Figure 1.
Genetic modification of the β-oxidation pathway in Saccharomyces cerevisiae.
The dashed line represents the original pathway; the solid line represents the modified pathway. The only acyl-CoA oxidase (encoded by the gene POX1) in the S. cerevisiae genome was deleted, and the POX2 gene from Yarrowia lipolytica, which encodes acyl-CoA oxidase with a preference for long chain acyl-CoAs, was expressed. To unblock the β-oxidation pathway, peroxisomal carnitine octanoyltransferase (CROT) from Mus musculus was also expressed to transport medium chain fatty acyl-CoAs out of peroxisomes.
Figure 2.
A. Expression of Aoxp2 in WT [pox2+], Δpox1 [pox2+] and Δpox1 [pox2+, crot+]. B. Expression of Crot in Δpox1 [pox2+, crot+].
Figure 3.
Growth tests on the engineered strains and the WT strain in YNBO medium.
WT and Δpox1 transformed with the empty vector pvtu260, WT transformed with pvtu260-pox2, Δpox1 transformed with pvtu260-pox2 and pvtu260-pox2-crot were all cultured overnight in liquid YNBD medium at 30°C. Yeast pellets were collected and resuspended at a cell density of 5×103 cells/µl after being washed twice in sterile distilled H2O. To test growth, 5 µl were spotted on YNBO-agar plates. The first drop contained 2.5×104 cells, and each subsequent drop was diluted six fold more than the previous one.
Figure 4.
Growth curves of the engineered strains and the WT strain.
A. Growth curves of the engineered strains and the WT strain in YNBD, B. Growth curves of the engineered strains and the WT strain in YNBD0.5O3. The results are the mean values of three independent experiments.
Figure 5.
MCFAs analysis of the cell extracts.
A. MCFAs content in the cell extracts from the engineered strains and the WT strain after 240.5O3 medium. B. Composition of MCFAs from the total fatty acids in the cell extracts from the engineered strains and the WT strain at 24 h when cultured in YNBD0.5O3 medium. Cells were collected after 24 h of growth in YNBD0.5O3 medium, and the cell pellet was separated by centrifugation. The total fatty acids were extracted and detected. There was C12:0 but not C8:0 and C10:0 in the cell extract, so the MCFAs were only C12:0 in this case.
Table 1.
Fatty acid production in the cell extract of the WT and the engineered strainsa.
Table 2.
Fatty acid composition changes in cell extract comparing the WT and the engineered strains in YNBD0.5O3 mediumb.
Figure 6.
MCFAs content in the culture supernatant of the engineered strains and the WT strain grown for 240.5O3 medium.
Cells were collected after growth for 240.5O3 medium, and the culture supernatant was separated by centrifugation. MCFAs were detected with the SPE method (detailed information referred to in the fatty acids analysis section). Data are the mean values of three independent experiments. The error bar indicates the S.D. C8:0: open bar, C10:0: striped bars, C12:0: gridded bar, MCFAs: solid bar.
Figure 7.
The representative GC-MS spectra for azelaic acid derived from total ion chromatograms.
Intracellular metabolites were extracted from the engineered strains and the WT strain, and metabolic profiling was conducted. WT (black), Δpox1 (blue), Δpox1 [pox2+] (red), and Δpox1[pox2+, crot+] (green).
Figure 8.
Differential expression levels of intracellular metabolites in the engineered strains compared to the WT strain.