Resistance Mechanism in a Terbinafine-Resistant Strain of <Emphasis Type="Italic">Microsporum canis</Emphasis>

To clarify the terbinafine (TRF) resistance mechanism in a TRF-resistant strain of Microsporum canis, the expression of the pleiotropic drug resistance (PDR1), multidrug resistance (MDR1), MDR2 and MDR4 genes were investigated by real-time quantitative PCR (RT-qPCR) analysis, given the known interaction of the corresponding proteins with antifungals and with the efflux blocker FK506. The expression of the PDR1, MDR1, MDR2 and MDR4 genes was 2–4 times higher in the TRF-resistant strain grown in the presence of 0.14 µg/mL of TRF than in TRF-susceptible strains cultured in the absence of TRF. The TRF-resistant strain exhibited MICs of > 32 µg/mL for TRF alone; this resistance was attenuated to an MIC of 8 µg/mL in the presence of FK506, indicating that the TRF inhibitory concentration index value was < 0.75. The additive effect of the efflux blocker FK506 on TRF resistance was detected in the TRF-resistant strain. These results indicated that the TRF resistance in this strain reflects overexpression of genes encoding ABC transporter proteins.     

Combining metabolomics and network analysis to improve tacrolimus production in <Emphasis Type="Italic">Streptomyces tsukubaensis</Emphasis> using different exogenous feedings

Tacrolimus is widely used as an immunosuppressant in the treatment of various autoimmune diseases. However, the low fermentation yield of tacrolimus has thus far restricted its industrial applications. To solve this problem, the time-series response mechanisms of the intracellular metabolism that were highly correlated with tacrolimus biosynthesis were investigated using different exogenous feeding strategies in S. tsukubaensis. The metabolomic datasets, which contained 93 metabolites, were subjected to weighted correlation network analysis (WGCNA), and eight distinct metabolic modules and seven hub metabolites were identified to be specifically associated with tacrolimus biosynthesis. The analysis of metabolites within each metabolic module suggested that the pentose phosphate pathway (PPP), shikimate and aspartate pathway might be the main limiting factors in the rapid synthesis phase of tacrolimus accumulation. Subsequently, all possible key-limiting steps in the above metabolic pathways were further screened using a genome-scale metabolic network model (GSMM) of S. tsukubaensis. Based on the prediction results, two newly identified targets (aroC and dapA) were overexpressed experimentally, and both of the engineered strains showed higher tacrolimus production. Moreover, the best strain, HT-aroC/dapA, that was engineered to simultaneously enhanced chorismate and lysine biosynthesis was able to produce 128.19 mg/L tacrolimus, 1.64-fold higher than control (78.26 mg/L). These findings represent a valuable addition to our understanding of tacrolimus accumulation in S. tsukubaensis, and pave the way to further production improvements.     

The biosynthesis of gibberellic acids by the transformants of orchid-associated <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

The genus Fusarium, including multiple strains in the Gibberella fujikuroi species complex (GFC), is well known for its production of diverse secondary metabolites. F. fujikuroi, associated with the “bakanae” disease of rice, is an active producer of gibberellins (GAs), a wide class of plant hormones. In addition to some members of the GFC, the GA biosynthetic gene cluster, or parts of it, occurs also in some isolates of the closely related species of F. oxysporum, which does not belong to the GFC. However, production of GAs has never been observed in any F. oxysporum strain. In this study, we report on the GA biosynthetic activity in an orchid-associated F. oxysporum strain by transforming a cosmid with the entire F. fujikuroi GA gene cluster. Southern and Northern blot analyses confirmed not only the integration of the entire gene cluster into the genome but also the active expression of the seven GA biosynthetic genes under nitrogen-limiting conditions. The transformants produced GAs at levels similar to those of F. fujikuroi. These data show that the regulatory network for expression of GA genes is fully active in the F. oxysporum background.     

