JIMB:中科院合肥研究院郑之明研究组发表细胞工厂构建与维生素
2017年3月3日，国际期刊《Journal of Industrial Microbiology & Biotechnology》在线发表了中国科学院合肥物质科学研究院技术生物与农业工程研究所郑之明研究团队的一篇研究论文，论文报道了在细胞工厂构建与维生素K2合成研究方面取得进展。
维生素K2作为一种典型的高值化学品，结构复杂、功能独特、代谢途径冗长。通过构建细胞工厂实现维生素K2生物合成是规模化生产的关键。通过重组毕赤酵母，实现甲萘醌的聚异戊二烯侧链化，并以此为基础催化合成维生素K2。
课题组通过功能基因组学数据挖掘，在筛选的几十种放线菌来源的维生素K2合成关键基因基础上，以雪色链霉菌(S.niveus)异戊烯基转移酶基因novQ为目的基因，构建重组载体pPIC9-novQ，经过拼接组装和优化，将目的基因成功转化到底盘生物毕赤酵母，再经组氨酸营养缺陷、菌落PCR、dot-blot蛋白点杂交等定向和精确筛选，获得NovQ高产细胞株。研究发现，NovQ在酸性和弱碱性环境中催化活性较高，Mg2+、聚氧乙烯油醚POE-20能有效促进NovQ发挥作用，而Cu2+、SDS强烈抑制酶的活性。在对表达的酶蛋白结构和功能研究基础上，为了提升细胞生长能力和产物合成能力，建立了全细胞催化技术体系，维生素K2合成能力达到189mg/L。
该研究工作为进一步采用合成生物学理念，通过底盘到元件、器件或模块的改造和优化，系统构建细胞工厂合成维生素K2系列物提供了有益的尝试。
毕赤酵母NovQ蛋白表达载体构建
原文链接：
Biotransformation of menadione to its prenylated derivative MK-3 using recombinant Pichia pastoris
原文摘要：
Prenylated quinones, especially menaquinones, have significant physiological activities, but are arduous to synthesize efficiently. Due to the relaxed aromatic substrate specificity and prenylation regiospecificity at the ortho- site of the phenolic hydroxyl group, the aromatic prenyltransferase NovQ from Streptomyces may be useful in menaquinone synthesis from menadione. In this study, NovQ was overexpressed in Pichia pastoris. After fermentation optimization, NovQ production increased by 1617%. Then the different effects of metal ions, detergents and pH on the activity of purified NovQ were investigated to optimize the prenylation reaction. Finally, purified NovQ and cells containing NovQ were used for menadione prenylation in vitro and in vivo, respectively. Menaquinone-1 (MK-1) was detected as the only product in vitro with γ,γ-dimethylallyl pyrophosphate and menadione hydroquinol substrates. MK-3 at a concentration of 90.53 mg/L was detected as the major product of whole cell catalysis with 3-methyl-2-buten-1-ol and menadione hydroquinol substrates. This study realized whole cell catalysis converting menadione to menaquinones.
作者：郑之明
