Influence of Cmr1 in the Regulation of Antioxidant Function Melanin Biosynthesis in Aureobasidium pullulans.

We have successfully identified the transcription factor Cmr1 from the fungus Aureobasidium pullulans Hit-lcy3T, which regulates melanin biosynthesis genes. Bioinformatics analysis revealed that the Cmr1 gene encodes a protein of 945 amino acids, containing two Cys2His2 zinc finger domains and a Zn(II)2Cys6 binuclear cluster domain located at the N-terminus of Cmr1. To investigate the function of the Cmr1 gene, we performed gene knockout and overexpression experiments. Our results showed that Cmr1 is a key regulator of melanin synthesis in Hit-lcy3T, and its absence caused developmental defects. Conversely, overexpression of Cmr1 significantly increased the number of chlamydospores in Hit-lcy3T and improved melanin production. RT-qPCR analysis further revealed that overexpression of Cmr1 enhanced the expression of several genes involved in melanin biosynthesis, including Cmr1, PKS, SCD1, and THR1. Melanin extracted from the Hit-lcy3T was characterized using UV and IR spectroscopy. Furthermore, we assessed the antioxidant properties of Hit-lcy3T melanin and found that it possesses strong scavenging activity against DPPH·, ABTS·, and OH·, but weaker activity against O2-·. These findings suggest that Hit-lcy3T melanin holds promise for future development as a functional food additive.

The role of pH transcription factor Appacc in upregulation of pullulan biosynthesis in Aureobasidium pullulans using potato waste as a substrate.

pH is one of the important environmental factors affecting the growth, development and secondary metabolites of fungi. To better utilize potato waste for the production of pullulan by fermentation, in this study, the amino acid sequence and structural domain of pH transcription factor Appacc were analyzed using the bioinformatics methods. Appacc showed three typically conserved zinc finger domains, with the closest homology to Zymoseptoria brevis. The function of Appacc was characterized by ΔAppacc and OEXpacc mutants. The mycelium growth of ΔApacc mutants was inhibited, especially, under alkaline conditions. Furthermore, the pullulan production of ΔAppacc mutant was reduced and the expression of pullulan synthetic genes also decreased. Moreover, the OEXpacc mutant further demonstrated that pacc could regulate the expression of pullulan synthesis genes. The yield of pullulan polysaccharide increased from 13.6 g/L to 17.8 g/L by direct fermentation without changing the pH of potato waste. These results suggest that Appacc played a vital role in the growth of Aureobasidium pullulans and that the production of pullulan from potato waste can be increased by overexpression of pacc gene.

Area Gene Regulates the Synthesis of ß-Glucan with Antioxidant Activity in the Aureobasidium pullulans.

The ability of the fungus to regulate metabolism on various nitrogen sources makes it survive and metabolize in different environments. The biomass and the ß-glucan yield of Aureobasidium pullulans are closely associated with the nitrogen source. This study found the only GATA nitrogen source activation regulating factor Area in HIT-LCY3. In order to testify the Area function, we amplified its conserved domain to build a silencing vector and used the RNAi to obtain the Area silent strain, and then explored its effect on the phenotype of A. pullulans and the yield of ß-glucan. We found that the biomass and ß-glucan yield of the silent strain decreased significantly after culturing with different nitrogen sources, in particular when using sodium nitrate and glutamate as the source. However, the ß-glucan yield increased significantly after overexpression of Area, reaching 5.2 g/L when glutamine was the nitrogen source. In addition, the strain morphology changed as well under different nitrogen sources. At last, we investigated the antioxidant activity in vitro of ß-glucan and found that it has a significant clearance effect on OH·, DPPH·, and ABTS·, being best with ABTS. Therefore, this study believed that the Area gene has a certain regulation function on the synthesis of ß-glucan with antioxidant activity.

Identification of a Novel Pleiotropic Transcriptional Regulator Involved in Sporulation and Secondary Metabolism Production in Chaetomium globosum.

