A Polyketide Synthase Encoded by the Gene An15g07920 Is Involved

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A polyketide synthase encoded by the gene An15g07920 is involved in the biosynthesis of ochratoxin A in Aspergillus niger Jian Zhang, Liuyang Zhu, Haoyu Chen, Min Li, Xiaojuan Zhu, Qiang Gao, Depei Wang, and Ying Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03907 • Publication Date (Web): 14 Nov 2016 Downloaded from http://pubs.acs.org on November 19, 2016

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Journal of Agricultural and Food Chemistry

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A polyketide synthase encoded by the gene An15g07920 is involved in the

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biosynthesis of ochratoxin A in Aspergillus niger

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Jian Zhang,† Liuyang Zhu, †

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and Ying Zhang *,‡

Haoyu Chen,† Min Li,† Xiaojuan Zhu,† Qiang Gao,†

Depei Wang,†

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†

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Science and Technology, Tianjin 300457, China

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‡

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Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin University of

Key Laboratory of Food Nutrition and Safety, Ministry of Education, Tianjin University of Science and

Technology, Tianjin 300457, China

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* Corresponding author:

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Ying Zhang (E-mail: [email protected]; Tel: +86-022-60912431)

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ACS Paragon Plus Environment


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ABSTRACT: The polyketide synthase gene An15g07920 was known in Aspergillus niger CBS 513.88

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as putatively involved in the production of ochratoxin A (OTA). Genome re-sequencing analysis

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revealed that the gene An15g07920 is also present in the ochratoxin-producing A. niger strain 1062.

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Disruption of An15g07920 in A. niger 1062 removed its capacity to biosynthesize ochratoxin β (OTβ),

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ochratoxin α (OTα) and OTA. These results indicate that the polyketide synthase encoded by

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An15g07920 is a crucial player in the biosynthesis of OTA, in the pathway prior to the phenylalanine

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ligation step. The gene An15g07920 reached its maximum transcription level before OTA accumulation

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reached its highest level, confirming that gene transcription precedes OTA production. These findings

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will not only help explain the mechanism of OTA production in A. niger, but also provide necessary

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information for the development of effective diagnostic, preventive and control strategies to reduce the

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risk of OTA contamination in foods.

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KEYWORDS: Ochratoxin A, Aspergillus niger, Kinetics, Gene expression, Biosynthesis

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INTRODUCTION

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Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus and Penicillium spp. fungi. It has been

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demonstrated to have nephrotoxic, immunotoxic, genotoxic, neurotoxic and teratogenic properties; it

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was classified as a renal carcinogen of animals and a possible (Group 2B) carcinogen of humans by the

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International Agency for Research on Cancer (IARC).1 This compound is found widely in cereals and

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their derivatives, wine, coffee, beer, nuts, dried fruits, and even meat products.2 Due to the

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well-established routes of human exposure and abundance of toxicological data from animal studies, the

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European Union has established stringent regulatory limits for OTA in a wide range of food products

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such as raw cereal grains (5 µg/kg), products derived from cereals (3 µg/kg), dried fruits (10 µg/kg),

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roasted and instant coffee (5 and 10 µg/kg, respectively), beverages containing wine and grape juice (2

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µg/kg), baby food (0.5µg/kg) and infant formula (0.5 µg/kg).3

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Chemically, OTA is composed of a chlorinated type I polyketide dihydroisocoumarin moiety linked to l-

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phenylalanine by an amide bond. Early feeding and chemical degradation experiments showed that the

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isocoumarin moiety is produced from five acetate units and a single methionine-derived carbon added at

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the C-7 position, while the l- phenylalanine part stems from the normal shikimic acid pathway.4

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According to OTA structure and its proposed biosynthetic pathway, the synthesis can be expected to

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require several proteins, including a polyketide synthase (PKS) for the biosynthesis of the polyketide

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dihydroisocoumarin, a nonribosomal peptide synthetase (NRPS) for the ligation of the amino acid

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phenylalanine to the polyketide moiety, and a halogenase for the chlorination step. There are three types

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of known PKSs—modular type I PKSs, iterative type I PKSs, and type II PKSs. Fungi mainly utilize the

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iterative type I PKSs, which have a modular organization similar to the modular type I PKSs, whereby

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the catalytic reaction of each domain can be engaged repeatedly in an iterative fashion. Typical

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representatives of iterative type I PKS structures include ketosynthase (KS), acyl transferase (AT),

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ketoreductase (KR), dehydratase (DH), enoyl reductase (ER), methyltransferase (MT), thioesterase (TE),

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and acyl carrier protein (ACP) domains.5

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Most of the molecular aspects of OTA biosynthesis have been elucidated in Penicillium species. In P.

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nordicum, a putative OTA biosynthetic cluster has been identified which contains biosynthetic genes

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encoding PKS, NRPS, as well as sequences with homology to chlorinating enzymes and homology to a

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transporter protein, respectively.6 The orthologues of these genes have also been identified in the closely

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related species P. verrucosum.7 Within Aspergillus spp., the pks gene, the nrps gene and halogenase gene,

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have been fully characterized and demonstrated to be required for ochratoxin production in A.

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carbonarius.

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characterized in A. ochraceus.

