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.

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.

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.

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.