Disruption or reduced expression of the orotidine-5'-decarboxylase gene pyrG increases citric acid production: a new discovery during recyclable genome editing in Aspergillus niger
Lihui Zhang,Lihui Zhang,Xiaomei Zheng,Timothy C. Cairns,Zhidan Zhang,Depei Wang,Ping Zheng,Jibin Sun +7 more
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TLDR
The hypothesis that uridine/pyrimidine biosynthetic pathway offer future avenues for strain engineering efforts is supported, and it is demonstrated for the first time that A. niger pyrG is a promising genetic lead for generating citric acid hyper-producing strains.Abstract:
Aspergillus niger is a filamentous fungus used for the majority of global citric acid production. Recent developments in genome editing now enable biotechnologists to engineer and optimize A. niger. Currently, however, genetic-leads for maximizing citric acid titers in industrial A. niger isolates is limited. In this study, we try to engineer two citric acid A. niger production isolates, WT-D and D353, to serve as platform strains for future high-throughput genome engineering. Consequently, we used genome editing to simultaneously disrupt genes encoding the orotidine-5′-decarboxylase (pyrG) and non-homologous end-joining component (kusA) to enable use of the pyrG selection/counter selection system, and to elevate homologous recombination rates, respectively. During routine screening of these pyrG mutant strains, we unexpectedly observed a 2.17-fold increase in citric acid production when compared to the progenitor controls, indicating that inhibition of uridine/pyrimidine synthesis may increase citric acid titers. In order to further test this hypothesis, the pyrG gene was placed under the control of a tetracycline titratable cassette, which confirmed that reduced expression of this gene elevated citric acid titers in both shake flask and bioreactor fermentation. Subsequently, we conducted intracellular metabolomics analysis, which demonstrated that pyrG disruption enhanced the glycolysis flux and significantly improved abundance of citrate and its precursors. In this study, we deliver two citric acid producing isolates which are amenable to high throughput genetic manipulation due to pyrG/kusA deletion. Strikingly, we demonstrate for the first time that A. niger pyrG is a promising genetic lead for generating citric acid hyper-producing strains. Our data support the hypothesis that uridine/pyrimidine biosynthetic pathway offer future avenues for strain engineering efforts.read more
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Comparative genomics of citric-acid producing Aspergillus niger ATCC 1015 versus enzyme-producing CBS 513.88 [working title: Aspergillus niger strain evolution]
Susan Lucas,Igor V. Grigoriev,Harris Shapiro,Andrea Aerts,Alex Atkins,Asaf Salamov,Erika Lindquist,Jasmyn Pangilinan,Yunian Lou,Jane Grimwood,Mikael Rørdam Andersen,Margarita Salazar,Peter J. Schaap,P de Vondervoot,David E. Culley,Scott E. Baker,Jette Thykaer,Jens Christian Frisvad,Kristian Fog Nielsen,Richard Albang,Kaj Albermann,Randy M. Berka,Gerhard H. Braus,Susanna A. Braus-Stromeyer,Luis M. Corrochano,Ziyu Dai,P van Dijck,Gerald Hofmann,Linda L. Lasure,J Magnusson,Susan Lisette Meijer,Jens Nielsen,Michael Lynge Nielsen,A van Ooyen,K Panther,Herman Jan Pel,Lars Kongsbak Poulsen,Rob Samson,Hein Stam,Adrian Tsang,J den Brink,Christian P. Kubicek,Diego Martinez,N van Peij,Johannes Andries Roubos +44 more
TL;DR: In this paper, the authors performed whole-genome sequencing of the Aspergillus niger wild-type strain (ATCC 1015) and produced a genome sequence of very high quality.
Journal ArticleDOI
Citric acid from Aspergillus niger: a comprehensive overview
TL;DR: The availability of genome sequence of this organism has facilitated numerous studies in gene function, gene regulation, primary and secondary metabolism, and an attempt has been also made to address the molecular mechanisms and application of recent advanced techniques such as CRISPR/Cas9 systems in enhancement of citric acid production.
Journal ArticleDOI
Microbial citric acid: Production, properties, application, and future perspectives
Journal ArticleDOI
Applications of CRISPR/Cas9 in the Synthesis of Secondary Metabolites in Filamentous Fungi.
TL;DR: In this article, the authors introduce the CRISPR/Cas9 system in detail and summarize the latest applications of CCA-mediated genome editing in filamentous fungi, and highlight and discuss some of the challenges and deficiencies of using the CAA-based genome editing technology in research on the biosynthesis of secondary metabolites.
Journal ArticleDOI
Something old, something new: challenges and developments in Aspergillus niger biotechnology.
TL;DR: In this article, the authors summarize the current state-of-the-art for Aspergillus niger biotechnology with special focus on organic acids (citric acid, malic acid, gluconic acid and itaconic acid), secreted proteins and secondary metabolites.
