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.     

The global regulator LaeA controls penicillin biosynthesis, pigmentation and sporulation, but not roquefortine C synthesis in Penicillium chrysogenum

The biosynthesis of the beta-lactam antibiotic penicillin is an excellent model for the study of secondary metabolites produced by filamentous fungi due to the good background knowledge on the biochemistry and molecular genetics of the beta-lactam producing microorganisms. The three genes (pcbAB, pcbC, penDE) encoding enzymes of the penicillin pathway in Penicillium chrysogenum are clustered, but no penicillin pathway-specific regulators have been found in the genome region that contains the penicillin gene cluster. The biosynthesis of this beta-lactam is controlled by global regulators of secondary metabolism rather than by a pathway-specific regulator. In this work we have identified the gene encoding the secondary metabolism global regulator LaeA in P. chrysogenum (PcLaeA), a nuclear protein with a methyltransferase domain. The PclaeA gene is present as a single copy in the genome of low and high-penicillin producing strains and is not located in the 56.8-kb amplified region occurring in high-penicillin producing strains. Overexpression of the PclaeA gene gave rise to a 25% increase in penicillin production. PclaeA knock-down mutants exhibited drastically reduced levels of penicillin gene expression and antibiotic production and showed pigmentation and sporulation defects, but the levels of roquefortine C produced and the expression of the dmaW involved in roquefortine biosynthesis remained similar to those observed in the wild-type parental strain. The lack of effect on the synthesis of roquefortine is probably related to the chromatin arrangement in the low expression roquefortine promoters as compared to the bidirectional pbcAB-pcbC promoter region involved in penicillin biosynthesis. These results evidence that PcLaeA not only controls some secondary metabolism gene clusters, but also asexual differentiation in P. chrysogenum.     

LaeA在丝状真菌次级代谢、生长发育和其他重要生物过程中的作用

LaeA是在构巢曲霉中首次鉴定的一种全局调控因子,其同源蛋白在丝状真菌中广泛存在,具有高度的保守性。LaeA及其同源蛋白序列存在S-腺苷甲硫氨酸结合基序,是一种甲基转移酶,可能影响组蛋白修饰,导致染色体结构的改变,进而调控一系列基因的表达。大量研究表明,LaeA及其同源蛋白参与调控丝状真菌多种次级代谢产物的生物合成,影响丝状真菌的生长发育和形态分化,甚至在丝状真菌生产有机酸和一些工业酶的过程中也发挥着重要作用。本文综述了LaeA及其同源蛋白的作用机制,以及该蛋白在丝状真菌次级代谢、生长发育和其他重要生物过程中的作用,并对存在的问题及应用前景进行讨论与展望。     

Key role of LaeA and velvet complex proteins on expression of β-lactam and PR-toxin genes in <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis>: cross-talk regulation of secondary metabolite pathways

Penicillium chrysogenum is an excellent model fungus to study the molecular mechanisms of control of expression of secondary metabolite genes. A key global regulator of the biosynthesis of secondary metabolites is the LaeA protein that interacts with other components of the velvet complex (VelA, VelB, VelC, VosA). These components interact with LaeA and regulate expression of penicillin and PR-toxin biosynthetic genes in P. chrysogenum. Both LaeA and VelA are positive regulators of the penicillin and PR-toxin biosynthesis, whereas VelB acts as antagonist of the effect of LaeA and VelA. Silencing or deletion of the laeA gene has a strong negative effect on penicillin biosynthesis and overexpression of laeA increases penicillin production. Expression of the laeA gene is enhanced by the P. chrysogenum autoinducers 1,3 diaminopropane and spermidine. The PR-toxin gene cluster is very poorly expressed in P. chrysogenum under penicillin-production conditions (i.e. it is a near-silent gene cluster). Interestingly, the downregulation of expression of the PR-toxin gene cluster in the high producing strain P. chrysogenum DS17690 was associated with mutations in both the laeA and velA genes. Analysis of the laeA and velA encoding genes in this high penicillin producing strain revealed that both laeA and velA acquired important mutations during the strain improvement programs thus altering the ratio of different secondary metabolites (e.g. pigments, PR-toxin) synthesized in the high penicillin producing mutants when compared to the parental wild type strain. Cross-talk of different secondary metabolite pathways has also been found in various Penicillium spp.: P. chrysogenum mutants lacking the penicillin gene cluster produce increasing amounts of PR-toxin, and mutants of P. roqueforti silenced in the PR-toxin genes produce large amounts of mycophenolic acid. The LaeA-velvet complex mediated regulation and the pathway cross-talk phenomenon has great relevance for improving the production of novel secondary metabolites, particularly of those secondary metabolites which are produced in trace amounts encoded by silent or near-silent gene clusters.     

Role of alternative sigma factor 54 (RpoN) from Vibrio anguillarum M3 in protease secretion, exopolysaccharide production, biofilm formation, and virulence

The sigma factor σ⁵⁴ (RpoN) is an important regulator of bacterial response to environmental stresses. Here, we demonstrate the roles of RpoN in Vibrio anguillarum M3 by comparative investigation of physiological phenotypes and virulence of the wild-type, an rpoN mutant, and an rpoN complemented strain. Disruption of rpoN was found to decrease biofilm formation, production of exopolysaccharides, and production of the metalloproteases EmpA and PrtV. Injection experiments in fish showed that the M3 ΔrpoN mutant was attenuated in virulence when administrated either by intramuscular injection or by immersion challenge. Slower proliferation of the mutant in fish was also observed. Complementation of the mutant strain with rpoN restored some of the phenotypes to wild-type levels. RpoN was involved in regulation of some virulence-associated genes, as shown by real-time quantitative reverse PCR analysis. These results revealed a pleiotropic regulatory role of RpoN in biofilm formation, production of proteases and exopolysaccharides, and virulence in V. anguillarum M3.     

