Screening and genetic engineering of marine-derived Aspergillus terreus for high-efficient production of lovastatin.

BACKGROUND: Lovastatin has widespread applications thanks to its multiple pharmacological effects. Fermentation by filamentous fungi represents the major way of lovastatin production. However, the current lovastatin productivity by fungal fermentation is limited and needs to be improved. RESULTS: In this study, the lovastatin-producing strains of Aspergillus terreus from marine environment were screened, and their lovastatin productions were further improved by genetic engineering. Five strains of A. terreus were isolated from various marine environments. Their secondary metabolites were profiled by metabolomics analysis using Ultra Performance Liquid Chromatography-Mass spectrometry (UPLC-MS) with Global Natural Products Social Molecular Networking (GNPS), revealing that the production of secondary metabolites was variable among different strains. Remarkably, the strain of A. terreus MJ106 could principally biosynthesize the target drug lovastatin, which was confirmed by High Performance Liquid Chromatography (HPLC) and gene expression analysis. By one-factor experiment, lactose was found to be the best carbon source for A. terreus MJ106 to produce lovastatin. To improve the lovastatin titer in A. terreus MJ106, genetic engineering was applied to this strain. Firstly, a series of strong promoters was identified by transcriptomic and green fluorescent protein reporter analysis. Then, three selected strong promoters were used to overexpress the transcription factor gene lovE encoding the major transactivator for lov gene cluster expression. The results revealed that compared to A. terreus MJ106, all lovE over-expression mutants exhibited significantly more production of lovastatin and higher gene expression. One of them, LovE-b19, showed the highest lovastatin productivity at a titer of 1512 mg/L, which represents the highest production level reported in A. terreus. CONCLUSION: Our data suggested that combination of strain screen and genetic engineering represents a powerful tool for improving the productivity of fungal secondary metabolites, which could be adopted for large-scale production of lovastatin in marine-derived A. terreus.

Development of a rapid detection method for Karenia mikimotoi by using CRISPR-Cas12a.

Harmful algal blooms (HABs), mainly formed by dinoflagellates, have detrimental effects on marine ecosystems and public health. Therefore, detecting HABs is crucial for early warning and prevention of HABs as well as the mitigation of their adverse effects. Although various methods, such as light microscopy, electron microscopy, real-time PCR, and microarrays, have already been established for the detection of HABs, they are still cumbersome to be exploited in the field. Therefore, rapid nucleic detection methods such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP)-lateral flow dipstick (LFD) have been developed for monitoring bloom-forming algae. However, the CRISPR/Cas-based detection of HABs has yet to be applied to this field. In this study, we developed a method for detecting Karenia mikimotoi (K. mikimotoi), a typical ichthyotoxic dinoflagellate responsible for global blooms. Our method utilized Cas12a from Lachnospiraceae bacterium ND2006 (LbCas12a) to target and cleave the internal transcribed spacer (ITS) of K. mikimotoi, guided by RNA. We leveraged the target-activated non-specific single-stranded deoxyribonuclease cleavage activity of LbCas12a to generate signals that can be detected using fluorescence-read machines or LFDs. By combining RPA and LbCas12a with reporters, we significantly enhanced the sensitivity, enabling the detection of ITS-harboring plasmids at concentrations as low as 9.8 aM and genomic DNA of K. mikimotoi at levels as low as 3.6 × 10-5 ng/µl. Moreover, we simplified the genomic DNA extraction method using cellulose filter paper (CFP) by directly eluting the DNA into RPA reactions, reducing the extraction time to < 30 s. The entire process, from genomic DNA extraction to result reporting, takes less than an hour, enabling the identification of nearly a single cell. In conclusion, our method provided an easy, specific, and sensitive approach for detecting K. mikimotoi, offering the potential for efficient monitoring and management of K. mikimotoi blooms.

The Histone Deacetylase HstD Regulates Fungal Growth, Development and Secondary Metabolite Biosynthesis in Aspergillus terreus.

