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1.
PLoS Genet ; 19(7): e1010867, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-37523410

RESUMEN

Many filamentous fungi produce plant-polysaccharide-degrading enzymes (PPDE); however, the regulatory mechanism of this process is poorly understood. A Gal4-like transcription factor, CxrA, is essential for mycelial growth and PPDE production in Penicillium oxalicum. Its N-terminal region, CxrAΔ207-733 is required for the regulatory functions of whole CxrA, and contains a DNA-binding domain (CxrAΔ1-16&Δ59-733) and a methylated arginine (R) 94. Methylation of R94 is mediated by an arginine N-methyltransferase, PRMT2 and appears to induce dimerization of CxrAΔ1-60. Overexpression of prmt2 in P. oxalicum increases PPDE production by 41.4-95.1% during growth on Avicel, compared with the background strain Δku70;hphR+. Another arginine N-methyltransferase, PRMT3, appears to assist entry of CxrA into the nucleus, and interacts with CxrAΔ1-60 in vitro under Avicel induction. Deletion of prmt3 resulted in 67.0-149.7% enhanced PPDE production by P. oxalicum. These findings provide novel insights into the regulatory mechanism of fungal PPDE production.


Asunto(s)
Penicillium , Proteína-Arginina N-Metiltransferasas , Proteína-Arginina N-Metiltransferasas/genética , Penicillium/genética , Celulosa , Arginina
2.
Appl Environ Microbiol ; 90(1): e0130023, 2024 01 24.
Artículo en Inglés | MEDLINE | ID: mdl-38112424

RESUMEN

Streptomyces bingchenggensis is an industrial producer of milbemycins, which are important anthelmintic and insecticidal agents. Two-component systems (TCSs), which are typically situated in the same operon and are composed of a histidine kinase and a response regulator, are the predominant signal transduction pathways involved in the regulation of secondary metabolism in Streptomyces. Here, an atypical TCS, AtcR/AtcK, in which the encoding genes (sbi_06838/sbi_06839) are organized in a head-to-head pair, was demonstrated to be indispensable for the biosynthesis of multiple secondary metabolites in S. bingchenggensis. With the null TCS mutants, the production of milbemycin and yellow compound was abolished but nanchangmycin was overproduced. Transcriptional analysis and electrophoretic mobility shift assays showed that AtcR regulated the biosynthesis of these three secondary metabolites by a MilR3-mediated cascade. First, AtcR was activated by phosphorylation from signal-triggered AtcK. Second, the activated AtcR promoted the transcription of milR3. Third, MilR3 specifically activated the transcription of downstream genes from milbemycin and yellow compound biosynthetic gene clusters (BGCs) and nanR4 from the nanchangmycin BGC. Finally, because NanR4 is a specific repressor in the nanchangmycin BGC, activation of MilR3 downstream genes led to the production of yellow compound and milbemycin but inhibited nanchangmycin production. By rewiring the regulatory cascade, two strains were obtained, the yield of nanchangmycin was improved by 45-fold to 6.08 g/L and the production of milbemycin was increased twofold to 1.34 g/L. This work has broadened our knowledge on atypical TCSs and provided practical strategies to engineer strains for the production of secondary metabolites in Streptomyces.IMPORTANCEStreptomyces bingchenggensis is an important industrial strain that produces milbemycins. Two-component systems (TCSs), which consist of a histidine kinase and a response regulator, are the predominant signal transduction pathways involved in the regulation of secondary metabolism in Streptomyces. Coupled encoding genes of TCSs are typically situated in the same operon. Here, TCSs with encoding genes situated in separate head-to-head neighbor operons were labeled atypical TCSs. It was found that the atypical TCS AtcR/AtcK played an indispensable role in the biosynthesis of milbemycin, yellow compound, and nanchangmycin in S. bingchenggensis. This atypical TCS regulated the biosynthesis of specialized metabolites in a cascade mediated via a cluster-situated regulator, MilR3. Through rewiring the regulatory pathways, strains were successfully engineered to overproduce milbemycin and nanchangmycin. To the best of our knowledge, this is the first report on atypical TCS, in which the encoding genes of RR and HK were situated in separate head-to-head neighbor operons, involved in secondary metabolism. In addition, data mining showed that atypical TCSs were widely distributed in actinobacteria.


