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A food-grade expression system for d-psicose 3-epimerase production in Bacillus subtilis using an alanine racemase-encoding selection marker

BACKGROUND: Food-grade expression systems require that the resultant strains should only contain materials from food-safe microorganisms, and no antibiotic resistance marker can be utilized. To develop a food-grade expression system for d-psicose 3-epimerase production, we use an alanine racemase-en...

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Autores principales: Chen, Jingqi, Jin, Zhaoxia, Gai, Yuanming, Sun, Jibin, Zhang, Dawei
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer Berlin Heidelberg 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5274643/
https://www.ncbi.nlm.nih.gov/pubmed/28191448
http://dx.doi.org/10.1186/s40643-017-0139-7
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author Chen, Jingqi
Jin, Zhaoxia
Gai, Yuanming
Sun, Jibin
Zhang, Dawei
author_facet Chen, Jingqi
Jin, Zhaoxia
Gai, Yuanming
Sun, Jibin
Zhang, Dawei
author_sort Chen, Jingqi
collection PubMed
description BACKGROUND: Food-grade expression systems require that the resultant strains should only contain materials from food-safe microorganisms, and no antibiotic resistance marker can be utilized. To develop a food-grade expression system for d-psicose 3-epimerase production, we use an alanine racemase-encoding gene as selection marker in Bacillus subtilis. RESULTS: In this study, the d-alanine racemase-encoding gene dal was deleted from the chromosome of B. subtilis 1A751 using Cre/lox system to generate the food-grade host. Subsequently, the plasmid-coded selection marker dal was complemented in the food-grade host, and RDPE was thus successfully expressed in dal deletion strain without addition of d-alanine. The selection appeared highly stringent, and the plasmid was stably maintained during culturing. The highest RDPE activity in medium reached 46 U/ml at 72 h which was comparable to RDPE production in kanamycin-based system. Finally, the capacity of the food-grade B. subtilis 1A751D2R was evaluated in a 7.5 l fermentor with a fed-batch fermentation. CONCLUSION: The alanine racemase-encoding gene can be used as a selection marker, and the food-grade expression system was suitable for heterologous proteins production in B. subtilis. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s40643-017-0139-7) contains supplementary material, which is available to authorized users.
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spelling pubmed-52746432017-02-10 A food-grade expression system for d-psicose 3-epimerase production in Bacillus subtilis using an alanine racemase-encoding selection marker Chen, Jingqi Jin, Zhaoxia Gai, Yuanming Sun, Jibin Zhang, Dawei Bioresour Bioprocess Research BACKGROUND: Food-grade expression systems require that the resultant strains should only contain materials from food-safe microorganisms, and no antibiotic resistance marker can be utilized. To develop a food-grade expression system for d-psicose 3-epimerase production, we use an alanine racemase-encoding gene as selection marker in Bacillus subtilis. RESULTS: In this study, the d-alanine racemase-encoding gene dal was deleted from the chromosome of B. subtilis 1A751 using Cre/lox system to generate the food-grade host. Subsequently, the plasmid-coded selection marker dal was complemented in the food-grade host, and RDPE was thus successfully expressed in dal deletion strain without addition of d-alanine. The selection appeared highly stringent, and the plasmid was stably maintained during culturing. The highest RDPE activity in medium reached 46 U/ml at 72 h which was comparable to RDPE production in kanamycin-based system. Finally, the capacity of the food-grade B. subtilis 1A751D2R was evaluated in a 7.5 l fermentor with a fed-batch fermentation. CONCLUSION: The alanine racemase-encoding gene can be used as a selection marker, and the food-grade expression system was suitable for heterologous proteins production in B. subtilis. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s40643-017-0139-7) contains supplementary material, which is available to authorized users. Springer Berlin Heidelberg 2017-01-28 2017 /pmc/articles/PMC5274643/ /pubmed/28191448 http://dx.doi.org/10.1186/s40643-017-0139-7 Text en © The Author(s) 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
spellingShingle Research
Chen, Jingqi
Jin, Zhaoxia
Gai, Yuanming
Sun, Jibin
Zhang, Dawei
A food-grade expression system for d-psicose 3-epimerase production in Bacillus subtilis using an alanine racemase-encoding selection marker
title A food-grade expression system for d-psicose 3-epimerase production in Bacillus subtilis using an alanine racemase-encoding selection marker
title_full A food-grade expression system for d-psicose 3-epimerase production in Bacillus subtilis using an alanine racemase-encoding selection marker
title_fullStr A food-grade expression system for d-psicose 3-epimerase production in Bacillus subtilis using an alanine racemase-encoding selection marker
title_full_unstemmed A food-grade expression system for d-psicose 3-epimerase production in Bacillus subtilis using an alanine racemase-encoding selection marker
title_short A food-grade expression system for d-psicose 3-epimerase production in Bacillus subtilis using an alanine racemase-encoding selection marker
title_sort food-grade expression system for d-psicose 3-epimerase production in bacillus subtilis using an alanine racemase-encoding selection marker
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5274643/
https://www.ncbi.nlm.nih.gov/pubmed/28191448
http://dx.doi.org/10.1186/s40643-017-0139-7
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