Alternative splicing of lncRNA LAIR fine-tunes the regulation of neighboring yield-related gene LRK1 expression in rice.

The diversity in alternative splicing of long noncoding RNAs (lncRNAs) poses a challenge for functional annotation of lncRNAs. Moreover, little is known on the effects of alternatively spliced lncRNAs on crop yield. In this study, we cloned nine isoforms resulting from the alternative splicing of the lncRNA LAIR in rice. The LAIR isoforms are generated via alternative 5'/3' splice sites and different combinations of specific introns. All LAIR isoforms activate the expression of the neighboring LRK1 gene and enhance yield-related rice traits. In addition, there are slight differences in the binding ability of LAIR isoforms to the epigenetic modification-related proteins OsMOF and OsWDR5, which affect the enrichment of H4K16ac and H3K4me3 at the LRK1 locus, and consequently fine-tune the regulation of LRK1 expression and yield-related traits. These differences in binding may be caused by polymorphic changes to the RNA secondary structure resulting from alternative splicing. It was also observed that the composition of LAIR isoforms was sensitive to abiotic stress. These findings suggest that the alternative splicing of LAIR leads to the formation of a functional transcript population that precisely regulates yield-related gene expression, which may be relevant for phenotypic polymorphism-based crop breeding under changing environmental conditions.

A key O-demethylase in the degradation of guaiacol by Rhodococcus opacus PD630.

Rhodococcus opacus PD630 is a high oil-producing strain with the ability to convert lignin-derived aromatics to high values, but limited research has been done to elucidate its conversion pathway, especially the upper pathways. In this study, we focused on the upper pathways and demethylation mechanism of lignin-derived aromatics metabolism by R. opacus PD630. The results of the aromatic carbon resource utilization screening showed that R. opacus PD630 had a strong degradation capacity to the lignin-derived methoxy-containing aromatics, such as guaiacol, 3,4-veratric acid, anisic acid, isovanillic acid, and vanillic acid. The gene of gcoAR, which encodes cytochrome P450, showed significant up-regulation when R. opacus PD630 grew on diverse aromatics. Deletion mutants of gcoAR and its partner protein gcoBR resulted in the strain losing the ability to grow on guaiacol, but no significant difference to the other aromatics. Only co-complementation alone of gcoAR and gcoBR restored the strain's ability to utilize guaiacol, demonstrating that both genes were equally important in the utilization of guaiacol. In vitro assays further revealed that GcoAR could convert guaiacol and anisole to catechol and phenol, respectively, with the production of formaldehyde as a by-product. The study provided robust evidence to reveal the molecular mechanism of R. opacus PD630 on guaiacol metabolism and offered a promising study model for dissecting the demethylation process of lignin-derived aromatics in microbes.IMPORTANCEAryl-O-demethylation is believed to be the key rate-limiting step in the catabolism of heterogeneous lignin-derived aromatics in both native and engineered microbes. However, the mechanisms of O-demethylation in lignin-derived aromatic catabolism remain unclear. Notably, guaiacol, the primary component unit of lignin, lacks in situ demonstration and illustration of the molecular mechanism of guaiacol O-demethylation in lignin-degrading bacteria. This is the first study to illustrate the mechanism of guaiacol metabolism by R. opacus PD630 in situ as well as characterize the purified key O-demethylase in vitro. This study provided further insight into the lignin metabolic pathway of R. opacus PD630 and could guide the design of an efficient biocatalytic system for lignin valorization.

RNA-Seq Transcriptome Analysis and Evolution of OsEBS, a Gene Involved in Enhanced Spikelet Number per Panicle in Rice.

