Back to Search Start Over

Modular pathway engineering of Bacillus subtilis for improved N-acetylglucosamine production.

Authors :
Liu Y
Zhu Y
Li J
Shin HD
Chen RR
Du G
Liu L
Chen J
Source :
Metabolic engineering [Metab Eng] 2014 May; Vol. 23, pp. 42-52. Date of Electronic Publication: 2014 Feb 19.
Publication Year :
2014

Abstract

In previous work, we constructed a recombinant Bacillus subtilis strain for microbial production of N-acetylglucosamine (GlcNAc), which has applications in nutraceuticals and pharmaceuticals. In this work, we improve GlcNAc production through modular engineering of B. subtilis. Specifically, the GlcNAc synthesis-related metabolic network in B. subtilis was divided into three modules-GlcNAc synthesis, glycolysis, and peptidoglycan synthesis. First, two-promoter systems with different promoter types and strengths were used for combinatorial assembly of expression cassettes of glmS (encoding GlcN-6-phosphate synthase) and GNA1 (encoding GlcNAc-6-phosphate N-acetyltransferase) at transcriptional levels in the GlcNAc synthesis module, resulting in a 32.4% increase in GlcNAc titer (from 1.85g/L to 2.45g/L) in shake flasks. In addition, lactate and acetate synthesis were blocked by knockout of ldh (encoding lactate dehydrogenase) and pta (encoding phosphotransacetylase), leading to a 44.9% increase in GlcNAc production (from 2.45g/L to 3.55g/L) in shake flasks. Then, various strengths of the glycolysis and peptidoglycan synthesis modules were constructed by repressing the expression of pfk (encoding 6-phosphofructokinase) and glmM (encoding phosphoglucosamine mutase) via the expression of various combinations of synthetic small regulatory RNAs and Hfq protein. Next, GlcNAc, glycolysis, and peptidoglycan synthesis modules with various strengths were assembled and optimized via a module engineering approach, and the GlcNAc titer was improved to 8.30g/L from 3.55g/L in shake flasks. Finally, the GlcNAc titer was further increased to 31.65g/L, which was 3.8-fold that in the shake flask, in a 3-L fed-batch bioreactor. This work significantly enhanced GlcNAc production through modular pathway engineering of B. subtilis, and the engineering strategies used herein may be useful for the construction of versatile B. subtilis cell factories for the production of other industrially important chemicals.<br /> (Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Details

Language :
English
ISSN :
1096-7184
Volume :
23
Database :
MEDLINE
Journal :
Metabolic engineering
Publication Type :
Academic Journal
Accession number :
24560814
Full Text :
https://doi.org/10.1016/j.ymben.2014.02.005