Histone acetylation modification significantly affects secondary metabolism in filamentous fungi. However, how histone acetylation regulates secondary metabolite synthesis in the lovastatin (a lipid-lowering drug) producing Aspergillus terreus remains unknown because protein is involved and has been identified in this species. Here, the fungal-specific histone deacetylase gene, hstD, was characterized through functional genomics in two marine-derived A. terreus strains, Mj106 and RA2905. The results showed that the ablation of HstD resulted in reduced mycelium growth, less conidiation, and decreased lovastatin biosynthesis but significantly increased terrein biosynthesis. However, unlike its homologs in yeast, HstD was not required for fungal responses to DNA damage agents, indicating that HstD likely plays a novel role in the DNA damage repair process in A. terreus. Furthermore, the loss of HstD resulted in a significant upregulation of H3K56 and H3K27 acetylation when compared to the wild type, suggesting that epigenetic functions of HstD, as a deacetylase, target H3K27 and H3K56. Additionally, a set of no-histone targets with potential roles in fungal growth, conidiation, and secondary metabolism were identified for the first time using acetylated proteomic analysis. In conclusion, we provide a comprehensive analysis of HstD for its targets in histone or non-histone and its roles in fungal growth and development, DNA damage response, and secondary metabolism in A. terreus.

Sclerotioloids A-C: Three New Alkaloids from the Marine-Derived Fungus Aspergillus sclerotiorum ST0501.

Alkaloids, as one of the largest classes of natural products with diverse structures, are an important source of innovative medicines. Filamentous fungi, especially those derived from the marine environment, are one of the major producers of alkaloids. In this study, three new alkaloids, sclerotioloids A-C (1-3), along with six known analogs (4-9), were obtained under the guidance of the MS/MS-based molecular networking from the marine-derived fungus, Aspergillus sclerotiorum ST0501, collected from the South China Sea. Their chemical structures were elucidated by comprehensive analysis of the spectroscopic data, including 1D and 2D NMR and HRESIMS. Additionally, the configuration of compound 2 was unambiguously determined by X-ray single crystal diffraction, and that of compound 3 was determined by the TDDFT-ECD approach. Sclerotioloid A (1) represents the first example of 2,5-diketopiperazine alkaloid with a rare terminal alkyne. Sclerotioloid B (2) showed the inhibition of NO production induced by lipopolysaccharide (LPS), with an inhibition rate of 28.92% higher than that of dexamethasone (25.87%). These results expanded the library of fungal-derived alkaloids and further prove the potential of marine fungi in the generation of alkaloids with new scaffolds.

Bioactive Alkaloids from the Marine-Derived Fungus Metarhizium sp. P2100.

The Metarhizium fungal species are considered the prolific producers of bioactive secondary metabolites with a variety of chemical structures. In this study, the biosynthetic potential of marine-derived fungus Metarhizium sp. P2100 to produce bioactive alkaloids was explored by using the one strain many compounds (OSMAC) strategy. From the rice solid medium (mixed with glucose peptone and yeast broth (GPY)), wheat solid medium (mixed with Czapek) and GPY liquid medium, one rare N-butenone spiroquinazoline alkaloid, N-butenonelapatin A (1), together with nine known compounds (2-10), were isolated and identified. Their structures were elucidated by analysis of the comprehensive spectroscopic data, including 1D and 2D NMR and HRESIMS, and the absolute configuration of 1 was determined by a single-crystal X-ray crystallographic experiment. N-butenonelapatin A (1) represents the first example of N-butenone spiroquinazoline with a rare α, ß-unsaturated ketone side chain in the family of spiroquinazoline alkaloids. Compound 4 displayed antibacterial activity against Vibrio vulnificus MCCC E1758 with a minimum inhibitory concentration (MIC) value of 6.25 µg/mL. Compound 7 exhibited antibacterial activities against three aquatic pathogenic bacteria, including V. vulnificus MCCC E1758, V. rotiferianus MCCC E385 and V. campbellii MCCC E333 with the MIC values of 12.5, 12.5 and 6.25 µg/mL, respectively. Compounds 3 and 6 demonstrated anti-inflammatory activity against NO production induced by lipopolysaccharide (LPS) with the IC50 values of 37.08 and 37.48 µM, respectively. In addition, compound 1 showed weak inhibitory activity against the proliferation of tumor cell lines A-375 and HCT 116. These findings further demonstrated that fungi of the Metarhizium species harbor great potentials in the synthesis of a variety of bioactive alkaloids.

