Effective surface display of sucrose isomerase PalI on the cells of Yarrowia lipolytica

Abstract:

[Background] Bioconversion via sucrose isomerase (PalI) is currently a preferred method to produce isomaltulose. However, this method still has some shortcomings, such as the formation of multiple products and the tedious purification process of PalI from cell extracts. [Objective] We sought to achieve efficient isomaltulose production via constructing a surface display strain for PalI on Y. lipolytica Po1g and reduce the proportion of by-products and purification cost. [Methods] The gene PalI encoding sucrose isomerase PalI from Klebsiella singaporensis LX3 was fused with the gene Pir1 encoding the anchoring protein Pir1 from the yeast cell wall by overlap extension PCR. The fused gene was then transferred into Y. lipolytica Po1g for the surface display of PalI. The enzymatic properties of the surface-displayed PalI were investigated via colorimetry with 3,5-dinitrosalicylic acid (DNS), and the products of sucrose conversion were detected by high performance liquid chromatography. [Results] The PalI surface display strain Pir1-PalI/Po1g was successfully constructed, and the highest activity of the displayed PalI reached (4 694.6±56.6) U/g of dry cell weight. The displayed PalI was stable at a broad range of 20-55 ℃ and pH 3.5-9.0, with the best performance at pH 6.0 and 45 ℃. Moreover, the displayed PalI led to significantly lower proportion of monosaccharide by-products during the bioconversion process than the free PalI. [Conclusion] Using Y. lipolytica Po1g as the host, we constructed a recombinant strain for the surface display of PalI, which provided a basis for the industrial production of isomaltulose.

Key words:sucrose isomerase;Yarrowia lipolytica;surface display system;yeast cell wall-anchoring protein Pir1;enzymatic properties