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

Tobias Bruun Pedersen,Kåre Lehmann Nielsen,Eva Mie Lang Spedtsberg,Sebastian Birkedal Kristensen,Trine Sørensen,Mikkel Rank Nielsen,Donald Max Gardiner,Jens Muff,Reinhard Wimmer,Celine Petersen,Teis Esben Sondergaard,Jens Laurids Sørensen

doi:10.1186/s12934-021-01734-9

Tobias Bruun Pedersen, Kåre Lehmann Nielsen + Show 10 more

Open Access

https://doi.org/10.1186/s12934-021-01734-9

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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.

Highlights

The chemical group of compounds known as polyketides exhibit an abundance in structural and bioactive diversity, making them some of the foremost interesting compounds for researching natural products, with a plethora of applications ranging from antibiotics, immunosuppressants, anti-cancer to fungicides and perhaps even as a solution to energy storage in the future [1,2,3,4,5]
The two target polyketides were successfully expressed in S. cerevisiae alongside the seven phosphopantetheinyl transferases (PPTases)
Of the 14 polyketide synthases (PKS) and PPTase combinations, 13 showed successful interactions resulting in production of the target metabolite and one resulting in miniscule production of target polyketide

Summary

Introduction

The chemical group of compounds known as polyketides exhibit an abundance in structural and bioactive diversity, making them some of the foremost interesting compounds for researching natural products, with a plethora of applications ranging from antibiotics, immunosuppressants, anti-cancer to fungicides and perhaps even as a solution to energy storage in the future [1,2,3,4,5]. Polyketides are attractive targets for large-scale production due to their bioactive properties, which can be achieved in their natural host or in heterologous hosts [4, 6]. Three conserved domains are found in all iterative PKSs, the β-ketosynthase (KS), acyltransferase (AT) and acyl-carrier protein (ACP), which together constitute a minimal PKS [13]. The majority of studies with heterologous production of polyketides in S. cerevisiae have utilized the same few standard PPTases, Sfp from Bacillus subtilis or NpgA from Aspergillus nidulans [10, 20], opening the possibility for titer increase through substitution of PPTases. Through a substitutional approach we investigated the standard PPTases alongside five additional PPTase of different origins. Three different fungal PPTase were investigated to determine if an evolutionary advantage of PPTases originating from fungi natively expressing these compounds, was evident

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