Systematic Engineering to Enhance 8-Hydroxygeraniol Production in Yeast

Abstract

8-Hydroxygeraniol, an important component of insect sex pheromones and defensive secretions, can be used as a potential biological insect repellent in agriculture. Microbial production provides sustainable and green means to efficiently gain 8hydroxygeraniol. The conversion of geraniol to 8-hydroxygeraniol by P450 geraniol-8-hydroxylase (G8H) was regarded as the bottleneck for 8-hydroxygeraniol production. Herein, an integrated strategy consisting of the fitness between G8H and cytochrome P450 reductase (CPR), endoplasmic reticulum (ER) engineering, and reduced nicotinamide adenine dinucleotide phosphate (NADPH) supply is implemented to enhance the production of 8-hydroxygeraniol in Saccharomyces cerevisiae. The titer of 8hydroxygeraniol was gradually increased by 2.1-fold (up to 158.1 mg/L). Moreover, dehydrogenase ADH6 and reductase ARI1 responsible for the reduction of 8-hydroxygeraniol toward shunt products were also deleted, elevating 8-hydroxygeraniol production to 238.9 mg/L at the shake flask level. Consequently, more than 1.0 g/L 8-hydroxygeraniol in S. cerevisiae was achieved in 5.0 L fedbatch fermentation by a carbon restriction strategy, which was the highest-reported titer in microbes so far. Our work not only provides a sustainable way for de novo biosynthesis of 8-hydroxygeraniol but also sets a good reference in P450 engineering in microbes.

Systems Metabolic Engineering for Efficient Violaxanthin Production in Yeast

“…In yeast, the NADH kinase (POS5) can convert NADH to NADPH, while glucose-6-phosphate dehydrogenase (ZWF1) responsible for the reaction from d -glucopyranose 6-phosphate to 6-phospho d -glucono-1,5-lactone and 6-phosphogluconate dehydrogenase (GND1) responsible for the reaction from d -gluconate 6-phosphate to D-ribulose 5-phosphate in the pentose phosphate pathway provide the major source of NADPH required for reductive biosynthetic reactions (Figure A). , POS5 and ZWF1 overexpression have been widely used to induce terpenoid production in S. cerevisiae. , Specifically, POS5Δ17 (lacking the coding sequence for the first 17 amino acid residues) can efficiently regenerate NADPH from NADH during the conversion of ATP into ADP . Thus, POS5Δ17 , ZWF1 , and GND1 were chosen for NADPH regeneration by an extra copy of yeast endogenous enzymes.…”

“…33,34 POS5 and ZWF1 overexpression have been widely used to induce terpenoid production in S. cerevisiae. 17,18 Specifically, POS5Δ17 (lacking the coding sequence for the first 17 amino acid residues) can efficiently regenerate NADPH from NADH during the conversion of ATP into ADP. 18 Thus, POS5Δ17, ZWF1, and GND1 were chosen for NADPH regeneration by an extra copy of yeast endogenous enzymes.…”

“…Moreover, the cofactor pairs NADP + /NADPH, FMN/FMNH 2 , and FAD/FADH 2 , as important components for electron transfer, are probably essential for enhancing violaxanthin biosynthesis efficiency. Wang et al successfully improved the production of 8-hydroxygeraniol by 2.1-fold by overexpressing ZWF1 and POS5 to increase NADPH supply .…”

Balancing Pyruvate Node Based on a Dual-Layered Dynamic Regulation System to Improve the Biosynthesis of Caffeic Acid in Candida glycerinogenes

“…9 Synthetic biology is a green and sustainable approach that can be used to achieve sustainable production of high-value-added compounds using simple carbon sources. 10,11 Microbial cell factory based on metabolic engineering strategies is a green and efficient way to produce high-valueadded products. 12,13 It can effectively modify cellular metabolism to maximize the yield of the target product.…”

“…However, the efficient extraction of CA is seriously hampered by the low CA content in plants, the slow growth rate of the plants concerned, and the complexity of the isolation process. , Furthermore, traditional chemical synthesis is energy intensive and unsustainable . Synthetic biology is a green and sustainable approach that can be used to achieve sustainable production of high-value-added compounds using simple carbon sources. , …”