Asunto(s)
Éteres , Macrólidos , Compuestos de Espiro , Streptomyces , Histidina Quinasa/metabolismo , Streptomyces/genética , Proteínas Bacterianas/genética
3.
Arch Microbiol ; 204(10): 631, 2022 Sep 19.
Artículo en Inglés | MEDLINE | ID: mdl-36121479

RESUMEN

Streptomyces bingchenggensis is the main industrial producer of milbemycins, which are a group of 16-membered macrocylic lactones with excellent insecticidal activities. In the past several decades, scientists have made great efforts to solve its low productivity. However, a lack of understanding of the regulatory network of milbemycin biosynthesis limited the development of high-producing strains using a regulatory rewiring strategy. SARPs (Streptomyces Antibiotic Regulatory Proteins) family regulators are widely distributed and play key roles in regulating antibiotics production in actinobacteria. In this paper, MilR3 (encoded by sbi_06842) has been screened out for significantly affecting milbemycin production from all the 19 putative SARP family regulators in S. bingchenggensis with the DNase-deactivated Cpf1-based integrative CRISPRi system. Interestingly, milR3 is about 7 Mb away from milbemycin biosynthetic gene cluster and adjacent to a putative type II PKS (the core minimal PKS encoding genes are sbi_06843, sbi_06844, sbi_06845 and sbi_06846) gene cluster, which was proved to be responsible for producing a yellow pigment. The quantitative real-time PCR analysis proved that MilR3 positively affected the transcription of specific genes within milbemycin BGC and those from the type II PKS gene cluster. Unlike previous "small" SARP family regulators that played pathway-specific roles, MilR3 was probably a unique SARP family regulator and played a pleotropic role. MilR3 was an upper level regulator in the MilR3-MilR regulatory cascade. This study first illustrated the co-regulatory role of this unique SARP regulator. This greatly enriches our understanding of SARPs and lay a solid foundation for milbemycin yield enhancement in the near future.


Asunto(s)
Regulación Bacteriana de la Expresión Génica , Streptomyces , Antibacterianos/metabolismo , Desoxirribonucleasas/genética , Streptomyces/genética , Streptomyces/metabolismo
4.
Arch Microbiol ; 203(10): 5849-5857, 2021 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-34550409

RESUMEN

Milbemycins, a group of 16-membered macrocylic lactones with excellent acaricidal, insecticidal and anthelmintic activities, can be produced by several Streptomyces species. For the reason that they have low toxicity in mammals, milbemycins and their derivatives are widely used in agricultural, medical and veterinary industries. Streptomyces bingchenggensis, one of milbemycin-producing strains, has been sequenced and intensively investigated in the past decades. In this mini-review, we comprehensively revisit the progress that has been made in research efforts to elucidate the biosynthetic pathways and regulatory networks for the cellular production of milbemycins. The advances in the development of production strains for milbemycin and its derivatives are discussed along the strain-generation technical approaches of random mutagenesis, metabolic engineering and combinatorial biosynthesis. The research progress made so far indicates that strain improvement and generation of novel milbemycin derivatives will greatly benefit from future development of enabling technologies and deeper understanding of the fundamentals of biosynthesis of milbemycin and the regulation of its production in S. bingchenggensis. This mini-review also proposes that the overproduction of milbemycins could be greatly enhanced by genome minimization, systematical metabolic engineering and synthetic biology approaches in the future.


