The Influence of Homologous Arm Length on Homologous Recombination Gene Editing Efficiency Mediated by SSB/CRISPR-Cas9 in Escherichia coli.

The precise editing of genes mediated by CRISPR-Cas9 necessitates the application of donor DNA with appropriate lengths of homologous arms and fragment sizes. Our previous development, SSB/CRISPR-Cas9, has demonstrated high efficiency in homologous recombination and non-homologous end joining gene editing within bacteria. In this study, we optimized the lengths and sizes of homologous arms of the donor DNA within this system. Two sets of donor DNA constructs were generated: one set comprised donors with only 10-100 bp homologous arms, while the other set included donors with homologous arms ranging from 10-100 bp, between which was a tetracycline resistance expression cassette (1439 bp). These donor constructs were transformed into Escherichia coli MG1655 cells alongside pCas-SSB/pTargetF-lacZ. Notably, when the homologous arms ranged from 10 to 70 bp, the transformation efficiency of non-selectable donors was significantly higher than that of selectable donors. However, within the range of 10-100 bp homologous arm lengths, the homologous recombination rate of selectable donors was significantly higher than that of non-selectable donors, with the gap narrowing as the homologous arm length increased. For selectable donor DNA with homologous arm lengths of 10-60 bp, the homologous recombination rate increased linearly, reaching a plateau when the homologous arm length was between 60-100 bp. Conversely, for non-selectable donor DNA, the homologous recombination rate increased linearly with homologous arm lengths of 10-90 bp, plateauing at 90-100 bp. Editing two loci simultaneously with 100 bp homologous arms, whether selectable or non-selectable, showed no difference in transformation or homologous recombination rates. Editing three loci simultaneously with 100 bp non-selectable homologous arms resulted in a 45% homologous recombination rate. These results suggest that efficient homologous recombination gene editing mediated by SSB/CRISPR-Cas9 can be achieved using donor DNA with 90-100 bp non-selectable homologous arms or 60-100 bp selectable homologous arms.

Single-Stranded DNA-Binding Proteins Mediate DSB Repair and Effectively Improve CRISPR/Cas9 Genome Editing in Escherichia coli and Pseudomonas.

Single-stranded DNA-binding proteins (SSBs) are essential for all living organisms. Whether SSBs can repair DNA double-strand breaks (DSBs) and improve the efficiency of CRISPR/Cas9-mediated genome editing has not been determined. Here, based on a pCas/pTargetF system, we constructed pCas-SSB and pCas-T4L by replacing the λ-Red recombinases with Escherichia coli SSB and phage T4 DNA ligase in pCas, respectively. Inactivation of the E. coli lacZ gene with homologous donor dsDNA increased the gene editing efficiency of pCas-SSB/pTargetF by 21.4% compared to pCas/pTargetF. Inactivation of the E. coli lacZ gene via NHEJ increased the gene editing efficiency of pCas-SSB/pTargetF by 33.2% compared to pCas-T4L/pTargetF. Furthermore, the gene-editing efficiency of pCas-SSB/pTargetF in E. coli (ΔrecA, ΔrecBCD, ΔSSB) with or without donor dsDNA did not differ. Additionally, pCas-SSB/pTargetF with donor dsDNA successfully deleted the wp116 gene in Pseudomonas sp. UW4. These results demonstrate that E. coli SSB repairs DSBs caused by CRISPR/Cas9 and effectively improves CRISPR/Cas9 genome editing in E. coli and Pseudomonas.

Dynamic succession of microbial compost communities and functions during Pleurotus ostreatus mushroom cropping on a short composting substrate.

