Enhanced production of coenzyme Q₁₀ by self‐regulating the engineered MEP pathway in Rhodobacter sphaeroides

Abstract

Fine-tuning the expression level of an engineered pathway is crucial for the metabolic engineering of a host toward a desired phenotype. However, most engineered hosts suffer from nonfunctional protein expression, metabolic imbalance, cellular burden or toxicity from intermediates when an engineered pathway is first introduced, which can decrease production of the desired product. To circumvent these obstacles, we developed a self-regulation system utilizing the trc/tac promoter, LacI(q) protein and ribosomal binding sites (RBS). With the purpose of improving coenzyme Q10 (CoQ10 ) production by increasing the decaprenyl diphosphate supplement, enzymes DXS, DXR, IDI, and IspD were constitutively overexpressed under the control of the trc promoter in Rhodobacter sphaeroides. Then, a self-regulation system combining a set of RBSs for adjusting the expression of the LacI(q) protein was applied to tune the expression of the four genes, resulting in improved CoQ10 production. Finally, another copy of the tac promoter with the UbiG gene (involved in the ubiquinone pathway of CoQ10 biosynthesis) was introduced into the engineered pathway. By optimizing the expression level of both the upstream and downstream pathway, CoQ10 production in the mutants was improved up to 93.34 mg/L (7.16 mg/g DCW), about twofold of the wild-type (48.25 mg/L, 3.24 mg/g DCW).

Quantitative characterization of gene regulation by Rho dependent transcription termination

“…They also noted that increasing the translation initiation rate caused a smaller fold change in the mRNA concentration compared to the protein concentration, which is also consistent with our observations for RTT. Other regulatory mechanisms that have been quantified using fluorescent proteins [35,36] have been shown to be applicable to at least some non-fluorescent reporter genes [77][78][79][80]. There is no apparent reason why this should not also be the case with our observed relationship between the protein and full length mRNA concentrations due to RTT.…”

“…Secondly, the model could be combined with tools such as the "RBS calculator" [64] in future investigations to determine if it is possible to accurately predict how changes in the TIR affect full length mRNA and protein concentrations in genes with RTT. Thirdly, it can be used to optimize the translation initiation rates and order of genes in synthetic and native operons so that the proteins generated from the operon occur in specific ratios that improve the yield and efficiencies of metabolic pathways and multi-protein complexes and decrease the concentrations of toxic intermediates [77][78][79][80].…”

Cellular factories for coenzyme Q10 production

“…Promoter-based balancing of metabolic flux increased the production to 7.16–8.7 mg/g DCW [60, 98], and a recent study reported production as high as 12.96 mg/g DCW [87] (Table 1). However, other efforts to increase MEP pathway flux did not translate well into increased CoQ 10 production, probably due to an accumulation of toxic intermediates [99]. R. sphaeroides , however, is reported to have limited growth rates, even when grown in optimal fermentation conditions [84].…”

Synthetic Biology for Specialty Chemicals

“…A self-regulation system was developed using the trc/tac promoter in R. sphaeroides. The expression of four genes was fine-tuned through the use of ribosomal binding sites to improve coenzyme Q 10 production to 93.34 mg/L, or twofold over that of wild type (4). Opiates are commonly used to treat human disease in the form of morphine, codeine, oxycodone, and hydrocodone, to name just a few.…”