Selective production of retinol by engineered Saccharomyces cerevisiae through the expression of retinol dehydrogenase

Abstract

Retinol is a fat-soluble vitamin A that is widely used in the food and pharmaceutical industries. Currently, retinol is commercially produced by chemical synthesis. Microbial production of retinol has been alternatively explored but restricted to a mixture of retinoids including retinol, retinal, and retinoic acid. Thus, we introduced heterologous retinol dehydrogenase into retinoids mixture-producing Saccharomyces cerevisiae for the selective production of retinol using xylose. Expression of human RDH10 and Escherichia coli ybbO led to increase in retinol production, but retinal remained as a major product. In contrast, S. cerevisiae harboring human RDH12 produced retinol selectively with negligible production of retinal. The resulting strain (SR8A-RDH12) produced retinol only. However, more glycerol was accumulated due to intracellular redox imbalance. Therefore, Lactococcus lactis noxE coding for H 2 Oforming NADH oxidase was additionally introduced to resolve the redox imbalance.The resulting strain produced 52% less glycerol and more retinol with a 30% higher yield than a parental strain. As the produced retinol was not stable, we examined culture and storage conditions including temperature, light, and antioxidants for the optimal production of retinol. In conclusion, we achieved selective production of retinol efficiently from xylose by introducing human RDH12 and NADH oxidase into S. cerevisiae.

Cas9-Based Metabolic Engineering of Issatchenkia orientalis for Enhanced Utilization of Cellulosic Hydrolysates

“…All transformation methods used in this study were based on the previous lithium acetate/single-stranded carrier DNA/poly­(ethylene glycol) method using a CRISPR/Cas9 system with some modifications. , For obtaining the ADE2-disrupted strains, a repair DNA was designed by combining the 150 bp upstream and 150 bp downstream sequences of ADE2 . The 300 bp DNA fragment was synthesized using the gBlock service of IDT (Integrated DNA Technologies).…”

“…Using this fragment as a template, a repair DNA was generated by PCR amplification with the primers of F_check_ADE2 and R_check_ADE2 (Table S1). For transformation, 0.7–1 μg of pCast_ADE2 and 1–1.5 μg of the repair DNA fragment were cotransformed into the SD108, IO21, IO45, and IO46 strains using a lithium acetate transformation method with some modifications. , Heat-shock treatment was maintained at 42 °C for 90 min, which is much longer than the method described in a previous study . The heat-shocked cells were cultured in 2× YP medium for 4 h for recovery before planting the cells on YPD agar plates with 120 μg/mL of nourseothricin (cloNAT).…”

75 Years of Vitamin A Production: A Historical and Scientific Overview of the Development of New Methodologies in Chemistry, Formulation, and Biotechnology

“…Briefly, the second phase acts as a sink to remove the product from the biocatalyst and drive the reaction based on Le Chatelier‘s principle. Mineral oils, silicone oils, olive oil, and dodecane have demonstrated utility in the sequestration of retinol in fermentations. , The authors indicate that the use of this second phase can lead to the stability of the product. Others have found that the addition of antioxidative adjuncts like BHT and carnosic acid led to the stabilization of retinoids. , For downstream processing, the second phase can be further purified from the biomass and fermentation broth by decantation, and subsequently, the product can then be separated from this aliphatic phase by standard methods like distillation or crystallization, which is an active research topic.…”

Multi-level engineering of Saccharomyces cerevisiae for the synthesis and accumulation of retinal

“…The microbial synthesis of retinoids has been established in Escherichia coli , Saccharomyces cerevisiae and Yarrowia lipolytica by introducing the heterologous β-carotene biosynthetic pathway and β-carotene 15,15′-monooxygenase (BCMO, encoded by the BLH gene) from the marine bacterium 66A03 or the halophilic bacterium Salinibacter ruber. 7,11–14 The heterologous biosynthesis of retinoids was first reported in β-carotene-producing E. coli by introducing the BLH gene from the archaeon Halobacterium sp. NRC-1, and the resulting strain produced 136 mg L −1 retinoids with a mixture of retinol, retinal and retinyl acetate.…”