Fungus-Specific Sirtuin HstD Coordinates Secondary Metabolism and Development through Control of LaeA

The sirtuins are members of the NAD⁺-dependent histone deacetylase family that contribute to various cellular functions that affect aging, disease, and cancer development in metazoans. However, the physiological roles of the fungus-specific sirtuin family are still poorly understood. Here, we determined a novel function of the fungus-specific sirtuin HstD/Aspergillus oryzae Hst4 (AoHst4), which is a homolog of Hst4 in A. oryzae yeast. The deletion of all histone deacetylases in A. oryzae demonstrated that the fungus-specific sirtuin HstD/AoHst4 is required for the coordination of fungal development and secondary metabolite production. We also show that the expression of the laeA gene, which is the most studied fungus-specific coordinator for the regulation of secondary metabolism and fungal development, was induced in a ΔhstD strain. Genetic interaction analysis of hstD/Aohst4 and laeA clearly indicated that HstD/AoHst4 works upstream of LaeA to coordinate secondary metabolism and fungal development. The hstD/Aohst4 and laeA genes are fungus specific but conserved in the vast family of filamentous fungi. Thus, we conclude that the fungus-specific sirtuin HstD/AoHst4 coordinates fungal development and secondary metabolism via the regulation of LaeA in filamentous fungi.     

The α-1,6-mannosyltransferase VdOCH1 plays a major role in microsclerotium formation and virulence in the soil-borne pathogen Verticillium dahliae

Sunflower yellow wilt is a widespread and destructive disease caused by the soil-borne pathogen Verticillium dahliae (V. dahliae). To better understand the pathogenesis mechanism of V. dahliae in sunflower, T-DNA insertion library was generated via Agrobacterium tumefaciens mediated transformation system (ATMT). Eight hundred positive transformants were obtained. Transformants varied in colony morphology, growth rate, conidia production and pathogenicity in sunflower compared to the wild type strain. A mutant, named VdGn3-L2, was chosen for further analysis based on its deprivation on microsclerotia formation. The flanking sequence of T-DNA insertion site of VdGn3-L2 was identified via hiTAIL-PCR, and the interrupted gene encoded an initiation-specific α-1, 6-mannosyltransferase, named as VdOCH1. The deletion mutant ΔVdOCH1 was impaired in certain characteristics such as fungal growth, conidia production, and microsclerotia formation. Also, ΔVdOCH1 mutants were more sensitive to the cell wall perturbing reagents, such as SDS and Congo red, lost their penetration ability through cellophane membrane, and exhibited dramatically decreased pathogenicity to sunflower. The impaired phenotypes could be restored to the wild type level by complementation of the deletion mutant with full-length VdOCH1 gene. In conclusion, VdOCH1, encoded α-1,6-mannosyltransferase, manipulating the biological characteristics, microsclerotia formation and pathogenic ability of V. dahliae in sunflower.     

The global regulator LaeA controls production of citric acid and endoglucanases in <Emphasis Type="Italic">Aspergillus carbonarius</Emphasis>

The global regulatory protein LaeA is known for regulating the production of many kinds of secondary metabolites in Aspergillus species, as well as sexual and asexual reproduction, and morphology. In Aspergillus carbonarius, it has been shown that LaeA regulates production of ochratoxin. We have investigated the regulatory effect of LaeA on production of citric acid and cellulolytic enzymes in A. carbonarius. Two types of A. carbonarius strains, having laeA knocked out or overexpressed, were constructed and tested in fermentation. The knockout of laeA significantly decreased the production of citric acid and endoglucanases, but did not reduce the production of beta-glucosidases or xylanases. The citric acid accumulation was reduced with 74–96 % compared to the wild type. The endoglucanase activity was reduced with 51–78 %. Overexpression of LaeA seemed not to have an effect on citric acid production or on cellulose or xylanase activity.     

The Putative Protein Methyltransferase LAE1 of Trichoderma atroviride Is a Key Regulator of Asexual Development and Mycoparasitism

In Ascomycota the protein methyltransferase LaeA is a global regulator that affects the expression of secondary metabolite gene clusters, and controls sexual and asexual development. The common mycoparasitic fungus Trichoderma atroviride is one of the most widely studied agents of biological control of plant-pathogenic fungi that also serves as a model for the research on regulation of asexual sporulation (conidiation) by environmental stimuli such as light and/or mechanical injury. In order to learn the possible involvement of LAE1 in these two traits, we assessed the effect of deletion and overexpression of lae1 gene on conidiation and mycoparasitic interaction. In the presence of light, conidiation was 50% decreased in a Δ lae1 and 30–50% increased in lae1-overexpressing (OElae1) strains. In darkness, Δ lae1 strains did not sporulate, and the OElae1 strains produced as much spores as the parent strain. Loss-of-function of lae1 also abolished sporulation triggered by mechanical injury of the mycelia. Deletion of lae1 also increased the sensitivity of T. atroviride to oxidative stress, abolished its ability to defend against other fungi and led to a loss of mycoparasitic behaviour, whereas the OElae1 strains displayed enhanced mycoparasitic vigor. The loss of mycoparasitic activity in the Δ lae1 strain correlated with a significant underexpressionn of several genes normally upregulated during mycoparasitic interaction (proteases, GH16 ß-glucanases, polyketide synthases and small cystein-rich secreted proteins), which in turn was reflected in the partial reduction of formation of fungicidal water soluble metabolites and volatile compounds. Our study shows T. atroviride LAE1 is essential for asexual reproduction in the dark and for defense and parasitism on other fungi.