Cultivating oyster mushrooms (Pleurotus ostreatus), a typical primary decomposer of lignocellulose, on a short composting substrate is a novel procedure which possesses energy conserves, reduced the chance of infection by competitive species, shorter production duration and achieved high production efficiency. However, the microbiome and microbial metabolic functions in the composting substrate during the mushroom cropping is unknown. In the present study, the contents of hemicellulose, cellulose and lignin and the activities of protease, laccase and cellulase were evaluated in the corncob short composting substrate from before oyster mushroom spawning to first flush fructification; meanwhile the changes in the microbiome and microbial metabolic functions were surveyed by using metagenomic sequencing. Results showed that the hemicellulose, cellulose and lignin in the short composting substrate were decomposed of 42.76, 34.01, and 30.18%, respectively, during the oyster mushroom cropping process. In addition, the contents of hemicellulose, cellulose and lignin in the composting substrate were reduced rapidly and negatively correlated with the abundance of the Actinobacteria phylum. The activities of protease, laccase and cellulase fastly increased in the period of before oyster mushroom spawning to full colonization and were positively correlated to the abundance of Actinobacteria phylum. The total abundance of bacteria domain gradually decreased by only approximately 15%, while the abundance of Actinobacteria phylum increased by 68% and was positively correlated with that of oyster mushroom. The abundance of oyster mushroom increased by 50 times from spawning to first flush fructification. The dominant genera, all in the order of Actinomycetales, were Cellulosimicrobium, Mycobacterium, Streptomyces and Saccharomonospora. The total abundance of genes with functions annotated in the Clusters of Orthologous Groups of proteins (COG) for Bacteria and Archaea and Kyoto Encyclopedia of Genes and Genomes (KEGG) database for all three life domains was positively correlated. The three metabolic pathways for carbohydrates, amino acids and energy were the primary enrichment pathways in KEGG pathway, accounting for more than 30% of all pathways, during the mushroom cropping in which the glycine metabolic pathway, carbon fixation pathways in prokaryotes and methane metabolism were all dominated by bacteria. The genes of cellulolytic enzymes, hemicellulolytic enzymes, laccase, chitinolytic enzymes, peptidoglycanlytic enzymes and ammonia assimilation enzymes with abundances from 0.28 to 0.24%, 0.05 to 0.02%, 0.02 to 0.01%, 0.14 to 0.08%, 0.39 to 0.16%, and 0.13 to 0.12% during the mushroom cropping identified in the Evolutionary Genealogy of Genes: Non-supervised Orthologous Groups (eggNOG) database for all three life domains were all aligned to COG database. These results indicated that bacteria, especially Actinomycetales, were the main metabolism participants in the short composting substrate during the oyster mushroom cropping. The relationship between oyster mushrooms and bacteria was cooperative, Actinomycetales were oyster mushroom growth promoting bacteria (OMGPB).

Two Hybrid Histidine Kinases Involved in the Ethylene Regulation of the Mycelial Growth and Postharvest Fruiting Body Maturation and Senescence of Agaricus bisporus.

Ethylene regulates mycelial growth, primordium formation, and postharvest mushroom maturation and senescence in the white button mushroom, Agaricus bisporus. However, it remains unknown how ethylene is detected by the mushroom. In this study, we found that two hybrid histidine kinases in the mushroom, designated AbETR1 and AbETR2, showed domain structures similar to those of plant ethylene receptors. The transmembrane helices of AbETR1 and AbETR2 were expressed in yeast cells and showed ethylene-binding activities. Mushroom strains with downregulated expressions of AbETR1 and AbETR2 showed reduced sensitivity to the ethylene inhibition of mycelial growth, ethylene regulation of their own synthesis, postharvest mushroom maturation, and senescence and expression of maturation- and senescence-related genes. Therefore, AbETR1 and AbETR2 are expected to be biologically functional ethylene receptors and exhibit a different mode of action from that of the receptors of plants. Here, we fill gaps in the knowledge pertaining to higher fungus ethylene receptors, discover a novel mode of action of ethylene receptors, confirm ethylene as a novel fungal hormone, and provide a facilitated approach for preventing the maturation and senescence of postharvest button mushrooms. IMPORTANCE Ethylene regulates diverse physiological activities in bacteria, cyanobacteria, fungi, and plants, but how to perceive ethylene by fungi only remains unknown. In this study, we identify two biologically functional ethylene receptors in the basidiomycete fungus Agaricus bisporus, which fills the gaps of deficient fungal ethylene receptors. Furthermore, we found that decreased expression of the ethylene receptors facilitates preventing the maturation and senescence of postharvest button mushrooms, indicating that the two fungal ethylene receptors positively regulate the ethylene response, in contrast to that in plants.

The Biosynthesis of 1-octene-3-ol by a Multifunctional Fatty Acid Dioxygenase and Hydroperoxide Lyase in Agaricus bisporus.

