Tr-milRNA1 Contributes to Lignocellulase Secretion under Heat Stress by Regulating the Lectin-Type Cargo Receptor Gene Trvip36 in Trichoderma guizhouence NJAU 4742

Background: MicroRNA plays an important role in multifarious biological processes by regulating their corresponding target genes. However, the biological function and regulatory mechanism of fungal microRNA-like RNAs (milRNAs) remain poorly understood. Methods: In this study, combined with deep sequencing and bioinformatics analysis, milRNAs and their targets from Trichoderma guizhouence NJAU 4742 were isolated and identified under solid-state fermentation (SSF) by using rice straw as the sole carbon source at 28 °C and 37 °C, respectively. Results: A critical milRNA, TGA1_S04_31828 (Tr-milRNA1), was highly expressed under heat stress (37 °C) and adaptively regulated lignocellulase secretion. Overexpression of Tr-milRNA1 (OE-Tr-milRNA1) did not affect vegetative growth, but significantly increased lignocellulose utilization under heat stress. Based on the bioinformatics analysis and qPCR validation, a target of Tr-milRNA1 was identified as Trvip36, a lectin-type cargo receptor. The expression of Tr-milRNA1 and Trvip36 showed a divergent trend under SSF when the temperature was increased from 28 °C to 37 °C. In addition, the expression of Trvip36 was suppressed significantly in Tr-milRNA1 overexpression strain (OE-Tr-milRNA1). Compared with the wild type, deletion of Trvip36 (ΔTrvip36) significantly improved the secretion of lignocellulases by reducing the retention of lignocellulases in the ER under heat stress. Conclusions: Tr-milRNA1 from NJAU 4742 improved lignocellulose utilization under heat stress by regulating the expression of the corresponding target gene Trvip36. These findings might open avenues for exploring the mechanism of lignocellulase secretion in filamentous fungi.

Function of ORFC of the polyketide synthase gene cluster on fatty acid accumulation in Schizochytrium limacinum SR21

Background As a potential source of polyunsaturated fatty acids (PUFA), Schizochytrium sp. has been widely used in industry for PUFA production. Polyketide synthase (PKS) cluster is supposed to be the primary way of PUFA synthesis in Schizochytrium sp. As one of three open reading frames (ORF) in the PKS cluster, ORFC plays an essential role in fatty acid biosynthesis. However, the function of domains in ORFC in the fatty acid synthesis of Schizochytrium sp. remained unclear. Results In this study, heterologous expression and overexpression were carried out to study the role of ORFC and its domains in fatty acid accumulation. Firstly, ORFC was heterologously expressed in yeast which increased the PUFA content significantly. Then, the dehydratase (DH) and enoyl reductase (ER) domains located on ORFC were overexpressed in Schizochytrium limacinum SR21, respectively. Fatty acids profile analysis showed that the contents of PUFA and saturated fatty acid were increased in the DH and ER overexpression strains, respectively. This indicated that the DH and ER domains played distinct roles in lipid accumulation. Metabolic and transcriptomic analysis revealed that the pentose phosphate pathway and triacylglycerol biosynthesis were enhanced, while the tricarboxylic acid cycle and fatty acids oxidation were weakened in DH-overexpression strain. However, the opposite effect was found in the ER-overexpression strain. Conclusion Therefore, ORFC was required for the biosynthesis of fatty acid. The DH domain played a crucial role in PUFA synthesis, whereas the ER domain might be related to saturated fatty acids (SFA) synthesis in Schizochytrium limacinum SR21. This research explored the role of ORFC in the PKS gene cluster in Schizochytrium limacinum and provided potential genetic modification strategies for improving lipid production and regulating PUFA and SFA content.

Effects of acuC on the growth development and spinosad biosynthesis of Saccharopolyspora spinosa

Background Acetoin utilization protein (acuC) is a type I histone deacetylase which is highly conserved in bacteria. The acuC gene is related to the acetylation/deacetylation posttranslational modification (PTM) system in S. spinosa. Spinosyns, the secondary metabolites produced by Saccharopolyspora spinosa, are the active ingredients in a family of insect control agents. However, the specific functions and influences of acuC protein in S. spinosa are yet to be characterized. Results The knockout strain and overexpression strain were constructed separately with the shuttle vector pOJ260. The production of spinosyns A and D from S. spinosa-acuC were 105.02 mg/L and 20.63 mg/L, which were 1.82-fold and 1.63-fold higher than those of the wild-type strain (57.76 mg/L and 12.64 mg/L), respectively. The production of spinosyns A and D from S. spinosa-ΔacuC were 32.78 mg/L and 10.89 mg/L, respectively. The qRT-PCR results of three selected genes (bldD, ssgA and whiA) confirmed that the overexpression of acuC affected the capacities of mycelial differentiation and sporulation. Comparative proteomics analysis was performed on these strains to investigate the underlying mechanism leading to the enhancement of spinosad yield. Conclusions This study first systematically analysed the effects of overexpression acuC on the growth of S. spinosa and the production of spinosad. The results identify the differentially expressed proteins and provide evidences to understand the acetylation metabolic mechanisms which can lead to the increase of secondary metabolites.

