原儿茶酸脱羧关键酶挖掘改造优化儿茶酚生物合成  

Screening and engineering of a protocatechuic acid decarboxylase for efficient biosynthesis of catechol

作　　者：田立岩 李静[1,2,3] 江小龙 宋国田[2,3] 路福平 戴宗杰 李庆刚[1] 王钦宏 TIAN Liyan;LI Jing;JIANG Xiaolong;SONG Guotian;LU Fuping;DAI Zongjie;LI Qinggang;WANG Qinhong(School of Biological Engineering,Tianjin University of Science&Technology,Tianjin 300457,China;Key Laboratory of Engineering Biology for Low-carbon Manufacturing,Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin 300308,China;National Center of Technology Innovation for Synthetic Biology,Tianjin 300308,China)

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]中国科学院天津工业生物技术研究所低碳合成工程生物学重点实验室,天津300308 [3]国家合成生物技术创新中心,天津300308

出　　处：《生物工程学报》2024年第9期3057-3071,共15页Chinese Journal of Biotechnology

基　　金：国家重点研发计划(2018YFA0901400);国家自然科学基金(32200063);天津市合成生物技术创新能力提升行动(TSBICIP-CXRC-057);中国博士后科学基金(2022M723339)。

摘　　要：儿茶酚(catechol,CA)是一种重要的化工和医药中间体,应用范围极为广泛。目前制备CA的方法是苯酚羟化法,但由于该方法存在对环境污染大、石化资源不可再生等问题,使得生物合成儿茶酚的方法逐渐受到关注。但受制于原儿茶酸脱羧酶的活性不够高且生物合成儿茶酚生产效率较低,难以满足大规模工业生产的要求。为了进一步提高儿茶酚生产效率,本研究首先对不同来源的21个原儿茶酸(protocatechuic acid,PCA)脱羧酶进行筛选,发现理研菌(Rikenellaceae)来源的RbAroY脱羧酶性能最好,含有该酶的全细胞生物催化剂ER11能够合成13.54 g/L儿茶酚。然后利用在线工具HotSpotWizard对该酶进行稳定性计算,选取自由能下降最多的10个潜在突变位点进行验证,发现含有突变子RbAroYG99A的全细胞生物催化剂ERT01能够催化合成15.16 g/L儿茶酚,较野生型产量提高12%。之后对生物催化条件进一步优化,以原儿茶酸为底物,全细胞生物催化剂ERT01能够催化合成25.70 g/L儿茶酚。最后,以3-脱氢莽草酸发酵液为底物,同时表达3-脱氢莽草酸脱水酶和原儿茶酸脱羧酶的全细胞生物催化剂DER03,催化合成29.55 g/L儿茶酚,为目前国内外报道的生物合成的最高产量。本研究为儿茶酚生物合成工业生产提供了重要参考。Catechol(CA)is an important chemical and pharmaceutical intermediate with wide applications.At present,CA is produced by phenol hydroxylation with non-renewable petrochemical resources,which causes serious environmental pollution.Hence,the biosynthesis of CA attracts much attention recently.However,due to the low activities of protocatechuic acid(PCA)decarboxylases,the production efficiency of biosynthetic catechol is too low to meet the requirements of large-scale industrial production.To improve the yield of CA,we screened 21 PCA decarboxylases from different species.RbAroY originated from Rikenellaceae had the best catalytic performance.The whole-cell biocatalyst ER11 with RbAroY was able to produce CA at a titer of 13.54 g/L.Then,the online tool HotSpot Wizard was employed to measure the enzyme stability,which revealed 10 potential mutation sites causing significant decreases in Gibbs free energy.The whole-cell biocatalyst ERT01 with the mutated RbAroYG99A could produce CA at a titer of 15.16 g/L,which increased by 12%compared with that of the wild-type whole-cell biocatalyst.After optimization of the biocatalytic conditions,the whole-cell biocatalyst ERT01 was able to produce CA at a titer of 25.70 g/L with PCA as the substrate.Finally,with the fermentation broth of 3-dehydroshikimate as the substrate,the whole-cell biocatalyst DER03 expressing both 3-dehydroshikimate dehydratase and PCA decarboxylase realized the production CA at a titer of 29.55 g/L,which is currently the highest biosynthetic titer reported.This study provides a reference for the industrial production of CA by biosynthesis.

关 键 词：生物合成 全细胞催化 原儿茶酸脱羧酶 儿茶酚 

分 类 号：Q789[生物学—分子生物学] TQ920.6[轻工技术与工程—发酵工程]