Spikelet number per panicle (SNP) is one of the most important yield components in rice. Rice ENHANCING BIOMASS AND SPIKELET NUMBER (OsEBS), a gene involved in improved SNP and yield, has been cloned from an accession of Dongxiang wild rice. However, the mechanism of OsEBS increasing rice SNP is poorly understood. In this study, the RNA-Seq technology was used to analyze the transcriptome of wildtype Guichao 2 and OsEBS over-expression line B102 at the heading stage, and analysis of the evolution of OsEBS was also conducted. A total of 5369 differentially expressed genes (DEGs) were identified between Guichao2 and B102, most of which were down-regulated in B102. Analysis of the expression of endogenous hormone-related genes revealed that 63 auxin-related genes were significantly down-regulated in B102. Gene Ontogeny (GO) enrichment analysis showed that the 63 DEGs were mainly enriched in eight GO terms, including auxin-activated signaling pathway, auxin polar transport, auxin transport, basipetal auxin transport, and amino acid transmembrane transport, most of which were directly or indirectly related to polar auxin transport. Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway analysis further verified that the down-regulated genes related to polar auxin transport had important effects on increased SNP. Analysis of the evolution of OsEBS found that OsEBS was involved in the differentiation of indica and japonica, and the differentiation of OsEBS supported the multi-origin model of rice domestication. Indica (XI) subspecies harbored higher nucleotide diversity than japonica (GJ) subspecies in the OsEBS region, and XI experienced strong balancing selection during evolution, while selection in GJ was neutral. The degree of genetic differentiation between GJ and Bas subspecies was the smallest, while it was the highest between GJ and Aus. Phylogenetic analysis of the Hsp70 family in O. sativa, Brachypodium distachyon, and Arabidopsis thaliana indicated that changes in the sequences of OsEBS were accelerated during evolution. Accelerated evolution and domain loss in OsEBS resulted in neofunctionalization. The results obtained from this study provide an important theoretical basis for high-yield rice breeding.

The kinase OsSK41/OsGSK5 negatively regulates amylose content in rice endosperm by affecting the interaction between OsEBP89 and OsBP5.

Amylose content (AC) is the main factor determining the palatability, viscosity, transparency, and digestibility of rice (Oryza sativa) grains. AC in rice grains is mainly controlled by different alleles of the Waxy (Wx) gene. The AP2/EREBP transcription factor OsEBP89 interacts with the MYC-like protein OsBP5 to synergistically regulate the expression of Wx. Here, we determined that the GLYCOGEN SYNTHASE KINASE 5 (OsGSK5, also named SHAGGY-like kinase 41 [OsSK41]) inhibits the transcriptional activation activity of OsEBP89 in rice grains during amylose biosynthesis. The loss of OsSK41 function enhanced Wx expression and increased AC in rice grains. By contrast, the loss of function of OsEBP89 reduced Wx expression and decreased AC in rice grains. OsSK41 interacts with OsEBP89 and phosphorylates four of its sites (Thr-28, Thr-30, Ser-238, and Thr-257), which makes OsEBP89 unstable and attenuates its interaction with OsBP5. Wx promoter activity was relatively weak when regulated by the phosphomimic variant OsEBP89E -OsBP5 but relatively strong when regulated by the nonphosphorylatable variant OsEBP89A -OsBP5. Therefore, OsSK41-mediated phosphorylation of OsEBP89 represents an additional layer of complexity in the regulation of amylose biosynthesis during rice grain development. In addition, our findings provide four possible sites for regulating rice grain AC via precise gene editing.

Auxin signaling module OsSK41-OsIAA10-OsARF regulates grain yield traits in rice.

Auxin is an important phytohormone in plants, and auxin signaling pathways in rice play key roles in regulating its growth, development, and productivity. To investigate how rice grain yield traits are regulated by auxin signaling pathways and to facilitate their application in rice improvement, we validated the functional relationships among regulatory genes such as OsIAA10, OsSK41, and OsARF21 that are involved in one of the auxin (OsIAA10) signaling pathways. We assessed the phenotypic effects of these genes on several grain yield traits across two environments using knockout and/or overexpression transgenic lines. Based on the results, we constructed a model that showed how grain yield traits were regulated by OsIAA10 and OsTIR1, OsAFB2, and OsSK41 and OsmiR393 in the OsSK41-OsIAA10-OsARF module and by OsARF21 in the transcriptional regulation of downstream auxin response genes in the OsSK41-OsIAA10-OsARF module. The population genomic analyses revealed rich genetic diversity and the presence of major functional alleles at most of these loci in rice populations. The strong differentiation of many major alleles between Xian/indica and Geng/japonica subspecies and/or among modern varieties and landraces suggested that they contributed to improved productivity during evolution and breeding. We identified several important aspects associated with the genetic and molecular bases of rice grain and yield traits that were regulated by auxin signaling pathways. We also suggested rice auxin response factor (OsARF) activators as candidate target genes for improving specific target traits by overexpression and/or editing subspecies-specific alleles and by searching and pyramiding the 'best' gene allelic combinations at multiple regulatory genes in auxin signaling pathways in rice breeding programs.

