Engineering Industrial Strain of Acremonium chrysogenum for Deacetoxycephalosporin C Overproduction Using CRISPR/Cas9

TL;DR

Researchers used CRISPR/Cas9 to create a high-yield strain of Acremonium chrysogenum for making a key antibiotic precursor, DAOC, in a single step.

Contribution

A single-step CRISPR/Cas9 method for simultaneous gene deletion and overexpression in industrial A. chrysogenum strains.

Findings

The edited strain achieved a DAOC titer of 12.4 ± 0.2 g/L, suitable for industrial production.

The method improved DAOC production compared to the initial strain (7.2 ± 0.22 g/L).

CRISPR/Cas9 offers a strategy for enhancing other cephalosporin precursor production.

Abstract

Background: The fungus Acremonium chrysogenum is crucial for producing cephalosporin antibiotics. While CRISPR/Cas9 has been developed for this species, it has not been applied to first-line industrial strains, to the best of our knowledge. For example, engineering industrial A. chrysogenum for overproducing deacetoxycephalosporin C (DAOC, an important precursor for clinically used cephalosporin antibiotics) is currently often a multi-step and inefficient process. Methods: Here, we applied CRISPR/Cas9 to create a DAOC overproducer in a single step. Our method uses a donor template to simultaneously delete and overexpress genes, offering a simple, efficient, and time-saving solution. Results: Furthermore, through methodological optimization, the final homozygous multigene-edited strain achieved an industrial-scale DAOC titer of 12.4 ± 0.2 g/L, representing a significant improvement over the initial edited strain (7.2 ± 0.22 g/L). Conclusions: This demonstrates that CRISPR/Cas9 can effectively edit industrial A. chrysogenum, providing a strategy to enhance the production of other cephalosporin precursors.

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Full text

1. Introduction

Deacetoxycephalosporin C (DAOC) is the core precursor for 7-aminodeacetoxycephalosporanic acid (7-ADCA), a key intermediate in the industrial synthesis of cephalosporin antibiotics such as cefradine and cephalexin [1,2]. Through the side-chain modifications of 7-ADCA, a broad spectrum of clinically important antibiotics can be derived, with approximately one-third of global cephalosporin production relying on this route [3,4]. The conversion from DAOC to 7-ADCA has been streamlined using engineered CPC acylase, enabling the efficient one-step hydrolysis of the amide bond in DAOC’s side chain [5] (Figure 1A).

In the fungus A. chrysogenum, DAOC is also a central biosynthetic intermediate in the cephalosporin C (CPC) pathway. CPC biosynthesis begins in the cytoplasm with the formation of the tripeptide LLD-ACV, which is cyclized to isopenicillin N by PcbC. After translocation into peroxisomes and conversion to penicillin N by CefD, the bifunctional enzyme CefEF catalyzes the expansion of the thiazolidine ring to form DAOC and its hydroxylated derivative, deacetylcephalosporin C (DCPC). DAOC is subsequently converted to CPC by CefG [6] (Figure 1B,C).

Therefore, DAOC occupies a critical junction: it is both the direct precursor to the high-value industrial building block 7-ADCA and an obligatory intermediate in the native CPC pathway of A. chrysogenum. Metabolic analysis confirms that DAOC yield directly dictates the final output of 7-ADCA [7,8]. Consequently, metabolically engineering the industrial workhorse A. chrysogenum to construct a high-yielding DAOC strain represents a pivotal objective for the antibiotic industry.

This specific engineering strategy is as follows (Figure 1D): AccefEF gene encoding the bifunctional expandase/hydroxylase is disrupted to block the conversion of penicillin N to deacetylcephalosporin C (DCPC) [9,10], while penicillin N expandase encoding genes cefE from Streptomyces clavuligerus [7], Mycobacterium abscessus or Sphingomonas dokdonensis [5] and the isopenicillin N epimerase encoding gene SccefD from S. clavuligerus [5,11,12] are randomly integrated into the genome of A. chrysogenum strains to redirect metabolic flux toward DAOC accumulation, achieving DAOC titers in the range of 8.66–10.16 g/L [5]. This method combines two distinct editing tools: the former (gene deletion) relies on homologous recombination, while the latter (gene overexpression) is achieved through random plasmid integration.

