Pneumocandin B0 Research Articles

Exploration of the pneumocandin biosynthetic gene cluster based on efficient CRISPR/Cas9 gene editing strategy in Glarea lozoyensis

Pneumocandin B0 (PB0) is a lipopeptide produced by the fungus Glarea lozoyensis. The existing challenges with the low-yield and the extended-fermentation cycle emphasize necessity for strain improvement. In this study, we optimized conditions to obtain high-quality protoplasts and screened effective selection markers, leading to the construction of three CRISPR/Cas9 gene editing systems. Utilizing a constitutive Cas9 expression recipient strain, combined with dual sgRNAs targeting, we achieved highly efficient editing of target genes. We successfully knocked out 10 genes within the pneumocandin putative biosynthetic gene cluster and analyzed their roles in PB0 production. Our findings reveal that 4 of 10 genes are directly involved in PB0 production. Specially, the deletion of gltrt or gl10050 resulted in reduced PB0 production, while the absence of glhyp or glhtyC led to the complete loss of PB0 biosynthesis. Notably, the deletion of glhyp caused the silencing of nearly all cluster genes, whereas overexpression of glhyp led to a 2.38-fold increase in PB0 production. Therefore, this study provides the first comprehensive exploration of the functions of 10 genes within the pneumocandin putative biosynthetic gene cluster. Our findings provide valuable technical strategies for constructing bioengineering strains with purposefully enhanced PB0 production.

Insight into advances for the biosynthetic progress of fermented echinocandins of antifungals.

Invasive fungal infections have increased remarkably, which have become unprecedented concern to human health. However, the effectiveness of current antifungal drugs is limited due to drug resistance and toxic side-effects. It is urgently required to establish the effective biosynthetic strategy for developing novel and safe antifungal molecules economically. Echinocandins become a promising option as a mainstay family of antifungals, due to specifically targeting the fungal specific cell wall. To date, three kinds of echinocandins for caspofungin, anidulafungin, and micafungin, which derived from pneumocandin B0 , echinocandin B, and FR901379, are commercially available in clinic and have shown potential in managing invasive fungal infections in a cost-effective manner. However, current echinocandins-derived precursors all are produced by environmental fungal isolates with long fermentation cycle and low yields, which challenge the production efficacy of these precursors in industry. Therefore, understanding their biosynthetic machinery is of great importance for improving antifungal titres and creating new echinocandins-derived products. With the development of genome-wide sequencing and establishment of gene-editing technology, there are a growing number of reports on echinocandins-derived products and their biosynthetic gene clusters. This review briefly summarizes the discovery and development history of echinocandins, compares their structural characteristics and biosynthetic processes, and sums up existed strategies for improving their production. Moreover, the genomic analysis of related biosynthetic gene clusters of echinocandins is discussed, highlighting the similarities and differences among the clusters. Last, the biosynthetic processes of echinocandins are compared, focusing on the activation and attachment of side-chains and the formation of the hexapeptide core. This review aims to provide insights into the development and production of new echinocandin drugs by modifying the structure of echinocandin-derived precursors and/or optimizing the fermentation processes; and achieve a new microbial chassis for efficient production of echinocandins in heterologous hosts.

Effect of fatty acids on intracellular pneumocandin B0 storage in the fermentation of Glarea lozoyensis

The natural product pneumocandin B0 is the precursor of the antifungal drug caspofungin. To explore the relationship between pneumocandin B0 and oil. We found that the addition of 1 g/L of oil to the fermentation medium is more conducive to the production of pneumocandin B0. The metabolic reaction mechanism was explored using different fatty acids and the results showed that stearic acid and acetic acid increased the total production of pneumocandin B0 by 22.98% and 9.08%, respectively, as well as increasing the content of intracellular lipid droplets. We also analyzed gene expression and pathway differences between the two different fatty acids using transcriptome analyses. The addition of both acetic acid and stearic acid promoted an active pentose phosphate pathway, providing cells with higher intracellular reducing power. We found that the addition of fatty acids can lead to lipid accumulation, and lipid droplets can sequester lipophilic secondary metabolites such as pneumocandin B0 to reduce cell damage. These results provide novel insights into the relationship between pneumocandin B0 biosynthesis and fatty acids in G. lozoyensis. In addition, this study provides important genetic information for improving the yield of pneumocandin B0 through a strategy of metabolic engineering in the future.Graphical

