Identification and characterization of the vanillin dehydrogenase YfmT in Bacillus subtilis 3NA.

With vanillin as one of the most important flavoring agents, many efforts have been made to optimize its biotechnological production from natural abundant substrates. However, its toxicity against the hosts results in rather low yields and product concentrations. Bacillus subtilis as a soil-dwelling bacterium is a possible lignin-derived compound-degrading microorganism. Therefore, its vanillin and ferulic acid metabolism was investigated. With a rather high tolerance for vanillin up to 20 mM, it is a promising candidate to produce natural vanillin. In this study, the well-studied phenolic acid decarboxylases PadC and BsdBCD could be ascribed to function as the only enzymes in B. subtilis 3NA converting ferulic acid to 4-vinylguaiacol and vanillic acid to guaiacol, respectively. As vanillin also becomes converted to guaiacol, a previous conversion to vanillic acid was assumed. Usage of bioinformatic tools revealed YfmT, which could be shown to function as the only vanillin dehydrogenase in B. subtilis 3NA. Thus, YfmT was further characterized regarding its temperature and pH optima as well as its substrate range. Vanillin and ferulic acid metabolic routes in the tested B. subtilis strain were revealed, a direct conversion of ferulic acid to vanillin, however, could not be found.

Analysis of the molecular response of Pseudomonas putida KT2440 to the next-generation biofuel n-butanol.

To increase the efficiency of biocatalysts a thorough understanding of the molecular response of the biocatalyst to precursors, products and environmental conditions applied in bioconversions is essential. Here we performed a comprehensive proteome and phospholipid analysis to characterize the molecular response of the potential biocatalyst Pseudomonas putida KT2440 to the next-generation biofuel n-butanol. Using complementary quantitative proteomics approaches we were able to identify and quantify 1467 proteins, corresponding to 28% of the total KT2440 proteome. 256 proteins were altered in abundance in response to n-butanol. The proteome response entailed an increased abundance of enzymes involved in n-butanol degradation including quinoprotein alcohol dehydrogenases, aldehyde dehydrogenases and enzymes of fatty acid beta oxidation. From these results we were able to construct a pathway for the metabolism of n-butanol in P. putida. The initial oxidation of n-butanol is catalyzed by at least two quinoprotein ethanol dehydrogenases (PedE and PedH). Growth of mutants lacking PedE and PedH on n-butanol was significantly impaired, but not completely inhibited, suggesting that additional alcohol dehydrogenases can at least partially complement their function in KT2440. Furthermore, phospholipid profiling revealed a significantly increased abundance of lyso-phospholipids in response to n-butanol, indicating a rearrangement of the lipid bilayer. BIOLOGICAL SIGNIFICANCE: n-butanol is an important bulk chemical and a promising alternative to gasoline as a transportation fuel. Due to environmental concerns as well as increasing energy prices there is a growing interest in sustainable and cost-effective biotechnological production processes for the production of bulk chemicals and transportation fuels from renewable resources. n-butanol fermentation is well established in Clostridiae, but the efficiency of n-butanol production is mainly limited by its toxicity. Therefore bacterial strains with higher intrinsic tolerance to n-butanol have to be selected as hosts for n-butanol production. Pseudomonas bacteria are metabolically very versatile and exhibit a high intrinsic tolerance to organic solvents making them suitable candidates for bioconversion processes. A prerequisite for a potential production of n-butanol in Pseudomonas bacteria is a thorough understanding of the molecular adaption processes caused by n-butanol and the identification of enzymes involved in n-butanol metabolization. This work describes the impact of n-butanol on the proteome and the phospholipid composition of the reference strain P. putida KT2440. The high proteome coverage of our proteomics survey allowed us to reconstruct the degradation pathway of n-butanol and to monitor the changes in the energy metabolism of KT2440 induced by n-butanol. Key enzymes involved in n-butanol degradation identified in study will be interesting targets for optimization of n-butanol production in Pseudomonads. The present work and the identification of key enzymes involved in butanol metabolism may serve as a fundament to develop new or improve existing strategies for the biotechnological production of the next-generation biofuel n-butanol in Pseudomonads.

Genetic engineering of Pseudomonas putida KT2440 for rapid and high-yield production of vanillin from ferulic acid.