However, this method has the following limitations: (1) The sequential use of two different editing tools is time-consuming, and (2) both editing tools rely on traditional approaches, which may be inefficient. More importantly, the CRISPR-Cas9 system has only been applied for the genetic engineering of the A. chrysogenum wild type and C10 strain, to the best of our knowledge [13,14]. We expected that, if the CRISPR-Cas9 system is introduced for the construction of a DAOC industrial strain, due to the Cas9 induced-double-strand breaks (DSBs) that would greatly enhance the efficiency of Homology-Directed Repair (HDR) [15,16], gene deletion and overexpression could be achieved in a single step by using a donor template containing both the homologous arms and the overexpression cassette (Figure 1E).

In the current study, we systematically optimized the PEG/CaCl_2_-mediated protoplast transformation protocols for the CRISPR-Cas9 genome editing system. Further genome editing toward the first-line industrial strain A. chrysogenum using CRISPR/Cas9 coupled with HDR, enables the generation of DAOC overproducing strain in a single step transformation, which circumvents the drawbacks of the known method [14], including inefficient and time-consuming multi-step transformations with the requirement for multiple genetic tools and difficulties in subsequent strain improvement (Figure 1E). Our study would not only establish the CRISPR-Cas9 editing system for the first time in the industrial strain of A. chrysogenum and directly yield a high-producing DAOC strain, but more importantly, it would provide a new technical pathway for further upgradations of this high-producing strain and for the targeted engineering of industrial strains producing other cephalosporin antibiotics.

2. Results

2.1. Optimization Fungal Transformation for CRISPR/Cas9 Engineering

Although previous studies have described the split-marker homologous recombination strategy [17], our application of this method failed to yield effective mutants (0% replacement efficiency), indicating the limitations of the conventional genetic manipulation method in the multinucleate Mc-1 strain. Therefore, we employed the Streptococcus pyogenes Cas9 nuclease (SpCas9) developed in our laboratory to induce targeted double-strand breaks (DSBs) and used linear donor templates to drive homology-directed repair (HDR), thereby establishing a precise genome-editing system.

To establish a robust transformation system, we systematically optimized two key parameters: protoplast preparation and transformation conditions. First, by evaluating various enzyme combinations and digestion conditions, we identified an optimal formulation consisting of 0.5 mg/mL snailase and 0.5 mg/mL yatalase dissolved in K-buffer. This optimization significantly increased protoplast yield to 1.35 × 10^8^ protoplasts per milliliter of YPS culture, representing a 20-fold increase over the initial protocol (Table S4, Figure S1).

Second, PEG-mediated transformation conditions were refined using the CRISPR-Cas9 plasmid pDC-1. The CRISPR-Cas9 plasmid pDC-1 incorporating the SpCas9 gene driven by the strong promoter Ptef1, a specific sgRNA driven by the PafU6/tRNA chimeric promoter, and a hygromycin resistance cassette (Hph^R^) (Figure 2A). This plasmid was then used to refine the PEG-mediated transformation protocol. Through a comparison of different PEG concentrations, 45% PEG6000 was determined to yield the highest transformation efficiency, as assessed by transformant growth (Figure S2).

2.2. Construction of DAOC Strain Using Linear Donor Templates

Building upon this foundation, this study employed Gibson Assembly to synthesize the linear HDR donor template dsDC-1, which directed the integration of the exogenous SccefE gene expression cassette (containing the PgpdA promoter and Ttef1 terminator) and the glufosinate resistance (Bar^R^) gene expression cassette into the AccefEF locus (Figure 2B). This enabled the knockout of the endogenous AccefEF gene (999 bp) in A. chrysogenum and simultaneous site-specific integration of the S. clavuligerus-derived SccefE gene (933 bp) at the locus, achieving gene replacement [18]. Consequently, this approach precisely blocks the cephalosporin C (CPC) biosynthesis pathway, facilitating the efficient synthesis of deacetoxycephalosporin C (DAOC).

