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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.
Highlights
Over 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
This study aims to establish the solvent tolerant, genome-sequenced Pseudomonas sp. strain VLB120 [29,32] as a platform biocatalyst for the fermentative production of isobutyric acid
Overexpression of a 2-keto acid decarboxylase encoding gene in Pseudomonas sp. strain VLB120 allows the synthesis of isobutyric acid and isobutanol To evaluate the application of Ps. sp. strain VLB120 for the synthesis of isobutyric acid, the 2-keto acid decarboxylase Kivd from Lactococcus lactis was produced using a pCOM10 expression system
Summary
Over 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. The production of isobutyric acid was demonstrated in Escherichia coli with remarkable titers and yields These examples suffer from byproduct formation due to the fermentative growth mode of the respective organism. A major bottleneck of current bioprocesses is the susceptibility of microorganisms to toxic compounds [9,10] This results in low product titers and limited process stabilities having a negative impact on overall productivity. Pseudomonas species are known to exhibit mechanism enabling the adaptation to toxic environmental conditions [12] 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 [13]. Biofilms provide increased process stability and potentially allow, in contrast to commonly used planktonic cells, a continuous process mode [14,15]
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