Metabolic Engineering Of Escherichia Coli For Production Enhancement Of Biosynthesized Lycopene Based On Heterogenous MVA Pathway

Lycopene,a kind of dark red carotenoid,belongs to C40 terpenoids.As an essential nutrient substance for the human body,lycopene has become one of best-selling health products because of possessing many beneficial functions,such as anti-oxidation,immunity enhancement,and prevention of ocular and cardiovascular diseases.The engineered Escherichia coli is one of the excellent lycopene producers due to the high yield of lycopene and easy cultivation.Engineering heterogenous MVA pathway is a popular strategy for lycopene overproduction to avoid from the native regulatory mechanisms and physiological controls.However,till now it has been reported that the plasmid systems were utilized to produce lycopene with synergy of multiple operons and genes,which may cause instability and a long period of lag stage of strains.To overcome these shortcomings,engineering integrated expression pathways was an alternative strategy.In this study,three models of expression pathway were constructed:(1)Two plasmids system with uncontrolled copy number of expression pathway,(2)Copy controlled model of MVA pathway,and(3)Copy controlled model of expression pathway.A high level lycopene-producing Escherichia coli carrying integrated heterogenous MVA pathway was engineered based on the third model plus chromosomal multiple position integration strategy,which was relatively stable.(1)Two plasmids system carries MVA lower pathway and lycopene expression pathway respectively for lycopene production.The E.coli DHGT7E carrying integrated MVA upper pathway can produce high level of mevalonate.After transformed with two plasmids carrying artificial synthetic MVA lower pathway and lycopene expression pathway respectively,the strain achieved the ability to produce lycopene.In addition,the two plasmids system was optimized that eight sub models were generated by using combination of p15A ori,pBR322 ori,T7 promoter,and T7lac promoter.However,these sub models didn't enhance the lycopene yield(3 mg/g DCW or less).(2)Enhancement of lycopene yield in an integrated strain plus plasmid system.The MVA upper pathway,MVA lower pathway,lycopene expression pathway,and T7 RNA polymerase gene were integrated into the 57th nonessential region,64th nonessential region,23th nonessential region,and araB site in genome of E.coli DH6(a reduced-genome E.coli DH1),generating DH411.However,the lycopene yield of strain DH411 was only 0.66 mg/g DCW in test tube experiments.To enhance the lycopene yield,based on strain DH411,the MVA lower pathway and lycopene expression pathway were overexpressed by using plasmid system.After optimizing the plasmid system,the resulting strain D111,which has two copies of MVA lower pathway,produced 27.43 mg/g DCW of lycopene.Thus the copy mumber of MVA pathway was controlled.(3)Developing the lycopene producer strains based on chromosomal multiple position integration strategy of MVA upper pathway.At first,strain DH6T7 was chosen as the host for investigation of the integrated site of MVA upper pathway impact on lycopene production.Plasmid pCANLEfG was employed for MVA lower pathway and lycopene expression pathway overexpression.The 64th region,58th region,44th region,and lpxM site were chosen for MVA upper pathway integration as they were distributed throughout the chromosome.Integration of the 58th region has the best performance for lycopene production,followed by lpxM site.Then,based on strain D111 the MVA upper pathway was further overexpressed.The resulting strain D311 and D511 had one copy MVA upper pathway more than D111 into 58th region and lpxM site respectively.The lycopene yield of D311 was 29.6 mg/g DCW,however,D511 had the best growth ability,nearly 2.2-fold higher than D311,and 2.0-fold higher than D111.After the combinatorial effect of the stated advantages,the resulting strain D711 had three copies of MVA upper pathway with a better lycopene yield of 34.52 mg/g DCW.(4)Constructing the integrated metabolic pathway by using copy controlled model of expression pathway plus chromosomal multiple position integration strategy.The best combination was one copy of MVA upper pathway,two copies of MVA lower pathway,and three copies of lycopene expression pathway by utilization of the copy controlled model of expression pathway.In addition,chromosomal multiple position integration of lycopene expression pathway was used for optimizing lycopene production.The 8th region,44th region,58trh region,and lpxM site were chosen for lycopene expression pathway integration.Interestingly,the integration of the 58th region still has the best performance for lycopene yield,and integration of the lpxM site has the best growth ability.Based on these two strategies,the final integrated strain DH416 had a better lycopene yield and lycopene level of 35.34 mg/g DCW and 92.66 mg/L respectively.After optimizing the addition time of L-arabinose,the lycopene level of DH416 was up to 393.24 mg/L.A lycopene titer of 1.22 g/L and a mean productivity of 61.0 mg/L/h were achieved in a fermentation process.To the best of our knowledge,this is the highest mean productivity for lycopene production.