Streamlining heterologous expression of top carbonic anhydrases in Escherichia coli: bioinformatic and experimental approaches

Abstract

Background: Carbonic anhydrase (CA) enzymes facilitate the reversible hydration of CO₂ to bicarbonate ions and protons. Identifying efficient and robust CAs and expressing them in model host cells, such as Escherichia coli, enables more efficient engineering of these enzymes for industrial CO₂ capture. However, expression of CAs in E. coli is challenging due to the possible formation of insoluble protein aggregates, or inclusion bodies. This makes the production of soluble and active CA protein a prerequisite for downstream applications.

Results: In this study, we streamlined the process of CA expression by selecting seven top CA candidates and used two bioinformatic tools to predict their solubility for expression in E. coli. The prediction results place these enzymes in two categories: low and high solubility. Our expression of high solubility score CAs (namely CA5-SspCA, CA6-SazCAtrunc, CA7-PabCA and CA8-PhoCA) led to significantly higher protein yields (5 to 75 mg purified protein per liter) in flask cultures, indicating a strong correlation between the solubility prediction score and protein expression yields. Furthermore, phylogenetic tree analysis demonstrated CA class-specific clustering patterns for protein solubility and production yields. Unexpectedly, we also found that the unique N-terminal, 11-amino acid segment found after the signal sequence (not present in its homologs), was essential for CA6-SazCA activity.

Conclusions: Overall, this work demonstrated that protein solubility prediction, phylogenetic tree analysis, and experimental validation are potent tools for identifying top CA candidates and then producing soluble, active forms of these enzymes in E. coli. The comprehensive approaches we report here should be extendable to the expression of other heterogeneous proteins in E. coli.

Keywords: Escherichia coli; Carbonic anhydrase; Phylogenetic tree analysis; Protein expression; Protein solubility prediction.

Fig. 1

Plasmid construction for N-terminal (A) and C-terminal (B) His-tagged CAs. It involved cloning the synthetic genes into pET-28( +) between the NcoI and XhoI restriction sites. The expression of CA proteins was driven by the T7 promoter (T7 Prom) and terminated by the T7 terminator (T7 Term). Lac O denotes the Lac operator, and RBS indicates the ribosome binding site

Fig. 2

SDS-PAGE analysis of the cell lysates from the overexpression of CA1, CA2, and CA4 to CA8 in E. coli. CA1-TaCA with N-terminal his-tag had lower expression level than CA4 to CA8 (based on the protein bands that indicated by the red circles in individual lanes). CA1’s expression was confirmed by purification using Ni–NTA and SEC (see Supplementary Figure S1). The SDS-PAGE analysis of the cell lysate from the overexpression of CA3 was show in lane 1 of Supplementary Figure S2C

Fig. 3

CA6-SazCAtruc protein sequence feature and purification. A Amino acid sequence alignment for the CDS of full length SspCA and SazCAfull, including their signal sequences. A 11-amino acid sequence (highlighted in yellow) was removed from SazCAfull to obtain CA6-SazCAtrunc (in blue text). B SEC chromatogram for CA6-SazCAtrunc. C SDS PAGE for fractions B9-C5 from CA6-SazCAtruc SEC purification

Fig. 4

Multiple sequence alignment of CA1 to CA8 using Clustal Omega

Fig. 5

Phylogenetic analysis of CAs. The amino acid sequences were aligned, and the phylogenetic tree was built by using Clustal Omega. Neighbour-joining tree without distance corrections was selected. CA1-TaCA and CA2-TaCA represent the TaCA expressed with N- and C-terminal His-tag, respectively