Yao Zhang

Two fatty acid synthase genes (fas1 and fas2) were individually deleted in oleaginous fungus Mucor circinelloides WJ11 in our previous work. In this study, an in-depth analysis on supplement of sodium citrate and soybean oil to achieve high yields of lipid and β-carotene was investigated in fas-knockout M. circinelloides strains. The effects of different concentrations of sodium citrate or soybean oil on cell growth, lipid and β-carotene accumulation in fas-knockout strains were measured, and it was determined that the optimal addition concentrations of sodium citrate and soybean oil were both 15 g/L. Combination use of 15 g/L sodium citrate and 15 g/L soybean oil as additives had a synergistic effect on the cell growth, lipid and β-carotene accumulation in fas-knockout strains, especially for the Mc-fas2Δ strain. The maximum yields of biomass, lipid and β-carotene in Mc-fas2Δ reached 19 g/L, 33.6% and 328 µg/g respectively, which were 25%, 34.4% and 2.2- fold higher than those of the control strain. Moreover, the transcriptional levels of the key genes involved in fatty acid and β-carotene synthesis in the engineered strains were significantly upregulated after adding sodium citrate and soybean oil. This is the first study to systematically evaluate the combined effects of soybean oil and sodium citrate on both lipid and β-carotene production in fas-knockout M. circinelloides strains.

Two fas genes (namely fas1 and fas2) of Mucor circinelloides WJ11 were deleted through homologous recombination technology. The deletion of the fas gene has a considerable influence on lipid content and fatty acid composition. Compared with the control strain, the lipid content at fermentation of 96 h was increased by 9.1% for the fas1-knockout strain Mc-fas1Δ and 18.4% for the fas2-knockout strain Mc-fas2Δ, respectively. Possibly due to the compensatory effect between fas1 and fas2, the expression of fas2 was improved 4.3-fold in Mc-fas1Δ compared with the control strain, and that of fas1 was significantly improved 14.3-fold in Mc-fas2Δ, aligning with the elevated FAS activity in both mutant strains. Meanwhile, the deletion of the fas gene also drives the enzymatic activity and gene expression of key enzymes that provide substrates and reducing power for fatty acid synthesis. This is the first report demonstrating the correlation between the fas gene and lipid synthesis in the oleaginous fungus M. circinelloides.

Fatty acid synthase (FAS) is one of the most important enzymes in lipid biosynthesis, which can catalyze the reaction of acetyl-CoA and malonyl-CoA to produce fatty acids. However, the structure, function, and molecular mechanism of FAS regulating lipid synthesis in the fungus Mucor circinelloides are unclear. In the present study, two encoding fas genes in the high lipid-producing strain WJ11 and low lipid-producing strain CBS277.49 from M. circinelloides based on their genome database were identified and annotated respectively. Bioinformatic analysis confirmed the presence of typical conserved domains in FAS proteins. Phylogenetic analysis revealed the evolutionary relationship of these FAS proteins, and the FAS proteins from WJ11 have similar properties as their counterparts in CBS277.49. Furthermore, transcriptional profiling displayed marked differences in the expression of these fas genes, and fas1 was analyzed to predict the possible functions in lipid metabolism in the high lipid-producing strain WJ11. This is the first report on the structures and functions of FAS proteins in M. circinelloides. This research has suggested the association of fas genes with lipid metabolism at the transcriptional level and contributed to the selection of candidates related to lipid metabolism for further study.

Microbial lipases could be used to hydrolyze or recombine fats and oils, and had great applications in food processing, bioenergy and chemical industry. Mucor circinelloides was an important gamma-linolenic acid producing strain, and its genome was predicted to contain a large number of genes encoding lipases, the key enzymes in lipid metabolism. In the present study, a potential lipase WJ_23 from Mucor circinelloides WJ11 was cloned for the first time and heterologously expressed and purified to homogeneity in Pichia pastoris. By SDS-PAGE analysis, the molecular weight of the recombinant lipase was estimated to be ~ 34 kDa. The optimal temperature and pH of the recombinant lipase were 50 °C and 9.0, respectively. The recombinant lipase had good thermal stability at 50 °C with a broad pH stability from 6.0 to 11.0. After incubation at 37 °C for 24 h, the activity of the recombinant lipase remained over 95% between pH 7.0 and 9.0. The recombinant lipase possessed a preference for the long chain substrates. Using p-NPP as substrate, the measured kinetic parameters V_max and K_m were 94.34 U/mg and 6.37 mmoL/L, respectively. In addition, the activity of the recombinant lipase was not affected obviously by various metal ions, and it exhibited certain stability and tolerance towards several kinds of organic solvents. This research might provide a basis for the further industrial application of recombinant M. circinelloides lipase.

