## 2 Result and Discussion
### Metabolic Engineering of Aurantiochytrium sp. SK4 for Carotenoid Production  
The low carotenoid contents of _Aurantiochytrium_ sp. SK4 could result from insufficient GGPP precursor for \(\beta\)-carotene formation due to the competition of FPP for squalene formation (Figure 2A). There is substantial squalene (>10 mg \(\times\) g\({}^{-1}\)) which consumes much FPP (Table S3) that as the intermediates of IPP to GGPP was the precursor of carotenoids. The _Archaeoglobus_ homologous enzyme (GPS) was demonstrated to effectively catalyze GGPP synthesis from IPP and GGPP to produce high amounts of \(\beta\)-carotene. [34; 35]. Thus we intended to express the _Archaeoglobus GPS_ in _Aurantiochytrium_ sp. SK4 thus as to further investigate the biosynthesis of carotenoids. The _GPS_ was linked downstream to an _E. coli IDI_ gene via a 2A sequence [36], which was further inserted into the _Aurantiochytrium_ expression construct [17]. The resulting vector was named as p-VBIG (Figure 3A). The linear DNA fragments of p-VBIG were introduced into _Aurantiochytrium_ sp. SK4 by an electroporation approach. The _ble_ gene conferring zeocin resistance was used as a selectable marker to screen putative transformants. Zeocin-resistant transformants were further detected by PCR to confirm the presence of the transgenes (Figure 3B). One transformant (AT26) was selected for further investigation. The transgenes seemed not to disturb the growth of AT26 as demonstrated by the similar growth curves of wild type (WT) and the transformant (Figure 3C). Figure 3: The information of transformant AT26. (**A**) Schematic diagram of the p-VBIG construct containing the endogenous tubulin promoter, \(-\)_VHb\(-\)ble\(-\)_2A-_IDI_-2A-_GPS_ (VBIG) genes, and the SV40 terminator. (**B**) Genomic PCR detection of VBIG in the transformants resistance to zeocin. (**C**) Growth curves of _Aurantiochytrium_ sp. SK4 and its transformant AT26. Data are shown as mean \(\pm\) SD, n = 3. However, the transgenes greatly enhanced the biosynthesis of both carotenoids and fatty acids (Figure 4, Table 1). AT26 produced 1.78-fold total carotenoids of WT, with \(\beta\)-carotene the predominant carotenoid (66.9% of total carotenoids). In addition, AT26 accumulated 5.02-fold astaxanthin of WT, possibly resulting from the expression of _VHb_ as shown by [PERSON] et al. (2017). The transformant AT26 produced 141.1 \(\pm\) 6.5 \(\mathrm{\SIUnitSymbolMicro g}\times\mathrm{g}^{-1}\) astaxanthin which was much higher than _Schizochytrium_ sp. SH104 and its mutant SHG104 ([PERSON] et al., 2017). However, astaxanthin only accounted for 7.87% of total carotenoids (Table 1), suggesting that the ketolation and hydroxylation of \(\beta\)-carotene are the limiting steps for astaxanthin formation. If this bottleneck can be removed, the astaxanthin production might be increased to a level that has the potential to meet the market demand. The increased production of carotenoids might result from the cooperation of the _VHb_, _IDI_, and _GPS_, which could decrease the FPP levels and hence decrease the production of squalene. We detected the contents of squalene in WT and AT26 and found that the transformant produced squalene at the amount of less than 1/20 of WT (Table S3). To investigate if the biosynthesis of fatty acids is coupled with carotenoids, we analyzed the fatty acid compositions and contents in AT26. Interestingly, the expression of VBIG triggered great increases of total fatty acids and PUFA, especially DHA both in contents and proportions (Figure 4B and Table 1). The contents of total fatty acids and DHA in AT26 were 2.40-, and 2.74-fold of WT. These results supported that the biosynthesis of carotenoids and fatty acids might be coordinated as reported previously ([PERSON] et al., 2017; [PERSON] et al., 2017). The overexpression of _VHb_ was previously found to enhance the production of astaxanthin at the expense of \(\beta\)-carotene, echinenone, and canthaxanthin in _Aurantiochytrium_ sp. SK4 ([PERSON] et al., 2017).