基于异戊烯基焦磷酸异构酶的改造及表达优化提高异戊二烯产量

Modification and expression optimization of isopentenyl pyrophosphate isomerase to improve isoprene production

异戊二烯是最简单的萜烯类化合物,是橡胶、食品、医药和化妆品工业的重要原料。在微生物中构建甲羟戊酸(mevalonate,MVA)代谢途径是目前生产异戊二烯及其衍生萜烯的有效途径,但关键酶的活性和表达水平会限制异戊二烯的合成。针对关键酶-异戊烯基焦磷酸异构酶(isopentenyl diphosphate isomerase,IDI)的活性低等问题,该研究从酶工程、启动子工程和核糖体结合位点(ribosome binding site,RBS)工程3个方面对IDI的表达进行了优化。首先在大肠杆菌中建立了异戊二烯生产体系,挑选肺炎链球菌来源的IDI进行蛋白质建模和结构分析,计算得到了活性热点Met146并对其进行点突变。经过改造得到1株突变体Met146His,其将异戊二烯的产量提升至820.46 mg/L。接着针对该突变株进行启动子优化和RBS优化,最终得到的优化菌株将异戊二烯的产量提升至862.79 mg/L,是出发菌株的2.58倍。结果证明,对于IDI进行酶分子改造和表达优化以提高酶活性并调控表达水平,是提高异戊二烯产量的有效策略。IDI的优化也可以应用于异戊二烯的下游产物,如倍半萜和二萜等的生物合成。

Isoprene is the simplest terpene and an important raw material for the rubber,food,pharmaceutical,and cosmetic industries.The construction of the mevalonate metabolic pathway in microorganisms is currently an effective way to produce isoprene and its derived terpenes,however,it can be limited by the activity and expression levels of key enzymes.Aiming at the low activity of isopentenyl pyrophosphate isomerase(IDI)and other problems,in this study,the expression of IDI was optimized from three aspects:enzyme engineering,promoter engineering and ribosome binding site(RBS)engineering.Firstly,an isoprene production system was established in Escherichia coli.IDI from Streptococcus pneumoniae was then selected for protein modeling and structural analysis,and the functional hotspot Met146 was computationally obtained and subjected to site-directed mutagenesis.The modification resulted in a mutant strain Met146His,which enhanced isoprene production to 820.46 mg/L and was 2.45 times higher than of the wild type.Next,the promoter optimization and the RBS optimization were performed for this mutant,and a final optimized strain obtained enhanced isoprene production to 862.79 mg/L,which was 2.58 times higher than that of the starting strain.The results demonstrate it is an efficient strategy to improve isoprene production by performing enzyme molecular modification and expression optimization for IDI to improve enzyme activity and regulate expression levels.The modification of IDI can also be applied to the biosynthesis of downstream products such as sesquiterpenes and diterpenes.