Azotobacter vinelandii is a soil bacterium related to the Pseudomonas genus that fixes nitrogen under aerobic conditions while simultaneously protecting nitrogenase from oxygen damage. In response to carbon availability, this organism undergoes a simple differentiation process to form cysts that are resistant to drought and other physical and chemical agents. Here we report the complete genome sequence of A. vinelandii DJ, which has a single circular genome of 5,365,318 bp. In order to reconcile an obligate aerobic lifestyle with exquisitely oxygen-sensitive processes, A. vinelandii is specialized in terms of its complement of respiratory proteins. It is able to produce alginate, a polymer that further protects the organism from excess exogenous oxygen, and it has multiple duplications of alginate modification genes, which may alter alginate composition in response to oxygen availability. The genome analysis identified the chromosomal locations of the genes coding for the three known oxygen-sensitive nitrogenases, as well as genes coding for other oxygen-sensitive enzymes, such as carbon monoxide dehydrogenase and formate dehydrogenase. These findings offer new prospects for the wider application of A. vinelandii as a host for the production and characterization of oxygen-sensitive proteins.
Alginate is a copolymer of beta-D-mannuronic acid and alpha-L-guluronic acid (GulA), linked together by 1-4 linkages. The polymer is a well-established industrial product obtained commercially by harvesting brown seaweeds. Some bacteria, mostly derived from the genus Pseudomonas and belonging to the RNA superfamily I, are also capable of producing copious amounts of this polymer as an exopolysaccharide. The molecular genetics, regulation and biochemistry of alginate biosynthesis have been particularly well characterized in the opportunistic human pathogen Pseudomonas aeruginosa, although the biochemistry of the polymerization process is still poorly understood. In the last 3 years major aspects of the molecular genetics of alginate biosynthesis in Azotobacter vinelandii have also been reported. In both organisms the immediate precursor of polymerization is GDP-mannuronic acid, and the sugar residues in this compound are polymerized into mannuronan. This uniform polymer is then further modified by acetylation at positions O-2 and/or O-3 and by epimerization of some of the residues, leading to a variable content of acetyl groups and GulA residues. In contrast, seaweed alginates are not acetylated. The nature of the epimerization steps are more complex in A. vinelandii than in P. aeruginosa, while other aspects of the biochemistry and genetics of alginate biosynthesis appear to be similar. The GulA residue content and distribution strongly affect the physicochemical properties of alginates, and the epimerization process is therefore of great interest from an applied point of view. This article presents a survey of our current knowledge of the molecular genetics and biochemistry of bacterial alginate biosynthesis, as well as of the biotechnological potential of such polymers.
A complete set of genes responsible for the biosynthesis of the antifungal polyene antibiotic nystatin in S. noursei ATCC 11455 has been cloned and analyzed. This represents the first example of the complete DNA sequence analysis of a polyene antibiotic biosynthetic gene cluster. Manipulation of the genes identified within the cluster may potentially lead to the generation of novel polyketides and yield improvements in the production strains.
Bacterial alginates are produced as 1-4-linked -D-mannuronan, followed by epimerization of some of the mannuronic acid residues to ␣-L-guluronic acid. Here we report the isolation of four different epimerizationdefective point mutants of the periplasmic Pseudomonas fluorescens mannuronan C-5-epimerase AlgG. All mutations affected amino acids conserved among AlgG-epimerases and were clustered in a part of the enzyme also sharing some sequence similarity to a group of secreted epimerases previously reported in Azotobacter vinelandii. An algG-deletion mutant was constructed and found to produce predominantly a dimer containing a 4-deoxy-L-erythro-hex-4-enepyranosyluronate residue at the nonreducing end and a mannuronic acid residue at the reducing end. The production of this dimer is the result of the activity of an alginate lyase, AlgL, whose in vivo activity is much more limited in the presence of AlgG. A strain expressing both an epimerase-defective (point mutation) and a wild-type epimerase was constructed and shown to produce two types of alginate molecules: one class being pure mannuronan and the other having the wild-type content of guluronic acid residues. This formation of two distinct classes of polymers in a genetically pure cell line can be explained by assuming that AlgG is part of a periplasmic protein complex.
BackgroundProduction of recombinant proteins in bacteria for academic and commercial purposes is a well established field; however the outcomes of process developments for specific proteins are still often unpredictable. One reason is the limited understanding of the performance of expression cassettes relative to each other due to different genetic contexts. Here we report the results of a systematic study aiming at exclusively comparing commonly used regulator/promoter systems by standardizing the designs of the replicon backbones.ResultsThe vectors used in this study are based on either the RK2- or the pMB1- origin of replication and contain the regulator/promoter regions of XylS/Pm (wild-type), XylS/Pm ML1-17 (a Pm variant), LacI/PT7lac, LacI/Ptrc and AraC/PBAD to control expression of different proteins with various origins. Generally and not unexpected high expression levels correlate with high replicon copy number and the LacI/PT7lac system generates more transcript than all the four other cassettes. However, this transcriptional feature does not always lead to a correspondingly more efficient protein production, particularly if protein functionality is considered. In most cases the XylS/Pm ML1-17 and LacI/PT7lac systems gave rise to the highest amounts of functional protein production, and the XylS/Pm ML1-17 is the most flexible in the sense that it does not require any specific features of the host. The AraC/PBAD system is very good with respect to tightness, and a commonly used bioinformatics prediction tool (RBS calculator) suggested that it has the most translation-efficient UTR. Expression was also studied by flow cytometry in individual cells, and the results indicate that cell to cell heterogeneity is very relevant for understanding protein production at the population level.ConclusionsThe choice of expression system needs to be evaluated for each specific case, but we believe that the standardized vectors developed for this study can be used to more easily identify the nature of case-specific bottlenecks. By then taking into account the relevant characteristics of each expression cassette it will be easier to make the best choice with respect to the goal of achieving high levels of protein expression in functional or non-functional form.
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