Protein structure-based enhancement of enzyme performance for food and bioproduct applications using X-ray crystallography, protein modification and metabolic engineering methods

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The overarching goal of my DG research program is to take a structure-function approach to enhance the performance of enzymes as processing aids for food and bioproduct processing. We have worked on proline-specific peptidases in this context. Proline has a side chain attached to its nitrogen atom that makes it structurally distinct from the other amino acids and resistant to proteolysis. When foods are fermented, an accumulation of proline-containing peptides (Pro-peptides) occurs to elicit undesirable bitter tastes. Bitterness of Pro-peptides can be reduced through liberation of proline. At least 9 proline-specific peptidases are known and studied, with the smallest units of Pro-peptides being hydrolyzed by two principle enzymes: Xaa-Pro dipeptidase (X-Pase) and Pro-Xaa dipeptidase (P-Xase). X-Pases are also known to degrade organophosphorus (OP; compounds containing C-P (or S-P) bonds; used as pesticides and nerve gas agents). For this DG research (2016-2021), these two dipeptidases were chosen as model systems for investigating their structure-function relationships for applications in food and bioproduct processing. In this proposed plan, I aim to achieve the following three objectives: 1) To characterize the structure of Lactobacillus plantarum P-Xase using X-ray crystallography that will be the first structure model of X-Pases, 2) to protein engineer P-Xase based on its crystal structure, and 3) to metabolic engineer Lactococcus lactis with mutant X-Pase and P-Xase. A Ph.D. student (HQP-1) will work on the X-ray crystallography of P-Xase in year 1 and 2. The revealed structural models will be examined to find the residues responsible for substrate specificity and catalytic activity of P-Xase. Roles of these residues will be examined and modified using site-directed mutagenesis. The mutated enzymes will be examined by X-ray crystallography to confirm the relationship between structure and function. In year 3, HQP-1 and the second Ph.D. student (HQP-2), who will be recruited at the beginning of year 3, will collaboratively start this investigation of protein engineering, and HQP-2 will continue it into year 4. Starting in year 3, parallel to the protein engineering work, HQP-2 will work on the metabolic manipulation of L. lactis through cloning of engineered X-Pase (from my previous study) and P-Xase (this proposed study). Engineered L. lactis strains will be examined for their ability to reduce bitterness and OP in year 4 and 5. The research program will train two Ph.D. students in biotechnology fields in a multidisciplinary manner and will contribute to growing biotechnology industries in Canada. Beyond 5-year plan, the structure-function relationship information of proline-specific dipeptidases can contribute to the development of bioproducts, such as bioactive peptide production.