Biosynthesis mechanism, genome mining and artificial construction of echinocandin O-sulfonation

Micafungin, a semisynthetic derivative of the cyclic hexapeptide FR901379 produced by Coleophoma empetri fermentation, is the only O-sulfonated echinocandin-type antifungal drug. However, the detailed formation mechanism of O-sulfonate group, whether before or after the assembly of hexapeptide, remains elusive. Here, we confirmed that O-sulfonylation occurs after hexapeptide assembly as a kind of postmodification in the biosynthesis of FR901379. The released cyclic hexapeptide was hydroxylated by cytochrome P450 McfP and successively sulfonated by sulfotransferase McfS. And other three echinocandin sulfotransferases were identified through genome mining by using McfS as a sequence probe. Moreover, pneumocandin B0, the precursor of caspofungin, could be O-sulfonated by heterologously introducing the McfP-McfS into the pneumocandin B0-producing species Glarea lozoyensis. The water-solubility of sulfonated pneumocandin B0 is 4000 times higher than that of pneumocandin B0. The revealed O-sulfonation mechanism will provide new insights into the design and production of novel sulfonated echinocandins by metabolic engineering.

Glyap1 regulates pneumocandin B0 synthesis by controlling the intracellular redox balance in Glarea lozoyensis.

Pneumocandin B0, the precursor of the antifungal drug caspofungin, is a lipohexapeptide produced by the fungus Glarea lozoyensis. Oxidative stress and the resulting production of reactive oxygen species (ROS) are known to be involved in the regulation of pneumocandin B0 biosynthesis. In this study, the Glyap1 gene of Glarea lozoyensis, a homologue of the yeast redox regulator YAP1, was knocked out. The intracellular ROS levels of the resulting ΔGlyap1 strain were higher than in the wild-type strain, which was caused by the downregulated expression of superoxide dismutase (SOD) and catalase (CAT). Compared with the wild-type strain, ΔGlyap1 exhibited an oxidative phenotype throughout its life cycle, which resulted in significantly higher pneumocandin B0 production per unit biomass. In addition, ΔGlyap1 showed growth inhibition and decreased pneumocandin B0 production in the presence of CCl4, which leads to strong oxidative stress. To overcome the strain's sensitivity, a three-stage antioxidant addition strategy was developed. This approach significantly improved the growth of ΔGlyap1 while maintaining a high pneumocandin B0 production per unit biomass, which reached 38.78mg/g DCW. Notably, this result represents a 50% increase over the wild-type strain. These findings provide new insights into the regulatory mechanisms that control pneumocandin B0 production under oxidative stress, which may be applied to improve the production of other secondary metabolites. KEY POINTS: • Glyap1 is involved in expression of redox and pneumocandin B0 synthesis-related genes. • Addition of a three-stage antioxidant alleviated the sensitivity of ΔGlyap1 strain. • The yield of pneumocandin B0 per unit biomass of ΔGlyap1 strain was 38.78mg/g DCW.

In silico analysis of echinocandins binding to the main proteases of coronaviruses PEDV (3CLpro) and SARS-CoV-2 (Mpro).

The porcine epidemic diarrhea virus (PEDV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are two highly pathogenic viruses causing tremendous damages to the swine and human populations, respectively. Vaccines are available to prevent contamination and to limit dissemination of these two coronaviruses, but efficient and widely affordable treatments are needed. Recently, four natural products targeting the 3C-like protease (3CLpro) of PEDV and inhibiting replication of the virus in vitro have been identified: tomatidine, epigallocatechin-3-gallate, buddlejasaponin IVb and pneumocandin B0. We have evaluated the interaction of these compounds with 3CLpro of PEDV and with the structurally similar main protease (Mpro) of SARS-CoV-2. The molecular docking analysis indicated that the echinocandin-type lipopeptide pneumocandin B0 can generate much more stable complexes with both proteases compared to tomatidine. The empirical energy of interaction (ΔE) calculated with pneumocandin B0 bound to Mpro is extremely high, comparable to that measured with known antiviral drugs. Pneumocandin B0 and its analogue capsofungin appeared a little less adapted to interact with 3CLpro compared to Mpro. In contrast, the antifungal drug micafungin bearing an unfused tricyclic side chain, emerges as a better ligand of 3CLpro of PEDV compared to Mpro of SARS-CoV-2, based on our calculations. Collectively, the analysis underlines the benefit of echinocandin-type antifungal drugs as potential inhibitors of PEDV and SARS-CoV-2 main proteases. These clinically important antifungal natural products deserve further studies as antiviral agents.