Using a PEG/CaCl_2_-mediated protoplast transformation system and dual antibiotic screening (300 μg/mL hygromycin + 100 μg/mL glufosinate), 120–160 transformants were obtained. Subsequent gradient resistance screening (200 μg/mL glufosinate) yielded 10 strains. PCR validation (Figure 2B) identified three positive mutants with successful AccefEF gene replacement. This method achieved an editing efficiency of 30% (3/10), representing a significant improvement over the split-marker homologous recombination strategy.

However, this approach also introduced challenges. PCR amplification of the target region revealed residual bands in some strains, indicating incomplete homologous recombination and the presence of heterokaryotic mutants (ΔAccefEF::SccefE/+AccefEF). The recombinant positive strain DAOC-1 was selected for flask fermentation. Sampling at 144 h (Figure 3A,B), when both biomass and DAOC production peaked, showed a DAOC titer of 7.2 ± 0.22 g/L (Figure 3C). This represents a 34.1-fold increase over the original Mc-1 strain (0.21 ± 0.1 g/L), while the mutant strain retained CPC synthesis capability (Figure 3D,E and Figure S3).

2.3. Construction of DAOC Strain Using Circular Donor Templates

Although this study found that CRISPR-Cas9-mediated homologous recombination significantly improved gene replacement efficiency compared to the split-marker strategy (30% vs. 0%) and enabled one-step completion, thereby shortening the transformation cycle, the genetic heterogeneity caused by incomplete recombination could affect the consistency of edited strains and easily lead to strain degeneration. To address this, we further explored circular HDR donor templates to enhance recombination efficiency, laying a stable genetic foundation for constructing high-yield DAOC strains.

To overcome incomplete gene knockout due to multinucleation, this study further developed a dual-plasmid synergistic editing system to optimize recombination efficiency (Figure 2C). Based on retaining the elements of dsDC-1, a circular HDR donor template plasmid, pDC-2, was constructed. Using the same method as in Section 2.2, protoplasts were transformed, and dual antibiotic screening yielded 150–200 transformants. Subsequent gradient resistance screening produced 11 strains. PCR validation (Figure 2C) successfully identified four positive mutants with AccefEF gene replacement, achieving an editing efficiency of 37% (4/11) with this method.

With the optimization using the circular HDR donor template, PCR results of the target region showed residual amplification bands in only one strain (a heterokaryotic mutant), while the other three strains (DAOC-2, DAOC-3, DAOC-4) showed no detection of the AccefEF gene fragment. This indicates successful synchronous genome editing in multinucleate filamentous fungi, resulting in homokaryotic mutants (ΔAccefEF::SccefE).

Flask fermentation and metabolic product analysis of the three homokaryotic mutants were performed. When sampling at 144 h, when both biomass and DAOC production peaked (Figure 3A,B), LC-MS detection revealed a complete loss of CPC synthesis capability in the mutants (no characteristic CPC peaks detected), confirming the complete replacement of the AccefEF gene (Figure 3D,E and Figure S3). HPLC quantitative analysis showed that DAOC titers were significantly increased to 8.1 ± 0.2 g/L (DAOC-2), 10.0 ± 1.1 g/L (DAOC-3), and 10.1 ± 1.3 g/L (DAOC-4) (Figure 3C). Among them, DAOC-4 exhibited a significant 19.6% increase (p < 0.05) compared to the heterokaryotic mutant DAOC-1 (7.16 ± 0.3 g/L).

The dual-plasmid system, by enhancing the stability of the HDR donor template, significantly reduced the proportion of heterokaryotic transformants and enabled the precise and complete replacement of the AccefEF gene, providing an efficient technical solution for metabolic flux reprogramming in industrial strains.

