Engineering Pseudomonas putida KT2440 for the production of isobutanol.

Robert Nitschel,Ian Fotheringham,Christoph Massner,Andreas Ankenbauer,Stephen Mccolm,Frédéric Borges,Nicolas T Wirth,Ilona Welsch,Ralf Takors,Bastian Blombach,Naveed Ahmad

doi:10.1002/elsc.201900151

Abstract

We engineered P. putida for the production of isobutanol from glucose by preventing product and precursor degradation, inactivation of the soluble transhydrogenase SthA, overexpression of the native ilvC and ilvD genes, and implementation of the feedback‐resistant acetolactate synthase AlsS from Bacillus subtilis, ketoacid decarboxylase KivD from Lactococcus lactis, and aldehyde dehydrogenase YqhD from Escherichia coli. The resulting strain P. putida Iso2 produced isobutanol with a substrate specific product yield (Y Iso/S) of 22 ± 2 mg per gram of glucose under aerobic conditions. Furthermore, we identified the ketoacid decarboxylase from Carnobacterium maltaromaticum to be a suitable alternative for isobutanol production, since replacement of kivD from L. lactis in P. putida Iso2 by the variant from C. maltaromaticum yielded an identical YIso/S. Although P. putida is regarded as obligate aerobic, we show that under oxygen deprivation conditions this bacterium does not grow, remains metabolically active, and that engineered producer strains secreted isobutanol also under the non‐growing conditions.

Highlights

Biofuel production from renewable feed stocks is of special importance because of the finite nature of the currently used crude oil derivatives and growing concerns about climate change [1]
We identified the ketoacid decarboxylase from Carnobacterium maltaromaticum to be a suitable alternative for isobutanol production, since replacement of kivD from L. lactis in P. putida Iso2 by the variant from C. maltaromaticum yielded an identical YIso/S
We identified KivD from Carnobacterium maltaromaticum as a suitable alternative to KivD from L. lactis to drive the decarboxylation of 2-ketoisovalerate and we showed that isobutanol production can be achieved under oxygen deprivation conditions with this obligate aerobic bacterium

Summary

Introduction

Biofuel production from renewable feed stocks is of special importance because of the finite nature of the currently used crude oil derivatives and growing concerns about climate change [1]. Isobutanol is an attractive alternative to the employed fossil fuels. It has several advantages such as a higher energy density, compatibility with existing engines, lower vapor pressure and volatility, as well as a lower corrosivity compared to bio-ethanol [2,3]. Isobutanol can be synthesized via the branched-chain amino acid biosynthesis and the so-called Ehrlich pathway to convert pyruvate to isobutanol (Figure 1). The first step in this route is the conversion of two pyruvate molecules to Abbreviations: 2-KIV, 2-ketoisovalerate; AlsS, acetolactate synthase; BHI, brain–heart infusion; KDC, ketoacid decarboxylase; LB, Lysogeny broth

Methods

Results

Conclusion