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 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 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.

Keywords: Pseudomonas putida; isobutanol; ketoacid decarboxylase; metabolic engineering; microaerobic.

Figure 1

The central metabolism of P. putida KT2440 with the Ehrlich pathway. Abbreviations (coding genes are given in brackets): G6P: glucose‐6‐phosphate 2‐KG: 2‐ketogluconate, 2‐K6PG: 2‐keto‐6‐phosphogluconate, 6‐PG: 6‐phosphogluconate, 2‐KDPG: 2‐keto‐3‐deoxy‐6‐phosphogluconate, G3P: glyceraldehyde‐3‐phosphate, 1,3‐bPG: 1,3‐bisphosphoglycerate, 3‐PG: 3‐phosphoglycerate, 2‐PG: 2‐phosphoglycerate, PEP: phosphoenolpyruvate, DHAP: dihydroxyacetone‐phosphate, F‐1,6‐bP: fructose‐1,6‐bisphosphate, F6P: fructose‐6‐phosphate, CoA: co‐enzyme A, Gcd: glucose dehydrogenase (gcd), gad: gluconate 2‐dehdyrogenase (gad), PQQ: pyrroloquinoline quinone, Glk: glucokinase (glk), Zwf: glucose‐6‐phosphate 1‐dehydrogenase (zwf‐1, zwf‐2, zwf‐3), GnuK: gluconate kinase (gnuK), KguD: 2‐6‐phosphoketogluconate reductase (kguD), KguK: 2‐ketogluconate kinase (kguK), Edd: 6‐phosphogluconate dehydratase (edd), Eda: 2‐keto‐3‐deoxy‐6‐phosphogluconate aldolase (eda), IlvHI/AlsS: acetolactacte synthase (ilvHI/alsS), IlvC: ketolacid reductoisomerase (ilvC), IlvD: dihydroxyacid dehydratase (ilvD), KivD: ketoacid decarboxylase (kivD), Bkd: branched‐chain ketoacid dehydrogenase complex (bkd), Yqhd: aldehyde reductase (yqhD), AldH: aldehdye dehdyrogenases, PntAB: pyridine nucleotide transhydrogenase (membrane bound) (pntAB), SthA: pyridine nucleotide transhydrogenase (soluble) (sthA)

Figure 2

(A) Growth of P. putida KT2440 and P. putida GN346 in DeBont minimal medium containing 0.5 g/L isobutanol (filled symbols) or 5.4 g/L glucose (open symbols). (B) Growth of P. putida and P. putida EP1 in DeBont minimal medium containing 2.9 g/L 2‐ketoisovalerate. Experiments were performed in triplicates and error bars represent the corresponding standard deviation

Figure 3

Growth (black circles), glucose consumption (grey circles), isobutanol production (white bars) and 2‐KG formation (grey bars) of P. putida Iso2 in DeBont minimal medium containing glucose. Experiments were performed in triplicates and error bars represent the corresponding standard deviation