Metabolic Engineering Of Escherichia Coli For Efficient Production Of Menaquinone-7

Menaquinone-7(MK-7),a highly valuable member of the vitamin K2 series,is an essential nutrient for humans.It has important medical functions and broad market prospects.However,the low natural content of MK-7 and the high cost of industrial production have led to the current MK-7 being expensive and limited in application.In order to reduce the production cost of MK-7 and achieve green,sustainable and efficient production of MK-7,this study intends to fundamentally solve the problem of low production efficiency of MK-7.To overcome the problems of the long cycle and low production efficiency of the current production of MK-7 by Bacillus subtilis,Escherichia coli was used as a cell factory to construct efficient synthetic MK-7 industrial strains.The reason why there are few studies on the synthesis of MK-7 in E.coli is mainly based on the following challenges in the synthesis of MK-7:(1)Under aerobic conditions,E.coli mainly synthesizes ubiquinone Q-8,only a small amount of menadione MK-8 is synthesized,and MK-7 cannot be detected,so it is needed to find a heterogenous heptaprenyl pyrophosphate(Hep PP)synthetase(Hep PPS)that can efficiently synthesize Hep PP in E.coli;(2)It is needed to overcome the competition of ubiquinone synthesis in E.coli under aerobic conditions by metabolic engineering;(3)Due to the long and complicated synthetic pathway of MK-7,complex pathway optimization and module balance are needed to overcome the accumulation of intermediate products.In this study,firstly,the bottleneck of MK-7 synthesis in E.coli was explored,and MK-7 pathway was initially constructed in E.coli.Secondly,based on the experience of preliminary exploration,the MK-7 synthesis pathway was further modularized and optimized through metabolic engineering.So the balance of intra-module pathways and the balance among modules was achieved and the engineered E.coli gained high production of MK-7 from glucose;Thirdly,to reduce the cost of raw materials and achieve waste utilization,E.coli was metabolized to synthesize MK-7 from fatty acids.At the same time,to increase the accumulation of MK-7 in E.coli and promote the synthesis of MK-7,membrane metabolism was engineered for MK-7 production,and the accumulation of MK-7 was further improved in E.coli.To realize the industrial application of high-yield MK-7engineering E.coli,the high-yield MK-7 strain was fermented,and the MK-7 production reached 2074 μM(1.35 g/L)after 52 h fermentation,and the maximum titer of MK-7reached 2086 μM after 62 h of fermentation,which is the highest titer of MK-7 ever reported.This study has successfully obtained an engineered E.coli with high production of MK-7 through multi-level metabolic engineering,which is expected to solve the bottleneck of MK-7 industry.1.According to the metabolic characteristics of E.coli,the synthesis route and potential competition pathway of MK-7 in E.coli were preliminarily designed.The heterogenous MVA pathway was first introduced to supply IPP for MK-7 synthesis,and the introduction of B.subtilis-derived Hep PPS(Bs Hep PPS)is used for the synthesis of Hep PP in E.coli.Further optimization of key enzymes in the MVA pathway and Bs Hep PPS resulted in a 22-fold increase in MK-7 production.The analysis of by-products and the knock-out of competitive pathways showed that ubiquinone Q-7,Q-8and intermediate DMK-7 of MK-7 were the main by-products in E.coli.Further blocking the competition bypass revealed that the knockout of Isp B in the competition pathway could not reduce the flux from IPP toward Oct PP.Then,the knockout of Ubi CA could completely block the synthesis competition of ubiquinone,but seriously affected the growth of E.coli,thus leading to the decrease of MK-7 production.The above results indicated that the increase in the production of MK-7 cannot be achieved by knocking out the competitive pathway,but the expression of the corresponding enzymes Men A and Ubi E of the competitive target needs to be strengthened to increase the synthetic fluxes of MK-7.2.The de novo biosynthesis pathway of MK-7 was rewrote and divided into three modules.Module 1 is the MVA pathway,which is responsible for the supply of precursors IPP and FPP.Module 2 is the 1,4-dihydroxy-2-naphthoate(DHNA)pathway,responsible for supplying the precursor DHNA.Module 3 is the MK-7 pathway,which is the main pathway for MK-7 synthesis and the core module.Based on the results of the first part,Men A and Ubi E was overexpressed step by step and the MK-7 pathway was completely constructed.Then,combinatorial screening of Men A and Ubi E and expression optimization of the three key enzymes in the module were performed.As a result,the best source combination and expression order was achieved: Bacillus subtilis-derived Hep PPS-E.coli-derived Men A-Bacillus subtilis-derived Ubi E.In addition,the MVA module was further optimized and the screening and overexpression of Idi increased MK-7.Finally,the DHNA synthesis module was optimized.Through expression optimization and module balance,the main synthesis of quinone was successfully changed from Q-8 to MK-7 under aerobic conditions,with the accumulation of the main by-products of Q-7,Q-8 and DMK-7 reduced,and the anabolic flux of MK-7increased.The high-efficiency full biosynthesis of MK-7 from glucose was achieved and yielded a high-titer strain MK17,which produced 157 μM MK-7 after 10 h whole cell conversion.3.In order to promote the use of waste oil and increase the theoretical yield of MK-7,an attempt was made to synthesize MK-7 using fatty acid.For the first time,the engineered E.coli can synthesize MK-7 from fatty acid.Considering MK-7 is fat-soluble,which is mainly stored on the cell membrane.In order to further increase the accumulation of MK-7,membrane engineering was processed to increase the storage space of MK-7 and the titer of MK-7 was increased from 157 μM to 200 μM through membrane engineering.4.Through further screening of fermentation strains and optimization of fermentation conditions in the 5 L fermentor,the importance of the balance of module expression intensity on the MK-7 fermentation yield was determined,and the efficient synthesis of MK-7 from glucose by engineered E.coli was achieved.After 52 h fermentation,the biomass reached OD600 =122,and the titer of 2074 μM(1.35 g/L)MK-7 was achieved with a productivity of 26 mg/L/h.The maximum titer of MK-7reached 2086 μM after 62 h of fermentation.In summary,this study was the first to synthesize MK-7 using E.coli with the heterologous MVA pathway.The synthetic pathway of MK-7 in E.coli was innovatively de novo designed,modularized and balanced.The MVA pathway was first tried to supply IPP for MK-7 synthesis.The balance of IPP module,DHNA module and MK-7 module was achieved,overcoming the bottleneck of MK-7 synthesis.Further through 5 L fermentation,the optimal engineered E.coli produced high titer of MK-7 with high efficiency from glucose under aerobic conditions,which is the highest titer of MK-7 ever reported.The above results further confirm that E.coli,as a cell factory for many chemical products,is actually a very malleable chassis.This study has important reference significance for the biosynthesis of other chemical products,and is expected to solve the current bottleneck of MK-7 industrial production.It provides an important experimental and theoretical basis for the synthesis of similar products by metabolic engineering in E.coli.