Comparative Transcriptome Analysis Demonstrates the Positive Effect of the Cyclic AMP Receptor Protein Crp on Daptomycin Biosynthesis in Streptomyces roseosporus

Daptomycin, which is produced by Streptomyces roseosporus, has been characterized as a novel cyclic lipopeptide antibiotic that is effective against Gram-positive bacteria. The biosynthesis of daptomycin is regulated by various factors. In the present study, we demonstrated that the cyclic AMP receptor protein (Crp) plays an important role in producing daptomycin in the S. roseosporus industrial strain. We found that daptomycin production from the crp deletion strain decreased drastically, whereas production from the crp overexpression strain increased by 22.1%. Transcriptome and qPCR analyses showed that some genes related to the daptomycin biosynthetic gene cluster (dpt) and the pleiotropic regulator (adpA) were significantly upregulated. RNA-seq also shows Crp to be a multifunctional regulator that modulates primary metabolism and enhances precursor flux to secondary metabolite biosynthesis. These results provide guidance for the development and improvement of potential natural products.

A Novel Protein AoHPS1 Is Involved In Oxidative Stress and Kojic Acid Synthesis in Aspergillus Oryzae

Aspergillus oryzae usually suffers from oxidative stress during the process of aerobic fermentation. However, there is little information on what genes involve in oxidative stress of A. oryzae. Here, we found that the expression of a novel gene Aohps1 was induced during the growth and development of A. oryzae with and without oxidative stress. Sequence analysis revealed that Aohps1 has four transmembrane regions and is conserved in Aspergillus species. Overexpression and deletion of Aohpi1 caused the growth defects with and without oxidative stress, including mycelium growth, conidia formation and biomass. Meanwhile, the Aohpi1 overexpression strain exhibited more sensitivity to oxidative stress than the Aohpi1 disrupted mutant. Furthermore, overexpression and disruption of Aohps1 resulted in the inhibition of kojic acid production with and without oxidative stress, consistent with the reduced expression of kojA that directly contributed to the synthesis of kojic acid. Additionally, the yield of kojic acid is less in the Aohps1 overexpression strain than in the Aohps1 deletion mutant under oxidative stress. Collectively, the discovery of Aohps1 provides new insights into oxidative stress and kojic acid synthesis in A. oryzae.

Identification of Transcription Factors and the Regulatory Genes Involved in Triacylglycerol Accumulation in the Unicellular Red Alga Cyanidioschyzon merolae

Microalgal triacylglycerols (TAGs) are a good feedstock for liquid biofuel production. Improving the expression and/or function of transcription factors (TFs) involved in TAG accumulation may increase TAG content; however, information on microalgae is still lacking. In this study, 14 TFs in the unicellular red alga Cyanidioschyzon merolae were identified as candidate TFs regulating TAG accumulation using available transcriptome and phosphoproteome data under conditions driving TAG accumulation. To investigate the roles of these TFs, we constructed TF-overexpression strains and analyzed lipid droplet (LD) formation and TAG contents in the cells grown under standard conditions. Based on the results, we identified four TFs involved in LD and TAG accumulation. RNA-Seq analyses were performed to identify genes regulated by the four TFs using each overexpression strain. Among the TAG biosynthesis-related genes, only the gene encoding the endoplasmic reticulum-localized lysophosphatidic acid acyltransferase 1 (LPAT1) was notably increased among the overexpression strains. In the LPAT1 overexpression strain, TAG accumulation was significantly increased compared with the control strain under normal growth conditions. These results indicate that the four TFs positively regulate TAG accumulation by changing their target gene expression in C. merolae.

mRNA-Seq reveals the quorum sensing system luxS gene contributes to the environmental fitness of Streptococcus suis type 2

Background Streptococcus suis type 2 (SS2) is an important zoonotic pathogen. We have previously reported the structure of LuxS protein and found that the luxS gene is closely related to biofilm, virulence gene expression and drug resistance of SS2. However, the mechanism of luxS mediated SS2 stress response is unclear. Therefore, this experiment performed stress response to luxS mutant (ΔluxS) and complement strain (CΔluxS), overexpression strain (luxS+) and wild-type SS2 strain HA9801, and analyzed the differential phenotypes in combination with transcriptome data. Results The results indicate that the luxS gene deletion causes a wide range of phenotypic changes, including chain length. RNA sequencing identified 278 lx-regulated genes, of which 179 were up-regulated and 99 were down-regulated. Differential genes focus on bacterial growth, stress response, metabolic mechanisms and drug tolerance. Multiple mitotic genes were down-regulated; while the ABC transporter system genes, cobalamin /Fe3+-iron carrier ABC transporter ATPase and oxidative stress regulators were up-regulated. The inactivation of the luxS gene caused a significant reduction in the growth and survival in the acid (pH = 3.0, 4.0, 5.0) and iron (100 mM iron chelator 2,2′-dipyridyl) stress environments. However, the mutant strain ΔluxS showed increased antioxidant activity to H2O2 (58.8 mmol/L). Conclusions The luxS gene in SS2 appears to play roles in iron metabolism and protective responses to acidic and oxidative environmental conditions.

