First-class - biosynthesis of 6-MSA and bostrycoidin type I polyketides in Yarrowia lipolytica.

Fungal polyketides are a large group of secondary metabolites, valuable due to their diverse spectrum of pharmacological activities. Polyketide biosynthesis in filamentous fungi presents some challenges: small yield and low-purity titers. To tackle these issues, we switched to the yeast Yarrowia lipolytica, an easily cultivable heterologous host. As an oleaginous yeast, Y. lipolytica displays a high flux of acetyl- and malonyl-CoA precursors used in lipid synthesis. Likewise, acetyl- and malonyl-CoA are the building blocks of many natural polyketides, and we explored the possibility of redirecting this flux toward polyketide production. Despite its promising prospect, Y. lipolytica has so far only been used for heterologous expression of simple type III polyketide synthases (PKSs) from plants. Therefore, we decided to evaluate the potential of Y. lipolytica by targeting the more complex fungal polyketides synthesized by type I PKSs. We employed a CRISPR-Cas9-mediated genome editing method to achieve markerless gene integration of the genes responsible for bostrycoidin biosynthesis in Fusarium solani (fsr1, fsr2, and fsr3) and 6-methylsalicylic acid (6-MSA) biosynthesis in Aspergillus hancockii (6MSAS). Moreover, we attempted titer optimization through metabolic engineering by overexpressing two enzymes, TGL4 and AOX2, involved in lipid ß-oxidation, but we did not observe an effect on polyketide production. With maximum titers of 403 mg/L 6-MSA and 35 mg/L bostrycoidin, the latter being substantially higher than our previous results in Saccharomyces cerevisiae (2.2 mg/L), this work demonstrates the potential of Y. lipolytica as a platform for heterologous production of complex fungal polyketides.

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.

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.

Production and Selectivity of Key Fusarubins from Fusarium solani due to Media Composition.

Natural products display a large structural variation and different uses within a broad spectrum of industries. In this study, we investigate the influence of carbohydrates and nitrogen sources on the production and selectivity of production of four different polyketides produced by Fusarium solani, fusarubin, javanicin, bostrycoidin and anhydrofusarubin. We introduce four different carbohydrates and two types of nitrogen sources. Hereafter, a full factorial design was applied using combinations of three levels of sucrose and three levels of the two types of nitrogen. Each combination displayed different selectivity and production yields for all the compounds of interest. Response surface design was utilized to investigate possible maximum yields for the surrounding combinations of media. It was also shown that the maximum yields were not always the ones illustrating high selectivity, which is an important factor for making purification steps easier. We visualized the production over time for one of the media types, illustrating high yields and selectivity.

Heterologous Expression of the Core Genes in the Complex Fusarubin Gene Cluster of Fusarium Solani.

Through stepwise recreation of the biosynthetic gene cluster containing PKS3 from Fusarium solani, it was possible to produce the core scaffold compound of bostrycoidin, a red aza-anthraquinone pigment in Saccharomyces cerevisiae. This was achieved through sequential transformation associated recombination (TAR) cloning of FvPPT, fsr1, fsr2, and fsr3 into the pESC-vector system, utilizing the inducible bidirectional galactose promoter for heterologous expression in S. cerevisiae. The production of the core metabolite bostrycoidin was investigated through triplicate growth cultures for 1-4 days, where the maximum titer of bostrycoidin was achieved after 2 days of induction, yielding 2.2 mg/L.

Simulation of electrochemical properties of naturally occurring quinones.

Quinones are produced in organisms and are utilized as electron transfer agents, pigments and in defence mechanisms. Furthermore, naturally occurring quinones can also be cytotoxins with antibacterial properties. These properties can be linked to their redox properties. Recent studies have also shown that quinones can be utilized in flow battery technology, though naturally occurring quinones have not yet been investigated. Here, we have analyzed the properties of 990 different quinones of various biological sources through a computation approach to determine their standard reduction potentials and aqueous solubility. The screening was performed using the PBE functional and the 6-31G** basis set, providing a distribution of reduction potentials of the naturally occurring quinones varying from - 1.4 V to 1.5 V vs. the standard hydrogen electrode. The solvation energy for each quinone, which indicates the solubility in aqueous solution, was calculated at the same level. A large distribution of solubilities was obtained, containing both molecules that show tendencies of good solubilities and molecules that do not. The solubilities are dependent on the nature of the side groups and the size of the molecules. Our study shows that the group containing the quinones of fungal origin, which is also the largest of the groups considered, has the largest antimicrobial and electrochemical potential, when considering the distribution of reduction potentials for the compounds.

There it is! Fusarium pseudograminearum did not lose the fusaristatin gene cluster after all.

Fusarium pseudograminearum is a significant pathogen of cereals in arid regions worldwide and has the ability to produce numerous bioactive secondary metabolites. The genome sequences of seven F. pseudograminearum strains have been published and in one of these strains, C5834, we identified an intact gene cluster responsible for biosynthesis of the cyclic lipopeptide fusaristatin A. The high level of sequence identity of the fusaristatin cluster remnant in strains that do not produce fusaristatin suggests that the absence of the cluster evolved once, and subsequently the resulting locus with the cluster fragments became widely dispersed among strains of F. pseudograminearum in Australia. We examined a selection of 99 Australian F. pseudograminearum isolates to determine how widespread the ability to produce fusaristatin A is in F. pseudograminearum. We identified 15 fusaristatin producing strains, all originating from Western Australia. Phylogenetic analyses could not support a division of F. pseudograminearum into fusaristatin producing and nonproducing populations, which could indicate the loss has occurred relatively recent.