Swollenin is a protein from Trichoderma reesei that has a unique activity for disrupting cellulosic materials, and it has sequence similarity to expansins, plant cell wall proteins that have a loosening effect that leads to cell wall enlargement. In this study we cloned a gene encoding a swollenin-like protein, Swo1, from the filamentous fungus Aspergillus fumigatus, and designated the gene Afswo1. AfSwo1 has a bimodular structure composed of a carbohydrate-binding module family 1 (CBM1) domain and a plant expansin-like domain. AfSwo1 was produced using Aspergillus oryzae for heterologous expression and was easily isolated by celluloseaffinity chromatography. AfSwo1 exhibited weak endoglucanase activity toward carboxymethyl cellulose (CMC) and bound not only to crystalline cellulose Avicel but also to chitin, while showing no detectable affinity to xylan. Treatment by AfSwo1 caused disruption of Avicel into smaller particles without any detectable reducing sugar. Furthermore, simultaneous incubation of AfSwo1 with a cellulase mixture facilitated saccharification of Avicel. Our results provide a novel approach for efficient bioconversion of crystalline cellulose into glucose by use of the cellulose-disrupting protein AfSwo1.Cellulose is the primary polysaccharide of plant cell wall and the most abundant renewable biomass resource. Biological degradation of cellulose to soluble sugars has long been considered an alternative to the use of starch feedstocks for bioethanol production. Natural cellulose is an ordered, linear polymer of thousands of D-glucose residues linked by ␤-1,4-glucosidic bonds. Spontaneous crystallization of cellulose molecules due to chemical uniformity of glucose units and the high degree of hydrogen bonding in cellulose can often result in the formation of tightly packed microfibrils (8), which remain inaccessible to cellulolytic enzymes. No single enzyme is able to hydrolyze crystalline cellulose microfibrils completely. Synergistic effects of cellulase mixtures on crystalline cellulose degradation are well known (1,7,21). Nevertheless, cost-competitive technology for overcoming the recalcitrance of cellulosic biomass to enhance enzymatic saccharification is still a major impediment to the utilization of cellulosic materials in bioenergy generation.Expansins are plant cell wall proteins that cause cell wall enlargement by a unique loosening effect in an acid-induced manner (15,20). They are also involved in many physiological processes where cell wall extension occurs, such as pollination, fruit ripening, organ abscission, and seed germination (13,14). It has been proposed that plant expansins disrupt hydrogen bonding between cellulose microfibrils and other cell wall polysaccharides without hydrolytic activity, causing sliding of cellulose fibers or expansion of the cell wall (18,19,27). Swollenin, an expansin-like protein, was isolated and characterized from the cellulolytic filamentous fungus Trichoderma reesei. It has a bimodular structure consisting of a carbohydrate-binding module fami...

BackgroundStreptomyces chattanoogensis L10 is the industrial producer of natamycin and has been proved a highly efficient host for diverse natural products. It has an enormous potential to be developed as a versatile cell factory for production of heterologous secondary metabolites. Here we developed a genome-reduced industrial Streptomyces chassis by rational ‘design-build-test’ pipeline.ResultsTo identify candidate large non-essential genomic regions accurately and design large deletion rationally, we performed genome analyses of S. chattanoogensis L10 by multiple computational approaches, optimized Cre/loxP recombination system for high-efficient large deletion and constructed a series of universal suicide plasmids for rapid loxP or loxP mutant sites inserting into genome. Subsequently, two genome-streamlined mutants, designated S. chattanoogensis L320 and L321, were rationally constructed by depletion of 1.3 Mb and 0.7 Mb non-essential genomic regions, respectively. Furthermore, several biological performances like growth cycle, secondary metabolite profile, hyphae morphological engineering, intracellular energy (ATP) and reducing power (NADPH/NADP+) levels, transformation efficiency, genetic stability, productivity of heterologous proteins and secondary metabolite were systematically evaluated. Finally, our results revealed that L321 could serve as an efficient chassis for the production of polyketides.ConclusionsHere we developed the combined strategy of multiple computational approaches and site-specific recombination system to rationally construct genome-reduced Streptomyces hosts with high efficiency. Moreover, a genome-reduced industrial Streptomyces chassis S. chattanoogensis L321 was rationally constructed by the strategy, and the chassis exhibited several emergent and excellent performances for heterologous expression of secondary metabolite. The strategy could be widely applied in other Streptomyces to generate miscellaneous and versatile chassis with minimized genome. These chassis can not only serve as cell factories for high-efficient production of valuable polyketides, but also will provide great support for the upgrade of microbial pharmaceutical industry and drug discovery.Electronic supplementary materialThe online version of this article (10.1186/s12934-019-1055-7) contains supplementary material, which is available to authorized users.

Due to the plethora natural products made by Streptomyces, the regulation of its metabolism are of great interest, whereas there is a lack of detailed understanding of the role of posttranslational modifications (PTM) beyond traditional transcriptional regulation. Herein with Streptomyces roseosporus as a model, we showed that crotonylation is widespread on key enzymes for various metabolic pathways, and sufficient crotonylation in primary metabolism and timely elimination in secondary metabolism are required for proper Streptomyces metabolism. Particularly, the glucose kinase Glk, a keyplayer of carbon catabolite repression (CCR) regulating bacterial metabolism, is identified reversibly crotonylated by the decrotonylase CobB and the crotonyl-transferase Kct1 to negatively control its activity. Furthermore, crotonylation positively regulates CCR for Streptomyces metabolism through modulation of the ratio of glucose uptake/Glk activity and utilization of carbon sources. Thus, our results revealed a regulatory mechanism that crotonylation globally regulates Streptomyces metabolism at least through positive modulation of CCR.

