. doi: 10.1016/j.jbiotec.2018.05.007 URL pmid: 29738785
[30]	Moosmann D, Mokeev V, Kulik A, et al. Genetic engineering approaches for the fermentative production of phenylglycines[J]. Appl Microbiol Biotechnol, 2020,104(8):3433-3444. doi: 10.1007/s00253-020-10447-9 URL pmid: 32078019
[31]	Liu Q, Zhou J, Yang T, et al. Efficient biosynjournal of L-phenylglycine by an engineered Escherichia coli with a tunable multi-enzyme-coordinate expression system[J]. Applied Microbiology & Biotechnology, 2018,102:2129-2141. doi: 10.1007/s00253-018-8741-y URL pmid: 29352398
[32]	Broussy S, Cheloha RW, Berkowitz DB. Enantioselective, ketoreductase-based entry into pharmaceutical building blocks:ethanol as tunable nicotinamide reductant[J]. Organic Letters, 2009,11(2):305-308. doi: 10.1021/ol802464g URL pmid: 19128188
[33]	Li J, Pan J, Zhang J, et al. Stereoselective synjournal of l-tert-leucine by a newly cloned leucine dehydrogenase from Exiguobacterium sibiricum[J]. J Mol Catal B:Enzy, 2014,105:11-17.
[34]	Chen F, Liu Y, Zheng G, et al. Asymmetric amination of secondary alcohols by using a redox-neutral two-enzyme cascade[J]. ChemCatChem, 2015,7(23):3838-3841. doi: 10.1002/cctc.201500785 URL
[35]	刘巧利, 杨套伟, 等. 酶法高效转化苯乙酮酸合成L-苯甘氨酸[J]. 应用与环境生物学报, 2019,25(2):451-456.
	Liu QL, Yang TW, et al. Efficient enzymatic synjournal of L-phenylglycine from benzoylformic acid[J]. Chinese Journal of Applied and Environmental Biology, 2019,25(2):451-456.
[36]	Qi Y, Yang T, Zhou J, et al. Development of a multi-enzymatic desymmetrization and its application for the biosynjournal of L-norvaline from DL-norvaline[J]. Process Biochemistry, 2017,55:104-109. doi: 10.1016/j.procbio.2017.01.022 URL
[37]	Ou Z, Shi H, Sun X, et al. Synjournal of S-licarbazepine by asymmetric reduction of oxcarbazepine with Saccharomyces cerevisiae CGMCC No. 2266[J]. Journal of Molecular Catalysis B Enzymatic, 2011,72(3-4):294-297. doi: 10.1016/j.molcatb.2011.07.004 URL
[38]	Cheng J, Xu G, Han R, et al. Efficient access to L-phenylglycine using a newly identified amino acid dehydrogenase from Bacillus clausii[J]. RSC Adv, 2016,6(84):80557-80563. doi: 10.1039/C6RA17683F URL
[39]	Fan CW, Bai YP, Ma BD, et al. A novel D-mandelate dehydrogenase used in three-enzyme cascade reaction for highly efficient synjournal of non-natural chiral amino acids[J]. Journal of Biotechnology, 2015,195:67-71. doi: 10.1016/j.jbiotec.2014.10.026 URL pmid: 25449542
[40]	贾园园, 李祥, 张振华, 等. 重组大肠杆菌全细胞催化D, L-扁桃酸对映选择性制备L-苯甘氨酸[J]. 食品科学, 2021,42(2), 83-89.
	Jia YY, Li X, Zhang ZH, et al. Enantioselective production of L-phenylglycine from D, L-mandelic acid catalyzed by recombinant E. coli whole cells[J]. Food Science, 2021,42(2), 83-89.
[41]	Cardillo AB, Perassolo M, Sartuqui M, et al. Production of tropane alkaloids by biotransformation using recombinant, Escherichia coli, whole cells[J]. Biochem Engi J, 2017,125:180-189.
[42]	Wang P, Yang X, Lin B, et al. Cofactor self-sufficient whole-cell biocatalysts for the production of 2-phenylethanol[J]. Metab Eng, 2017,44:143-149. doi: 10.1016/j.ymben.2017.09.013 URL pmid: 28951189
[43]	Wachtmeister J, Rother D. Recent advances in whole cell biocatalysis techniques bridging from investigative to industrial scale[J]. Curr Opin Biotechnol, 2016,42:169-177. doi: 10.1016/j.copbio.2016.05.005 URL pmid: 27318259
[44]	Liu SP, Liu RX, El-Rotail, et al. Heterologous pathway for the production of L-phenylglycine from glucose by E. coli[J]. Biotechnol, 2014,186:91-97.
[45]	Wiyakrutta S, Meevootisom V. A stereo-inverting D-phenylglycine aminotransferase from Pseudomonas stutzeri ST-201:purification, characterization and application for D-phenylglycine synjournal[J]. J Biotechnol, 1997,55(3):193-203. doi: 10.1016/s0168-1656(97)00075-8 URL pmid: 9249994
[46]	刘双平. 大肠杆菌中L-苯甘氨酸生物合成途径的架构及系统改造[D]. 无锡:江南大学, 2015.
	Liu SP. Construction and systematical engineering of L-phenylglycine biosynthetic pathway in E. coli[D]. Wuxi:JiangNan University, 2015.