重组大肠杆菌全细胞催化高效合成胆绿素  

Efficient production of biliverdin through whole-cell biocatalysis using recombinant Escherichia coli

作　　者：闫思翰 邵明龙[1] 徐美娟[1] 张显[1] 杨套伟[1] 饶志明[1] YAN Sihan;SHAO Minglong;XU Meijuan;ZHANG Xian;YANG Taowei;RAO Zhiming(Key Laboratory of Industrial Biotechnology of Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,Jiangsu,China)

机构地区：[1]江南大学生物工程学院工业生物技术教育部重点实验室,江苏无锡214122

出　　处：《生物工程学报》2022年第7期2581-2593,共13页Chinese Journal of Biotechnology

基　　金：国家重点研发计划(2019YFA0905300);国家自然科学基金(31700041);天津市合成生物技术创新能力提升行动项目(TSBICIP-KJGG-001-14);新疆生产建设兵团科技攻关计划项目(2019AB009);江南大学基本科研计划青年基金(JUSRP12017)。

摘　　要：胆绿素作为一种重要的保护细胞的抗氧化剂,其传统生产方法主要由胆红素的化学氧化产生,但过程复杂、纯度不高。本研究提出了一种高效、绿色、安全的生产胆绿素的方法。通过比较,筛选得到了破伤风梭状芽孢杆菌(Clostridium tetani)来源的血红素加氧酶(heme oxygenase,HO)基因,并成功构建具备转化血红素合成胆绿素能力的重组大肠杆菌(Escherichiacoli)BL21/pETDuet-hoCt。在pH 7.0、35℃、100 mg/L底物浓度条件下胆绿素产量为32.9 mg/L。为提高还原力,构建了基于谷氨酸脱氢酶(glutamate dehydrogenase,GdhA)的NADPH辅酶再生系统,获得重组菌E.coli BL21/pETDuet-gdhAEc-hoCt,胆绿素产量为71.5 mg/L。此外,通过引入膜表面展示系统,构建重组菌E.coli BL21/pETDuet-gdhAEc-blc/hoCt,缩短转化时间的同时,胆绿素产量进一步得到提高,达到76.3 mg/L,是目前生物法合成胆绿素的最高研究报道。本研究为胆绿素的绿色生产奠定了良好的基础。Biliverdin is an important cellular antioxidant.Traditionally,biliverdin is produced by chemical oxidation of bilirubin,which is a complex process and the final product is of low purity.Here we report an efficient,green and safe process for biotechnological production of biliverdin.A heme oxygenase(HO)gene from Clostridium tetani was screened,and a recombinant strain Escherichia coli BL21/pETDuet-hoCt with the ability of transforming heme into biliverdin was constructed.A biliverdin yield of 32.9 mg/L from 100 mg/L substrate was achieved under pH 7.0 and 35℃.In order to improve the supply of reducing power,an NADPH regeneration system using glutamate dehydrogenase(GdhA)was constructed,resulting in a recombinant strain E.coli BL21/pETDuet-gdhAEc-hoCt which was capable of producing 71.5 mg/L biliverdin.Moreover,through introduction of a membrane surface display system,a recombinant strain E.coli BL21/pETDuet-gdhAEc-blc/hoCt was constructed to shorten the transformation time,and the production of biliverdin was further increased to 76.3 mg/L,this is the highest titer of biosynthesized biliverdin reported to date,and the research may thus facilitate the green production of biliverdin.

关 键 词：血红素加氧酶 谷氨酸脱氢酶 锚定蛋白 胆绿素IXα 生物催化 合成生物学 

分 类 号：Q78[生物学—分子生物学] TQ920.6[轻工技术与工程—发酵工程]