Novel homologous lactate transporter improves l-lactic acid production from glycerol in recombinant strains of Pichia pastoris
| Autor: | Beatriz Simas Magalhães, Nadiele Tamires Moreira Melo, Gisele Menino, Virgilio H. de Castro, Nádia Skorupa Parachin, Lucas Silva Carvalho, Eduardo Mulinari, Gustavo H. Goldman, Thaila Fernanda dos Reis, Pollyne Borborema Almeida de Lima, Kelly C.L. Mulder |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
Glycerol
Monocarboxylic Acid Transporters 0106 biological sciences 0301 basic medicine Leveduras Bioengineering Biology 01 natural sciences Applied Microbiology and Biotechnology Pichia Pichia pastoris 03 medical and health sciences Acetic acid chemistry.chemical_compound Bioreactors Pichia (Komagataella) pastoris 010608 biotechnology Lactate dehydrogenase Ácido lático Animals Lactic Acid Acetic Acid FERMENTAÇÃO L-Lactate Dehydrogenase Research food and beverages Metabolism biology.organism_classification Yeast Lactic acid 030104 developmental biology Metabolic Engineering chemistry Biochemistry Biofuels l-Lactic acid Fermentation Lactate transporter Cattle Oxygen limited fermentation Biodiesel Lactic acid fermentation Biotechnology |
| Zdroj: | Repositório Institucional da UnB Universidade de Brasília (UnB) instacron:UNB Microbial Cell Factories Repositório Institucional da USP (Biblioteca Digital da Produção Intelectual) Universidade de São Paulo (USP) instacron:USP |
| ISSN: | 1475-2859 |
| DOI: | 10.1186/s12934-016-0557-9 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|