Construction And Improvement Of Recombinant Escherichia Coli For The Production Of Riboflavin And β-carotene

In this study, we firstly used the strategies of modification of central metabolism, riboflavin biosynthesis pathway and optimization of the fermentation condition to study the metabolic capacity of E. coli for the riboflavin production. Secondly, we used a CRISPR/Cas9 assisted method to introduce the β-carotene synthetic pathway into E. coli genome and optimize the methylerythritol-phosphate(MEP) pathway and central metabolic pathways for the β-carotene overproduction.Firstly, the genes of the riboflavin synthetic pathway(ribA, ribB, ribD, ribE, ribC) from E. coli were assembled into an artificial operon, under the control of the inducible trc promoter(Ptrc), and inserted into plasmids with different copy numbers. Then, we further tested the productivity of the riboflavin pathway from B. subtilis. The results indicated that higher copy number of riboflavin synthetic genes from E.coli was beneficial for riboflavin overproduction.Ribulose 5-phosphate is an important precursor for riboflavin biosynthesis. The central metabolism, including oxidative pentose phosphate pathway, glycolytic pathways-Embden-Meyerhof pathway(EMP), Entner-Doudoroff Pathway(EDP), were further engineered to improve the availability of ribulose 5-phosphate. The resulting strain with the overexpression of mutant genes zwf(A243T) and gnd(S361F) from C. glutamicum showed only a slight increase in riboflavin titer, compared to the reference strains. We further overexpressed pgl(encoding 6-phosphogluconolactonase) from E. coli along with zwf and gnd in RF01 S, which led to significantly improve the riboflavin production. The strains RF02 S with the disruption of pgi in glycolytic pathway accumulated 394.8±19.6 mg/L riboflavin, which was 72.4% higher than that of the reference strain RF01 S. The strains RF03 S with the disruption of ED pathway produced 559.88± 8.99 mg/L riboflavin, which increased by 41.8% compared to that of RF02 S. Overexpressing the acs gene led to significant decrease in the acetate production of strains RF05 S and produced 585.2 ±13.6 mg/L riboflavin in LB medium with 10 g/L glucose, which did not change significantly compared to RF03 S.Riboflavin is the direct precursor of FMN and FAD. Therefore, we speculate that reducing the expression level ribF might reduce the conversion of riboflavin to FMN/FAD and increase riboflavin production. With modulating the expression of ribF, strain RF05S-M40 produced 1036.1±54.6 mg/L riboflavin, which was the highest among all selected strains, and did not significantly change of cell growth. After optimizing the fermentation conditions and the composition of medium, strain RF05S-M40 produced 2702.8 mg/L riboflavin with a yield of 137.5 mg riboflavin/g glucose. To our best of knowledge, this was the highest yield among all the reported riboflavin production strain.In addition, we used a CRISPR/Cas9 base method to introduce the β-carotene synthetic pathway into E. coli genome and optimize the methylerythritol-phosphate(MEP) pathway to improve the isopentenyl pyrophosphate(IPP) and dimethylallyl pyrophosphate(DMAPP) availability for the β-carotene overproduction, creating strains ZF43. Glyceraldehyde-3-phosphate and pyruvate are the direct precursor of MEP pathway, and NADPH is an important cofactor for the β-carotene production. To improve the availability of these precursors, the central metabolic modules(including pentose phosphate pathway, Embden-Meyerhof pathway(EMP), Entner-Doudoroff Pathway(EDP)), MEP pathway and β-carotene synthetic pathway were further engineered to increaseβ-carotene production. A genetically-defined β-carotene production strain ZF237 T was obtained by combined engineering of β-carotene de novo synthetic pathway, MEP pathway and central metabolic modules, and produced 2.03g/L β-carotene. For the first time, we used a CRISPR/Cas9 base method for iterative genome editing and metabolic engineering and have successfully obtained a high-yielding strain.