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 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.

Bioisobutanol as a Promising Feedstock for Production of “Green” Hydrocarbons and Petrochemicals (A Review)

“…Nitschel et al [32,33] used for isobutanol production modified Pseudomonas putida strains under anaerobic conditions. They obtained the modified P. putida strain KT2440 giving the isobutanol yield of 22 ± 2 mg per gram of glucose under anaerobic conditions [32].…”

“…They obtained the modified P. putida strain KT2440 giving the isobutanol yield of 22 ± 2 mg per gram of glucose under anaerobic conditions [32]. Later this process was scaled for a 30-L bioreactor [33]. In the two-step bioprocess with the separated steps of bacterial growth and isobutanol production under microaerobic conditions, the isobutanol yield reached 60 mg per gram of glucose, and undesirable carbon loss in the form of 2-ketogluconic acid was prevented.…”

Engineering of Pseudomonas putida for accelerated co-utilization of glucose and cellobiose yields aerobic overproduction of pyruvate explained by an upgraded metabolic model

“…P. putida also has a broad potential to valorize lignocellulosic substrates (Anna Weimer et al, 2020). The rich metabolic network of the bacterium has been tailored for the bioproduction of polyhydroxyalkanoates (Poblete-Castro et al, 2013; Salvachúa et al, 2020), rhamnolipids (Tiso et al, 2020, p. 2), cis , cis -muconic acid (Bentley et al, 2020; Kohlstedt et al, 2018), pyruvate and lactate (Johnson and Beckham, 2015), isobutanol (Ankenbauer et al, 2021), or even fluorinated building-blocks (Calero et al, 2020). These and other advantageous characteristics including HV1 safety certification (Kampers et al, 2019), resistance to oxidative stress (Elmore et al, 2020; Guarnieri et al, 2017), suitability for large-scale aerobic fermentations (Ankenbauer et al, 2020), availability of high-quality genome-scale metabolic reconstruction (Nogales et al, 2020), or amenability to genetic manipulations with an available broad palette of engineering tools (Martínez-García and de Lorenzo, 2017) predetermine P. putida for the biotechnological upcycling of lignocellulosic substrates.…”

Listeria monocytogenes is a solvent tolerant organism secreting a solvent stable lipase: potential biotechnological applications

“…Currently, significant effort is being expended to either identify new solvent tolerant organisms or to engineer existing organisms for specific solvent environments (Mohedano et al 2022 ; Tian et al 2022 ; Kyoungseon et al 2021 ; Schalck et al 2021 ; Srivastava et al 2021 ; Wang et al 2021 ; de Carvalho et al 2019 ; Wynands et al 2019 ). Extensive engineering of Pseudomonads , for example, has yielded a solvent stable iso- butanol (biofuel) producing strain and a strain capable of converting cyclohexane to 6-hydroxyhexanoic acid, a polycaprolactone monomer (Ankenbauer et al 2021 ; Bretschneider et al 2021 ). Another area that requires robust solvent tolerant organisms is the processing of wastewater; specifically, there is a need for organisms that produce extracellular lipases to degrade wastewater lipids (Aktar et al 2021 ; Priyanka et al 2019a , 2019b ).…”