## 3 Discussion
### Association and Differences between Exogenous and Exogenous BKT Gene Overexpression
Combined with the physiological data, we speculate that the high similarity in the amino acid sequences of these two proteins makes the expression of _CrBKT_ and _PrBKT_ have similar effects. However, by analyzing the transcriptome and metabolome, the two strains C18 vs. P1 showed partially different expression levels with metabolites on other metabolic pathways, which we further discuss in the following two sections. Transcriptome and Metabolome Analysis and Discussion of the Carotenoid Metabolic Pathways in the C18 and P1 Strain  
The function of \(\beta\)-carotene ketolase is mainly to ketolate the ring end groups of \(\beta\)-carotene. Some studies have shown that the upstream steps of carotenoid synthesis are key steps for \(\beta\)-carotene and astaxanthin biosynthesis [21]. However, by modifying the _BKT_ gene, astaxanthin production can also be greatly increased. Functionally, if a large amount of astaxanthin needs to be synthesized in _Chlamydomonas reinhardtii_, not only does the expression level of \(\beta\)-carotene ketolase need to be increased, but so does the coordination of \(\beta\)-carotene carboxylase [22; 23]. Astaxanthin synthesis in _Chlamydomonas reinhardtii_ belongs to the bicyclic pathway, but this green microcalga itself hardly synthesizes astaxanthin, because its \(\beta\)-carotene ketolase (_CrBKT_) gene expression level is very low, and it contains an uncommon C-terminal extension, which may be a pseudogene [17]. The genes for synthesizing \(\beta\)-carotene in _Phaffia rhodozyma_ are crtYB, crtl, crtO, crtS and so on. Among them, crtl catalyzes carotenoid conversion into \(\beta\)-carotene. Subsequently, \(\beta\)-carotene can be converted into astaxanthin by _BKT_ and _CHYB_. It can also be converted into astaxanthin by CrtR and CrtO. At the same time, CrtS, the protein translated after astaxanthin synthetase), can continuously catalyze the 4-ketolation and 3-hydroxylation of \(\beta\)-carotene, to synthesize astaxanthin [24]. However, according to [PERSON] et al. 's study, in three different _P.Hodozyma_ strains, the CrtS gene was poorly correlated with astaxanthin synthesis [8; 25]. However, their relationship and regulatory mechanism are not clear. The \(\beta\)-carotene ketolase protein (CDZ97953.1) of _Phaffia rhodozyma_ consists of 328 amino acids, with a molecular weight of 36.8 kDa and an isoelectric point of 5.18. There are 250 residues, of which 116 are polar residues and 134 are non-polar residues. Similarly, the \(\beta\)-carotene ketolase (\(\beta\)-carotene ketolase) (X_001698699.2) of _Chlamydomonas reinhardtii_ consists of 400 amino acids, with a molecular weight of 39.3 kDa and an isoelectric point of 5.03, of which 108 are polar residues and 259 are non-polar residues. Therefore, whether it is _Phaffia rhodozyma_ or _Chlamydomonas reinhardtii_'s _BKT_, they are relatively conservative and function-specific. As shown in Figure 12, we present the carotenoid pathway of C18 and P1, and indicate the transcriptome and metabolome data. In C18, the overexpression of _CrBKT_ only up-regulated the transcription level of _BKT_ and _LCYE_. However, in P1, the overexpression of _PrBKT_ led to the up-regulation of _BKT_, _LCYE_, _ZDS_ and _CHYB_. The \(\beta\)-carotene synthesis pathway in _Chlamydomonas reinhardtii_ mainly consists of two branches; one is the \(\beta\)-cyclization branch catalyzed by lycopene cyclase (_LCYE_), which can convert \(\beta\)-carotene into \(\beta\)-cryptoxanthin, and then into astaxanthin, and the other is the \(\epsilon\)-cyclization branch catalyzed by \(\beta\)-carotene hydroxylase (BCH), which can convert \(\beta\)-carotene into echinenone, and then that into canthaxanthin. The _BKT_ gene belongs to monocyclic pathway, and \(\beta\)-carotene is synthesized into astaxanthin via the ketolation and carboxylation of _BKT_ and _CHYB_. We overexpressed the _BKT_ gene to enhance monocyclic pathway. The carotenoid synthesis of zeaxanthin is a bicyclic pathway. How these two pathways regulate each other is not clear.