The biosynthetic pathway from linoleic acid to 1-octen-3-ol in Agaricus bisporus has long been established, in which linoleic acid is converted to 10-hydroperoxide (10-HPOD) by deoxygenation, and 10-HPOD is subsequently cleaved to yield 1-octene-3-ol and 10-oxodecanoic acid. However, the corresponding enzymes have not been identified and cloned. In the present study, four putative genes involved in oxylipid biosynthesis, including one lipoxygenase gene named AbLOX, two linoleate diol synthase genes named AbLDS1 and AbLDS2, and one hydroperoxide lyase gene named AbHPL were retrieved from the A. bisporus genome by a homology search and cloned and expressed prokaryotically. AbLOX, AbLDS1, and AbLDS2 all exhibited fatty acid dioxygenase activity, catalyzing the conversion of linoleic acid to generate hydroperoxide, and AbHPL showed a cleaving hydroperoxide activity, as was determined by the KI-starch method. AbLOX and AbHPL catalyzed linoleic acid to 1-octen-3-ol with an optimum temperature of 35 °C and an optimum pH of 7.2, whereas AbLDS1, AbLDS2, and AbHPL catalyzed linoleic acid without 1-octen-3-ol. Reduced AbLOX expression in antisense AbLOX transformants was correlated with a decrease in the yield of 1-octen-3-ol. AbLOX and AbHPL were highly homologous to the sesquiterpene synthase Cop4 of Coprinus cinerea and the yeast sterol C-22 desaturase, respectively. These results reveal that the enzymes for the oxidative cleavage of linoleic acid to synthesize 1-octen-3-ol in A. bisporus are the multifunctional fatty acid dioxygenase AbLOX and hydroperoxide lyase AbHPL.

Phylogenesis of the Functional 1-Aminocyclopropane-1-Carboxylate Oxidase of Fungi and Plants.

The 1-aminocyclopropane-1-carboxylic acid (ACC) pathway that synthesizes ethylene is shared in seed plants, fungi and probably other organisms. However, the evolutionary relationship of the key enzyme ACC oxidase (ACO) in the pathway among organisms remains unknown. Herein, we cloned, expressed and characterized five ACOs from the straw mushroom (Volvariella volvacea) and the oyster mushroom (Pleurotus ostreatus): VvACO1-4 and PoACO. The five mushroom ACOs and the previously identified AbACO of the button mushroom contained all three conserved residues that bound to Fe(II) in plant ACOs. They also had variable residues that were conserved and bound to ascorbate and bicarbonate in plant ACOs and harbored only 1-2 of the five conserved ACO motifs in plant ACOs. Particularly, VvACO2 and AbACO had only one ACO motif 2. Additionally, VvACO4 shared 44.23% sequence identity with the cyanobacterium Hapalosiphon putative functional ACO. Phylogenetic analysis showed that the functional ACOs of monocotyledonous and dicotyledonous plants co-occurred in Type I, Type II and Type III, while putative functional gymnosperm ACOs also appeared in Type III. The putative functional bacterial ACO, functional fungi and slime mold ACOs were clustered in ancestral Type IV. These results indicate that ACO motif 2, ACC and Fe(II) are essential for ACO activity. The ACOs of the other organisms may come from the horizontal transfer of fungal ACOs, which were found ordinarily in basidiomycetes. It is mostly the first case for the horizontal gene transfers from fungi to seed plants. The horizontal transfer of ACOs from fungi to plants probably facilitates the fungal-plant symbioses, plant-land colonization and further evolution to form seeds.

Taisui TS-2007S, a Large Microbial Mat Discovered in Soil in China.

In this study, Taisui TS-2007S, a previously unidentified biological object discovered in soil in China, was identified. TS-2007S was shown to contain abundant carbohydrates but a scarcity of protein, fat, and minerals. The exopolymers of TS-2007S showed FT-IR spectra that were similar to those of xanthan gum (XG) but that were dissimilar to those of polyvinyl alcohol (PVA). The NMR spectra of TS-2007S exopolymers in D2O were similar to those of PVA but differed from those of xanthan gum. Unlike PVA, TS-2007S exopolymers and xanthan gum were not soluble in dimethyl sulfoxide (DMSO). Furthermore, the exopolymers contained many monosaccharide components, including fucose, rhamnose, mannose, and glucuronic acid in a molar ratio of 87.90:7.49:4.45:0.15. The exopolymers also included traces of glucuronic acid, galactose, and xylose. Taken together, these results suggest that the exopolymers are microbial extracellular polymeric substances (EPSs). The microbial community structure in TS-2007S showed that the predominant bacterial, archaeal, and fungal phyla were Proteobacteria, Euryarchaeota, and Ascomycota at high relative abundances of 90.77, 97.15, and 87.43%, respectively, different from those observed in water and soil environments. Based on these results, we strongly propose that TS-2007S should be defined as a microbial mat formed in soil.

