Enhanced vitamin A production by engineering transporters, ATP and precursor supply

Vitamin A (retinoids) is essential for human metabolism and has extensive applications in medicine, health, and cosmetics. Microbial cell factories have been developed for retinoid biosynthesis, coupled with two-phase fermentation for <i>in situ</i> extraction. Given that notable portions of retinoids remained in the cells, promotion of retinoid secretion is expected for further production improvements. This study investigates the potential of yeast endogenous PDR family proteins to enhance retinoid efflux by overexpressing them in retinoid-producing <i>Saccharomyces cerevisiae</i> strains. Among the PDR proteins tested, the transcriptional factor Pdr3p and the transporters Pdr10p and Snq2p significantly enhanced retinol and retinal secretion and production, while the transcriptional factor Pdr8p and the transporters Pdr11p, Pdr12p, Pdr18p, and Aus1p markedly increased retinoic acid production. <i>PDR3</i>/<i>PDR10</i> co-overexpression improved retinal production to a record 638.12 ​mg/L, while <i>PDR8</i> overexpression led to 106.75 ​mg/L retinoic acid production in shake flasks. For retinol, synergistic overexpression of <i>PDR3</i> and <i>PDR10</i> elevated the extracellular proportion to 96.7 ​%. Given the ATP requirement of PDR protein-mediated transportation, ATP supply was strengthened by overexpressing the mitochondrial fusion-related gene <i>MGM1</i> and introducing hemoglobin Vgb, both enhancing retinol secretion and production. Further precursor supply enhancement resulted in 727.30 ​mg/L retinol from 20 ​g/L glucose in shake flasks, with a carbon conversion rate of 7.62 ​%. These results confirm the combination of transport engineering, energy regulation and precursor supply enhancement as a pivotal strategy for augmenting vitamin A production.