Development of a versatile shuttle vector for gene expression in Geobacillus spp.

Taylor, Mark Paul; Esteban, Carlos David; Leak, David J.

doi:10.1016/j.plasmid.2008.04.001

Cited by 70 publications

(78 citation statements)

References 35 publications

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“…For ZpaPDC, activity was absent at growth temperatures above 45°C (Taylor et al, 2008), while in this study, the GoxPDC WT showed no activity at 45°C (see above). These data collectively suggest that an analysis of the number of rare or infrequently used codons is insufficient to accurately predict the ability of genes to express in heterologous hosts.…”

Section: Assessing Codon Usage and Predicting A Gene Sequence For Harcontrasting

confidence: 53%

“…The reasons for the low levels of activity are not fully understood (Taylor et al, 2008;Thompson et al, 2008), but protein misfolding resulting in inactive protein has been proposed (Thompson et al ., 2008). Attempts to express these proteins in mesophilic Gram positive hosts (notably lactic acid bacteria and Bacillus megatarium) have also had limited success (Gold et al, 1996;Bongers et al, 2005;Kaczowka et al, 2005;Liu et al, 2005;Talarico et al, 2005;Liu et al, 2006;Liu et al, 2007;Orencio-Trejo et al, 2008;Bi et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

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Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius

Zyl

Taylor

Eley

et al. 2013

Appl Microbiol Biotechnol

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This study reports the expression, purification and kinetic characterization of a PDC from Gluconobacter oxydans. Kinetic analyses showed the enzyme to have high affinity for pyruvate (120µM at pH 5), high catalytic efficiency (4.75 x 10 5 M -1 s -1 at pH 5), a pH opt of approximately 4.5 and an in vitro temperature optimum at approximately 55°C (the highest yet reported for a b a c t e r i a l P D C ) . D u e t o g o o d in vitro thermostablity (approximately 40% enzyme activity retained after 30 minutes at 65°C) this PDC was considered to be a suitable candidate for heterologous expression in the thermophile Geobacillus thermoglucosidasius. Initial studies using a variety of methods failed to detect activity at any growth temperature. However, the application of codon harmonization (i.e., mimicry of the heterogeneous host's transcription and translational rhythm) yielded a protein that was fully functional in the thermophilic strain at 45°C (as determined by enzyme activity, Western blot, mRNA detection and ethanol productivity). Here we describe the successful expression of PDC in a true thermophile. Yields as high as 0.35 g/g ±0.04 ethanol per gram of glucose consumed were detected, highly competitive to those reported in ethanologenic thermophilic mutants. Although activities could not be detected at temperatures approaching the growth optimum for the strain, this study highlights that the possibility that previously unsuccessful expression of pdcs in Geobacillus spp. may be the result of ineffective transcription / translation coupling.

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Section: Assessing Codon Usage and Predicting A Gene Sequence For Harcontrasting

confidence: 53%

Section: Introductionmentioning

confidence: 99%

Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius

Zyl

Taylor

Eley

et al. 2013

Appl Microbiol Biotechnol

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“…The 44 selected pure cultures were tested for their transformation efficiency by electroporation. Depending on the species, slightly modified versions of either a Bacillus (29) or a Geobacillus (24) protocol were used to prepare competent cells. Subsequently, the obtained cell suspensions were electroporated using E. coli-Geobacillus/Bacillus shuttle vector pNW33n.…”

Section: Resultsmentioning

confidence: 99%

“…Depending on their 16S rRNA genebased genus identification, strains selected in the initial selection were subjected to a transformation protocol for either Geobacillus (24) or Bacillus (29), with slight modifications as follows. Strains were grown overnight at their isolation temperature in 10 ml medium in a 50-ml Greiner tube, and the next morning, cultures were diluted to an OD 600 of 0.08 in 100 ml medium for Bacillus species and 50 ml medium for Geobacillus species.…”

