A Strategy for Scleroglucan Production in Sclerotium Rolfsii: Lowering pH in Fermentation Process by Manipulating Oxalate Metabolic Pathway With CRISPR/Cas9 Tool

Background: Sclerotium rolfsii is a potent producer of many secondary metabolites, one of which like scleroglucan is an exopolysaccharide (EPS) appreciated as a multipurpose compound applicable in many industrial elds. Results: We chose AAT1 gene in oxalate metabolic pathway as target of CRISPR/Cas9. When the AAT1 gene is disrupted, oxalate was not converted to α-ketoglutarate (AKG), but accumulated. So AAT1-mutant serves to lower the pH (pH 3-4) to increase the production of the pH-sensitive metabolite scleroglucan to be 21.03 g l -1 with productivity reached 0.25 g/(L·h). Conclusions: We established a platform for gene editing to rapidly generate and select mutants, and provide a new benecial strain of S. rolfsii as a scleroglucan hyper-producer which could also reduce the cost of controlling optimum pH condition in fermentation industry.


Background
Microbial biopolymers have been discovered as novel materials to replace plant gums 30 years ago [1].
The advantages of microbial polysaccharides are sustainable production and high quality [2]. In addition, EPS from microorganism is often easy to recover [3]. So it has attracted widespread attention in recent years. For example, pullulan from Aureobasidium pullulans, xanthan from Xanthomonas sp., hyaluronic acid from Streptococcus zooepidemicus, scleroglucan from Sclerotium rolfsii (Teleomorph: Athelia rolfsii) have been reported [4][5][6]. Because of unique structure and high molecular weight, scleroglucan has many properties so that it could be applied in oil recovery [7], food industry [8] and the pharmaceutical industry [9]. Scleroglucan is produced mainly via microbial fermentation. Meanwhile, it has many limits such as low yield and high production cost, which severely hinder scleroglucan application in a wider range of industries [10].
Other factors such as phosphate levels or initial pH do in uence scleroglucan production in a much lesser extent [7]. Many researchers have attempted to select the type and concentration of carbon source to affect the production of scleroglucan. The mechanism of scleroglucan production is not clear. Although, some studies have also demonstrated that pH plays a signi cant role in the fermentation process [11], there is no research of increasing the production of scleroglucan by manipulating relative metabolic pathways at the genomic level.
Oxalic acid is the main acidic metabolite in S. rolfsi, and in many other fungi such as Sclerotinia sclerotiorum [12]. Oxalic acid is also reported to be directly toxic to plant tissue [13], for it is a strong acid among organic acids and is 10,000 times more acidic than acetic acid. So it could also in uence the pH in the process of liquid fermentation to produce scleroglucan. And we also performed bioassays of fungus mutants for indicating more oxalic acid could be produced. For generating a new strain of S. rolfsii that is more suitable for fermentation industrial, an appropriate adjustment of oxalate biosynthesis pathway at the genetic level to further increase the production of scleroglucan is in high demand.
The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (CRISPR-Cas9) system has rapidly progressed as an e cient genome editing technique in various organisms, including many ascomycetes and several basidiomycete fungi [14]. Currently, there is limited information about manipulating S. rolfsii at genetic level, owing to technical limitations including lack of wellannotated genome and e cient recombinant DNA methodologies. Few reports described the transgenic strains based on protoplast transformation [15][16]. However, there is no gene disruption linked with precise phenotype analysis reported. Recently the draft of S. rolfsii genome was published [17]. By combining the transcriptome data [18] and cross-species sequence homology analysis, we were able to obtain the transcripts for essential enzymes for oxalate synthesis, which offered a roadmap for establishing mutant strains that is of indirect relevance for production of scleroglucan with high yields.

Results
Identi cation of transformation for pDHt/sk-PE plasmid by PCR and uorescence microscopy To test whether transgene line could be established in S. rolfsii, we performed the pDHt/sk-PE plasmid transformation with the PEG based method. After 5 days in hygromycin-PDA medium, the eGFP signal can be consistently detected. The PCR analysis with eGFP primers showed the positive band ( Figure 1A).
Then, we observed the bright uorescent signal in hypha of transformants by uorescence microscopy ( Figure 1B), indicating the transformed strains contain the functional elements. Altogether, we have established the convenient method for PEG-based pDHt/sk-PE transformation.

