Micro‐aerobic production of isobutanol with engineered Pseudomonas putida

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 p...

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Bibliographic Details
Published in:Engineering in life sciences 2021-07, Vol.21 (7), p.475-488
Main Authors: Ankenbauer, Andreas, Nitschel, Robert, Teleki, Attila, Müller, Tobias, Favilli, Lorenzo, Blombach, Bastian, Takors, Ralf
Format: Article
Language:English
Subjects:
2‐ketogluconic acid
Adenosine triphosphate
Aerobic conditions
Alcohols
Aromatic compounds
Biofuels
Biomass
Bioreactors
Carbon
Dehydrogenases
Ecological footprint
Glucose
Growth rate
Isobutanol
Metabolism
Metabolites
Microorganisms
micro‐aerobic
NAD
NADH
NADPH
Nicotinamide adenine dinucleotide
Nucleotides
Pseudomonas putida
Pyridine nucleotides
Pyridines
Raw materials
Substrate preferences
Substrates
Trace elements
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Summary: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 gisobutanol 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 mgisobutanol gglucose–1 while preventing undesired carbon loss as 2‐ketogluconic acid.
ISSN:1618-0240
1618-2863
DOI:10.1002/elsc.202000116