title: Novel homologous lactate transporter improves L-lactic acid production from glycerol in recombinant strains of Pichia pastoris.
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.
authors.affiliation: Grupo de Engenharia Metabólica Aplicada a Bioprocessos, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, CEP 70.790-900, Brazil.
authors.forename: Pollyne Borborema Almeida
authors.initials: PB
authors.lastname: de Lima
authors.affiliation: Integra Bioprocessos e Análises, Campus Universitário Darcy Ribeiro, Edifício CDT, Sala AT-36/37, Brasília, DF, CEP 70.904-970, Brazil.
authors.forename: Kelly Cristina Leite
authors.initials: KC
authors.lastname: Mulder
authors.affiliation: Pós-Graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, Brasília, DF, CEP 70.790-160, Brazil.
authors.forename: Nadiele Tamires Moreira
authors.initials: NT
authors.lastname: Melo
authors.affiliation: Pós-Graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, Brasília, DF, CEP 70.790-160, Brazil.
authors.forename: Lucas Silva
authors.initials: LS
authors.lastname: Carvalho
authors.affiliation: Integra Bioprocessos e Análises, Campus Universitário Darcy Ribeiro, Edifício CDT, Sala AT-36/37, Brasília, DF, CEP 70.904-970, Brazil.
authors.forename: Gisele Soares
authors.initials: GS
authors.lastname: Menino
authors.affiliation: Integra Bioprocessos e Análises, Campus Universitário Darcy Ribeiro, Edifício CDT, Sala AT-36/37, Brasília, DF, CEP 70.904-970, Brazil.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Mulinari
authors.affiliation: Integra Bioprocessos e Análises, Campus Universitário Darcy Ribeiro, Edifício CDT, Sala AT-36/37, Brasília, DF, CEP 70.904-970, Brazil.
authors.forename: Virgilio H
authors.initials: VH
authors.lastname: de Castro
authors.affiliation: Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, São Paulo, CEP 14.040-903, Brazil.
authors.forename: Thaila F
authors.initials: TF
authors.lastname: Dos Reis
authors.affiliation: Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, São Paulo, CEP 14.040-903, Brazil.
authors.forename: Gustavo Henrique
authors.initials: GH
authors.lastname: Goldman
authors.affiliation: Pós-Graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, Brasília, DF, CEP 70.790-160, Brazil.|Integra Bioprocessos e Análises, Campus Universitário Darcy Ribeiro, Edifício CDT, Sala AT-36/37, Brasília, DF, CEP 70.904-970, Brazil.
authors.forename: Beatriz Simas
authors.initials: BS
authors.lastname: Magalhães
authors.affiliation: Grupo de Engenharia Metabólica Aplicada a Bioprocessos, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, CEP 70.790-900, Brazil. nadiasp@gmail.com.
authors.forename: Nádia Skorupa
authors.identifier: http://orcid.org/0000-0001-7405-9855
authors.initials: NS
authors.lastname: Parachin
mesh_terms: D019342:Acetic Acid / Q000378:metabolism; D000818:Animals; D056804:Biofuels; D019149:Bioreactors; D002417:Cattle; D005285:Fermentation; D005990:Glycerol / Q000378:metabolism*; D007770:L-Lactate Dehydrogenase / Q000235:genetics; D019344:Lactic Acid / Q000378:metabolism*; D060847:Metabolic Engineering; D027501:Monocarboxylic Acid Transporters / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010843:Pichia / Q000235:genetics* / Q000378:metabolism
chemical_list: D056804:Biofuels; D027501:Monocarboxylic Acid Transporters; D019344:Lactic Acid; D007770:L-Lactate Dehydrogenase; D005990:Glycerol; D019342:Acetic Acid
keywords: L-Lactic acid; Lactate dehydrogenase; Lactate transporter; Oxygen limited fermentation; Pichia (Komagataella) pastoris