Cargando…

Enhancing the Heterologous Fructosyltransferase Activity of Kluyveromyces lactis: Developing a Scaled-Up Process and Abolishing Invertase by CRISPR/Cas9 Genome Editing

The enzymatic production of prebiotic fructo-oligosaccharides (FOS) from sucrose involves fructosyltransferases (FFTs) and invertases, both of which catalyze forward (transferase) and reverse (hydrolysis) reactions. FOS yields can therefore be increased by favoring the forward reaction. We investiga...

Descripción completa

Detalles Bibliográficos
Autores principales: Burghardt, Jan Philipp, Fan, Rong, Baas, Markus, Eckhardt, Dustin, Gerlach, Doreen, Czermak, Peter
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7724039/
https://www.ncbi.nlm.nih.gov/pubmed/33324627
http://dx.doi.org/10.3389/fbioe.2020.607507
_version_ 1783620469400797184
author Burghardt, Jan Philipp
Fan, Rong
Baas, Markus
Eckhardt, Dustin
Gerlach, Doreen
Czermak, Peter
author_facet Burghardt, Jan Philipp
Fan, Rong
Baas, Markus
Eckhardt, Dustin
Gerlach, Doreen
Czermak, Peter
author_sort Burghardt, Jan Philipp
collection PubMed
description The enzymatic production of prebiotic fructo-oligosaccharides (FOS) from sucrose involves fructosyltransferases (FFTs) and invertases, both of which catalyze forward (transferase) and reverse (hydrolysis) reactions. FOS yields can therefore be increased by favoring the forward reaction. We investigated process conditions that favored transferase activity in the yeast strain Kluyveromyces lactis GG799, which expresses a native invertase and a heterologous FFT from Aspergillus terreus. To maximize transferase activity while minimizing native invertase activity in a scaled-up process, we evaluated two reactor systems in terms of oxygen input capacity in relation to the cell dry weight. In the 0.5-L reactor, we found that galactose was superior to lactose for the induction of the LAC4 promoter, and we optimized the induction time and induction to carbon source ratio using a response surface model. Based on the critical parameter of oxygen supply, we scaled up the process to 7 L using geometric similarity and a higher oxygen transport rate, which boosted the transferase activity by 159%. To favor the forward reaction even more, we deleted the native invertase gene by CRISPR/Cas9 genome editing and compared the ΔInv mutant to the original production strain in batch and fed-batch reactions. In fed-batch mode, we found that the ΔInv mutant increased the transferase activity by a further 66.9%. The enhanced mutant strain therefore provides the basis for a highly efficient and scalable fed-batch process for the production of FOS.
format Online
Article
Text
id pubmed-7724039
institution National Center for Biotechnology Information
language English
publishDate 2020
publisher Frontiers Media S.A.
record_format MEDLINE/PubMed
spelling pubmed-77240392020-12-14 Enhancing the Heterologous Fructosyltransferase Activity of Kluyveromyces lactis: Developing a Scaled-Up Process and Abolishing Invertase by CRISPR/Cas9 Genome Editing Burghardt, Jan Philipp Fan, Rong Baas, Markus Eckhardt, Dustin Gerlach, Doreen Czermak, Peter Front Bioeng Biotechnol Bioengineering and Biotechnology The enzymatic production of prebiotic fructo-oligosaccharides (FOS) from sucrose involves fructosyltransferases (FFTs) and invertases, both of which catalyze forward (transferase) and reverse (hydrolysis) reactions. FOS yields can therefore be increased by favoring the forward reaction. We investigated process conditions that favored transferase activity in the yeast strain Kluyveromyces lactis GG799, which expresses a native invertase and a heterologous FFT from Aspergillus terreus. To maximize transferase activity while minimizing native invertase activity in a