MilliporeSigma
  • Home
  • Search Results
  • A synthetic O2 -tolerant butanol pathway exploiting native fatty acid biosynthesis in Escherichia coli.

A synthetic O2 -tolerant butanol pathway exploiting native fatty acid biosynthesis in Escherichia coli.

Biotechnology and bioengineering (2014-07-02)
András Pásztor, Pauli Kallio, Dávid Malatinszky, M Kalim Akhtar, Patrik R Jones
ABSTRACT

Several synthetic metabolic pathways for butanol synthesis have been reported in Escherichia coli by modification of the native CoA-dependent pathway from selected Clostridium species. These pathways are all dependent on the O2 -sensitive AdhE2 enzyme from Clostridium acetobutylicum that catalyzes the sequential reduction of both butyryl-CoA and butyraldehyde. We constructed an O2 -tolerant butanol pathway based on the activities of an ACP-thioesterase, acting on butyryl-ACP in the native fatty acid biosynthesis pathway, and a promiscuous carboxylic acid reductase. The pathway was genetically optimized by screening a series of bacterial acyl-ACP thioesterases and also by modification of the physical growth parameters. In order to evaluate the potential of the pathway for butanol production, the ACP-dependent butanol pathway was compared with a previously established CoA-dependent pathway. The effect of (1) O2 -availability, (2) media, and (3) co-expression of aldehyde reductases was evaluated systematically demonstrating varying and contrasting functionality between the ACP- and CoA-dependent pathways. The yield of butanol from the ACP-dependent pathway was stimulated by enhanced O2 -availability, in contrast to the CoA-dependent pathway, which did not function well under aerobic conditions. Similarly, whilst the CoA-dependent pathway only performed well in complex media, the ACP-dependent pathway was not influenced by the choice of media except in the absence of O2 . A combination of a thioesterase from Bacteroides fragilis and the aldehyde reductase, ahr, from E. coli resulted in the greatest yield of butanol. A product titer of ~300 mg/L was obtained in 24 h under optimal batch growth conditions, in most cases exceeding the performance of the reference CoA-pathway when evaluated under equivalent conditions.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Glycerol solution, puriss. p.a., 86-89% (T)
Sigma-Aldrich
Ammonium chloride, ReagentPlus®, ≥99.5%
Sigma-Aldrich
1-Butanol, ACS reagent, ≥99.4%
Sigma-Aldrich
1-Butanol, 99.9%
Sigma-Aldrich
Glycerol solution, puriss., meets analytical specification of Ph. Eur., BP, 84-88%
Sigma-Aldrich
Ammonium chloride, puriss., meets analytical specification of Ph. Eur., BP, USP, FCC, 99.5-100.5% (calc. to the dried substance)
Sigma-Aldrich
1-Butanol, BioRenewable, ACS reagent, ≥99.4%
Sigma-Aldrich
1-Butanol, suitable for HPLC, ≥99.7%
Sigma-Aldrich
1-Butanol, puriss. p.a., ACS reagent, reag. ISO, reag. Ph. Eur., ≥99.5% (GC)
Sigma-Aldrich
Magnesium sulfate solution, for molecular biology, 1.00 M±0.04 M
Sigma-Aldrich
Ammonium chloride, for molecular biology, suitable for cell culture, ≥99.5%
Supelco
1-Butanol, analytical standard
Sigma-Aldrich
1-Butanol, anhydrous, 99.8%
Sigma-Aldrich
Glycerin, meets USP testing specifications
Sigma-Aldrich
1-Butanol, for molecular biology, ≥99%
Sigma-Aldrich
Ammonium chloride, 99.99% trace metals basis
Supelco
1-Butanol, suitable for HPLC, 99.8%
Sigma-Aldrich
Magnesium sulfate solution, BioUltra, for molecular biology
Sigma-Aldrich
Ammonium chloride, 99.998% trace metals basis
Supelco
Calcium standard for AAS, analytical standard, 1.000 g/L Ca+2 in hydrochloric acid, traceable to BAM
Sigma-Aldrich
Ammonium chloride, BioUltra, for molecular biology, ≥99.5% (AT)
Sigma-Aldrich
Ammonium chloride, tested according to Ph. Eur.
Sigma-Aldrich
Glycerol solution, 83.5-89.5% (T)
Supelco
Glycerin, Pharmaceutical Secondary Standard; Certified Reference Material
Supelco
Ammonium chloride, Pharmaceutical Secondary Standard; Certified Reference Material
USP
Glycerin, United States Pharmacopeia (USP) Reference Standard
Supelco
1-Butanol, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
Magnesium sulfate, puriss. p.a., drying agent, anhydrous, ≥98.0% (KT), powder (very fine)
Sigma-Aldrich
Magnesium sulfate, anhydrous, free-flowing, Redi-Dri, ReagentPlus®, ≥99.5%
Sigma-Aldrich
Magnesium sulfate, anhydrous, free-flowing, Redi-Dri, reagent grade, ≥97%