Metabolic evolution and a comparative omics analysis of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> for putrescine production

Putrescine is widely used in the industrial production of bioplastics, pharmaceuticals, agrochemicals, and surfactants. Because the highest titer of putrescine is much lower than that of its precursor l-ornithine reported in microorganisms to date, further work is needed to increase putrescine production in Corynebacterium glutamicum. We first compared 7 ornithine decarboxylase genes and found that the Enterobacter cloacae ornithine decarboxylase gene speC1 was most suitable for putrescine production in C. glutamicum. Increasing NADPH availability and blocking putrescine oxidation and acetylation were chosen as targets for metabolic engineering. The putrescine producer C. glutamicum PUT4 was first constructed by deleting puo, butA and snaA genes, and replacing the fabG gene with E. cloacae speC1. After adaptive evolution with C. glutamicum PUT4, the evolved strain C. glutamicum PUT-ALE, which produced an 96% higher amount of putrescine compared to the parent strain, was obtained. The whole genome resequencing indicates that the SNPs located in the odhA coding region may be associated with putrescine production. The comparative proteomic analysis reveals that the pentose phosphate and anaplerotic pathway, the glyoxylate cycle, and the ornithine biosynthetic pathway were upregulated in the evolved strain C. glutamicum PUT-ALE. The aspartate family, aromatic, and branched chain amino acid and fatty acid biosynthetic pathways were also observed to be downregulated in C. glutamicum PUT-ALE. Reducing OdhA activity by replacing the odhA native start codon GTG with TTG and overexpression of cgmA or pyc458 further improved putrescine production. Repressing the carB, ilvH, ilvB and aroE expression via CRISPRi also increased putrescine production by 5, 9, 16 and 19%, respectively.     

Isolation and characterization of <Emphasis Type="Italic">Aspergillus flavus</Emphasis> strains in China

Important staple foods (peanuts, maize and rice) are susceptible to contamination by aflatoxin (AF)-producing fungi such as Aspergillus flavus. The objective of this study was to explore non-aflatoxin-producing (atoxigenic) A. flavus strains as biocontrol agents for the control of AFs. In the current study, a total of 724 A. flavus strains were isolated from different regions of China. Polyphasic approaches were utilized for species identification. Non-aflatoxin and non-cyclopiazonic acid (CPA)-producing strains were further screened for aflatoxin B₁ (AFB₁) biosynthesis pathway gene clusters using a PCR assay. Strains lacking an amplicon for the regulatory gene aflR were then analyzed for the presence of the other 28 biosynthetic genes. Only 229 (32%) of the A. flavus strains were found to be atoxigenic. Smaller (S) sclerotial phenotypes were dominant (51%) compared to large (L, 34%) and non-sclerotial (NS, 15%) phenotypes. Among the atoxigenic strains, 24 strains were PCR-negative for the fas-1 and aflJ genes. Sixteen (67%) atoxigenic A. flavus strains were PCRnegative for 10 or more of the biosynthetic genes. Altogether, 18 new PCR product patterns were observed, indicating great diversity in the AFB₁ biosynthesis pathway. The current study demonstrates that many atoxigenic A. flavus strains can be isolated from different regions of China. In the future laboratory as well as field based studies are recommended to test these atoxigenic strains as biocontrol agents for aflatoxin contamination.     

<Emphasis Type="Italic">Lactobacillus paracasei</Emphasis> strain 06TCa19 suppresses inflammatory chemokine induced by <Emphasis Type="Italic">Helicobacter pylori</Emphasis> in human gastric epithelial cells

Helicobacter (H.) pylori infection is an important risk factor for gastric cancer that causes gastric inflammation. Inflammatory chemokines such as interleukin (IL)-8 and regulated on activation normal T cell expressed and secreted (RANTES) are elevated in the gastric mucosa by H. pylori. This study aimed to investigate the effects of Lactobacillus paracasei strain 06TCa19, a probiotic strain, on IL-8 and RANTES expression and production induced by H. pylori using human gastric epithelial cell lines. Strain 06TCa19 was shown to suppress H. pylori-mediated elevation of gene expression related to these chemokines in MKN45 cells. The strain also suppressed the increase in IL-8 and RANTES products induced by H. pylori in AGS cells as well as in MKN45 cells. In MKN45 cells inoculated with H. pylori, strain 06TCa19 was shown to downregulate the activation of NF-κB and p38 MAPK signaling pathways. Additionally, the level of the CagA virulence protein of H. pylori in the MKN45 cells and the number of viable H. pylori adhering to MKN45 cells decreased with the addition of strain 06TCa19. Moreover, the strain 06TCa19 notably increased lactic acid in the supernatant of MKN45 cells. Thus, lactic acid released from strain 06TCa19 might have inhibited the adhesion of H. pylori to MKN45 cells and prevented the insertion of H. pylori CagA into the cells, and elevation of IL-8 and RANTES genes and proteins might be suppressed by downregulating the NF-κB and p38 MAPK pathways. Therefore, use of strain 06TCa19 may prevent H. pylori-associated gastric inflammation.     

