Abstract: Abstract: Mucor circinelloides recombinant strain Mc-MT-2 is generally responsible for the trans-membrane transport of malate and citric acid in lipid synthesis. A preliminary research has been carried out to successfully construct the M. circinelloides recombinant strain Mc-MT-2 overexpressing the malate transporter gene. It was found that the strain can be expected to behave potential applications in lipid production, indicating that the lipid content of the strain was 70% higher than that of the control strain. In addition, the lipid accumulation level of microorganisms depends mainly on the strain characteristics, growth environment, and fermentation. In this study, a regulation strategy of the Mc-MT-2 strain was presented to produce the lipid, ranging from the selection of medium composition to microbial growth control and fermentation mode. An investigation was made to clarify the effects of nitrogen/carbon sources and pH values on the growth and sporulation of recombinant strain Mc-MT-2. An optimal condition was achieved for the growth and sporulation culture of the Mc-MT-2 strain, where the compound carbon source of glucose and malate with the compound ratio of 9:1, tryptone as the nitrogen source, other components as same as Kendirck medium, and pH 5. The yield of spores significantly increased under optimized conditions, about 4 times the initial one. A systematic optimization was also performed on the plate culture conditions, such as nitrogen/carbon source, and pH value. A batch cultivation was further carried out in a 1.5 L fermenter to determine the optimal fermentation of recombinant strain Mc-MT-2 in lipid production. The fermentation conditions were set as follows: a temperature of 30°C, pH value of 5.0, aeration of 0.5 L/(L·min), a rotation speed of 500 rpm, and a fermentation time of 96 h. The biomass, lipid content, and yield after batch cultivation reached 11.2 g/L, 24%, and 2.6 g/L, respectively. A fed-batch fermentation culture of the Mc-MT-2 strain was carried out in a 3 L fermenter to further increase the lipid production. The fermentation conditions were set as follows: a temperature of 30°C, pH of 5.0, aeration of 0.5 L/(L·min), a rotation speed of 500 rpm, and a fermentation time of 120 h. The feed solution was added every 24 hours, and the glucose concentration was maintained at about 10-15 g/L, after the glucose was exhausted (the dissolved oxygen rose sharply). The maximum biomass, lipid content, and yield of the Mc-MT-2 strain in the fed-batch culture were 15.4 g/L, 28.6%, and 4.4 g/L, which were improved by 1.38-, 1.19-, and 1.69-fold, respectively, compared with the batch culture. There was an obvious difference in the composition of fatty acid for the Mc-MT-2 strain during fermentation, with a high content of oleic acid (C18: 1), while low contents of linoleic acid and linolenic acid (C18: 2, C18: 3), as well as no or less tetradecanoic acid (C14:0), and palmitic acid (C16:1). This finding can offer a potential theoretical foundation for the M. circinelloides recombinant strain Mc-MT-2 to be further applied in the lipid production suitable for industrial requirements.