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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 $$Saitozyma \ podzolica$$ S a i t o z y m a p o d z o l i c a zwy-2-3. Results Compared with WT, $$\Delta gat1$$ Δ g a t 1 , 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 $$\Delta gat1$$ Δ g a t 1 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.

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.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.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.

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 β-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.

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.

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.