pNEB193-derived suicide plasmids for gene deletion and protein expression in the methane-producing archaeon, Methanosarcina acetivorans

Shea, Mitchell T.; Walter, Mary E.; Duszenko, Nikolas; Ducluzeau, Anne Lise; Aldridge, Jared; King, Shannon K.; Buan, Nicole R.

doi:10.1016/j.plasmid.2016.02.003

Cited by 10 publications

(9 citation statements)

References 82 publications

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“…To generate isoprene-synthesizing methanogens, we cloned the codon-optimized ispS gene from poplar (Populus alba) into the Methanosarcina spp. overexpression suicide vector pNB730 (Figure 2a) (30,31). Once transfected into cells, the resulting plasmid pJA2 integrates into the chromosome, resulting in constitutive overexpression of synthetic ispS from the methyl coenzyme M (29).…”

Section: Resultsmentioning

confidence: 99%

“…All plasmids were verified by sequencing (Eurofins, Louisville, KY). Plasmid pNB730 was used as a parent vector (31). Key features of pNB730 include: (i) pUC ori for highcopy replication in E. coli; (ii) w C31 phage recombinase att site for chromosomal insertion of the vector into the host genome; (iii) resistance to ampicillin for selection during amplification in Escherichia coli; and (iv) puromycin resistance for selection in Methanosarcina spp.…”

Section: Methodsmentioning

confidence: 99%

“…Electroporated E. coli cells were plated on lysis broth (LB) agarose plates with 100 mg liter 21 ampicillin and colonies were selected after overnight growth at 37°C (53). Plasmids were screened by PCR as described and sequenced (31). Plasmids were transfected into Methanosarcina spp.…”

Section: Methodsmentioning

confidence: 99%

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Anaerobic Production of Isoprene by Engineered Methanosarcina Species Archaea

Aldridge

Carr

Weber

et al. 2021

Appl Environ Microbiol

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Isoprene is a valuable petrochemical used for a wide variety of consumer goods such as adhesives and synthetic rubber. We were able to achieve high yield of renewable isoprene by taking advantage of the naturally high-flux mevalonate lipid synthesis pathway in anaerobic methane-producing archaea (methanogens). Our study illustrates that by genetically manipulating Methanosarcina spp. methanogens it is possible to create organisms that grow by producing the hemiterpene isoprene. Mass balance measurements show engineered methanogens direct up to 4% total carbon flux to isoprene, demonstrating methanogens produce higher isoprene yields than engineered yeast, bacteria, or cyanobacteria from inexpensive feedstocks. Expression of isoprene synthase resulted in increased biomass and changes in gene expression that indicate isoprene synthesis depletes membrane precursors and redirects electron flux enabling isoprene to be a major metabolic product. Our results demonstrate methanogens are a promising engineering chassis for renewable isoprene synthesis. IMPORTANCE A significant barrier to implementing renewable chemical technologies is high production costs versus petroleum-derived products. Existing technologies using engineered organisms have difficulty competing with petroleum-derived chemicals due to the cost of feedstocks (such as glucose), product extraction, and purification. The hemiterpene monomer isoprene is one such chemical that cannot currently be produced using cost-competitive renewable biotechnologies. To reduce the cost of renewable isoprene, we have engineered methanogens to synthesize it from inexpensive feedstocks such as methane, methanol, acetate, and carbon dioxide. The "isoprenogen" strains we developed have potential to be used for industrial production of inexpensive renewable isoprene.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Anaerobic Production of Isoprene by Engineered Methanosarcina Species Archaea

Aldridge

Carr

Weber

et al. 2021

Appl Environ Microbiol

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“…Sequences were synthesized from Eurofins Scientific and are given in Supplementary file 1. Classical cloning was used to clone the synthesized fragments into pBN730 (47) for expression in M. acetivorans and pMEV4 (48) for expression in M. maripaludis . The generated plasmids were named pNB730::tdFAST2 and pMEV4::tdFAST2.…”

