Novel genetic tools improve Penicillium expansum patulin synthase production in Aspergillus niger

Since the first CRISPR (clustered regularly interspaced short palindromic repeats)‐Cas (CRISPR‐associated) system was developed for creating double‐stranded DNA breaks, it has been adapted and improved for different biotechnological applications. In this issue of The FEBS Journal, Arentshorst et al. developed a novel approach to enhance transgene expression of a specific protein, patulin synthase (PatE) from Penicillium expansum, in the important industrial filamentous fungus Aspergillus niger. Their technique involved the disruption of selected genes with counter‐effects on targeted protein production and simultaneous integration of glucoamylase landing sites into the disrupted gene locus such as protease regulator (prtT) in an ATP‐dependent DNA helicase II subunit 1 (kusA or ku70)‐deletion strain. Multiple copies of the PatE transgene expression cassette were introduced by CRISPR‐Cas9‐mediated insertion. The purified PatE was further used for structural and functional studies, and the technique laid the foundation for elevating the overall production of various proteins or chemicals in those industrially important fungi.

Since the first CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system was developed for creating double-stranded DNA breaks, it has been adapted and improved for different biotechnological applications.In this issue of The FEBS Journal, Arentshorst et al. developed a novel approach to enhance transgene expression of a specific protein, patulin synthase (PatE) from Penicillium expansum, in the important industrial filamentous fungus Aspergillus niger.Their technique involved the disruption of selected genes with counter-effects on targeted protein production and simultaneous integration of glucoamylase landing sites into the disrupted gene locus such as protease regulator (prtT) in an ATP-dependent DNA helicase II subunit 1 (kusA or ku70)-deletion strain.Multiple copies of the PatE transgene expression cassette were introduced by CRISPR-Cas9-mediated insertion.The purified PatE was further used for structural and functional studies, and the technique laid the foundation for elevating the overall production of various proteins or chemicals in those industrially important fungi.

Introduction
Traditionally, a transgene expression chassis consists of two expression cassettes: one for the gene of interest and the other for the selectable marker gene.When eukaryotic cells are transinfected with the transgene expression chassis, the integration event mainly occurs randomly.To increase homologous recombination events, a common strategy involves suppressing or disrupting the nonhomologous end-joining (NHEJ) pathway, which was first identified in Hela cells [1] and further examined in different organisms [2][3][4][5].The Ku-dependent nonhomologous end-joining (NHEJ) pathway for gene modifications has been well established in different fungal systems, including the industrially important filamentous fungus Aspergillus niger [6,7].
In 2013, the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPRassociated) system was developed to produce doublestranded DNA breaks in eukaryotic cells [8].The CRISPR-Cas9 technique was successfully demonstrated early in Saccharomyces cerevisiae [9], Aspergillus aculeatus [10], and Trichoderma reesei [11].The intact plasmids prepared for CRISPR-Cas9 expression in S. cerevisiae or A. aculeatus contained extrachromosomal 2l or AMA1 (autonomous maintenance in Aspergillus) for effective plasmid replication in extrachromosomal locations.In contrast, the transgene expression cassette for T. reesei was prepared for chromosomal integration.The advantage of the 2l or AMA1-based episomal vector is that the plasmid can be eliminated by culturing cells without antibiotic selection.This leads to markerfree transgene expression.The AMA1-based episomal vector has been applied to a wide variety of filamentous fungi, including A. niger.
Aspergillus niger is one of the most important industrial filamentous fungi and is known for its applications in the production of various proteins and organic acids.More than 80% of global citric acid production is obtained by A. niger fermentation [12].Aspergillus niger is being explored for heterologous protein production, such as amylase, chymosin, glucose oxidase, lipase, pectinase, proteases, and xylanases.Although common molecular biology techniques have been adapted and developed for A. niger, a further expansion of the molecular toolbox is needed to accommodate the rapidly growing bioeconomy.
When A. niger is adapted for heterologous production of certain chemicals or proteins, the general considerations are the promoters, codon usage, transcriptional terminators, and transgene integration location.The effectiveness of the production is governed by many endogenous and exogenous factors, such as various regulatory proteins or metabolites inside the producing cells.To achieve optimal production, multiomics data would need to be generated from diverse culture conditions and genotypes [13].Such omics data would form the foundation to understanding, via machine learning, how the endogenous factors exert their effects on the production of a specific protein or chemical.Downregulating certain genes can be accomplished by the CRISPR-Cas system.In contrast, gene upregulation will require thorough considerations for optimal transgene expression, such as random versus site-specific homologous integration, the number of transgene expression cassettes to be integrated, and the use of native versus synthetic promoters/transcriptional terminators.