Asunto(s)
Macrólidos , Streptomyces , Animales , Vías Biosintéticas/genética , Streptomyces/genética
5.
Appl Environ Microbiol ; 85(24)2019 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-31604764

RESUMEN

Transcriptional regulation of cellulolytic and xylolytic genes in ascomycete fungi is controlled by specific carbon sources in different external environments. Here, comparative transcriptomic analyses of Penicillium oxalicum grown on wheat bran (WB), WB plus rice straw (WR), or WB plus Avicel (WA) as the sole carbon source under solid-state fermentation (SSF) revealed that most of the differentially expressed genes (DEGs) were involved in metabolism, specifically, carbohydrate metabolism. Of the DEGs, the basic core carbohydrate-active enzyme-encoding genes which responded to the plant biomass resources were identified in P. oxalicum, and their transcriptional levels changed to various extents depending on the different carbon sources. Moreover, this study found that three deletion mutants of genes encoding putative transcription factors showed significant alterations in filter paper cellulase production compared with that of a parental P. oxalicum strain with a deletion of Ku70 (ΔPoxKu70 strain) when grown on WR under SSF. Importantly, the ΔPoxAtf1 mutant (with a deletion of P. oxalicumAtf1, also called POX03016) displayed 46.1 to 183.2% more cellulase and xylanase production than a ΔPoxKu70 mutant after 2 days of growth on WR. RNA sequencing and quantitative reverse transcription-PCR revealed that PoxAtf1 dynamically regulated the expression of major cellulase and xylanase genes under SSF. PoxAtf1 bound to the promoter regions of the key cellulase and xylanase genes in vitro This study provides novel insights into the regulatory mechanism of fungal cellulase and xylanase gene expression under SSF.IMPORTANCE The transition to a more environmentally friendly economy encourages studies involving the high-value-added utilization of lignocellulosic biomass. Solid-state fermentation (SSF), that simulates the natural habitat of soil microorganisms, is used for a variety of applications such as biomass biorefinery. Prior to the current study, our understanding of genome-wide gene expression and of the regulation of gene expression of lignocellulose-degrading enzymes in ascomycete fungi during SSF was limited. Here, we employed RNA sequencing and genetic analyses to investigate transcriptomes of Penicillium oxalicum strain EU2101 cultured on medium containing different carbon sources and to identify and characterize transcription factors for regulating the expression of cellulase and xylanase genes during SSF. The results generated will provide novel insights into genetic engineering of filamentous fungi to further increase enzyme production.


Asunto(s)
Factor de Transcripción Activador 1/metabolismo , Ascomicetos/enzimología , Ascomicetos/genética , Celulasa/genética , Fermentación , Regulación Fúngica de la Expresión Génica , Xilosidasas/genética , Ascomicetos/crecimiento & desarrollo , Biomasa , Celulasa/metabolismo , Medios de Cultivo/química , ADN de Hongos/genética , Eliminación de Gen , Genes Fúngicos/genética , Lignina/metabolismo , Penicillium/enzimología , Penicillium/genética , Penicillium/crecimiento & desarrollo , Regiones Promotoras Genéticas , ARN de Hongos/genética , Microbiología del Suelo , Xilosidasas/metabolismo
6.
Appl Environ Microbiol ; 84(18)2018 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-29980558

RESUMEN

Soil fungi produce a wide range of chemical compounds and enzymes with potential for applications in medicine and biotechnology. Cellular processes in soil fungi are highly dependent on the regulation under environmentally induced stress, but most of the underlying mechanisms remain unclear. Previous work identified a key GATA-type transcription factor, Penicillium oxalicum NsdD (PoxNsdD; also called POX08415), that regulates the expression of cellulase and xylanase genes in P. oxalicum PoxNsdD shares 57 to 64% identity with the key activator NsdD, involved in asexual development in Aspergillus In the present study, the regulatory roles of PoxNsdD in P. oxalicum were further explored. Comparative transcriptomic profiling revealed that PoxNsdD regulates major genes involved in starch, cellulose, and hemicellulose degradation, as well as conidiation and pigment biosynthesis. Subsequent experiments confirmed that a ΔPoxNsdD strain lost 43.9 to 78.8% of starch-digesting enzyme activity when grown on soluble corn starch, and it produced 54.9 to 146.0% more conidia than the ΔPoxKu70 parental strain. During cultivation, ΔPoxNsdD cultures changed color, from pale orange to brick red, while the ΔPoxKu70 cultures remained bluish white. Real-time quantitative reverse transcription-PCR showed that PoxNsdD dynamically regulated the expression of a glucoamylase gene (POX01356/Amy15A), an α-amylase gene (POX09352/Amy13A), and a regulatory gene (POX03890/amyR), as well as a polyketide synthase gene (POX01430/alb1/wA) for yellow pigment biosynthesis and a conidiation-regulated gene (POX06534/brlA). Moreover, in vitro binding experiments showed that PoxNsdD bound the promoter regions of the above-described genes. This work provides novel insights into the regulatory mechanisms of fungal cellular processes and may assist in genetic engineering of Poxalicum for potential industrial and medical applications.IMPORTANCE Most filamentous fungi produce a vast number of extracellular enzymes that are used commercially for biorefineries of plant biomass to produce biofuels and value-added chemicals, which might promote the transition to a more environmentally friendly economy. The expression of these extracellular enzyme genes is tightly controlled at the transcriptional level, which limits their yields. Hitherto our understanding of the regulation of expression of plant biomass-degrading enzyme genes in filamentous fungi has been rather limited. In the present study, regulatory roles of a key regulator, PoxNsdD, were further explored in the soil fungus Penicillium oxalicum, contributing to the understanding of gene regulation in filamentous fungi and revealing the biotechnological potential of Poxalicum via genetic engineering.