CRISPR/Cas9-Based Genome Editing in the Filamentous Fungus Glarea lozoyensis and Its Application in Manipulating gloF.

Glarea lozoyensis is an important industrial fungus that produces the pneumocandin B0, which is used for the synthesis of antifungal drug caspofungin. However, because of the limitations and complications of traditional genetic tools, G. lozoyensis strain engineering has been hindered. In this study, we established an efficient CRISPR/Cas9-based gene editing tool in G. lozoyensis SIPI1208. With this method, gene mutagenesis efficiency in the target locus can be up to 80%, which enables the rapid gene knockout. According to the reports, GloF and Ap-HtyE, proline hydroxylases involved in pneumocandin and Echinocandin B biosynthesis, respectively, can catalyze the proline to generate different ratios of trans-3-hydroxy-l-proline to trans-4-hydroxy-l-proline. Heterologous expression of Ap-HtyE in G. lozoyensis decreased the ratio of pneumocandin C0 to (pneumocandin B0 + pneumocandin C0) from 33.5% to 11% without the addition of proline to the fermentation medium. Furthermore, the gloF was replaced by ap-htyE to study the production of pneumocandin C0. However, the gene replacement has been hampered by traditional gene tools since gloF and gloG, two contiguous genes indispensable in the biosynthesis of pneumocandins, are cotranscribed into one mRNA. With the CRISPR/Cas9 strategy, ap-htyE was knocked in and successfully replaced gloF, and results showed that the knock-in strain retained the ability to produce pneumocandin B0, but the production of pneumocandin C0 was abolished. Thus, this strain displayed a competitive advantage in the industrial production of pneumocandin B0. In summary, this study showed that the CRISPR/Cas9-based gene editing tool is efficient for manipulating genes in G. lozoyensis.

Comparative Transcriptomics Analysis of the Responses of the Filamentous Fungus Glarea lozoyensis to Different Carbon Sources

The natural product pneumocandin B0 is the precursor of the antifungal drug caspofungin. We found that replacing glucose in the initial fermentation medium with 20 g/L fructose is more conducive to pneumocandin B0 production and biomass accumulation. In order to explore the mechanism of the different metabolic responses to fructose and glucose, we used each as the sole carbon source, and the results showed that fructose increased the total pneumocandin B0 yield and biomass by 54.76 and 13.71%, respectively. Furthermore, we analyzed the differences of gene expression and metabolic pathways between the two different carbon sources by transcriptomic analysis. When fructose was used as the carbon source, genes related to the pentose phosphate pathway (PPP), glycolysis and branched-chain amino acid metabolism were significantly upregulated, resulting in increased intracellular pools of NADPH and acetyl-CoA in Glarea lozoyensis for cell growth and pneumocandin B0 product synthesis. Interestingly, the pneumocandin B0 biosynthetic gene cluster and the genes of the TCA cycle were significantly downregulated, while the FAS genes were significantly upregulated, indicating that more acetyl-CoA was used for fatty acid synthesis. In particular, we found that excessive synthesis of fatty acids caused lipid accumulation, and lipid droplets can sequester lipophilic secondary metabolites such as pneumocandin B0 to reduce cell damage, which may also be an important reason for the observed increase of pneumocandin B0 yield. These results provide new insights into the relationship between pneumocandin B0 biosynthesis and carbon sources in G. lozoyensis. At the same time, this study provides important genomic information for improving pneumocandin B0 production through metabolic engineering strategies in the future.

Effect of SDS on release of intracellular pneumocandin B0 in extractive batch fermentation of Glarea lozoyensis

Pneumocandin B0 is a hydrophobic secondary metabolite that accumulates in the mycelia of Glarea lozoyensis and inhibits fungal 1,3-β-glucan synthase. Extractive batch fermentation can promote the release of intracellular secondary metabolites into the fermentation broth and is often used in industry. The addition of extractants has been proven as an effective method to attain higher accumulation of hydrophobic secondary metabolites and circumvent troublesome solvent extraction. Various extractants exerted significant but different influences on the biomass and pneumocandin B0 yields. The maximum pneumocandin B0 yield (2528.67 mg/L) and highest extracellular pneumocandin B0 yield (580.33 mg/L) were achieved when 1.0 g/L SDS was added on the 13th day of extractive batch fermentation, corresponding to significant increases of 37.63 and 154% compared with the conventional batch fermentation, respectively. The mechanism behind this phenomenon is partly attributed to the release of intracellular pneumocandin B0 into the fermentation broth and the enhanced biosynthesis of pneumocandin B0 in the mycelia.

