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. 2015 Jan;81(2):597-603.
doi: 10.1128/AEM.02885-14. Epub 2014 Nov 7.

Engineering Neurospora Crassa for Improved Cellobiose and Cellobionate Production

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Engineering Neurospora Crassa for Improved Cellobiose and Cellobionate Production

Amanda Hildebrand et al. Appl Environ Microbiol. .
Free PMC article

Abstract

We report engineering Neurospora crassa to improve the yield of cellobiose and cellobionate from cellulose. A previously engineered strain of N. crassa (F5) with six of seven β-glucosidase (bgl) genes knocked out was shown to produce cellobiose and cellobionate directly from cellulose without the addition of exogenous cellulases. In this study, the F5 strain was further modified to improve the yield of cellobiose and cellobionate from cellulose by increasing cellulase production and decreasing product consumption. The effects of two catabolite repression genes, cre-1 and ace-1, on cellulase production were investigated. The F5 Δace-1 mutant showed no improvement over the wild type. The F5 Δcre-1 and F5 Δace-1 Δcre-1 strains showed improved cellobiose dehydrogenase and exoglucanase expression. However, this improvement in cellulase expression did not lead to an improvement in cellobiose or cellobionate production. The cellobionate phosphorylase gene (ndvB) was deleted from the genome of F5 Δace-1 Δcre-1 to prevent the consumption of cellobiose and cellobionate. Despite a slightly reduced hydrolysis rate, the F5 Δace-1 Δcre-1 ΔndvB strain converted 75% of the cellulose consumed to the desired products, cellobiose and cellobionate, compared to 18% converted by the strain F5 Δace-1 Δcre-1.

Figures

FIG 1
FIG 1
CDH and exoglucanase activities for ace-1 and/or cre-1 knockout strains at selected time points. The values shown are the means for biological triplicates. Error bars are the standard deviations.
FIG 2
FIG 2
Cellobionate consumption by the ndvB knockout F5 Δace-1 Δcre-1 ΔndvB strain compared to the parent F5 Δace-1 Δcre-1 strain. The values shown are the means for biological triplicates for each strain. Error bars are the standard deviations for the biological triplicates.
FIG 3
FIG 3
Cellobiose (A) and cellobionate (B) production by the F5 Δace-1 Δcre-1 and F5 Δace-1 Δcre-1 ΔndvB strains grown on 20 g/liter Avicel. The values shown are the means for biological triplicates. Error bars are the standard deviations.

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