title: In silico and in vivo stability analysis of a heterologous biosynthetic pathway for 1,4-butanediol production in metabolically engineered E. coli.
abstract: Recently, several approaches have been published in order to develop a functional biosynthesis route for the non-natural compound 1,4-butanediol (BDO) in E. coli using glucose as a sole carbon source or starting from xylose. Among these studies, there was reported as high as 18 g/L product concentration achieved by industrial strains, however BDO production varies greatly in case of the reviewed studies. Our motivation was to build a simple heterologous pathway for this compound in E. coli and to design an appropriate cellular chassis based on a systemic biology approach, using constraint-based flux balance analysis and bi-level optimization for gene knock-out prediction. Thus, the present study reports, at the "proof-of concept" level, our findings related to model-driven development of a metabolically engineered E. coli strain lacking key genes for ethanol, lactate and formate production (ΔpflB, ΔldhA and ΔadhE), with a three-step biosynthetic pathway. We found this strain to produce a limited quantity of 1,4-BDO (.89 mg/L BDO under microaerobic conditions and .82 mg/L under anaerobic conditions). Using glycerol as carbon source, an approach, which to our knowledge has not been tackled before, our results suggest that further metabolic optimization is needed (gene-introductions or knock-outs, promoter fine-tuning) to address the redox potential imbalance problem and to achieve development of an industrially sustainable strain. Our experimental data on culture conditions, growth dynamics and fermentation parameters can consist a base for ongoing research on gene expression profiles and genetic stability of such metabolically engineered E. coli strains.
authors: a Department of Bioengineering , Sapientia Hungarian University of Transylvania , Libertatii Square, no. 1, 530104 Miercurea Ciuc , Romania.|b Faculty of Applied Chemistry and Materials Science , Politehnica University of Bucharest , Bucharest , Romania.
authors: Ildikó
authors: I
authors: Miklóssy
authors: a Department of Bioengineering , Sapientia Hungarian University of Transylvania , Libertatii Square, no. 1, 530104 Miercurea Ciuc , Romania.
authors: Zsolt
authors: Z
authors: Bodor
authors: a Department of Bioengineering , Sapientia Hungarian University of Transylvania , Libertatii Square, no. 1, 530104 Miercurea Ciuc , Romania.|b Faculty of Applied Chemistry and Materials Science , Politehnica University of Bucharest , Bucharest , Romania.
authors: Réka
authors: R
authors: Sinkler
authors: a Department of Bioengineering , Sapientia Hungarian University of Transylvania , Libertatii Square, no. 1, 530104 Miercurea Ciuc , Romania.
authors: Kálmán Csongor
authors: KC
authors: Orbán
authors: a Department of Bioengineering , Sapientia Hungarian University of Transylvania , Libertatii Square, no. 1, 530104 Miercurea Ciuc , Romania.
authors: Szabolcs
authors: S
authors: Lányi
authors: a Department of Bioengineering , Sapientia Hungarian University of Transylvania , Libertatii Square, no. 1, 530104 Miercurea Ciuc , Romania.
authors: Beáta
authors: B
authors: Albert
mesh_terms: D001420:Bacteria, Aerobic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053898:Biosynthetic Pathways / Q000235:genetics; D002072:Butylene Glycols / Q000737:chemistry / Q000378:metabolism*; D003198:Computer Simulation; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics*; D005285:Fermentation; D055786:Gene Knockout Techniques; D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D005990:Glycerol / Q000737:chemistry; D060847:Metabolic Engineering; D014994:Xylose / Q000737:chemistry / Q000235:genetics
chemical_list: D002072:Butylene Glycols; C039681:1,4-butanediol; D014994:Xylose; D005947:Glucose; D005990:Glycerol
keywords: 1,4-butanediol; E. coli; FBA; fermentation; metabolic engineering