Induction of Secondary Metabolite Biosynthesis by Deleting the Histone Deacetylase HdaA in the Marine-Derived Fungus Aspergillus terreus RA2905.

Aspergillus terreus is well-known for its ability to biosynthesize valuable pharmaceuticals as well as structurally unique secondary metabolites. However, numerous promising cryptic secondary metabolites in this strain regulated by silent gene clusters remain unidentified. In this study, to further explore the secondary metabolite potential of A. terreus, the essential histone deacetylase hdaA gene was deleted in the marine-derived A. terreus RA2905. The results showed that HdaA plays a vital and negative regulatory role in both conidiation and secondary metabolism. Loss of HdaA in A. terreus RA2905 not only resulted in the improvement in butyrolactone production, but also activated the biosynthesis of new azaphilone derivatives. After scaled fermentation, two new azaphilones, asperterilones A and B (1 and 2), were isolated from ΔhdaA mutant. The planar structures of compounds 1 and 2 were undoubtedly characterized by NMR spectroscopy and mass spectrometry analysis. Their absolute configurations were assigned by circular dichroism spectra analysis and proposed biosynthesis pathway. Compounds 1 and 2 displayed moderate anti-Candida activities with the MIC values ranging from 18.0 to 47.9 µM, and compound 1 exhibited significant cytotoxic activity against human breast cancer cell line MDA-MB-231. This study provides novel evidence that hdaA plays essential and global roles in repressing secondary metabolite gene expression in fungi, and its deletion represents an efficient strategy to mine new compounds from A. terreus and other available marine-derived fungi.

Enhanced production of terrein in marine-derived Aspergillus terreus by refactoring both global and pathway-specific transcription factors.

BACKGROUND: Terrein, a major secondary metabolite from Aspergillus terreus, shows great potentials in biomedical and agricultural applications. However, the low fermentation yield of terrein in wild A. terreus strains limits its industrial applications. RESULTS: Here, we constructed a cell factory based on the marine-derived A. terreus RA2905, allowing for overproducing terrein by using starch as the sole carbon source. Firstly, the pathway-specific transcription factor TerR was over-expressed under the control of a constitutive gpdA promoter of A. nidulans, resulting in 5 to 16 folds up-regulation in terR transcripts compared to WT. As expected, the titer of terrein was improved in the two tested terR OE mutants when compared to WT. Secondly, the global regulator gene stuA, which was demonstrated to suppress the terrein synthesis in our analysis, was deleted, leading to greatly enhanced production of terrein. In addition, LS-MS/MS analysis showed that deletion of StuA cause decreased synthesis of the major byproduct butyrolactones. To achieve an optimal strain, we further refactored the genetic circuit by combining deletion of stuA and overexpression of terR, a higher terrein yield was achieved with a lower background of byproducts in double mutants. In addition, it was also found that loss of StuA (both ΔstuA and ΔstuA::OEterR) resulted in aconidial morphologies, but a slightly faster growth rate than that of WT. CONCLUSION: Our results demonstrated that refactoring both global and pathway-specific transcription factors (StuA and TerR) provides a high-efficient strategy to enhance terrein production, which could be adopted for large-scale production of terrein or other secondary metabolites in marine-derived filamentous fungi.

Development of versatile and efficient genetic tools for the marine-derived fungus Aspergillus terreus RA2905.

