Beyond rational-biosensor-guided isolation of 100 independently evolved bacterial strain variants and comparative analysis of their genomes.

BACKGROUND: In contrast to modern rational metabolic engineering, classical strain development strongly relies on random mutagenesis and screening for the desired production phenotype. Nowadays, with the availability of biosensor-based FACS screening strategies, these random approaches are coming back into fashion. In this study, we employ this technology in combination with comparative genome analyses to identify novel mutations contributing to product formation in the genome of a Corynebacterium glutamicum L-histidine producer. Since all known genetic targets contributing to L-histidine production have been already rationally engineered in this strain, identification of novel beneficial mutations can be regarded as challenging, as they might not be intuitively linkable to L-histidine biosynthesis. RESULTS: In order to identify 100 improved strain variants that had each arisen independently, we performed > 600 chemical mutagenesis experiments, > 200 biosensor-based FACS screenings, isolated > 50,000 variants with increased fluorescence, and characterized > 4500 variants with regard to biomass formation and L-histidine production. Based on comparative genome analyses of these 100 variants accumulating 10-80% more L-histidine, we discovered several beneficial mutations. Combination of selected genetic modifications allowed for the construction of a strain variant characterized by a doubled L-histidine titer (29 mM) and product yield (0.13 C-mol C-mol-1) in comparison to the starting variant. CONCLUSIONS: This study may serve as a blueprint for the identification of novel beneficial mutations in microbial producers in a more systematic manner. This way, also previously unexplored genes or genes with previously unknown contribution to the respective production phenotype can be identified. We believe that this technology has a great potential to push industrial production strains towards maximum performance.

Combinatorial optimization of synthetic operons for the microbial production of p-coumaryl alcohol with Escherichia coli.

BACKGROUND: Microbes are extensively engineered to produce compounds of biotechnological or pharmaceutical interest. However, functional integration of synthetic pathways into the respective host cell metabolism and optimization of heterologous gene expression for achieving high product titers is still a challenging task. In this manuscript, we describe the optimization of a tetracistronic operon for the microbial production of the plant-derived phenylpropanoid p-coumaryl alcohol in Escherichia coli. RESULTS: Basis for the construction of a p-coumaryl alcohol producing strain was the development of Operon-PLICing as method for the rapid combinatorial assembly of synthetic operons. This method is based on the chemical cleavage reaction of phosphorothioate bonds in an iodine/ethanol solution to generate complementary, single-stranded overhangs and subsequent hybridization of multiple DNA-fragments. Furthermore, during the assembly of these DNA-fragments, Operon-PLICing offers the opportunity for balancing gene expression of all pathway genes on the level of translation for maximizing product titers by varying the spacing between the Shine-Dalgarno sequence and START codon. With Operon-PLICing, 81 different clones, each one carrying a different p-coumaryl alcohol operon, were individually constructed and screened for p-coumaryl alcohol formation within a few days. The absolute product titer of the best five variants ranged from 48 to 52 mg/L p-coumaryl alcohol without any further optimization of growth and production conditions. CONCLUSIONS: Operon-PLICing is sequence-independent and thus does not require any specific recognition or target sequences for enzymatic activities since all hybridization sites can be arbitrarily selected. In fact, after PCR-amplification, no endonucleases or ligases, frequently used in other methods, are needed. The modularity, simplicity and robustness of Operon-PLICing would be perfectly suited for an automation of cloning in the microtiter plate format.

Putting bugs to the blush: metabolic engineering for phenylpropanoid-derived products in microorganisms.

Phenylpropanoids and phenylpropanoid-derived phenolic compounds such as flavonoids, anthocyanins, or stilbenes are secondary plant metabolites which serve as pigments and scent compounds or provide protection against environmental stress. Due to their antioxidant properties they also have been widely recognized for their benefit on human health. Traditionally, such compounds are extracted from their natural plant sources, but this approach is limited by low abundance and environmental, seasonal as well as regional variations in yield. Chemical synthesis is not a true alternative for the large scale production of more complex phenylpropanoid-derived substances since chemical synthesis becomes commercially unfeasible as the structural complexity of these plant natural products increases. In the last years, many biosynthetic pathways for plant natural products have been elucidated through the advancements in DNA sequencing technologies. In combination with new recombinant DNA technologies this technical progress opens the door toward the functional integration of full biosynthetic pathways for the synthesis of phenylpropanoids and phenylpropanoid-derived compounds in microorganisms. We believe that this approach has great potential to provide sufficient quantities of the desired plant natural product from cheap and renewable resources. This commentary highlights recent advances in the microbial production of phenylpropanoid-derived compounds with an emphasis on flavonoids and stilbenes.

Driving unidirectional molecular rotary motors with visible light by intra- and intermolecular energy transfer from palladium porphyrin.

Driving molecular rotary motors using visible light (530-550 nm) instead of UV light was achieved using palladium tetraphenylporphyrin as a triplet sensitizer. Visible light driven rotation was confirmed by UV/vis absorption, circular dichroism and (1)H NMR spectroscopy and the rotation was confirmed to be unidirectional and with similar photostationary states, despite proceeding via a triplet instead of a singlet excited state of the molecular motor. Energy transfer proceeds in both inter- and intramolecular fashion from the triplet state of the porphyrin to the motor. Stern Volmer plots show that the rate of intermolecular quenching of the porphyrin excited state by the molecular motor is diffusion-controlled.

On the effect of donor and acceptor substituents on the behaviour of light-driven rotary molecular motors.

Light-driven rotary molecular motors based on overcrowded alkenes can be substituted with electron-donating and electron-withdrawing substituents (R = OMe, Cl and CN) in direct conjugation with the central double bond (the axis of rotation) without having a significant influence on the rate-limiting, thermal isomerisation step of their rotary cycle. This indicates that in this system, it is predominantly steric factors that determine the barrier to the thermal helix inversion. In contrast, the quantum yield and photoequilibria in the photochemical step were found to be quite sensitive to the combination of substituent and solvent employed.