Phospholipid and antioxidant responses of oleaginous fungus Cunninghamella echinulata against hydrogen peroxide stress.

Hydrogen peroxide (H2O2) is one of the major oxidative stress intracellularly and extracellularly, which may affect lipid membrane or cell membrane. However, the mechanism remains unclear. The present study investigated phospholipid and antioxidant responses of Cunninghamella echinulata under exogenous H2O2 stress by integrating lipidomics and transcriptomics. H2O2 significantly affected phospholipid profile of C. echinulata exposed to exogenous H2O2. The phospholipid content was reduced from 6.41% to 2.47% on the first day, and to 1.03% on the 7th day, which was 5-6 times lower than that in the control. Phosphatidyl choline was reduced significantly from 29.71% to 2.73% on the 7th day. The lipid-related metabolic maps of C. echinulata responding to H2O2 were constructed based on transcriptomics, lipidomics and biochemical analysis. Results showed that H2O2 almost mobilized all the signaling pathways in the cell, especially the AMPK and cAMP signaling pathway, which regulated the metabolism of proteins and fatty acids. H2O2-stress triggered the high expression of heat shock genes. The antioxidant enzymes were activated to supply more NADPH, which contributed to the modulation of intracellular redox balance, and continuously scavenged active substances, thus improving the mycelial resistance to oxidative stress.

A New RING Finger Protein, PLANT ARCHITECTURE and GRAIN NUMBER 1, Affects Plant Architecture and Grain Yield in Rice.

Developing methods for increasing the biomass and improving the plant architecture is important for crop improvement. We herein describe a gene belonging to the RING_Ubox (RING (Really Interesting New Gene) finger domain and U-box domain) superfamily, PLANT ARCHITECTURE and GRAIN NUMBER 1 (PAGN1), which regulates the number of grains per panicle, the plant height, and the number of tillers. We used the CRISPR/Cas9 system to introduce loss-of-function mutations to OsPAGN1. Compared with the control plants, the resulting pagn1 mutant plants had a higher grain yield because of increases in the plant height and in the number of tillers and grains per panicle. Thus, OsPAGN1 may be useful for the genetic improvement of plant architecture and yield. An examination of evolutionary relationships revealed that OsPAGN1 is highly conserved in rice. We demonstrated that OsPAGN1 can interact directly with OsCNR10 (CELL NUMBER REGULATOR10), which negatively regulates the number of rice grains per panicle. A transcriptome analysis indicated that silencing OsPAGN1 affects the levels of active cytokinins in rice. Therefore, our findings have clarified the OsPAGN1 functions related to rice growth and grain development.

Lytic polysaccharide monooxygenases promote oxidative cleavage of lignin and lignin-carbohydrate complexes during fungal degradation of lignocellulose.

Overcoming lignocellulosic biomass recalcitrance, especially the cleavage of cross-linkages in lignin-carbohydrate complexes (LCCs) and lignin, is essential for both the carbon cycle and industrial biorefinery. Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that play a key role in fungal polysaccharide oxidative degradation. Nevertheless, comprehensive analysis showed that LPMOs from a white-rot fungus, Pleurotus ostreatus, correlated well with the Fenton reaction and were involved in the degradation of recalcitrant nonpolysaccharide fractions in this research. Thus, LPMOs participated in the extracellular Fenton reaction by enhancing iron reduction in quinone redox cycling. A Fenton reaction system consisting of LPMOs, hydroquinone, and ferric iron can efficiently produce hydroxy radicals and then cleave LCCs or lignin linkages. This finding indicates that LPMOs are underestimated auxiliary enzymes in eliminating biomass recalcitrance.