2.4. Enhancing DAOC Production by Overexpressing SccefD

Given that the conversion of isopenicillin N to penicillin N is a critical rate-limiting step in DAOC synthesis (Figure 1B,C) [6,11,12], integrating cefD and cefE into the Mc-1 strain not only directly enhances the DAOC synthesis pathway but also alleviates precursor supply limitations at this step. Moreover, since the reaction catalyzed by CefD primarily occurs in peroxisomes, fusing the Peroxisomal Targeting Signal 1 (PTS1) with CefD enables the construction of an efficient ‘synthesis-transport’ microdomain [19,20]. This strategy optimizes the metabolic flux for DAOC synthesis while mitigating potential cytotoxicity caused by intermediate metabolites. To this end, this study employs a circular dual-plasmid co-expression system to further optimize the DAOC biosynthetic pathway.

Specifically, the codon-optimized SccefD gene expression cassette—with its C-terminus fused to the PTS1 sequence from the AccefD1 gene of A. chrysogenum and regulated by the PpcbC promoter—was inserted into the circular HDR donor template plasmid pDC-3, successfully constructing the versatile expression plasmid pDC-4 (Figure 2D). Following protoplast transformation, dual antibiotic screening yielded 150–200 transformants. Subsequent gradient resistance screening produced 20 strains. PCR validation (Figure 2D) successfully identified two positive mutants with AccefEF gene replacement, corresponding to an editing efficiency of 10% (2/20) with this method.

PCR analysis of the target region showed residual amplification bands in one strain (a heterokaryotic mutant, ΔAccefEF::[SccefE::SccefD]/+AccefEF), while strain DAOC-5 completely lacked the AccefEF gene (a homokaryotic mutant, ΔAccefEF::[SccefE-SccefD]). Flask fermentation analysis of DAOC-5 was conducted. When sampling at 144 h, when both biomass and DAOC production peaked (Figure 3A,B), LC-MS detection confirmed the complete disappearance of characteristic CPC peaks, verifying the full replacement of the AccefEF gene (Figure 3D,E and Figure S3). HPLC quantitative analysis revealed a DAOC titer of 12.4 ± 0.2 g/L (Figure 3C), representing a 22.4% increase (p < 0.05) compared to the homokaryotic mutant DAOC-4 expressing only SccefE (10.1 ± 1.3 g/L).

These results indicate that the dual-plasmid system, by co-expressing the SccefE and SccefD genes, effectively alleviates the rate-limiting step in DAOC synthesis (penicillin N accumulation). Furthermore, CRISPR-Cas9-mediated targeted integration achieves directed reprogramming of metabolic flux. This strategy provides a novel approach for multi-gene stacking and pathway enhancement in the engineering of secondary metabolite production in filamentous fungi.

Through the continuous optimization of the gene-editing tools developed in this study, the shift from linear to circular vectors effectively resolved the issue of heterokaryotic edited strains (Figure S5), while simultaneously increasing DAOC production significantly from 7.2 ± 0.22 g/L to 10.1 ± 1.3 g/L. Further advancing from single-gene replacement to multigene editing via circular vectors not only strengthened DAOC synthesis but also enhanced precursor supply, ultimately raising the titer to 12.4 ± 0.2 g/L. For the first time in an industrial strain, the knockout of one gene and the overexpression of two genes were accomplished in a single step, resulting in a high-yielding and genetically stable homozygous edited strain.

2.5. Transcriptional Analysis of Engineered Strains

After confirming the high DAOC production capability of the mutant strains, transcriptional analysis was performed to assess gene expression levels. Mycelial samples were collected at different fermentation time points and subjected to real-time quantitative PCR (qRT-PCR) analysis. β-actin was used as the internal reference to evaluate the expression of the endogenous AccefEF gene as well as the heterologous SccefE and SccefD genes.