Marine-derived Aspergillus terreus produces a variety of structurally novel secondary metabolites, most of which show unique biological activities. However, the lack of efficient genetic tools limits the discovery of new compounds, the elucidation of involved biosynthesis mechanism, as well as the strain engineering efforts. Therefore, in this study, we first established both an effective PEG-mediated chemical transformation system of protoplasts and an electroporation system of conidia in a marine-derived fungus A. terreus RA2905. To overcome the insensitivity of RA2905 to fungicides, the uracil auxotrophy strain (pyrG gene deletion mutant, ΔpyrG) was constructed using PEG-mediated transformation system, and using ΔpyrG as the genetic background, the methyltransferase gene laeA-overexpression transformants were further constructed through both PEG- and electroporation-mediated transformations, which showed enhanced terrein production. Besides, in this study, an efficient CRISPR/Cas9 genome-editing system was established for the first time in A. terreus, and a higher gene deletion efficiency of 71% for APSES transcription factor gene stuA could be achieved when using short homologous arms compared with conventional long homologous ones. In addition, using a non-integrative Cas9 plasmid, another efficient and marker-free genome-editing system was established, which allowing repeatable and unlimited genetic manipulation in A. terreus. Using the marker-free genome-editing system, we successfully developed the ΔpyrGΔku70 double-deletion mutant in RA2905, which could further improve gene deletion efficiency. In conclusion, efficient genetic manipulation systems along with a variety of functional mutants were developed in this study, which would significantly expedite both theoretical and applied researches in not only A. terreus but also other marine-derived filamentous fungi.

Bioactive Alpha-Pyrone and Phenolic Glucosides from the Marine-Derived Metarhizium sp. P2100.

Glycoside compounds have attracted great interest due to their remarkable and multifarious bioactivities. In this study, four hitherto unknown 4-methoxy-ß-D-glucosyl derivatives were obtained and identified from the marine-derived fungus Metarhizium sp. P2100, including three alpha-pyrone glycosides (1-3) and one phenolic glycoside (4). Their planar structures were elucidated by comprehensive spectroscopic analysis, including 1D/2D NMR and HRESIMS. The absolute configurations of 1-3 were determined by a single-crystal X-ray crystallographic experiment, a comparison of the experimental, and a calculated electronic circular dichroism (ECD) spectra, respectively. Compounds 2 and 3 are a pair of rare epimeric pyranoside glycosides at C-7 with a core of aglycone as 2H-pyrone. Compounds 1-4 exhibited weak anti-inflammatory activities. In particular, compounds 1-3 displayed inhibitory activities against α-amylase, showing a potential for the development of a new α-amylase inhibitor for controlling diabetes.

Subchronic toxicity of dietary sulfamethazine and nanoplastics in marine medaka (Oryzias melastigma): Insights from the gut microbiota and intestinal oxidative status.

Antibiotics and nanoplastics are two prevalent pollutants in oceans, posing a great threat to marine ecosystems. As antibiotics and nanoplastics are highly bioconcentrated in lower trophic levels, evaluating their impacts on marine organisms via dietary exposure route is of great importance. In this study, the individual and joint effects of dietborne sulfamethazine (SMZ) and nanoplastic fragments (polystyrene, PS) in marine medaka (Oryzias melastigma) were investigated. After 30 days of dietary exposure, 4.62 mg/g SMZ decreased the Chao1 index (60.86% for females and 26.85% for males) and the Shannon index (68.95% for females and 65.05% for males) and significantly altered the structure of gut microbial communities in both sexes. The female fish exposed to 4.62 mg/g SMZ exhibited higher intestinal sod (43.5%), cat (38.5%) and gpx (39.6%) transcripts, indicating oxidative stress in the gut. PS alone at 3.45 mg/g slightly altered the composition of the gut microbiota. Interestingly, the mixture of SMZ and PS caused more modest effects on the gut microbiota and intestinal antioxidant physiology than the SMZ alone, suggesting that the presence of PS might alleviate the intestinal toxicity of SMZ in a scenario of dietary co-exposure. This study helps better understand the risk of antibiotics and nanoplastics to marine ecosystems.

