Engineering of New Pneumocandin Side-Chain Analogues from Glarea lozoyensis by Mutasynthesis and Evaluation of Their Antifungal Activity

Abstract

Pneumocandins are lipohexapeptides of the echinocandin family that inhibit fungal 1,3-β-glucan synthase. Most of the pathway steps have been identified previously. However, the lipoinitiation reaction has not yet been experimentally verified. Herein, we investigate the lipoinitiation step of pneumocandin biosynthesis in Glarea lozoyensis and demonstrate that the gene product, GLligase, catalyzes this step. Disruption of GLHYD, a gene encoding a putative type II thioesterase and sitting upstream of the pneumocandin acyl side chain synthase gene, GLPKS4, revealed that GLHYD was necessary for optimal function of GLPKS4 and to attain normal levels of pneumocandin production. Double disruption of GLHYD and GLPKS4 did not affect residual function of the GLligase or GLNRPS4. Mutasynthesis experiments with a gene disruption mutant of GLPKS4 afforded us an opportunity to test the substrate specificity of GLligase in the absence of its native polyketide side chain to diversify pneumocandins with substituted side chains. Feeding alternative side chain precursors yielded acrophiarin and four new pneumocandin congeners with straight C14, C15, and C16 side chains. A comprehensive biological evaluation showed that one compound, pneumocandin I (5), has elevated antifungal activity and similar hemolytic activity compared to pneumocandin B0, the starting molecule for caspofungin. This study demonstrates that the lipoinitiation mechanism in pneumocandin biosynthesis involves interaction among a highly reducing PKS, a putative type II thioesterase, and an acyl AMP-ligase. A comparison of the SAR among pneumocandins with different-length acyl side chains demonstrated the potential for using GLligase for future engineering of new echinocandin analogues.

Effects of Cotton Seed Powder as the Seed Medium Nitrogen Source on the Morphology and Pneumocandin B0 Yield of Glarea lozoyensis

“…The accumulated isocitrate is converted to citric acid and transported through the malate/citrate transporter on the mitochondrial membrane to the outside of the mitochondria ( Palmieri et al, 1996 ). Then, under the action of ATP:citrate lyase, it is cleaved to form acetyl-CoA, the key two-carbon metabolite in several metabolic processes, particularly in terms of precursor supply for the 10R,12S-dimethylmyristylside chain of pneumocandin B 0 , which determines the start of peptide elongation, directly affecting pneumocandin B 0 biosynthesis ( Chen et al, 2016 ). In addition to the sufficient supply of acetyl-CoA, it is well known that the supply of reducing power in form of NADPH has a critical effect on fatty acid biosynthesis ( Sun et al, 2016 ).…”

Cryptic Production of trans -3-Hydroxyproline in Echinocandin B Biosynthesis

“…Regarding echinocandin biosynthesis in general, 4-Me-3-Hyp (and not 3-Hyp) is found at position 6 in most structures (13), although the production of the precursor 4-Me-Pro requires additional metabolic energy. Therefore, it can be hypothesized that 4-Me-3-Hyp confers an advantage over 3-Hyp to the producer strain in its natural habitat, even though a strong bioactivity has been found for echinocandins with 3-Hyp under laboratory conditions (27). The preference for 4-Me-3-Hyp is also reflected in the significantly lower K m values of the PHs for 4-Me-Pro than for Pro.…”

Genomics‐driven discovery of a novel self‐resistance mechanism in the echinocandin‐producing fungus Pezicula radicicola

“…Each of the echinocandin gene clusters shares core conserved architecture, and variations in gene content closely track with the chemical structures of pathway products. Among A novel echinocandin resistance mechanism in fungi 7 V C 2018 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 00, 00-00 these clusters, the gene clusters for pneumocandin and echinocandin B have undergone extensive functional analysis via gene disruption studies and in vitro expression of pathway enzymes, thus contributing to understanding the underlying chemical logic (Cacho et al, 2012;Jiang et al, 2013;Chen et al, 2015;2016b;Li et al, 2015). A plethora of fungal genome data has also stimulated research on drug resistance of fungal pathogens against echinocandins (Singh-Babak et al, 2012).…”