Part:BBa_K3578010
AnGlu Starch degradation
EXP, AnGlu, XPR2t part will be digested by BsaI and then assembled as the module 1 through the T4 ligase. The module 1 and Leu selection marker will be digested by SapI and then assembled as “EXP-AnGlu-XPR2t-Leu” expression cassette through the T4 ligase. The cassette can be transformed into the Yarrowia lipolytica plotoplast and integrated into the genome for AnGlu efficient expression.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 3683
Illegal EcoRI site found at 3733
Illegal EcoRI site found at 4695
Illegal SpeI site found at 756 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 3683
Illegal EcoRI site found at 3733
Illegal EcoRI site found at 4695
Illegal NheI site found at 817
Illegal SpeI site found at 756 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 3683
Illegal EcoRI site found at 3733
Illegal EcoRI site found at 4695
Illegal BglII site found at 2537
Illegal BglII site found at 5964
Illegal BamHI site found at 3157
Illegal XhoI site found at 4678
Illegal XhoI site found at 4707 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 3683
Illegal EcoRI site found at 3733
Illegal EcoRI site found at 4695
Illegal SpeI site found at 756 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 3683
Illegal EcoRI site found at 3733
Illegal EcoRI site found at 4695
Illegal SpeI site found at 756
Illegal NgoMIV site found at 3821
Illegal AgeI site found at 3364
Illegal AgeI site found at 5292 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
Yarrowia lipolytica can use starch as sole carbon sources when the AnGlu is expressed successfully, which means the AnGlu has the ability to degrade the starch efficiently.
Results
1.Plasmid Construction
We constructed the plasmids according to DESIGN page. There are four parts in our plasmid library: a promoter-EXP (BBa_K3578000), a terminator-XPR2t (BBa_K3578002), expression genes glucoamylase (BBa_K3578001), and the Leu selection marker (BBa_K3578004). By BsaI and SapI IIs enzymes digestion and T4 ligase, we successfully constructed the plasmids which separately carried the AnGlu expression cassette with Leu selection marker (Figure 1). Then plasmids were linearizd with EcoR I and transformed into Y. lipolytica plotoplast. The AnGlu expression cassette with Leu selection marker DNA fragment will be randomly integrated into genome loci.
By the PCR experiment, we randomly selected 10 strains in which AnGlu and Leu expression cassette have been successfully integrated into the genome. We designed the primers(F/R) to identify the Anglu expression cassette. 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 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
In order to verify whether the AnGlu possess the ability for starch degradation. Engineered Y. lipolytica expressing the AnGlu (Polg-AnGlu) individually were cultured in the starch medium in which the 30 g/L starch was used as the sole carton source. In addition, the strain Po1g was also cultured in the starch medium with the addition of 0.4g/L leucine as the control (Polg-Leu). The OD was measured every 24 hours. As shown in Figure 3, Po1g-AnGlu strain can grow well in the starch medium, it means that AnGlu had the relatively higher starch utilization efficiency. The result can also be directly observed as shown in Figure 4, in which Polg-AnGlu possess a much higher cell density. In summary, the expression of AnGlu in Y. lipolytica is sufficient to allow growth on starch.
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.
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).
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