## 3 Discussion
### Other Metabolic Pathway Changes in C18 and P1 Strain
#### 3.3.4 Glycolysis and Unsaturated Fatty Acids  
In _Chlamydomonas reinhardtii_, an increase in carotenoid content can promote sugar metabolism. Studies have shown that cells can regulate lipid and carotenoid metabolism to cope with environmental changes [34; 35]. Under heterotrophic conditions, lipid content increased by about 1.5-fold and \(\beta\)-carotene content increased by about 3-fold in _Chlamydomonas reinhardtii_[36]. Another study found that interfering with the activity of starch branching enzyme (DBE) via genetic engineering methods can increase lipid and carotenoid accumulation in _Chlamydomonas reinhardtii_ under high-light conditions [37]. This is because DBE interference can promote carbon resource transfer from starch to lipids and carotenoids. Some studies have found that carotenoid and sugar contents are positively correlated in some plants [38]. For example, in tomatoes, high-carotenoid varieties also have high sugar content [31]. In kale, carotenoid accumulation is associated with an increase in soluble sugars in leaves [39]. In summary, in _Chlamydomonas reinhardtii_, carotenoids are not only important pigments and antioxidants, but also regulators of sugar metabolism and carbon allocation. Therefore, in this study, we hypothesized that the increase in carotenoid content caused more carbon to be allocated to sugar metabolism, which was also consistent with the transcriptomic and metabolomic data. Most genes of the glycolysis and pentose phosphate pathway were up-regulated, and soluble sugars were measured to find that C18 and P1 had higher soluble sugar content than did the WT (Figure 4h). Additionally, in _Chlamydomonas reinhardtii_, the regulation of sugar and unsaturated fatty acids in the heterotrophic mode seems to be positively correlated (efficient heterotrophic cultivation of _Chlamydomonas reinhardtii_), especially under heterotrophic culture where cells do not need to perform photosynthesis and have more carbon available (Figure 14). The Combination of Genetic Engineering and Heterotrophy for the Commercialization of Chlamydomonas reinhardtii  
The production of microalgal carotenoids has been a hot topic in recent years, and the common targets of genetic engineering are _Haematococcus pluvialis_, _Dualiella salina_, _Chorella vulgaris_ and others [40]. Compared with traditional plant carotenoid production, natural microalgal carotenoids have short production cycles, low costs, and high sustainability [38; 41]. In this study, we combined the heterotrophic cultivation of _Chlamydomonas reinhardtii_ with genetic engineering in the hope of obtaining some novel data. The advantages of the heterotrophic cultivation of microalgae have been well-known due to the recent years of research [42]. Compared with open cultivation, heterotrophic fermentation in industrialization can obtain a high density of microalgal cultures, and the whole process is safe and controllable [43]. Since the fermentation environment can be controlled via modern industrial integration, the culture conditions can be precisely adjusted to increase the biomass and achieve better culture outcomes. In addition, the closed system of the ferment can also effectively avoid external contamination and ensure the safety of the culture [44]. With ordinary heterotrophy, in as little as 6-7 days, the cell density of _C. reinhardtii_ will reach more than 7-8 \(\times\) 10\({}^{6}\) cell/mL. If a high-density culture is performed, the cell density  
Figure 14: Metabolic pathway changes in _PrBKT_ and _CrBKT_ overexpression strains C18 and P1 vs. WT. The orange font with the blue background represents the compound up-regulated and the white font with the blue background represents the compound down-regulated. Compounds with * indicate that the differential compound was detected only in P1. Data were derived from the biochemical experiments of this experiment and the \(\log^{2}\) FC (differential fold change of metabolites in different groups) of the metabolome.