No abstract

Thioesterases play an essential role in the metabolism of fatty acids since they are considered one of the key enzymes to change the total amount and composition of fatty acid in an organism’s cells. Acyl-coenzyme A thioesterase 8 (ACOT8) exhibits substrate specificity mainly for short- to long-chain acyl-CoA. To identify and characterize the ACOT8 enzyme’s superfamily in Mucor circinelloides, three genes were characterized and homologously expressed in M. circinelloides WJ11 which has been used as a model organism to investigate the mechanism of lipid accumulation. Multiple sequence alignment showed that McACOT8s had significant conserved motifs in the ACOT8 family. Experimental data indicated that the biomass of the three recombinant strains (McACOT8a, McACOT8b and McACOT8c) was slightly lower than that of the control strain Mc2075, but the total fatty acid (TFA) content was significantly increased by 30.3, 21.5 and 23.9%, respectively, and the free fatty acid production increased from 12.9% (control strain) to 19.2, 25.2 and 26.2% (recombinant types), respectively, with a maximum increase of 103.1% in McACOT8c. The three strains did not show any significant improvement in fatty acid composition compared to the control strain. However, in terms of fatty acid composition of free fatty acid, McACOT8b and McACOT8c contained γ-linolenic acid (C18:3), which was not detected in the control strain or in McACOT8a, indicating that ACOT8b and ACOT8c had substrate specificity for C18:3. These results displayed that ACOT8 can increase TFA accumulation and that it may be an important target of genetic manipulation for microbial oil production.

Diacylglycerol acyltransferase (DGAT) catalyzes the binding of acyl-CoA to diacylglycerol to form triacylglycerol (TAG). Previous studies strongly indicate that DGAT2, rather than DGAT1, is crucial for TAG accumulation in the oleaginous fungus Mucor circinelloides. To increase the lipid content of M. circinelloides WJ11, McDGAT2 was overexpressed by homologous recombination; compared to the control strain Mc2075, transformants McDGAT2d showed a significant increase in biomass for both spores and mycelia (from 87.7 to 101.2 mg/g in spores and from 75.6 to 93.1 mg/g in mycelia). McDGAT2 overexpression under static solid fermentation gave a greater boost to lipid accumulation in mycelia than in spores. Total fatty acid content in mycelia increased by 68.0% (from 13.6 to 22.8%) and in spores by 26.3% (from 10.6 to 13.4%). However, under submerged fermentation, the lipid content of McDGAT2d was the same as the control, while biomass was slightly reduced. Transcriptomics showed that NADPH was derived mainly from the pentose phosphate pathway, acetyl-CoA was from multiple pathways, and leucine metabolism played an important role in substrate supply for fatty acid biosynthesis. Static solid fermentation may be the more suitable fermentation method for microbial oil production by filamentous fungi due to its lower fermentation costs.

Oxygen availability is a limiting factor for lipid biosynthesis in eukaryotic microorganisms. Two bacterial hemoglobins from Vitreoscilla sp. (VHb) and Shinorhizobium meliloti (SHb), which could deliver the oxygen to the respiratory chain to produce more ATP, were introduced into Mucor circinelloides to alleviate oxygen limitation, thereby improving cell growth and fatty acid production. VHb and SHb genes were integrated into the M. circinelloides MU402 genome through homologous recombination, and their protein expression was verified by carbon monoxide difference spectrum (CO-difference spectrum)analysis. SHb-expressing strain showed higher biomass than VHb-expressing strain. The biomass of the SHb-expressing strain was increased by about 50% and the total fatty acid (TFA) content was as high as 15.7% of the dry cell weight which was about 40% higher than that of the control strain in flask conditions. In the fermenter, the maximum biomass and TFA content was obtained in SHb-expressing strains, with the biomass being 12.1 g/L and the TFA being 21.1% of the dry cell weight. VHb and SHb expression also affected the fatty acid composition with the proportion of polyunsaturated fatty acids being increased. Over-expression of bacterial hemoglobins, especially SHb increased cell growth and TFA content in M. circinelloides at low and high aeration, suggesting that SHb is better than VHb in improving the fatty acid production in oleaginous microorganisms.

Significance and Impact of the Study: Solid-state fermentation is an efficient and economical method for lipid accumulation in the oleaginous microorganism. In this study, engineered strains Mucor circinelloides McD15D with overexpressing the delta15-desaturase were able to accumulate more stearidonic acid (SDA) under solid-state fermentation than submerged fermentation, providing a new strategy for fermentation technology to enhance SDA production by oleaginous microorganisms.