Modular Low-Copy-Number Plasmid Vectors for Rhodobacterales with Extended Host Range in Alphaproteobacteria.

Members of the alphaproteobacterial order Rhodobacterales are metabolically diverse and highly abundant in the ocean. They are becoming increasingly interesting for marine biotechnology, due to their ecological adaptability, wealth of versatile low-copy-number plasmids, and their ability to produce secondary metabolites. However, molecular tools for engineering strains of this bacterial lineage are limited. Here, we expand the genetic toolbox by establishing standardized, modular repABC-based plasmid vectors of four well-characterized compatibility groups from the Roseobacter group applicable in the Rhodobacterales, and likely in further alphaproteobacterial orders (Hyphomicrobiales, Rhodospirillales, Caulobacterales). We confirmed replication of these newly constructed pABC vectors in two members of Rhodobacterales, namely, Dinoroseobacter shibae DFL 12 and Rhodobacter capsulatus B10S, as well as in two members of the alphaproteobacterial order Hyphomicrobiales (synonym: Rhizobiales; Ensifer meliloti 2011 and "Agrobacterium fabrum" C58). Maintenance of the pABC vectors in the biotechnologically valuable orders Rhodobacterales and Hyphomicrobiales facilitates the shuttling of genetic constructs between alphaproteobacterial genera and orders. Additionally, plasmid replication was verified in one member of Rhodospirillales (Rhodospirillum rubrum S1) as well as in one member of Caulobacterales (Caulobacter vibrioides CB15N). The modular construction of pABC vectors and the usage of four compatible replication systems, which allows their coexistence in a host cell, are advantageous features for future implementations of newly designed synthetic pathways. The vector applicability was demonstrated by functional complementation of a nitrogenase mutant phenotype by two complementary pABC-based plasmids in R. capsulatus.

Structural insights into the iron nitrogenase complex.

Nitrogenases are best known for catalyzing the reduction of dinitrogen to ammonia at a complex metallic cofactor. Recently, nitrogenases were shown to reduce carbon dioxide (CO2) and carbon monoxide to hydrocarbons, offering a pathway to recycle carbon waste into hydrocarbon products. Among the three nitrogenase isozymes, the iron nitrogenase has the highest wild-type activity for the reduction of CO2, but the molecular architecture facilitating these activities has remained unknown. Here, we report a 2.35-Å cryogenic electron microscopy structure of the ADP·AlF3-stabilized iron nitrogenase complex from Rhodobacter capsulatus, revealing an [Fe8S9C-(R)-homocitrate] cluster in the active site. The enzyme complex suggests that the iron nitrogenase G subunit is involved in cluster stabilization and substrate channeling and confers specificity between nitrogenase reductase and catalytic component proteins. Moreover, the structure highlights a different interface between the two catalytic halves of the iron and the molybdenum nitrogenase, potentially influencing the intrasubunit 'communication' and thus the nitrogenase mechanism.

The Conversion of Carbon Monoxide and Carbon Dioxide by Nitrogenases.

Nitrogenases are the only known family of enzymes that catalyze the reduction of molecular nitrogen (N2 ) to ammonia (NH3 ). The N2 reduction drives biological nitrogen fixation and the global nitrogen cycle. Besides the conversion of N2 , nitrogenases catalyze a whole range of other reductions, including the reduction of the small gaseous substrates carbon monoxide (CO) and carbon dioxide (CO2 ) to hydrocarbons. However, it remains an open question whether these 'side reactivities' play a role under environmental conditions. Nonetheless, these reactivities and particularly the formation of hydrocarbons have spurred the interest in nitrogenases for biotechnological applications. There are three different isozymes of nitrogenase: the molybdenum and the alternative vanadium and iron-only nitrogenase. The isozymes differ in their metal content, structure, and substrate-dependent activity, despite their homology. This minireview focuses on the conversion of CO and CO2 to methane and higher hydrocarbons and aims to specify the differences in activity between the three nitrogenase isozymes.

Boron-Doped Polycyclic π-Electron Systems with an Antiaromatic Borole Substructure That Forms Photoresponsive B-P Lewis Adducts.

Heteroatom doping is a powerful strategy to alter the electronic structure of polycyclic aromatic hydrocarbons (PAHs). Especially boron doping endows PAH scaffolds with electron-accepting character and Lewis acidic centers. Herein, we report that embedding a five-membered borole ring into a polycyclic skeleton imparts the π-system with antiaromatic character and thereby induces unique properties and behavior. A series of borole-embedded π-conjugated compounds were synthesized from teraryl precursors via a borylation/intramolecular electrophilic C-H borylation sequence. The obtained compounds exhibit planar structures with distorted geometries around the boron center and form columnar slipped face-to-face π-stacked structures. Among these compounds, a pyrene-fused derivative shows an intense emission with a high quantum yield in solution. This compound also exhibits high Lewis acidity, which reflects the antiaromatic character and strained structure of the borole substructure. This compound forms a Lewis acid-base adduct even with weakly Lewis basic phosphorus-containing polycyclic π-systems. Analyzing the crystal structure of the thus-obtained adduct revealed a complex between the boron- and phosphorus-embedded π-systems with a direct B-P dative bond. This complex undergoes photodissociation in the excited state and exhibits an emission exclusively from the base-free borole-embedded π-system.