Genomic and Chemical Investigation of Bioactive Secondary Metabolites From a Marine-Derived Fungus Penicillium steckii P2648.

Marine fungi of the genus Penicillium are rich resources of secondary metabolites, showing a variety of biological activities. Our anti-bacterial screening revealed that the crude extract from a coral-derived fungus Penicillium steckii P2648 showed strong activity against some pathogenic bacteria. Genome sequencing and mining uncovered that there are 28 secondary metabolite gene clusters in P2648, potentially involved in the biosynthesis of antibacterial compounds. Chemical isolation and structural determination suggested citrinin is the dominant component of the crude extracts of P2648, and our further tests confirmed that citrinin showed excellent activities against various pathogenic bacteria. Moreover, the gene cluster containing a homolog of the polyketide synthase CitS was identified as the citrinin biosynthesis gene cluster through genetic analysis. Interestingly, three isoquinoline alkaloids were unexpectedly activated and isolated from the Δcits mutant and structural determination by using high-resolution electron spray ionization mass spectroscopy (HRESIMS), 1D, and 2D NMR. Further antibacterial assays displayed that compounds 1 and 2, but not compound 3, showed moderate activities against two antibiotic-resistant pathogenic bacteria with minimum inhibitory concentration (MIC) of 16-32 µg/ml. In conclusion, our results demonstrated that citrinin and isoquinoline alkaloids represent as the major antibacterial agents in the coral-associated fungus P. steckii P2648, and our genomic and chemical analyses present evidence in support of P. steckii P2648 as a potent natural products source for anti-bacterial drug discovery.

Benzyl Furanones and Pyrones from the Marine-Derived Fungus Aspergillus terreus Induced by Chemical Epigenetic Modification.

Chemical epigenetic modification on a marine-derived fungus Aspergillus terreus RA2905 using a histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), resulted in a significantly changed metabolic profile. A chemical investigation of its ethyl acetate (EtOAc) extract led to the isolation of a racemate of benzyl furanone racemate (±)-1, which further separated chirally as a pair of new enantiomers, (+)- and (-)-asperfuranone (1), together with two new benzyl pyrones, asperpyranones A (2) and B (3). Their structures were elucidated by analysis of the comprehensive spectroscopic data, including one-dimensional (1D) and two-dimensional (2D) NMR, and HRESIMS. The absolute configurations were determined by electronic circular dichroism (ECD) calculation and single-crystal X-ray crystallographic experiment. The structures with benzyl furanone or benzyl pyrone skeletons were discovered from natural products for the first time. Compounds (±)-1, (+)-1, (-)-1, and 2 displayed the antifungal activities against Candida albicans with MIC values of 32, 16, 64, and 64 µg/mL and PTP1B inhibitory activities with the IC50 values of 45.79, 17.32, 35.50, and 42.32 µM, respectively. Compound 2 exhibited antibacterial activity against Pseudomonas aeruginosa with the MIC value of 32 µg/mL.

New Thiodiketopiperazine and 3,4-Dihydroisocoumarin Derivatives from the Marine-Derived Fungus Aspergillus terreus.

Aspergillus terreus has been reported to produce many secondary metabolites that exhibit potential bioactivities, such as antibiotic, hypoglycemic, and lipid-lowering activities. In the present study, two new thiodiketopiperazines, emestrins L (1) and M (2), together with five known analogues (3-7), and five known dihydroisocoumarins (8-12), were obtained from the marine-derived fungus Aspergillus terreus RA2905. The structures of the new compounds were elucidated by analysis of the comprehensive spectroscopic data, including high-resolution electrospray ionization mass spectrometry (HRESIMS), one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR), and electronic circular dichroism (ECD) data. This is the first time that the spectroscopic data of compounds 3, 8, and 9 have been reported. Compound 3 displayed antibacterial activity against Pseudomonas aeruginosa (minimum inhibitory concentration (MIC) = 32 µg/mL) and antifungal activity against Candida albicans (MIC = 32 µg/mL). In addition, compound 3 exhibited an inhibitory effect on protein tyrosine phosphatase 1 B (PTP1B), an important hypoglycemic target, with an inhibitory concentration (IC)50 value of 12.25 µM.

