A Set of Versatile Brick Vectors and Promoters for the Assembly, Expression, and Integration of Synthetic Operons in <i>Methylobacterium extorquens</i> AM1 and Other Alphaproteobacteria

Borzyskowski, Lennart Schada von; Remus-Emsermann, Mitja N. P.; Weishaupt, Ramon; Vorholt, Julia A.; Erb, Tobias J.

doi:10.1021/sb500221v

Cited by 52 publications

(46 citation statements)

References 71 publications

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“…2B and D). The vector backbone of pREDSIX contains mCherry constitutively expressed from the M. extorquens Tu translation elongation factor gene promoter (PtuF), which functions efficiently in a broad range of bacterial species (30). Accordingly, clones that have undergone only one homologous recombination event have the entire plasmid inserted in the genome and therefore exhibit red fluorescence upon appropriate excitation.…”

Section: Resultsmentioning

confidence: 99%

Versatile Vectors for Efficient Mutagenesis of Bradyrhizobium diazoefficiens and Other Alphaproteobacteria

Ledermann

Strebel

Kampik

et al. 2016

Appl Environ Microbiol

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Analysis of bacterial gene function commonly relies on gene disruption or replacement followed by phenotypic characterization of the resulting mutant strains. Deletion or replacement of targeted regions is commonly achieved via two homologous recombination (HR) events between the bacterial genome and a nonreplicating plasmid carrying DNA fragments flanking the region to be deleted. The counterselection of clones that have integrated the entire plasmid in their genome via a single HR event is crucial in this procedure. Various genetic tools and well-established protocols are available for this type of mutagenesis in model bacteria; however, these methods are not always efficiently applicable in less established systems. Here we describe the construction and application of versatile plasmid vectors pREDSIX and pTETSIX for marker replacement and markerless mutagenesis, respectively. Apart from an array of restriction sites optimized for cloning of GC-rich DNA fragments, the vector backbone contains a constitutively expressed gene for mCherry, enabling the rapid identification of clones originating from single or double HR events by fluorescence-assisted cell sorting (FACS). In parallel, we constructed a series of plasmids from which gene cassettes providing resistance against gentamicin, kanamycin, hygromycin B, streptomycin and spectinomycin, or tetracycline were excised for use with pREDSIX-based marker replacement mutagenesis. In proof-of-concept mutagenesis experiments, we demonstrated the potential for the use of the developed tools for gene deletion mutagenesis in the nitrogen-fixing soybean symbiont Bradyrhizobium diazoefficiens (formerly Bradyrhizobium japonicum) and three additional members of the alphaproteobacteria. IMPORTANCEMutation and phenotypic analysis are essential to the study of gene function. Efficient mutagenesis protocols and tools are available for many bacterial species, including various model organisms; however, genetic analysis of less-well-characterized organisms is often impaired by the lack of efficient methods. Here we describe a set of novel genetic tools for facilitated mutagenesis of the nitrogen-fixing soybean symbiont Bradyrhizobium diazoefficiens and related alphaproteobacteria. We demonstrated their usefulness by generating several mutant strains lacking defined genes. Isolation of both antibiotic resistance gene-containing and markerless deletion mutants is greatly facilitated because undesired clones which contain the entire mutagenic plasmid integrated in the genome can be identified on the basis of their fluorescent phenotype derived from the mCherry gene carried by the vector backbone. The possibility to generate markerless mutants assists with the isolation of strains carrying multiple deletions, which can be crucial while studying functionally redundant genes. Many functional studies of biological phenomena rely on the isolation of mutants and the availability of the respective genetic tools. Mutants are preferably constructed in a targeted manner, which traditi...

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Section: Resultsmentioning

confidence: 99%

Versatile Vectors for Efficient Mutagenesis of Bradyrhizobium diazoefficiens and Other Alphaproteobacteria

Ledermann

Strebel

Kampik

et al. 2016

Appl Environ Microbiol

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“…Consequently, for each new metabolic gene, not only expression levels but also enzyme activities and the impact on fitness have to be tested. Therefore, besides P mxaF and P lac , a couple of other promoters with different strengths are already available for heterologous expression in strain AM1 (32,33). In addition, inducible vectors allowing titratable activation of promoters have been developed (34).…”

Section: Discussionmentioning

confidence: 99%

Production of 2-Hydroxyisobutyric Acid from Methanol by Methylobacterium extorquens AM1 Expressing ( R )-3-Hydroxybutyryl Coenzyme A-Isomerizing Enzymes

