Simultaneously promoting gellan lyase expression and gellan oligosaccharide production by gellan gum co-feeding during Pichia pastoris fermentation

Abstract

The enzymatic hydrolysis of gellan gum (GG) using gellan lyase presents an efficient and practical method for generating gellan oligosaccharides (GOSs). GOSs have promising applications due to their diverse biological functions, including beneficial properties and immunoreactivity. They are characterized by low molecular weight, high water solubility, and easy absorption and utilization by the body. In addition, GOSs exhibit properties such as plant-induced disease resistance and enhanced immune activity. However, current degradation methods for producing GOSs remain limited. In this study, recombinant Bacillus sp. GL1-derived gellan lyase was successfully expressed in Pichia pastoris under the control of the AOX1 promoter. DO-stat carbon source of waste mixed sugar used as a substitute for glycerol, feeding strategy was adopted for scale-up cultivation in a 7 L fermenter. The highest dry cell weight (DCW) and enzyme activity reached 68.7 g L⁻¹ and 1473.5 U L⁻¹, respectively. Furthermore, a novel co-feeding fermentation approach was developed to enable the one-step production of GOSs by directly supplementing GG into the fermentation medium. During the induction phase, a co-feeding strategy of methanol and 0.5% GG solution (2:3 ratio) was adopted, achieving a gellan lyase activity of 1728.1 U L⁻¹, which increased 17.3%, compared to the enzyme activity without supplementation of GG. Notably, the co-feeding method not only enhanced enzyme induction efficiency but also selectively generated GOSs with a degree of polymerization (DP) of three. This study provides a direct and simple way to produce oligosaccharides, as well as a novel fermentation method for enhancing gellan lyase production as well as GOSs production simultaneously.

Keywords

Gellan gum / Gellan oligosaccharides / Co-feeding

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Funding

the General Program of National Natural Science Foundation of China(32372468)

the National First-class Discipline Program of Light Industry Technology and Engineering (QGJC20230201)

The Basic Research Program of Jiangsu and supported by the Jiangsu Basic Research Center for Synthetic Biology(BK20233003)

The Inner Mongolia Science and Technology Projects(NMKJXM202207)

RIGHTS & PERMISSIONS

Jiangnan University