Epigenetic Agents Trigger the Production of Bioactive Nucleoside Derivatives and Bisabolane Sesquiterpenes From the Marine-Derived Fungus Aspergillus versicolor.

Epigenetic agents, histone deacetylase inhibitor (SAHA) and DNA methyltransferase inhibitor (5-Aza), were added to Czapek-Dox medium to trigger the chemical diversity of marine-derived fungus Aspergillus versicolor XS-20090066. By HPLC and 1H NMR analysis, the diversity of fungal secondary metabolites was significantly increased compared with the control. With the aid of MS/MS-based molecular networking, two new nucleoside derivatives, kipukasins K (1) and L (2) were obtained. Meanwhile, the yields of four known nucleoside derivatives were significantly enhanced. In addition, one new bisabolane sesquiterpene, aspergillusene E (7), along with ten known derivatives were also isolated. The structures were elucidated by comprehensive spectroscopic methods of NMR and HRESIMS analysis. Compounds 1 and 7 displayed antibacterial activities against Staphylococcus epidermidis and Staphylococcus aureus with the MIC values of 8-16 µg/mL. Our study revealed that the fungus A. versicolor XS-20090066 has been effectively induced by chemical epigenetic manipulation with a combination of SAHA and 5-Aza to produce new metabolites.

Exploration of the Regulatory Mechanism of Secondary Metabolism by Comparative Transcriptomics in Aspergillus flavus.

Mycotoxins cause a huge threaten to agriculture, food safety, and human and animal life. Among them, aflatoxins (AFs) have always been considered the most potent carcinogens, and filamentous fungi from Aspergillus genus are their major producers, especially A. flavus. Although the biosynthesis path of these chemicals had been well-identified, the regulatory mechanisms controlling expression of AF gene cluster were poorly understood. In this report, genome-wide transcriptome profiles of A. flavus from AF conducing [yeast sucrose media (YES)] and non-conducing [yeast peptone media (YEP)] conditions were compared by using deep RNA sequencing (RNA-seq), and the results revealed that AF biosynthesis pathway and biosynthesis of amino acids were significantly upregulated in YES vs. YEP. Further, a novel LaeA-like methyltransferase AFLA_121330 (Lael1) was identified for the first time, to play a specific role in the regulation of AF biosynthesis. Contrary to LaeA, which gene deletion reduced the level, lael1 deletion resulted in a significant increase in AF production. Further, co-expression network analysis revealed that mitochondrial pyruvate transport and signal peptide processing were potentially involved in AF synthesis for the first time, as well as biological processes of ribosome, branched-chain amino acid biosynthetic process and translation were co-regulated by AfRafA and AfStuA. To sum up, our analyses could provide novel insights into the molecular mechanism for controlling the AF and other secondary metabolite synthesis, adding novel targets for plant breeding and making fungicides.

Contribution of peroxisomal protein importer AflPex5 to development and pathogenesis in the fungus Aspergillus flavus.

Peroxisomes are important organelles that have diverse metabolic functions and participate in the pathogenicity of fungal pathogens. Previous studies indicate that most functions of peroxisomes are dependent on peroxisomal matrix proteins, which are delivered from the cytoplasm into peroxisomes by peroxisomal protein importers. In this study, the roles of peroxisomal protein importer AflPex5 were investigated in Aspergillus flavus with the application of gene disruption. AflPex5 deletion mutants failed to localize the fluorescently fused peroxisomal targeting signal 1 (PTS1) proteins to peroxisomes. Deletion of AflPex5 caused defects in sporulation, sclerotial formation, aflatoxin biosynthesis, stress response, and plant infection. Moreover, AflPex5 null mutants exhibited a significant defect in carbon metabolism and oxidants' clearance. These results indicate that the PTS1 pathway mediated by AflPex5 serves as an important role in the development, metabolism, and pathogenesis of A. flavus.