Rohde

Tischer

Harms

et al. 2017

Appl Environ Microbiol

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The biotechnological production of the methyl methacrylate precursor 2-hydroxyisobutyric acid (2-HIBA) via bacterial poly-3-hydroxybutyrate (PHB) overflow metabolism requires suitable (R)-3-hydroxybutyryl coenzyme A (CoA)-specific coenzyme B 12 -dependent mutases (RCM). Here, we characterized a predicted mutase from Bacillus massiliosenegalensis JC6 as a mesophilic RCM closely related to the thermophilic enzyme previously identified in Kyrpidia tusciae DSM 2912 (M.-T. Weichler et al., Appl Environ Microbiol 81:4564 -4572, 2015, https://doi.org/10.1128/ AEM.00716-15). Using both RCM variants, 2-HIBA production from methanol was studied in fed-batch bioreactor experiments with recombinant Methylobacterium extorquens AM1. After complete nitrogen consumption, the concomitant formation of PHB and 2-HIBA was achieved, indicating that both sets of RCM genes were successfully expressed. However, although identical vector systems and incubation conditions were chosen, the metabolic activity of the variant bearing the RCM genes from strain DSM 2912 was severely inhibited, likely due to the negative effects caused by heterologous expression. In contrast, the biomass yield of the variant expressing the JC6 genes was close to the wild-type performance, and 2-HIBA titers of 2.1 g liter Ϫ1 could be demonstrated. In this case, up to 24% of the substrate channeled into overflow metabolism was converted to the mutase product, and maximal combined 2-HIBA plus PHB yields from methanol of 0.11 g g Ϫ1 were achieved. Reverse transcription-quantitative PCR analysis revealed that metabolic genes, such as methanol dehydrogenase and acetoacetyl-CoA reductase genes, are strongly downregulated after exponential growth, which currently prevents a prolonged overflow phase, thus preventing higher product yields with strain AM1.IMPORTANCE In this study, we genetically modified a methylotrophic bacterium in order to channel intermediates of its overflow metabolism to the C 4 carboxylic acid 2-hydroxyisobutyric acid, a precursor of acrylic glass. This has implications for biotechnology, as it shows that reduced C 1 substrates, such as methanol and formic acid, can be alternative feedstocks for producing today's commodities. We found that product titers and yields depend more on host physiology than on the activity of the introduced heterologous function modifying the overflow metabolism. In addition, we show that the fitness of recombinant strains substantially varies when they express orthologous genes from different origins. Further studies are needed to extend the overflow production phase in methylotrophic microorganisms for the implementation of biotechnological processes.KEYWORDS acyl-CoA mutase, bulk chemicals, fed-batch bioreactor, overflow metabolism, polyhydroxybutyrate

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“…[16] This strain can grow on minimal medium with succinate as sole carbon source,but not with methanol as long as it lacks an operational Ccr to restore the ethylmalonyl-CoA pathway,w hich is indispensable for growth on C1 substrates. [16] This strain can grow on minimal medium with succinate as sole carbon source,but not with methanol as long as it lacks an operational Ccr to restore the ethylmalonyl-CoA pathway,w hich is indispensable for growth on C1 substrates.…”

Section: Methodsmentioning

confidence: 99%

“…Thes ubstrate spectrum of the enzyme increased incrementally from individual single mutants to the triple mutant (Figure 4), the latter being essentially indistinguishable from at rue member of the ECR-2 subfamily.T he CcrCc triple mutant converted short-chain (1), long-chain (11), branchedchain (8), and also bulky substrates (16), demonstrating the importance of all three candidate residues in opening up the substrate binding site. .…”

Section: Angewandte Chemiementioning

confidence: 99%

Screening and Engineering the Synthetic Potential of Carboxylating Reductases from Central Metabolism and Polyketide Biosynthesis

et al. 2015

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Carboxylating enoyl-thioester reductases (ECRs) are a recently discovered class of enzymes. They catalyze the highly efficient addition of CO2 to the double bond of α,β-unsaturated CoA-thioesters and serve two biological functions. In primary metabolism of many bacteria they produce ethylmalonyl-CoA during assimilation of the central metabolite acetyl-CoA. In secondary metabolism they provide distinct α-carboxyl-acyl-thioesters to vary the backbone of numerous polyketide natural products. Different ECRs were systematically assessed with a diverse library of potential substrates. We identified three active site residues that distinguish ECRs restricted to C4 and C5-enoyl-CoAs from highly promiscuous ECRs and successfully engineered a selected ECR as proof-of-principle. This study defines the molecular basis of ECR reactivity, allowing for predicting and manipulating a key reaction in natural product diversification.

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A Set of Versatile Brick Vectors and Promoters for the Assembly, Expression, and Integration of Synthetic Operons in Methylobacterium extorquens AM1 and Other Alphaproteobacteria

Cited by 52 publications

References 71 publications

Versatile Vectors for Efficient Mutagenesis of Bradyrhizobium diazoefficiens and Other Alphaproteobacteria

Versatile Vectors for Efficient Mutagenesis of Bradyrhizobium diazoefficiens and Other Alphaproteobacteria

Production of 2-Hydroxyisobutyric Acid from Methanol by Methylobacterium extorquens AM1 Expressing ( R )-3-Hydroxybutyryl Coenzyme A-Isomerizing Enzymes

Screening and Engineering the Synthetic Potential of Carboxylating Reductases from Central Metabolism and Polyketide Biosynthesis

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