The results show that the expression of the AccefEF gene peaked at 72 h of fermentation in both the parental Mc-1 strain and the heterokaryotic mutant DAOC-1 (Figure 4A). In the engineered strains DAOC-1 and the homozygous mutant DAOC-4, the transcription of the bacterial-derived SccefE gene reached its highest level at 48 h. In contrast, the multigene-edited strain DAOC-5 maintained high SccefE transcript levels throughout early (24 h), middle (72 h), and late (144 h) fermentation stages (Figure 4B), corresponding to its highest final DAOC titer. Furthermore, in strain DAOC-5, SccefD expression peaked at 96 h (Figure 4C), a period during which SccefE expression was relatively low, suggesting that SccefD activity ensured sufficient precursor supply for subsequent DAOC biosynthesis.

These transcriptional data demonstrate that the DAOC-5 strain, obtained through iterative integration of different methodological optimizations, not only achieved one-step gene replacement but also yielded a homozygous edited strain carrying multiple genetic modifications. Compared to the parental strain, DAOC-5 showed a substantial increase in product titer, representing a novel breakthrough in the gene editing of industrial fungal strains.

3. Discussion

The efficient biomanufacturing of deacetoxycephalosporin C (DAOC) holds strategic significance for ensuring the supply chain security of β-lactam antibiotics and addressing the risks of global drug shortages [21]. In fermentation production, the performance of the microbial strain is the primary factor determining yield. An excellent strain should possess characteristics such as a high titer, genetic stability, strong resistance to degeneration, and good adaptability to raw materials. Therefore, the selection and breeding of high-yield, stable strains that simultaneously enhance the DAOC synthesis pathway and precursor supply, are of considerable industrial value.

The CRISPR-Cas9 strategy coupled with Homology-Directed Repair (HDR) employed in this study effectively circumvented the adverse effects of the Non-Homologous End Joining (NHEJ)-dominant repair pathway in filamentous fungi on linear DNA donor templates [22]. Similar studies have also been validated in Aspergillus nidulans, where the knockout of ku70—responsible for Non-Homologous End Joining (NHEJ)—led to enhanced gene editing efficiency, with 90% or more of the transformants carrying a single insertion of the transforming DNA at the correct site [23,24]. By optimizing from a linear HDR template (30% editing efficiency) to a circular donor plasmid, the rate of homokaryotic mutants increased to 75%. This not only significantly improved editing efficiency but also enhanced the genetic stability of the exogenous constructs. Building on this, the further use of a circular multigene editing vector to simultaneously strengthen the DAOC synthesis pathway and precursor supply significantly increased the titer from 10.1 ± 1.3 g/L to 12.4 ± 0.2 g/L, achieving systematic optimization of the metabolic pathway.

Compared to traditional random integration and mutagenesis methods, the precise editing strategy established in this study offers multiple advantages: Firstly, it avoids expression variability caused by the insertion of foreign genes into silent regions or unstable copy numbers. Secondly, it significantly reduces heterokaryon formation, improving strain genetic consistency. Thirdly, it drastically reduces the screening scale, requiring only 3–4 transformants to obtain the target high-yield strain, thereby shortening the R&D cycle and lowering screening costs.

This strategy not only provides efficient strain resources for the stable production of β-lactam antibiotics, aiding in addressing public health challenges like antimicrobial resistance [25,26,27], but also offers a transferable technical approach for metabolic engineering in other multinucleate industrial strains for antibiotic production, where the CRISPR-Cas9 genetic tools are less used. For instance, in penicillin-producing strains (Penicillium chrysogenum) [28,29], erythromycin-producing strains (Saccharopolyspora erythraea) [30,31,32], and avermectin-producing strains (Streptomyces avermitilis) [33,34], CRISPR-based precise editing could systematically alleviate metabolic bottlenecks, knockout byproduct pathways, or enhance precursor supply. This would increase the target product yield while reducing byproduct formation, offering significant advantages in efficiency, precision, and reproducibility compared to traditional mutagenesis and random integration methods.

In the current study, residual selectable marker genes in the genome may increase the metabolic burden on the host. Additionally, recent research has demonstrated that the use of endogenous 5S rRNA promoters in A. chrysogenum enables 100% single-gene deletion and large-scale chromosomal deletions without the need for processing elements or donor DNA [35]. Future work could integrate novel CRISPR-Cas9 tools or site-specific recombination techniques (e.g., Cre-loxP, FLP-FRT, pyrG) [36,37,38] to develop marker-free genome editing strategies, thereby further optimizing the metabolic network of A. chrysogenum.

