Enhanced degradation of exogenetic citrinin by glycosyltransferases in the oleaginous yeast Saitozyma podzolica zwy-2-3.

The contamination by the toxin citrinin (CIT), produced by fungi, has been reported in agricultural foods and is known to be nephrotoxic to humans. In this study, we found that CIT could be effectively degraded by the oleaginous yeast Saitozyma podzolica zwy-2-3. Four genes encoding glycosyltransferases (GTs) in S. podzolica zwy-2-3 (SPGTs) were identified by evolutionary and structural analyses. The overexpression of SPGTs enhanced CIT degradation to 0.56 mg/L/h in S. podzolica zwy-2-3 by increasing ATP and glutathione (GSH) contents to oxidize CIT and scavenge reactive oxygen species (ROS). Besides, SPGTs promoted lipid synthesis by 9.3 % of S. podzolica zwy-2-3 under CIT stress. These results suggest that SPGTs in oleaginous yeast play a pivotal role in enhancing CIT degradation and lipid accumulation. These findings provide a valuable basis for the application of GTs in oleaginous yeast to alleviate CIT contamination in agricultural production, which may contribute to food safety.

Enhancement of lipid synthesis by the transcription factor Asg1 in Saitozyma podzolica zwy-2-3 under dissolved oxygen stress.

Microbial oils have been of considerable interest as food additives and biofuel resources due to high lipid contents, but lipid accumulation of oleaginous microorganisms can be induced by environmental stresses, such as dissolved oxygen (DO), which limit large-scale lipid production. Here, DO stress gave rise to the endogenous nitric oxide (NO) level to mediate S-nitrosylation of SpAsg1, regulating the lipid accumulation in Saitozyma podzolica zwy-2-3. Notably, qRT-PCR, yeast one-hybrid, dual-luciferase reporter assays, and metabolomics analysis exhibited that overexpression of SpAsg1 promoted lipid synthesis by directly regulation of glucose metabolism, enhancing glucose uptake, ATP and NADPH contents under DO stress. Meanwhile, SpAsg1 improved the antioxidant capacity to reduce the intracellular reactive oxygen species (ROS) and NO levels. Overall, we systematically investigated the regulation of SpAsg1 on lipid metabolism of S. podzolica zwy-2-3 under DO stress, which sheds light on further studies for alleviating oxygen limitation of lipid production in microbial industry.

Novel extracellular lipase gene Lip1728 influences nutrient-dependent performance bacterial quorum sensing of Burkholderia pyrrocinia WZ10-3.

Quorum sensing (QS) is a cellular communication mechanism in which bacteria secrete and recognize signaling molecules to regulate group behavior. Lipases provide energy for bacterial cell growth but it is unknown whether they influence nutrient-dependent QS by hydrolyzing substrate. A high-yield lipase-producing strain, Burkholderia pyrrocinia WZ10-3, was previously identified in our laboratory, but the composition of its crude enzymes was not elucidated. Here, we identified a key extracellular lipase, Lip1728, in WZ10-3, which accounts for 99 % of the extracellular lipase activity. Lip1728 prefers to hydrolyze triglycerides at sn-1,3 positions, with pNP-C16 being its optimal substrate. Lip1728 exhibited activity at pH 5.0-10.0 and regardless of the presence of metal ions. It had strong resistance to sodium dodecyl sulfate and short-chain alcohols and was activated by phenylmethanesulfonylfluoride (PMSF). Lip1728 knockout significantly affected lipid metabolism and biofilm formation in the presence of olive oil. Finally, oleic acid, a hydrolysate of Lip1728, influenced the production of the signal molecule N-acyl homoserine lactone (AHL) and biofilm formation by downregulating the AHL synthetase gene pyrI. In conclusion, Lip1728, as a key extracellular lipase in B. pyrrocinia WZ10-3, exhibits superior properties that make it suitable for biodiesel production and plays a crucial role in QS.

Characterization and expression profiles of toll-like receptor genes (TLR2 and TLR5) in immune tissues of hybrid yellow catfish under bacterial infection.

