Research group of Zhang Haibo, Qingdao Institute of energy, Chinese Academy of Sciences improves the efficiency of synthesis of mevalonate from terpenes by point line combination

Terpenes, including isoprene and pinene, have great application value in materials, energy and medicine Using renewable sugar as raw material, using green and sustainable microbial metabolism engineering to synthesize terpenoids is the current research focus in the field of biochemistry Among them, the microbial available exogenous mevalonate (MVA) pathway has high efficiency and good controllability, which is the current research hotspot The synthesis route of MVA pathway from precursor acetyl coenzyme A to dimethylallylpyrophosphate (dmapp) involves seven reactions and seven enzymes How to break through the point limiting the efficiency of the pathway and balance and coordinate the synthesis route of these seven enzymes, so as to improve the metabolic efficiency, balance the metabolic flow, and prevent the toxicity of intermediate products to host cells is the breakthrough point and difficulty of the pathway Zhang Haibo, research group of Qingdao Institute of bioenergy and process, Chinese Academy of Sciences, has been committed to the biosynthesis of terpenes for a long time, focusing on the enzyme mevalonate kinase (MK) and isopentene pyrophosphate isomerase (IDI) of MVA pathway ）And terpene synthetase and other restrictive points, at the same time, coordinate metabolism flow and toxicity of intermediate products, balance the whole synthesis route, and make a new breakthrough in the combination of point and line to improve the efficiency of MVA pathway of terpene synthesis Firstly, aiming at the MK and IDI in MVA pathway, through different species source selection, random mutation and semi rational design, directional evolution and optimization are carried out to obtain MK and IDI with increased enzyme activity, so as to reduce the blocking of metabolic flow at the MK and IDI sites, and ultimately improve the yield of terpenes Secondly, coordinate MVA The pathway involves multi-step reactions, and the regulation of enzyme expression involves multiple levels In this study, we selected the translation level regulation method to avoid the instability of RNA and the burden of bacterial metabolism after translation level regulation By adjusting the ribosome binding site (RBS) sequence of different enzymes, we regulated the expression of different enzymes and balanced the whole metabolic route The RBS sequence of mvae and erg19 was regulated to reduce T.I.R and protein expression For the restriction enzymes MK and IDI, the RBS sequence of them was regulated to improve their T.I.R and protein expression Through the regulation of protein expression of different enzymes in mevalonate pathway, the metabolic balance was achieved, and the yield of terpenes was finally increased.