Effective disruption and homologous replacement of multiple genes
In this issue of The FEBS Journal, Arentshorst et al. [14] described a novel CRISPR-Cas9-based multicopy integration method to augment transgene expression of Penicillium expansum patulin synthase (PatE) in A. niger (Fig. 1).The resulting heterologously expressed PatE was purified from A. niger and used for structural elucidation and functional characterization, which is described in the same issue of The FEBS Journal [15].In their efforts to increase PatE production in A. niger, 12 selected genes were disrupted: two aspartic proteases, two aspartic peptidases, a carboxypeptidase, transcriptional activator of proteases, amylase, glucoamylase, acid amylase, alpha-glucosidase, glucose oxidase, and oxaloacetate hydrolase.The aspartic proteases and peptidases, carboxypeptidase, and transcriptional activator of proteases are key enzymes in cellular protein degradation, whereas amylase and acid amylase, glucoamylase, and a-glucosidase are abundant proteins produced in A. niger.In A. niger cultures, glucose oxidase hydrolyzes the glucose for D-gluconic acid accumulation or oxaloacetate hydrolase for oxalic acid production, which leads to low pH and an increase in the activities of related proteases [16].
Arentshorst et al. [14] effectively introduced up to 10 glucoamylase landing sites (GLSs) targeted to the above-selected gene-coding region in the genome by combining both ATP-dependent DNA helicase II subunit 1 (kusA) disruption and the plasmid-based CRISPR-Cas9 technique.Each GLS contains the glucoamylase (gla1) promoter, unique dubbed KORE sequence for single guide RNA (sgRNA) targeting, and its transcriptional terminator, which was designed for homologous recombination at the coding region of selected genes.After several repeats of transformed events, 10 GLSs were introduced at the predetermined sites in a final marker-free transgenic strain.Similarly, co-transformation with plasmid-based CRISPR-Cas9 with a specific KORE sgRNA targeting doublestranded breaks, the transgene expression cassette glaP-PatE-6xHis-glaT was integrated into three specific GLSs.After several repeats of transformation, the PatE transgene expression cassette integrated into 10 different GLSs was confirmed at the final transgenic strain.About 70 mgÁL À1 PatE was produced in this selected strain.
Penicillium expansum PatE is known to be involved in the biosynthesis of the mycotoxin patulin.It contains seven predicted N-glycosylation sites, and the purified PatE was about 41% larger than the PatE without glycosylation [15].The PatE derived from de-glycosylation was still larger than the expected molecular weight, suggesting the incomplete removal of N-glycan chains.Nevertheless, this de-glycosylated PatE can be used for crystal structure analyses and functional characterization.

Conclusion
The novel molecular toolbox for genetic manipulation presented by Arentshorst et al. in this issue of The FEBS Journal demonstrated that the combination of CRISPR-Cas9-based double-strand break formation and kusA disruption of the NHEJ pathway enhances the efficiency of gene replacement at the specific chromosomal locus with the targeted transgene fragment containing a unique GLS.Along with gene-specific GLSs, multiple copies of the transgene expression cassette can be integrated into the chromosomes after repeated CRISPR-Cas9-mediated transformations.De-glycosylation of PatE proteins produced in A. niger can be used for protein structure and function characterization.This will pave the way for production of various proteins or chemicals at a high production titer, rate, and yield in A. niger.