Identification of lactic acid bacteria proteases for plant-based food fermentation

The global demand for sustainable foods has expanded the interest in alternative protein sources. The shift to plant-based foods as the primary source of proteins offers environmental and health benefits. However, the current plant-derived substrate often suffers from suboptimal organoleptic qualities, including inconsistent texture, off-flavor, and limited digestibility. These shortcomings hinder consumer acceptance and underscore the need for innovative approaches to improve food quality, functionality, and safety of plant-based foods.

Nitrogen metabolism is central to microbial fermentation, providing amino acids and peptides that influence flavor, texture, and nutritional value of fermented foods. Lactic acid bacteria (LAB) have a long history of being integral to food production, particularly due to their robust proteolytic systems. Among these are cell envelope proteases (CEPs), membrane-anchored serine proteases, notable for their selectivity and specificity on protein hydrolysis, distinguishing them from broader-acting proteases like subtilisin. This is deemed to be of technological importance and well-explored and leveraged in dairy fermentation, yet their potential in plantbased fermentation remains underexplored. Various pioneering research have uncovered gaps in our understanding of the metabolism of LAB as well as the underlying mechanisms of selectivity and overall activity of CEPs in plant-based matrices. As we push towards sustainable food production, there is an urgent need to understand, adapt, and optimize starter cultures to cater production demands and consumer demands and a need to overcome hurdles attributed to the limited tool kit constrain our progress.

This thesis addresses these challenges by identifying LAB proteases and characterizing their activities on plant-derived proteins. Specifically, this work expands available toolkits from streamlined genetic engineering of CEPs to optimization of biochemical assays for mechanistic profiling of these enzymes in plant-derived substrates, thereby laying groundwork for precision fermentation attributed to LAB proteases in these next generation food matrices.

Chapter 2 (Manuscript 1) begins with the identification of proteolytic LAB isolates from diverse species and ecological niche. A survey of their CEP homologs was conducted, followed by comprehensive characterization of enzyme activity on both milk and soy protein substrates to pinpoint key features of plant protein-specific proteases. This work underscores the significance of expanding starter culture screening beyond traditional dairy starter strains by profiling CEP activities across these two distinct substrates.

To fully elucidate the functionality of these plant-adapted proteases, their activity must be isolated in an isogenic background. The existing toolkit for studying these large membrane-bound proteases has been limited to plasmid-based assays in Lactococcus hosts. The limitations of this technique challenge the efficiency of advancing discovery in this field of study, leading the subsequent chapter (Chapter 3: Manuscript 2) to explore heterologous expression of CEPs in an alternative dairy-associated host, Streptococcus thermophilus LMD-9. This work identifies and resolves the key bottlenecks of using this strain for CEP engineering and records the first successful heterologous expression of PrtP in S. thermophilus. It also highlights the critical role of the cognate peptidyl-prolyl cis/trans isomerase (prtM) in producing active PrtP enzyme.

Yet, the confounding role of the cognate maturase enzyme associated with CEPs requires further investigation. This leads to Chapter 4 (Manuscript 3), which focuses on designing a more suitable isogenic host for protease phenotyping using S. thermophilus LMG18311, an intrinsically protease-negative strain. This work centers on identifying an optimal engineering locus, selecting an isogenic promoter, and strategically organizing gene cassettes. These considerations aim to eliminate inherent issues such as transcriptional interference caused by varying promoter strengths and promoter collisions from opposing genes.

However, the efforts to fully evaluate the protease enzyme functionality were hindered by limitations in the available assay media. As a result, Chapter 5 (Manuscript 4) shifts focus to the development of a suitable soy-based medium for multipurpose biochemical assays. Specifically, this work aimed to establish a straightforward way of fractionating plant-proteins adaptable for various applications. The versatility of the designed medium was demonstrated through characterizing plant-adapted proteases expressed in Lactococcus cremoris and in optimizing droplet-based microfluidics strain-development assays.

In conclusion, this thesis provides critical insights into the role of LAB proteases, particularly in soy-based dairy alternatives. Findings from this work contribute to a more precise and informed approach of screening and selecting starter strains for plant-based foods. It also underscores the potential of CEPs in the targeted hydrolysis of plant proteins for product development, especially in improving texture, flavor, and nutritional value. Additionally, this work expands the toolkit available for studying membrane-bound proteases by overcoming key challenges in genetic engineering, emphasizing the essential role of the cognate maturase enzyme (PrtM), and establishing an alternative host system for heterologous expression. Moreover, a novel and accessible method was developed for accurate protease phenotyping in plant-based substrates, enabling more precise functional characterization of these enzymes.