## 2 Results
### Growth and Carotenoid Production in C. reinhardtii Colonies and Wild Type  
Heterotrophic cultivation was performed for CC-125, C18 and P1. The growth cycle of these three groups was about 15 days, and the initial cell density was \(\sim\)1 \(\times\) 10\({}^{5}\) cell/mL. The results showed that after Day 10, the growth of these three groups entered a stable phase, so a period of 0-10 days for heterotrophic cultivation was chosen to study the growth and pigment contents. In the CC-125, C18 and P1 culture, the cell density increased from \(\sim\)0.7 \(\times\) 10\({}^{5}\) cell/mL to \(\sim\)80 \(\times\) 10\({}^{5}\) cell/mL after 10 days. The growth rate and cell density of CC-125 were slightly greater than those of C18 and P1, and reached 8.87 \(\times\) 10\({}^{6}\) cell/mL at Day 10. Meanwhile, the cell density of C18 and P1 reached 6.64 \(\times\) 10\({}^{6}\) cell/mL and 7.52 \(\times\) 10\({}^{6}\) cell/mL, respectively (Figure 4a). For _Chlamydomonas reinhardtii_ cell growth, we also examined the OD\({}_{750}\) of the  
Figure 3: Heatmap for showing changes in gene transcript levels. (**a**) Heatmap of _CrBKT_ gene transcript level changes in Group C. (**b**) Heatmap of _PrBKT_ gene transcript level changes in Group P.  
Figure 2: Agarose gel electrophoreses of _CrBKT_ (**a**) and _PrBKT_ (**b**) PCR products. (**a**) _CrBKT_ PCR products signed at 660 bp. (**b**) _PrBKT_ PCR products signed at 684 bp. cultures. As shown in Figure 4b, the OD\({}_{750}\) values of the three groups stayed at a stable level from Day 4, and maintained between 0.44 and 0.66 overall. Figure 4: Growth curves and carotenoid production figures of CC-125, C18 and P1. (**a**) Growth curves. (**b**) Line chart for OD\({}_{750}\) of the three groups. (**c**) Bar graph of total chlorophyll content. (**d**) Bar graph of total carotenoid content. (**e**) Line chart of astaxanthin content. (**f**) Line chart of \(\beta\)-carotenoid content. (**g**) Bar graph of soluble protein content. (**h**) Bar graph of soluble sugar content. For pigment content, we used both a UV spectrophotometer (for total chlorophyll content and total carotenoid content) and HPLC (for astaxanthin content and \(\beta\)-carotenoid content) for detection. The total chlorophyll content was maintained between 2 and 3 \(\mathrm{\SIUnitSymbolMicro g}\)/mL for all three groups under heterotrophy, indicating that the cells lacked photosynthesis and that the chlorophyll content did not change with increasing days of cultivation (Figure 4c). However, there was an obvious change in the total carotenoid content, and we can see that the total carotenoid content of C18 and P1 reached a maximum (0.1825 and 0.1889 \(\mathrm{\SIUnitSymbolMicro g}\)/10\({}^{6}\) cell, respectively) at Day 4, followed by a slow decrease at Day 4-10 (Figure 4d), while the total carotenoid content of CC-125 was constantly decreasing. After Day 4, the total carotenoid content in both the C18 and P1 groups was greater than that of the CC-125 strain. Taking Day 4 as a node, the total carotenoid contents of C18 and P1 were 2.13-and 2.20-fold that of CC-125, respectively. Qualitative and quantitative analysis of astaxanthin and \(\beta\)-carotenoid from cultures of CC-125, C18 and P1 was performed via HPLC. The standard curve of astaxanthin and \(\beta\)-carotenoid and a figure of the absorption peak for cultures are shown in Supplementary Material S3. Due to the low pigment content of partial samples at Day 2, astaxanthin and \(\beta\)-carotenoid were not detected. Therefore, we chose to show the data from Day 4 to 10. At Day 4, the contents of astaxanthin and \(\beta\)-carotenoid in all three groups were highest, and then decreased slowly. For astaxanthin (Figure 4e), C18 had the highest content, P1 was next and CC-125 had the lowest. Additionally, taking Day 4 as a node, the astaxanthin contents of C18 and P1 were 0.047 and 0.049 \(\mathrm{\SIUnitSymbolMicro g}\)/10\({}^{6}\) cells, respectively. Meanwhile, the astaxanthin content of CC125 was 0.039 \(\mathrm{\SIUnitSymbolMicro g}\)/10\({}^{6}\) cells.