Abstract
BackgroundThe 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.ResultsExpression 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.ConclusionThis work represents the first step towards a heterologous ABA cell factory for the commercially relevant sesquiterpenoid.
Highlights
The sesquiterpenoid abscisic acid (ABA) is mostly known for regulating developmental processes and abiotic stress responses in higher plants
Establishment of ABA production in three different S. cerevisiae strains To investigate if ABA can be produced in S. cerevisiae with the B. cinerea genes described in the literature far, we first integrated all heterologous genes in the genome that were reported to be involved in the pathway: bcaba1, bcaba2, bcaba3, bcaba4, bcaba5 and bcceP450 [19, 21]
In B. cinerea, bcaba1, bcaba2, bcaba3 and bcaba4 are located in the ABA gene cluster [19], while bcaba5 and bcceP450 are colocalized on a different chromosome [21]
Summary
The sesquiterpenoid abscisic acid (ABA) is mostly known for regulating developmental processes and abiotic stress responses in higher plants. We constructed and characterised an ABA-producing S. cerevisiae strain using the ABA biosynthetic pathway from Botrytis cinerea. Since its discovery in 1963 [1] the signalling of abscisic acid (ABA) in plants has been studied extensively. As one of the major plant hormones, ABA fulfils a pivotal role in regulating processes like seed dormancy, germination and abiotic stress resistance [2,3,4]. Even though the signalling properties of ABA seem to be conserved between the kingdoms of life, the biosynthetic pathway differs between organisms. B. cinerea utilises a pathway in which the sesquiterpenoid is produced directly from the C15 molecule farnesyl
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