Asunto(s)
Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Penicillium/metabolismo , Pigmentos Biológicos/biosíntesis , Esporas Fúngicas/crecimiento & desarrollo , Factores de Transcripción/metabolismo , Biodegradación Ambiental , Celulasa/genética , Celulasa/metabolismo , Celulosa/metabolismo , Proteínas Fúngicas/genética , Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Glucano 1,4-alfa-Glucosidasa/genética , Glucano 1,4-alfa-Glucosidasa/metabolismo , Penicillium/enzimología , Penicillium/genética , Penicillium/crecimiento & desarrollo , Sintasas Poliquetidas/genética , Sintasas Poliquetidas/metabolismo , Esporas Fúngicas/genética , Esporas Fúngicas/metabolismo , Factores de Transcripción/genética , alfa-Amilasas/genética , alfa-Amilasas/metabolismo
7.
Appl Microbiol Biotechnol ; 102(8): 3739-3753, 2018 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-29511847

RESUMEN

High-mobility group (HMG)-box proteins are involved in chromatin organization in eukaryotes, especially in sex determination and regulation of mitochondrial DNA compaction. Although a novel HMG-box protein, PoxHmbB, had been initially identified to be required for filter paper cellulase activity by Penicillium oxalicum, the biological roles of HMG-box proteins in biomass-degrading enzyme production have not been systematically explored. The P. oxalicum mutant ∆PoxHmbB lost 34.7-86.5% of cellulase (endoglucanase, p-nitrophenyl-ß-cellobiosidase, and p-nitrophenyl-ß-glucopyranosidase) activities and 60.3% of xylanase activity following Avicel induction, whereas it exhibited about onefold increase in amylase activity following soluble corn starch induction. Furthermore, ∆PoxHmbB presented delayed conidiation and hyphae growth. Transcriptomic profiling and real-time quantitative reverse transcription-PCR revealed that PoxHmbB regulated the expression of major genes encoding plant biomass-degrading enzymes such as PoxCel7A-2, PoxCel5B, PoxBgl3A, PoxXyn11B, and PoxGA15A, as well as those involved in conidiation such as PoxBrlA. In vitro binding experiments further confirmed that PoxHmbB directly binds to the promoter regions of these major genes. These results further indicate the diversity of the biological functions of HMG-box proteins and provide a novel and promising engineering target for improving plant biomass-degrading enzyme production in filamentous fungi.