The yellow catfish (Pelteobagrus fulvidraco) is one of the most economically important freshwater species in Asia. However, pathogenic bacterial infections often cause high rates of mortality and economic losses in practical aquaculture. Previous studies in mammals have shown that Toll-like receptor 2 (TLR2) and Toll-like receptor 5 (TLR5) are involved in the recognition of cell wall components such as lipopolysaccharides and flagella of various bacteria, thereby acting as key regulators in the innate immunity response. However, TLR2 and TLR5 in yellow catfish have not been characterized. In the present study, TLR2 and TLR5 were examined through comparative genomic approaches. The gene structure, collinearity, protein spatial structure, and phylogenetic relationships were compared with those in multiple representative vertebrates. Meanwhile, quantitative real-time PCR was conducted to explore transcriptional changes in TLR2 and TLR5 in immune tissues after infection with exogenous A. hydrophila and E. tarda. The results demonstrated the presence of TLR2 and TLR5 in yellow catfish. However, a systematic analysis showed that TLR2 was not associated with the arrangement of diverse neighboring genes. The expression of hybrid yellow catfish TLR2 transcripts in multiple tissues (including liver, spleen, kidney, and intestine) was significantly up-regulated after infection with A. hydrophila and E. tarda, suggesting that hybrid yellow catfish TLR2 and TLR5 may participate in the immune process. Taken together, the results indicate that TLR2 and TLR5 are conserved in terms of evolution and possess significant antibacterial activity as well as regulatory properties in immune-related tissues and thus play key roles in host defense against pathogen invasion.

Molecular characterization and expression profiling of two flavohemoglobin genes play essential roles in dissolved oxygen and NO stress in Saitozyma podzolica zwy2-3.

Flavohemoglobins (Fhbs) are key enzymes involved in microbial nitrosative stress resistance and nitric oxide degradation. However, the roles of Fhbs in fungi remain largely unknown. In this study, SpFhb1 and SpFhb2, two flavohemoglobin-encoding genes in Saitozyma podzolica zwy2-3 were characterized. Protein structure analysis and molecular docking showed that SpFhbs were conserved in bacteria and fungi. Phylogenetic analysis revealed that SpFhb2 may be acquired through the transfer event of independent horizontal genes from bacteria. The expression levels of SpFhb1 and SpFhb2 showed opposite trend under high/low dissolved oxygen, implying that they may exhibited different functions. Through deletion and overexpression of SpFhbs, we confirmed that SpFhbs were conducive to lipid accumulation under high stress. The sensitivities of ΔFhb mutants to NO stress were significantly increased compared with that in the WT, indicating that they were required for NO detoxification and nitrosative stress resistance in S. podzolica zwy2-3. Furthermore, SpAsg1 was identified that simultaneously regulates SpFhbs, which functions in the lipid accumulation under high/low dissolved oxygen and NO stress in S. podzolica zwy2-3. Overall, two different SpFhbs were identified in this study, providing new insights into the mechanism of lipid accumulation in fungi under high/low dissolved oxygen and NO stress.

Potential xylose transporters regulated by CreA improved lipid yield and furfural tolerance in oleaginous yeast Saitozyma podzolica zwy-2-3.

Lignocellulose's hydrolysate, a significant renewable source, contains xylose and furfural, making it challenging for industrial production of oleaginous yeast. On xylose fermentation with furfural treatment, OE::DN7263 and OE::DN7661 increased lipid yield and furfural tolerance versus WT, while, which of OE::CreA were decreased owing to CreA regulating DN7263 and DN7661 negatively. OE::CreA generated reactive oxygen species (ROS) causing oxidative damage. OE::DN7263, OE::DN7661, and ΔCreA reduced furfural via NADH; while ΔCreA produced less ROS and OE::DN7263, and OE::DN7661 scavenged ROS quickly, minimizing oxidative damage. Overall, CreA knockout increased DN7263 and DN7661 expression to facilitate xylose assimilation, enhancing NADH generation and ROS clearance. Finally, with mixed sugar fermentation, ΔCreA and OE::DN7263's biomass and lipid yield rose without furfural addition, while that of ΔCreA remained higher than WT after furfural treatment. These findings revealed how oleaginous yeast zwy-2-3 resisted furfural stress and indicated ΔCreA and OE::DN7263 might develop into robust industrial chassis strains.

