# Enhancement of Lycopene Biosynthesis Using Self-Assembled Multi-Enzymic Protein Cages
## 1 Introduction
Recently, engineered protein nanocages based on an \(\alpha\)-carboxysome shell containing specific cargo loading sites using the SpyTag/SpyCatcher system were constructed and exhibited potential in enzyme immobilization and catalysis [19], which shows application potential in the field of natural product synthesis. Terpenoids are widely distributed secondary metabolites in bacteria, plant, archaea, and fungi, among others [20; 21], encompassing over 50,000 known types characterized by their diverse molecular structures and biological functions [22; 23; 24]. Many tetraterpenes (carotenoids) such as lycopene and \(\beta\)-carotene possess significant application value in the food and pharmaceutical industries [25; 26]. There are two primary methods for obtaining tetraterpenes--biosynthesis and chemical synthesis [27]. Microbial biosynthesis of lycopene represents a sustainable and environmentally friendly production strategy. Consequently, the development of efficient microbial cell factories for lycopene synthesis has emerged as a significant research focus for sustainable production of the product. In bacteria, the lycopene synthesis pathway usually involves multiple enzymes using isopentenyl pyrophosphate (IPP) as C5 precursor substrate [28]. IPP and its allylic isomer, dimethylallyl pyrophosphate (DMAPP), serve as precursors in terpenoid biosynthesis. Geranylgeranyl diphosphate synthase (CrtE) catalyzes the head-to-tail condensation of these C5 building blocks to yield isoprenoid precursors, including geranyl diphosphate (GPP, C10), farnesyl diphosphate (FPP, C15), and geranylgeranyl diphosphate (GGPP, C20). The subsequent enzymes CrtB and CrtI are responsible for the conversion of GGPP to produce phytoene and then lycopene [29; 30]. Moreover, IPP can be produced through the isoprenoid alcohol pathway [31; 32; 33; 34], which is an artificial designed enzyme cascade involving choline kinase from _Saccharomyces cerevisiae_ (ScCK) and isopentenyl phosphate kinase from _Arabidopsis thaliana_ (AtIPK) using exogenous isoprenol as the substrate [31; 32; 33; 34]. In recent years, with the rapid advancements in synthetic biology and metabolic engineering, synthesis of lycopene has been achieved in model microorganisms such as _E. coli_ and yeast [35; 36; 37]. However,enzymes, metabolites, and cofactors associated with lycopene synthesis might disperse within a cell and are limited by local concentration of the enzymes, which impacts the efficiency of product synthesis [38; 39; 40]. The strategy of cellular compartmentalization employs organelles or subcellular structures for the reconstruction of terpenoid metabolic pathways in microbial cell factories will effectively address the aforementioned challenges. Previous studies have indicated that co-localizing the cascade enzymes involved in the biosynthesis of lycopene within the protein cage Mi3 can enhance lycopene production [41]. The Mi3 is an engineered dodecahedral protein cage derived from the aldolase of the hyperthermophilic bacterium _Thermotoga maritima_, comprising 60 identical subunits [42; 43]. In this study, we developed an IPP-synthetic protein nanocage based on \(\alpha\)-carboxysome of the marine bacterium _Prochlorococcus marinus_ MED4. We co-immobilized the key enzymes of IPP synthesis (ScCK, AtIPK and MXanIDI) within the protein nanocage, which were co-expressed with CrtE, CrtB, and CrtI in _Escherichia coli_ to enhance the production of lycopene. The function of the IPP-synthetic protein nanocage for lycopene synthesis was evaluated.