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Integration of in vivo and in silico metabolic fluxes for improvement of recombinant protein production

Paper ID Volume ID Publish Year Pages File Format Full-Text
31628 44825 2012 12 PDF Available
Title
Integration of in vivo and in silico metabolic fluxes for improvement of recombinant protein production
Abstract

The filamentous fungus Aspergillus niger is an efficient host for the recombinant production of the glycosylated enzyme fructofuranosidase, a biocatalyst of commercial interest for the synthesis of pre-biotic sugars. In batch culture on a minimal glucose medium, the recombinant strain A. niger SKAn1015, expressing the fructofuranosidase encoding suc1 gene secreted 45 U/mL of the target enzyme, whereas the parent wild type SKANip8 did not exhibit production. The production of the recombinant enzyme induced a significant change of in vivo fluxes in central carbon metabolism, as assessed by 13C metabolic flux ratio analysis. Most notably, the flux redistribution enabled an elevated supply of NADPH via activation of the cytosolic pentose phosphate pathway (PPP) and mitochondrial malic enzyme, whereas the flux through energy generating TCA cycle was reduced. In addition, the overall possible flux space of fructofuranosidase producing A. niger was investigated in silico by elementary flux mode analysis. This provided theoretical flux distributions for multiple scenarios with differing production capacities. Subsequently, the measured flux changes linked to improved production performance were projected into the in silico flux space. This provided a quantitative evaluation of the achieved optimization and a priority ranked target list for further strain engineering. Interestingly, the metabolism was shifted largely towards the optimum flux pattern by sole expression of the recombinant enzyme, which seems an inherent attractive property of A. niger. Selected fluxes, however, changed contrary to the predicted optimum and thus revealed novel targets—including reactions linked to NADPH metabolism and gluconate formation.

► Coupling of 13C flux and elementary flux mode analysis for systems analysis. ► Novel approach integrating in vivo and in silico flux analysis for strain design. ► Optimization of recombinant enzyme production in filamentous fungi. ► Identification of NADPH metabolism as key target towards improved protein production.

Keywords
Fructofuranosidase; Aspergillus niger; 13C metabolic flux; Metabolic engineering; Filamentous fungi; Elementary mode
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Integration of in vivo and in silico metabolic fluxes for improvement of recombinant protein production
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Publisher
Database: Elsevier - ScienceDirect
Journal: Metabolic Engineering - Volume 14, Issue 1, January 2012, Pages 47–58
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us