Matching Items (4)

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Activity of Lactobacillus brevis Alcohol Dehydrogenase on Primary and Secondary Alcohol Biofuel Precursors

Description

The R-specific alcohol dehydrogenase (ADH) from Lactobacillus brevis LB19 (LbADH) was studied with respect to its ability to reduce a series of 3- through 5-carbon 2-alkanones and aldehydes of relevance

The R-specific alcohol dehydrogenase (ADH) from Lactobacillus brevis LB19 (LbADH) was studied with respect to its ability to reduce a series of 3- through 5-carbon 2-alkanones and aldehydes of relevance as biofuel precursors. Although active on all substrates tested, LbADH displays a marked preference for longer chain substrates. Interestingly, however, 2-alkanones were found to impose substrate inhibition towards LbADH, whereas aldehyde substrates rendered no such effect. Inhibition caused by 2-alkanones was furthermore found to intensify with increasing chain length. Despite demonstrating both primary and secondary ADH activities, a preliminary sequence analysis suggests that LbADH remains distinct from other, previously characterized primary-secondary ADHs. In addition to further characterizing the substrate range of this industrially important enzyme, this study suggests that LbADH has the potential to serve as a useful enzyme for the engineering of various novel alcohol biofuel pathways.

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  • 2015-08-05

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Characterizing the activity of alcohol dehydrogenase from Lactobacillus brevis on primary and secondary alcohol biofuel precursors

Description

The R-specific alcohol dehydrogenase (RADH or LVIS_0347) from Lactobacillus brevis LB19 was found to possess activity on several short chain aldehydes and ketones. This broad substrate specificity was previously uncharacterized.

The R-specific alcohol dehydrogenase (RADH or LVIS_0347) from Lactobacillus brevis LB19 was found to possess activity on several short chain aldehydes and ketones. This broad substrate specificity was previously uncharacterized. To demonstrate its relevance to the biofuels industry as well as its broader utility for chiral reductions, a detailed characterization was performed to further investigate the activity and function of RADH.

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Date Created
  • 2015-05

Rational and combinatorial approaches to engineering styrene production by Saccharomyces cerevisiae

Description

Background
Styrene is an important building-block petrochemical and monomer used to produce numerous plastics. Whereas styrene bioproduction by Escherichia coli was previously reported, the long-term potential of this approach will

Background
Styrene is an important building-block petrochemical and monomer used to produce numerous plastics. Whereas styrene bioproduction by Escherichia coli was previously reported, the long-term potential of this approach will ultimately rely on the use of hosts with improved industrial phenotypes, such as the yeast Saccharomyces cerevisiae.
Results
Classical metabolic evolution was first applied to isolate a mutant capable of phenylalanine over-production to 357 mg/L. Transcription analysis revealed up-regulation of several phenylalanine biosynthesis pathway genes including ARO3, encoding the bottleneck enzyme DAHP synthase. To catalyze the first pathway step, phenylalanine ammonia lyase encoded by PAL2 from A. thaliana was constitutively expressed from a high copy plasmid. The final pathway step, phenylacrylate decarboxylase, was catalyzed by the native FDC1. Expression of FDC1 was naturally induced by trans-cinnamate, the pathway intermediate and its substrate, at levels sufficient for ensuring flux through the pathway. Deletion of ARO10 to eliminate the competing Ehrlich pathway and expression of a feedback-resistant DAHP synthase encoded by ARO4[subscript K229L] preserved and promoted the endogenous availability precursor phenylalanine, leading to improved pathway flux and styrene production. These systematic improvements allowed styrene titers to ultimately reach 29 mg/L at a glucose yield of 1.44 mg/g, a 60% improvement over the initial strain.
Conclusions
The potential of S. cerevisiae as a host for renewable styrene production has been demonstrated. Significant strain improvements, however, will ultimately be needed to achieve economical production levels.

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Date Created
  • 2014-08-21

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Biosynthetic production of aromatic fine chemicals

Description

This dissertation focuses on the biosynthetic production of aromatic fine chemicals in engineered Escherichia coli from renewable resources. The discussed metabolic pathways take advantage of key metabolites in the shikimic

This dissertation focuses on the biosynthetic production of aromatic fine chemicals in engineered Escherichia coli from renewable resources. The discussed metabolic pathways take advantage of key metabolites in the shikimic acid pathway, which is responsible for the production of the aromatic amino acids phenylalanine, tyrosine, and tryptophan. For the first time, the renewable production of benzaldehyde and benzyl alcohol has been achieved in recombinant E. coli with a maximum titer of 114 mg/L of benzyl alcohol. Further strain development to knockout endogenous alcohol dehydrogenase has reduced the in vivo degradation of benzaldehyde by 9-fold, representing an improved host for the future production of benzaldehyde as a sole product. In addition, a novel alternative pathway for the production of protocatechuate (PCA) and catechol from the endogenous metabolite chorismate is demonstrated. Titers for PCA and catechol were achieved at 454 mg/L and 630 mg/L, respectively. To explore potential routes for improved aromatic product yields, an in silico model using elementary mode analysis was developed. From the model, stoichiometric optimums maximizing both product-to-substrate and biomass-to-substrate yields were discovered in a co-fed model using glycerol and D-xylose as the carbon substrates for the biosynthetic production of catechol. Overall, the work presented in this dissertation highlights contributions to the field of metabolic engineering through novel pathway design for the biosynthesis of industrially relevant aromatic fine chemicals and the use of in silico modelling to identify novel approaches to increasing aromatic product yields.

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Date Created
  • 2016