Enhancing the 1-Aminocyclopropane-1-Carboxylate Metabolic Rate of Pseudomonas sp. UW4 Intensifies Chemotactic Rhizocompetence.

1-aminocyclopropane-1-carboxylic acid (ACC) is a strong metabolism-dependent chemoattractant for the plant beneficial rhizobacterium Pseudomonas sp. UW4. It is unknown whether enhancing the metabolic rate of ACC can intensify the chemotaxis activity towards ACC and rhizocompetence. In this study, we selected four promoters to transcribe the UW4 ACC deaminase (AcdS) gene in the UW4 ΔAcdS mutant. PA is the UW4 AcdS gene promoter, PB20, PB10 and PB1 are synthetic promoters. The order of the AcdS gene expression level and AcdS activity of the four strains harboring the promoters were PB20 > PA > PB10 > PB1. Interestingly, the AcdS activity of the four strains and their parent strain UW4 was significantly positively correlated with their chemotactic activity towards ACC, rhizosphere colonization, roots elongation and dry weight promotion. The results released that enhancing the AcdS activity of PGPRenable them to achieve strong chemotactic responses to ACC, rhizocompetence and plant growth promotion.

Identification of two Pleurotus ostreatus blue light receptor genes (PoWC-1 and PoWC-2) and in vivo confirmation of complex PoWC-12 formation through yeast two hybrid system.

Blue light is necessary for initiation of mushroom formation of Pleurotus ostreatus. In this study, we isolated homologues of the blue light receptor genes wc-1 and wc-2 from P. ostreatus, PoWC-1 and PoWC-2. The PoWC-1 contained three typical PAS domains and one PAS domain exhibited significant similarity to the LOV domain of known blue light receptors. The PoWC-2 had one typical PAS domain and one ZnF domain. The qRT-PCR analysis showed that PoWC-1 and PoWC-2 expression increased in a short time, and the final level tended to be stable along with the light illumination. The PoWC-1 and PoWC-2 expression levels of the primordium period was higher than that of mature fruiting-body period; and in the pileus were the highest, followed by the stipe and the gills. The expression of PoWC-1 and PoWC-2 in pre-primordial mycelia was induced by light exposure. In vivo analysis through yeast two-hybrid experiment disclosed that PoWC-1 and PoWC-2 could form heterologous complex to activate the reporter genes and the complex perform the transcription factor function requiring the addition of FAD.

Pseudomonas sp. UW4 acdS gene promotes primordium initiation and fruiting body development of Agaricus bisporus.

To simplify industrial mushroom cultivation, we introduced a bacterial Pseudomonas sp. UW4 acdS gene, encoding 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase (AcdS), into fungus Agaricus bisporus. Transformant A. bisporus-acdS14 cased with sterilized-vermiculite generated primordia 5 days sooner than wild-type strain, confirming the specific role of the AcdS enzyme. Being consistent with the AcdS enzyme activity increased by 84%, the mycelium growth rate was increased by 25%; but, the ACC and ethylene concentrations were reduced by 71% and 36%, respectively, in the A. bisporus-acdS14 transformant. And the bacterium P. sp. UW4 attachment on the mycelium of the A. bisporus-acdS14 transformant was drastically reduced. We conclude that the heterogeneously expressed bacterial acdS gene degrades ACC and reduces ethylene-synthesis, eliminating ethylene inhibition on the mycelium growth and primordium formation in A. bisporus. Our results provide new insights into the mechanism underlying casing soil bacterium, and help formulate a casing-less cultivation for the next-generation mushroom industry.

Heterologous Expression of Rhizopus Oryzae CYP509C12 Gene in Rhizopus Nigricans Enhances Reactive Oxygen Species Production and 11α-Hydroxylation Rate of 16α, 17-Epoxyprogesterone.