CRISPR/Cas9 mediated gene inactivation in S. rolfsii
To transfer Cas9 RNPs (ribonucleoprotein complex) targeting AAT1, we used the PEG-mediated transformation method to deliver RNP into fungal protoplasts, with selecting plasmid Htb2-GFP which carrying the hygro cassette. And we also co-transformed Cas9 expressing plasmid pDHt/sk-PE and guide RNA complex to knock out AAT1 simultaneously. AAT1-MT colonies were con rmed by sequencing ( Figure 2A). In fact, they could be quickly selected by the yellow color they caused in hygromycin-BPDA medium among hundreds of colonies ( Figure 2B). Because the early appearance of yellow color is due to that AAT1-MT colonies have produced more acid metabolites than WT. HPLC-MS was used to identify which acid they secreted here played a major role.
Disruption AAT1 leads to elevated both oxalic acid and scleroglucan production Metabolic pathway of AAT1-MT is showed in Figure 3A. Based on the measurement of the oxalic acid peak area ( Figure 3B), we calculate that the concentrations of oxalic acid in WT and AAT1-MT samples are 843.32 µg ml-1 and 2854.42 µg ml-1, respectively. And we also analyzed AKG, the close related metabolite in this pathway (supplementary gure s3 . The mass spectrograms are showed in supplementary gure s4. Above all, the mutant strains secreted 3 times more of oxalate than WT and signi cantly less of AKG, indicating the disruption of AAT1 leads to the metabolic ow towards the oxaloacetate direction then produce more oxalic acid. Clearly, due to the inactivation of AAT1 and the accumulation of oxalic acid, the lesion area in mutant strains was more signi cant compared to control strains, which is shown with brown color in the lea ets ( Figure 3C).
The line chart ( Figure 3D) about concentrations of oxalic acid and scleroglucan in fermentation broth showed that the ability of AAT1-MT for producing scleroglucan (21.03 g l-1) is stronger than WT (12.11 g l-1). The scleroglucan production of AAT1-MT is increased by 73.66%, when compared with WT. Dry cell weight (DCW) concentration and pH in the fermentation process was showed in Figure 3E. After 48h, the pH could keep at about 3.37 and the growth of oxalate accumulation slowed. This slow-down coincided with a drop in culture pH to less than 3.5 [19]. This phenomenon could possibly be due to the preactivation of some degradative enzymes, such as oxalate decarboxylase and oxalate oxidase [18], by a mass of oxalic acid. In other fungi such as Coriolus versicofor and Collybia velutipes, the optimum pH of these enzymes has been reported to be 3 [20][21]. The highest DCW (14.26g l-1) of AAT1-MT was achieved when pH was at 4 after 36 h, while the DCW of the WT is 10.23 g-1. When pH is between 3 and 4, both cell growth and polysaccharide synthesis is fast-growing. And the scleroglucan productivity reached 0.25 g/(L•h) which increased by 78.57% when compared with that obtained from WT (0.14g/(L•h)).

Discussion
Scleroglucan is a polysaccharide composed of a linear chain of β-(1,3)-linked D-glucopyranosyl residues with single β-(1,6)-linked D-glucopyranosyl groups attached to about every third residue of the main chain [22]. Many excellent properties, including water solubility, pseudoplasticity, moisture retention, salt tolerance, and viscosity stability, deserve us thinking about how to solve its application problem fundamentally. This is the rst study of exploring CRISPR-Cas9 system in S. rolfsii. In the CRISPR/Cas9 system, the Cas9 endonuclease catalyzes a DNA double-strand break (DSB) aided by a single-guide RNA (sgRNA) containing a 20-nt sequence that matches the sequence upstream of the protospacer-adjacent motif (PAM; 5'-NGG-3') site on the target locus. The DSB can then be followed by deletion, insertion, or substitution of the sequence using homology recombination or non-homologous end joining (NHEJ) pathway. Therefore, we aimed at establishing the CRISPR/Cas9 system to change metabolic pathway then more oxalic acid could be produced to change the pH of fermentation liquid that is in favour of scleroglucan production.
We also tried with sgRNA transformation together with Cas9 plasmid. However we only got one mutant colony. Using the Cas9 protein/sgRNA ribonucleoproteins (RNPs) to perform genome editing has several advantages compared with co-transforming of Cas9 expression plasmid and sgRNA. A major advantage is transformation of Cas9 RNPs alleviates the possibility of integration of genetic material to a nontargeted region of the genome [23][24]. Additionally, Cas9 and sgRNA are able to form a stable ribonucleoprotein in vitro, so there is less likelihood of RNA degradation compared with Cas9 mRNA/sgRNA transformation.