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there are other reports which inferred that some genes (such as those encoding PKS, NRPS, P450,

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phenylalanine tRNA synthetase and methylase) were related to OTA synthesis based on the correlation

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of gene expression profiles and data obtained using genome sequencing and bioinformatics methods.13-17

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Aspergillus niger is an important industrial “workhorse” organism, with extensive applications in the

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production of industrial enzymes, heterologous proteins and organic acids, among others.18 Whereas this

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species has gained the generally recognized as safe (GRAS) status from the US Food and Drug

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Administration, there have been increasingly frequent reports that some isolates of A. niger are able to

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produce OTA, which represents a matter of great concern for its industrial application.19 Genome

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sequencing of A. niger strain CBS 513.88 revealed the presence of the pks gene An15g07920, that has a

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strong similarity to the pks gene of A. ochraceus involved in OTA biosynthesis.15 What’s more, Ferracin

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et al. have found a positive association between the presence of strain-specific pks genes such as

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The pks gene has also been partially characterized in A. westerdijkiae,11 and fully 12

In addition to characterizing the genes involved in OTA biosynthesis,

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An15g07920 and the capability of the respective A. niger strains to produce OTA.20 Castella and

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Cabanes showed that a fragment of the gene An15g07920 was specific for ochratoxin-producing strains

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of A. niger and developed a real-time PCR protocol for the detection of OTA-producing strains of the A.

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niger aggregate.21 Recently, the correlation between pks gene expression and OTA production in A. niger

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has also been reported.22 However, this gene has to our best knowledge not been disrupted and its role in

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OTA biosynthesis has not been confirmed in A. niger.

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In this work, we re-sequenced the genome of A. niger 1062 in order to find the corresponding gene

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homologous to An15g07920, after which we have characterized and disrupted this gene by targeted gene

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replacement using Agrobacterium tumefaciens-mediated transformation (ATMT). We further analyzed

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the phenotypic characteristics of the wild-type and the mutant strains to explore the function of

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An15g07920. Finally, we utilized a qRT-PCR assay to analyze An15g07920 expression. Our results

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reveal for the first time that the gene An15g07920 is involved in the biosynthetic pathway of OTA in A.

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niger and that it plays a crucial role in the biosynthesis pathway that precedes phenylalanine ligation.

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MATERIALS AND METHODS

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Strains and preservation conditions

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The ochratoxin-producing wild-type strain A. niger 1062 used in this study, was previously isolated from

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Chinese vineyard and deposited in China General Microbiological Culture Collection as No.9668. The

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mutant strain used in this study was derived from strain 1062 and is indicated as ∆pks. A. tumefaciens

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strain AGL-1 was kindly provided by Prof. Depei Wang (Tianjin University of Science and Technology,

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Tianjin, China). Escherichia coli DH5α (Takara, Shiga, Japan) was used for the construction and

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propagation of vectors. Both A. niger strains (1062 and ∆pks) were grown at 25°C on potato dextrose

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agar plates (PDA) (Fisher Labosi) for 5 days, after which spores were collected by washing with a sterile

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solution of 0.9% (v/v) normal saline (Fisher Labosi) and stored at -80°C in 25% (v/v) of glycerol (Fisher

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Labosi) for further use.

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DNA and RNA Extraction, cDNA Synthesis, qPCR and qRT-PCR

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A. niger conidia (106/mL) were used to inoculate 250 mL Erlenmeyer flasks containing 100 mL of

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Czapek yeast autolysate (CYA) liquid medium, and incubated at 25 °C without shaking for 7 days. The

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mycelia were harvested by filtration through a 0.45 µm filter (Millipore), frozen in liquid nitrogen and

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stored at -80 °C for further analysis. Genomic DNA of A. niger 1062 was extracted using the E.Z.N.A.

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Fungal DNA kit (Omega Bio-Tek, Doraville, GA, USA) according to the manufacturer’s protocol.

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Total RNA was extracted using the Trizol Reagent (Invitrogen,CA, USA) according to the

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manufacturer's protocol. RNA concentrations were quantified using a NanoDrop ND-1000

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spectrophotometer (Thermo Fisher, MA, USA), and adjusted to 0.5 mg/mL. The cDNA was synthesized

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with the RNA samples as templates using the ReverTra Ace qPCR RT Master Mix with gDNA Remover

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(Toyobo, Osaka, Japan).

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Real-time genomic PCR (qPCR) was carried out in order to determine the copy numbers of integrated

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T-DNA cassettes in the genomes of transformants. The primer pair HPHq-F/R (Table 1) was designed to

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amplify the selection marker. The single-copy A. niger actin gene served as a reference and was

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amplified using the ACTq-F/R primers (Table 1)23. Reactions were performed in a final volume of 20 µL,

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comprising 10 µL of SYBR PremixEx TaqII (Takara, Tokyo, Japan), 0.5 µL of each primer (10 µM), and

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1 µL of genomic DNA, made up to volume with PCR-grade ultrapure water. The amplification program

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was conducted with an initial step of 10 min at 95°C, followed by 40 cycles of 10 s at 95°C and 34 s at

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60°C.