The 11α-hydroxylation of 16α, 17-epoxyprogesterone (EP) catalyzed by Rhizopus nigricans is crucial for the steroid industry. However, lower conversion rate of the biohydroxylation restricts its potential industrial application. The 11α-steroid hydroxylase CYP509C12 from R. oryzae were reported to play a crucial role in the 11α-hydroxylation in recombinant fission yeast. In the present study, the CYP509C12 of R. oryzae (RoCYP) was introduced into R. nigricans using the liposome-mediated mycelial transformation. Heterologous expression of RoCYP resulted in increased fungal growth and improved intracellular reactive oxygen species content in R. nigricans. The H2O2 levels in RoCYP transformants were approximately 2-folder that of the R. nigricans wild type (RnWT) strain, with the superoxide dismutase activities increased approximately 45% and catalase activities decreased approximately 68%. Furthermore, the 11α-hydroxylation rates of EP in RoCYP transformants (C4, C6 and C9) were 39.7%, 38.3% and 38.7%, which were 12.1%, 8.2% and 9.4% higher than the rate of the RnWT strain, respectively. This paper investigated the effect of heterologous expression of RoCYP in R. nigricans, providing an effective genetic method to construct the engineered strains for steroid industry.

Development of nucleic acid isolation by non-silica-based nanoparticles and real-time PCR kit for edible vegetable oil traceability.

For efficient extraction of amplifiable DNA from edible vegetable oils, we developed a novel DNA extraction approach based on the non-silica-based dipolar nanocomposites. The nanoparticle comprises a hydrophilic polymethyl methacrylate core with abundant capillaries, hydrophilic vesicles decorated with molecules having DNA affinity and a coating hydrophobic polystyrene layer. The nanoparticles are soluble in oil, adsorb the DNA from the aqueous phase and gave a high DNA recovery ratio. All DNA extracts from fully refined vegetable oil soybean, peanut, rapeseed, and cottonseed oils, including their blends, were sufficiently pure to be amplified by real-time PCR targeting the chloroplast ribulose-1,5-bisphosphate gene (rbcL), therefore, the species of origin and their ratios in mixed vegetable oils blended from two or three oil-species could be determined. These results indicate that the novel DNA isolation and real-time PCR kit is a simple, sensitive and efficient tool for the species identification and traceability in refined vegetable oils.

Promotion of the growth and plant biomass degrading enzymes production in solid-state cultures of Lentinula edodes expressing Vitreoscilla hemoglobin gene.

Vitreoscilla hemoglobin (VHb), encoded by the Vitreoscilla hemoglobin gene (vgb), is highly effective at binding oxygen and delivering it to both prokaryotes and eukaryotes under hypoxic conditions. In this study, we introduced the vgb gene into shiitake mushrooms, and the mycelia of the transformatants grew faster. In particular, they spread into the solid substrate located in the lower part of the test tubes and bags where the oxygen was hypoxic and produced more ß-glucan and plant biomass degrading enzymes compared to the original strain. The maximum growth rate of the transformants was 8.5%-15.9% higher than that of the original strain on sawdust-based cultures in plastic bags. The laccase and amylase activities were 17.7%-40.3% and 16.7%-37.9% higher than that of the original strain, respectively. In addition, the ß-glucan contents of the transformant mycelia from the submerged fermentation were 12.9%-24.0% higher than that of the original strain. These results reveal that the expression of VHb in mushroom fungi promots the mycelial growth in solid-state cultures under the hypoxic condition as well as enhances ß-glucan and plant biomass degrading enzymes production.

Identification and expression analysis of Pofst3 suggests a role during Pleurotus ostreatus primordia formation.

Only a few transcription factors involved in mushroom development have been reported till now, Therefore, identification of transcription factors in common edible mushroom has commercial and scientific importance. In this study, the Pofst3 gene from Pleurotus ostreatus was cloned and characterized. Bioinformatics analysis showed that Pofst3 protein had 71% sequence similarity with fst3 of model mushroom Schizophyllum commune. Furthermore, the function of Pofst3 gene was analyzed by overexpression and antisense silencing in P. ostreatus via Agrobacterium-mediated transformation. Expression verification of Pofst3 in transformants through qRT-PCR showed that compared with the wild type strains, the transcription level was about 1.26 ∼ 9.59 and 0.01 ∼ 0.30 fold in Pofst3 overexpressing and silencing strains, respectively. Petri dish and bag cultivation tests of transgenic strains showed that the number of primordia and the type of fruiting bodies of Pofst3 overexpressing strains were consistent with the wild type strains, i.e. fewer primordia and larger fruiting bodies; the number of primordia formed by Pofst3 silencing strains were more than those of wild type strains, but fruiting bodies were smaller. It was very likely that Pofst3 was involved in the regulation of P. ostreatus development through inhibiting the formation of clusters of primordia.