Integrative Analysis of Selected Metabolites and the Fungal Transcriptome during the Developmental Cycle of Ganoderma lucidum Strain G0119 Correlates Lignocellulose Degradation with Carbohydrate and Triterpenoid Metabolism.

To systemically understand the biosynthetic pathways of bioactive substances, including triterpenoids and polysaccharides, in Ganoderma lucidum, the correlation between substrate degradation and carbohydrate and triterpenoid metabolism during growth was analyzed by combining changes in metabolite content and changes in related enzyme expression in G. lucidum over 5 growth phases. Changes in low-polarity triterpenoid content were correlated with changes in glucose and mannitol contents in fruiting bodies. Additionally, changes in medium-polarity triterpenoid content were correlated with changes in the lignocellulose content of the substrate and with the glucose, trehalose, and mannitol contents of fruiting bodies. Weighted gene coexpression network analysis (WGCNA) indicated that changes in trehalose and polyol contents were related to carbohydrate catabolism and polysaccharide synthesis. Changes in triterpenoid content were related to expression of the carbohydrate catabolic enzymes laccase, cellulase, hemicellulase, and polysaccharide synthase and to the expression of several cytochrome P450 monooxygenases (CYPs). It was concluded that the products of cellulose and hemicellulose degradation participate in polyol, trehalose, and polysaccharide synthesis during initial fruiting body formation. These carbohydrates accumulate in the early phase of fruiting body formation and are utilized when the fruiting bodies mature and a large number of spores are ejected. An increase in carbohydrate metabolism provides additional precursors for the synthesis of triterpenoids. IMPORTANCE Most studies of G. lucidum have focused on its medicinal function and on the mechanism of its activity, whereas the physiological metabolism and synthesis of bioactive substances during the growth of this species have been less studied. Therefore, theoretical guidance for cultivation methods to increase the production of bioactive compounds remains lacking. This study integrated changes in the lignocellulose, carbohydrate, and triterpenoid contents of G. lucidum with enzyme expression from transcriptomics data using WGCNA. The findings helped us better understand the connections between substrate utilization and the synthesis of polysaccharides and triterpenoids during the cultivation cycle of G. lucidum. The results of WGCNA suggest that the synthesis of triterpenoids can be enhanced not only through regulating the expression of enzymes in the triterpenoid pathway, but also through regulating carbohydrate metabolism and substrate degradation. This study provides a potential approach and identifies enzymes that can be targeted to regulate lignocellulose degradation and accelerate the accumulation of bioactive substances by regulating substrate degradation in G. lucidum.

Exosome-Mediated miR-29 Transfer Reduces Muscle Atrophy and Kidney Fibrosis in Mice.

Our previous study showed that miR-29 attenuates muscle wasting in chronic kidney disease. Other studies found that miR-29 has anti-fibrosis activity. We hypothesized that intramuscular injection of exosome-encapsulated miR-29 would counteract unilateral ureteral obstruction (UUO)-induced muscle wasting and renal fibrosis. We used an engineered exosome vector, which contains an exosomal membrane protein gene Lamp2b that was fused with the targeting peptide RVG (rabies viral glycoprotein peptide). RVG directs exosomes to organs that express the acetylcholine receptor, such as kidney. The intervention of Exo/miR29 increased muscle cross-sectional area and decreased UUO-induced upregulation of TRIM63/MuRF1 and FBXO32/atrogin-1. Interestingly, renal fibrosis was partially depressed in the UUO mice with intramuscular injection of Exo/miR29. This was confirmed by decreased TGF-ß, alpha-smooth muscle actin, fibronectin, and collagen 1A1 in the kidney of UUO mice. When we used fluorescently labeled Exo/miR29 to trace the Exo/miR route in vivo and found that fluorescence was visible in un-injected muscle and in kidneys. We found that miR-29 directly inhibits YY1 and TGF-ß3, which provided a possible mechanism for inhibition of muscle atrophy and renal fibrosis by Exo/miR29. We conclude that Exo/miR29 ameliorates skeletal muscle atrophy and attenuates kidney fibrosis by downregulating YY1 and TGF-ß pathway proteins.