Essential APSES Transcription Factors for Mycotoxin Synthesis, Fungal Development, and Pathogenicity in Aspergillus flavus.

Aflatoxins are a potent carcinogenic mycotoxin and has become a research model of fungal secondary metabolism (SM). Via systematically investigating the APSES transcription factors (TFs), two APSES proteins were identified: AfRafA and AfStuA. These play central roles in the synthesis of mycotoxins including aflatoxin and cyclopiazonic acid, and fungal development and are consequently central to the pathogenicity of the aflatoxigenic A. flavus. Loss of AfRafA not only dramatically suppressed aflatoxin cluster expression, subsequently reducing toxin synthesis both in vitro and in vivo, but also impaired conidia and sclerotia development. More importantly, aflatoxin biosynthesis as well as conidia and sclerotia development were fully blocked in ΔAfStuA. In addition, our results supported that AfStuA regulated the aflatoxin synthesis in an AflR-dependent manner. Intriguingly, it was revealed that AfRafA and AfStuA exert an antagonistic role in the regulation of biosynthesis of cyclopiazonic acid. In summary, two global transcriptional regulators for fungal development, mycotoxin production, and seed pathogenicity of the A. flavus system have been established. The two novel regulators of mycotoxins are promising targets for future plant breeding and for the development of fungicides.

Putative methyltransferase LaeA and transcription factor CreA are necessary for proper asexual development and controlling secondary metabolic gene cluster expression.

The morphological development of fungi is a complex process and is often coupled with secondary metabolite production. In this study, we assessed the function of putative methyltransferase LaeA and transcription factor CreA in controlling asexual development and secondary metabolic gene cluster expression in Penicillium oxalicum. The deletion of laeA (ΔlaeA) impaired the conidiation in P. oxalicum, with a downregulated expression of brlA. Overexpression of P. oxalicum brlA in ΔlaeA could upregulate brlA and abaA remarkably, but could not rescue the conidiation defect; therefore, brlA and abaA expression were necessary but not sufficient for conidiation. Deletion of creA in ΔlaeA background (ΔlaeAΔcreA) blocked conidiation with a white fluffy phenotype. Nutrient-rich medium could not rescue developmental defects in ΔlaeAΔcreA mutant but could rescue defects in ΔlaeA. Expression of 10 genes, namely, albA/wA, abrB/yA, arpA, aygA, arpA-like, arpB, arpB-like, rodA, rodA-like, and rodB, for pigmentation and spore wall protein genes was silenced in ΔlaeAΔcreA, whereas only six of them were downregulated in ΔlaeA. Among the 28 secondary metabolism gene clusters in P. oxalicum, four secondary metabolism gene clusters were silenced in ΔlaeA and two were also silenced in ΔbrlA mutant. A total of 10 physically linked and coregulated genes were distributed over five chromosomes in ΔlaeA. Six of these genes were located in subtelomeric regions, thus demonstrating a positional bias for LaeA-regulated clusters toward subtelomeric regions. All of silenced clusters located in subtelomeric regions were derepressed in ΔlaeAΔcreA, hence showing that lack of CreA could remediate the repression of gene clusters in ΔlaeA background. Results show that both putative methyltransferase LaeA and transcription factor CreA are necessary for proper asexual development and controlling secondary metabolic gene cluster expression.

Production of a high-efficiency cellulase complex via ß-glucosidase engineering in Penicillium oxalicum.