This study establishes, for the first time, a CRISPR-Cas9 genome editing platform in a first-line industrial strain of A. chrysogenum. To overcome the inefficiency of traditional two-step engineering for DAOC overproduction, we developed a precise one-step strategy using a single donor template for simultaneous gene knockout and multigene overexpression. Iterative optimization from linear to circular donor templates significantly enhanced editing efficiency and genetic stability. The final homozygous engineered strain, DAOC-5, achieved a high titer of 12.4 g/L. This work provides an efficient and transferable toolkit for metabolic engineering in industrial filamentous fungi.

4. Materials and Methods

4.1. Strains and Culture Condition

The industrial strain Mc-1 of A. chrysogenum was preserved in the laboratory at the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences. The genetic backgrounds, genotypic characteristics, and specific applications of all strains used in this study are summarized in Table S1.

The culture medium and solutions used in this study are as follows:

YPS medium: sucrose (Solarbio, Beijing, China) 20.00 g/L, yeast extract (Oxoid Ltd., Basingstoke, UK) 5.00 g/L, polypeptone (Oxoid Ltd., UK) 20.00 g/L, K_2_HPO_4_ (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) 1.00 g/L, and MgSO_4_·7H_2_O (Sinopharm Chemical Reagent Co., Ltd.) 1.00 g/L, pH 7.0.

Regeneration medium: wort 10% (v/v), maltose 40.00 g/L, polypeptone 20.00 g/L, sucrose 20.00 g/L, KCl (Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China) 0.6 M, CaCl_2_ (Sinopharm Chemical Reagent Co., Ltd.) 25 mM, MgCl_2_ (Sinopharm Chemical Reagent Co., Ltd.) 10 mM, and agar 20.00 g/L, pH 7.0.

Primary seed medium: CaCO_3_ (Sinopharm Chemical Reagent Co., Ltd.) 10.00 g/L, yeast extract 10.00 g/L, glucose 5.00 g/L, sucrose 10.00 g/L, pH 7.0.

Secondary seed medium: CaCO_3_ 10.55 g/L, yeast extract 18.00 g/L, corn flour 10.00 g/L, glucose 10.00 g/L, and sucrose 20.00 g/L, pH 7.0.

Fermentation medium: CaCO_3_ 10.55 g/L, CaSO_4_·2H_2_O (Sinopharm Chemical Reagent Co., Ltd.) 3.00 g/L, MgSO_4_ (Sinopharm Chemical Reagent Co., Ltd.) 2.00 g/L, peanut powder (Solarbio) 5.00 g/L, yeast extract 40.00 g/L, corn flour (Solarbio) 10.00 g/L, and corn slurry (Solarbio) 20.00 g/L, pH 7.0.

4.2. Plasmid Construction

Competent Escherichia coli DH5α cells were procured from Tsingke Biotechnology Co., Ltd. (Beijing, China). PCR product purification, gel extraction, and plasmid miniprep kits were obtained from TIANGEN Biotech Co., Ltd. (Beijing, China). Plasmids and primers used in this work are listed in Tables S1 and S2, with primers synthesized by Tsingke Biotechnology Co., Ltd. (Beijing, China). The CRISPR-Cas9 backbone vector pAN7-Cas9 was maintained in our laboratory.

To design the targeting construct, the AccefEF gene reference sequence (GenBank No. M91649.1) was aligned with the full genome sequence of strain Mc-1. A specific sgRNA spacer (5′-CATGCGGAGGGATAAGCCGG-3′) was designed using the online tool ‘Sequence Scan for CRISPR’ (http://cistrome.org/SSC/, accessed on 8 November 2024) (Figure S6). The sgRNA expression cassette was amplified with primers cefEF-sgRNA-F/R and assembled into the linearized pAN7-Cas9 vector (amplified with primers Cas9-F/R) via Gibson Assembly, yielding the CRISPR-Cas9 plasmid pDC-1.

