Spatial modulation of key pathway enzymes by DNA-guided scaffold system and respiration chain engineering for improved N-acetylglucosamine production by bacillus subtilis.
Previously we constructed a Bacillus subtilis strain for efficient production of N-acetylglucosamine (GlcNAc) by engineering of GlcNAc synthetic and catabolic pathways. However, the further improvement of GlcNAc titer is limited by the intrinsic inefficiency of GlcNAc synthetic pathway and undesirable cellular properties including sporulation and high maintenance metabolism. In this work, we further improved GlcNAc titer through spatial modulation of key pathway enzymes and by blocking sporulation and decreasing maintenance metabolism. Specifically, a DNA-guided scaffold system was firstly used to modulate the activities of glucosamine-6-phosphate synthase and GlcNAc-6-phosphate N-acetyltransferase, increasing the GlcNAc titer from 1.83g/L to 4.55g/L in a shake flask. Next, sporulation was blocked by respectively deleting spo0A (gene encoding the initiation regulon of sporulation) and sigE (gene encoding RNA polymerase sporulation-specific sigma factor). Deletion of sigE more effectively blocked sporulation without altering cell growth or GlcNAc production. The respiration chain was then engineered to decrease the maintenance metabolism of recombinant B. subtilis by deleting cydB and cydC, genes encoding cytochrome bd ubiquinol oxidase (subunit II) and ATP-binding protein for the expression of cytochrome bd, respectively. The respiration-engineered B. subtilis produced 6.15g/L GlcNAc in a shake flask and 20.58g/L GlcNAc in a 3-L fed-batch bioreactor. To the best of our knowledge, this report is the first to describe the modulation of pathway enzymes via a DNA-guided scaffold system in B. subtilis. The combination of spatial modulation of key pathway enzymes and optimization of cellular properties may be used to develop B. subtilis as a well-organized cell factory for the production of the other industrially useful chemicals. Copyright \copyright 2014. Published by Elsevier Inc.
%0 Journal Article
%1 Liu2014Spatial
%A Liu, Yanfeng
%A Zhu, Yanqiu
%A Ma, Wenlong
%A Shin, Hyun-Dong D.
%A Li, Jianghua
%A Liu, Long
%A Du, Guocheng
%A Chen, Jian
%D 2014
%J Metabolic engineering
%K scaffolds synthetic-biology
%T Spatial modulation of key pathway enzymes by DNA-guided scaffold system and respiration chain engineering for improved N-acetylglucosamine production by bacillus subtilis.
%U http://view.ncbi.nlm.nih.gov/pubmed/24815549
%X Previously we constructed a Bacillus subtilis strain for efficient production of N-acetylglucosamine (GlcNAc) by engineering of GlcNAc synthetic and catabolic pathways. However, the further improvement of GlcNAc titer is limited by the intrinsic inefficiency of GlcNAc synthetic pathway and undesirable cellular properties including sporulation and high maintenance metabolism. In this work, we further improved GlcNAc titer through spatial modulation of key pathway enzymes and by blocking sporulation and decreasing maintenance metabolism. Specifically, a DNA-guided scaffold system was firstly used to modulate the activities of glucosamine-6-phosphate synthase and GlcNAc-6-phosphate N-acetyltransferase, increasing the GlcNAc titer from 1.83g/L to 4.55g/L in a shake flask. Next, sporulation was blocked by respectively deleting spo0A (gene encoding the initiation regulon of sporulation) and sigE (gene encoding RNA polymerase sporulation-specific sigma factor). Deletion of sigE more effectively blocked sporulation without altering cell growth or GlcNAc production. The respiration chain was then engineered to decrease the maintenance metabolism of recombinant B. subtilis by deleting cydB and cydC, genes encoding cytochrome bd ubiquinol oxidase (subunit II) and ATP-binding protein for the expression of cytochrome bd, respectively. The respiration-engineered B. subtilis produced 6.15g/L GlcNAc in a shake flask and 20.58g/L GlcNAc in a 3-L fed-batch bioreactor. To the best of our knowledge, this report is the first to describe the modulation of pathway enzymes via a DNA-guided scaffold system in B. subtilis. The combination of spatial modulation of key pathway enzymes and optimization of cellular properties may be used to develop B. subtilis as a well-organized cell factory for the production of the other industrially useful chemicals. Copyright \copyright 2014. Published by Elsevier Inc.
@article{Liu2014Spatial,
abstract = {Previously we constructed a Bacillus subtilis strain for efficient production of N-acetylglucosamine ({GlcNAc}) by engineering of {GlcNAc} synthetic and catabolic pathways. However, the further improvement of {GlcNAc} titer is limited by the intrinsic inefficiency of {GlcNAc} synthetic pathway and undesirable cellular properties including sporulation and high maintenance metabolism. In this work, we further improved {GlcNAc} titer through spatial modulation of key pathway enzymes and by blocking sporulation and decreasing maintenance metabolism. Specifically, a {DNA}-guided scaffold system was firstly used to modulate the activities of glucosamine-6-phosphate synthase and {GlcNAc}-6-phosphate N-acetyltransferase, increasing the {GlcNAc} titer from {1.83g/L} to {4.55g/L} in a shake flask. Next, sporulation was blocked by respectively deleting {spo0A} (gene encoding the initiation regulon of sporulation) and {sigE} (gene encoding {RNA} polymerase sporulation-specific sigma factor). Deletion of {sigE} more effectively blocked sporulation without altering cell growth or {GlcNAc} production. The respiration chain was then engineered to decrease the maintenance metabolism of recombinant B. subtilis by deleting {cydB} and {cydC}, genes encoding cytochrome bd ubiquinol oxidase (subunit {II}) and {ATP}-binding protein for the expression of cytochrome bd, respectively. The respiration-engineered B. subtilis produced {6.15g/L} {GlcNAc} in a shake flask and {20.58g/L} {GlcNAc} in a {3-L} fed-batch bioreactor. To the best of our knowledge, this report is the first to describe the modulation of pathway enzymes via a {DNA}-guided scaffold system in B. subtilis. The combination of spatial modulation of key pathway enzymes and optimization of cellular properties may be used to develop B. subtilis as a well-organized cell factory for the production of the other industrially useful chemicals. Copyright {\copyright} 2014. Published by Elsevier Inc.},
added-at = {2018-12-02T16:09:07.000+0100},
author = {Liu, Yanfeng and Zhu, Yanqiu and Ma, Wenlong and Shin, Hyun-Dong D. and Li, Jianghua and Liu, Long and Du, Guocheng and Chen, Jian},
biburl = {https://www.bibsonomy.org/bibtex/27d2b5324fbab0ab9e26779b2776ad49a/karthikraman},
citeulike-article-id = {13171168},
citeulike-linkout-0 = {http://view.ncbi.nlm.nih.gov/pubmed/24815549},
citeulike-linkout-1 = {http://www.hubmed.org/display.cgi?uids=24815549},
day = 6,
interhash = {cd9e1c8b8f93b5b918e03b538839f48e},
intrahash = {7d2b5324fbab0ab9e26779b2776ad49a},
issn = {1096-7184},
journal = {Metabolic engineering},
keywords = {scaffolds synthetic-biology},
month = may,
pmid = {24815549},
posted-at = {2014-05-15 06:47:11},
priority = {2},
timestamp = {2018-12-02T16:09:07.000+0100},
title = {Spatial modulation of key pathway enzymes by {DNA}-guided scaffold system and respiration chain engineering for improved N-acetylglucosamine production by bacillus subtilis.},
url = {http://view.ncbi.nlm.nih.gov/pubmed/24815549},
year = 2014
}