Pathway engineering in yeast for synthesizing the complex polyketide bikaverin

Abstract

Fungal polyketides display remarkable structural diversity and bioactivity, and therefore the biosynthesis and engineering of this large class of molecules is therapeutically significant. Here, we successfully recode, construct and characterize the biosynthetic pathway of bikaverin, a tetracyclic polyketide with antibiotic, antifungal and anticancer properties, in S. cerevisiae. We use a green fluorescent protein (GFP) mapping strategy to identify the low expression of Bik1 (polyketide synthase) as a major bottleneck step in the pathway, and a promoter exchange strategy is used to increase expression of Bik1 and bikaverin titer. Then, we use an enzyme-fusion strategy to directly couple the monooxygenase (Bik2) and methyltransferase (Bik3) to efficiently channel intermediates between modifying enzymes, leading to an improved titer of bikaverin at 202.75 mg/L with flask fermentation (273-fold higher than the initial titer). This study demonstrates that the biosynthesis of complex fungal polyketides can be established and efficiently engineered in S. cerevisiae, highlighting the potential for natural product synthesis and large-scale fermentation in yeast.

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

“…By expressing the aurZ and aurJ genes in addition to PKS12 in a S. cerevisiae strain carrying PPTase from Aspergillus fumigatus, Rugbjerg et al achieved the recombinant production of 1.1 mg/l rubrofusarin [34]. More recently, Zhao et al demonstrated how S. cerevisiae could be employed both for the whole pathway validation and engineered for an improved recombinant production of bikaverin, a red-colored tetracyclic polyketide with antibacterial and anticancer activities [35]. The authors first introduced four elements from a putative gene cluster for bikaverin synthesis, which included Type I PKS Bik1, FAD-dependent monooxygenase Bik2, O-methyltransferase Bik3, permease Bik6, and PPTase from Fusarium fujikuroi, as well as npgA from A. nidulans, into the S. cerevisiae [35].…”

“…More recently, Zhao et al demonstrated how S. cerevisiae could be employed both for the whole pathway validation and engineered for an improved recombinant production of bikaverin, a red-colored tetracyclic polyketide with antibacterial and anticancer activities [35]. The authors first introduced four elements from a putative gene cluster for bikaverin synthesis, which included Type I PKS Bik1, FAD-dependent monooxygenase Bik2, O-methyltransferase Bik3, permease Bik6, and PPTase from Fusarium fujikuroi, as well as npgA from A. nidulans, into the S. cerevisiae [35]. With the resulting strain, the authors tested the essentiality of each element and validated the order of the biocatalytic reactions (see the detailed reaction route in Figure 4 in [35]).…”

“…The authors first introduced four elements from a putative gene cluster for bikaverin synthesis, which included Type I PKS Bik1, FAD-dependent monooxygenase Bik2, O-methyltransferase Bik3, permease Bik6, and PPTase from Fusarium fujikuroi, as well as npgA from A. nidulans, into the S. cerevisiae [35]. With the resulting strain, the authors tested the essentiality of each element and validated the order of the biocatalytic reactions (see the detailed reaction route in Figure 4 in [35]). Furthermore, by swapping the promoter for Bik genes to the GAL1 promoter and expressing a fused gene of Bik2 and Bik3, the authors improved the bikaverin production by 273-fold to 0.2 g/l [35].…”

“…With the resulting strain, the authors tested the essentiality of each element and validated the order of the biocatalytic reactions (see the detailed reaction route in Figure 4 in [35]). Furthermore, by swapping the promoter for Bik genes to the GAL1 promoter and expressing a fused gene of Bik2 and Bik3, the authors improved the bikaverin production by 273-fold to 0.2 g/l [35].…”

Practical Enzymatic Production of Carbocycles

“…A recent study by Boeke and co‐workers [135] demonstrated the viability of establishing a heterologous expression system and subsequent enzyme/metabolic engineering for fungal polyketide production in S. cerevisiae (Scheme 33). Bikaverin ( 7.33 ), a red‐pigmented tetracyclic xanthone with antibiotic and immunosuppressive activities, [136] was used as a case study.…”