Section: Methodsmentioning

confidence: 99%

Application of the fluorescence-activating and absorption-shifting tag (FAST) for flow cytometry in methanogenic archaea

Adlung

Scheller²

2022

Preprint

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Methane-producing archaea play a crucial role in the global carbon cycle and are used for biotechnological fuel production. Methanogenic model organisms such as Methanococcus maripaludis and Methanosarcina acetivorans are biochemically characterized and can be genetically engineered using a variety of molecular tools. Methanogens’ anaerobic lifestyle and autofluorescence, however, restrict the use of common fluorescent reporter proteins (e.g., GFP and derivatives) which require oxygen for chromophore maturation. Here, we employ the tandem activation and absorption-shifting tag protein 2 (tdFAST2) which is fluorescent when the cell-permeable fluorescent ligand (fluorogen) 4-hydroxy-3,5-dimethoxybenzylidene rhodanine (HBR-3,5DOM) is present. tdFAST2 expression in M. acetivorans and M. maripaludis is not cytotoxic and tdFAST2:HBR-3,5DOM fluorescence can be clearly distinguished from the autofluorescence. In flow cytometry experiments, mixed methanogen cultures can be clearly distinguished which allows high-throughput investigations of dynamics within single and mixed cultures.

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“…The cdh operon promoter has also been used successfully to drive acetate-dependent overexpression of a carbonic anhydrase in M. acetivorans ( Macauley et al, 2009 ). More recent work has generated a new series of suicide plasmids that simplify the cloning process and allow for facile expression and purification of tagged proteins in Methanosarcina ( Shea et al, 2016 ). Additionally, an efficient CRISPR/Cas9 system has been developed for M. acetivorans ( Nayak and Metcalf, 2017 ).…”

Section: Methanogens As Hosts For the Heterologous Production Of Reco...mentioning

confidence: 99%

Overview of Diverse Methyl/Alkyl-Coenzyme M Reductases and Considerations for Their Potential Heterologous Expression

Gendron

Allen

2022

Front. Microbiol.

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Methyl-coenzyme M reductase (MCR) is an archaeal enzyme that catalyzes the final step of methanogenesis and the first step in the anaerobic oxidation of methane, the energy metabolisms of methanogens and anaerobic methanotrophs (ANME), respectively. Variants of MCR, known as alkyl-coenzyme M reductases, are involved in the anaerobic oxidation of short-chain alkanes including ethane, propane, and butane as well as the catabolism of long-chain alkanes from oil reservoirs. MCR is a dimer of heterotrimers (encoded by mcrABG) and requires the nickel-containing tetrapyrrole prosthetic group known as coenzyme F430. MCR houses a series of unusual post-translational modifications within its active site whose identities vary depending on the organism and whose functions remain unclear. Methanogenic MCRs are encoded in a highly conserved mcrBDCGA gene cluster, which encodes two accessory proteins, McrD and McrC, that are believed to be involved in the assembly and activation of MCR, respectively. The requirement of a unique and complex coenzyme, various unusual post-translational modifications, and many remaining questions surrounding assembly and activation of MCR largely limit in vitro experiments to native enzymes with recombinant methods only recently appearing. Production of MCRs in a heterologous host is an important step toward developing optimized biocatalytic systems for methane production as well as for bioconversion of methane and other alkanes into value-added compounds. This review will first summarize MCR catalysis and structure, followed by a discussion of advances and challenges related to the production of diverse MCRs in a heterologous host.

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pNEB193-derived suicide plasmids for gene deletion and protein expression in the methane-producing archaeon, Methanosarcina acetivorans

Cited by 10 publications

References 82 publications

Anaerobic Production of Isoprene by Engineered Methanosarcina Species Archaea

Anaerobic Production of Isoprene by Engineered Methanosarcina Species Archaea

Application of the fluorescence-activating and absorption-shifting tag (FAST) for flow cytometry in methanogenic archaea

Overview of Diverse Methyl/Alkyl-Coenzyme M Reductases and Considerations for Their Potential Heterologous Expression

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