Systematic metabolic engineering enables highly efficient production of vitamin A in Saccharomyces cerevisiae

Vitamin A is a micronutrient critical for versatile biological functions and has been widely used in the food, cosmetics, pharmaceutical, and nutraceutical industries. Synthetic biology and metabolic engineering enable microbes, especially the model organism <i>Saccharomyces cerevisiae</i> (generally recognised as safe) to possess great potential for the production of vitamin A. Herein, we first generated a vitamin A-producing strain by mining β-carotene 15,15'-mono(di)oxygenase from different sources and identified two isoenzymes <i>Mbblh</i> and <i>Ssbco</i> with comparable catalytic properties but different catalytic mechanisms. Combinational expression of isoenzymes increased the flux from β-carotene to vitamin A metabolism. To modulate the vitamin A components, retinol dehydrogenase 12 from <i>Homo sapiens</i> was introduced to achieve more than 90 % retinol purity using shake flask fermentation. Overexpressing <i>POS5Δ17</i> enhanced the reduced nicotinamide adenine dinucleotide phosphate pool, and the titer of vitamin A was elevated by almost 46 %. Multi-copy integration of the key rate-limiting step gene <i>Mbblh</i> further improved the synthesis of vitamin A. Consequently, the titer of vitamin A in the strain harbouring the Ura3 marker was increased to 588 mg/L at the shake-flask level. Eventually, the highest reported titer of 5.21 g/L vitamin A in <i>S. cerevisiae</i> was achieved in a 1-L bioreactor. This study unlocked the potential of <i>S. cerevisiae</i> for synthesising vitamin A in a sustainable and economical way, laying the foundation for the commercial-scale production of bio-based vitamin A.