Open AccessMicro‐aerobic production of isobutanol with engineered Pseudomonas putida
Author(s) -
Ankenbauer Andreas,
Nitschel Robert,
Teleki Attila,
Müller Tobias,
Favilli Lorenzo,
Blombach Bastian,
Takors Ralf
Publication year - 2021
Publication title -
engineering in life sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.547
H-Index - 57
eISSN - 1618-2863
pISSN - 1618-0240
DOI - 10.1002/elsc.202000116
Subject(s) - isobutanol , pseudomonas putida , bioprocess , nad+ kinase , biochemistry , metabolic engineering , chemistry , catabolism , biology , metabolism , ethanol , enzyme , paleontology
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.