Título :	Novel homologous lactate transporter improves l‑lactic acid production from glycerol in recombinant strains of Pichia pastoris
Autor :	Lima, Pollyne Borborema Almeida de Mulder, Kelly Cristina Leite Melo, Nadiele Tamires Moreira Carvalho, Lucas Silva Menino, Gisele Soares Mulinari, Eduardo Castro, Virgilio H. de Reis, Thaila Fernanda dos Goldman, Gustavo Henrique Magalhães, Beatriz Simas Parachi, Nádia Skorupa
Assunto::	Biodiesel Ácido lático Leveduras
Fecha de publicación :	15-sep-2016
Editorial :	BioMed Central
Citación :	LIMA, Pollyne Borborema Almeida de et al. Novel homologous lactate transporter improves l‑lactic acid production from glycerol in recombinant strains of Pichia pastoris. Microb Cell Fact, v. 15, Article 158, 15 set. 2016. Disponível em:<https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-016-0557-9>. Acesso em: 23 jun. 2017. doi: https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-016-0557-9.
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 heterologous l-lactic acid production using glycerol as a single carbon source. For that, the Bos taurus lactate dehydrogenase gene was introduced into P. pastoris. Moreover, a heterologous and a novel homologous lactate transporter have been evaluated for l-lactic acid production. Results: Batch fermentation of the P. pastoris X-33 strain producing LDHb allowed for lactic acid production in this yeast. Although P. 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 from P. pastoris named PAS has been identified by search similarity with the lactate transporter from Saccharomyces cerevisiae Jen1p. Both heterologous and homologous transporters, Jen1p and PAS, were evaluated in one strain already containing LDH activity. Fed-batch experiments of P. 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 that P. pastoris has a great potential as a fermentative organism for producing l-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 the S. cerevisiae transporter. Finally, it was also shown that increased lactic acid production was concomitant to reduction of acetic acid formation by half.
Licença::	© 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
DOI:	https://dx.doi.org/10.1186/s12934-016-0557-9
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