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Construction of Recombinant Klebsiella pneumoniae to Increase Ethanol Production on Residual Glycerol Fed-Batch Cultivations

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Abstract

K. pneumoniae BLh-1 strain was genetically modified aiming at obtaining high ethanol productivity in cultivations using residual glycerol from biodiesel synthesis as substrate. The recombinant strain K. pneumoniae Kp17 was obtained by inserting the multicopy plasmid pTOPOBL17 containing the AdhE gene, and its own promoter, from K. pneumoniae BLh-1. Influence of Fe2+ supplementation and initial glycerol concentration on culture conditions were analyzed, both in rotatory shaker and in batch bioreactors. In the bioreactor cultures, K. pneumoniae Kp17 strain produced 4.5 g L−1 of ethanol (productivity of 0.50 g L−1 h−1 and yields of 0.15 g g−1) after 24-h cultivation, corresponding to an increase of approximately 40% in ethanol concentration compared to wild strain, K. pneumoniae BLh-1. Best conditions were then applied in exponential fed-batch bioreactors, with final ethanol concentration of 17.30 g L−1 (productivity of 0.59 g L−1 h−1 and yields of 0.16 g g−1) after 30 h of feeding, representing 11.5% of increment in titer of ethanol compared to the wild strain. Mutant cells kept 92.5% of the plasmids under batch in 24 h, and 71.9% under fed-batch after 27 h of exponential feeding. The findings in this work show the possibility of using a simple approach to genetically modify K. pneumoniae to be employed this versatile bacterium for the bioconversion of residual glycerol into ethanol.

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Acknowledgments

This work was supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil) and from the Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS, RS). Scholarships were also provided by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Finance Code 001).

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  1. Biotechnology & Biochemical Engineering Laboratory (BiotecLab), Federal University of Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil

    Vanessa Zimmer da Silva, Laura Jensen Ourique, Cíntia de David & Marco Antonio Zachia Ayub

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da Silva, V.Z., Ourique, L.J., de David, C. et al. Construction of Recombinant Klebsiella pneumoniae to Increase Ethanol Production on Residual Glycerol Fed-Batch Cultivations. Appl Biochem Biotechnol 192, 1147–1162 (2020). https://doi.org/10.1007/s12010-020-03397-5

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