# Enhancement of Lycopene Biosynthesis Using Self-Assembled Multi-Enzymic Protein Cages
## 1 Introduction  
Cell compartmentalization spatially segregates various cellular functions within the cell into distinct compartments, providing a structural framework for the regulation and optimization of intracellular metabolic functions and efficiencies. This organization enables the simultaneous occurrence of diverse or cascading complex biochemical reactions without interference [1]. Cell compartmentalization offers numerous advantages, such as increasing the local concentrations of enzymes and substrates to improve catalytic efficiency, reducing the accumulation of toxic intermediates to minimize cytotoxic effects, and eliminating interference from competing pathways [2, 3]. In recent years, the strategy of cellular compartmentalization has played a crucial role in the development of microbial cell factories for natural product synthesis. Prokaryotes possess intricate proteinaceous cellular structures known as bacterial microcompartments (BMCs) [4], which are widely distributed across various bacteria and exhibit functionalities akin to eukaryotic organelles [5, 6]. Some BMCs can be formed through the self-assembly of a series of homologous shell proteins, encapsulating enzymes associated with specific metabolic pathways [3, 7]. Their remarkable potential in the design and fabrication of novel protein nanoreactors stems from intrinsic properties such as self-assembly, selective permeability, enzyme encapsulation efficiency, and high biocompatibility [8, 9]. For instance, BMCs were used for the biosynthesis of ethanol in _Escherichia coli_[10]. Among these proteinaceous BMCs, carboxysomes are found in cyanobacteria andcertain chemolithouattotrophic organisms that function as CO\({}_{2}\) fixation mechanisms, playing a crucial role in the global carbon cycle [11]. The carboxosome encapsulates carbonic anhydrase (CA) and the primary carbon dioxide-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) within a protein shell. This protein shell facilitates the selective entry of HCO\({}_{3}\)\({}^{-}\), which is converted into CO\({}_{2}\) by the CA in proximity to RuBisCO. Subsequently, RubisCO fixes the locally generated CO\({}_{2}\) onto ribulose-1,5-bisphosphate (RuBP) molecules, yielding two molecules of 3-phosphoglycerate (3 PGA) [12]. Concurrently, the protein shell restricts the passage of CO\({}_{2}\), resulting in its efficient accumulation within the shell, thereby enhancing RuBisCO carboxylation and reducing photorespiration [13]. Based on the phylogeny of RuBisCO, the types of carboxysomes are usually classified into \(\alpha\)-carboxysome found in marine cyanobacteria and \(\beta\)-carboxysome, which are mainly present in freshwater and estuarine cyanobacteria [14]. The assembly of \(\beta\)-carboxysomes occurs in an inward-out manner, necessitating the gradual formation of the RuBisCO core before the assembly of the shell [15; 16]. In contrast, \(\alpha\)-carboxysomes utilize strategies where the shell is formed first or where RuBisCO and shell proteins are assembled simultaneously [17; 18]. Due to the inherent compartmentalization of \(\alpha\)-carboxysomes, leveraging their protein shells to integrate diverse metabolic pathways facilitates the construction of innovative biological nanoreactors. Previous studies have demonstrated the potential of utilizing the \(\alpha\)-carboxysome shell to encapsulate oxygen-sensitive hydrogenases for the development of hydrogen-producing nanoreactors [9]. However, despite the significant potential of carboxysome shells in various biotechnological applications, the complexity of their components presents challenges. Simplifying the carboxysome shell architecture and achieving tunable and efficient enzyme loading strategies are critical issues for the rational design and engineering of carboxysome-based nanostructures.