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Real-time quantitative reverse transcription PCR (qRT-PCR) was performed to examine the expression

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of An15g07920 at different times during growth of A. niger in CYA liquid medium, using the primers

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KSqrt-F/R designed based on the KS domain of the An15g07920 gene (Table 1). The constitutively

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expressed β-tubulin gene (primers Tubqrt-F/R) served as an internal reference to normalize gene

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expression. The amplification reactions were performed in reaction volumes of 20 µL, comprising 10 µL

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of SYBR PremixEx TaqII (Takara, Tokyo, Japan), 0.5 µL of each primer (10 µM) and 2 µL of cDNA

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template, made up to volume with PCR-grade ultrapure water. Amplification was conducted using an

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initial denaturation step at 95 °C for 3 min, followed by 40 cycles of 10 s at 95 °C, 30 s at 60 °C and 30 s

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at 72 °C. The specificity of amplification was confirmed by dissociation curve analysis.

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qPCR and qRT-PCR assays were performed and monitored using an ABI PRISM 7900HT system

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(Applied Biosystems, Spain). The relative quantitative gene expression data were established using the

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comparative 2−∆∆CT method. PCR efficiency of each oligonucleotide pair was calculated using linear

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regression based on standard curves. All experiments were performed in three biological replicates, and

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each sample was tested in triplicate, in addition to a no-template control included for each primer pair.

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Genome re-sequencing, assembly and sequence analysis

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Genome re-sequencing of A. niger 1062 was performed using an IRoche GS FLX system by Shanghai

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Personalbio Biotechnology (Shanghai, China). The reads were mapped against the reference genome (A.

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niger CBS513.88) using the program bwa

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developed by 454 Life Sciences (Branford, Connecticut, USA), to yield a number of contigs. The

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and were assembled de novo using Newbler (version 2.6)

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resulting contigs were extended to obtain longer scaffolds using SSPACE (Version 2.0). The software

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GapFiller was used to close gaps.25 The deduced amino acid sequence was determined using the translate

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tool from Expasy (http://web.expasy.org/translate/) while protein–protein Blast (Blastp) searches were

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conducted in GenBank (http://www.ncbi.nlm.nih.gov). Mulitple sequence alignments were calculated

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using MultAlin (http://prodes.toulouse.inra.fr/multalin/multalin.html). Conserved functional domains

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were analyzed using InterProScan (http://www.ebi.ac.uk/interpro/) with default parameter settings.

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Construction of gene-disruption vector

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The plasmid p44 which was used for the construction of the gene-disruption vector was maintained in E.

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coli DH5α. Restriction endonucleases and DNA-modifying enzymes were purchased from Takara (Shiga,

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Japan). The upstream (646 bp) and downstream (507 bp) fragments flanking the hygromycin-resistance

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gene in the p44 vector were amplified from the genomic DNA of A. niger 1062 using PCR with GXL

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high-fidelity DNA polymerase (Takara, Shiga, Japan), with the primer sets KS5F/R and KS3F/R (Table

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1), respectively, and the corresponding fragments purified using the MiniBEST agarose gel DNA

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extraction kit (Takara, Shiga, Japan). The purified upstream fragment was inserted into the PstI and

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HindIII digested p44 vector using the In-Fusion cloning kit (Takara, Shiga, Japan) to produce p44-HR1.

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Similarly, the purified downstream fragment was inserted between the BamHI and KpnI sites of

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p44-HR1 to generate the completed disruption vector p44-HR1-HR2 (Figure 1A). An aliquot of vector

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DNA was used to transform E. coli DH5α chemically competent cells without prior ligation.

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Kanamycin-resistant transformants were further screened using PCR and cloning junctions were

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confirmed by DNA sequencing (Solarbio, China). Finally, the resulting vector p44-HR1-HR2 was

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introduced into electrocompetent A. tumefaciens AGL-1 cells as described previously.26

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Table 1

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A. tumefaciens-mediated transformation of A. niger

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A. tumefaciens AGL-1 cells carrying the vector p44-HR1-HR2 were cultured in 3 mL of Luria Bertani

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(LB, Miller, Solarbio, China) medium on a rotatory shaker at 180 rpm and 28 °C for 10 h. A. tumefaciens

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cells were collected by centrifugation (2,200×g,10 min) and resuspended in fresh induction medium24

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(IM) containing 200 µmol/L acetosyringone (AS, Solarbio, China) at a cell density corresponding to an

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optical density at 600nm (OD600) of 0.15. After pre-incubation for 6 h under constant orbital shaking at

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28 °C to an OD600 of 0.7 to 0.8, a 5mL aliquot of the cell suspension was mixed with an equal volume of

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5-day-old A. niger spores (2×107/mL), and the mixture was subsequently spread onto a nitrocellulose

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filter membrane (0.45 µm pore and 45 mm diameter, Whatman, GE Life Sciences, USA) and placed on

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an IM agar plate containing 200 µmol/L AS. The plates were incubated at 25 °C in the dark for 48h.

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After co-cultivation, the membrane was transferred onto a complete medium27 (CM) agar plate

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containing hygromycin B (Solarbio, China; 100 µg/mL) as the selection agent for fungal transformants,

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and cefotaxime (Solarbio,China; 200 µg/mL) to inhibit growth of A. tumefaciens cells.

