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

ABSTRACT: BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

SUBMITTER: Lang K

PROVIDER: S-EPMC3897908 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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

Lang Karsten K Zierow Jessica J Buehler Katja K Schmid Andreas A

Microbial cell factories 20140107

<h4>Background</h4>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 isobu ...[more]

PMID: 24397404

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Project description:BackgroundFilamentous fungi can each produce dozens of secondary metabolites which are attractive as therapeutics, drugs, antimicrobials, flavour compounds and other high-value chemicals. Furthermore, they can be used as an expression system for eukaryotic proteins. Application of most fungal secondary metabolites is, however, so far hampered by the lack of suitable fermentation protocols for the producing strain and/or by low product titers. To overcome these limitations, we report here the engineering of the industrial fungus Aspergillus niger to produce high titers (up to 4,500 mg • l-1) of secondary metabolites belonging to the class of nonribosomal peptides.ResultsFor a proof-of-concept study, we heterologously expressed the 351 kDa nonribosomal peptide synthetase ESYN from Fusarium oxysporum in A. niger. ESYN catalyzes the formation of cyclic depsipeptides of the enniatin family, which exhibit antimicrobial, antiviral and anticancer activities. The encoding gene esyn1 was put under control of a tunable bacterial-fungal hybrid promoter (Tet-on) which was switched on during early-exponential growth phase of A. niger cultures. The enniatins were isolated and purified by means of reverse phase chromatography and their identity and purity proven by tandem MS, NMR spectroscopy and X-ray crystallography. The initial yields of 1 mg • l-1 of enniatin were increased about 950 fold by optimizing feeding conditions and the morphology of A. niger in liquid shake flask cultures. Further yield optimization (about 4.5 fold) was accomplished by cultivating A. niger in 5 l fed batch fermentations. Finally, an autonomous A. niger expression host was established, which was independent from feeding with the enniatin precursor d-2-hydroxyvaleric acid d-Hiv. This was achieved by constitutively expressing a fungal d-Hiv dehydrogenase in the esyn1-expressing A. niger strain, which used the intracellular ?-ketovaleric acid pool to generate d-Hiv.ConclusionsThis is the first report demonstrating that A. niger is a potent and promising expression host for nonribosomal peptides with titers high enough to become industrially attractive. Application of the Tet-on system in A. niger allows precise control on the timing of product formation, thereby ensuring high yields and purity of the peptides produced.

Project description:The current chassis organisms or various types of cell factories have considerable advantages and disadvantages. Therefore, it is necessary to develop various chassis for an efficient production of different bioproducts from renewable resources. In this context, synthetic biology offers unique potentialities to produce value-added products of interests. Microbial genome reduction and modification are important strategies for constructing cellular chassis and cell factories. Many genome-reduced strains from Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum and Streptomyces, have been widely used for the production of amino acids, organic acids, and some enzymes. Some Pseudomonas strains could serve as good candidates for ideal chassis cells since they grow fast and can produce many valuable metabolites with low nutritional requirements and strong environmental adaptability. Pseudomonas chlororaphis GP72 is a non-pathogenic plant growth-promoting rhizobacterium that possesses capacities of tolerating various environmental stresses and synthesizing many kinds of bioactive compounds with high yield. These include phenazine-1-carboxylic acid (PCA) and 2-hydroxyphenazine (2-OH-PHZ), which exhibit strong bacteriostatic and antifungal activity toward some microbial pathogens.We depleted 685 kb (10.3% of the genomic sequence) from the chromosome of P. chlororaphis GP72(rpeA-) by a markerless deletion method, which included five secondary metabolic gene clusters and 17 strain-specific regions (525 non-essential genes). Then we characterized the 22 multiple-deletion series (MDS) strains. Growth characteristics, production of phenazines and morphologies were changed greatly in mutants with large-fragment deletions. Some of the genome-reduced P. chlororaphis mutants exhibited more productivity than the parental strain GP72(rpeA-). For example, strain MDS22 had 4.4 times higher production of 2-OH-PHZ (99.1 mg/L) than strain GP72(rpeA-), and the specific 2-OH-PHZ production rate (mmol/g/h) increased 11.5-fold. Also and MDS10 had the highest phenazine production (852.0 mg/L) among all the studied strains with a relatively high specific total phenazine production rate (0.0056 g/g/h).In conclusion, P. chlororaphis strains with reduced genome performed better in production of secondary metabolites than the parent strain. The newly developed mutants can be used for the further genetic manipulation to construct chassis cells with the less complex metabolic network, better regulation and more efficient productivity for diverse biotechnological applications.

Dataset Information

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

Publications

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

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