Chaetoglobosin A (CheA), a well-known macrocyclic alkaloid with prominently highly antimycotic, antiparasitic, and antitumor properties, is mainly produced by Chaetomium globosum. However, a limited understanding of the transcriptional regulation of CheA biosynthesis has hampered its application and commercialization in agriculture and biomedicine. Here, a comprehensive study of the CgXpp1 gene, which encodes a basic helix-loop-helix family regulator with a putative role in the regulation of fungal growth and CheA biosynthesis, was performed by employing CgXpp1-disruption and CgXpp1-complementation strategies in the biocontrol species C. globosum. The results suggest that the CgXpp1 gene could be an indirect negative regulator in CheA production. Interestingly, knockout of CgXpp1 considerably increased the transcription levels of key genes and related regulatory factors associated with the CheA biosynthetic. Disruption of CgXpp1 led to a significant reduction in spore production and attenuation of cell development, which was consistent with metabolome analysis results. Taken together, an in-depth analysis of pleiotropic regulation influenced by transcription factors could provide insights into the unexplored metabolic mechanisms associated with primary and secondary metabolite production.

New insight into the production improvement and resource generation of chaetoglobosin A in Chaetomium globosum.

Chaetoglobosin A is a complex macrocyclic alkaloid with potent antimycotic, antiparasitic and antitumor properties. However, the low output and high cost of chaetoglobosin A biosynthesis have hampered the application and commercialization of chaetoglobosin A in agriculture and biomedicine. Here, the CgMfs1 gene, which encodes the major facilitator superfamily secondary transporter, was identified based on bioinformatics analysis, and an intensive study of its effects on chaetoglobosin A biosynthesis and secretion was performed using CgMfs1-silencing and CgMfs1-overexpression strategies. Inactivation of CgMfs1 caused a notable decrease in chaetoglobosin A yield from 58.66 mg/L to 19.95 mg/L (MFS1-3) and 17.13 mg/L (MFS1-4). The use of an efficient expression plasmid in Chaetomium globosum W7 to generate the overexpression mutant OEX13 resulted in the highest chaetoglobosin A increase to 298.77 mg/L. Interestingly, the transcription level of the polyketide synthase gene significantly fluctuated with the change in CgMfs1, confirming that the predicted efflux gene CgMfs1 could play a crucial role in chaetoglobosin A transportation. Effective efflux of chaetoglobosin A could possibly alleviate feedback inhibition, resulting in notable increase in the expression of the polyketide synthase gene. Furthermore, we utilized cornstalk as the fermentation substrate to produce chaetoglobosin A, and scanning electron microscopy and Fourier transform-infrared spectroscopy revealed that the strain OEX13 could well degrade cornstalk, presenting significant increases in the chaetoglobosin A yield, when compared with that produced by the wild-type strain (from 40.32 to 191.90 mg/L). Thus, this research provides a novel analogous engineering strategy for the construction of high-yielding strain and offers new insight into large-scale chaetoglobosin A production.

Screening and identification of a strain of Aureobasidium pullulans and its application in potato starch industrial waste.

Pullulan and melanin have become important secondary metabolites that are now widely studied. In this study, a strain of Aureobasidium pullulans HIT-LCY3T was used to ferment potato starch industrial waste to produce pullulan and melanin. After optimization, the culture conditions for the fermentation medium were obtained: inoculum age of 48 h, initial pH of 6.0, inoculation quantity of 1.5%, temperature of 26 °C, fermentation time of 5 d and speed of 160 rpm. Under these conditions, the yield of pullulan was 23.47 g/L with a molecular weight (MW) of 1.21 × 106 Da and the yield of melanin was 18.98 g/L. In addition, the adaptive evolution could significantly increase the yield of pullulan and melanin and the air-floating fermenters was more conductive to product accumulation. Through the 5 L small-scale test and 1000 L pilot test, the yield of pullulan reached 16.52 g/L with molecular weight of 0.92 × 106 Da and the yield of melanin was 12.08 g/L at the trial production of 30,000 L. This work could provide strong support for industrial production and new guidance for waste utilization and environmental protection.

