Cargando…

Screening for optimal protease producing Bacillus licheniformis strains with polymer-based controlled-release fed-batch microtiter plates

BACKGROUND: Substrate-limited fed-batch conditions have the favorable effect of preventing overflow metabolism, catabolite repression, oxygen limitation or inhibition caused by elevated substrate or osmotic concentrations. Due to these favorable effects, fed-batch mode is predominantly used in indus...

Descripción completa

Detalles Bibliográficos
Autores principales: Habicher, Tobias, Klein, Tobias, Becker, Jacqueline, Daub, Andreas, Büchs, Jochen
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7903736/
https://www.ncbi.nlm.nih.gov/pubmed/33622330
http://dx.doi.org/10.1186/s12934-021-01541-2
_version_ 1783654795164254208
author Habicher, Tobias
Klein, Tobias
Becker, Jacqueline
Daub, Andreas
Büchs, Jochen
author_facet Habicher, Tobias
Klein, Tobias
Becker, Jacqueline
Daub, Andreas
Büchs, Jochen
author_sort Habicher, Tobias
collection PubMed
description BACKGROUND: Substrate-limited fed-batch conditions have the favorable effect of preventing overflow metabolism, catabolite repression, oxygen limitation or inhibition caused by elevated substrate or osmotic concentrations. Due to these favorable effects, fed-batch mode is predominantly used in industrial production processes. In contrast, screening processes are usually performed in microtiter plates operated in batch mode. This leads to a different physiological state of the production organism in early screening and can misguide the selection of potential production strains. To close the gap between screening and production conditions, new techniques to enable fed-batch mode in microtiter plates have been described. One of these systems is the ready-to-use and disposable polymer-based controlled-release fed-batch microtiter plate (fed-batch MTP). In this work, the fed-batch MTP was applied to establish a glucose-limited fed-batch screening procedure for industrially relevant protease producing Bacillus licheniformis strains. RESULTS: To achieve equal initial growth conditions for different clones with the fed-batch MTP, a two-step batch preculture procedure was developed. Based on this preculture procedure, the standard deviation of the protease activity of glucose-limited fed-batch main culture cultivations in the fed-batch MTP was ± 10%. The determination of the number of replicates revealed that a minimum of 6 parallel cultivations were necessary to identify clones with a statistically significant increased or decreased protease activity. The developed glucose-limited fed-batch screening procedure was applied to 13 industrially-relevant clones from two B. licheniformis strain lineages. It was found that 12 out of 13 clones (92%) were classified similarly as in a lab-scale fed-batch fermenter process operated under glucose-limited conditions. When the microtiter plate screening process was performed in batch mode, only 5 out of 13 clones (38%) were classified similarly as in the lab-scale fed-batch fermenter process. CONCLUSION: The glucose-limited fed-batch screening process outperformed the usual batch screening process in terms of the predictability of the clone performance under glucose-limited fed-batch fermenter conditions. These results highlight that the implementation of glucose-limited fed-batch conditions already in microtiter plate scale is crucial to increase the precision of identifying improved protease producing B. licheniformis strains. Hence, the fed-batch MTP represents an efficient high-throughput screening tool that aims at closing the gap between screening and production conditions.
format Online
Article
Text
id pubmed-7903736
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-79037362021-03-01 Screening for optimal protease producing Bacillus licheniformis strains with polymer-based controlled-release fed-batch microtiter plates Habicher, Tobias Klein, Tobias Becker, Jacqueline Daub, Andreas Büchs, Jochen Microb Cell Fact Research BACKGROUND: Substrate-limited fed-batch conditions have the favorable effect of preventing overflow metabolism, catabolite repression, oxygen limitation or inhibition caused by elevated substrate or osmotic concentrations. Due to these favorable effects, fed-batch mode is predominantly used in industrial production processes. In contrast, screening processes are usually performed in microtiter plates