Proof of Concept

The overall goal of the project was to construct Yarrowia lipolytica as an omega-3 fatty acids production cell factory through the synthetic biology. Firstly, the Δ-9 extension desaturase pathway was introduced into the genome of Y. lipolytica. In addition, we knock out the genes in β-oxidation pathway which control the conversion of free fatty acids to short-chain acyl-CoA. In order to make it possible to apply engineered Y. lipolytica to large-scale production of EPA, we screened different sources of elongase or desaturase, studied the optimal gene copy number and replaced the promoter to increase the content of EPA. In order to expand the application scope of the project and further convert EPA to DHA, we continued to transfer the enzyme genes required for this process into the strain to co-produce EPA and DHA using Y. lipolytica. The Fig1 shows the metatbolic engineering strategies, and Fig 2 shows the results obtained at each step.

Fig. 1 Metabolic engineering strategies Fig. 2 Results involved EPA content obtained at each step

In order to further investigate the performance of the engineered omega-3 producer, fed-batch fermentation was carried out using a 5-liter bioreactor. During fermentation, the initial medium contained 120 g/L glucose and 2.64 g/L ammonium sulfate, and 17.6 g/L ammonium sulfate and 800 g/L glucose were supplemented during fermentation. At 120h, the DCW and lipid yield was reached to 108 g/L and 42.1 g/L, respectively (Fig. 3A). Moreover, the 27% of EPA content in total lipid was obtained (Fig. 3B), which was higher than that of flask fermentation. Lastly, the EPA titer was reached to 11.4g/L, representing a higher titer in Yarrowia lipolytica (Fig. 4A and B).

Fig. 3 Dry cell weight (DCW), total fatty acids and glucose consumption in 5-liter bioreactor. (B)The percentage of EPA content produced by Yarrowia lipolytica in 5-liter bioreactor.

Fig. 4 (A) The fermentation situation in 5-liter bioreactor. (B) Pictures of oil produced in 5-liter bioreactor.

In order to investigate the possibility of making the engineered strain into feed, we developed a set of technological process to make it into fungi powder (Fig. 5), and detected the fatty acid components in the bacterial powder content. The results showed that the content of EPA in fungi powder reached 10%. Moreover, the 1kg of dry cell weight can be made to 1.1kg fungi powder.

Fig. 5 the production process of fungal powder