Asymmetric Synthesis of Chiral 1,3-Dimethyl Units Through a Double Michael Reaction of Nitromethane and Crotonaldehyde Catalyzed by Diphenylprolinol Silyl Ether

An efficient synthetic route to install chiral 1,3-dimethyl units through a double Michael reaction of crotonaldehyde and nitromethane catalyzed by diphenylprolinol silyl ether is developed. Either 1,3-syn- or 1,3-anti-dimethyl units are obtained selectively depending on the enantiomer of the diphenylprolinol silyl ether catalyst used. The side chain of pneumocandin B0 is synthesized enantioselectively by using the present method as a key step.

Enhancement of Pneumocandin B0 Production in Glarea lozoyensis by Low-Temperature Adaptive Laboratory Evolution.

The production of pneumocandin B0 is limited by feedback inhibition. Here, low-temperature adaptive laboratory evolution (ALE) was used to improve the production capacity of Glarea lozoyensis by enhancing its membrane permeability. After 50 cycles of ALE, the pneumocandin B0 production of the endpoint strain (ALE50) reached 2131 g/L, which was 32% higher than the starting strain (ALE0). ALE50 showed a changed fatty acid composition of the cell membrane, which-+h increased its permeability by 14%, which in turn increased the secretion ratio threefold. Furthermore, ALE50 showed increased intracellular proline and acetyl-CoA concentrations, superoxide dismutase (SOD), and catalase (CAT) activity, as well as total antioxidant capacity. The slight biomass decrease in ALE50 was accompanied by decreased isocitrate dehydrogenase (ICDH) and glucose-6-phosphate dehydrogenase (G6PDH) activity. Finally, a putative model of the accumulation and secretion of pneumocandin B0 in ALE50 was established. ALE is a promising method to release intracellular feedback inhibition.

Novel osmotic stress control strategy for improved pneumocandin B0 production in Glarea lozoyensis combined with a mechanistic analysis at the transcriptome level.

Pneumocandin B0, the precursor of the antifungal drug caspofungin, is a secondary metabolite of the fungus Glarea lozoyensis. In this study, we investigated the effects of mannitol as the sole carbon source on pneumocandin B0 production by G. lozoyensis. The osmotic pressure is more important in enhancing pneumocandin B0 production than is the substrate concentration. Based on the kinetic analysis, an osmotic stress control fed-batch strategy was developed. This strategy led to a maximum pneumocandin B0 concentration of 2711mg/L with a productivity of 9.05mg/L/h, representing 34.67 and 6.47% improvements, respectively, over the best result achieved by the one-stage fermentation. Furthermore, G. lozoyensis accumulated glutamate and proline as compatible solutes to resist osmotic stress, and these amino acids also provided the precursors for the enhanced pneumocandin B0 production. Osmotic stress also activated ROS (reactive oxygen species)-dependent signal transduction by upregulating the levels of related genes and increasing intracellular ROS levels by 20%. We also provided a possible mechanism for pneumocandin B0 accumulation based on signal transduction. These findings will improve our understanding of the regulatory mechanisms of pneumocandin B0 biosynthesis and may be applied to improve secondary metabolite production.

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

Pneumocandin B0 is an important antifungal drug precursor produced by filamentous fungus Glarea lozoyensis. The high broth viscosity of cultures of this organism results in lower oxygen solubility and higher energy consumption for agitation and aeration, which mostly caused by the morphologies of filamentous fungi in submerged culture. In this study, the effects of different seed medium nitrogen sources on morphology and fermentation behavior of G. lozoyensis were investigated, and cotton seed powder resulted in small, compact pellets. Moreover, pneumocandin B0 yield in Erlenmeyer flasks were increased by 22.9%. Furthermore, pneumocandin B0 yield in a 50-L fermenter reached 2,100 mg/L and the dissolved oxygen maintained above 30%. Additionally, activities of phosphofructokinase (PFK), isocitrate dehydrogenase (ICDH), glucose 6-phosphate dehydrogenase (G6PDH), and malic enzyme (ME) were increased by 87.5, 50, 41.6, and 10.7%, respectively. This study demonstrates the feasibility and advantages of using cotton seed powder for controlling the fungal morphology and improving the product yield in pneumocandin fermentations.

