BackgroundThe development of modern producer strains with metabolically engineered pathways poses special problems that often require manipulating many genes and expressing them individually at different levels or under separate regulatory controls. The construction of plasmid-less marker-less strains has many advantages for the further practical exploitation of these bacteria in industry. Such producer strains are usually constructed by sequential chromosome modifications including deletions and integration of genetic material. For these purposes complex methods based on in vitro and in vivo recombination processes have been developed.ResultsHere, we describe the new scheme of insertion of the foreign DNA for step-by-step construction of plasmid-less marker-less recombinant E. coli strains with chromosome structure designed in advance. This strategy, entitled as Dual-In/Out, based on the initial Red-driven insertion of artificial φ80-attB sites into desired points of the chromosome followed by two site-specific recombination processes: first, the φ80 system is used for integration of the recombinant DNA based on selective marker-carrier conditionally-replicated plasmid with φ80-attP-site, and second, the λ system is used for excision of inserted vector part, including the plasmid ori-replication and the marker, flanked by λ-attL/R-sites.ConclusionThe developed Dual-In/Out strategy is a rather straightforward, but convenient combination of previously developed recombination methods: phages site-specific and general Red/ET-mediated. This new approach allows us to detail the design of future recombinant marker-less strains, carrying, in particular, rather large artificial insertions that could be difficult to introduce by usually used PCR-based Recombineering procedure. The developed strategy is simple and could be particularly useful for construction of strains for the biotechnological industry.
The gene cluster for methylamine utilization (mau genes) has been cloned from the obligate methylotrophic bacterium Methylobacillus flagellatum KT. Partial sequence data showed that the organization of these genes was similar to that found in Methylophilus methylotrophus W3A1-NS, including the lack of a gene for amicyanin, which had been thought to be the electron acceptor for methylamine dehydrogenase in M. flagellatum KT. However, a gene encoding azurin was discovered at the 3 end of the mau gene cluster, transcribed in the opposite orientation. A mutant with a defect in this gene showed impaired growth on methylamine, suggesting that azurin is involved in methylamine oxidation in M. flagellatum KT.Methylamine dehydrogenase (MADH) is the enzyme that oxidizes methylamine to formaldehyde in many gram-negative bacteria that grow on methylamine (13,17,20,(21)(22)(23)(24)27). In all cases that have been studied, MADH has been a periplasmic protein consisting of two large subunits and two small subunits (17,20,(22)(23)(24)27). Each small subunit has a covalently bound prosthetic group called tryptophan tryptophylquinone synthesized from two tryptophans belonging to the small-subunit polypeptide chain (28). Two types of electron acceptors for MADH are known. MADH is thought to use a c-type cytochrome in Methylophilus strains (4, 6), whereas MADHs from the other methylotrophs are thought to use blue copper proteins, or cupredoxins, as electron acceptors (2,11,19,24,31,32,34). Three classes of cupredoxins are found in methylotrophs, amicyanins, azurins, and pseudoazurins. By definition, the cupredoxin that accepts electrons from MADH is usually termed amicyanin (2). However, for at least one methylotroph, Methylomonas sp. strain J, the amino acid composition shows this cupredoxin to be an azurin. Several methylotrophs, including Methylobacillus flagellatum KT, are known to have two cupredoxins (1,2,11,24). For M. flagellatum KT, one was methylamine inducible and was assumed to be amicyanin (11). In two strains, organism 4025 and M. flagellatum KT, cells can grow slowly on methylamine in a medium depleted of copper, conditions under which cupredoxins are absent (11,24). An unknown cytochrome was suspected to function under such growth conditions, since the known cytochrome c's in these bacteria do not accept electrons from MADH in vitro.Genetic analysis has shown that in the ␣-proteobacteria containing MADH, the genes required for synthesis of active MADH (mau genes) are clustered and include a gene encoding amicyanin (7,8,33,35). The mau gene cluster of Methylophilus methylotrophus W3A1-NS is similar except that it does not include the amicyanin gene, as expected, since this strain does not contain amicyanin (9). Since the electron acceptor for MADH in M. flagellatum KT is uncertain, we have analyzed the mau gene cluster of this bacterium to determine whether the amicyanin gene was present.Escherichia coli DH5␣ (New England Biolabs) was grown in Luria-Bertani medium in the presence of appropriate antibiotics as describ...
The organization of genes involved in utilization of methylamine (mau genes) was studied in the obligate methylotroph ' Methylobacillus flagellatum ' KT. Nine open reading frames were identified as corresponding to the genes mauFBEDAGLMN. In addition, an open reading frame (orf-1) encoding a polypeptide with unknown function was identified upstream of the rnau gene cluster. Subclones of the 'M. flagellatum KT gene cluster were used for complementation of a series of chemically induced mau mutants of 'M. flagellatum' KT. Mutants in mauF, mauB, mauEID, mauA, mauG, mauL and mauM were identified. Two mutants (mau-18 and mau-19) were not complemented b y the known mau genes. Since none of the chemically induced mutants studied had a defect in orf-1 or mauN, insertion mutants in these genes were constructed. Phenotypically the mutants fell into three groups. The mauF, mauB, mauEID, mauA, mauG, mauL and mauM mutants do not grow on methylamine as a source of carbon and lack methylamine dehydrogenase activity, but they synthesize both the large and the small subunit polypeptides albeit a t different ratios. The mau-18 and mau-19 mutants do not grow on methylamine as a source of carbon, and lack both methylamine dehydrogenase activity and the methylamine dehydrogenase subunits. The orf-1 and mauN mutants grow on methylamine as a source of carbon and synthesize wild-type levels of methylamine dehydrogenase. It has been shown earlier that the product of the mauM gene is not required for synthesis of active methylamine dehydrogenase in Methylobacterium extorquens AM1 and Paracoccus denitrificans. However, MauM is required for synthesis of functional methylamine dehydrogenase in 'M. flagellatum I .
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.