1-Aminocyclopropane-1-Carboxylate: A Novel and Strong Chemoattractant for the Plant Beneficial Rhizobacterium Pseudomonas putida UW4.

Plant growth-promoting rhizobacteria (PGPR) and fungi-bacterial biofilms are both important biofertilizer inoculants for sustainable agriculture. However, the strongest chemoattractant for bacteria to colonize the rhizosphere and mycelia is not clear. Coincidentally, almost all the PGPRs possess 1-aminocyclopropane-1-carboxylate (ACC) deaminase (AcdS) and can utilize ACC as the sole nitrogen source. Here, we found that ACC was a novel, metabolic dependent and methyl-accepting chemoreceptor-involved chemoattractant for Pseudomonas putida UW4. The chemotactic response of UW4 to ACC is significantly greater than that to the amino acids and organic acids identified in the plant root and fungal hyphal exudates. The colonization counts of the UW4 acdS or cheR deletion mutants in the wheat rhizosphere and on Agaricus bisporus mycelia were reduced one magnitude compared with those of UW4. The colonization counts of UW4 on A. bisporus antisense ACC oxidase mycelia with a high ACC production significantly increased compared with A. bisporus, followed by the UW4 cheR complementary strain and the ethylene chemoreceptor gene-deletion mutant. The colonization counts of the UW4 strains on A. bisporus acdS+ mycelia with a low ACC production decreased significantly compared with A. bisporus wild type. These results suggested that ACC and not ethylene should be the strongest chemoattractant for the PGPR that contain AcdS.

Identification of up-regulated transcripts during Pleurotus ostreatus primordium stage and characterization of PoALDH1.

In order to isolate the differentially expressed genes in the primordium stage of Pleurotus ostreatus, the SSH cDNA library was constructed using the cDNA from dikaryotic mycelium stage as a driver and the cDNA from primordium stage as a tester. There were 423 significantly differently expressed clones among 2055 positive clones after three times of reverse Northern blot differential screening. After the repeated sequences being removed, 46 genes were identified which were putatively involved in cell rescue and defense, energy metabolism, transcription and protein regulation, membrane proteins, and signal transduction; 18 genes encoding hypothetical proteins with unknown function; 5 genes without any homology. PoALDH1 and its full-length cDNA sequence were cloned using the Aldehyde dehydrogenase EST isolated from the library. The amino acid sequence of PoALDH1 contains conservative glutamic acid and cysteine residues active sites of aldehyde dehydrogenase family. When exposed to different concentrations of sodium chloride, the mycelium growth was inhibited and the expression level of PoALDH1 was significantly higher than that of the control one, which indicated that PoALDH1 may have the ability to relieve salt stress. The results of this study will provide useful information for isolating growth and development related genes of P. ostreatus.

The identification of transcriptional regulation related gene of laccase poxc through yeast one-hybrid screening from Pleurotus ostreatus.

Pleurotus ostreatus laccase gene poxc is transcriptionally induced by copper, and several putative MREs have been found and confirmed in its promoter region. However, the related transcription factors mediating copper response via MREs have not been reported. To isolate MRE binding proteins, we carried out yeast one-hybrid screens. Reporter genes containing two trimers of the cMRE2 and cMRE3 element were prepared and introduced into a yeast strain. The yeast was transformed with library cDNA that represents RNA isolated from CuSO4-treated fungi of P. ostreatus. From the screen of yeast transformants, we isolated ltf4 which encoded protein with HTH DNA binding domain. Electrophoretic mobility shift assay suggested direct and specific interaction of ltf4 with the MRE2 of poxc. Quantitative RT-PCR showed that the transcription of ltf4 was significantly up-regulated after the copper addition, and the expression trend was consistent with the poxc after copper introduction. The results indicated that ltf4 could interact with cMRE2 and thus participated in the regulation of copper mediated poxc transcription in P. ostreatus.

