重组枯草芽孢杆菌全细胞催化高效合成2′-脱氧腺苷  

Highly efficient production of 2′-deoxyadenosine by whole-cell catalysis of engineered Bacillus subtilis

作　　者：陈伟 陈令伟 李文超 郑玲辉 CHEN Wei;CHEN Lingwei;LI Wenchao;ZHENG Linghui(Hangzhou Hizyme Biotech Co.,Ltd.,Hangzhou 310011,Zhejiang,China;College of Biotechnology,Tianjin University of Science and Technology,Tianjin 300457,Tianjin,China)

机构地区：[1]杭州珲益生物科技有限公司,浙江杭州310011 [2]天津科技大学生物工程学院,天津300457

出　　处：《精细化工》2023年第11期2436-2444,共9页Fine Chemicals

基　　金：天津市教委科研计划项目(2019KJ237)。

摘　　要：以枯草芽孢杆菌168(简写为BS)为宿主,以脱氧胸苷和腺嘌呤为底物,异源表达组合来源于大肠杆菌(E.coli)的嘧啶核苷磷酸化酶(PyNP)与嘌呤核苷磷酸化酶(PNP)作为催化酶源,全细胞催化合成2′-脱氧腺苷(dA)。首先,以BS敲除内源性的PNP(由基因deoD编码,Gene ID:940038)后的菌株BS0为出发菌株,经过PyNP1酶(来源于E.coli,Gene ID:948901)与PNP3酶(来源于E.coli,Gene ID:945654)的重组表达得到CW3,再优化PyNP1与PNP3的对核糖体结合位点(RBS)序列得到重组菌株CW3-3,在CW3-3作用下,dA的产量为133.4 g/L,脱氧胸苷的转化率为64.3%;其次,以CW3-3为出发菌株,利用互作短肽构建自组装多酶复合物,经过PyNP1酶N端融合RIAD,PNP3酶C端融合RIDD(其中RIAD和RIDD为互作短肽标签)处理后,PyNP1酶N端融合4个RIAD标签得到重组菌株CW17,在其作用下,dA的产量达到179.6 g/L,显著提升了底物转化率;最后,对重组菌株CW17全细胞催化条件细胞添加质量浓度及催化反应温度进行了优化,在细胞添加质量浓度为150g/L,反应温度50℃条件下,dA的产量达到200.3g/L,脱氧胸苷的转化率为96.6%。2′-Deoxyadenosine(dA)was synthesized from the whole cell of Bacillus subtilis 168(BS for short),which were catalyzed by the heterologous enzymes of pyrimidine nucleoside phosphorylase(PyNP)and purine nucleoside phosphorylase(PNP)from Escherichia coli,using deoxythymidine and adenine as substrate.The recombinant B.subtilis CW3-3,which was obtained from recombinant expression of PyNP1 enzyme(from E.coli,Gene ID:948901)and PNP3 enzyme(from E.coli,Gene ID:945654)and optimization of the ribosome binding site(RBS)for PyNP1 and PNP3 enzyme using BS0 strain(BS strain knocked out of endogenous PNP and encoded by Gene deoD,Gene ID:940038)as starting strain,exhibited a yield of dA 133.4 g/L and a conversion rate of deoxythymidine 64.3%.Moreover,B.subtilis CW3-3 was further used as starting strain to construct a self-assembled multi-enzyme complex with interacting short peptides.After treatment with N-terminal fusion RIAD of PyNP1 enzyme and C-terminal fusion RIDD of PNP3 enzyme(RIAD and RIDD are interacting short peptide labels),the N-terminal fusion of PyNP1 enzyme fused with four RIAD labels to obtain recombinant strain B.subtilis CW17,which improved the yield of dA to 179.6 g/L.Finally,the cell addition mass concentration and catalytic reaction temperature conditions of the recombinant B.subtilis CW17 whole-cell catalysis were optimized and further boosted the production of dA to 200.3 g/L and the conversion rate of deoxythymidine to 96.6%.

关 键 词：2′-脱氧腺苷 嘧啶核苷磷酸化酶 嘌呤核苷磷酸化酶 互作短肽 枯草芽孢杆菌 全细胞催化 生物工程 

分 类 号：TQ460.1[化学工程—制药化工] TQ426.97