A PKS gene, pks-1, is involved in chaetoglobosin biosynthesis, pigmentation and sporulation in Chaetomium globosum

Chaetomium globosum is one of the most common fungi in nature. It is best known for producing chaetoglobosins; however, the molecular basis of chaetoglobosin biosynthesis is poorly understood in this fungus. In this study, we utilized RNA interference (RNAi) to characterize a polyketide synthase gene, pks-1, in C. globosum that is involved in the production of chaetoglobosin A. When pks-1 was knocked down by RNAi, the production of chaetoglobosin A dramatically decreased. Knock-down mutants also displayed a pigment-deficient phenotype. These results suggest that the two polyketides, melanin and chaetoglobosin, are likely to share common biosynthetic steps. Most importantly, we found that pks-1 also plays a critical role in sporulation. The silenced mutants of pks-1 lost the ability to produce spores. We propose that polyketides may modulate cellular development via an unidentified action. We also suggest that C. globosum pks-1 is unique because of its triple role in melanin formation, chaetoglobosin biosynthesis and sporulation. This work may shed light on chaetoglobosin biosynthesis and indicates a relationship between secondary metabolism and fungal morphogenesis.     

Significance of the Cgl1427 gene encoding cytidylate kinase in microaerobic growth of Corynebacterium glutamicum

The Cgl1427 gene was previously found to be relevant to the microaerobic growth of Corynebacterium glutamicum (Ikeda et al. Biosci Biotechnol Biochem 73:2806–2808, 2009). In the present work, Cgl1427 was identified as a cytidylate kinase gene (cmk) by homology analysis of its deduced amino acid sequence with that of other bacterial cytidylate kinases (CMP kinases) and on the basis of findings that deletion of Cgl1427 results in loss of CMP kinase activity. Deletion of the cmk gene significantly impaired the growth of C. glutamicum in oxygen-limiting static culture, and the impaired growth was restored by introducing a plasmid containing the cmk gene, suggesting that this gene plays an important role in the microaerobic growth of C. glutamicum. On the other hand, in the main culture with aerobic shaking, a prolonged lag phase was observed in the cmk disruptant, despite an unchanged growth rate, compared to the behavior of the wild-type strain. The prolongation was observed when using seed culture grown to later growth stages in which oxygen limitation occurred, but it was not observed when using seed culture grown to an earlier growth stage in which oxygen remained relatively plentiful. Since nucleotide biosynthesis in C. glutamicum requires oxygen, we hypothesized that the ability of the cmk disruptant to synthesize nucleotides was influenced by oxygen limitation in the later growth stages of the seed culture, which caused the prolongation of the lag phase in the following shaken culture. To verify this hypothesis, a plasmid containing genes encoding all components of a homologous ribonucleotide reductase, a key enzyme for nucleotide synthesis that requires oxygen for its reaction, was introduced into the cmk disruptant, which significantly ameliorated the lag phase prolongation. Furthermore, this experimental setup almost completely restored the growth of the cmk disruptant in the oxygen-limiting static culture. These results indicate that CMP kinase plays an important role in normal nucleotide biosynthesis under an oxygen-limiting environment.     

veA Is Required for Toxin and Sclerotial Production in Aspergillus parasiticus

It was long been noted that secondary metabolism is associated with fungal development. In Aspergillus nidulans, conidiation and mycotoxin production are linked by a G protein signaling pathway. Also in A. nidulans, cleistothecial development and mycotoxin production are controlled by a gene called veA. Here we report the characterization of a veA ortholog in the aflatoxin-producing fungus A. parasiticus. Cleistothecia are not produced by Aspergillus parasiticus; instead, this fungus produces spherical structures called sclerotia that allow for survival under adverse conditions. Deletion of veA from A. parasiticus resulted in the blockage of sclerotial formation as well as a blockage in the production of aflatoxin intermediates. Our results indicate that A. parasiticus veA is required for the expression of aflR and aflJ, which regulate the activation of the aflatoxin gene cluster. In addition to these findings, we observed that deletion of veA reduced conidiation both on the culture medium and on peanut seed. The fact that veA is necessary for conidiation, production of resistant structures, and aflatoxin biosynthesis makes veA a good candidate gene to control aflatoxin biosynthesis or fungal development and in this way to greatly decrease its devastating impact on health and the economy.     

