## 3 Discussion  
### Association and Differences between Exogenous and Exogenous BKT Gene Overexpression  
Overexpression in genetic engineering is a common approach to breeding improvement. In microalgae, the modification of a given gene's genome is achieved by changing its biological properties by increasing the amount of expression of that gene [19]. Overexpression enables the increased production of specific proteins, which in turn influences the characteristics of the microalgal strain. For instance, it can lead to a higher yield of specific metabolites, ultimately enhancing the nutritional value of microalgae. Therefore, overexpression technology serves as an effective approach for improving and selecting  
Figure 10: KEGG enrichment results of differential metabolites. (**a**) Scatter plot of KEGG enrichment results for differential positive ion metabolites in C18 vs. WT. (**b**) Scatter plot of KEGG enrichment results for differential negative ion metabolites in C18 vs. WT. (**c**) Scatter plot of KEGG enrichment results for differential positive ion metabolites in P1 vs. WT. (**d**) Scatter plot of KEGG enrichment results for differential negative ion metabolites in P1 vs. WT. commercially viable microalgal strains. _Chlamydomonas reinhardtii_ is considered a safe and edible microalga species with a higher protein content compared to that of _Chlorella vulgaris_. Additionally, it possesses an intact genome, making it a suitable candidate for genetic manipulation [20]. Within the carotenoid metabolism pathway of _Chlamydomonas reinhardtii_, \(\beta\)-cartene ketolases play a crucial role. These enzymes are responsible for converting \(\beta\)-carotenoids through ketification reactions, leading to the formation of valuable products such as echinenone, canthaxanthin and astaxanthin, among others. However, recent studies have focused on engineering the _CrBKT_ gene or co overexpressing multiple genes of the carotenoid synthesis pathway to achieve high carotenoid and astaxanthin production. This study mainly focused on the _BKT_ gene. We found that the _BKT_ gene of _Phaffia rhodozyma_ had the highest homology with _CrBKT_ via alignment in NCBI. We therefore decided to overexpress _CrBKT_ versus _PrBKT_ separately to investigate whether or not the overexpression results were differential. From the amino acid alignment results of these two proteins (Figure 11), we can see that _PrBKT_ has a longer stretch of sequences than _CrBKT_ does at the beginning, and _CrBKT_ has a longer stretch of sequences than _PrBKT_ does at the C' end. In the study by [PERSON] et al., they found that there was a long, seemingly nonsense repeat in the \(\beta\)-carotenoid ketolase gene of _Chlamydomonas reinhardtii_. The expression of _CrBKT_ will be optimized after these redundant sequences are removed, promoting astaxanthin synthesis. However, _PrBKT_ has an extended sequence in the N-segment, which we consider relevant for carotenoid synthesis in yeast, and does not belong to a 'pseudogene'. So, when initially designing the experiments, we speculated that the overexpression effect of _PrBKT_ would be better than that of _CrBKT_. Indeed, the carotenoid production level of C18 was comparable to that P1, even after codon optimization. We speculate that the copy number and insertion site of the exogenous _BKT_ gene may be different from that of the endogenous gene. It contains copies of a foreign gene within _Chlamydomonas reinhardtii_ or a different chromosomal location that affects its expression level and stability. Second, the translated protein of _PrBKT_ may be subject to splicing and modification effects different from those of endogenous genes, such as  
Figure 11: Alignment of amino acid sequences of \(\beta\)-carotenoid ketogenes from _Chlamydomonas reinhardtii_ (XP_001698699.2) and _Phaffia rhodozyma_ (CDZ97953.1). methylation, acetylation, phosphorylation, etc., which can affect its post-transcriptional levels and function.