Metabolic engineering of Geobacillus thermoglucosidasius for high yield ethanol production

Cripps, R. E.; Eley, Kirstin L.; Leak, David J.; Rudd, Brian A. M.; Taylor, Mark Paul; Todd, Matthew J.; Boakes, Steven; Martin, Steve; Atkinson, Tony

doi:10.1016/j.ymben.2009.08.005

Cited by 237 publications

(316 citation statements)

References 34 publications

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“…The lack of improved ethanol yields at 52°C cannot be attributed to inadequate in vitro GoxPDC thermostability at that temperature (GoxPDC retains 100% enzymic activity at 55°C after 1 hour incubation The high ethanol yield at 45°C is significant and consistent with yields reported in other multiply mutated thermophiles (Shaw et al, 2008;Cripps et al, 2009). It has been demonstrated that effective partitioning of carbon into biosynthesis and fermentation is critical in achieving optimal production of ethanol under fermentative conditions (Underwood e t a l .…”

Section: Expression Of Goxpdc Wt and Goxpdc Opt In E Coli And G Thesupporting

confidence: 61%

“…thermoglucosidasius (Cripps et al, 2009). The promoter region was amplified from pTMO49 using the LDHF and LDHR primers and cloned into pGEM-T Easy.…”

Section: Construction Of the Pdc Knock-in Vectors And Transformationmentioning

confidence: 99%

“…Sequencing confirmed that no DNA base changes had occurred. Pldh was cloned into pGO (Table 1) thermoglucosidasius competent cells were prepared and transformed (Cripps et al, 2009).…”

Section: Construction Of the Pdc Knock-in Vectors And Transformationmentioning

confidence: 99%

“…There has been an increased interest in the use of thermophiles, such as Geobacillus thermoglucosidasius, for ethanol production, primarily because of their catabolic promiscuity, an important benefit for a second generation bioprocess design (Cripps et al, 2009;. Other advantages include improved product removal, reduced incidence of contamination and high ethanol yields in selectively mutated strains ).…”

Section: Introductionmentioning

confidence: 99%

“…Ethanol production in G. thermoglucosidasius and mutants with enhanced ethanologenic phenotypes relies on endogenous metabolic pathways, generating acetyl CoA via pyruvate dehydrogenase and its subsequent conversion to acetaldehyde and ethanol by aldehyde dehydrogenase and alcohol dehydrogenase, respectively (Cripps et al, 2009). An alternative to further develop G. thermoglucosidasius as an ethanologenic strain is to engineer the expression of the PDC pathway.…”

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: Expression Of Goxpdc Wt and Goxpdc Opt In E Coli And G Thesupporting

confidence: 61%

“…thermoglucosidasius (Cripps et al, 2009). The promoter region was amplified from pTMO49 using the LDHF and LDHR primers and cloned into pGEM-T Easy.…”

Section: Construction Of the Pdc Knock-in Vectors And Transformationmentioning

confidence: 99%

“…Sequencing confirmed that no DNA base changes had occurred. Pldh was cloned into pGO (Table 1) thermoglucosidasius competent cells were prepared and transformed (Cripps et al, 2009).…”

Section: Construction Of the Pdc Knock-in Vectors And Transformationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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