Diversity and characterization of cultivable oleaginous yeasts isolated from mangrove forests

A total of 198 yeasts were isolated from 140 samples collected from 7 mangrove forests in 4 provinces of Thailand, and were found to belong to 30 genera, 45 described species and at least 12 undescribed species based on their 26S rRNA (D1/D2 domain) gene sequence. The most prevalent species was Candida tropicalis, followed by Candida pseudolambica and Rhodosporidium paludigena. Lipid accumulation, as determined by Nile red staining, of the isolated yeasts revealed that 69 and 18 strains were positive and strongly positive, respectively, while quantitative analysis of the intracellular lipid accumulated in the latter indicated that 10 of these strains, Pseudozyma tsukubaensis (YWT7-2 and YWT7-3), Rhodotorula sphaerocarpa (YWW6-1 and SFL14-1SF), Saitozyma podzolica (YWT1-1, NS3-3 and NS10-2), Prototheca zopfii var. hydrocarbonea OMS6-1 and Prototheca sp. (YMTW3-1 and YMTS5-2), were oleaginous. In this study we found that under nitrogen depletion condition (155 C/N ratio) Pseudozyma tsukubaensis YWT7-2 accumulated the highest level of intracellular lipid at 32.4% (w/w, dry cell weight), with a broadly similar fatty acid composition to that in palm oil.     

Identification and genomic analysis of antifungal property of a tomato root endophyte <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. p21

Pseudomonas sp., which occupy a variety of ecological niches, have been widely studied for their versatile metabolic capacity to promote plant growth, suppress microbial pathogens, and induce systemic resistance in plants. In this study, a Pseudomonas sp. strain p21, which was isolated from tomato root endophytes, was identified as having antagonism against Aspergillus niger. Further analysis showed that this strain had the ability to biosynthesise siderophores and was less effective in inhibiting the growth of A. niger with the supplementation of Fe³⁺ in the agar medium. Genomic sequencing and the secondary metabolite cluster analysis demonstrated that Pseudomonas sp. p21 harboured 2 pyoverdine biosynthetic gene clusters, which encode compounds with predicted core structures and two variable tetra-peptide or eleven-peptide chains. The results indicated that siderophore-mediated competition for iron might be an important mechanism in Pseudomonas suppression of the fungal pathogen A. niger and in microbe-pathogen-plant interactions.     

Biosynthesis of poly(2-hydroxybutyrate-co-lactate) in metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

We have previously reported in vivo biosynthesis of polyhydroxyalkanoates containing 2-hydroxyacid monomers such as lactate and 2-hydroxybutyrate in recombinant Escherichia coli strains by the expression of evolved Clostridium propionicum propionyl-CoA transferase (PctCp) and Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1 _Ps6-19). Here, we report the biosynthesis of poly(2-hydroxybutyrate-co-lactate)[P(2HB-co-LA)] by direct fermentation of metabolically engineered E. coli strain. Among E. coli strains WL3110, XL1-Blue, and BL21(DE3), recombinant E. coli XL1-Blue strain expressing PhaC1437 and Pct540 produced P(76.4mol%2HB-co-23.6mol%LA) to the highest content of 88 wt% when it was cultured in a chemically defined medium containing 20 g/L of glucose and 2 g/L of sodium 2-hydroxybutyrate. When recombinant E. coli XL1-Blue strain expressing PhaC1437 and Pct540 was cultured in a chemically defined medium containing 20 g/L of glucose and varying concentration of sodium 2-hydroxybutyrate, 2HB monomer fraction in P(2HB-co-LA) increased proportional to the concentration of sodium 2-hydroxybutyrate added to the culture medium. P(2HB-co-LA)] could also be produced from glucose as a sole carbon source without sodium 2-hydroxybutyrate into the culture medium. Recombinant E. coli XL1-Blue strain expressing the phaC1437, pct540, cimA3.7, and leuBCD genes together with the L. lactis Il1403 panE gene, successfully produced P(23.5mol%2HB-co-76.5mol%LA)] to the polymer content of 19.4 wt% when it cultured in a chemically defined medium containing 20 g/L of glucose. The metabolic engineering strategy reported here should be useful for the production of novel copolymer P(2HB-co-LA)].