Response of Trametes hirsuta to hexavalent chromium promotes laccase-mediated decolorization of reactive black 5.

The recalcitrant azo dyes combined with heavy metals constitute a major challenge for the bioremediation of industrial effluents. This study aimed to investigate the effect and mechanism of action of a white-rot fungus Trametes hirsuta TH315 on the simultaneous removal of hexavalent chromium [Cr(VI)] and azo dye (Reactive Black 5, RB5). Here, this study discovered that toxic Cr(VI) (1 mM) greatly promoted RB5 decolorization (from 57.15% to 83.65%) by white-rot fungus Trametes hirsuta with high Cr(VI)-reducing ability (>96%), resulting in the simultaneous removal of co-contaminants. On the basis of transcriptomic and biochemical analysis, our study revealed that the oxidative stress in co-contaminants mainly caused by Cr(VI), and a number of dehydrogenases and oxidases showed up-regulation in response to Cr(VI) stress. It was noteworthy that the oxidative stress caused by Cr(VI) in co-contaminants can both significantly induce glutathione S-transferase and laccase expression. Glutathione S-transferase potentially involved in antioxidation against Cr(VI) stress. Laccase was found to play a key role in RB5 decolorization by T. hirsuta. These results suggested that the simultaneous removal of co-contaminants by T. hirsuta could be achieved with Cr(VI) exposure. Overall, the elucidation of the molecular basis in details will help to advance the general knowledge about the fungus by facing harsh environments, and put forward a further possible application of fungi on environmental remediation.

Role of PKC and AMPK in hypertonicity-stimulated water reabsorption in rat inner medullary collecting ducts.

Hypertonicity increases water permeability, independently of vasopressin, in the inner medullary collecting duct (IMCD) by increasing aquaporin-2 (AQP2) membrane accumulation. We investigated whether protein kinase C (PKC) and adenosine monophosphate kinase (AMPK) are involved in hypertonicity-regulated water permeability. Increasing perfusate osmolality from 150 to 290 mosmol/kgH2O and bath osmolality from 290 to 430 mosmol/kgH2O significantly stimulated osmotic water permeability. The PKC inhibitors chelerythrine (10 µM) and rottlerin (50 µM) significantly reversed the increase in osmotic water permeability stimulated by hypertonicity in perfused rat terminal IMCDs. Chelerythrine significantly increased phosphorylation of AQP2 at S261 but not at S256. Previous studies show that AMPK is stimulated by osmotic stress. We tested AMPK phosphorylation under hypertonic conditions. Hypertonicity significantly increased AMPK phosphorylation in inner medullary tissues. Blockade of AMPK with Compound C decreased hypertonicity-stimulated water permeability but did not alter phosphorylation of AQP2 at S256 and S261. AICAR, an AMPK stimulator, caused a transient increase in osmotic water permeability and increased phosphorylation of AQP2 at S256. When inner medullary tissue was treated with the PKC activator phorbol dibutyrate (PDBu), the AMPK activator metformin, or both, AQP2 phosphorylation at S261 was decreased with PDBu or metformin alone, but there was no additive effect on phosphorylation with PDBu and metformin together. In conclusion, hypertonicity regulates water reabsorption by activating PKC. Hypertonicity-stimulated water reabsorption by PKC may be related to the decrease in endocytosis of AQP2. AMPK activation promotes water reabsorption, but the mechanism remains to be determined. PKC and AMPK do not appear to act synergistically to regulate water reabsorption.

