Engineering Pseudomonas putida KT2440 for the production of isobutanol

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 feedbackresistant 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 Y Iso/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. K E Y W O R D Sisobutanol, ketoacid decarboxylase, metabolic engineering, microaerobic, Pseudomonas putida Abbreviations: 2-KIV, 2-ketoisovalerate; AlsS, acetolactate synthase; BHI, brain-heart infusion; KDC, ketoacid decarboxylase; LB, Lysogeny broth.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Micro‐aerobic production of isobutanol with engineered Pseudomonas putida

“…The P. putida Iso2 strain that was engineered for isobutanol production from glucose [23] was applied in all experiments. The strain is based on the GN346 variant [13] of P. putida KT2440 with additional deletions of bkdAA and sthA .…”

“…These metabolic advantages render P. putida a suitable microbial platform for isobutanol production which was successfully shown in shake flask cultures [23]. The authors engineered the P. putida GN346 mutant [13], that is unable to degrade n‐butanol, for the overproduction of isobutanol from glucose via the NADPH dependent Ehrlich pathway.…”

“…The authors engineered the P. putida GN346 mutant [13], that is unable to degrade n‐butanol, for the overproduction of isobutanol from glucose via the NADPH dependent Ehrlich pathway. Besides isobutanol, high accumulation of the by‐product 2‐ketogluconic acid (2‐KG) (>0.4 g 2‐KG g glc ‐1 ) was observed for P. putida Iso2 and engineered derivatives [23]. The problem of unwanted 2‐KG formation also occurred with P. putida engineered for cis,cis ‐muconic acid production [24, 25].…”

“…P. putida KT2440 is known to oxidize glucose to gluconate (glt) and to 2‐KG in the periplasm via glucose dehydrogenase (Gcd) and gluconate dehydrogenase (Gad) [21, 26]. However, the deletion of gcd in the isobutanol producer P. putida Iso2 not only stopped 2‐KG production but isobutanol formation, too [23].…”

“…Since anaerobic conditions improved isobutanol production in E. coli [6] and C. glutamicum [9] through increased supply of NADPH, Nitschel et al. [23] tested P. putida's capability of converting glucose into isobutanol during micro‐aerobic conditions in sealed shake flasks. The authors observed minimal metabolic activity even though P. putida KT2440 is not capable of anaerobic respiration to control its energy and redox balance in the absence of oxygen [15, 27].…”