Selective biosynthesis of retinol in S. cerevisiae

Abstract

The vitamin A component retinol has become an increasingly sought-after cosmetic ingredient. In previous efforts for microbial biosynthesis of vitamin A, a mixture of retinoids was produced. In order to efficiently produce retinol at high purity, the precursor and NADPH supply was first enhanced to improve retinoids accumulation in the S. cerevisiae strain constructed from a β-carotene producer by introducing β-carotene 15,15ʹ-dioxygenase, following by screening of heterologous and endogenous oxidoreductases for retinal reduction. Env9 was found as an endogenous retinal reductase and its activity was verified in vitro. By co-expressing Env9 with the E. coli ybbO, as much as 443.43 mg/L of retinol was produced at 98.76% purity in bi-phasic shake-flask culture when the antioxidant butylated hydroxytoluene was added to prevent retinoids degradation. The retinol titer reached 2479.34 mg/L in fed-batch fermentation. The success in selective biosynthesis of retinol would lay a solid foundation for its biotechnological production. Graphical Abstract

Production of retinoic acid by engineered Saccharomyces cerevisiae using an endogenous aldehyde dehydrogenase

“…Since deletion of ENV9 and DPP1 had no effect on RA production in the strain Y03 (Supporting Information: Figure 1), they were used as the integration sites. phase (Hu et al, 2022), which was in consistence with the findings of other studies (Jang et al, 2011(Jang et al, , 2015Sun et al, 2019). To our surprise, when we changed the composition of the mobile phase to 92.5:7.5 acetonitrile and acetic acid solution (2% v/v), three compounds were eluted from the culture of Y03, with a newly emerged peak occurring at the retention time of 13.55 min besides retinal and retinol (Figure 2b).…”

“…to have a weaker extraction capacity for retinol and retinal than dodecane (Hu et al, 2022). Compared with dodecane, both the extracellular production of RA (45.45,58.22,61.36,78.09,80.73 mg/L) and its proportions in total retinoids (31.17%, 34.80%, 37.73%, 47.54%, 50.69% vs. 18.85%, 25.28%, 27.18%, 32.68%, 36.11%) were improved for all five strains when olive oil was used as the extractant (Figure 5b).…”

“…In our previous study, we found both retinol and retinal could be extracted to the dodecane layer in bi-phasic cultures, leading to improvement in their production (Hu et al, 2022). Therefore, we also explored bi-phasic fermentation of RA.…”

“…Retinoids were extracted according to the previous protocol (Hu et al, 2022) Triple-TOF/MS was used for the identification of RA. In the negative ionization mode, the mass spectrometer was scanned from m/z 100 to 1500.…”

“…Heterologous retinal production has been achieved by introducing a β‐carotene dioxygenase into Escherichia coli (Jang et al, 2011) or Saccharomyces cerevisiae (Sun et al, 2019) harboring an exogenous β‐carotene synthesis pathway. Further studies identified ybbO and Env9 as endogenous enzymes with alcohol dehydrogenase activities in E. coli (Jang et al, 2015) and S. cerevisiae (Hu et al, 2022), respectively, overexpression of which enabled selective production of retinol in these microbial chasses. In contrast, microbial production of RA is rarely reported.…”