SUMMARYThe origin recognition complex (ORC) is a pivotal element in DNA replication, heterochromatin assembly, checkpoint regulation and chromosome assembly. Although the functions of the ORC have been determined in yeast and model animals, they remain largely unknown in the plant kingdom. In this study, Oryza sativa Origin Recognition Complex subunit 3 (OsORC3) was cloned using map-based cloning procedures, and functionally characterized using a rice (Oryza sativa) orc3 mutant. The mutant showed a temperaturedependent defect in lateral root (LR) development. Map-based cloning showed that a G?A mutation in the 9th exon of OsORC3 was responsible for the mutant phenotype. OsORC3 was strongly expressed in regions of active cell proliferation, including the primary root tip, stem base, lateral root primordium, emerged lateral root primordium, lateral root tip, young shoot, anther and ovary. OsORC3 knockdown plants lacked lateral roots and had a dwarf phenotype. The root meristematic zone of ORC3 knockdown plants exhibited increased cell death and reduced vital activity compared to the wild-type. CYCB1;1::GUS activity and methylene blue staining showed that lateral root primordia initiated normally in the orc3 mutant, but stopped growing before formation of the stele and ground tissue. Our results indicate that OsORC3 plays a crucial role in the emergence of lateral root primordia.

Plant roots move through the soil by elongation. This is vital to their ability to anchor the plant and acquire water and minerals from the soil. In order to identify new genes involved in root elongation in rice, we screened an ethyl methane sulfonate (EMS)-mutagenized rice library, and isolated a short root mutant, Osglu3-1. The map-based cloning results showed that the mutant was due to a point mutation in OsGLU3, which encodes a putative membrane-bound endo-1,4-β-glucanase. Osglu3-1 displayed less crystalline cellulose content in its root cell wall, shorter root cell length, and a slightly smaller root meristem as visualized by restricted expression of OsCYCB1,1:GUS. Exogenous application of glucose can suppress both the lower root cell wall cellulose content and short root phenotypes of Osglu3-1. Consistently, OsGLU3 is ubiquitously expressed in various tissues with strong expression in root tip, lateral root, and crown root primodia. The fully functional OsGLU3-GFP was detected in plasma membrane, and FM4-64-labeled compartments in the root meristem and elongation zones. We also found that phosphate starvation, an environmental stress, altered cell wall cellulose content to modulate root elongation in a OsGLU3-dependant way.

Efficient genome editing is a prerequisite of genetic engineering in synthetic biology, which has been recently achieved by the powerful CRISPR/Cas9 system. However, the toxicity of Cas9, due to its abundant intracellular expression, has impeded its extensive applications. Here we constructed a genetic cassette with triple controls of Cas9 activities at transcriptional, translational and protein levels, together with over-expression of the ATP synthase β-subunit AtpD, for the efficient genome editing in Streptomyces. By deletion of actII-ORF4 in Streptomyces coelicolor as a model, we found that constitutive expression of cas9 had about 90% editing efficiency but dramatically reduced transformation efficiency by 900-fold. However, triple controls of Cas9 under non-induction conditions to reduce its activity increased transformation efficiency over 250-fold, and had about 10% editing efficiency if combined with atpD overexpression. Overall, our strategy accounts for about 30-fold increased possibility for successful genome editing under the non-induction condition. In addition, about 80% editing efficiency was observed at the actII-ORF4 locus after simultaneous induction with thiostrepton, theophylline and blue light for Cas9 activity reconstitution. This improved straightforward efficient genome editing was also confirmed in another locus redD. Thus, we developed a new strategy for efficient genome editing, and it could be readily and widely adaptable to other Streptomyces species to improve genetic manipulation for rapid strain engineering in Streptomyces synthetic biology, due to the highly conserved genetic cassettes in this genus.

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Background: Secondary metabolites as natural products from endophytic fungi are important sources of pharmaceuticals. However, there is currently little understanding of endophytic fungi at the omics levels about their potential in secondary metabolites. Calcarisporium arbuscula, an endophytic fungus from the fruit bodies of Russulaceae, produces a variety of secondary metabolites with anti-cancer, anti-nematode and antibiotic activities. A comprehensive survey of the genome and transcriptome of this endophytic fungus will help to understand its capacity to biosynthesize secondary metabolites and will lay the foundation for the development of this precious resource. Results: In this study, we reported the high-quality genome sequence of C. arbuscula NRRL 3705 based on Single Molecule Real-Time sequencing technology. The genome of this fungus is over 45 Mb in size, larger than other typical filamentous fungi, and comprises 10,001 predicted genes, encoding at least 762 secretory-proteins, 386 carbohydrate-active enzymes and 177 P450 enzymes. 398 virulence factors and 228 genes related to pathogen-host interactions were also predicted in this fungus. Moreover, 65 secondary metabolite biosynthetic gene clusters were revealed, including the gene cluster for the mycotoxin aurovertins. In addition, several gene clusters were predicted to produce mycotoxins, including aflatoxin, alternariol, destruxin, citrinin and isoflavipucine. Notably, two independent gene clusters were shown that are potentially involved in the biosynthesis of alternariol. Furthermore, RNA-Seq assays showed that only expression of the aurovertin gene cluster is much stronger than expression of the housekeeping genes under laboratory conditions, consistent with the observation that aurovertins are the predominant metabolites. Gene expression of the remaining 64 gene clusters for compound backbone biosynthesis was all lower than expression of the housekeeping genes, which partially explained poor production of other secondary metabolites in this fungus. Conclusions: Our omics data, along with bioinformatics analysis, indicated that C. arbuscula NRRL 3705 contains a large number of biosynthetic gene clusters and has a huge potential to produce a profound number of secondary metabolites. This work also provides the basis for development of endophytic fungi as a new resource of natural products with promising biological activities.