Codon-optimized SccefE (GenBank No. M32324.1) and SccefD (GenBank No. M32324.1) genes from S. clavuligerus were synthesized by Tsingke Biotechnology Co., Ltd. A peroxisomal targeting signal 1 (PTS1) sequence (Table S3) was appended to the C-terminus of SccefD as described previously [5], generating plasmids pET30a-SccefE and pET30a-SccefD.

The 1.5 kb upstream and downstream homologous arms of AccefEF were amplified with primers cefEF-1F/R and cefEF-2F/R. These were ligated with the SccefE expression cassette (driven by the A. chrysogenum PgpdA promoter and terminated by the A. nidulans Ttef1 terminator), amplified from pET30a-SccefE with primers cefE-F/R. The Bar^R^ expression cassette (containing the A. nidulans PtrpC promoter and TtrpC terminator) was amplified from the pBAR plasmid using primers Bar-F/R. These fragments were assembled by Gibson Assembly to generate the linear HDR template dsDC-1, which was subsequently cloned into the pUC19 backbone (amplified with primers Amp-F/R) to produce the circular HDR donor plasmid pDC-2.

Finally, the SccefD expression cassette (under the control of the PpcbC promoter and the A. nidulans Ttef1 terminator) was amplified from pET30a-SccefD with primers cefD-F/Rand inserted into the linearized pDC-2 vector (amplified with primers cefE-Donor-F/R) via Gibson Assembly, resulting in the multi-gene expression plasmid pDC-3.

4.3. Assessment and Characterization of Mutant Strains

The Mc-1 strain was inoculated on solid regeneration medium and cultured at 30 °C for 7 d. Subsequently, a 1 cm^2^ slant culture was inoculated into 100 mL YPS liquid medium and incubated at 30 °C, with shaking at 120 rpm for 4 d. The harvested mycelia were washed three times with K-buffer (0.6 mol/L KCl, 20.0 mmol/L CaCl_2_, 10.0 mmol/L MgCl_2_) and incubated in a 10 mL enzyme lysis solution (a mixture of cellulase, snailase, and yatalase) (Table S4) at 30 °C and 100 r/min for 3 h. After enzymatic digestion, the mixture was washed with K-buffer and centrifuged at 424× g for 5 min to collect protoplasts. A total of 1 × 10^7^ protoplasts were incubated on ice with 1 μg of CRISPR-Cas9 plasmid pDC-2 and 2 μg donor DNA (molar ratio 1:2) for 30 min. A total of 4.5 mL 45% (w/v) PEG 6000 solution was added to induce DNA uptake (25 °C, 30 min). After recovery in 5 mL liquid regeneration medium (30 °C, 4 h), the transformation system was mixed with 5 mL of soft agar (0.6%) in K-buffer containing 300 μg/mL hygromycin and 100 μg/mL glufosinateammonium, evenly spread on the regeneration medium plates with the same antibiotics. The plate was incubated upright at 30 °C for 5–7 days and then inverted until the transformants formed. The transformants were subsequently transferred using a sterile toothpick to regeneration II medium containing 200 μg/mL glufosinateammonium at 30 °C for 7 days for gradient resistance strengthening screening.

Single colonies from the resistance screening plates were subjected to genomic DNA extraction using the 50 mM NaOH alkaline lysis method (100 °C, 10 min), followed by centrifugation at 6796× g for 10 min, and the supernatant was used as PCR template. Three pairs of specific primers (Table S2) were used for multi-region verification: (I) Upstream homologous recombination region verification: primers1-F/R amplified the junction region between the outer genomic sequence of the upstream homologous arm and the promoter of the expression cassette (with a 2.2 Kb expected product). (II) Downstream integration verification: primer pairs 2-F/R and 2-1F/R amplified the junction between the outer genomic sequence of the downstream homologous arm and the terminator of the expression cassette (with 2.1 Kb and 2.5 Kb expected products separately). (III) Target gene knockout verification: the primer pair 0-F/R was used to amplify a 564 bp internal fragment within the target gene AccefEF and no amplification should be detected in positive clones.

