CA is one of the most ancient enzymes on Earth, widely distributed across both eukaryotic and prokaryotic organisms [1]. To date, eight distinct classes of CA have been identified—α, β, γ, δ, ζ, η, θ, and ι. Despite their low sequence homology and structural divergence, all CA classes catalyze the same fundamental reaction [2]. Among them, α-class CAs (α-CAs) are the most extensively studied and are found in animals, plants, algae, and many Gram-negative bacteria. Most α-CAs exist as monomers while the typical α-CA structure consists of a central core of 10 antiparallel β-strands and is surrounded by several α-helices or β-sheet elements. A zinc ion is positioned at the center of the active site, coordinated by three conserved histidine residues and a hydroxide ion [3]. Two additional conserved residues, i.e., threonine and glutamate, interact with water molecules, enhancing the nucleophilicity of the hydroxide ion and thereby facilitating the catalytic conversion of CO₂ [4].

Currently, addressing the global challenge of carbon emissions and advancing biotechnological innovation, CA has emerged as a vital biocatalyst with diverse and impactful applications. This zinc metalloenzyme catalyzes the rapid interconversion of carbon dioxide (CO₂) and bicarbonate (HCO₃⁻), playing a pivotal role in natural and engineered carbon capture systems [5], [6]. In the industrial sector, CA has been explored as a promising catalyst for carbon capture technologies, where it can accelerate the hydration of CO₂ in flue gases, facilitating its transformation into bicarbonate for subsequent sequestration or reuse [7]. In biotechnological applications, genetically engineered microalgae expressing elevated levels of CA have demonstrated enhanced CO₂ assimilation capacity, leading to improved biomass, lutein and lipid production [8]. Furthermore, CA has been adapted to function as a metal ion biosensor, leveraging its metal-dependent activity and structural sensitivity to zinc and other divalent cations [9]. This feature enables its integration into engineered circuits for monitoring intracellular or environmental metal concentrations.

CAs from different organisms display remarkable diversity in stability and activity. For example, SyCA from the thermophilic bacterium Sulfurihydrogenibium yellowstonense retains full activity even at 100 ℃ [10]. MlCA from Mesorhizobium loti exhibits extraordinarily high CO₂ hydration activity, with a specific activity of 2 × 106 μmol/min/mg using pure enzyme [11], [12]. However, producing MlCA in its native host is challenging due to low enzyme yields [11]. Escherichia coli, with its rapid growth rate and extensive genetic toolkit, is the most widely used chassis for recombinant protein production. Nevertheless, recombinant production of MlCA continues to face challenges, including difficulties of over-production and poor solubility. Fusion of the thioredoxin A (TrxA) tag to MlCA has proven beneficial in enhancing both protein expression and solubility [13]. Co-expression of molecular chaperones such as GroELS, either via auxiliary plasmids or genomic integration, has further improved the solubility of MlCA [14].

Protein expression is the most energy-intensive processes in a cell, consuming approximately 85 % of the nutrients taken up and around 67 % of the ATP generated through respiration [15]. The expression of heterologous genes places an additional burden on the host’s cellular machinery, competing directly with its native metabolic processes. When the demand for biosynthetic resources exceeds the cell’s capacity, it can trigger stress responses that impair growth and reduce biomass accumulation, that ultimately leads to decreased protein production [16], [17]. A comparative study on E. coli genes showed that highly expressed genes tend to favor the use of metabolically inexpensive amino acids, suggesting that expression efficiency is strongly influenced by biosynthetic cost [18]. Similarly, smaller proteins, with an average molecular weight of around 23 kDa, were found to be expressed more efficiently and soluble than larger proteins, which exceed 40 kDa [19]. Therefore, reducing protein size may offer an alternative to enhance expression.

In this study, we aimed at exploring the potential of protein minimization to improve the expression and solubility of MlCA. A series of fusion tags of varying sizes to investigate the relationship between protein size and expression levels are evaluated. In addition, fusion tags that affected the mRNA secondary structure of the MlCA variants are discussed. Finally, a de novo protein design approach, assisted by molecular dynamics simulations, was employed to create a compact and soluble protein scaffold capable of accommodating the MlCA active site.