Achim Heck

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.

Flavin Mononucleotide-Based Fluorescent Reporter Proteins Outperform Green Fluorescent Protein-Like Proteins as Quantitative In Vivo Real-Time Reporters

ABSTRACT 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.

FMN-based fluorescent reporter proteins outperform GFP-like proteins as quantitative in vivo real-time reporters

ABSTRACT 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.

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 DFL12T. 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 DFL12T might have been obtained from cyanobacteria by horizontal gene transfer.

Engineering Photosynthetic α-Proteobacteria for the Production of Recombinant Proteins and Terpenoids

Phototrophic non-sulfur purple α-proteobacteria are able to harvest sunlight and to fix atmospheric carbon dioxide and dinitrogen. Consequently, these microbes are used as model organisms for the investigation of regulation and activity of the photosynthesis complexes, the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) as well as the nitrogenase and hydrogenase enzyme complexes. In addition, this group of prokaryotic phototrophs has emerged as microbial production chassis for the synthesis of recombinant proteins and natural products. To this end, a versatile set of different expression tools has been developed allowing the functional expression of single genes as well as the transfer of complete metabolic pathways. This review provides an overview of different strategies to engineer photosynthetic α-proteobacteria, especially the two most commonly employed representatives Rhodobacter capsulatus and Rhodobacter sphaeroides, for the production of difficult-to-express proteins and terpenoids. Unique physiological properties of these alternative production hosts are discussed in the context of respective production processes. Furthermore, synthetic biology tools applicable for heterologous gene expression and establishment of combinatorial biosynthetic pathways in phototrophic α-proteobacteria are described. Finally, the potential of phototrophic bacteria in future bioeconomic production routes is briefly discussed.

Expressionsoptimierung in Mikroorganismen

The genome era lead to the identification of a tremendous number of genes encoding potentially scientific and biotechnological relevant proteins. Moreover, the number of these proteins is still exponentially growing. Nevertheless, the corresponding genes have to be expressed to produce functional proteins thereby constituting a huge scientific, industrial and economic challenge. This challenge was taken by the CLIB2021 Technology Platform Protein Expression and Optimization (ExpressO).