Improving the expression of recombinant pullulanase by increasing mRNA stability in Escherichia coli

Background: Pullulanase production in both wild-type strains and recombinantly engineered strains remains low. The Shine-Dalgarno (SD) sequence and stem-loop structure in the 5' or 3' untranslated region (UTR) are well-known determinants of mRNA stability. This study investigated the effect of mRNA stability on pullulanase heterologous expression. Results: We constructed four DNA fragments, pulA, SD-pulA, pulA-3t, and SD-pulA-3t, which were cloned into the expression vector pHT43 to generate four pullulanase expression plasmids. The DNA fragment pulA was the coding sequence (CDS) of pulA in Klebsiella variicola Z-13. SD-pulA was constructed by the addition of the 5' SD sequence at the 5' UTR of pulA. pulA-3t was constructed by the addition of a 3' stem-loop structure at the 3' UTR of pulA. SD-pulA-3t was constructed by the addition of the 5' SD sequence at the 5' UTR and a 3' stem-loop structure at the 3' UTR of pulA. The four vectors were transformed into Escherichia coli BL21(DE3). The pulA mRNA transcription of the transformant harboring pHT43-SD-pulA-3t was 338.6%, 34.9%, and 79.9% higher than that of the other three transformants, whereas the fermentation enzyme activities in culture broth and intracellularly were 107.0 and 584.1 times, 1.2 and 2.0 times, and 62.0 and 531.5 times the amount of the other three transformants (pulA, SD-pulA, and pulA-3 t), respectively. Conclusion: The addition of the 5' SD sequence at the 5' UTR and a 3' stem-loop structure at the 3' UTR of the pulA gene is an effective approach to increase pulA gene expression and fermentation enzyme activity.

Genome-wide gene expression patterns in dikaryon of the basidiomycete fungus Pleurotus ostreatus

Abstract Dikarya is a subkingdom of fungi that includes Ascomycota and Basidiomycota. The gene expression patterns of dikaryon are poorly understood. In this study, we bred a dikaryon DK13 × 3 by mating monokaryons MK13 and MK3, which were from the basidiospores of Pleurotus ostreatus TD300. Using RNA-Seq, we obtained the transcriptomes of the three strains. We found that the total transcript numbers in the transcriptomes of the three strains were all more than ten thousand, and the expression profile in DK13 × 3 was more similar to MK13 than MK3. However, the genes involved in macromolecule utilization, cellular material synthesis, stress-resistance and signal transduction were much more up-regulated in the dikaryon than its constituent monokaryons. All possible modes of differential gene expression, when compared to constituent monokaryons, including the presence/absence variation, and additivity/nonadditivity gene expression in the dikaryon may contribute to heterosis. By sequencing the urease gene poure sequences and mRNA sequences, we identified the monoallelic expression of the poure gene in the dikaryon, and its transcript was from the parental monokaryon MK13. Furthermore, we discovered RNA editing in the poure gene mRNA of the three strains. These results suggest that the gene expression patterns in dikaryons should be similar to that of diploids during vegetative growth.

Genome-wide gene expression patterns in dikaryon of the basidiomycete fungus Pleurotus ostreatus.

Dikarya is a subkingdom of fungi that includes Ascomycota and Basidiomycota. The gene expression patterns of dikaryon are poorly understood. In this study, we bred a dikaryon DK13×3 by mating monokaryons MK13 and MK3, which were from the basidiospores of Pleurotus ostreatus TD300. Using RNA-Seq, we obtained the transcriptomes of the three strains. We found that the total transcript numbers in the transcriptomes of the three strains were all more than ten thousand, and the expression profile in DK13×3 was more similar to MK13 than MK3. However, the genes involved in macromolecule utilization, cellular material synthesis, stress-resistance and signal transduction were much more up-regulated in the dikaryon than its constituent monokaryons. All possible modes of differential gene expression, when compared to constituent monokaryons, including the presence/absence variation, and additivity/nonadditivity gene expression in the dikaryon may contribute to heterosis. By sequencing the urease gene poure sequences and mRNA sequences, we identified the monoallelic expression of the poure gene in the dikaryon, and its transcript was from the parental monokaryon MK13. Furthermore, we discovered RNA editing in the poure gene mRNA of the three strains. These results suggest that the gene expression patterns in dikaryons should be similar to that of diploids during vegetative growth.