CgVeA, a light signaling responsive regulator, is involved in regulation of chaetoglobosin A biosynthesis and conidia development in Chaetomium globosum.

Cytochalasans, with diverse structures and pharmacological activities, are a class of compounds containing isoindolinone moieties fused to the tricyclic or tetracyclic ring system. Chaetoglobosin A (cheA), mainly produced by Chaetomium globosum, is the most abundant cytochalasan. However, limited understanding of transcriptional regulation of morphological development and cheA biosynthesis in C. globosum has hindered cheA application in agriculture and biomedical field. This study examined the regulatory role of CgVeA gene in C. globosum. CgVeA had significant effect on secondary metabolites production in C. globosum, similar to that reported in other filamentous fungi. Inactivation of CgVeA caused an obvious decrease in cheA production from 51.32 to 19.76 mg/L under dark conditions. In contrast, CgVeA overexpression resulted in a dramatic increase in cheA production, reaching 206.59 mg/L under light conditions, which was higher than that noted under dark condition. The RT-qPCR results confirmed that CgVeA, as a light responsive regulator, positively regulated cheA biosynthesis by controlling the expression of core genes of the cheA biosynthetic gene cluster and other relevant regulators. Electrophoretic mobility shift assays proved that CgVeA directly regulated LaeA, cheR, and p450, and indirectly regulated PKS. Moreover, CgVeA had a significant effect on the regulation of asexual spores production. When compared with wild-type C. globosum, CgVeA-silenced and CgVeA overexpression mutants presented remarkable differences in sporulation, irrespective of light or dark condition. Besides, CgVeA expression was speculated to negatively regulate spore formation. These findings illustrated the regulatory mechanism of a hypothetical global regulator, CgVeA, in C. globosum, suggesting its potential application in industrial-scale cheA biosynthesis.

Streptomyces luteolifulvus sp. nov., a novel actinomycete isolated from soil in Nanjing, China.

During the investigation of exploring potential sources of novel species and natural bioactives, a novel actinomycete, designated strain HIT-DPA4T, was isolated from a soil sample, which was collected from Nanjing, Jiangsu Province, PR China and characterized using a polyphasic approach. On the basis of 16S rRNA gene sequence similarities and the result of phylogenetic analysis, strain HIT-DPA4T was most closely related to Streptomyces cyaneus CGMCC 4.1671 T, and shared the highest sequence similarity of 98.76%. In addition, the cell walls of the species HIT-DPA4T contained LL-diaminopimelic acid as the diagnostic diamino acid and the whole-cell hydrolysates were identified as glucose and ribose, and the principal phospholipids were found to be diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol mannoside and phosphatidylmonomethylethanolamine. MK-9(H6) and MK-9(H4) were predominant menaquinones; and C16:0, anteiso-C15:0 and C15:0 as major cellular fatty acids of the organism HIT-DPA4T. Gene Ontology database analysis and antiSMASH server predicted results displayed that strain HIT-DPA4T was a promising classification units, which has various types of functions and contains multiple biosynthetic gene clusters with the similarity more than 80%. Multilocus sequence analysis (MLSA) of five housekeeping genes (atpD, gyrB, recA, rpoB and trpB) illustrated that Streptomyces luteolifulvus formed a separate branch in the genus Streptomyces. However, a combination of low level of DNA-DNA relatedness and physiological properties indicated that strain HIT-DPA4T can be distinguished from its phylogenetically related species Streptomyces cyaneus CGMCC 4.1671 T. Moreover, gene synteny research could be further differed organism HIT-DPA4T from similarity species. Therefore, the strain is concluded to represent a novel species of the genus Streptomyces, for which the name Streptomyces luteolifulvus sp. nov. is proposed. The type strain is HIT-DPA4T (= CGMCC 4.7558 T = TISTR 2751 T).