A Lytic Polysaccharide Monooxygenase from a White-Rot Fungus Drives the Degradation of Lignin by a Versatile Peroxidase.

Lytic polysaccharide monooxygenases (LPMOs), a class of copper-dependent enzymes, play a crucial role in boosting the enzymatic decomposition of polysaccharides. Here, we reveal that LPMOs might be associated with a lignin degradation pathway. An LPMO from white-rot fungus Pleurotus ostreatus, LPMO9A (PoLPMO9A), was shown to be able to efficiently drive the activity of class II lignin-degrading peroxidases in vitro through H2O2 production regardless of the presence or absence of a cellulose substrate. An LPMO-driven peroxidase reaction can degrade ß-O-4 and 5-5' types of lignin dimer with 46.5% and 37.7% degradation, respectively, as well as alter the structure of natural lignin and kraft lignin. H2O2 generated by PoLPMO9A was preferentially utilized for the peroxidase from Physisporinus sp. strain P18 (PsVP) reaction rather than cellulose oxidation, indicating that white-rot fungi may have a strategy for preferential degradation of resistant lignin. This discovery shows that LPMOs may be involved in lignin oxidation as auxiliary enzymes of lignin-degrading peroxidases during the white-rot fungal decay process.IMPORTANCE The enzymatic biodegradation of structural polysaccharides is affected by the degree of delignification of lignocellulose during the white-rot fungal decay process. The lignin matrix decreases accessibility to the substrates for LPMOs. H2O2 has been studied as a cosubstrate for LPMOs, but the formation and utilization of H2O2 in the reactions still represent an intriguing focus of current research. Lignin-degrading peroxidases and LPMOs usually coexist during fungal decay, and therefore, the relationship between H2O2-dependent lignin-degrading peroxidases and LPMOs should be considered during the wood decay process. The current study revealed that white-rot fungal LPMOs may be involved in the degradation of lignin through driving a versatile form of peroxidase activity in vitro and that H2O2 generated by PoLPMO9A was preferentially used for lignin oxidation by lignin-degrading peroxidase (PsVP). These findings reveal a potential relationship between LPMOs and lignin degradation, which will be of great significance for further understanding the contribution of LPMOs to the white-rot fungal decay process.

Carbaryl biodegradation by Xylaria sp. BNL1 and its metabolic pathway.

Although ascomycetes occupy a vaster niche in soil than the well-studied basidiomycetes, they have received limited attention in studies related to bioremediation. In this study, the degradation of carbaryl by Xylaria sp. was studied in different culture conditions and its possible metabolic pathway was elucidated. In liquid culture, 99% of the added carbaryl was eliminated when cytochrome P450 (CYP450) was active, which was similar to the degradation rate of Pleurotus ostreatus, a fungus with strong bioremediation ability. Mn2+ is beneficial to the degradation of carbaryl. Compared to the 72.17% degradation rate in sterile soil, 59.0% carbaryl was eliminated in non-sterile soil, which suggested that Xylaria sp. BNL1 can resist microorganismal infection. Furthermore, the intracellular fractions containing laccase, CYP450, and carbaryl esterase efficiently degraded carbaryl. The presence of carbaryl metabolites suggested that Xylaria sp. BNL1 initiated its attack on carbaryl via carbaryl esterase to release α-naphthol, which was further degraded to 1,4-naphthoquinone and benzoic acid by CYP450 and laccase. Thus, our study highlights the potential of using Xylaria sp. for bioremediation.

Biodegradation of n-hexadecane by Aspergillus sp. RFC-1 and its mechanism.

