Systematic assessment of Pichia pastoris system for optimized β -galactosidase production

Abstract

β- Galactosidase (commonly known as β-lactase; EC 3.2.1.23) is a multifunctional enzyme that can catalyze the hydrolysis of terminal non-reducing β-d-galactose residues in β-d-galactosides or transfer the galactosyl residue to saccharide acceptors to yield galacto-oligosaccharides (GOS). β-Galactosidase has a variety of applications in food and medical industries such as hydrolysis of lactose in milk, manufacture of galactooligosaccharides (GOS) and treatment of lactose malabsorption [1]. Although β-galactosidase is an ubiquitous enzyme existing in plants, animals and microorganisms, only a few β-galactosidases from Kluveromyces lactis, Aspergillus niger and Aspergillus oryzae are regarded as safe for food related industry applications. To achieve commercial scale production of β-galactosidase, heterologous expression systems were applied including Saccharomyces cerevisiae and Pichia pastoris [1]. P. pastoris is a methylotrophic yeast with great protein expression potential, and has been used as host for expression of many proteins both experimentally and industrially. P. pastoris has also been used for the extracellular expression of β-galactosidase from Paecilomyces aerugineus [2], Lactobacillus crispatus [3] and strains belonging to Aspergillus spp [4]. Despite its advantage in expression of proteins, P. pastoris system usually needs to be optimized to achieve maximum possible production level for a given protein. In achieving this, potential expression bottlenecks are analyzed and alleviated through perturbing and engineering of P. pastoris at different levels. And this process is often performed in a protein-specific manner, depending on the inherent nature and applications of target protein as well as its interaction with P. pastoris host. Take β-galactosidase for example, while some previous works have reported its successful expression in P. pastoris with reasonably high level, there are still several concerns needing to be addressed before further optimization, for instance: 1) Which kind of promoters are suitable to express β-galactosidase, the inducible or constitutive promoters? The strong AOX1 (alcohol oxidase I) promoter has been the most frequently used one. Nevertheless, the adoption of constitutive promoters has been appreciated in recent years because it does not need methanol to induce the expression, and therefore is safer (especially for food-grade β-galactosidase production) and eases the process control during the fermentation. 2) Unexpected N-glycosylation of foreign proteins are very commonly observed in P. pastoris system and its effects on the activity of expressed proteins remain unpredictable. In some cases, glycosylation is essential for maintaining the activities of expressed enzymes [5], [6], [7], while in other cases, glycosylation can negatively affect the enzyme activity [8], [9], [10]. Although β-galactosidase possesses multiple potential N-glycosylation sites, the effects of N-glycosylation on β-galactosidase activity were rarely investigated. 3) Of thousands of proteins that have been expressed using P. pastoris, the protein expression levels can range from tens of milligrams to tens of grams per liter. β-galactosidase can easily reach several grams per liter of production, which is obviously near the high end of this range. This raises the concern that the high enzyme yield may saturate the protein secretion ability of P. pastoris and thus limit the further improvement of its production level. In order to address the above concerns and systematically assess P. pastoris system for optimized β-galactosidase, we expressed β-galactosidase from K. lactis and A. oryzae in P. pastoris, evaluated different constitutive promoters in addition to AOX1 promoter and examined co-expression of different chaperones in hope of enhancing the secretion of β-galactosidase in P. pastoris. We equally assess the effect of glycosylation on β-galactosidase activity using OCH1 disrupted strain. Combining these strategies, the production level of β-galactosidase from A. Oryzae reached 1434.75 U/mL in 1 L fermentor, which therefore provided a basis for further optimization and industrial scale production of β-galactosidase in future works.