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...
Autores principales: | , , , , , |
---|---|
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 |