Fungi can use n-hexadecane (HXD) as a sole carbon source. But the mechanism of HXD degradation remains unclear. This work mainly aimed to study the degradation of HXD by Aspergillus sp. RFC-1 obtained from oil-contaminated soil. The HXD content, medium acidification and presence of hexadecanoic acid in the medium were determined by gas chromatography-mass spectrometry, and fungal growth was observed. Enzyme and gene expression assays suggested the involvement of an alkane hydroxylase, an alcohol dehydrogenase, and a P450 enzyme system in HXD degradation. A biosurfactant produced by the strain RFC-1 was also characterized. During 10 days of incubation, 86.3% of HXD was degraded by RFC-1. The highest activities of alkane hydroxylase (125.4 µmol mg-1 protein) and alcohol dehydrogenase (12.5 µmol mg-1 proteins) were recorded. The expression level of cytochrome P450 gene associated with oxidation was induced (from 0.94-fold to 5.45-fold) under the HXD condition by Real-time PCR analysis. In addition, HXD accumulated in inclusion bodies of RFC-1with the maximum of 5.1 g L-1. Results of blood agar plate and thin-layer chromatography analysis showed RFC-1 released high lipid and emulsification activity in the fungal culture. Induced cell surface hydrophobicity and reduced surface tension also indicated the RFC-1-mediated biosurfactant production, which facilitated the HXD degradation and supported the degradation process.

Directed Evolution of a Homodimeric Laccase from Cerrena unicolor BBP6 by Random Mutagenesis and In Vivo Assembly.

Laccases have great potential for industrial applications due to their green catalytic properties and broad substrate specificities, and various studies have attempted to improve the catalytic performance of these enzymes. Here, to the best of our knowledge, we firstly report the directed evolution of a homodimeric laccase from Cerrena unicolor BBP6 fused with α-factor prepro-leader that was engineered through random mutagenesis followed by in vivo assembly in Saccharomyces cerevisiae. Three evolved fusion variants selected from ~3500 clones presented 31- to 37-fold increases in total laccase activity, with better thermostability and broader pH profiles. The evolved α-factor prepro-leader enhanced laccase expression levels by up to 2.4-fold. Protein model analysis of these variants reveals that the beneficial mutations have influences on protein pKa shift, subunit interaction, substrate entrance, and C-terminal function.

Differential expression of desaturase genes and changes in fatty acid composition of Mortierella sp. AGED in response to environmental factors.

BACKGROUND: Some oleaginous fungi can produce large amounts of polyunsaturated fatty acids (PUFAs) which serve many physiological functions. Numerous desaturases are critical for the synthesis of PUFAs. This study aimed to investigate the regulation of lipid production and desaturase gene expression in Mortierella sp. AGED in response to different environmental factors, and the relationships between lipid production and desaturase gene expression. RESULTS: The fatty acid composition and mRNA levels of desaturase genes were significantly changed under low temperatures. With the exception of Δ5-desaturase, the transcript levels of all desaturase genes increased at a temperature of 20 °C. Changes in content of lipid and PUFAs responding to low temperature were consistent with desaturase gene expression. Time course studies on gene expression showed that mRNA levels of four desaturase genes increased rapidly after transferring the cells to low temperature. Ethanol (1.5% v/v) increased the transcript levels of Δ9-, Δ6- and Δ5-desaturase genes significantly and of Δ12-desaturase gene slightly. Different metal ions such as Ca2+ , Zn2+ and Fe3+ could stimulate PUFA synthesis and up-regulate desaturase gene transcription, while Cu2+ inhibited desaturase gene expression and lipid accumulation. CONCLUSION: This study should enable us to understand the regulatory mechanism of desaturase gene expression and lipid synthesis. It is helpful to improve PUFA productivity in Mortierella sp. AGED. © 2016 Society of Chemical Industry.

Cloning and functional analysis of Δ6-desaturase gene and its upstream region from Mortierella sp. AGED.

