# Enhancement of Lycopene Biosynthesis Using Self-Assembled Multi-Enzymic Protein Cages
## 3 Results
### Expression and Characterization of Carboxysome Nanocage Scaffold in E. coli
(**c**) Comparison of the average diameter of CBs in CBs1 with that in CBS0 (control strain) (\(n=50\)). expression of IDI from MXAN (MXanIDI) or DRP (DrpIDI) in the strain CBs-SC+IDI-ST resulted in the higher yield of lycopene compared with the strains with unimmobilized IDI (CBs+IDI-ST), indicating that the strain with IDI immobilized on the CB nanocage could improve the lycopene production (Figure 3b). Therefore, the immobilization of rate-limiting enzymes within carboxysome nanocages is advantageous for product synthesis compared to free enzymes. Sufficient supply of IPP and DMAPP is crucial for the effective biosynthesis of terpenoids [47]. Furthermore, we immobilized the ScCK and AtIPK from the isoprenoid alcohol pathway for the IPP synthesis on the CB nanocages containing MXanIDI from MXAN. By fusing ST to the genes, we generated the fusion genes for expression of ScCK-ST and AtIPK-ST, respectively, resulting in IPP synthetic nanocages assembled with ScCK, AtIPK and MXanIDI. Then, we constructed a plasmid containing the three genes involved in IPP (isopentenyl pyrophosphate) synthesis and co-transformed it with CBs-SC into the ly001 strain to enhance lycopene biosynthesis. Two strains were constructed: CBIUP\({}_{1}\), which contained multi-enzymatic IPP synthetic cages, and the control strain CBIUP\({}_{0}\), which contained only \"free\" enzymes. The CBIUP1 strain expresses the ST-linked enzymes and CBs-SC, facilitating the spontaneous attachment of the enzymes to the exterior of the CB protein cage, resulting in the assembly of the ScCK/AtIPK/MXanIDI-CBs (Figure 1b). The distinction between CBIUP\({}_{1}\) and CBIUP\({}_{0}\) is the absence of ST in ScCK, AtIPK, and MXanIDI. The addition of isoprenol might influence the growth and metabolism of the cells [48]. However, it was observed that the addition of 15 mM isoprenol resulted in little impact on the CBIUP0 strain's growth (Figure 4a). Additionally, we compared the lycopene yield of CBIUP1 and CBIUP0 during fermentation. After 48 h of fermentation, the CBIUP1 achieved a lycopene production of 12.6 mg/g DCW for strain, which is 1.7 times greater than that of CBIUP0 (7.3 mg/g DCW) (Figure 4c). We monitored the levels of metabolites involved in the biosynthesis of lycopene. Notably, the IPP levels in the CBIUP1 strain increased about 11.8-fold compared with the CBIUP0 after 24 h of induction (Figure 4b). There were no significant changes in the transcription level of the relevant biosynthetic genes, which could eliminate the possibility that variations in expression level of enzymes contributed to differences in lycopene production (Figure 4d). Therefore, the increase in lycopene yield can  
Figure 3: Immobilization of IDI on carboxysome nanocages improved lycopene production in _E. coli._ (**a**) Comparison of cell pigment of _E. coli_ ly001 strains co-expressed with CBs-SC+IDI or CBs-SC+IDI-ST, respectively. The IDI was from DR, DRP, or MXAN; (**b**) lycopene analysis of the _E. coli_ strains using HPLC. Data are presented as mean \(\pm\) standard deviation (\(\mu\) = 3). be attributed to the assembly of IPP synthetic enzymes on the nanocages. The assembled IPP synthetic CB cages might accumulate related enzymes and enhance metabolic flux for lycopene production. The enzymes might be attached spontaneously to the exterior of the CB nanocages through the SC-ST system. The detail and ratio of binding for the ScCK/AtIPK/MxanIDI to CB nanocages need to be investigated in future study.