Integration of a multi-step heterologous pathway in Saccharomyces cerevisiae for the production of abscisic acid

Abstract

Background: The sesquiterpenoid abscisic acid (ABA) is mostly known for regulating developmental processes and abiotic stress responses in higher plants. Recent studies show that ABA also exhibits a variety of pharmacological activities. Affordable and sustainable production will be required to utilize the compound in agriculture and as a potential pharmaceutical. Saccharomyces cerevisiae is an established workhorse for the biotechnological production of chemicals. In this study, we constructed and characterised an ABA-producing S. cerevisiae strain using the ABA biosynthetic pathway from Botrytis cinerea. Results: Expression of the B. cinerea genes bcaba1, bcaba2, bcaba3 and bcaba4 was sufficient to establish ABA production in the heterologous host. We characterised the ABA-producing strain further by monitoring ABA production over time and, since the pathway contains two cytochrome P450 enzymes, by investigating the effects of overexpressing the native S. cerevisiae or the B. cinerea cytochrome P450 reductase. Both, overexpression of the native or heterologous cytochrome P450 reductase, led to increased ABA titres. We were able to show that ABA production was not affected by precursor or NADPH supply, which suggested that the heterologous enzymes were limiting the flux towards the product. The B. cinerea cytochrome P450 monooxygenases BcABA1 and BcABA2 were identified as pathway bottlenecks and balancing the expression levels of the pathway enzymes resulted in 4.1-fold increased ABA titres while reducing by-product formation. Conclusion: This work represents the first step towards a heterologous ABA cell factory for the commercially relevant sesquiterpenoid.

Harnessing the yeast Saccharomyces cerevisiae for the production of fungal secondary metabolites

“…The secretion of metabolites by cell factories is preferred as secretion can relieve the cellular feedback inhibition and simplify the downstream product recovery [97]. While some of the FSMs (such as ABA [42], psilocybin [46]) are mostly secreted to the extracellular medium in S. cerevisiae, others (such as ergothionine [47], betaxanthins [77]) are partly retained in the cell. The substrate specificity of S. cerevisiae transporter proteins to xenobiotic compounds is unclear, which complicates rational transporter engineering to facilitate the export.…”

“…cytochrome P450 monooxygenase (bcaba1 bcab2), sesquiterpene cyclase (bcaba3), short-chain dehydrogenase/reductase (bcaba4), as well as the cytochrome P450 reductase (bccpr1) from B. cinerea, into S. cerevisiae. They further deleted lipid phosphate phosphatase (LPP1) and diacylglycerol pyrophosphate (DGPP) phosphatase (DPP1) genes, swapped ERG9 promoter to glucose-dependent HXT1 promoter, and overexpressed ERG20 and tHMG1 genes, leading to a strain that produced 11 mg/l ABA [42].…”

Heterologous Metabolic Pathways: Strategies for Optimal Expression in Eukaryotic Hosts

“…Initially, biosynthetic pathway metabolite production rises with increasing gene dosage; however, overexpression of heterologous proteins leads to a significant drop in the metabolic pathway output, since intracellular accumulation of metabolites can trigger cellular stress responses, and the metabolic efflux to the heterologous pathway cannot be balanced by the host cells [ 25 ]. Thus, addition of extra gene copies is useful only in case of genes encoding the rate-limiting enzymes, while changes in the copy number of other pathway genes have little or no effect on the final product titers [ 82 ]. The gene dosage approach has proved to be effective in heterologous β-carotenoid biosynthesis in Yarrowia lipolitica [ 83 ].…”

“…Another key requirement for a sustainable and effective heterologous pathway expression is precursor availability. The deficit of ATP [ 91 ], CoA derivatives [ 92 ], NADH [ 93 ], NADPH [ 82 ], FMN [ 94 ], which are involved in the vast majority of biosynthetic pathways, was shown to be the limiting factor for heterologous metabolite production. In order to remove this bottleneck, the precursors and cofactors may be added exogenously or biosynthesized by the host.…”

Transcription Factor Engineering Harnesses Metabolic Networks to meeting industrial requirements in Yeast

“…Yeast is a valuable microbial cell factory in biosynthesis of industrial products, such as biofuels, organic acids terpenoids, abscisic acid and flavonoids [1][2][3][4][5]. However, cell growth rate and product yield are limited in industrial fermentation processes, due to the issues such as heat shock, product toxicities and oxidative stress [6].…”