BACKGROUND: Δ6-desaturase belonging to membrane-bound enzyme is a key enzyme involved in the synthesis of polyunsaturated fatty acids (PUFAs). This study aimed to clone and characterise Δ6-desaturase gene and its upstream regulatory region of Mortierella sp. AGED. RESULTS: Glucose and soybean meal are best for lipid and arachidonic acid accumulation of Mortierella sp. AGED. A 1375-bp Δ6-desaturase gene AGfad6 which contains a 1275-bp open reading frame encoding 424 amino acids without signal peptide was cloned. The putative protein contained three conserved histidine-rich motifs and a conserved cytochrome b5 HPGG (H: Histine, P: Proline, G: Glycine, G: Glycine) motif, with a mass of 48.3 kDa and an isoelectric point of 5.96. AGfad6 was successfully expressed in Pichia pastoris GS115, which exerted the effect on converting linoleic acid to γ-linolenic acid. The 1712-bp upstream region contained basic transcriptional elements including TATA, GC and GATA box, putative target-binding sites for transcription factors such as TATA binding protein, transcription activator, CCAAT-enhancer-binding protein, activator protein 1, alcohol dehydrogenase gene regulator 1 and metabolic regulators p40x in fungi, stress-related elements including GT-1 (light-responsive, salicylic acid-inducible), stress response element, heat stress-responsive element, which might participate in regulation of PUFAs synthesis. CONCLUSION: The present finding could enable us to understand the evolution and regulatory mechanism of Δ6-desaturase gene.

OsEXPA7 Encoding an Expansin Affects Grain Size and Quality Traits in Rice (Oryza sativa L.).

BACKGROUND: Yield and quality are the two most important traits in crop breeding. Exploring the regulatory mechanisms that affect both yield and quality traits is of great significance for understanding the molecular genetic networks controlling these key crop attributes. Expansins are cell wall loosening proteins that play important roles in regulating rice grain size. RESULTS: We investigated the effect of OsEXPA7, encoding an expansin, on rice grain size and quality. OsEXPA7 overexpression resulted in increased plant height, panicle length, grain length, and thousand-grain weight in rice. OsEXPA7 overexpression also affected gel consistency and amylose content in rice grains, thus affecting rice quality. Subcellular localization and tissue expression analyses showed that OsEXPA7 is localized on the cell wall and is highly expressed in the panicle. Hormone treatment experiments revealed that OsEXPA7 expression mainly responds to methyl jasmonate, brassinolide, and gibberellin. Transcriptome analysis and RT-qPCR experiments showed that overexpression of OsEXPA7 affects the expression of OsJAZs in the jasmonic acid pathway and BZR1 and GE in the brassinosteroid pathway. In addition, OsEXPA7 regulates the expression of key quantitative trait loci related to yield traits, as well as regulates the expression levels of BIP1 and bZIP50 involved in the seed storage protein biosynthesis pathway. CONCLUSIONS: These results reveal that OsEXPA7 positively regulates rice yield traits and negatively regulates grain quality traits by involving plant hormone pathways and other trait-related pathway genes. These findings increase our understanding of the potential mechanism of expansins in regulating rice yield and quality traits and will be useful for breeding high-yielding and high-quality rice cultivars.

Lignin valorization to bioplastics with an aromatic hub metabolite-based autoregulation system.

Exploring microorganisms with downstream synthetic advantages in lignin valorization is an effective strategy to increase target product diversity and yield. This study ingeniously engineers the non-lignin-degrading bacterium Ralstonia eutropha H16 (also known as Cupriavidus necator H16) to convert lignin, a typically underutilized by-product of biorefinery, into valuable bioplastic polyhydroxybutyrate (PHB). The aromatic metabolism capacities of R. eutropha H16 for different lignin-derived aromatics (LDAs) are systematically characterized and complemented by integrating robust functional modules including O-demethylation, aromatic aldehyde metabolism and the mitigation of by-product inhibition. A pivotal discovery is the regulatory element PcaQ, which is highly responsive to the aromatic hub metabolite protocatechuic acid during lignin degradation. Based on the computer-aided design of PcaQ, we develop a hub metabolite-based autoregulation (HMA) system. This system can control the functional genes expression in response to heterologous LDAs and enhance metabolism efficiency. Multi-module genome integration and directed evolution further fortify the strain's stability and lignin conversion capacities, leading to PHB production titer of 2.38 g/L using heterologous LDAs as sole carbon source. This work not only marks a leap in bioplastic production from lignin components but also provides a strategy to redesign the non-LDAs-degrading microbes for efficient lignin valorization.