The elucidation of phosphosugar stress response in Bacillus subtilis guides strain engineering for high N-acetylglucosamine production.
Bacillus subtilis
N-acetylglucosamine
metabolic engineering
phosphosugar stress
Journal
Biotechnology and bioengineering
ISSN: 1097-0290
Titre abrégé: Biotechnol Bioeng
Pays: United States
ID NLM: 7502021
Informations de publication
Date de publication:
01 2021
01 2021
Historique:
received:
15
07
2020
revised:
16
09
2020
accepted:
20
09
2020
pubmed:
24
9
2020
medline:
26
11
2021
entrez:
23
9
2020
Statut:
ppublish
Résumé
Bacillus subtilis is a preferred microbial host for the industrial production of nutraceuticals and a promising candidate for the synthesis of functional sugars, such as N-acetylglucosamine (GlcNAc). Previously, a GlcNAc-overproducer B. subtilis SFMI was constructed using glmS ribozyme dual-regulatory tool. Herein, we further engineered to enhance carbon flux from glucose towards GlcNAc synthesis. As a result, the increased flux towards GlcNAc synthesis triggered phosphosugar stress response, which caused abnormal cell growth. Unfortunately, the mechanism of phosphosugar stress response had not been elucidated in B. subtilis. To reveal the stress mechanism and overcome its negative effect in bioproduction, we performed comparative transcriptome analysis. The results indicate that cells slow glucose utilization by repression of glucose import and accelerate catabolic reactions of phosphosugar. To verify these results, we overexpressed the phosphatase YwpJ, which relieved phosphosugar stress and allowed us to identify the enzyme responsible for GlcNAc synthesis from GlcNAc 6-phosphate. In addition, the deletion of nagBB and murQ, responsible for GlcNAc precursor degradation, further improved GlcNAc synthesis. The best engineered strain, B. subtilis FMIP34, increased GlcNAc titer from 11.5 to 26.1 g/L in shake flasks and produced 87.5 g/L GlcNAc in 30-L fed-batch bioreactor. Our results not only elucidate, for the first time, the phosphosugar stress response mechanism in B. subtilis, but also demonstrate how the combination of rational metabolic engineering with novel insights into physiology and metabolism allows the construction of highly efficient microbial cell factories for the production of high-value chemicals.
Substances chimiques
Bacterial Proteins
0
Acetylglucosamine
V956696549
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
383-396Subventions
Organisme : National Natural Science Foundation of China
ID : 31671845
Organisme : National Natural Science Foundation of China
ID : 21676119
Organisme : National Natural Science Foundation of China
ID : 31600068
Organisme : Natural Science Foundation of Jiangsu Province
ID : BK20160176
Organisme : 111 Project
ID : 111-2-06
Informations de copyright
© 2020 Wiley Periodicals LLC.
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