scaled-up process, we evaluated two reactor systems in terms of oxygen input capacity in relation to the cell dry weight. In the 0.5-L reactor, we found that galactose was superior to lactose for the induction of the LAC4 promoter, and we optimized the induction time and induction to carbon source ratio using a response surface model. Based on the critical parameter of oxygen supply, we scaled up the process to 7 L using geometric similarity and a higher oxygen transport rate, which boosted the transferase activity by 159%. To favor the forward reaction even more, we deleted the native invertase gene by CRISPR/Cas9 genome editing and compared the ΔInv mutant to the original production strain in batch and fed-batch reactions. In fed-batch mode, we found that the ΔInv mutant increased the transferase activity by a further 66.9%. The enhanced mutant strain therefore provides the basis for a highly efficient and scalable fed-batch process for the production of FOS. Frontiers Media S.A. 2020-11-25 /pmc/articles/PMC7724039/ /pubmed/33324627 http://dx.doi.org/10.3389/fbioe.2020.607507 Text en Copyright © 2020 Burghardt, Fan, Baas, Eckhardt, Gerlach and Czermak. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Bioengineering and Biotechnology
Burghardt, Jan Philipp
Fan, Rong
Baas, Markus
Eckhardt, Dustin
Gerlach, Doreen
Czermak, Peter
Enhancing the Heterologous Fructosyltransferase Activity of Kluyveromyces lactis: Developing a Scaled-Up Process and Abolishing Invertase by CRISPR/Cas9 Genome Editing
title Enhancing the Heterologous Fructosyltransferase Activity of Kluyveromyces lactis: Developing a Scaled-Up Process and Abolishing Invertase by CRISPR/Cas9 Genome Editing
title_full Enhancing the Heterologous Fructosyltransferase Activity of Kluyveromyces lactis: Developing a Scaled-Up Process and Abolishing Invertase by CRISPR/Cas9 Genome Editing
title_fullStr Enhancing the Heterologous Fructosyltransferase Activity of Kluyveromyces lactis: Developing a Scaled-Up Process and Abolishing Invertase by CRISPR/Cas9 Genome Editing
title_full_unstemmed Enhancing the Heterologous Fructosyltransferase Activity of Kluyveromyces lactis: Developing a Scaled-Up Process and Abolishing Invertase by CRISPR/Cas9 Genome Editing
title_short Enhancing the Heterologous Fructosyltransferase Activity of Kluyveromyces lactis: Developing a Scaled-Up Process and Abolishing Invertase by CRISPR/Cas9 Genome Editing
title_sort enhancing the heterologous fructosyltransferase activity of kluyveromyces lactis: developing a scaled-up process and abolishing invertase by crispr/cas9 genome editing
topic Bioengineering and Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7724039/
https://www.ncbi.nlm.nih.gov/pubmed/33324627
http://dx.doi.org/10.3389/fbioe.2020.607507
work_keys_str_mv AT burghardtjanphilipp enhancingtheheterologousfructosyltransferaseactivityofkluyveromyceslactisdevelopingascaledupprocessandabolishinginvertasebycrisprcas9genomeediting
AT fanrong enhancingtheheterologousfructosyltransferaseactivityofkluyveromyceslactisdevelopingascaledupprocessandabolishinginvertasebycrisprcas9genomeediting
AT baasmarkus enhancingtheheterologousfructosyltransferaseactivityofkluyveromyceslactisdevelopingascaledupprocessandabolishinginvertasebycrisprcas9genomeediting
AT eckhardtdustin enhancingtheheterologousfructosyltransferaseactivityofkluyveromyceslactisdevelopingascaledupprocessandabolishinginvertasebycrisprcas9genomeediting
AT gerlachdoreen enhancingtheheterologousfructosyltransferaseactivityofkluyveromyceslactisdevelopingascaledupprocessandabolishinginvertasebycrisprcas9genomeediting
AT czermakpeter enhancingtheheterologousfructosyltransferaseactivityofkluyveromyceslactisdevelopingascaledupprocessandabolishinginvertasebycrisprcas9genomeediting