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Characterization of transformants

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The transformants were screened for homologous recombination by PCR using primers KS5-F and

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KS3-R. The parental strain had an amplicon of about 1.8 kb, and successful transformants had amplicons

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larger than 2.5 kb. Transformants with a correct amplicon size, as predicted based on the insertion

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cassette, were collected for further qPCR analysis (see below). A melting curve was determined for each

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sample at the end of each run to ensure the purity of the amplified product. qPCR was performed in five

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Replicates. The copy number was calculated according to a previously published 28 modified formula:

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X 0 R0 = 10

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where I X and I R represent the intercepts of the relative standard curves of target and reference genes,

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SX

C and S R represent the slopes of the standard curves of target and reference genes, and T X and

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CT R

are the detected threshold cycles for the amplification of the target and reference genes of a tested

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sample, respectively.

( CTX − I X ) S X  − ( CTR − I R ) SR 

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Analysis of fungal growth and conidiogenesis

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For growth assessment, CYA agar plates were inoculated centrally with 5 µL of a 106 /mL suspension of

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conidia of A. niger 1062 and the ∆pks mutant strains, respectively. Cultures were incubated at 25 °C for

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7 days. Colony morphology was recorded and the number of conidia was determined at 24 h intervals

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for 7 days. The conidia were washed in 10 mL of sterile water, followed by filtration through four layers

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of lens tissues (Solarbio, China) to remove mycelial debris. The concentrations of the conidial

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suspensions were determined using a Thoma cell counting chamber (Qiujing, Shanghai, China).

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Six-day-old samples of A. niger conidia were prepared for scanning electron microscopy (SEM) as

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described previously by Darah et al.29 and were observed under a Leo-Supra 50VP field emission

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scanning electron microscope (FESEM, Carl Zeiss, Germany).

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Chemical analysis of culture supernatants by HPLC-FLD and HPLC-MS 10


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For the determination of the production of OTA and its metabolites, conidia samples comprising 105

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spores in 100 µL of water were used to inoculate Erlenmeyer flasks containing 20 mL of CYA liquid

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medium. Incubation was carried out at 25 °C in the dark under stationary conditions for 9 days. The

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supernatants of the liquid cultures of the two tested strains were filtered through a 0.22 µm pore filter

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(Sartorius AG, Goettingen, Germany) and analyzed for their content of OTA and its metabolites by

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high-performance liquid chromatography with fluorescence detection (HPLC-FLD). Liquid cultures of

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1062 and ∆pks were also analyzed by high-performance liquid chromatography coupled to a mass

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spectrometry system (HPLC-MS) to confirm the results and to identify other metabolites belonging to

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the OTA biosynthetic pathway.

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HPLC-FLD analysis was carried out on an Agilent 1100 series system equipped with a G1312A binary

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pump, a G1313A autosampler, a G1321A fluorescence detector set to 333 nm excitation and 460 nm

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emission wavelength, and an Agilent Chemstation G2170AA Windows 2000 operating system (Agilent,

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Waldbronn, Germany). The column was a KromstarTM C18 (250 × 4.6 mm, 5 µm particles), and was

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preceded by a Rheodyne guard filter (3 mm, 0.45 µm pore size). The mobile phase was an isocratic

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mixture of acetonitrile/water/acetic acid (99:99:2, v/v/v) and the analytes were eluted at a flow rate of

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1.0 mL/min.

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HPLC-MS analysis was performed on an Agilent 1200 series system connected to an Agilent 6110 single

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quadrupole mass spectrometer (Agilent, MA, USA) via an ESI source. The ESI was operated in negative

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mode to detect OTα and OTβ, in positive mode to detect OTA. The parameters used for the mass

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spectrometer in all experiments were as follows: 350 °C gas temperature, 13.0 L/min drying gas (nitrogen)

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flow, 3.5 kg/cm2 nebulizer gas pressure and 3500 V capillary voltage. The molecular ions [M+H]+ were

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monitored at m/z 404 for OTA. The molecular ions [M-H]- were monitored at m/z 255 for OTα, and m/z

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221 for OTβ.

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The compounds were identified by comparing the retention times and mass spectra obtained from

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samples with those obtained from pure authentic standards injected under the same conditions. OTA and

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OTB were purchased from a commercial source (Sigma-Aldrich, St. Louis, MO, USA). OTα was

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synthesized by hydrolysis of OTA as described by Xiao et al.30 OTβ was prepared by acid hydrolysis of

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OTB.31 Quantitation of the compounds was performed based on measurements of peak areas.

256 257

Statistical analysis

258 259

All statistical analyses were performed using the SPSS software package, version 17.0 for Windows

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(IBM, Chicago, IL). The OTA contents and the An15g07920 gene expression analyses were evaluated

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using one-way analysis of variance (ANOVA). Mean differences were determined using Tukey’s

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post-hoc test (p = 0.01). All figures were plotted using Origin software (Version 8.5, OriginLab Corp.,

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Northampton, MA, USA).

