Worldshaper-Nanjing Engineering Success

Engineering Success

Summary

At present, we independently expressed the glucoamylase gene (AnGlu) and α-Amylase gene (AnAmyA) from the Aspergillus niger in the Yarrowia lipolytica Po1g. Their capability for starch degradation was verified by measuring the OD of the two strains in the fermentation medium using the starch as the sole carton source. The results showed that Y. lipolytica expressing AnGlu can degrade the starch efficiently and AnAmyA did not work well. Subsequently, we regulated the C/N ratio by adding different concentrations of starch to verify whether the C/N ratio had the influence on the oil biosynthesis, the results proved that high C/N ratio is more conducive to oil accumulation.

1. Plasmid Construction

We constructed the plasmids according to DESIGN page. There are five parts in our plasmid library: a promoter-EXP (BBa_K3578000), a terminator-XPR2t (BBa_K3578002), two expression genes-α-amylase (BBa_K3578003), glucoamylase (BBa_K3578001), and the Leu selection marker (BBa_K3578004). By BsaI and SapI IIs enzymes digestion and T4 ligase, we successfully constructed two plasmids which separately carried the AnGlu and AnAmyA expression cassette with Leu selection marker (Figure 1). Then two plasmids were linearizd with EcoR I and transformed into Y. lipolytica plotoplast. The AnGlu/AnAmyA expression cassette with Leu selection marker DNA fragment will be randomly integrated into genome loci.

Figure 1 Schematic diagram of plasmid construction process.

By the PCR experiment, we randomly selected 10 strains in which AnGlu and Leu expression cassette have been successfully integrated into the genome. In addition, 10 strains integrating AnAmyA and Leu expression cassette were picked up for the following experiments.

Figure 2 Primer design and agarose gel electrophoresis for plasmid identification

We designed the primers(F/R) to identify the two plasmids. The primer design was shown in the figure 2, and the predicted PCR result is 2736 bp. The electrophoresis results showed that about 3000 bp bands were obtained by using AnGlu/AnAmyA expression cassette as template, the electrophoresis results were consistent with our expectation (Figure 2).

2. The expression of AnGlu in Y. lipolytica is sufficient to allow growth on starch

Figure 3 Growth curve in starch medium as sole carbon source

Figure 4 Photos of Y. lipolytica solution after culturing 96 hours.

3. High C/N ratio is more conducive to oil accumulation

Since the Polg-AnGlu strain can growth well on the starch medium, we then measured the lipid titer and content. The Polg-AnGlu can produce up to 0.76 ± 0.04 g/L lipid and 14.66 ± 0.43% of DCW as fatty acids in the starch media and a C/N ratio of 30. The titer and content were relatively low. Previous researchers proved that higher C/N ratio is more conducive to lipid accumulation when glucose, xylose and glycerin were used as the carbon source. In order to verify whether the higher C/N ratio in the starch media also help improve the lipid biosynthesis capability, we regulated the C/N ratio by adding different concentrations of starch (Figure 5). The C/N ratio was 30, 60, 90. These statistics showed that when C/N ratio was increased to 60, the lipid titer increased from 0.76 ± 0.04 g/L to 1.07 ± 0.02 g/L, a 55% improvement. when C/N ratio was increased to 90, the lipid titer further increased to 1.17 ± 0.02 g/L. Similar results about the increasing lipid content were also observed. The lipid content increased from 14.66 ± 0.43% to 24.06 ± 0.13% of DCW, a 64% improvement. In summary, it shows that improving C/N ratio is an effective strategy to strengthen lipid accumulation.

Figure 5 Lipid production and content in the starch medium with different C/N ratio

Finally, in order to verify the above conclusion, lipid bodies visualization was carried out by addition of 1 mg/mL Nile red to the cell suspension. The results showed that the color of lipid bodies is brighter and the size is relatively bigger when C/N ratio was controlled as 90 rather than 30. The above results indicate that the ratio of C/N is closely related to the lipid accumulation (Figure 6).

Figure 6 Fluorescence micrograph of Y. lipolytica cell.

4. Future work

Due to the Covid-19 epidemic, the experimental time of this project is limited, and lots of following plans have not yet been carried out. According to the experimental results, we are ready to further increase AnGlu gene copy number to accelerate starch degradation rate. In addition, the strategies for improving the lipid titer, yield and rate were also taken into consideration, including disrupting the fatty acid oxidation pathway, increasing the content of acetyl coenzyme A in cytoplasm, simultaneously overexpressing the rate-limiting genes-diacylglycerol acyltransferase and acetyl-CoA carboxylase, as well as balancing the cofactor supply.

At last, according to the Human Practices survey results, we found that the biggest contradiction in the production of biodiesel and even biofuels is the lack of cheap raw materials. Therefore, in the future, we need to further expand the scope of our usable waste substrates to better realize the sustainable development goals of protecting the global environment and waste utilization.