Biosynthesis of pneumocandin lipopeptides and perspectives for its production and related echinocandins

Fungal diseases are a global public health problem. Invasive fungal infections pose a serious threat to patients with compromised immune systems, such as those undergoing organ or bone marrow transplants, cancer, or HIV/AIDS. Pneumocandins are antifungal lipohexapeptides of the echinocandin family that noncompetitively inhibit of 1,3-β-glucan synthase of fungal cell wall and provide the precursor for the semisynthesis of caspofungin, which is widely used as first-line therapy for invasive fungal infections. Recently, the biosynthetic steps leading to formation of pneumocandin B0 and echinocandin B have been elucidated, and thus, provide a framework and attractive model for further design new antifungal therapeutics around natural variations in echinocandin structural diversities via genetic and chemical tools. In this article, we analyze the biosynthetic pathway of pneumocandins and other echinocandins, provide an update on the array of pneumocandin analogues generated by genetic manipulation, and summarize advances in the enhancement of pneumocandin B0 production by random mutagenesis and fermentation optimization. We also give offer advice on the development of improved pneumocandin drug candidates and more efficient production of pneumocandin B0.

Metabolomics profiling reveals the mechanism of increased pneumocandin B0 production by comparing mutant and parent strains

Metabolic profiling was used to discover mechanisms of increased pneumocandin B0 production in a high-yield strain by comparing it with its parent strain. Initially, 79 intracellular metabolites were identified, and the levels of 15 metabolites involved in six pathways were found to be directly correlated with pneumocandin B0 biosynthesis. Then by combining the analysis of key enzymes, acetyl-CoA and NADPH were identified as the main factors limiting pneumocandin B0 biosynthesis. Other metabolites, such as pyruvate, α-ketoglutaric acid, lactate, unsaturated fatty acids and previously unreported metabolite γ-aminobutyric acid were shown to play important roles in pneumocandin B0 biosynthesis and cell growth. Finally, the overall metabolic mechanism hypothesis was formulated and a rational feeding strategy was implemented that increased the pneumocandin B0 yield from 1821 to 2768mg/L. These results provide practical and theoretical guidance for strain selection, medium optimization, and genetic engineering for pneumocandin B0 production.

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

Echinocandins are antifungal nonribosomal hexapeptides produced by fungi. Two of the amino acids are hydroxy-l-prolines: trans-4-hydroxy-l-proline and, in most echinocandin structures, (trans-2,3)-3-hydroxy-(trans-2,4)-4-methyl-l-proline. In the case of echinocandin biosynthesis by Glarea lozoyensis, both amino acids are found in pneumocandin A0, while in pneumocandin B0 the latter residue is replaced by trans-3-hydroxy-l-proline (3-Hyp). We have recently reported that all three amino acids are generated by the 2-oxoglutarate-dependent proline hydroxylase GloF. In echinocandin B biosynthesis by Aspergillus species, 3-Hyp derivatives have not been reported. Here we describe the heterologous production and kinetic characterization of HtyE, the 2-oxoglutarate-dependent proline hydroxylase from the echinocandin B biosynthetic cluster in Aspergillus pachycristatus Surprisingly, l-proline hydroxylation with HtyE resulted in an even higher proportion (∼30%) of 3-Hyp than that with GloF. This suggests that the selectivity for methylated 3-Hyp in echinocandin B biosynthesis is due solely to a substrate-specific adenylation domain of the nonribosomal peptide synthetase. Moreover, we observed that one product of HtyE catalysis, 3-hydroxy-4-methyl-l-proline, is slowly further oxidized at the methyl group, giving 3-hydroxy-4-hydroxymethyl-l-proline, upon prolonged incubation with HtyE. This dihydroxylated amino acid has been reported as a building block of cryptocandin, an echinocandin produced by CryptosporiopsisIMPORTANCE Secondary metabolites from bacteria and fungi are often produced by sets of biosynthetic enzymes encoded in distinct gene clusters. Usually, each enzyme catalyzes one biosynthetic step, but multiple reactions are also possible. Pneumocandins A0 and B0 are produced by the fungus Glarea lozoyensis They belong to the echinocandin family, a group of nonribosomal cyclic lipopeptides that exhibit a strong antifungal activity. Chemical derivatives are important drugs for the treatment of systemic fungal infections. We have recently shown that in the biosynthesis of pneumocandins A0 and B0, three hydroxyproline building blocks are provided by one proline hydroxylase. Here we demonstrate that the proline hydroxylase from echinocandin B biosynthesis in Aspergillus pachycristatus produces the same hydroxyprolines, with an increased proportion of trans-3-hydroxyproline. However, echinocandin B biosynthesis does not require trans-3-hydroxyproline; its formation remains cryptic. While one can only speculate on the evolutionary background of this unexpected finding, proline hydroxylation in G. lozoyensis and A. pachycristatus provides an unusual insight into peptide antibiotic biosynthesis-namely, the complex interplay between the selectivity of a hydroxylase and the substrate specificity of a nonribosomal peptide synthetase.