operated in batch mode. This leads to a different physiological state of the production organism in early screening and can misguide the selection of potential production strains. To close the gap between screening and production conditions, new techniques to enable fed-batch mode in microtiter plates have been described. One of these systems is the ready-to-use and disposable polymer-based controlled-release fed-batch microtiter plate (fed-batch MTP). In this work, the fed-batch MTP was applied to establish a glucose-limited fed-batch screening procedure for industrially relevant protease producing Bacillus licheniformis strains. RESULTS: To achieve equal initial growth conditions for different clones with the fed-batch MTP, a two-step batch preculture procedure was developed. Based on this preculture procedure, the standard deviation of the protease activity of glucose-limited fed-batch main culture cultivations in the fed-batch MTP was ± 10%. The determination of the number of replicates revealed that a minimum of 6 parallel cultivations were necessary to identify clones with a statistically significant increased or decreased protease activity. The developed glucose-limited fed-batch screening procedure was applied to 13 industrially-relevant clones from two B. licheniformis strain lineages. It was found that 12 out of 13 clones (92%) were classified similarly as in a lab-scale fed-batch fermenter process operated under glucose-limited conditions. When the microtiter plate screening process was performed in batch mode, only 5 out of 13 clones (38%) were classified similarly as in the lab-scale fed-batch fermenter process. CONCLUSION: The glucose-limited fed-batch screening process outperformed the usual batch screening process in terms of the predictability of the clone performance under glucose-limited fed-batch fermenter conditions. These results highlight that the implementation of glucose-limited fed-batch conditions already in microtiter plate scale is crucial to increase the precision of identifying improved protease producing B. licheniformis strains. Hence, the fed-batch MTP represents an efficient high-throughput screening tool that aims at closing the gap between screening and production conditions. BioMed Central 2021-02-23 /pmc/articles/PMC7903736/ /pubmed/33622330 http://dx.doi.org/10.1186/s12934-021-01541-2 Text en © The Author(s) 2021 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Research
Habicher, Tobias
Klein, Tobias
Becker, Jacqueline
Daub, Andreas
Büchs, Jochen
Screening for optimal protease producing Bacillus licheniformis strains with polymer-based controlled-release fed-batch microtiter plates
title Screening for optimal protease producing Bacillus licheniformis strains with polymer-based controlled-release fed-batch microtiter plates
title_full Screening for optimal protease producing Bacillus licheniformis strains with polymer-based controlled-release fed-batch microtiter plates
title_fullStr Screening for optimal protease producing Bacillus licheniformis strains with polymer-based controlled-release fed-batch microtiter plates
title_full_unstemmed Screening for optimal protease producing Bacillus licheniformis strains with polymer-based controlled-release fed-batch microtiter plates
title_short Screening for optimal protease producing Bacillus licheniformis strains with polymer-based controlled-release fed-batch microtiter plates
title_sort screening for optimal protease producing bacillus licheniformis strains with polymer-based controlled-release fed-batch microtiter plates
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7903736/
https://www.ncbi.nlm.nih.gov/pubmed/33622330
http://dx.doi.org/10.1186/s12934-021-01541-2
work_keys_str_mv AT habichertobias screeningforoptimalproteaseproducingbacilluslicheniformisstrainswithpolymerbasedcontrolledreleasefedbatchmicrotiterplates
AT kleintobias screeningforoptimalproteaseproducingbacilluslicheniformisstrainswithpolymerbasedcontrolledreleasefedbatchmicrotiterplates
AT beckerjacqueline screeningforoptimalproteaseproducingbacilluslicheniformisstrainswithpolymerbasedcontrolledreleasefedbatchmicrotiterplates
AT daubandreas screeningforoptimalproteaseproducingbacilluslicheniformisstrainswithpolymerbasedcontrolledreleasefedbatchmicrotiterplates
AT buchsjochen screeningforoptimalproteaseproducingbacilluslicheniformisstrainswithpolymerbasedcontrolledreleasefedbatchmicrotiterplates