Micro-aerobic production of isobutanol with engineered Pseudomonas putida

Abstract

Pseudomonas putida KT2440 is emerging as a promising microbial host for biotechnological industry due to its broad range of substrate affinity and resilience to physicochemical stresses. Its natural tolerance towards aromatics and solvents qualifies this versatile microbe as promising candidate to produce next generation biofuels such as isobutanol. In this study, we scaled-up the production of isobutanol with P. putida from shake flask to fed-batch cultivation in a 30 L bioreactor. The design of a two-stage bioprocess with separated growth and production resulted in 3.35 g_isobutanol L^-1. Flux analysis revealed that the NADPH expensive formation of isobutanol exceeded the cellular catabolic supply of NADPH finally causing growth retardation. Concomitantly, the cell counteracted to the redox imbalance by increased formation of 2-ketogluconic thereby providing electrons for the respiratory ATP generation. Thus, P. putida partially uncoupled ATP formation from the availability of NADH. The quantitative analysis of intracellular pyridine nucleotides NAD(P)⁺ and NAD(P)H revealed elevated catabolic and anabolic reducing power during aerobic production of isobutanol. Additionally, the installation of micro-aerobic conditions during production doubled the integral glucose-to-isobutanol conversion yield to 60 mg_isobutanol g_glucose ^-1 while preventing undesired carbon loss as 2-ketogluconic acid.

Keywords: 2‐ketogluconic acid; NADPH; Pseudomonas putida; isobutanol; micro‐aerobic.

FIGURE 1

(A) Schematic overview of the three different process strategies applied in this study. Process R serves as reference process. After the batch and fed‐batch phase, plasmid was induced and isobutanol production was monitored. After 8 h, submerse aeration was switched to headspace aeration. In contrast, fast growing cells were induced in Process F and micro‐aerobic condition was installed after 6 h. Additionally, constant micro‐aerobic condition was installed in Process MA 20 min after plasmid induction. The time span of each phase is given in brackets. (B) Time course of biomass concentration, extracellular concentrations of isobutanol, 2‐KG, l‐valine, 2‐ketoisovalerate (KIV), and isobutanol per glucose yield Y_{isobutanol/glucose}. Each plot displays the reference Process R (grey square), the Process F (black triangle), and one replicate of the Process MA (circle). The process time (h) is given in relation to the induction of the plasmid (dotted vertical line)

FIGURE 2

Uptake (negative) rates of carbon sources and formation (positive) rates of products (in mmol g_X ^–1 h^–1) as well as growth rates during production of isobutanol in the reference Process R (grey bar), in Process F (dark grey bar) and in one replicate of Process MA (black bar). Time point 0 h equals the reference condition before induction of the plasmid. Micro‐aerobic condition was installed in Process R 8 h, in Process F 6 h, and in Process MA 20 min after plasmid induction. Abbreviations (coding genes are given in parentheses): C‐6: difference of glucose uptake and gluconate and 2‐ketogluconate formation, G6P: glucose‐6‐phosphate, 2‐K6PG: 2‐keto‐6‐phosphogluconate, 6PG: 6‐phosphogluconate, Gcd: glucose dehydrogenase (gcd), Gad: gluconate 2‐dehdyrogenase complex (PP3382‐PP3384, PP3623, PP4232), 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), AlsS: acetolactacte synthase (ilvHI/alsS), IlvC: ketol‐acid reductoisomerase (ilvC), IlvD: dihydroxyacid dehydratase (ilvD), KivD: alpha‐ketoisovalerate decarboxylase (kivD), Yqhd: aldehyde reductase (yqhD)

FIGURE 3

(A) Carbon share of the products KIV, l‐valine, isobutanol, and CO₂ per uptake of substrates in Process R (grey bar) and Process F (dark grey bar). (B) NADPH demand for production of KIV, l‐valine and isobutanol as percentage of the total NADPH supply in Process R (grey bar) and Process F (dark grey bar). The dotted line represents the theoretical NADPH surplus in P. putida WT. (C‐D) Biomass specific ATP formation rates based on oxidative phosphorylation (q_ATP,O, sum of dark blue and light blue bar) and substrate level phosphorylation (q_ATP,S, green bar) in (C) the reference Process R and (D) the Process F. The dark blue bar represents the ATP generation based on the electron transport via oxidation of glucose to 2‐KG (q_ATP,alt). The light blue bar represents the remaining part of oxidative phosphorylation via NADH and FADH₂ (=q_ATP,O – q_ATP,alt). The production time in (A‐D) is given with respect to induction of the plasmid whereas 0 h equals the reference condition before plasmid induction

FIGURE 4

Intracellular concentrations (in μmol g_X  ^–1) of the redox cofactors NAD⁺, NADP⁺, NADH and NADPH and the respective redox ratios during the production phase in the reference Process R (grey circle) and in Process F (black circle). Time point 0 h equals the reference condition before induction of the plasmid

FIGURE 5

Schematic overview of assumed metabolic fluxes that are related to the intracellular pools of ATP, NADH, and NADPH (A) before induction and (B) during isobutanol formation in P. putida Iso2. The thickness of the arrows indicates the intensity of the related flux