Characterization of TLR1 and expression profiling of TLR signaling pathway related genes in response to Aeromonas hydrophila challenge in hybrid yellow catfish (Pelteobagrus fulvidraco ♀ × P. vachelli ♂).

Toll-like receptor 1 (TLR1) mediates the innate immune response to a variety of microbes through recognizing cell wall components (such as bacterial lipoproteins) in mammals. However, the detailed molecular mechanism of TLR1 involved in pathogen immunity in the representative hybrid yellow catfish (Pelteobagrus fulvidraco ♀ × P. vachelli ♂) has not been well studied. In the present study, we identified the TLR1 gene from the hybrid yellow catfish, and further comparative synteny data from multiple species confirmed that the TLR1 gene is highly conserved in teleosts. Phylogenetic analysis revealed distinguishable TLR1s in diverse taxa, suggesting consistence in evolution of the TLR1 proteins with various species. Structural prediction indicated that the three-dimensional structures of TLR1 proteins are relatively conserved among different taxa. Positive selection analysis showed that purifying selection dominated the evolutionary process of TLR1s and TLR1-TIR domain in both vertebrates and invertebrates. Expression pattern analysis based on the tissue distribution showed that TLR1 mainly transcribed in the gonad, gallbladder and kidney, and the mRNA levels of TLR1 in kidney were remarkably up-regulated after Aeromonas hydrophila stimulation, indicating that TLR1 participates in the inflammatory responses to exogenous pathogen infection in hybrid yellow catfish. Homologous sequence alignment and chromosomal location indicated that the TLR signaling pathway is very conserved in the hybrid yellow catfish. The expression patterns of TLR signaling pathway related genes (TLR1- TLR2 - MyD88 - FADD - Caspase 8) were consistent after pathogen stimulation, revealing that the TLR signaling pathway is triggered and activated after A. hydrophila infection. Our findings will lay a solid foundation for better understanding the immune roles of TLR1 in teleosts, as well as provide basic data for developing strategies to control disease outbreak in hybrid yellow catfish.

GATA-type transcriptional factor SpGAT1 interacts with SpMIG1 and promotes lipid accumulation in the oleaginous yeast [Formula: see text] zwy-2-3.

BACKGROUND: In oleaginous yeast, nitrogen limitation is a critical parameter for lipid synthesis. GATA-family transcriptional factor GAT1, a member of the target of rapamycin (TOR) pathway and nitrogen catabolite repression (NCR), regulates nitrogen uptake and utilization. Therefore, it is significant to study the SpGAT1 regulatory mechanism of lipid metabolism for conversion of biomass to microbial oil in [Formula: see text] zwy-2-3. RESULTS: Compared with WT, [Formula: see text], and OE::gat1, the lipid yield of OE::gat1 increased markedly in the low carbon and nitrogen ratio (C/N ratio) mediums, while the lipid yield and residual sugar of [Formula: see text] decreased in the high C/N ratio medium. According to yeast two-hybrid assays, SpGAT1 interacted with SpMIG1, and its deletion drastically lowered SpMIG1 expression on the high C/N ratio medium. MIG1 deletion has been found in earlier research to affect glucose metabolic capacity, resulting in a prolonged lag period. Therefore, we speculated that SpGAT1 influenced glucose consumption rate across SpMIG1. Based on yeast one-hybrid assays and qRT-PCR analyses, SpGAT1 regulated the glyoxylate cycle genes ICL1, ICL2, and pyruvate bypass pathway gene ACS, irrespective of the C/N ratio. SpGAT1 also could bind to the ACAT2 promoter in the low C/N medium and induce sterol ester (SE) accumulation. CONCLUSION: Our findings indicated that SpGAT1 positively regulated lipid metabolism in S.podzolica zwy-2-3, but that its regulatory patterns varied depending on the C/N ratio. When the C/N ratio was high, SpGAT1 interacted with SpMIG1 to affect carbon absorption and utilization. SpGAT1 also stimulated lipid accumulation by regulating essential lipid anabolism genes. Our insights might spur more research into how nitrogen and carbon metabolism interact to regulate lipid metabolism.