Asunto(s)
Celulasa/biosíntesis , Celulasa/genética , Proteínas HMGB/metabolismo , Penicillium/enzimología , Penicillium/genética , Biomasa , Celulasa/metabolismo , Regulación Fúngica de la Expresión Génica/genética , Mutación
8.
Biotechnol Biofuels ; 12: 105, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31073329

RESUMEN

BACKGROUND: Soil ascomycete fungi produce plant-biomass-degrading enzymes to facilitate nutrient and energy uptake in response to exogenous stress. This is controlled by a complex signal network, but the regulatory mechanisms are poorly understood. An essential Zn2Cys6 transcription factor (TF) PoxCxrA was identified to be required for cellulase and xylanase production in Penicillium oxalicum. The genome-wide regulon and DNA binding sequences of PoxCxrA were further identified through RNA-Sequencing, DNase I footprinting experiments and in vitro electrophoretic mobility shift assays. Moreover, a minimal DNA-binding domain in PoxCxrA was recognised. RESULTS: A PoxCxrA regulon of 1970 members was identified in P. oxalicum, and it was displayed that PoxCxrA regulated the expression of genes encoding major plant cell wall-degrading enzymes, as well as important cellodextrin and/or glucose transporters. Interestingly, PoxCxrA positively regulated the expression of a known important TF PoxClrB. DNase I footprinting experiments and in vitro electrophoretic mobility shift assays further revealed that PoxCxrA directly bound the promoter regions of PoxClrB and a cellobiohydrolase gene cbh1 (POX05587/Cel7A-2) at different nucleic acid sequences. Remarkably, PoxCxrA autoregulated its own PoxCxrA gene expression. Additionally, a minimal 42-amino-acid PoxCxrA DNA-binding domain was identified. CONCLUSION: PoxCxrA could directly regulate the expression of cellulase genes and the regulatory gene PoxClrB via binding their promoters at different nucleic acid sequences. This work expands the diversity of DNA-binding motifs known to be recognised by Zn2Cys6 TFs, and demonstrates novel regulatory mechanisms of fungal cellulase gene expression.

9.
Biotechnol Biofuels ; 10: 279, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-29201143

RESUMEN

BACKGROUND: The transition to a more environmentally friendly economy has prompted studies of modern biorefineries, including the utilization of low-value lignocellulose. The major challenge facing the widespread application of biorefineries is the high cost of enzymes that can efficiently hydrolyze recalcitrant cellulose to sugars. Penicillium oxalicum produces large amounts of plant-cell-wall-degrading enzymes, but their production is tightly controlled by complex regulatory networks, resulting in low yields of the native enzymes. Regulatory genes have been the targets of genetic engineering to improve enzyme production in microorganisms. In this study, we used transcriptomic profiling and genetic analyses to screen for and identify novel key regulators of cellulase and xylanase gene expression in P. oxalicum. RESULTS: A comparative analysis of the transcriptomes of P. oxalicum HP7-1 on different carbon sources, including glucose, wheat bran, and wheat bran plus Avicel, identified 40 candidate genes regulating the expression of cellulolytic enzyme genes. Deletion mutants of 31 candidate genes were constructed in P. oxalicum ∆PoxKu70 and 11 resultant mutants showed significant changes in their filter-paper cellulase production compared with the parental strain ∆PoxKu70. Among these 11 mutants, ΔPoxCxrA, ΔPoxCxrB, and ΔPoxNsdD showed the most significant reduction in the enzyme production (96.8, 75.9, and 58.5%, respectively). Ten of these 11 genes are here reported to be involved in cellulase production for the first time. Further tests revealed that ΔPoxCxrA, ΔPoxCxrB, and ΔPoxNsdD displayed significantly reduced xylanase production, whereas ΔPoxCxrA produced negligible xylanase. Interestingly, ΔPoxCxrB and ΔPoxNsdD showed significantly increased ß-glucosidase production. Real-time quantitative reverse transcription-PCR and an electrophoretic mobility shift assay (EMSA) showed that PoxCxrA, PoxCxrB, and PoxNsdD regulate the expression of one another, but the mode of regulation changes dynamically during the growth of fungal cells in the presence of cellulose. EMSA showed that PoxCxrA, PoxCxrB, and PoxNsdD directly bind the putative promoters of major cellulase and xylanase genes. CONCLUSIONS: We have detected and identified three key new regulatory genes, PoxCxrA, PoxCxrB, and PoxNsdD, that directly and indirectly regulate the expression of cellulase and xylanase genes in P. oxalicum. This study provides novel insights into the regulatory mechanisms of fungal cellulase and xylanase gene expression.

