A novel N-terminal region of the membrane β-hexosyltransferase: its role in secretion of soluble protein by Pichia pastoris

Abstract

The b-hexosyltransferase (BHT) from Sporobolomyces singularis is a membrane-bound enzyme that catalyses transgalactosylation reactions to synthesize galacto-oligosaccharides (GOSs). To increase the secretion of the active soluble version of this protein, we examined the uncharacterized novel N-terminal region (amino acids 1-110), which included two predicted endogenous structural domains. The first domain (amino acids 1-22) may act as a classical leader while a non-classical signal was located within the remaining region (amino acids 23-110). A functional analysis of these domains was performed by evaluating the amounts of the rBHT forms secreted by recombinant P. pastoris strains carrying combinations of the predicted structural domains and the a mating factor (MFa) from Saccharomyces cerevisiae as positive control. Upon replacement of the leader domain (amino acids 1-22) by MFa (MFa-rBht (23-594) ), protein secretion increased and activity of both soluble and membrane-bound enzymes was improved 53-and 14-fold, respectively. Leader interference was demonstrated when MFa preceded the putative classical rBHT leader (amino acids 1-22), explaining the limited secretion of soluble protein by P. pastoris (GS115 : : MFa-rBht (1-594) ). To validate the role of the N-terminal domains in promoting protein secretion, we tested the domains using a non-secreted protein, the anti-b-galactosidase single-chain variable antibody fragment scFv13R4.The recombinants carrying chimeras of the N-terminal 1-110 regions of rBHT preceding scFv13R4 correlated with the secretion strength of soluble protein observed with the rBHT recombinants. Finally, soluble bioactive HIS-tagged and non-tagged rBHT (purified to homogeneity) obtained from the most efficient recombinants (GS115 : : MFa-rBht (23-594) -HIS and GS115 : : MFa-rBht ) showed comparable activity rates of GOS generation.

Safety and Modulatory Effects of Humanized Galacto-Oligosaccharides on the Gut Microbiome

“…Membrane-bound β-hexosyl transferase from Hamamotoa (Sporobolomyces) singularis in Komagataella ( Pichia ) pastoris was produced as previously described ( 13 , 14 ). The standard transgalactosylation reaction utilizing Komagataella ( Pichia ) pastoris resting cells (harboring membrane-bound enzyme) was initiated by adding standardized amounts of enzyme (1 U g −1 lactose) in 5 mM sodium phosphate buffer (pH 5.0) to a similarly buffered solution containing lactose (200 g liter −1 ) and N-Acetylglucosamine (25 g liter −1 ) at 30°C.…”

“…Synbiotics (combinations of prebiotics and probiotics) are also emerging as a focal point of GI biology research, as each component, individually and synergistically, could provide unique benefits reestablishing community resilience and host physiology ( 11 , 12 ). In previous studies we evaluated highly pure β(1–4) galacto-oligosaccharides (GOS) formulations produced by the optimized version of the hexosyl-transferase gene from Hamamotoa (Sporobolomyces) singularis heterologously expressed in Komagataella ( Pichia ) pastoris ( 13 , 14 ). This enzyme is one of the most promising catalysts in the field of glycobiology due to its high stability, highly desirable enzymatic properties, and the metabolism of its reaction products (GOS) by specific members of the gut microbial community, impacting its composition and function ( 15 , 16 ).…”

“…In this study, we describe a novel biological synthesis solution to produce N-Acetyl-lactosamine (LacNAc)-enriched GOS (which we refer to as humanized GOS, hGOS) using optimized Hamamotoa (Sporobolomyces) singularis β-hexosyl transferase [rBHT ( 13 , 14 )]. The enzyme generates LacNAc-enriched GOS as the product of the reaction between lactose as a galactose donor and N-Acetylglucosamine as acceptor.…”

Crystal structure of a GH1 β‐glucosidase from Hamamotoa singularis

“…This binding appears to be relatively tight because the linker loop had a temperature factor that was only moderately higher than the average for all residues (averages of 55.1 Å 2 for the linker loop and 47.3 Å 2 for the whole chain) and is fully traceable in the electron density map (Figure 1e). Given that another group previously attempted but failed to express a variant of MFα-HsBglA (23-594) -His that lacked the Nterminal region (Val23-Lys110), 7 we presume that either or both the N-terminal short α-helix and linker loop are essential for proper folding and/or maintaining the native structure of HsBglA.…”

“…HsBglA is an extracellular membrane‐bound enzyme, and the N‐terminal leader segment was predicted to act as a membrane anchor (Figure 1b). 7 The C‐terminal catalytic domain of HsBglA has no detectable homology to β‐galactosidases in the glycosyl hydrolase families of GH2, GH35 and GH42, but it is similar to the catalytic domains of fungal and plant β‐glucosidases in the GH1 family (Figure S1). 3 Although to date more than 60 structures of GH1 β‐glycosidases have been determined according to the CAZy database (http://www.cazy.org/CAZY/), we can still find only structures with low‐to‐moderate homology to HsBglA.…”