Dongsheng Wei

Identification and characterization of a novel yeast ω3‐fatty acid desaturase acting on long‐chain n‐6 fatty acid substrates from Pichia pastoris

A cDNA sequence putatively encoding a ω3‐fatty acid desaturase gene was isolated from methylotrophic yeast Pichia pastoris GS115. The deduced amino acid sequence of this cloned cDNA showed high identity to known fungal ω3‐fatty acid desaturases. Functional identification of this gene heterologously in Saccharomyces cerevisiae strain INVScl indicated that the deduced amino acid sequence exhibited ω3‐fatty acid desaturase activity. The newly identified ω3‐fatty acid desaturase, named Pp‐FAD3, is novel because it showed broad n‐6 fatty acid substrate specificity by its ability to convert all the 18‐carbon and 20‐carbon n‐6 substrates examined to the corresponding n‐3 fatty acids, with an approximately equivalent high conversion rate. Pp‐FAD3 is the first known yeast ω3‐fatty acid desaturase to act on long‐chain n‐6 fatty acid substrates. Heterologous expression of the newly identified ω3 desaturase in different hosts will be an alternative method to increase the flow of n‐6 fatty acid intermediates into their n‐3 derivatives. Pp‐fad3 has been assigned GenBank Accession No. EF116884. Copyright

Identification and Functional Characterization of the Delta 6‐Fatty Acid Desaturase Gene from Thamnidium elegans

ABSTRACT. A cDNA sequence was cloned from the filamentous fungus Thamnidium elegans As3.2806 using reverse transcription polymerase chain reaction (RT‐PCR) and rapid amplification of cDNA ends method (RACE). Sequence analysis indicated that this cDNA sequence has an open reading frame of 1,380 bp, which encodes a 52.4 kDa peptide of 459 amino acids. The designated amino acid sequence has high similarity with that found in fungal delta 6‐fatty acid desaturases: it shows three conserved histidine‐rich motifs and two hydrophobic domains. A cytochrome b5‐like domain was observed at the N‐terminus. To elucidate the function of this novel putative desaturase, the open reading frame was cloned into the intracellular expression vector pPIC3.5K and the gene was expressed heterologously in Pichia pastoris. Accumulation of γ‐linolenic acid to the level of 6.83% in total fatty acid demonstrated that the deduced amino acid sequence possesses of delta 6‐fatty acid desaturase activity.

A 'suicide' CRISPR-Cas9 system to promote gene deletion and restoration by electroporation in Cryptococcus neoformans.

Loss-of-function mutagenesis is an important tool used to characterize gene functions, and the CRISPR-Cas9 system is a powerful method for performing targeted mutagenesis in organisms that present low recombination frequencies, such as the serotype D strains of Cryptococcus neoformans. However, when the CRISPR-Cas9 system persists in the host cells, off-target effects and Cas9 cytotoxicity may occur, which might block subsequent genetic manipulation. Here, we report a method of spontaneously eliminating the CRISPR-Cas9 system without impairing its robust editing function. We successfully expressed single guide RNA under the driver of an endogenous U6 promoter and the human codon-optimized Cas9 endonuclease with an ACT1 promoter. This system can effectively generate an indel mutation and efficiently perform targeted gene disruption via homology-directed repair by electroporation in yeast. We then demonstrated the spontaneous elimination of the system via a cis arrangement of the CRISPR-Cas9 expression cassettes to the recombination construct. After a system-mediated double crossover, the CRISPR-Cas9 cassettes were cleaved and degraded, which was validated by Southern blotting. This ‘suicide’ CRISPR-Cas9 system enables the validation of gene functions by subsequent complementation and has the potential to minimize off-target effects. Thus, this technique has the potential for use in functional genomics studies of C. neoformans.

Role of Candida albicans Aft2p transcription factor in ferric reductase activity, morphogenesis and virulence.

The ability of Candida albicans to act as an opportunistic fungal pathogen is linked to its ability to switch among different morphological forms. This conversion is an important feature of C. albicans and is correlated with its pathogenesis. Many conserved positive and negative transcription factors regulate morphogenetic transition of C. albicans. Here, we show the results of functional analysis of CaAFT2, which is an orthologue of the Saccharomyces cerevisiae AFT2 gene. We have cloned CaAFT2 which has an ability to complement the S. cerevisiae aft1Δ mutant strain growth defect. Interestingly, although disruption of the AFT2 gene did not affect cell growth in solid and liquid iron-limited conditions, the cell surface ferric reductase activity was significantly decreased. Importantly, deletion of AFT2 in C. albicans led to growth of a smooth colony with no peripheral hyphae. Moreover, virulence of an aft2Δ/aft2Δ mutant was markedly attenuated in a mouse model. Our results suggest that CaAft2p represents a novel activator and that it functions in ferric reductase activity, morphogenesis and virulence in C. albicans.

Isolation and Identification of a Thermophilic Strain Producing Trehalose Synthase from Geothermal Water in China

A slightly thermophilic strain, CBS-01, producing trehalose synthase (TreS), was isolated from geothermal water in this study. According to the phenotypic characteristics and phylogenetic analysis of the 16s rRNA gene sequence, it was identified as Meiothermus ruber. The trehalose synthase gene of Meiothermus ruber CBS-01 was cloned by polymerase chain reaction and sequenced. The TreS gene consisted of 2,895 nucleotides, which specified a 964-amino-acid protein. This novel TreS catalyzed reversible interconversion of maltose and trehalose.

