Functional insights from recombinant production of bacterial proteases in Saccharomyces cerevisiae

Abstract

Background: Proteases are important enzymes in food and pharmaceutical industries, but challenges persist in their recombinant production due to host cell proteome hydrolysis and fitness loss. The development of recombinant expression systems for directed evolution of proteolytic enzymes, and industrial production are desirable. This study evaluated Saccharomyces cerevisiae as expression host for three bacterial proteases: BdpK (from Bdellovibrio bacteriovorus), IdeS, and SpeB (both from Streptococcus pyogenes), each with distinct peptide substrate scopes.

Results: We developed an experimental pipeline for analysis of protease gene expression levels and fitness effects on yeast cultures. Heterologous genes were fused with green fluorescent protein and their expression and effects on cell viability was monitored at the single-cell level by flow cytometry. IdeS-GFP fusion was produced efficiently with a gaussian distribution within the population and without compromising cell growth or viability. BdpK, on the other hand, displayed lower expression level and a more heterogenous distribution that was less stable over time. Production of SpeB was not feasible. Inserting the speB-GFP fusion gene resulted in complete growth inhibition and a significantly higher frequency of cells with compromised membrane integrity. Plasmid-based expression was compared with integrated-based expression, revealing higher total expression levels and lower degree of population heterogeneity for the latter.

Conclusions: S. cerevisiae was found to be an efficient expression host for the bacterial protease IdeS. In contrast, the expression of BdpK and SpeB faced significant challenges, including lack of activity for BdpK, or imposing a substantial fitness burden on the cells for SpeB, likely due to its broad substrate scope resulting in native protein degradation. The findings of this study provide valuable insights into the limitations and possibilities of yeast as an expression host for bacterial protease production and for studying their physiological effects using yeast as a model eukaryote.

Keywords: Saccharomyces cerevisiae host; Bacterial protease engineering; CRISPR/Cas9 integration; Protease expression; Recombinant enzyme production; Yeast expression system.

Fig. 1

Aerobic batch growth of engineered yeast a) plasmid-based, and b) integration-based strains in YNB medium before and after induction of the GAL1 promoter (GAL1p) controlling protease gene expression. The dashed vertical line indicates the time of induction where galactose was added to a final concentration of 20 g/L. Symbols represents the strains harbouring the genes for BdpK-GFP (□), IdeS-GFP (∆), SpeB-GFP (○), and GFP control (◊). Error bars correspond to standard deviation of biological triplicates.

Fig. 2

Flow cytometry analysis of yeast strains carrying genes for different proteases fused with GFP. Each dot represents a cell, and the distribution is plotted along with the histograms, showing the fluorescent intensity in the channels FL1-H (GFP) and FL3-H (PI). Q1: intact GFP + cells. Q2: permeable GFP + cells. Q3: permeable GFP- cells. Q4: intact GFP- cells. The green colour corresponds to the multi-copy plasmid-based strains and the blue colour corresponds to the single-copy integrated strains. Samples taken 6 h after induction are representative for three biological triplicates. a) BdpK-GFP, strain TMBTL017 (green) and TMBTL031 (blue). b) IdeS-GFP, strain TMBTL018 (green) and TMBTL032 (blue). c) SpeB-GFP, strain TMBTL019 (green) and TMBTL033 (blue). d) GFP control strains TMBTL016 (green) and TMBTL030 (blue).

Fig. 3

Mean Fluorescence Intensity of FL1-H (GFP) of total yeast populations after induction for a) plasmid-based strains, and b) integration-based strains. Percentage of GFP + population after induction for c) plasmid-based strains, and d) integration-based strains. Symbols represent the strains harbouring the genes for BdpK-GFP (□), IdeS-GFP (∆), SpeB-GFP (○), and GFP control (◊). Plus (+) represents the control strains, empty plasmid, or wild type. Error bars correspond to standard deviation of three biological replicates.