Vanillin is one of the most important flavoring agents used today. That is why many efforts have been made on biotechnological production from natural abundant substrates. In this work, the nonpathogenic Pseudomonas putida strain KT2440 was genetically optimized to convert ferulic acid to vanillin. Deletion of the vanillin dehydrogenase gene (vdh) was not sufficient to prevent vanillin degradation. Additional inactivation of a molybdate transporter, identified by transposon mutagenesis, led to a strain incapable to grow on vanillin as sole carbon source. The bioconversion was optimized by enhanced chromosomal expression of the structural genes for feruloyl-CoA synthetase (fcs) and enoyl-CoA hydratase/aldolase (ech) by introduction of the strong tac promoter system. Further genetic engineering led to high initial conversion rates and molar vanillin yields up to 86% within just 3 h accompanied with very low by-product levels. To our knowledge, this represents the highest productivity and molar vanillin yield gained with a Pseudomonas strain so far. Together with its high tolerance for ferulic acid, the developed, plasmid-free P. putida strain represents a promising candidate for the biotechnological production of vanillin.

Functional characterization and application of a tightly regulated MekR/P mekA expression system in Escherichia coli and Pseudomonas putida.

A methyl ethyl ketone (MEK)-inducible system based on the broad-host-range plasmid pBBR1MCS2 and on the P mekA promoter region of the MEK degradation operon of Pseudomonas veronii MEK700 was characterized in Escherichia coli JM109 and Pseudomonas putida KT2440. For validation, ß-galactosidase (lacZ) was used as a reporter. The novel system, which is positively regulated by MekR, a member of the AraC/XylS family of regulators, was shown to be subject to carbon catabolite repression by glucose, which, however, could not be attributed to the single action of the global regulators Crc and PtsN. An advantage is its extremely tight regulation accompanied with three magnitudes of fold increase of gene expression after treatment with MEK. The transcriptional start site of P mekA was identified by primer extension, thereby revealing a potential stem-loop structure at the 5' end of the mRNA. Since MekR was highly insoluble, its putative binding site was identified through sequence analysis. The operator seems to be composed of a 15-bp tandem repeat (CACCN5CTTCAA) separated by a 6-bp spacer region, which resembles known binding patterns of other members of the AraC/XylS family. Subsequent mutational modifications of the putative operator region confirmed its importance for transcriptional activation. As the -35 promoter element seems to be overlapped by the putative operator, a class II activation mechanism is assumed.

Ethylene glycol metabolism by Pseudomonas putida.

In this study, we investigated the metabolism of ethylene glycol in the Pseudomonas putida strains KT2440 and JM37 by employing growth and bioconversion experiments, directed mutagenesis, and proteome analysis. We found that strain JM37 grew rapidly with ethylene glycol as a sole source of carbon and energy, while strain KT2440 did not grow within 2 days of incubation under the same conditions. However, bioconversion experiments revealed metabolism of ethylene glycol by both strains, with the temporal accumulation of glycolic acid and glyoxylic acid for strain KT2440. This accumulation was further increased by targeted mutagenesis. The key enzymes and specific differences between the two strains were identified by comparative proteomics. In P. putida JM37, tartronate semialdehyde synthase (Gcl), malate synthase (GlcB), and isocitrate lyase (AceA) were found to be induced in the presence of ethylene glycol or glyoxylic acid. Under the same conditions, strain KT2440 showed induction of AceA only. Despite this difference, the two strains were found to use similar periplasmic dehydrogenases for the initial oxidation step of ethylene glycol, namely, the two redundant pyrroloquinoline quinone (PQQ)-dependent enzymes PedE and PedH. From these results we constructed a new pathway for the metabolism of ethylene glycol in P. putida. Furthermore, we conclude that Pseudomonas putida might serve as a useful platform from which to establish a whole-cell biocatalyst for the production of glyoxylic acid from ethylene glycol.

Development of a method for markerless gene deletion in Pseudomonas putida.

We developed a negative counterselection system for Pseudomonas putida based on uracil phosphoribosyltransferase (UPRTase) and sensitivity against the antimetabolite 5-fluorouracil (5-FU). We constructed a P. putida strain that is resistant to 5-FU and constructed vectors for the deletion of the surface adhesion protein gene, the flagellum biosynthesis operon, and two endonuclease genes. The genes were efficiently disrupted and left a markerless chromosomal in-frame deletion.