Requirement of LaeA for sporulation, pigmentation and secondary metabolism in Chaetomium globosum.

The global regulator LaeA has been confirmed to govern the production of secondary metabolites in fungi. Herein, we examined the role of LaeA in Chaetomium globosum. Similarly as observed in other filamentous, CgLaeA had a significant effect on the secondary metabolism. The ΔCglaeA mutant strain did not exhibit chaetoglobosin A, whereas its production was restored in the CglaeAC strain. In addition, CglaeA overexpression led to an increase in chaetoglobosin A production. Transcriptional examination of the mutants indicated that CgLaeA positively regulated the expression of pathway-specific transcription factor CgcheR, while another global regulator CgvelB was negatively regulated by CgLaeA. Furthermore, CgLaeA also affected the morphological phenotypes of fungi. The ΔCglaeA mutant strains exhibited decreased sporulation and pigmentation compared with the wild-type strain, whereas the phenotypes were restored in the CglaeAC strain. Moreover, OE::CglaeA exhibited increased levels of sporulation and pigmentation. Moreover, inhibition activity against phytopathogenic fungi affected by decreased mycotoxin production of the ΔCglaeA mutant strain.

Functional analysis of a chaetoglobosin A biosynthetic regulator in Chaetomium globosum.

Cytochalasins are a group of fungal secondary metabolites with diverse structures and bioactivities, including chaetoglobosin A production. Chaetoglobosin A is produced by Chaetomium globosum and has potential antifungal activity. Bioinformatics analysis of the chaetoglobosin A gene cluster (che) showed it that consists of nine open reading frames, including those encoding polyketide synthases (PKSs), PKS extender units, post-PKS modifications, and proposed regulators. Here, the role of the CgcheR regulator was investigated using gene disruption experiments. The CgcheR disruptant (ΔCgcheR) completely abolished the production of chaetoglobosin A, which was restored by the introduction of a copy of the wild-type CgcheR gene, suggesting that CgcheR is involved in chaetoglobosin A biosynthesis. A transcriptional analysis of the CgcheR disruptant indicated that CgCheR activates the transcription of chaetoglobosin biosynthetic genes in a pathway-specific manner. Furthermore, constitutive overexpression of CgcheR significantly improved the production of chaetoglobosin A from 52 to 260 mg/L. Surprisingly, CgcheR also played a critical role in sporulation; the CgcheR disruptant lost the ability to produce spores, suggesting that the regulator modulates cellular development. Our results not only shed light on the regulation of chaetoglobosin A biosynthesis, but also indicate a relationship between secondary metabolism and fungal morphogenesis.

ATP-Binding Cassette Transporter Regulates N,N'-diacetylchitobiose Transportation and Chitinase Production in Trichoderma asperellum T4.

ATP-binding cassette (ABC) transporters are a superfamily of proteins that transport nutrient substances and secondary metabolites through cell membranes. They also act as an uptake system for N,N'-diacetylchitobiose (GlcNAc)2 in Streptomyces coelicolor. (GlcNAc)2 is an important inducer of chitinase. However, whether the ABC transporter in Trichoderma spp. is also responsible for (GlcNAc)2 uptake and chitinase induction has not yet been confirmed. In this study, we applied RNA interference and overexpression technologies to alter the expression level of the ABC-B transporter in order to detect changes in its transportation ability and the expression level of inducible endo-chitinase ECH42-an important biocontrol enzyme in Trichoderma asperellum. The results revealed that, after interference with the expression of the ABC-B transporter, T. asperellum T4 was only able to grow normally when glucose was the only carbon source. Compared with the wild-type, the efficiency of (GlcNAc)2 by the overexpression strain evidently increased, along with the activity level of ECH42. In conclusion, one of the functions of the ABC-B transporter in T. asperellum is the uptake and transport of (GlcNAc)2 into cells, and chitobiose is a strong inducer of ECH42 in T. asperellum T4.