Engineering the Methylerythritol Phosphate Pathway and Using a Temporal Promoter for Enhanced Lycopene Production in <i>Rhodobacter sphaeroides</i> HY01

<i>Rhodobacter sphaeroides</i> HY01 is a high-yield strain for industrial production of coenzyme Q₁₀ (Q₁₀), indicating its potential for producing other terpenoids. However, the production of Q₁₀ substantially depletes isoprene precursors, nearly eliminating other terpenoids like spheroidene and spheroidenone commonly found in wild-type <i>R. sphaeroides</i>. Lycopene was used as an example to demonstrate its potential for terpenoid biosynthesis. By refactoring the methylerythritol phosphate (MEP) pathway, such as overexpressing <i>crtE</i> and introducing <i>crtI</i>₄, lycopene production reached 126.1 mg/L in HY01. However, further overexpression of the deoxy-d-xylulose-5-phosphate synthase, 1-deoxy-d-xylulose 5-phosphate reductoisomerase, and isopentenyl-diphosphate isomerase genes led to strain degradation, significantly reducing lycopene production. Fine-tuning the engineered PrrAB two-component system, which upregulated the MEP pathway, increased lycopene production to 154.9 mg/L. Inspired by this result, a series of native promoters with varying strengths were identified and characterized through transcriptomic analysis during the late fermentation stage. Using these temporal promoters to control genes in the MEP pathway ultimately increased lycopene production to 283.1 mg/L, the highest reported in <i>R. sphaeroides</i>. These results underscore the potential of HY01 as a chassis for terpenoid biosynthesis.