Multiplex metabolic engineering of yeast for high-efficiency all-trans-retinoic acid production

Abstract

Background: All-trans-retinoic acid (ATRA) is a pivotal signaling molecule with multifaceted roles in cellular regulation and has broad valuable applications in pharmacology and dermatology. The demand for ATRA is growing, but there is a significant gap in research on its effective biosynthesis. Results: In this study, a β-carotene-producing chassis strain was firstly constructed by mining optimal Crt gene combinations, changing platform strains and adjusting the Crt gene copy numbers. Then, the optimal gene combination of the ATRA module was screened, and the subcellular locations of the enzymes were identified. To boost the expression of ER-localized proteins in the ATRA pathway, INO2 was employed to expand the size of the endoplasmic reticulum. Furthermore, sthA was introduced to improve the cellular NADPH and NAD+ levels to meet the high demand for redox cofactors. The supply of oxygen was increased by inserting the Vitreoscilla hemoglobin gene (VHb). In addition, acetyl-CoA was enhanced by overexpressing IME4 and the ATRA biosynthesis module was further strengthened. Finally, the autotrophic markers were complemented and the ATRA titer reached 1.84 g/L in a 5-L bioreactor. Conclusion: This synthetic biology platform can serve as a basis for creating an industrial microbial source of ATRA and other vitamin A derivates.