Conclusions
So far, most studies mainly cover the feasibility of establishing the CRISPR/Cas9 system in lamentous fungi, only few works have explored the application of genome editing for fungal metabolic engineering [25]. The results of this study provided an effective strategy to indirectly control pH condition, thereby increasing the yield of sclerogulcan by using newly emerging gene editing tool CRISPR/ Cas9, and showed a potential strategy to radically decrease the cost of arti cial regulating pH in industrial sclerogulcan fermentation process.

Methods
Strain and culture condition Sclerotium rolfsii (Teleomorph: Athelia rolfsii) deposited in the Chinese Academy of Agricultural Sciences (CAAS), was used as the wild-type (WT) and cultured in potato dextrose agar (PDA; peeled potato 200 g, dextrose 20 g, agar 15 g, distilled water 1 l, pH=7.5) and in the fermentation medium (glucose 130 g, NaNO 3 3 g, yeast extract 1 g, KCl 0.5 g, KH 2 PO 4 1 g, MgSO 4 ·7H 2 O 0.5 g, distilled water 1 l, pH=7.5) at 30°C. Batch fermentations were performed in a 5-L fermentor containing 3L of fermentation medium. All the components were autoclaved for 20 min at 115°C. The strain was identi ed by ITS primers. All primers used in this study are listed in supplementary table s1.

AAT1 identi cation
To identify the AAT1 gene, we rst downloaded the assembled genome of Athelia rolfsii (GCA_000961905.2) from NCBI and annotated its protein-coding genes using GeneMark-ES [26] with opinions '--ES -fungus --sequence'. We also assembled a transcriptome data available from the NCBI SRA database for A. rolfsii (accession ID: SRS025455) using SPAdes [27] with opinions '--sc -s --careful -k 75', and also annotated the protein-coding genes. All the protein-coding genes were combined and renamed starting with A0000000.
We manually selected AAT1 genes from six well-annotated fungal genomes including Scheffersomyces stipitis, Emiliania huxleyi, Kluyveromyces marxianus, Saccharomyces cerevisiae, Pseudogymnoascus destructans and Candida albicans) and BLASTed their protein sequences against the identi ed proteincoding genes of A.rolfsii (supplementary gure s1 a). We identi ed gene "A0001768" from transcriptome data as the best hit. We then con rmed that "A0001768" could cover the full-length of AAT1 by building a multiple sequence alignment using the protein sequences of "A0001768" and the six fungal AAT1 proteins using an online version of Clustal Omega [28][29]. The result was visualized by an online version of Mview [30] later.
Finally, we obtained the gene structure (i.e., the delineation of its exons) of "A0001768" (supplementary gure s1 b) by mapping its coding sequences to its assembled genome using BLAT [31].
Incubate the mixture at 95 °C for 5 min and cool down at R.T. for 20 min. All plasmids used in this study and their purposes are listed in supplementary table s2 and they were all purchased from Addgene.

