## 4 Discussion  
Like most prokaryotic microorganisms, the newly isolated strain _C. sphaeroides_ DT.1 synthesizes lycopene via the MEP pathway [30]. The critical regions responsible for functional divergence in desaturation activity, key catalytic sites, and micro-evolutionary variations of Crtl enzymes across diverse biological species remain to be elucidated [31; 32; 33; 34]. Currently, the functional characteristics of Crtl can only be inferred from the products of enzymatic reactions [35]. Previous studies have shown that the Crtl in _C. sphaeroides_ ATH 2.4.1 functions as a three-step desaturase, catalyzing the conversion of phytoene to neurosoprene as the end product, and is thus incapable of synthesizing lycopene [14]. However, besides lycopene, torulene, a downstream product derived from the five-step desaturation of phytoene was also detected, indicating that Crtl in strain DT.1 possesses four-step or even five-step desaturation capabilities. [PERSON] et al. showed that Crtl from _Rhodobacter azotoformans_ can catalyze the three-step and four-step desaturation of phytoene to produce neurosoprene and lycopene, respectively, and under certain conditions, it can also perform five-step desaturation to form 3,4-didehydrolycopene [35]. In our study, the amino acid sequence similarity of Crtl between strain DT.1 and _Rhodobacter azotoformans_ was calculated to be as high as 92.47%, supporting the aforementioned hypothesis. Results from the present study show that the addition of glutamate or proline promoted the lycopene accumulation in either wildtype or \(\Delta\)_crtC_ mutant strains (Figure 6B) and this was achieved by downregulating the expression of the _crtC_ gene related to lycopene conversion (the first mechanism) and promoting the accumulation of lycopene synthesis precursors (second mechanism). This phenomenon has not been reported in previous literature. The addition of glucose or glycerol also enhanced lycopene accumulation through the second mechanism, as both substrates can be converted via glycolytic pathway to glyceraldehyde-3-phosphate (G-3-P), one of the key precursors for lycopene synthesis [13; 36]. Glutamate and proline exhibited similar promoting effects, as shown in Figures 3 and 6. This can be explained by the fact that these two amino acids can be interconverted and share the same precursor (\(\alpha\)-ketoglutate) (Figure 5). According to [PERSON] et al., glutamate can be reduced to proline through the catalytic action of gamma-glutamyl transferase (\(\gamma\)-GK) and pyrroline-5-carboxylate reductase (P5 CR), while proline can be oxidized to glutamate through the catalytic action of proline dehydrogenase (PRODH) and delta-1-pyrroline-5-carboxylate dehydrogenase (P5 CDH) [37]. Here PRODH and P5 CDH formed a bifunctional enzyme which is encoded by the _putA_ gene [38]. This has been confirmed by the successful annotation of the _putA_ gene in the genome of strain DT.1, as well as expression of this gene as revealed in the transcriptomic results. Cultivation of PNSB under anaerobic light conditions represents the traditional manner of lycopene production [13]. However, industrial scale-up of this process would be restricted by drawbacks such as long fermentation period and insufficient light intensity caused by high cell density. Theoretically, aerobic fermentation would be preferred to overcome this problem. However, photosynthesis genes, including those of carotenoids, are repressed by PpsR, leading to the inhibition of lycopene synthesis under aerobic conditions [39; 40]. Recently [PERSON] et al. obtained lycopene of 50.6 mg/g DCW in _E. coli_ by regulating the expression of exogenously introduced genes such as _dxs_, _idi_, _crtCDF_, _sucAB_, _sdhABCD_, and _talB_ [41]. [PERSON] et al. succeeded in promoting the lycopene yield by up to 10.32 mg/g DCW under aerobic conditions by knocking out the _crtC_ gene to reduce lycopene conversion, followed by knocking out _ppsR_ to activate the expression of PS genes in C. _sphaeroides_ RL1 [14].