Section: Mediamentioning

confidence: 99%

“…Electroporation settings were a voltage of 2.0 kV, a capacity of 25 F, and a resistance of 200 ⍀ in a 2-mm cuvette (based on reference 30) for all strains. Additional settings were tested as 1.5 kV, 25 F, and 600 ⍀ in a 1-mm cuvette (29) for Bacillus strains ET 138, ET 143, ET 224, ET 226, ET 229, ET 230-1, ET 239-2, ET 244, ET 261-1, and ET 263; 2.5 kV, 25 F, and 600 ⍀ in a 1-mm cuvette (24) for Geobacillus strains ET 006, ET 011-1, ET 036, ET 042, ET 050, and ET 208; and 2.5 kV, 25 F, and 600 ⍀ in a 2-mm cuvette (based on reference 24) for Geobacillus strains ET 006, ET 036, ET 042, ET 050, ET 072, ET 129, and ET 208. Recovery was performed at 3°C below the isolation temperatures for 3 h in RG2 medium (LB2 with 121 g/liter sucrose and 10 g/liter glucose) for Bacillus species and for 2 h in LB2 or TGP2 supplemented with 0.5% (wt/vol) glucose for Geobacillus species.…”

Section: Mediamentioning

confidence: 99%

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Isolation and Screening of Thermophilic Bacilli from Compost for Electrotransformation and Fermentation: Characterization of Bacillus smithii ET 138 as a New Biocatalyst

Bosma

Weijer

Daas

et al. 2015

Appl Environ Microbiol

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b Thermophilic bacteria are regarded as attractive production organisms for cost-efficient conversion of renewable resources to green chemicals, but their genetic accessibility is a major bottleneck in developing them into versatile platform organisms. In this study, we aimed to isolate thermophilic, facultatively anaerobic bacilli that are genetically accessible and have potential as platform organisms. From compost, we isolated 267 strains that produced acids from C 5 and C 6 sugars at temperatures of 55°C or 65°C. Subsequently, 44 strains that showed the highest production of acids were screened for genetic accessibility by electroporation. Two Geobacillus thermodenitrificans isolates and one Bacillus smithii isolate were found to be transformable with plasmid pNW33n. Of these, B. smithii ET 138 was the best-performing strain in laboratory-scale fermentations and was capable of producing organic acids from glucose as well as from xylose. It is an acidotolerant strain able to produce organic acids until a lower limit of approximately pH 4.5. As genetic accessibility of B. smithii had not been described previously, six other B. smithii strains from the DSMZ culture collection were tested for electroporation efficiencies, and we found the type strain DSM 4216 T and strain DSM 460 to be transformable. The transformation protocol for B. smithii isolate ET 138 was optimized to obtain approximately 5 ؋ 10 3 colonies per g plasmid pNW33n. Genetic accessibility combined with robust acid production capacities on C 5 and C 6 sugars at a relatively broad pH range make B. smithii ET 138 an attractive biocatalyst for the production of lactic acid and potentially other green chemicals. Green chemicals are sustainable bio-based alternatives for chemicals based on fossil resources. Biomass is considered an attractive renewable resource for the production of such green chemicals. Major challenges for using biomass are to develop costeffective production processes and to convert substrates that go beyond first-generation pure sugars. From this perspective, the use of microbial fermentation processes that convert lignocellulosic sugars is gaining considerable attention. For particular products natural producers can be used, but often Escherichia coli and Saccharomyces cerevisiae are used as platform organisms because of their well-known physiology and ease of engineering for the production of many different products (1, 2).In general, most organisms used for the production of green chemicals and fuels are mesophiles, and current processes still are based mainly on first-generation feedstocks, i.e., sucrose from sugar beet or sugarcane or glucose derived from starches from corn or tapioca. Although the engineering of platform organisms broadens the spectrum of possible substrates (3), the efficiency of the process could be increased by the use of organisms naturally capable of degrading lignocellulose-derived sugars (4). To further increase the efficiency and reduce the costs of the microbial production of green chemicals, modera...

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Extremophiles and their Application to Biofuel Research

Taylor¹,

Bauer²,

Mackay³

et al. 2012

Extremophiles

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Development of a versatile shuttle vector for gene expression in Geobacillus spp.

Cited by 70 publications

References 35 publications

Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius

Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius

Isolation and Screening of Thermophilic Bacilli from Compost for Electrotransformation and Fermentation: Characterization of Bacillus smithii ET 138 as a New Biocatalyst

Extremophiles and their Application to Biofuel Research

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