Induced biosyntheses of a novel butyrophenone and two aromatic polyketides in the plant pathogen Stagonospora nodorum

Fungal aromatic compounds comprise an important and structurally diverse group of secondary metabolites.Several genome sequencing projects revealed many putative biosynthetic gene clusters of fungal aromatic compounds,but many of these genes seem to be silent under typical laboratory culture conditions.To gain access to this untapped reservoir of natural products,we utilized chemical epigenetic modifiers to induce the expression of dormant biosynthetic genes.As a result,the concomitant supplementation of the histone deacetylase inhibitors suberoylanilide hydroxamic acid(500mM)and nicotinamide(50mM)to the culture medium of a fungal pathogen,Stagonospora nodorum,resulted in the isolation of three aromatic compounds(1-3),including a novel natural butyrophenone,(+)-4'-methoxy-(2S)-methylbutyrophenone(1),and two known polyketides,alternariol(2)and(-)-(3R)-mellein methyl ether(3).

Epigenetic modifier-induced biosynthesis of novel fusaric acid derivatives in endophytic fungi from Datura stramonium L.

The treatment of fungi with DNA methyltransferase(DNMT)and/or histone deacetylase(HDAC)inhibitors is a promising way to activate secondary metabolite biosynthetic pathways that are dormant under normal conditions.In this study,we included an HDAC inhibitor,suberoylanilide hydroxamic acid(SBHA),in the culture medium of endophytic fungi isolated from the medicinal plant Datura stramonium L.The production of two compounds was induced in the culture supplemented with SBHA,and their structures were determined to be the fusaric acid derivatives 5-butyl-6-oxo-1,6-dihydropyridine-2-carboxylic acid and 5-(but-9-enyl)-6-oxo-1,6-dihydropyridine-2-carboxylic acid.The result confirmed that the use of chemical epigenetic modifiers is an effective technique for promoting the expression of silent biosynthetic pathways to produce unique secondary metabolites.

Oxidative modification of free-standing amino acids by Fe(Ⅱ)/αKG-dependent oxygenases

Fe(Ⅱ)/α-ketoglutarate(αKG)-dependent oxygenases catalyze the oxidative modification of various molecules,from DNA,RNA,and proteins to primary and secondary metabolites.They also catalyze a variety of biochemical reactions,including hydroxylation,halogenation,desaturation,epoxidation,cyclization,peroxidation,epimeriza-tion,and rearrangement.Given the versatile catalytic capability of such oxygenases,numerous studies have been conducted to characterize their functions and elucidate their structure-function relationships over the past few decades.Amino acids,particularly nonproteinogenic amino acids,are considered as important building blocks for chemical synthesis and components for natural product biosynthesis.In addition,the Fe(Ⅱ)/αKG-dependent oxy-genase superfamily includes important enzymes for generating amino acid derivatives,as they efficiently modify various free-standing amino acids.The recent discovery of new Fe(Ⅱ)/αKG-dependent oxygenases and the repur-posing of known enzymes in this superfamily have promoted the generation of useful amino acid derivatives.Therefore,this study will focus on the recent progress achieved from 2019 to 2022 to provide a clear view of the mechanism by which these enzymes have expanded the repertoire of free amino acid oxidative modifications.

Biosynthesis of clinically used antibiotic fusidic acid and identification of two short-chain dehydrogenase/reductases with converse stereoselectivity

Fusidic acid is the only fusidane-type antibiotic that has been clinically used. However,biosynthesis of this important molecule in fungi is poorly understood. We have recently elucidated the biosynthesis of fusidane-type antibiotic helvolic acid, which provides us with clues to identify a possible gene cluster for fusidic acid(fus cluster). This gene cluster consists of eight genes, among which six are conserved in the helvolic acid gene cluster except fusC1 and fusB1. Introduction of the two genes into the Aspergillus oryzae NSAR1 expressing the conserved six genes led to the production of fusidic acid. A stepwise introduction of fusC1 and fusB1 revealed that the two genes worked independently without a strict reaction order. Notably, we identified two short-chain dehydrogenase/reductase genes fusC1 and fusC2 in the fus cluster, which showed converse stereoselectivity in 3-ketoreduction. This is the first report on the biosynthesis and heterologous expression of fusidic acid.

Extensive expansion of the chemical diversity of fusidane-type antibiotics using a stochastic combinational strategy

Fusidane-type antibiotics,represented by helvolic acid,fusidic acid and cephalosporin P1,are fungi-derived antimicrobials with little cross-resistance to commonly used antibiotics.Generation of new fusidane-type derivatives is therefore of great value,but this is hindered by available approaches.Here,we developed a stochastic combinational strategy by random assembly of all the post-tailoring genes derived from helvolic acid,fusidic acid,and cephalosporin P1 biosynthetic pathways in a strain that produces their common intermediate.Among a total of 27 gene combinations,24 combinations produce expected products and afford 58 fusidane-type analogues,of which 54 are new compounds.Moreover,random gene combination can induce unexpected activity of some post-tailoring enzymes,leading to a further increase in chemical diversity.These newly generated derivatives provide new insights into the structure-activity relationship of fusidane-type antibiotics.The stochastic combinational strategy established in this study proves to be a powerful approach for expanding structural diversity of natural products.

Molecular Basis for Sesterterpene Diversity Produced by Plant Terpene Synthases

Class I terpene synthase(TPS)generates bioactive terpenoids with diverse backbones.Sesterterpene synthase(sester-TPS,C25),a branch of class I TPSs,was recently identified in Brassicaceae.However,the catalytic mechanisms of sester-TPSs are not fully understood.Here,we first identified three nonclustered functional sester-TPSs(AtTPS06,AtTPS22,and AtTPS29)in Arabidopsis thaliana.AtTPS06 utilizes a type-B cyclization mechanism,whereas most other sester-TPSs produce various sesterterpene backbones via a type-A cyclization mechanism.We then determined the crystal structure of the AtTPS18–FSPP complex to explore the cyclization mechanism of plant sester-TPSs.We used structural comparisons and site-directed mutagenesis to further elucidate the mechanism:(1)mainly due to the outward shift of helix G,plant sester-TPSs have a larger catalytic pocket than do mono-,sesqui-,and di-TPSs to accommodate GFPP;(2)type-A sester-TPSs have more aromatic residues(five or six)in their catalytic pocket than classic TPSs(two or three),which also determines whether the type-A or type-B cyclization mechanism is active;and(3)the other residues responsible for product fidelity are determined by interconversion of AtTPS18 and its close homologs.Altogether,this study improves our understanding of the catalytic mechanism of plant sester-TPS,which ultimately enables the rational engineering of sesterterpenoids for future applications.

Microbial soluble aromatic prenyltransferases for engineered biosynthesis

Prenyltransferase(PTase)enzymes play crucial roles in natural product biosynthesis by transferring isoprene unit(s)to target substrates,thereby generating prenylated compounds.The prenylation step leads to a diverse group of natural products with improved membrane affinity and enhanced bioactivity,as compared to the nonprenylated forms.The last two decades have witnessed increasing studies on the identification,characterization,enzyme engineering,and synthetic biology of microbial PTase family enzymes.We herein summarize several examples of microbial soluble aromatic PTases for chemoenzymatic syntheses of unnatural novel prenylated compounds.