Identification and elimination of metabolic bottlenecks in the quinone modification pathway for enhanced coenzyme Q10 production in Rhodobacter sphaeroides

• UbiE and UbiH were identified as bottleneck enzymes in the CoQ10 biosynthesis.
• The quinone modification pathway (QMP) was finely tuned with different strategies.
• Accumulation of intermediate revealed the blocking in the pathway.
• Biomass and the productivity were the important parameters in metabolic engineering.
• 200% improvement in CoQ10 titer after co-engineering the MEP and QMP.

In this report, UbiE and UbiH in the quinone modification pathway (QMP) were identified in addition to UbiG as bottleneck enzymes in the CoQ10 biosynthesis by Rhodobacter sphaeroides. The CoQ10 content was enhanced after co-overexpression of UbiE and UbiG, however, accompanied by the accumulation of the intermediate 10P-MMBQ. UbiH was then co-overexpressed to pull the metabolic flux towards downstream, resulting in an elevated CoQ10 productivity and decreased biomass. On the other hand, the expression levels of UbiE and UbiG were tuned to eliminate the intermediate accumulation, however at the sacrifice of productivity. To alleviate the detrimental effect on either productivity or cell growth, we tried to fuse UbiG with UbiE and localize them onto the membrane to elevate intermediate conversion. By fusing UbiE and UbiG to pufX, CoQ10 was accumulated to 108.51±2.76 mg/L with a biomass of 12.2±0.9 g/L. At last, we combined the optimized QMP and the previously engineered 2-methyl-d-erythritol-4-phosphate pathway (MEP) to further boost CoQ10 biosynthesis, resulting in a strain with 138±2.64 mg/L CoQ10 production.