10.
Biotechnol Biofuels ; 9: 216, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27777618

RESUMEN

BACKGROUND: Starch is a very abundant and renewable carbohydrate and is an important feedstock for industrial applications. The conventional starch liquefaction and saccharification processes are energy-intensive, complicated, and not environmentally friendly. Raw starch-digesting glucoamylases are capable of directly hydrolyzing raw starch to glucose at low temperatures, which significantly simplifies processing and reduces the cost of producing starch-based products. RESULTS: A novel raw starch-digesting glucoamylase PoGA15A with high enzymatic activity was purified from Penicillium oxalicum GXU20 and biochemically characterized. The PoGA15A enzyme had a molecular weight of 75.4 kDa, and was most active at pH 4.5 and 65 °C. The enzyme showed remarkably broad pH stability (pH 2.0-10.5) and substrate specificity, and was able to degrade various types of raw starches at 40 °C. Its adsorption ability for different raw starches was consistent with its degrading capacities for the corresponding substrate. The cDNA encoding the enzyme was cloned and heterologously expressed in Pichia pastoris. The recombinant enzyme could quickly and efficiently hydrolyze different concentrations of raw corn and cassava flours (50, 100, and 150 g/L) with the addition of α-amylase at 40 °C. Furthermore, when used in the simultaneous saccharification and fermentation of 150 g/L raw flours to ethanol with the addition of α-amylase, the ethanol yield reached 61.0 g/L with a high fermentation efficiency of 95.1 % after 48 h when raw corn flour was used as the substrate. An ethanol yield of 57.0 g/L and 93.5 % of fermentation efficiency were achieved with raw cassava flour after 36 h. In addition, the starch-binding domain deletion analysis revealed that SBD plays a very important role in raw starch hydrolysis by the enzyme PoGA15A. CONCLUSIONS: A novel raw starch-digesting glucoamylase from P. oxalicum, with high enzymatic activity, was biochemically, molecularly, and genetically identified. Its efficient hydrolysis of raw starches and its high efficiency during the direct conversion of raw corn and cassava flours via simultaneous saccharification and fermentation to ethanol suggests that the enzyme has a number of potential applications in industrial starch processing and starch-based ethanol production.

11.
Biotechnol Biofuels ; 9: 203, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27688806

RESUMEN

BACKGROUND: The filamentous fungus Penicillium oxalicum is a potential alternative to Trichoderma reesei for industrial production of a complete cellulolytic enzyme system for a bio-refinery. Comparative omics approaches can support rational genetic engineering and/or breeding of filamentous fungi with improved cellulase production capacity. In this study, comparative genomic, transcriptomic and secretomic profiling of P. oxalicum HP7-1 and its cellulase and xylanase hyper-producing mutant EU2106 were employed to screen for novel regulators of cellulase and xylanase gene expression. RESULTS: The 30.62 Mb P. oxalicum HP7-1 genome was sequenced, and 9834 protein-coding genes were annotated. Re-sequencing of the mutant EU2106 genome identified 274 single nucleotide variations and 12 insertion/deletions. Comparative genomic, transcriptomic and secretomic profiling of HP7-1 and EU2106 revealed four candidate regulators of cellulase and xylanase gene expression. Deletion of these candidate genes and measurement of the enzymatic activity of the resultant mutants confirmed the identity of three regulatory genes. POX02484 and POX08522, encoding a putative Zn(II)2Cys6 DNA-binding domain and forkhead protein, respectively, were found to be novel, while PoxClrB is an ortholog of ClrB, a key transcriptional regulator of cellulolytic enzyme gene expression in filamentous fungi. ΔPOX02484 and ΔPOX08522 mutants exhibited significantly reduced ß-glucosidase activity, increased carboxymethylcellulose cellulase and xylanase activities, and altered transcription level of cellulase and xylanase genes compared with the parent strain ΔPoxKu70, with Avicel as the sole carbon source. CONCLUSIONS: Two novel genes, POX02484 and POX08522, were found and characterized to regulate the expression of cellulase and xylanase genes in P. oxalicum. These findings are important for engineering filamentous fungi to improve cellulase and xylanase production.

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