References
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Journal ArticleDOI
Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88
Herman Jan Pel,Johannes H. de Winde,Johannes H. de Winde,David B. Archer,Paul S. Dyer,Gerald Hofmann,Peter J. Schaap,Geoffrey Turner,Ronald P. de Vries,Richard Albang,Kaj Albermann,Mikael Rørdam Andersen,Jannick Dyrløv Bendtsen,Jacques A.E. Benen,Marco A. van den Berg,Stefaan Breestraat,Mark X. Caddick,Roland Contreras,Michael Cornell,Pedro M. Coutinho,Etienne Danchin,Alfons J. M. Debets,Peter J. T. Dekker,Piet W.M. van Dijck,Alard Van Dijk,Lubbert Dijkhuizen,Arnold J. M. Driessen,Christophe d'Enfert,Steven Geysens,Coenie Goosen,Gert S.P. Groot,Piet W. J. de Groot,Thomas Guillemette,Bernard Henrissat,Marga Herweijer,Johannes Petrus Theodorus Wilhelmus Van Den Hombergh,Cees A. M. J. J. van den Hondel,René T. J. M. van der Heijden,Rachel M. van der Kaaij,Frans M. Klis,Harrie J. Kools,Christian P. Kubicek,Patricia Ann van Kuyk,Jürgen Lauber,Xin Lu,Marc J. E. C. van der Maarel,Rogier Meulenberg,Hildegard Henna Menke,Martin Mortimer,Jens Nielsen,Stephen G. Oliver,Maurien M.A. Olsthoorn,K. Pal,K. Pal,Noël Nicolaas Maria Elisabeth Van Peij,Arthur F. J. Ram,Ursula Rinas,Johannes Andries Roubos,Cornelis Maria Jacobus Sagt,Monika Schmoll,Jibin Sun,David W. Ussery,János Varga,Wouter Vervecken,Peter J.J. Van De Vondervoort,Holger Wedler,Han A. B. Wösten,An-Ping Zeng,Albert J. J. van Ooyen,Jaap Visser,Hein Stam +70 more
TL;DR: The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes and organic acids, particularly citric acid, and the sequenced genome revealed a large number of major facilitator superfamily transporters and fungal zinc binuclear cluster transcription factors.
Journal ArticleDOI
A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi
TL;DR: A CRISPR-Cas9 based system adapted for use in filamentous fungi that performs RNA-guided mutagenesis in six species of which one has not previously been genetically engineered and demonstrates that the resulting strain can be used for iterative gene targeting.
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Development of a homologous transformation system for Aspergillus niger based on the pyrG gene
W. van Hartingsveldt,I. E. Mattern,C. M. J. Van Zeijl,P.H. Pouwels,C.A.M.J.J. van den Hondel +4 more
TL;DR: The pyrG transformation system was used for the introduction of a non-selectable gene into A. niger through the use of an orotidine-5′-phosphate decarboxylase deficient mutant (pyrG) and a vector, pAB4-1, which contains the functional A.Niger pyr G gene as a selection marker.
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sgRNAcas9: A Software Package for Designing CRISPR sgRNA and Evaluating Potential Off-Target Cleavage Sites
TL;DR: By identifying potential off-target sites in silico, the sgRNAcas9 allows the selection of more specific target sites and aids the identification of bona fide off- target sites, significantly facilitating the design of sg RNA for genome editing applications.
Journal ArticleDOI
Comparative genomics of citric-acid-producing Aspergillus niger ATCC 1015 versus enzyme-producing CBS 513.88
Mikael Rørdam Andersen,Margarita Salazar,Peter J. Schaap,Peter J. I. van de Vondervoort,David E. Culley,Jette Thykaer,Jens Christian Frisvad,Kristian Fog Nielsen,Richard Albang,Kaj Albermann,Randy M. Berka,Gerhard H. Braus,Susanna A. Braus-Stromeyer,Luis M. Corrochano,Ziyu Dai,Piet W.M. van Dijck,Gerald Hofmann,Linda L. Lasure,Jon K. Magnuson,Hildegard Henna Menke,M. Meijer,Susan Lisette Meijer,Jakob Blæsbjerg Nielsen,Michael Lynge Nielsen,Albert J. J. van Ooyen,Herman Jan Pel,Lars Kongsbak Poulsen,Rob Samson,Hein Stam,Adrian Tsang,Johannes Maarten Van Den Brink,Alex Atkins,Andrea Aerts,Harris Shapiro,Jasmyn Pangilinan,Asaf Salamov,Yigong Lou,Erika Lindquist,Susan Lucas,Jane Grimwood,Igor V. Grigoriev,Christian P. Kubicek,Diego Martinez,Noël Nicolaas Maria Elisabeth Van Peij,Johannes Andries Roubos,Jens Nielsen,Scott E. Baker +46 more
TL;DR: In this article, the authors performed whole-genome sequencing of the Aspergillus niger wild-type strain (ATCC 1015) and produced a genome sequence of very high quality.