EVALUATION OF GENETIC STRATEGIES FOR IMPROVING LACTIC ACID PRODUCTION IN KOMAGATAELLA PHAFFIII YEAST STRAINS

 

 

1. Mitochondrial pyruvate carrier 2. Glycerol-3-Phosphate dehydrogenase 3. catalase 4. hemoglobin 5. glycerol

Lactic acid can be classified as a platform chemical due to its numerous industrial applications. In recent years, most of the lactic acid production goes to the synthesis of polylactide (PLA), a thermostable and compostable bioplastic that can replace the use of conventional plastics derived from the petrochemical industry. One route to obtain lactic acid is through microbial fermentation using renewable carbon sources, such as sugar cane. Production via microbial fermentation requires improvements in the bioprocess for PLA to become commercially competitive. However, one of the main challenges during lactic acid production by microbial fermentation is to alleviate competitive metabolic routes that lead to the formation of by-products. In previous works by our group, the yeast Komagataella phaffii was used to construct L-lactate-producing strains from glycerol, reaching a maximum yield of 0.68 (g/g) in fed-batch mode. From assessment of engineered K. phaffii strains, there is evidence that the previous modifications, such as the deletion of pyruvate decarboxylase 1, result in an NADH/NAD+ redox imbalance when glycerol is used as a fuel, which could explain the production of arabitol as a co-product and the difficulty to improve lactic acid yield. Therefore, different gene targets for heterologous expression and knockout were evaluated in this work with the aim of (i) correcting the redox balance in the GLp strain to avoid redirecting the flow to competing pathways; (ii) enabling the production of lactic acid in aerobic conditions; (iii) expressing different LDHs along with overexpressing the native lactate membrane transporter. The proposed modifications may help to better understand the physiology of K. phaffii and thus improve the yield of lactate production from glycerol.