BACKGROUND: Trichoderma reesei is a widely used model cellulolytic fungus, supplying a highly effective cellulase production system. Recently, the biofuel industry discovered filamentous fungi from the Penicillium genus as a promising alternative to T. reesei. RESULTS: In our study, we present a systematic over-expression analysis of nine ß-glucosidase encoding genes in the wild-type strain 114-2 of Penicillium oxalicum. We found that the over-expression of BGL1, BGL4, or BGL5 significantly enhanced both ß-glucosidase activity and hydrolysis efficiency of the enzyme system on filter paper. We utilised two strategies to over-express ß-glucosidase in the strain RE-10 that-although over-producing cellulase, does so at the cost of the cellulase mixture deficiency. The constitutive promoter of gene pde_02864 encoding 40S ribosomal protein S8 was used to over-express three ß-glucosidases: BGL1, BGL4, and BGL5. We found that all mutants show significantly enhanced levels of ß-glucosidase at transcriptional, protein, and activity levels. Furthermore, the inducible promoter from bgl2 was used to conditionally over-express the ß-glucosidases BGL1 and BGL4. Surprisingly, this induced expression strategy enables significantly improved expression efficiency. The BGL1 over-expressing mutant I1-13 particularly improved the ß-glucosidase activity at a factor of 65-folds, resulting in levels of up to 150 U/ml. All our BGL over-expression mutants displayed significant enhancement of cellulolytic ability on both microcrystalline cellulose and filter paper. In addition, they substantially reduced the enzyme loads in the saccharification of a natural lignocellulose material delignified corncob residue (DCCR). The mutant I4-32 with over-expression of BGL4 achieved the highest glucose yield in the saccharification of DCCR at only 25 % enzyme load compared to the parental strain RE-10. CONCLUSIONS: In summary, genetically engineering P. oxalicum to significantly improve ß-glucosidase activity is a potent strategy to substantially boost the hydrolytic efficiency of the cellulase cocktail, which will ultimately lead to a considerable reduction of cost for biomass-based biofuel.

Proteomic analysis of the biomass hydrolytic potentials of Penicillium oxalicum lignocellulolytic enzyme system.

BACKGROUND: The mining of high-performance enzyme systems is necessary to develop industrial lignocellulose bioconversion. Large amounts of cellulases and hemicellulases can be produced by Penicillium oxalicum. Hence, the enzyme system of this hypercellulolytic fungus should be elucidated to help design optimum enzyme systems for effective biomass hydrolysis. RESULTS: The cellulolytic and xylanolytic activities of an SP enzyme system prepared from P. oxalicum JU-A10 were comparatively analyzed. Results indicated that the fungus possesses a complete cellulolytic-xylanolytic enzyme system. The cellobiohydrolase- and xylanase-specific activities of this system were higher than those of two other enzyme systems, i.e., ST from Trichoderma reesei SN1 and another commercial preparation Celluclast 1.5L. Delignified corncob residue (DCCR) could be hydrolyzed by SP to a greater extent than corncob residue (CCR). Beta-glucosidase (BG) supplemented in SP increased the ability of the system to hydrolyze DCCR and CCR, and resulted in a 64 % decrease in enzyme dosage with the same glucose yield. The behaviors of the enzyme components in the hydrolysis of CCR were further investigated by monitoring individual enzyme dynamics. The total protein concentrations and cellobiohydrolase (CBH), endoglucanase (EG), and filter paper activities in the supernatants significantly decreased during saccharification. These findings were more evident in SP than in the other enzyme systems. The comparative proteomic analysis of the enzyme systems revealed that both SP and ST were rich in carbohydrate-degrading enzymes and multiple non-hydrolytic proteins. A larger number of carbohydrate-binding modules 1 (CBM1) were also identified in SP than in ST. This difference might be linked to the greater adsorption to substrates and lower hydrolysis efficiency of SP enzymes than ST during lignocellulose saccharification, because CBM1 not only targets enzymes to insoluble cellulose but also leads to non-productive adsorption to lignin. CONCLUSIONS: Penicillium oxalicum can be applied to the biorefinery of lignocellulosic biomass. Its ability to degrade lignocellulosic substrates could be further improved by modifying its enzyme system on the basis of enzyme activity measurement and proteomic analysis. The proposed strategy may also be applied to other lignocellulolytic enzyme systems to enhance their hydrolytic performances rationally.