Effects of Acuc on the Growth Development and Spinosad Biosynthesis of Saccharopolyspora Spinosa

Background: The interaction between acuC and spinosad biosynthesis is complex. In this study, acetoin utilization protein (acuC) was characterized. It is a type I histone deacetylase that is highly conserved in bacteria. This study first explored the effect of acuC on the growth and development of secondary metabolites of S. spinosa. Results: The knockout strain and overexpression strain were constructed separately with the shuttle vector pOJ260. The overexpression of the acuC gene affects the growth and phenotype of S. spinosa. Moreover, the spore production ability of the S. spinosa-acuC strain on solid medium was weaker than that of the wild-type strain. HPLC analysis of the fermentation products for the wild-type and mutant strains demonstrated that the yield of the overexpression strain was 87% higher than that of the wild-type strain. Conclusions: We concluded that the overexpression of acuC positively regulated the biosynthesis of spinosad and affected the acetylation pathway and the growth of S. spinosa. A comparative proteomic analysis between the wild-type and overexpression strains revealed related genes in different metabolic pathways that were affected. We envision that these results can be extended to other actinomycetes for secondary metabolite improvement.

Disruption of protease A and B orthologous genes in the basidiomycetous yeast Pseudozyma antarctica GB-4(0) yields a stable extracellular biodegradable plastic-degrading enzyme

The yeast Pseudozyma antarctica (currently designated Moesziomyces antarcticus) secretes a xylose-induced biodegradable plastic-degrading enzyme (PaE). To suppress degradation of PaE during production and storage, we targeted the inhibition of proteolytic enzyme activity in P. antarctica. Proteases A and B act as upper regulators in the proteolytic network of the model yeast, Saccharomyces cerevisiae. We searched for orthologous genes encoding proteases A and B in the genome of P. antarctica GB-4(0) based on the predicted amino acid sequences. We found two gene candidates, PaPRO1 and PaPRO2, with conserved catalytically important domains and signal peptides indicative of vacuolar protease function. We then prepared gene-deletion mutants of strain GB-4(0), ΔPaPRO1 and ΔPaPRO2, and evaluated PaE stability in culture by immunoblotting analysis. Both mutants exhibited sufficient production of PaE without degradation fragments, while the parent strain exhibited the degradation fragments. Therefore, we concluded that the protease A and B orthologous genes are related to the degradation of PaE. To produce a large quantity of PaE, we made a PaPRO2 deletion mutant of a PaE-overexpression strain named XG8 by introducing a PaE high-production cassette into the strain GB-4(0). The ΔPaPRO2 mutant of XG8 was able to produce PaE without the degradation fragments during large-scale cultivation in a 3-L jar fermenter for 3 days at 30°C. After terminating the agitation, the PaE activity in the XG8 ΔPaPRO2 mutant culture was maintained for the subsequent 48 h incubation at 25°C regardless of remaining cells, while activity in the XG8 control was reduced to 55.1%. The gene-deleted mutants will be useful for the development of industrial processes of PaE production and storage.

The rnc Gene Regulates the Microstructure of Exopolysaccharide in the Biofilm of Streptococcus mutans through the β-Monosaccharides

Streptococcus mutans is known as the crucial pathogen of human dental caries, owing to its contribution to the biofilm development via the capacity of synthesizing exopolysaccharide (EPS), which mainly compose of α-glycosidic bond and β-glycosidic bond. β-glycosidic bond is less flexible than α-glycosidic bond because of differences between their configurational properties. Previous studies have shown that the rnc gene is implicated in the EPS formation and the cariogenicity of S. mutans. However, the effects of rnc on the microstructure of EPS have been not well-understood yet. Here, we further investigated how the rnc gene worked to modulate microstructural properties of the extracellular polysaccharide of S. mutans using glycomics methods. The gas chromatography-mass spectrometer showed that the proportion of glucose was decreased in water-soluble EPS and galactose was absent in water-insoluble EPS from the S. mutans rnc-deficient strain (Smurnc), compared with the isogenic wild-type strain (UA159). The composition of functional groups and the displacement of hydrogen bond were analyzed by infrared radiation and 1H nuclear magnetic resonance, respectively. In addition, phenotypic modulation of the biofilm matrix was assessed by microscopy. We found that the EPS of UA159 and the rnc overexpression strain (Smurnc+) mainly consisted of β-glycosidic bonds. Conversely, the EPS of Smurnc were made up of mostly α-glycosidic bonds, leading to the attenuation of biofilm biomass and bacterial adhesion. Furthermore, the existence of β-glycosidic bond was verified by enzyme digestion. Collectively, the rnc gene modulates the conversion of β-glycosidic bonds, which may play important roles in regulating the micromolecule structure of the EPS matrix, thus affecting the characteristics of S. mutans biofilm. These data illustrate that β-glycosidic bonds mediated by rnc may be potential targets for the prevention and treatment of dental caries.