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RESULTS

266 267

Sequence Analysis

268 269

In a previous study, we have screened the A. niger strain 1062, and found that it can produce OTA in

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significant amounts.32 In order to understand the mechanism of OTA production in A. niger 1062,

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genome re-sequencing of this strain was performed. According to the genome annotation of A. niger

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CBS 513.88, gene An15g07920, also termed ANI_1_1836134, encodes a hypothetical protein with

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strong similarity to the PKS fragment involved in OTA biosynthesis in A. ochraceus.15 Thus, we

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attempted to find the A. niger 1062 orthologue of An15g07920. Comparative analysis revealed a

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nucleotide fragment in A. niger 1062 showing 100% sequence similarity with the gene An15g07920

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from A. niger CBS 513.88, indicating that An15g07920 is also present in A. niger 1062. An15g07920 is

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8173 nt in length and encodes a predicted protein of 2554 amino acids, with a predicted molecular mass

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of 277.73 kDa and a predicted pI of 5.86. Conserved domain analysis demonstrated that the gene

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An15g07920 encoded seven highly symptomatic catalytic domains, including KS, AT, DH, C-MT, ER,

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KR and ACP (Figure 1B).

281 282

Disruption of An15g07920 in A. niger 1062

283 284

To confirm that the product of An15g07920 is indeed involved in OTA synthesis, the sequence fragment

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of An15g07920 encoding the KS domain was disrupted in A. niger 1062 by replacing it with a

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hygromycin resistance cassette. The gene replacement vector was constructed based on the upstream and

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downstream fragments of the target region, which were cloned into the vector p44 flanking the

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hygromycin resistance marker. The resulting disruption vector (p44-HR1-HR2) was used to obtain the

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partial KS domain coding region knockout (∆pks) mutant through ATMT (Figure 1 A and B). The

290

hygromycin B-resistant colonies appeared after approximately 4 days of incubation. Genomic DNA was

291

isolated from the transformants, and the site-specific insertions were confirmed by PCR analysis using

292

the primers KS5-F and KS3-R (Table 1) (Figure 1C). In most of the transformants, there were two

293

amplicons with different sizes - one corresponding to the wild-type locus (1776 bp) and the other to the

294

T-DNA construct (2550 bp). However, in the ∆pks strains, there was only one amplicon, which

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corresponded to the T-DNA, because the target gene was no longer present. Thus, 6 out of the 50

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analyzed transformants had integrated the T-DNA cassette at the partial KS domain locus via

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homologous recombination, which means that the frequency of homologous recombination among the

298

hygromycin-resistant transformants was approximately 12%. We subsequently used qPCR to determine

299

the copy number of the integrated T-DNA cassettes that had been integrated into the genome of A. niger

300

1062 (Table 2). If a ∆pks mutant has one or several extra copies of the T-DNA cassette integrated

301

elsewhere in the genome, the phenotype of the strain cannot be unambiguously attributed solely to the

302

inactivation of An15g07920. Therefore, we selected a single knock-out mutant clone containing a single

303

T-DNA integration for further analysis.

304

Figure 1

305 306

Table 2

307 308 309

Phenotypical analysis

310 311

No statistically significant differences in growth and colony morphology were observed in the ∆pks

312

mutant when compared to the wild-type strain on non-selective media (CYA agar plates) (Figure S1 A).

313

Thus, by day 7, the average colony diameter of the ∆pks mutant was 39.2 ± 0.6 mm and the wild-type

314

reached 38.9 ± 0.8 mm. Similarly, both the ∆pks mutant and wild-type strains exhibited similar

315

sporulation behavior, with 9.5×107 ± 2.6×105 conidia per mL in the wild-type and 9.0×107 ± 4.3×105

316

conidia per mL in the ∆pks mutant at day 7 (Figure S1 C). Additionally, no significant differences of

317

conidial morphology were visible upon SEM observation (Figure S1 B).

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Figure S1

320 321

Furthermore, in order to investigate the kinetics of the production of OTA and its metabolites, the ∆pks

322

mutant and wild-type strain were grown in CYA liquid medium and the culture supernatants analyzed

323

using HPLC-FLD. The results showed that the metabolite profile of the ∆pks mutant was significantly

324

different from that of the wild-type strain. Three peaks at 4.122, 5.869, and 8.911 min were present only

325

in the wild-type A. niger 1062, and were observable at 2-5 days of incubation, whereby the peak at 8.911

326

min disappeared after day 5. On the other hand, the ∆pks mutant lacked three of those peaks, as observed

327

at days 2-9 (Figure 2). The peaks eluting at 4.122, 5.869, and 8.911 min were identified as OTβ, OTα

328

and OTA, since their retention times were identical to those of the OTB hydrolysis product (OTβ), OTA

329

hydrolysis product (OTα) and OTA standard, respectively. Furthermore, HPLC-MS confirmed the bona

330

fide identities of these compounds. The peak at 4.122 showed a molecular ion [M-H]- at m/z 222.1,

331

corresponding to OTβ, the peak at 5.869 showed the molecular ions [M-H]- at m/z 255.0, corresponding

332

to OTα, and the peak at 8.911 showed a molecular ion [M+H]+ at m/z 404.1 corresponding to OTA

333

(Figure 2). Conversely, OTA was not detected at all in the culture supernatants of the ∆pks mutant,

334

indicating that the disruption of the KS domain of An15g07920 indeed resulted in the inability to

335

produce OTA, as expected. In wild-type A. niger 1062, OTA production was initially observed on day 2

336

and increased to its maximum value on day 3, after which the productions levels decreased markedly by

337

day 5 (Figure 3A). During the first 3 days of incubation, the amount of OTβ in the culture supernatants

338

steadily increased and after the third day its concentration remained practically unchanged. Similarly, the

339

amount of OTα increased until day 6 and remained stable thereafter (Figure 3A). Additionally, the

340

relative expression of the gene An15g07920 was also investigated. The highest expression level was

15



Page 16 of 31

341

detected on day 2 after inoculation and it decreased afterwards, showing a 3-fold higher expression at

342

day 2 compared to day 5 (Figure 3B).

