Engineering cofactor metabolism for improved protein and glucoamylase production in Aspergillus niger

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dc.contributor.authorSui, Yu-fei
dc.contributor.authorSchütze, Tabea
dc.contributor.authorOuyang, Li-ming
dc.contributor.authorLu, Hongzhong
dc.contributor.authorLiu, Peng
dc.contributor.authorXiao, Xianzun
dc.contributor.authorQi, Jie
dc.contributor.authorZhuang, Ying-Ping
dc.contributor.authorMeyer, Vera
dc.date.accessioned2021-04-08T07:02:34Z
dc.date.available2021-04-08T07:02:34Z
dc.date.issued2020-10-23
dc.description.abstractBackground: Nicotinamide adenine dinucleotide phosphate (NADPH) is an important cofactor ensuring intracellular redox balance, anabolism and cell growth in all living systems. Our recent multi-omics analyses of glucoamylase (GlaA) biosynthesis in the filamentous fungal cell factory Aspergillus niger indicated that low availability of NADPH might be a limiting factor for GlaA overproduction. Results: We thus employed the Design-Build-Test-Learn cycle for metabolic engineering to identify and prioritize effective cofactor engineering strategies for GlaA overproduction. Based on available metabolomics and 13 C metabolic flux analysis data, we individually overexpressed seven predicted genes encoding NADPH generation enzymes under the control of the Tet-on gene switch in two A. niger recipient strains, one carrying a single and one carrying seven glaA gene copies, respectively, to test their individual effects on GlaA and total protein overproduction. Both strains were selected to understand if a strong pull towards glaA biosynthesis (seven gene copies) mandates a higher NADPH supply compared to the native condition (one gene copy). Detailed analysis of all 14 strains cultivated in shake flask cultures uncovered that overexpression of the gsdA gene (glucose 6-phosphate dehydrogenase), gndA gene (6-phosphogluconate dehydrogenase) and maeA gene (NADP-dependent malic enzyme) supported GlaA production on a subtle (10%) but significant level in the background strain carrying seven glaA gene copies. We thus performed maltose-limited chemostat cultures combining metabolome analysis for these three isolates to characterize metabolic-level fluctuations caused by cofactor engineering. In these cultures, overexpression of either the gndA or maeA gene increased the intracellular NADPH pool by 45% and 66%, and the yield of GlaA by 65% and 30%, respectively. In contrast, overexpression of the gsdA gene had a negative effect on both total protein and glucoamylase production. Conclusions: This data suggests for the first time that increased NADPH availability can indeed underpin protein and especially GlaA production in strains where a strong pull towards GlaA biosynthesis exists. This data also indicates that the highest impact on GlaA production can be engineered on a genetic level by increasing the flux through the pentose phosphate pathway ( gndA gene) followed by engineering the flux through the reverse TCA cycle ( maeA gene). We thus propose that NADPH cofactor engineering is indeed a valid strategy for metabolic engineering of A. niger to improve GlaA production, a strategy which is certainly also applicable to the rational design of other microbial cell factories.en
dc.description.sponsorshipTU Berlin, Open-Access-Mittel – 2020en
dc.identifier.eissn1475-2859
dc.identifier.urihttps://depositonce.tu-berlin.de/handle/11303/12957
dc.identifier.urihttp://dx.doi.org/10.14279/depositonce-11752
dc.language.isoen
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/
dc.subject.ddc540 Chemie und zugeordnete Wissenschaftenen
dc.subject.otherAspergillus nigeren
dc.subject.otherchemostaten
dc.subject.otherCRISPR/Cas9en
dc.subject.othergenetic engineeringen
dc.subject.otherglucoamylaseen
dc.subject.othermetabolic engineeringen
dc.subject.otherNADPHen
dc.subject.othertet-onen
dc.titleEngineering cofactor metabolism for improved protein and glucoamylase production in Aspergillus nigeren
dc.typeArticleen
dc.type.versionpublishedVersionen
dcterms.bibliographicCitation.articlenumber198en
dcterms.bibliographicCitation.doi10.1186/s12934-020-01450-wen
dcterms.bibliographicCitation.issue1en
dcterms.bibliographicCitation.journaltitleMicrobial Cell Factoriesen
dcterms.bibliographicCitation.originalpublishernameSpringerNatureen
dcterms.bibliographicCitation.originalpublisherplaceLondon [u.a.]en
dcterms.bibliographicCitation.volume19en
tub.accessrights.dnbfreeen
tub.affiliationFak. 3 Prozesswissenschaften::Inst. Biotechnologie::FG Angewandte und Molekulare Mikrobiologiede
tub.affiliation.facultyFak. 3 Prozesswissenschaftende
tub.affiliation.groupFG Angewandte und Molekulare Mikrobiologiede
tub.affiliation.instituteInst. Biotechnologiede
tub.publisher.universityorinstitutionTechnische Universität Berlinen

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