Novel homologous lactate transporter improves l-lactic acid production from glycerol in recombinant strains of Pichia pastoris. Issue 1 (December 2016)

• Record Type: Journal Article
• Authors: Lima, Pollyne; Mulder, Kelly; Melo, Nadiele; Carvalho, Lucas; Menino, Gisele; Mulinari, Eduardo; Castro, Virgilio; Reis, Thaila; Goldman, Gustavo; Magalhães, Beatriz; Parachin, Nádia
• Is Part Of: Microbial cell factories. Volume 15:Issue 1(2016)
• Page Start: 1
• Page End: 9
• Publication Date: 2016-12
• Subjects: l-Lactic acid -- Pichia (Komagataella) pastoris -- Lactate transporter -- Oxygen limited fermentation -- Lactate dehydrogenase; Microbial biotechnology -- Periodicals; Recombinant proteins -- Synthesis -- Periodicals; 660.62
• DOI: 10.1186/s12934-016-0557-9
• Languages: English
• ISSNs: 1475-2859

Abstract

Abstract Background Crude glycerol is the main byproduct of the biodiesel industry. Although it can have different applications, its purification is costly. Therefore, in this study a biotechnological route has been proposed for further utilization of crude glycerol in the fermentative production of lactic acid. This acid is largely utilized in food, pharmaceutical, textile, and chemical industries, making it the hydroxycarboxylic acid with the highest market potential worldwide. Currently, industrial production of lactic acid is done mainly using sugar as the substrate. Thus here, for the first time, Pichia pastoris has been engineered for heterologousl -lactic acid production using glycerol as a single carbon source. For that, theBos taurus lactate dehydrogenase gene was introduced intoP. pastoris . Moreover, a heterologous and a novel homologous lactate transporter have been evaluated forl -lactic acid production. Results Batch fermentation of theP. pastoris X-33 strain producingLDHb allowed for lactic acid production in this yeast. AlthoughP. pastoris is known for its respiratory metabolism, batch fermentations were performed with different oxygenation levels, indicating that lower oxygen availability increased lactic acid production by 20 %, pushing the yeast towards a fermentative metabolism. Furthermore, a newly putative lactate transporter fromP. pastoris named PAS has been identified by search similarity with the lactate transporter fromSaccharomyces cerevisiae Jen1p. Both heterologous and homologous transporters, Jen1p and PAS, were evaluated in one strain already containing LDH activity. Fed-batch experiments ofP. pastoris strains carrying the lactate transporter were performed with the batch phase at aerobic conditions followed by an aerobic oxygen-limited phase where production of lactic acid was favored. The results showed that the strain containing PAS presented the highest lactic acid titer, reaching a yield of approximately 0.7 g/g. Conclusions We showed thatP. pastoris has a great potential as a fermentative organism for producingl -lactic acid using glycerol as the carbon source at limited oxygenation conditions (below 0.05 % DO in the bioreactor). The best strain had both the LDHb and the homologous lactate transporter encoding genes expressed, and reached a titer 1.5 times higher than the strain with theS. cerevisiae transporter. Finally, it was also shown that increased lactic acid production was concomitant to reduction of acetic acid formation by half.