343

Figure 2.

344 345

Figure 3.

346 347 348

DISCUSSION

349 350

When A. niger was given the GRAS status in numerous industrial processes, the potential for OTA

351

production by this species had not been known. With the increasing number of reports showing that

352

some A. niger strains can produce OTA, great concern has been raised about some industrial

353

processes.32-34 It is clear that a thorough characterization of the OTA biosynthetic genes is necessary to

354

determine the molecular triggers which control OTA biosynthesis in A. niger. However, to our best

355

knowledge, a clear involvement of any of the genes in OTA biosynthesis in A. niger had yet to be

356

established at the time of this study. In general, genes encoding enzymes involved in OTA biosynthesis

357

are located in physical proximity to each other, forming gene clusters that usually harbor genes for PKS,

358

NRPS, hydrolases, oxidases, methylases, transporters, and regulatory proteins.35 The in silico analyses

359

performed by Ferracin et al. have shown that the pks-locus tag An15g07920 located in the genome of the

360

ochratoxin-producing A. niger strain CBS 513.88 is absent in the non-ochratoxin-producing strain ATCC

361

1015.20 Moreover, an in vivo analysis of several Brazilian strains has shown that there is an association

362

between the presence of this particular pks gene and the corresponding strains’ capability to synthesize

363

OTA.20 All these clues suggested that An15g07920 in A. niger was closely related to the biosynthesis of

16


Page 17 of 31


364

OTA, but prior to this study the gene had not been disrupted and its role in OTA biosynthesis had not

365

been confirmed in A. niger. Thus, we used gene disruption to verify that An15g07920 is indeed required

366

for the biosynthesis of OTA. Subsequent metabolic profiling of the corresponding ∆pks mutant showed

367

that OTA, OTα and OTβ were indeed absent from its culture supernatants.

368

The gene An15g07920 is 8173 nt in length, which means that any homologous genes should most likely

369

also be greater than 8000bp in length. However, PCR-amplification and restriction-fragment based

370

cloning techniques are inefficient, inaccurate, and not always applicable with large DNA segments.

371

Therefore, genome re-sequencing of this strain was carried out in order to characterize the homologous

372

gene An15g07920 in the A. niger 1062 genome. A sequence alignment map was generated by aligning

373

reads from the sequenced pool to the A. niger CBS 513.88 reference sequence. A nucleotide fragment in

374

A. niger 1062 showing 100% similarity with the gene An15g07920 from A. niger CBS 513.88, was

375

found, demonstrating that a gene corresponding to An15g07920 is indeed present in A. niger 1062.

376

Whole genome re-sequencing aims to sequence the genome of a specific strain of an organism for which

377

a reference genome is already available, and in this study we used the genomic sequence of A. niger

378

CBS 513.88 as a basis for the re-sequencing of A. niger 1062. This has become an easy, fast and

379

low-cost method for the identification of target genes.

380

Conidial pigments of A. niger are formed from two precursor molecules - hexahydroxyl pentacyclic

381

quinoid and melanin - whereby the latter is formed from acetate, while OTA biosynthesis begins with

382

one acetate and four malonate molecules condensing to a mullein moiety.36,37 Therefore, acetate is a

383

shared precursor of conidial pigments and OTA. However, our results suggested that the biosynthesis of

384

OTA is independent of the pigments because colony color remained unchanged when An15g07920 was

385

disrupted. One could probably speculate that A. niger does not share enzymes responsible for OTA

386

biosynthesis and pigment synthesis, despite the shared precursor. Furthermore, no statistically significant

17



Page 18 of 31

387

differences in growth and colony morphology were observed in the ∆pks mutant, indicating that the

388

results of the gene disruption were quite well segregated from overall metabolism. However, the

389

metabolic network in question is complex and it deserves further study to determine whether or not OTA

390

synthesis gene-encoded enzymes play any additional roles in fungal growth and conidiogenesis.

391

Disruption of the gene An15g07920 completely eliminated the production of OTβ, OTα and OTA during

392

9 days of culture. This result confirmed that the gene An15g07920 is involved in the biosynthetic

393

pathway of OTA and that it plays a role before the step of phenylalanine ligation. We have detected OTB

394

only at a single time-point at day 4 in A. niger 1062 (data not shown). The possible reason is that OTB is

395

not stable and can only accumulate for a short period. We never found OTB ethyl ester or OTC in the

396

culture supernatants of both the 1062 and ∆pks strains. This result is in accordance with a previous report

397

on the intermittent detection of such metabolites in A. carbonarius.9 Esterification of phenylalanine

398

might protect its carboxyl group in the binding reaction leading to OTC.38 However, our results suggest

399

that esterification of phenylalanine is not necessary for the biosynthesis of OTA. Since only OTA was

400

obtained as a pure commercial product, OTβ and OTα were synthesized by ourselves. Consequently, we

401

did not perform a strict quantitative analysis for these compounds, but used the peak areas to quantitate

402

them relatively. Even so, relative quantitative kinetic curves were able to detect increasing OTα

403

production in parallel with a decrease of OTA. One likely explanation of this is that OTα formation is

404

happening directly at the expense of OTA degradation.