Butyryl/Caproyl-CoA:Acetate CoA-transferase: cloning, expression and characterization of the key enzyme involved in medium-chain fatty acid biosynthesis.

Coenzyme A transferases (CoATs) are important enzymes involved in carbon chain elongation, contributing to medium-chain fatty acid (MCFA) biosynthesis. For example, butyryl-CoA:acetate CoA transferase (BCoAT) is responsible for the final step of butyrate synthesis from butyryl-CoA. However, little is known about caproyl-CoA:acetate CoA-transferase (CCoAT), which is responsible for the final step of caproate synthesis from caproyl-CoA. In the present study, two CoAT genes from Ruminococcaceae bacterium CPB6 and Clostridium tyrobutyricum BEY8 were identified by gene cloning and expression analysis. Enzyme assays and kinetic studies were carried out using butyryl-CoA or caproyl-CoA as the substrate. CPB6-CoAT can catalyze the conversion of both butyryl-CoA into butyrate and caproyl-CoA into caproate, but its catalytic efficiency with caproyl-CoA as the substrate was 3.8-times higher than that with butyryl-CoA. In contrast, BEY8-CoAT had only BCoAT activity, not CCoAT activity. This demonstrated the existence of a specific CCoAT involved in chain elongation via the reverse ß-oxidation pathway. Comparative bioinformatics analysis showed the presence of a highly conserved motif (GGQXDFXXGAXX) in CoATs, which is predicted to be the active center. Single point mutations in the conserved motif of CPB6-CoAT (Asp346 and Ala351) led to marked decreases in the activity for butyryl-CoA and caproyl-CoA, indicating that the conserved motif is the active center of CPB6-CoAT and that Asp346 and Ala351 have a significant impact on the enzymatic activity. This work provides insight into the function of CCoAT in caproic acid biosynthesis and improves understanding of the chain elongation pathway for MCFA production.

Electrode-dependent ammonium oxidation with different low C/N ratios in single-chambered microbial electrolysis cells.

Alternative method should be found to solve the ammonia accumulation in anaerobic digestion. Herein, electrode-dependent ammonium oxidation was successfully achieved in anaerobic single-chambered microbial electrolysis cells (MECs)under different low C/N ratios (0, 1, and 1.5), with an applied voltage of 0.6 V as well as an initial NH4+-N and NO3--N concentration of 500 and 300 mg/L. The nitrogen removal performance of MECs and the controls indicated that applying a voltage stimulated nitrogen removal under low C/N ratios of 0, 1, and 1.5. However, the remaining organic carbon in MEC with a relatively higher C/N ratio of 3 inhibited the ammonium oxidation. Current changes and cyclic voltammetry demonstrated that the bioanode with several bioelectrochemical activities could promote ammonium oxidation. The dominant genera Truepera, Aquamicrobium, Nitrosomonas, Arenimonas, Comamonas, and Cryobacterium enriched on both electrodes could be the key functional taxa in MECs with C/N ratios of 0, 1, and 1.5. The remaining sodium acetate in MEC with C/N ratio of 3 inhibits microbial community structure and relative abundance, which may adversely affected nitrogen removal. Further caculation showed that nitrogen balance was essentially achieved, while electron balance was disrupted since electrons may be consumed through NO3--N recycle and cell synthesis, and finally caused low coulombic efficiency.

Integrative effect of citrate on Cr(â ¥) and total Cr removal using a sulfate-reducing bacteria consortium.

