Improvement of the phosphoenolpyruvate carboxylase activity of Phaeodactylum tricornutum PEPCase 1 through protein engineering

• Phaeodactylum tricornutum PEPCase 1 protein was expressed in the E. coli expression system.
• N-terminal truncated PtPEPCase 1 derivates showed high yield and activity.
• cPtPEPCase 1 could be a strong candidate for use as a platform technology to capture CO2.

In order to mitigate CO2 accumulation and decrease the rate of global warming and climate change, we previously presented a strategy for the development of an efficient CO2 capture and utilization system. The system employs two recombinant enzymes, carbonic anhydrase and phosphoenolpyruvate carboxylase, which were originated from microalgae. Although utilization of this integrated system would require a large quantity of high quality PEPCase protein, such quantities could be produced by increasing the solubility of the Phaeodactylum tricornutum PEPCase 1 (PtPEPCase 1) protein in the Escherichia coli heterologous expression system. We first expressed the putative mitochondria targeting peptide- and chloroplast transit peptide-truncated proteins of PtPEPCase 1, mPtPEPCase 1 and cPtPEPCase 1, respectively, in E. coli. After affinity chromatography, the amount of purified PEPCase protein from 500 mL of E. coli culture was greatest for cPtPEPCase 1 (1.99 mg), followed by mPtPEPCase 1 (0.82 mg) and PtPEPCase 1 (0.61 mg). Furthermore, the enzymatic activity of mPtPEPCase 1 and cPtPEPCase 1 showed approximately 1.6-fold (32.19 units/mg) and 3-fold (59.48 units/mg) increases, respectively. Therefore, cPtPEPCase 1 purified using the E. coli heterogeneous expression system could be a strong candidate for a platform technology to capture CO2 and produce value-added four-carbon platform chemicals.