405

qRT-PCR was carried out to analyze the expression profile of An15g07920 during the production of

406

OTA by A. niger grown in CYA liquid medium, and the results of this analysis demonstrated a clear

407

correlation between An15g07920 expression and OTA production. The maximum expression of the gene

408

An15g07920 was observed at day 2, while OTA accumulation reached its highest level a day later (day

409

3), confirming that gene transcription preceded OTA detection. A similar timing relationship between

18


Page 19 of 31


410

gene transcription and OTA production has also been observed in a number of other OTA-producing

411

Aspergilli. 8,11,22.39

412

In conclusion, inactivation of the gene An15g07920 allowed us to determine its functional role in OTA

413

biosynthesis of A. niger. Future analysis of the OTA gene cluster in this fungus will proceed with the

414

characterization of additional genes found in close proximity to the nrps and pks genes that may be

415

involved in mycotoxin biosynthesis. Complete elucidation of this biosynthetic pathway will not only

416

help explain OTA production, but will also provide necessary information for the development of

417

effective diagnostic, preventive and control strategies to reduce the risk of OTA contamination in foods

418

and other products that utilize this nominally GRAS-status organism in their production processes.

419 420

ASSOCIATED CONTENT

421

Supporting Information description

422

Figure S1. Phenotypic analysis of wild-type (WT) A. niger strain 1062 and the ∆pks mutant strains (A)

423

Colony view of the two strains inoculated in CYA agar plates at different time intervals after inoculation

424

(1 -7 days). (B) SEM images (14,000 × magnification) of conidia derived from CYA agar plates after 4

425

days inoculation. (C) Amount of conidia formation in CYA agar plates at different time intervals after

426

inoculation (1-7 days). The data are the mean ± SD of three determinations from three separate

427

experiments.

428

AUTHOR INFORMATION

429

Corresponding Author

430

* Tel: +86-022-60912431. E-mail: (Y.Z.) [email protected]

431

Notes

432

The authors declare no competing financial interest.

433

Funding Sources 19



Page 20 of 31

434

This work was supported by the National Natural Science Foundation of China (31471725, 31370075,

435

and 31201354 ), Tianjin enterprise postdoctoral innovation project (2015); Tianjin funded training

436

selected outstanding postdoctoral program of internationalization (2014).

437 438

REFERENCES

439

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440

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(2) Duarte, S.C.; Lino, C.M.; Pena, A. Mycotoxin food and feed regulation and the specific case of

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ochratoxin A: a review of the worldwide status. Food Addit Contam, Part A. 2010, 27, 1440-1450.

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(3) European Commission. Commission Regulation (EC) No 1881/2006 of 19 December 2006 setting

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Contreras, R.; Cornell, M.; Coutinho, P. M.; Danchin, E. G J.; Debets, van Dijk, A.; Dijkhuizen, L.; Driessen, A. J. M.;,d’Enfert, C.;

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Kaaij, R. M.; Klis, F. M.; Kools, H. J.; Kubicek, C. P.; van Kuyk, P. A .; Lauber, J.; Lu, X.; vander

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Maarel, M. J. E. C.; Meulenberg, R.; Menke, H.; Mortimer, M. A.; Nielsen, J.; Oliver, S. G.; Olsthoorn,

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M.; Pal, K.; van Peij, N. N. M. E.; Ram, A. F J.; Rinas, U.; Roubos, J. A.; Sagt, C. M. J.; Schmoll, M.;

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Sun, J.; Ussery, D.; Varga, J.; Vervecken, W.; van de Vondervoort, P. J. J.; Wedler, H.; Wo¨sten, H. A. B.;

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Zeng, A.P.; van Ooyen, A. J. J.; Visser, J.; Stam, H.Genome sequencing and analysis of the versatile cell

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factory Aspergillus niger CBS 513.88. Nat Biotechnol. 2007, 25, 221-231

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Food Microbiol. 2015, 214:102-108.

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and ochratoxin production in industrial Aspergillus niger strains. PLoS One. 2011, 6, e23496.

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Aspergillus niger. Int. J. Food Microbiol. 2012, 155, 137-145.

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A-producing strains of the Aspergillus niger aggregate. Food Control. 2011, 22, 1367-1372.

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acid production.PLoS One. 2015, 10: e0145459.

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Bioinformatics 2009, 25, 1754-1760.

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paired reads. BMC Bioinformatics. 2012, 13(Suppl 14), S8

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Soc. Perkin Trans. II 1989:1835-1839.

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A-producing Aspergillus niger strain. Microbiol. China, 2015, 42, 1010-1016

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coffee and its formation in relation to processing methods. Int J Food Microbiol. 2003, 82, 173-179

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Occurrence of black Aspergilli and ochratoxin A on grapes in Italy. Toxins, 2010, 2, 840-855

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(35) Turner, G. Genomics and secondary metabolism in Aspergillus. In: Machida, M.,Gomi, K. (Eds.),

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Aspergillus: Molecular Biology and Genomics. Caister Academic Press, Norfolk, 2010, pp. 139-155.