In controlling toxic Cr(Ⅵ) pollution, the sulfate-reducing bacteria (SRB) method-a bioresource technology-is considered more sustainable and stable than synthetic technologies; however, its mechanisms of metal removal are unclear. This study investigated the mechanism of the use of citrate as a carbon source in an SRB bioreactor for Cr(Ⅵ) removal by disassemble or simulation approach. We show that citrate can mask toxicity, whereby the IC50 value (inhibitory concentration affecting 50% of the test population) of citrate was higher than that of lactate, and that citrate can also protect water systems from oxidation. The anti-oxidation rate of citrate ranged from 76.00% to 90.92%; whereas for citrate‒Cr(Ⅲ), the oxidation rate was only 0.185%-0.587%. Citrate can up-regulate microbial genes and functions, causing acetate and sulfide (NaFeS2) accumulation. Acetate addition promoted Cr adsorption by sulfide (mainly NaFeS2) and promoted sulfide sedimentation. Moreover, in addition to Cr(Ⅵ) reduction and Cr(Ⅲ)‒sulfide generation, the addition of sulfide promoted sedimentation; the correlation coefficient between the sedimentation coefficient and the sulfur content was r = -0.88877 at p < 0.01. Therefore, citrate had a systemic radiative effect on every aspect of the SRB‒citrate system model for Cr(Ⅵ) removal. In addition to the reduction in the former simple model, an integrative effect (including adsorption, sedimentation, and metabolism) was combined with NaFeS2 for Cr removal, which was regulated by the SRB‒citrate system. Exploration and understanding of these mechanisms promote SRB‒citrate methods to be wider implications in practice.

Cloning and characterization of a L-lactate dehydrogenase gene from Ruminococcaceae bacterium CPB6.

Lactate are proved to be attractive electron donor for the production of n-caproic acid (CA) that is a high value-added fuel precursor and chemical feedstock, but little is known about molecular mechanism of lactate transformation. In the present study, the gene for L-lactate dehydrogenase (LDH, EC.1.1.1.27) from a Ruminococcaceae strain CPB6 was cloned and expressed in Escherichia coli BL21 (DE3) with plasmid pET28a. The recombinant LDH exhibited molecular weight of 36-38 kDa in SDS-PAGE. The purified LDH was found to have the maximal oxidation activity of 29.6 U/mg from lactate to pyruvate at pH 6.5, and the maximal reduction activity of 10.4 U/mg from pyruvate to lactate at pH 8.5, respectively. Strikingly, its oxidative activity predominates over reductive activity, leading to a 17-fold increase for the utilization of lactate in E. coli/pET28a-LDH than E. coli/pET28a. The CPB6 LDH gene encodes a 315 amino acid protein sharing 42.19% similarity with Clostridium beijerinckii LDH, and lower similarity with LDHs of other organisms. Significant difference were observed between the CPB6 LDH and C. beijerinckii and C. acetobutylicum LDH in the predicted tertiary structure and active center. Further, X-ray crystal structure analysis need to be performed to verify the specific active center of the CPB6 LDH and its role in the conversion of lactate into CA.

Ammonia oxidation and denitrification in a bio-anode single-chambered microbial electrolysis cell.

In this study, anodic ammonia oxidation and denitrification were performed in single-chamber bioelectrochemical systems at a wide range of anodic potentials (-400 to +400 mV) versus Ag/AgCl. The low coulombic efficiencies (~30.84%) in reactors were mainly due to electrons being transferred to atmospheric oxygen through the electrode and reversal of the electrode. The removal efficiencies of acetate, ammonia, and total nitrogen were 100%, 100%, and 40.44% at +200 mV and 100%, 100%, and 50.24% at -200 mV, respectively. The nitrogen-removal mechanisms were nitrogen respiration/nitrate reduction at +200 mV and denitrification at -200 mV, and ammonia oxidation occurred by coupling with sulfate-reducing at -300 and -400 mV. Thauera, Comamonas, Alicycliphilus, Nitrosomonas, Desulforhabdus, Dethiosulfatibacter, and Desulfomicrobium were the dominant genera at the anode which participated in the nitrification/denitrification or sulfate-reducing processes. In summary, ammonia oxidation and denitrification could be coupled with carbon-removal or sulfur-reduction using a bio-anode with a suitable anodic potential.