Preparation of protoplast
Procedures of protoplast formation were carried out as described in [34], with some modi ed details presented here. After 90 min of incubation for depriving the cell wall, we used sterile 100 μm Cell Strainer to lter out impurities of reaction mixture.
Transformation for S. rolfsii PEG-mediated fungal transformation was conducted according to previously described with modi cations [35]. Brie y, the RNPs (ribonucleoprotein complexes which are composed of Cas9 and sgRNA) and Htb2-GFP plasmid were prepared during generation of the protoplasts where the Cas9 RNPs were made as follows: 10 μl assembled guide RNA complex (described above) and 5 μl Cas9 protein (50 μM) were added into a 50 μl total volume with 5 μl 10× Cas9 Nuclease Reaction Buffer and DEPC-treated water. This mixture was incubated in a 37°C water bath for 25 min, and 100 µL of fungal protoplasts were mixed with 20 μl Cas9 RNPs and Htb2-GFP plasmid at room temperature for 20 min. Then 40 % PEG was added into the above system and incubated at room temperature for 20 min. After STC buffer (1.2 M Sorbitol, 10 mM pH 7.5 Tris-HCl, 10 mM CaCl 2 ) was mixing well by gently inverting the tubes several times, the total system was directly transferred into MGY regeneration medium (1% malt extract, 1% glucose, 0.1% yeast extract, 2% agar, pH 5.5) with 0.5M sucrose osmotic stabilizer. Four days later, protoplasts developed into incipient colonies observable with the naked eye, then, bottom agar was covered with 20ml top selective hygromycin-BPDA agar containing hygromycin (35 µg ml -1 ) and bromophenol blue (60 µg ml -1 ) which is a kind of indicator that changes color from yellow to blue at the pH from 3.0 to 4.6.

Fluorescence microscopy
The subcellular localization of eGFP was carried out using a Leica DMi8 uorescence microscope. The transformants containing pDHt/sk-PE were cultured in MGY agar plate in the dark incubator at 30°C for 7 days.

Analytical method
Mycelium was obtained by germination of water-preserved sclerotia on PDA agar plate and incubated at 30 °C as previously described [36]. Then, two 250-ml Erlenmeyer asks of 50 ml of liquid medium (MGY) were inoculated with ve mycelium covered agar discs (approx. 5 mm diameter) removed from the 2-dayold PDA culture of WT and AAT1 mutant type (MT), respectively, at 30 °C on an orbital shaker at 250 rev min -1 for 45 h waiting for HPLC-MS analysis. Mycelia were frozen in the refrigerator at -80 °C. After thawing, mycelia were grinded in a mortar until mycelium was completely broken and mushy. Equal volume of ethyl acetate was added to extract and collect the ethyl acetate phase under ultrasonic condition (at 100 kHz for 1h). Rotate evaporation was used to dry the phase at 55 ℃, and added 6 ml methanol in a volumetric ask waiting to be tested about metabolites from mycelium itself, like AKG. Then we also measured the scleroglucan production in the fermentation broth between WT and AAT1-MT. The fermentation broth was diluted 5 times with distilled water, heated at 70°C for 40min, and then centrifuged at 13400×g for 25min. The precipitate obtained was washed with distilled water and dried at 105°C. An equal volume of absolute ethanol was added to the supernatant to precipitate scleroglucan. The mixture stayed on ice for 12 h to completely precipitate. In the end, the scleroglucan was recovered by vacuum drying [37].

Bioassays of acid metabolites
In order to identify that AAT1-MT could produce more acid metabolites than WT, bioassays were performed using detached peanut lea ets inoculated with an agar plug of S. rolfsii mycelia. S. rolfsii cultures were grown on potato dextrose agar plates and 5-mm plugs taken from the actively growing edge. Lea ets were wounded with a knife for 5mm on the adaxial surface, near the midvein, and plugs were placed on the open wounds. Five lea ets were inoculated for each plant line tested using a minimal quantity of agar in each plug. The plates were incubated for 36 h at 30 °C, and lesion area shown brown blight color caused by oxalic acid.

Statistical analysis
All the WT and AAT1-MT mycelium samples were designed for three biological replicates. The data from repeated HPLC analyses were pooled and subjected to ANOVA for statistical signi cance by least signi cance difference (LSD) test at P=0.01. An independent sample t-test was used for statistical evaluations between the WT and AAT1-MT groups (P ≤ 0.05) by the SPSS 21.0 software (IBM, Chicago, IL, USA). Hyphae of WT could barely show green lamentous uorescent, except the background signal from agar. In contrast, bright GFP uorescent signal was shown in transformants