Seekles et al. Fungal Biol Biotechnol (2021) 8:4 https://doi.org/10.1186/s40694-021-00111-w Fungal Biology and Biotechnology RESEARCH Open Access Preservation stress resistance of melanin defcient conidia from Paecilomyces variotii and Penicillium roqueforti mutants generated via CRISPR/Cas9 genome editing Sjoerd J. Seekles1,2, Pepijn P. P. Teunisse1,2, Maarten Punt1,3, Tom van den Brule1,4, Jan Dijksterhuis1,4, Jos Houbraken1,4, Han A. B. Wösten1,3 and Arthur F. J. Ram1,2* Abstract Background: The flamentous fungi Paecilomyces variotii and Penicillium roqueforti are prevalent food spoilers and are of interest as potential future cell factories. A functional CRISPR/Cas9 genome editing system would be benefcial for biotechnological advances as well as future (genetic) research in P. variotii and P. roqueforti. Results: Here we describe the successful implementation of an efcient AMA1-based CRISPR/Cas9 genome edit- ing system developed for Aspergillus niger in P. variotii and P. roqueforti in order to create melanin defcient strains. Additionally, kusA− mutant strains with a disrupted non-homologous end-joining repair mechanism were created to further optimize and facilitate efcient genome editing in these species. The efect of melanin on the resistance of conidia against the food preservation stressors heat and UV-C radiation was assessed by comparing wild-type and melanin defcient mutant conidia. Conclusions: Our fndings show the successful use of CRISPR/Cas9 genome editing and its high efciency in P. vari- otii and P. roqueforti in both wild-type strains as well as kusA− mutant background strains. Additionally, we observed that melanin defcient conidia of three food spoiling fungi were not altered in their heat resistance. However, melanin defcient conidia had increased sensitivity towards UV-C radiation. Keywords: CRISPR/Cas9, Cell factory, Melanin, Food spoilage, Food spoiling fungi, Polyketide synthase, Conidia, Aspergillus niger, Penicillium roqueforti, Paecilomyces variotii Introduction Cas9 gene editing tool has been introduced in over 40 Te genome editing system by clustered regularly inter- species of flamentous fungi and oomycetes to date [5]. spaced short palindromic repeats (CRISPR) and CRISPR- In this paper, we describe a functional CRISPR/Cas9 associated protein 9 (Cas9) has proven to be a powerful genome editing protocol for two food spoilage fungi Pae- tool in flamentous fungi, providing new insights and cilomyces variotii and Penicillium roqueforti. opportunities within food, agricultural, clinical and bio- Te CRISPR/Cas9 genome editing system introduces a technological research [1–4]. Currently, the CRISPR/ double stranded break (DSB) on a specifc genomic DNA site. Fungi have two main DNA repair mechanisms that can restore the DSB created by CRISPR/Cas9, namely the *Correspondence: [email protected] non-homologous end-joining repair mechanism (NHEJ) 1 TIFN, Agro Business Park 82, 6708 PW Wageningen, The Netherlands and the homology directed repair mechanism (HDR). Full list of author information is available at the end of the article © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Seekles et al. Fungal Biol Biotechnol (2021) 8:4 Page 2 of 13 For genome editing purposes, many studies rely on the future research on food spoilage capabilities and poten- HDR mechanism in order to control genomic editing tial biotechnological advances in both P. variotii and P. (e.g. gene replacement studies), by providing the fungus roqueforti. with homologous DNA created in vitro [6]. Tis allows Many food spoiling fungi, such as P. variotii and P. for precise DNA insertion, replacement or removal in roqueforti, produce asexual derived spores (conidia) the genome. However, many flamentous fungi prefer that can withstand commonly used preservation treat- repair via NHEJ over HDR, which complicates this pre- ments such as UV radiation or heat [40, 41]. Recently, the cise genome editing. In order to promote DNA repair by conidia of P. variotii have been reported to survive 60 °C HDR in fungi, genes involved in the NHEJ repair mech- for 20 min, being the most heat resistant of this type of anism can be deleted. A mutant fungus with a deleted asexual spores [10]. Additionally, conidia of food spoil- kusA gene is defective in the NHEJ repair mechanism, age fungi are able to survive UV radiation levels used therefore a DSB can only be repaired by HDR as shown in for decontamination by food industry [42–45]. It is yet several flamentous fungi such including e.g. Neurospora unclear if