Heterologous Production of ß-Caryophyllene and Evaluation of Its Activity against Plant Pathogenic Fungi.

Terpenoids constitute one of the largest and most diverse groups within the class of secondary metabolites, comprising over 80,000 compounds. They not only exhibit important functions in plant physiology but also have commercial potential in the biotechnological, pharmaceutical, and agricultural sectors due to their promising properties, including various bioactivities against pathogens, inflammations, and cancer. In this work, we therefore aimed to implement the plant sesquiterpenoid pathway leading to ß-caryophyllene in the heterologous host Rhodobacter capsulatus and achieved a maximum production of 139 ± 31 mg L-1 culture. As this sesquiterpene offers various beneficial anti-phytopathogenic activities, we evaluated the bioactivity of ß-caryophyllene and its oxygenated derivative ß-caryophyllene oxide against different phytopathogenic fungi. Here, both compounds significantly inhibited the growth of Sclerotinia sclerotiorum and Fusarium oxysporum by up to 40%, while growth of Alternaria brassicicola was only slightly affected, and Phoma lingam and Rhizoctonia solani were unaffected. At the same time, the compounds showed a promising low inhibitory profile for a variety of plant growth-promoting bacteria at suitable compound concentrations. Our observations thus give a first indication that ß-caryophyllene and ß-caryophyllene oxide are promising natural agents, which might be applicable for the management of certain plant pathogenic fungi in agricultural crop production.

Discovery of the first light-dependent protochlorophyllide oxidoreductase in anoxygenic phototrophic bacteria.

In all photosynthetic organisms, chlorophylls function as light-absorbing photopigments allowing the efficient harvesting of light energy. Chlorophyll biosynthesis recurs in similar ways in anoxygenic phototrophic proteobacteria as well as oxygenic phototrophic cyanobacteria and plants. Here, the biocatalytic conversion of protochlorophyllide to chlorophyllide is catalysed by evolutionary and structurally distinct protochlorophyllide reductases (PORs) in anoxygenic and oxygenic phototrophs. It is commonly assumed that anoxygenic phototrophs only contain oxygen-sensitive dark-operative PORs (DPORs), which catalyse protochlorophyllide reduction independent of the presence of light. In contrast, oxygenic phototrophs additionally (or exclusively) possess oxygen-insensitive but light-dependent PORs (LPORs). Based on this observation it was suggested that light-dependent protochlorophyllide reduction first emerged as a consequence of increased atmospheric oxygen levels caused by oxygenic photosynthesis in cyanobacteria. Here, we provide experimental evidence for the presence of an LPOR in the anoxygenic phototrophic α-proteobacterium Dinoroseobacter shibae DFL12(T). In vitro and in vivo functional assays unequivocally prove light-dependent protochlorophyllide reduction by this enzyme and reveal that LPORs are not restricted to cyanobacteria and plants. Sequence-based phylogenetic analyses reconcile our findings with current hypotheses about the evolution of LPORs by suggesting that the light-dependent enzyme of D. shibae DFL12(T) might have been obtained from cyanobacteria by horizontal gene transfer.

Flavin mononucleotide-based fluorescent reporter proteins outperform green fluorescent protein-like proteins as quantitative in vivo real-time reporters.

Fluorescent proteins of the green fluorescent protein (GFP) family are commonly used as reporter proteins for quantitative analysis of complex biological processes in living microorganisms. Here we demonstrate that the fluorescence signal intensity of GFP-like proteins is affected under oxygen limitation and therefore does not reflect the amount of reporter protein in Escherichia coli batch cultures. Instead, flavin mononucleotide (FMN)-binding fluorescent proteins (FbFPs) are suitable for quantitative real-time in vivo assays under these conditions.

A novel T7 RNA polymerase dependent expression system for high-level protein production in the phototrophic bacterium Rhodobacter capsulatus.

The functional expression of heterologous genes using standard bacterial expression hosts such as Escherichia coli is often limited, e.g. by incorrect folding, assembly or targeting of recombinant proteins. Consequently, alternative bacterial expression systems have to be developed to provide novel strategies for protein synthesis exceeding the repertoire of the standard expression host E. coli. Here, we report on the construction of a novel expression system that combines the high processivity of T7 RNA polymerase with the unique physiological properties of the facultative photosynthetic bacterium Rhodobacter capsulatus. This system basically consists of a recombinant R. capsulatus T7 expression strain (R. capsulatus B10S-T7) harboring the respective polymerase gene under control of a fructose inducible promoter. In addition, a set of different broad-host-range vectors (pRho) was constructed allowing T7 RNA polymerase dependent and independent target gene expression in R. capsulatus and other Gram-negative bacteria. The expression efficiency of the novel system was studied in R. capsulatus and E. coli using the yellow fluorescent protein (YFP) as model protein. Expression levels were comparable in both expression hosts and yielded up to 80mg/l YFP in phototrophically grown R. capsulatus cultures. This result clearly indicates that the novel R. capsulatus-based expression system is well suited for the high-level expression of soluble proteins.

Reporter proteins for in vivo fluorescence without oxygen.

Fluorescent reporter proteins such as green fluorescent protein are valuable noninvasive molecular tools for in vivo real-time imaging of living specimens. However, their use is generally restricted to aerobic systems, as the formation of their chromophores strictly requires oxygen. Starting with blue-light photoreceptors from Bacillus subtilis and Pseudomonas putida that contain light-oxygen-voltage-sensing domains, we engineered flavin mononucleotide-based fluorescent proteins that can be used as fluorescent reporters in both aerobic and anaerobic biological systems.

Characterization of the human mitochondrial methionyl-tRNA synthetase.

Human mitochondrial methionyl-tRNA synthetase (human mtMetRS) has been identified from the human EST database. The cDNA encodes a 593 amino acid protein with an 18 amino acid mitochondrial import signal sequence. Sequence analysis indicates that this protein contains the consensus motifs characteristic of a class I aminoacyl-tRNA synthetase but lacks the Zn(2+) binding motif and C-terminal dimerization region found in MetRSs from various organisms. The mature form of human mtMetRS has been cloned and expressed in Escherichia coli. Gel filtration experiments indicate that this protein functions as a monomer with an apparent molecular mass of 67 kDa. The kinetic parameters for activation of methionine have been determined for the purified enzyme. The K(M) and k(cat) for aminoacylation of E. coli initiator tRNA(f)(Met) are reported. The kinetics of aminoacylation of an in vitro transcript of human mitochondrial tRNA(Met) (mtRNA(Met)) have been determined. To address the effects of the modification of mtRNA on recognition of the mitochondrial tRNA by human mtMetRS, the kinetics of aminoacylation of native bovine mtRNA(Met) and of an in vitro transcript of the bovine mtRNA(Met) have also been investigated.