The calculation formula of final positive transformant rate is as follows:

$$[eqn]$$

4.4. Fermentation and Determination of Growth Curves

Single colonies of A. chrysogenum strains were inoculated on sporulation medium plates and incubated at 28 °C with 50% relative humidity for 7–10 days. A 1 cm^2^ mycelial block was transferred into a 250 mL flask containing 50 mL of primary seed medium and cultured at 28 °C, 220 r/min for 72 h. Subsequently, the primary seed culture was then transferred at a 10% (v/v) inoculation ratio to a 250 mL flask containing 50 mL of secondary seed medium and incubated at 28 °C and 220 r/min for 48 h. Finally, the secondary seed culture was transferred at a 10% (v/v) inoculation ratio to a 250 mL flask containing 30 mL of fermentation medium, and the main fermentation was carried out at 28 °C and 220 r/min. After 48 h of cultivation, the temperature was lowered to 25 °C; 5% (v/v) soybean oil was added at 96 h of fermentation, and the fermentation was continued for another 48 h before termination [5]. Samples were taken every 24 h during fermentation to determine the DAOC content in the fermentation broth and the biomass of the mycelium, which was assessed by the dry cell weight (DCW) method.

4.5. Sample Treatment and Product Analysis

Following a simple centrifugation step, the fermentation broth supernatant was diluted 50-fold and filtered through a 0.22 μm membrane to prepare the sample. Qualitative analysis of the sample was then performed using a Waters ZQ 2000 liquid chromatography-mass spectrometry (LC-MS) system (Waters Corporation, Milford, MA, USA). The analysis employed a Phenomenex Gemini C18 column (4.6 × 250 mm, 5 μm) with a gradient elution of mobile phase A (0.1% formic acid in water) and mobile phase B (0.1% formic acid in acetonitrile), ramping from 10% to 100% B over 20 min. The flow rate was 1.0 mL/min, the column temperature was maintained at 30 °C, and the injection volume was 10 µL. Mass spectrometry was operated in ESI positive mode, with a scan range of m/z 200–600, specifically detecting DAOC ([M + H]^+^ m/z 358). Quantitative high-performance liquid chromatography (HPLC) analysis was performed using a Waters Alliance 2695 HPLC system (Waters Corporation, USA) equipped with a Hypersil ODS2 column (4.6 × 150 mm, 5 μm). Mobile phase A consisted of 20 mM KH_2_PO_4_ (pH 3.0 ± 0.1), and mobile phase B was acetonitrile containing 10% A and a 0→100% B gradient elution (0–20 min) was applied at a flow rate of 1.0 mL/min, with the column temperature at 30 °C, a 10 μL injection volume and detection at 254 nm. The calibration curve and regression equation for the DAOC reference standard (purchased from Yili Chuanning Biotechnology Co., Ltd., Yili, China) are presented in Figure S3.

GraphPad Prism software (version 5; GraphPad Software, San Diego, CA, USA) was used to perform the paired Student’s t-test and two-way ANOVA.

4.6. Transcriptional Validation of Mutant Strains

Mycelial samples were collected every 24 h during the fermentation process. After grinding the samples in liquid nitrogen, total RNAs was extracted following the instructions of the TransZol Up Plus RNA Kit (TransGen Biotech, Beijing, China). Subsequently, the total RNA samples were reverse transcribed into cDNA using the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) kit (Novozan BioTech, Nanjing, China). The resulting cDNA was used as the template for qRT-PCR amplification with a SYBR^®^ Green Premix qPCR kit (Yisheng Biotech, Shanghai, China). Each reaction was performed in triplicate.

The relative gene expression (fold change) was calculated by the $[eqn]$ method, where $[eqn]$ , $[eqn]$ , and the result was derived as $[eqn]$ .