pigmentation provides stress resistance against crassa [7], Aspergillus niger [8]. these preservation techniques in food spoiling fungi. In Te thermotolerant nature of P. variotii spores makes many ascomycetes, disruption of a specifc polyketide this fungus a relevant food spoiler [9–12]. P. variotii is a synthase (PKS) gene results in loss of conidial pigmen- known spoiler of fruit juices, sauce, canned products and tation. As a consequence, these transformants produce non-carbonized sodas [13, 14]. Additionally, P. variotii lighter or white conidia [46–49]. Comparing the conidia strains have been reported to produce industrially inter- of these mutants with their parental strain will lead to esting, often thermostable, enzymes such as tannases, new insights into the potential roles of melanin in preser- amylases, β-glucosidases and an alcohol oxidase [15–21]. vation stress resistance of conidia. Recently, a genome of P. variotii has been published In this research, a functional CRISPR/Cas9 genome [22] in which the frst method on targeted gene disrup- editing system for P. variotii and P. roqueforti is imple- tions in this fungus using Agrobacterium tumefaciens mented to create melanin defcient mutants of both is described. Although A. tumefaciens mediated trans- fungi, and subsequently comparing these mutants to their formations are shown to be efcient and benefcial over wild-type parental strains, using a recently described other transformation methods in certain cases [23], it CRISPR/Cas9 deletion system developed for A. niger does require optimization of multiple factors and can be [50] with minor adaptations. Tis CRISPR/Cas9 genome tedious compared to the relatively quick and easy to use editing system developed for A. niger is based on the PEG-mediated transformations [24]. expression of Cas9 driven from the tef1 promoter [51]. Te flamentous fungus P. roqueforti is best known as Te Cas9 expression cassette, together with the guide the ‘blue cheese’ fungus for its use in blue cheese pro- RNA expression cassette and the hygromycin selection duction [25]. However, P. roqueforti is also a known food marker are located on a plasmid that also contains the spoiler that can produce mycotoxins such as PR-toxin AMA1 sequence which enables autonomous replication and roquefortine-C, which form potential health risks for in Aspergillus species, thereby making integration of the humans [26–29]. As such, P. roqueforti has been inten- vector into the genome less likely [52]. Tis AMA1-based sively studied for its secondary metabolite production CRISPR/Cas9 genome editing system allows for the tem- and specifcally its mycotoxin production [30–33]. Addi- poral presence of the CRISPR/Cas9 plasmid and there- tionally, P. roqueforti has biotechnological potential as a fore marker-free genome editing [50]. Te CRISPR/Cas9 cell factory, as it produces proteolytic enzymes of interest genome editing method is considerably faster than the to the cheese-making industry and high-value second- already established marker-free genome editing method ary metabolites such as mycophenolic acid [33–37]. A which relies on recyclable markers. Te CRISPR/Cas9 CRISPR/Cas9 genome editing system has been described based genome editing method allows for the creation for Penicillium chrysogenum, a closely related species to of multiple mutations in a single transformation experi- P. roqueforti, using a similar approach as has been used ment, as demonstrated in A. niger [50], whereas the recy- for Aspergillus species by providing a CRISPR/Cas9 plas- cling method requires deletions to be performed one at a mid during PEG-mediated transformation [38]. Tis has time. led to the possibility of large scale genome re-engineering Understanding the resistance mechanisms of conidia making P. chrysogenum a useful platform organism as cell from food spoiling fungi will help us in designing novel factory for production of natural products [39]. Taken targeted preservation techniques able to inactivate together, a functional CRISPR/Cas9 targeted genome conidia without altering food favor profles. In order editing protocol based on PEG-mediated transforma- to investigate this, a working CRISPR/Cas9 genome tions of CRISPR/Cas9 plasmids would be benefcial for editing system has been developed for P. variotii and P. Seekles et al. Fungal Biol Biotechnol (2021) 8:4 Page 3 of 13 roqueforti. Tese genome editing systems could enhance A.ab future research and provide a stepping stone towards cre- ating novel biotechnologically relevant cell factories.