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(36) Jørgensen, T. R.; Park, J.; Arentshorst, M.; van Welzen, A. M.; Lamers, G.; Vankuyk, P.A.; Damveld,

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(37) Steyn, P. S.; Holzapfel, W. H. The biosynthesis of the ochratoxins, metabolites of Aspergillus

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(38) Huff, W. E.; Hamilton, P. B. Mycotoxins—their biosynthesis in fungi:ochratoxins—metabolites of

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combined pathways. J. Food Prot. 1979, 42, 815-820.

540

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541

Characterisation of a pks gene which is expressed during ochratoxin A production by Aspergillus

542

carbonarius. Int. J. Food Microbiol. 2009, 129, 8-15.

543

24


Page 25 of 31


Figure captions

Figure 1. Schematic diagram of gene An15g07920 disruption. (A) Map of plasmid p44-HR1-HR2. (B) Sketch map of the replacement of the targeting gene using a homologous recombination method. (C) Transformants confirmed by the PCR method: M, marker (DL5000); lanes 1 , wild-type strain; lanes 2 , ectopic transformant; lanes 3, ∆pks mutants.

Figure 2. HPLC-FLD chromatograms and mass spectra. (A) Culture medium control; (B) Culture supernatant of wild-type A. niger strain 1062; (C) Culture supernatant of the ∆pks mutant strain. Retention times: OTA: 8.911 min; OTα: 5.869 min; OTβ , 4.122 min.

Figure 3. Kinetics of OTA, OTβ and OTα production (A) and Time-profile of the expression of the gene An15g07920 (B) in A. niger 1062 during growth on CYA media

25



Page 26 of 31

Tables

Table 1 Primers used in this study Primers

Function

Sequence (5’ to 3’)

KS5-F

upstream fragment amplification

CCCAAGCTTTGCATGCAAGTACGCCAATGG

KS5-R

upstream fragment amplification

TGCACTGCAGTGGTGGGTAAGGCTATCAGAC

KS3-F

downstream fragment amplification

GGGGTACCGATTCCTCAACGCGATGACCT

KS3-R

downstream fragment amplification

CGGAATTCGAATGCTGCAAGGACTCGC

HPHq-F

qPCR for gene copy number (target gene)

GGCTCCAACAATGTCCTGAC

HPHq-R

qPCR for gene copy number (target gene)

CGTCTGCTGCTCCATACAAG

ACTq-F

qPCR for gene copy number (reference gene)

TTGCGGTACAGCCTCCATTG

ACTq-R

qPCR for gene copy number (reference gene)

CGCTTGGACTGTGCCTCATC

KSqrt-F

qRT-PCR (target gene)

CGGATGACCTCTAAAGCAG

KSqrt-R

qRT-PCR (target gene)

GAAGAATGTCCCACCACC

Tubqrt-F

qRT-PCR (reference gene)

CTTCTGACACGCAGGATAG

Tubqrt-R

qRT-PCR (reference gene)

ACGGCACGAGGAACATAC

26


Page 27 of 31


Table 2 Estimation of the number of T-DNA copies that have been integrated in the genome of the mutants Slope

Inter

Ct HYG

Ct actin

Copy number

HYG

Actin

HYG

Actin

Wild type

△pks

Wild type

△pks

Wild type

△pks

-4.117

-3.399

38.053

30.049

30.97

21.46

16.96

16.69

0.008

1.279

27



Page 28 of 31

Figure graphics

A

C

B

Figure 1

28


Page 29 of 31


LU 17.5 15 12.5 10 7.5 5 2.5

A

0 LU 17.5 15 12.5 10 7.5 5 2.5

2

4

B

8

OTα

OTβ

0 LU 17.5 15 12.5 10 7.5 5 2.5

6

10

min

OTA

2

4

6

8

10

min

2

4

6

8

10

min

C

221.1

0 100

OTβ

Max: 1978

80

60

368.1

293.1

278.8

255.1

216.9

198.8

179.0

187.0

169.0

158.9

125.1

20

132.9

40

0 200

250

300

OTα

m/z

350

255.0

150 100

Max: 1432

80

556.0

380.9

300.9

232.7

217.0

191.1

160.8

20

172.9

129.2

40

255.9

195.9

60

0 200

300

400

80

OTA

m/z

500

404.1

100

100

Max: 3471

429.4

414.1

365.3

377.3

328.2

346.0

295.4

314.3

270.2

230.9 226.3

243.9

194.0 205.1

170.1 181.1

150.2

136.1

120.8

103.9

40

20

406.0

60

0 100

200

300

400

500

m/z

Figure. 2.

29



B

700

OTA OTα OTβ

Peak area (LU*S)

600

1.2

Differences expressed in multiples

A

500

400

300

200

100

0

Page 30 of 31

1.0

0.8

0.6

0.4

0.2

0.0

1

2

3

4

5

6

7

8

9

10

2

Time (d)

3

4

5

6

Time (d)

Figure 3.

30


Page 31 of 31


TOC Graphic

31


A Polyketide Synthase Encoded by the Gene An15g07920 Is Involved

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