Disruption of the Fatty Acid Δ6-Desaturase Gene in the Oil-Producing Fungus Mortierella isabellina by Homologous Recombination

The γ-linolenic acid-producing fungus Mortierella isabellina 6-22 is an important industrial strain. To clarify the biosynthetic pathways for polyunsaturated fatty acids in this strain, a disruption vector pD4MI6, including 5′ and 3′ regions of the fatty acid Δ6-desaturase open reading frame as homologous recombination elements and the Escherichia coli hygromycin B (HmB) phosphotransferase gene (hph) as selectable marker, was successfully constructed. Following transformation of pD4MI6 into the hygromycin B-sensitive recipient strain M. isabellina 6-22-4, a Δ6-desaturase gene-defective mutant strain was selected that was unable to produce γ-linolenic acid as determined by gas chromatography and molecular analysis. The morphology and physiology of the mutant, such as colony shape, color, and growth rate, were changed dramatically compared with that of strain M. isabellina 6-22-4.

Orotidine 5′‐phosphate decarboxylase‐based reusable in situ genetic editing system: Development and application in taxol‐producing Pestalotiopsis microspora

Pestalotiopsis microspora NK17 produces the antitumor drug paclitaxel and pestalotiollide B, an analog of cholesterol ester transfer protein inhibitors. However, little is known about their biosynthesis due to the lack of molecular tools. We report here the functional identification of the orotidine 5′‐phosphate decarboxylase encoding gene, pm‐ura3, from this fungus. We utilized it as a marker in the construction of a reusable system for gene scarless deletion, restoration, and tagging. As a positive marker, pm‐ura3 can be used to targeted locus of interest in a start uracil auxotrophic host. It then can be replaced by any artificial DNA sequence, e.g. a mutated ORF, under the negative selection of 5‐fluoroorotic acid. By this method, we can edit genes of interest in situ and recycle the marker. No heterogeneous markers are needed, and the accumulation of foreign DNA sequences such as HisG, FRP, and LoxP is avoided. We applied the system to study the function of pm‐mus53 and found that the gene had little effect on the nonhomologous end‐joining process mediated by agrobacterium tumefaciens‐mediated transformation. We also investigated the expression of a putative taxadiene synthase in taxol biosynthetic pathways of P. microspora and found it was differentially expressed in solid and liquid medium.

Agrobacterium rhizogenes-mediated transformation of a high oil-producing filamentous fungus Umbelopsis isabellina

TheAgrobacterium rhizogenes-mediated transformation procedure was developed by using the hygromycin B phosphotransferase gene (hph) as a selective marker for the oil-producing fungusUmbelopsis isabellina. Different conditions were combined to increase the transformation efficiency. The highest efficiency was obtained by usingA. rhizogenes strain R105 and a vector with zygomycete promoter. Southern blot analysis demonstrated that 71 % of transformants contained random integrations of T-DNA sequences under optimal conditions. We randomly selected 115 positive transformants resistant to hygromycin to analyze the amount of total fatty acid and gamma-linolenic acid (GLA). Six transformants produced a higher amount of total fatty acids than the wild strain, and one transformant also produced a higher level of GLA than the wild strain in gas chromatography analysis. This is the first report about usingA. rhizogenes strain R105 and germinated conidia to transform successfully the recalcitrant zygomycetes and to obtain transformants with a stable phenotype.

The Gα1-cAMP signaling pathway controls conidiation, development and secondary metabolism in the taxol-producing fungus Pestalotiopsis microspora

G-protein-mediated signaling pathways regulate fungal morphogenesis, development and secondary metabolism. In this study, we report a gene, pgα1, that putatively encodes the α-subunit of a group I G protein in Pestalotiopsis microspora NK17, which is known to produce various secondary metabolites, including the antitumor drug taxol and pestalotiollide B (PB). Mutants of pgα1 showed retarded vegetative growth, aging of the mycelium, premature conidiation, deformed conidia, significantly increased melanin production, and a sharp decrease in PB production. The introduction of extra copies of pgα1 led to a different phenotype that was characterized by enhanced production of PB. qRT-PCR revealed that the expression of pks1, which encodes melanin polyketide synthase, an enzyme that is involved in 1, 8-dihydroxynaphthalene (DHN) melanin biosynthesis, was up regulated by 55-fold in the absence of pgα1. Changes in conidiation and PB production in pgα1 mutants were able to be restored by the addition of exogenous cAMP. The deficiencies of PB production and conidiation in Δpgα1 were not able to be rescued by deletion or overexpression of a previously reported histone deacetylase gene (hid1), suggesting that pgα1 is able to override the effect of hid1 on PB production and conidiation. Our results suggested that the G protein-cAMP pathway plays a critical role in vegetative growth as well as in asexual development of P. microspora.

Cryptococcal phosphoglucose isomerase is required for virulence factor production, cell wall integrity and stress resistance.

Regulation of virulence factor production in the pathogen Cryptococcus neoformans remains to be fully illustrated. We present here a finding that a gene, encoding the glycolysis enzyme phosphoglucose isomerase (Pgi1), is critical for the biosynthesis of melanin and capsule, cell wall integrity and resistance to stress conditions. A leaky mutant of the yeast, LZM19, resulted from an insertion of T-DNA in the PGI1 promoter region, expressed PGI1 at a level only 1.9% of the wild type. LZM19 could synthesize the pigment melanin in the presence of 2% glucose, suggesting a status of LAC1 derepression. Phenotypically, capsule biosynthesis in LZM19 was remarkably reduced. Integrity of the cell wall and plasma membrane of LZM19 were impaired based on its sensitivity to Congo red and SDS. Also, LZM19 exhibited hypersensitivity to osmotic stress generated by 2 M NaCl or 1 M KCl, indicating possible impairment in the HOG signaling pathway. Furthermore, LZM19 failed to utilize mannose and fructose, suggesting a possible involvement of Pgi1 in the breakdown of these two sugars. Our results revealed a crucial role of PGI1 in coordination of the production of virulence factors, cell wall integrity and stress response in C. neoformans.