In vivo functions of the γ-butyrolactone autoregulator receptor in <Emphasis Type="Italic">Streptomyces ambofaciens</Emphasis> producing spiramycin

A gene encoding a γ-butyrolactone autoregulator receptor was cloned in to E. coli from Streptomyces ambofaciens producing spiramycin, a macrolide antibiotic used in both veterinary medicine and human medicine. A 714-bp intact receptor gene (saaR) was obtained by PCR and genomic Southern hybridization with the 100-bp PCR product as a probe. To clarify the in vivo function of saaR, a saaR-disrupted strain was constructed by means of homologous recombination, and phenotypes were compared with those of the wild-type strain. The number of saaR-disruptant spores was 4-fold less than that of the wild-type strain. In addition, saaR deletion from the S. ambofaciens chromosome resulted in complete loss of spiramycin production suggesting that saaR is a rare positive regulator, controlling both spiramycin biosynthesis and sporulation.     

Dysregulated gliotoxin biosynthesis attenuates the production of unrelated biosynthetic gene cluster-encoded metabolites in Aspergillus fumigatus

Gliotoxin is an epipolythiodioxopiperazine (ETP) class toxin, contains a disulfide bridge that mediates its toxic effects via redox cycling and is produced by the opportunistic fungal pathogen Aspergillus fumigatus. The gliotoxin bis-thiomethyltransferase, GtmA, attenuates gliotoxin biosynthesis in A. fumigatus by conversion of dithiol gliotoxin to bis-thiomethylgliotoxin (BmGT). Here we show that disruption of dithiol gliotoxin bis-thiomethylation functionality in A. fumigatus results in significant remodelling of the A. fumigatus secondary metabolome upon extended culture. RP-HPLC and LC–MS/MS analysis revealed the reduced production of a plethora of unrelated biosynthetic gene cluster-encoded metabolites, including pseurotin A, fumagillin, fumitremorgin C and tryprostatin B, occurs in A. fumigatus ΔgtmA upon extended incubation. Parallel quantitative proteomic analysis of A. fumigatus wild-type and ΔgtmA during extended culture revealed cognate abundance alteration of proteins encoded by relevant biosynthetic gene clusters, allied to multiple alterations in hypoxia-related proteins. The data presented herein reveal a previously concealed functionality of GtmA in facilitating the biosynthesis of other BGC-encoded metabolites produced by A. fumigatus.     

Identification of <Emphasis Type="Italic">mokB</Emphasis> involved in monacolin K biosynthesis in <Emphasis Type="Italic">Monascus pilosus</Emphasis>

Monacolin K (MK), which is widely used as an antihypercholesterolemia medicine, is produced as a fungal secondary metabolite through the polyketide pathway. The MK biosynthetic gene cluster proposed for Monascus pilosus BCRC38072 was also identified in M. pilosus NBRC4480. The mokB gene, located at the end of the putative gene cluster and possibly encoding polyketide synthase, was disrupted. The mokB disruptant did not produce MK, but accumulated an intermediate that was confirmed to be monacolin J, indicating that mokB encodes the polyketide synthase responsible for the biosynthesis of side-chain diketide moiety.     

An integrative analysis of four CESA isoforms specific for fiber cellulose production between Gossypium hirsutum and Gossypium barbadense

Cotton fiber is an excellent model system of cellulose biosynthesis; however, it has not been widely studied due to the lack of information about the cellulose synthase (CESA) family of genes in cotton. In this study, we initially identified six full-length CESA genes designated as GhCESA5–GhCESA10. Phylogenetic analysis and gene co-expression profiling revealed that CESA1, CESA2, CESA7, and CESA8 were the major isoforms for secondary cell wall biosynthesis, whereas CESA3, CESA5, CESA6, CESA9, and CESA10 should involve in primary cell wall formation for cotton fiber initiation and elongation. Using integrative analysis of gene expression patterns, CESA protein levels, and cellulose biosynthesis in vivo, we detected that CESA8 could play an enhancing role for rapid and massive cellulose accumulation in Gossypium hirsutum and Gossypium barbadense. We found that CESA2 displayed a major expression in non-fiber tissues and that CESA1, a housekeeping gene like, was predominantly expressed in all tissues. Further, a dynamic alteration was observed in cell wall composition and a significant discrepancy was observed between the cotton species during fiber elongation, suggesting that pectin accumulation and xyloglucan reduction might contribute to cell wall transition. In addition, we discussed that callose synthesis might be regulated in vivo for massive cellulose production during active secondary cell wall biosynthesis in cotton fibers.