Microbial lipases serve as versatile biocatalysts for modifying lipid substrates, enabling diverse biotechnological applications spanning food technology, renewable energy production, and pharmaceutical sectors. Genomic analysis of Mucor circinelloides WJ11 revealed multiple lipase-encoding genes, with WJ_23 successfully cloned and heterologously expressed in Escherichia coli. Since the lipase expression was mainly concentrated in the cell pellets, appropriate induction strategies had been investigated to enhance the extracellular production of the lipase. The results showed that when 0.5 mmol/L Isopropyl-1-thio-β-D-Galactopyranoside (IPTG) and 0.8% Triton X-100 were simultaneously added in the later stage of logarithmic growth at 30°C, the peak extracellular lipase activity attained2580 U/L, demonstrating a 6.79-fold increase over the wild strain. This study provided a theoretical basis for further improving the extracellular production of other heterogonous enzymes in E. coli, and it also laid a foundation for the future application of lipase in M. circinelloides.

Previously, we constructed engineered M. circinelloides strains that can not only utilize cellulose, but also increase the yield of γ-linolenic acid (GLA). In the present study, an in-depth analysis of lipid accumulation by engineered M. circinelloides strains using corn straw was to be explored. When a two-stage temperature control strategy was adopted with adding 1.5% cellulase and 15% inoculum, the engineered strains led to increases in the lipid yield (up to 1.56 g per 100 g dry medium) and GLA yield (up to 274 mg per 100 g dry medium) of 1.8- and 2.3-fold, respectively, compared with the control strain. This study proved the engineered M. circinelloides strains, especially for Mc-C2PD6, possess advantages in using corn straw to produce GLA. This work provided a reference for transformation from agricultural cellulosic waste to functional lipid in one step, which might play a positive role in promoting the sustainable development of biological industry.

Sucrose non-fermenting 1 (SNF1) protein kinase plays the regulatory roles in the utilization of selective carbon sources and lipid metabolism. Previously, the role of β subunit of SNF1 in lipid accumulation was evaluated by overexpression and knockout of Snf-β in oleaginous fungus M. circinelloides. In the present study, the growth and lipid accumulation of Snf-β overexpression and knockout strains were further analyzed and compared with glucose or xylose as a single or mixed carbon sources. The results showed that the lipid contents in Snf-β knockout strain improved by 23.2% (for glucose), 28.4% (for xylose), and 30.5% (for mixed glucose and xylose) compared with that of the control strain, respectively. The deletion of Snf-β subunit also altered the transcriptional level of acetyl-CoA carboxylase (ACC). The highest transcriptional levels of ACC1 in Snf-β knockout strain at 24 h were increased by 2.4-fold (for glucose), 2.8-fold (for xylose), and 3.1-fold (for mixed glucose and xylose) compared with that of the control strain, respectively. Our results indicated that Snf-β subunit enhanced lipid accumulation through the regulation of ACC1 in response to xylose or mixed sugars of glucose and xylose more significantly than that of response to glucose. This is the first study to explore the effect of Snf-β subunit of M. circinelloides in regulating lipid accumulation responding to different carbon nutrient signals of glucose and xylose. This study provides a foundation for the future application of the Snf-β engineered strains in lipid production from lignocellulose.

Sucrose non-fermenting 1 (SNF1) protein kinase plays an important role in the utilization of selective carbon sources and regulation of lipid metabolism. In order to further explore the function of SNF1 in regulating lipid accumulation by responding nutritional signals from non-glucose carbon sources, in the present study, the lipid production and SNF1 transcriptional levels of Mucor circinelloides were analyzed and compared on different carbon sources. The results indicated that M. circinelloides could effectively utilize some secondary metabolic carbon sources of monosaccharides and disaccharides for growth and lipids production, such as fructose, maltose and galactose. Snf-β subunit was associated with the regulation of lipid metabolism in response to nutritional signals from different carbon sources. This is the first report on the transcriptional analysis of SNF1 subunits on different carbons metabolism in oleaginous filamentous fungi. This research has suggested that genetic engineering of SNF1 subunits will alter the lipid production of M. circinelloides from alternative carbon sources.

Despite the rapid development of various types of fruit vinegar or nutrient-fortified vinegar, the weakened functions or high cost is still the main limiting factor for healthy vinegar. In the present study, a new type of rose vinegar was obtained from raw grain supplemented with natural extract of roses through the cooperative fermentation of multiple microorganisms by adding Mucor circinelloides and lactic acid bacteria. The chemical composition analysis showed that this rose vinegar was rich in various organic acids and amino acids, which endowed the rose vinegar with a special taste and nutritional value. In particular, fatty acid content results revealed that the rose vinegar contained several long-chain unsaturated fatty acids, especially the valuable Gamma-Linolenic Acid (GLA), which was mainly due to the addition of M. circinelloides that could synthesize GLA during the fermentation of vinegar. The antioxidant activity of the rose vinegar indicated that the contents of gallic acid and vanillic acid were relatively high and thus it had a certain scavenging ability on DPPH free radicals, superoxide anion free radicals, and hydroxyl free radicals. This is the first study to develop a healthy vinegar containing GLA with antioxidant activity by combining the rose extract with a variety of beneficial microbial fermentation.