Metabolic engineering of Pseudomonas sp. strain VLB120 as platform biocatalyst for the production of isobutyric acid and other secondary metabolites

Abstract

BackgroundOver the recent years the production of Ehrlich pathway derived chemicals was shown in a variety of hosts such as Escherichia coli, Corynebacterium glutamicum, and yeast. Exemplarily the production of isobutyric acid was demonstrated in Escherichia coli with remarkable titers and yields. However, these examples suffer from byproduct formation due to the fermentative growth mode of the respective organism. We aim at establishing a new aerobic, chassis for the synthesis of isobutyric acid and other interesting metabolites using Pseudomonas sp. strain VLB120, an obligate aerobe organism, as host strain.ResultsThe overexpression of kivd, coding for a 2-ketoacid decarboxylase from Lactococcus lactis in Ps. sp. strain VLB120 enabled for the production of isobutyric acid and isobutanol via the valine synthesis route (Ehrlich pathway). This indicates the existence of chromosomally encoded alcohol and aldehyde dehydrogenases catalyzing the reduction and oxidation of isobutyraldehyde. In addition we showed that the strain possesses a complete pathway for isobutyric acid metabolization, channeling the compound via isobutyryl-CoA into valine degradation. Three key issues were addressed to allow and optimize isobutyric acid synthesis: i) minimizing isobutyric acid degradation by host intrinsic enzymes, ii) construction of suitable expression systems and iii) streamlining of central carbon metabolism finally leading to production of up to 26.8 ± 1.5 mM isobutyric acid with a carbon yield of 0.12 ± 0.01 g gglc-1.ConclusionThe combination of an increased flux towards isobutyric acid using a tailor-made expression system and the prevention of precursor and product degradation allowed efficient production of isobutyric acid in Ps. sp. strain VLB120. This will be the basis for the development of a continuous reaction process for this bulk chemicals.

Multistep Synthesis of (S)‐3‐Hydroxyisobutyric Acid from Glucose usingPseudomonas taiwanensisVLB120 B83 T7 Catalytic Biofilms

“…This value reflects the consumption of the precursor2-ketoisovalerate during cell growth. [23] Despite this shortcoming, these results show that P. taiwanensis VLB120B 83 is ap romising catalyst for the synthesis of (S)-3-HIBA. These experiments set the basis for the development of af ermentativep roductionsystemdirectly from glucose.…”

“…In addition, the ability of recombinant P. taiwanensis VLB120B 83 T7 to form stable,c atalytically active biofilms wasu sed for the continuous fermentativep roduction of value addedchemicals.O xygen transfer through the silicone membrane was identifieda sc rucial andf ound to be limiting in standard membrane reactors.T he demonstrated proof-of-concept is ab lueprint for the development of biofilm-based fermentation processes using specialized Pseudomonas strains tailored to the synthesis of specificproducts. [23] Experimental Section…”

“…Thei mportance of deleting ilvE and panB in P. taiwanensis VLB120t of urther improve the isobutytric acid production yield was successfully demonstrated earlier for ad ifferent variant of this strain. [23] Theo verall productivity couldb em aximized by the additional overexpression of the responsible genes encoding the valined egradationp athwayt oi ncrease the respective protein amount,a sa lready discussed for the biotransformation based approach.…”

“…Thei mportance of deleting ilvE and panB in the parent strain P. taiwanensis VLB120t of urtheri mprove isobutytric acid production yield was already successfully demonstrated. [23] Theo verall reactionr ate was limited by the CoA-esterification of isobutyric acid, as no further pathwayi ntermediates were detectable in the fermentation broth. This is reasonable as isobutyric acid is an on-natural substrate in the wild-type P. taiwanensis VLB120, whereass ubsequent processing of isobutyryl-CoA is achieved by the natural valine degradation pathway, with its evolutionary optimized kinetics.…”

Petroleum Hydrocarbon Spills in the Environment and Abundance of Microbial Community Capable of Biosurfactant Production

“…is a prolific producer of a number of biosurfactants and extra cellular enzymes (like lipase). Importantly, these organisms are able to form stable surface associated microbial communities (biofilms), which have been described as potent alternative to planktonic cells regarding their application as biocatalyst, especially when solvents or otherwise toxic compounds are involved in the processes [42]. The term 'biofilm' refers to surface-associated microbial communities as well as microbes forming flocks and aggregates.…”