isobutyryl-coenzyme-a and isobutyl-alcohol

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. 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.. The 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 g(glc)⁻¹.. The 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.

Topics: Acyl Coenzyme A; Aldehydes; Biocatalysis; Biotransformation; Butanols; Carboxy-Lyases; Hemiterpenes; Isobutyrates; Keto Acids; Lactococcus lactis; Metabolic Engineering; Oxidation-Reduction; Pseudomonas; Valine

Supply of isobutanol to enhance the production of anti-tumor agent ansamitocin P-3 (AP-3) in medium containing agro-industrial residues was investigated with analysis of gene transcription, enzyme activity, and intermediate accumulation. Under the optimal addition of isobutanol, about 4-fold improvement of AP-3 production was obtained, and the consumption of isobutanol and accumulation of isobutyrate, malonyl-CoA, and acetyl-CoA were observed. Compared to the control without isobutanol addition, activities of both isobutanol dehydrogenase and valine dehydrogenase were enhanced in isobutanol supplemented culture. Transcription level of genes in AP-3 biosynthetic and isobutyryl-CoA catabolic pathways responded to isobutanol addition in a similar way as AP-3 biosynthesis. It is concluded that isobutanol addition was an effective strategy for increasing AP-3 production via regulation of gene transcription and pools of precursors, and the information obtained might be helpful to the fermentation productivity improvement on large scale.

Topics: Actinomycetales; Acyl Coenzyme A; Butanols; Fermentation; Gene Expression Regulation, Bacterial; Genes, Bacterial; Maytansine; Oxidoreductases; Transcription, Genetic