Protoplast mutant selection of Glarea Lozoyensis and statistical optimization of medium for pneumocandin B0 yield-up.

A combination of microbial strain improvement and statistical optimization is investigated to maximize pneumocandin B0 production from Glarea lozoyensis ATCC 74030. Atmospheric and room temperature plasma (ARTP) was used to enhance G. lozoyensis ATCC 74030 in pneumocandin B0 yield. Mutant strain G. lozoyensis Q1 exhibited 1.39-fold increase in pneumocandin B0 production to 1134mg/L when compared with the parent strain (810mg/L). Further, the optimized medium provided another 1.65-fold in final pneumocandin B0 concentration to 1873mg/L compared to the original medium. The results of this study indicated the combined application of a classical mutation and medium optimization can improve effectively pneumocandin B0 production from G. lozoyensis and could be a tool to improve other secondary metabolites production by fungal strains.

Pneumocandin B0-imprinted Polymer Using Surface-imprinting Technique for Efficient Purification of Crude Product

In this work, we prepared surface molecularly imprinted polymer (MIP) for selective recognition of pneumocandin B0 (PNB0). Methacrylic acid (MAA) was first grafted onto silica gel particles (SiO2) in the manner of "grafting from" by using 3-methacryloxypropyl trimethoxysilane as intermedium, and then PNB0 molecules were imprinted on the surface of the obtained particles in the presence of ethylene glycol diglycidyl ether as the cross-linker. The prepared MIP-PMAA/SiO2 was characterized by scanning electron microscopy, Fourier transform infrared spectroscopy and thermo-gravimetric analysis, which confirmed the successful grafting of MAA onto SiO2 and the grafting degree was calculated to be 12.50 wt%. The binding properties of the products were investigated and it is found that the binding process of PNB0 followed the pseudo-second-order kinetic model. The as-prepared material also displays relatively quick adsorption kinetics and decent recognition affinity toward the template over its structurally related compound.

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

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.

Engineering of Glarea lozoyensis for exclusive production of the pneumocandin B0 precursor of the antifungal drug caspofungin acetate.

Pneumocandins produced by the fungus Glarea lozoyensis are acylated cyclic hexapeptides of the echinocandin family. Pneumocandin B0 is the starting molecule for the first semisynthetic echinocandin antifungal drug, caspofungin acetate. In the wild-type strain, pneumocandin B0 is a minor fermentation product, and its industrial production was achieved by a combination of extensive mutation and medium optimization. The pneumocandin biosynthetic gene cluster was previously elucidated by a whole-genome sequencing approach. Knowledge of the biosynthetic cluster suggested an alternative way to produce exclusively pneumocandin B0. Disruption of GLOXY4, encoding a nonheme, α-ketoglutarate-dependent oxygenase, confirmed its involvement in l-leucine cyclization to form 4S-methyl-l-proline. The absence of 4S-methyl-l-proline abolishes pneumocandin A0 production, and 3S-hydroxyl-l-proline occupies the hexapeptide core's position 6, resulting in exclusive production of pneumocandin B0. Retrospective analysis of the GLOXY4 gene in a previously isolated pneumocandin B0-exclusive mutant (ATCC 74030) indicated that chemical mutagenesis disrupted the GLOXY4 gene function by introducing two amino acid mutations in GLOXY4. This one-step genetic manipulation can rationally engineer a high-yield production strain.