Engineering Escherichia coli for autoinducible production of n-butanol

Qinglong Wang,Yongchang Xue,Li Liu,Jiping Shi,Junsong Sun,Yi Ding

doi:10.1016/j.ejbt.2015.01.003

Abstract

article Background: Escherichia coli does not produce n-butanol naturally, but can be butanologenic when related enzymes were expressed using inducible elements on plasmids. In this study we attempted to confer E. coli strain capability of automatic excretion of the chemical by employing a native anaerobic promoter. Also, a novel DNA kit was designed for PCR preparation of linear DNA fragments to perform strain modification. The kit is primarily composed of two mother vectors, co-transformation of linear DNAs into E. coli can simultaneously introduce two butanol synthetic operons into the chromosome and create two in-frame gene deletions at targeted native loci. Results: E. colistrain Bw2V carries plasmid pCNA-PHC and pENA-TA, both utilizes native anaerobic promoter Phya for the expression of butanol synthetic enzymes. When Bw2V was subjected in anaerobic fermentation using medium containing extra glucose, the accumulated n-butanol in the broth was up to 2.8 g/L in bioreactor; as the genetic element expressing the same pathway was introduced into the genome, the titer of butanol was 1.4 g/L. Conclusions: The expression system using Phya iseffective inapplications that involve expression plasmids as also applicable in ectopic expression as single copy on the chromosome. Results imply that Phya can be subjected for broader application in bioproduction of more feedstock chemicals.

Highlights

Biobutanol is regarded as one of potential clean biofuels, and has been the renewed focus of bioenergy studies in recent years due to incessantly fermented public concerns on energy crisis and the environmental problems incurred by excessive use of fossil oil
In the present work we investigated the possibility of employing Phya as an auto-inducible promoter for the production of n-butanol in engineered E. coli
As a growth phase inducible element, Phya has been previously applied in heterologous expression of Pyrococcus furiosus hydrogenase [12], but its capacity on transcriptional initiation has never been evaluated upon genomic integration

Summary

Background

Escherichia coli does not produce n-butanol naturally, but can be butanologenic when related enzymes were expressed using inducible elements on plasmids. The kit is primarily composed of two mother vectors, co-transformation of linear DNAs into E. coli can simultaneously introduce two butanol synthetic operons into the chromosome and create two in-frame gene deletions at targeted native loci. Results: E. coli strain Bw2V carries plasmid pCNA-PHC and pENA-TA, both utilizes native anaerobic promoter Phya for the expression of butanol synthetic enzymes. When Bw2V was subjected in anaerobic fermentation using medium containing extra glucose, the accumulated n-butanol in the broth was up to 2.8 g/L in bioreactor; as the genetic element expressing the same pathway was introduced into the genome, the titer of butanol was 1.4 g/L. Results imply that Phya can be subjected for broader application in bioproduction of more feedstock chemicals

Introduction

Materials and methods

Construction of plasmids and butanologenic strains

Quantification of n-butanol and other metabolites

Result

Discussion