Yeast recombinational cloning for heterologous biosynthesis of polyketides: a molecular microbiology laboratory module for undergraduate students

Recombinant plasmids are essential tools in molecular biotechnology, and reliable plasmid assembly methods have, therefore, become a prerequisite for the successful cloning and transfer of genes. Among the multitude of available plasmid assembly strategies, in vivo homologous recombinational cloning in yeast has emerged as a cost-effective and relatively simple method. Since we use this method routinely in our group for assembling large plasmids with secondary metabolite gene clusters and for direct heterologous production of polyketides in Saccharomyces cerevisiae, we developed an exercise module for undergraduate students where they would get hands-on experience with these molecular practices. The exercises target several molecular techniques, including PCR, restriction enzyme digestion, and yeast recombinational cloning. The students will learn about plasmid assembly and yeast transformation methods by performing these experiments while inherently acquiring new skills valuable for their subsequent laboratory work or projects. </p

Speed dating for enzymes! Finding the perfect phosphopantetheinyl transferase partner for your polyketide synthase

The biosynthetic pathways for the fungal polyketides bikaverin and bostrycoidin, from Fusarium verticillioides and Fusarium solani respectively, were reconstructed and heterologously expressed in S. cerevisiae alongside seven different phosphopantetheinyl transferases (PPTases) from a variety of origins spanning bacterial, yeast and fungal origins. In order to gauge the efficiency of the interaction between the ACP-domains of the polyketide synthases (PKS) and PPTases, each were co-expressed individually and the resulting production of target polyketides were determined after 48 h of growth. In co-expression with both biosynthetic pathways, the PPTase from Fusarium verticillioides (FvPPT1) proved most efficient at producing both bikaverin and bostrycoidin, at 1.4 mg/L and 5.9 mg/L respectively. Furthermore, the remaining PPTases showed the ability to interact with both PKS’s, except for a single PKS-PPTase combination. The results indicate that it is possible to boost the production of a target polyketide, simply by utilizing a more optimal PPTase partner, instead of the commonly used PPTases; NpgA, Gsp and Sfp, from Aspergillus nidulans, Brevibacillus brevis and Bacillus subtilis respectively. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-021-01734-9

Additional file 1 of Speed dating for enzymes! Finding the perfect phosphopantetheinyl transferase partner for your polyketide synthase

Additional file 1: Table S1. This table contains the primer sequences of both the primers used for gene-amplification and the primer used for initial sanger-sequencing in fragments of around 700 bp, containing at least 50 bp overlap between each fragment. Table S2. This table contains the different plasmids utilized in the project, both the native plasmids used as expression vectors, but also plasmids purchased containing the synthetically derived codon optimized genes. Figure S1. Phylogenetic tree of the PPTases used in the present study (bold) together with 22 additional published PPTases. Bootstrap values (&gt; 70%) from 1000 replications are indicated at the respective nodes. Figure S2. Predicted structure of sfp/ACP interaction with the CoA and Mg2+ ion highlighted by arrows. Figure S3. Production levels of bikaverin and bostrycoidin in the individual strains (relative to OD at 48 h) in the supernatant and pellets. The mean of the supernatant from BY4743::FvPPT1 was set to 100 for both compounds

Speed dating for enzymes! Finding the perfect phosphopantetheinyl transferase partner for your polyketide synthase

Abstract The biosynthetic pathways for the fungal polyketides bikaverin and bostrycoidin, from Fusarium verticillioides and Fusarium solani respectively, were reconstructed and heterologously expressed in S. cerevisiae alongside seven different phosphopantetheinyl transferases (PPTases) from a variety of origins spanning bacterial, yeast and fungal origins. In order to gauge the efficiency of the interaction between the ACP-domains of the polyketide synthases (PKS) and PPTases, each were co-expressed individually and the resulting production of target polyketides were determined after 48 h of growth. In co-expression with both biosynthetic pathways, the PPTase from Fusarium verticillioides (FvPPT1) proved most efficient at producing both bikaverin and bostrycoidin, at 1.4 mg/L and 5.9 mg/L respectively. Furthermore, the remaining PPTases showed the ability to interact with both PKS’s, except for a single PKS-PPTase combination. The results indicate that it is possible to boost the production of a target polyketide, simply by utilizing a more optimal PPTase partner, instead of the commonly used PPTases; NpgA, Gsp and Sfp, from Aspergillus nidulans, Brevibacillus brevis and Bacillus subtilis respectively

Speed dating for enzymes! Finding the perfect phosphopantetheinyl transferase partner for your polyketide synthase

Abstract The biosynthetic pathways for the fungal polyketides bikaverin and bostrycoidin, from Fusarium verticillioides and Fusarium solani respectively, were reconstructed and heterologously expressed in S. cerevisiae alongside seven different phosphopantetheinyl transferases (PPTases) from a variety of origins spanning bacterial, yeast and fungal origins. In order to gauge the efficiency of the interaction between the ACP-domains of the polyketide synthases (PKS) and PPTases, each were co-expressed individually and the resulting production of target polyketides were determined after 48 h of growth. In co-expression with both biosynthetic pathways, the PPTase from Fusarium verticillioides (FvPPT1) proved most efficient at producing both bikaverin and bostrycoidin, at 1.4 mg/L and 5.9 mg/L respectively. Furthermore, the remaining PPTases showed the ability to interact with both PKS’s, except for a single PKS-PPTase combination. The results indicate that it is possible to boost the production of a target polyketide, simply by utilizing a more optimal PPTase partner, instead of the commonly used PPTases; NpgA, Gsp and Sfp, from Aspergillus nidulans, Brevibacillus brevis and Bacillus subtilis respectively