# Enhancement of Lycopene Biosynthesis Using Self-Assembled Multi-Enzymic Protein Cages
## 3 Results
### Expression and Characterization of Carboxysome Nanocage Scaffold in E. coli  
First, we expressed the assembled CBs with green fluorescent protein (mGFP) as the cargo protein in _E. coli_. For visualization, we fused the red fluorescent protein (mCherry) with surface proteins of carboxysome shells. We co-expressed the ST-linked mGFP (mGFP-ST) and SC-linked CBs (CBs-mCherry-SC) in _E. coli_ (strain CBs1) (Figure 2a). A strain (CBs0) expressing CBs-mCherry-SC and mGFP was used as the control. The mGFP-ST was co-localized with the SC-linked CBs in the cells compared with the control (Figure 2a), indicating that the cargo protein can be immobilized on the CBs. The intact spherical carboxysome shells in CBs0 and CBs1 were visualized by transmission electron microscopy (Figure 2b). The average diameter of the naked CBs-mCherry-SC in the control strain CBs0 was 79.5 nm, whereas the average diameter of the assembled nanocage in the CBs1 increased to 107.9 nm (Figure 2c), indicating that the cargo proteins were successfully attached to the surface of the protein cage. In addition, we found that the expression of CBs and the assembled protein cages had little effect on cell growth, suggesting that the protein cages are biocompatible with _E. coli_ (Supplementary Figure S2). Figure 1: Schematic diagram of construction of IPP synthetic multi-enzyme nanocage for lycopene synthesis. (**a**) Enzyme cascade pathway for lycopene synthesis based on the isoprenoid alcohol pathway and endogenous MEP pathway. ScCK: choline kinase from _Saccharomyces cerevisiae_; AtIPK: isopentenyl phosphate kinase from _Arabidopsis thaliana_; McanIDI: IPP isomerase from _Myxococcus xanthus_; IP: isopentenyl monophosphate; IPP: isoprenyl pyrophosphate; DMBPP: dimethylallyl pyrophosphate from the MEP pathway; FPP: farnesyl diphosphate. (**b**) Assembly of IPP synthetic enzyme on \(\alpha\)-carboxysome shell using the SpyTag/SpyCatcher for lycopene synthesis. Immobilization of IPP Synthetic Enzymes on Nanocages Led to Increased Lycopene Production in E. coli  
To verify the functionality of enzyme immobilization on carboxosome nanocages, we first immobilized the IDI on the carboxosome nanocage, which is one of the rate-limiting enzymes for lycopene synthesis [46]. Exogenous IDI genes were screened in _Deinococcus radiodurans_ (DR), _Deinococcus radiopugnans_ (DRP), and _Myxococcus xanthus_ (MXAN) based on bioinformatics analysis. Subsequently, we immobilized the IDIs on the nanocages by the conjugation of the C-terminal regions of IDI with ST-linker to the SC-linked CBs, which were expressed in the _E. coli_ ly001 strain containing CrtE/CrtB/CrtI. All the strains were cultured to the OD\({}_{600}\) of 0.8, and the inducer IPTG was added. After 48 h of overexpression at 25 \({}^{\circ}\)C, the cell cultures were centrifuged, and pellets were collected. As illustrated in Figure 3a, the strain with immobilized IDI on CB cages (CBs-SC+IDI-ST) exhibited a deeper red pigment compared to the strains with unimmobilized IDI (CBs-SC+IDI). Moreover, carotenoids were extracted from the cells, followed by HPLC analysis. Lycopene was identified as the major carotenoid product (Supplementary Figure S4), which was quantified against a standard calibration curve for lycopene (Supplementary Figures S3 and S4). The  
Figure 2: Characterization of the assembled nanocage scaffold in _E. coli_. (**a**) Schematic representation of the gene constructs for protein nanocage labeled with fluorescent proteins. CBs0, the _E. coli_ strain expressing CBs-mCherry-SC and mGFP (control strain); CBs1, the _E. coli_ strain expressing both CBs-mCherry-SC and mGFP-ST (**Left**). Confocal imaging of the engineered CBs1 and CB80 (**Right**). Scale bar, 1 \(\mu\)m. (**b**) TEM images of engineered _E. coli_ CBS0 and CBS1. For the CBs0, the nanocages are shown in the middle panel presenting an enlarged view of the area demarcated in the left panel. The nanocages are indicated with yellow arrows. For theCBs1, the nanocages carrying cargo proteins are indicated with red arrows. Scale bar, 500 nm.