Improving the production of the micafungin precursor FR901379 in an industrial production strain

Men, **; Zhou, Yu; **e, Li; Zhang, Xuan; Zhang, Wei; Huang, Xuenian; Lu, Xuefeng

doi:10.1186/s12934-023-02050-0

Improving the production of the micafungin precursor FR901379 in an industrial production strain

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Published: 06 March 2023

Volume 22, article number 44, (2023)
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Improving the production of the micafungin precursor FR901379 in an industrial production strain

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** Men^1,2,3,4,
Yu Zhou^1,2,3,5,
Li **e^1,2,3,6,
Xuan Zhang^1,2,3,
Wei Zhang^1,2,3,4,
Xuenian Huang^1,2,3 &
…
Xuefeng Lu^1,2,3,4,7

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Abstract

Background

Micafungin is an echinocandin-type antifungal agent used for the clinical treatment of invasive fungal infections. It is semisynthesized from the sulfonated lipohexapeptide FR901379, a nonribosomal peptide produced by the filamentous fungus Coleophoma empetri. However, the low fermentation efficiency of FR901379 increases the cost of micafungin production and hinders its widespread clinical application.

Results

Here, a highly efficient FR901379-producing strain was constructed via systems metabolic engineering in C. empetri MEFC09. First, the biosynthesis pathway of FR901379 was optimized by overexpressing the rate-limiting enzymes cytochrome P450 McfF and McfH, which successfully eliminated the accumulation of unwanted byproducts and increased the production of FR901379. Then, the functions of putative self-resistance genes encoding β-1,3-glucan synthase were evaluated in vivo. The deletion of CEfks1 affected growth and resulted in more spherical cells. Additionally, the transcriptional activator McfJ for the regulation of FR901379 biosynthesis was identified and applied in metabolic engineering. Overexpressing mcfJ markedly increased the production of FR901379 from 0.3 g/L to 1.3 g/L. Finally, the engineered strain coexpressing mcfJ, mcfF, and mcfH was constructed for additive effects, and the FR901379 titer reached 4.0 g/L under fed-batch conditions in a 5 L bioreactor.

Conclusions

This study represents a significant improvement for the production of FR901379 and provides guidance for the establishment of efficient fungal cell factories for other echinocandins.

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Background

Echinocandin-type antifungal agents are semisynthesized from lipohexapeptides, nonribosomal peptides produced by filamentous fungi [1, 2]. As an inhibitor of β-1,3-glucan synthase, echinocandins can potently inhibit the growth of fungal pathogens by blocking the biosynthesis of β-1,3-glucan, which is a predominant and specific constituent of the cell wall in most fungi [1, 3]. Therefore, they exhibit pronounced activity against a broad range of Candida spp. and Asperillus spp., especially against azole-resistant strains [4,5,6,7]. To date, caspofungin, micafungin, and anidulafungin have been used as first-line drugs to treat invasive fungal infections [3, 6, 8, 9]. Unlike other echinocandin-type agents, micafungin exhibits excellent water solubility due to the sulfonate moiety originating from the precursor FR901379 [10, 11]. The high water solubility significantly improves the pharmacological efficacy and pharmacokinetic properties [12].

Industrial production of micafungin includes three steps: FR901379 is produced through Coleophoma empetri fermentation [11], then the palmitic acid side chain of FR901379 is deacylated and substituted with the optimized N-acyl side chain (Fig. 1) [13, 14]. The production of FR901379 is the key step in the manufacture of micafungin. However, the low titer, byproducts with similar structures, and poor mycelium pellet are obstacles to high yield during C. empetri cultivation, which increases the production cost and complicates large-scale purification processes [11, 15]. Although mutation breeding and fermentation optimization have been applied to improve the production of FR901379, the above problems remain unsolved [15, 16].

Metabolic engineering provides promising strategies to improve the production of natural products, such as enhancing and optimizing the biosynthesis pathway [17,18,19], eliminating the competitive pathway [18,

To verify the effect of CEfks1 and CEfks2 on the production of FR901379, these two genes were individually disrupted in C. empetri MEFC09 (Fig. 5B). The double-knockout strain could not be obtained. ∆CEfks1 and ∆CEfks2 exhibited different growth rates, pellet formation, and cell wall structures (Fig. 5). Deleting CEfks1 significantly decreased the growth rate on potato dextrose agar (PDA) plates, whereas the colony of ∆CEfks2 was larger and fluffier than that of C. empetri MEFC09 (Fig. 5D). The FR901379 titer of ∆CEfks1 was 30% higher than that of C. empetri MEFC09, which might be caused by improved mycelial morphogenesis (Fig. 5C). The mycelia pellets of ∆CEfks1 were smaller and more regular than those of MEFC09. However, no obvious change was observed in the strain ∆CEfks2 (Fig. 5D). Further scanning electron microscopy (SEM) imaging revealed that the structure of the cell wall changed remarkably in both mutant strains ∆CEfks1 and ∆CEfks2. These results demonstrated that these genes both contribute to cell wall formation and affect the morphogenesis and growth of C. empetri.

To improve the production of FR901379 by enhancing product resistance, the putative self-resistance gene CEfks2 was overexpressed in C. empetri MEFC09. However, the titer of FR901379 was decreased and accompanied by a slight slowdown in growth (Fig. 5C). This result indicated that the resistance of C. empetri MEFC09 to FR901379 was sufficient to protect it from the current concentration of FR901379. Enhancing product resistance is not an effective strategy to improve production. Unlike resistance genes of other natural products, β-1,3-glucan synthase plays an important role in fungal cell wall formation. Genetic engineering of β-1,3-glucan synthase genes would affect morphogenesis and growth. Therefore, metabolic engineering based on self-resistance genes in echinocandin-producing strains will be more complicated and requires a strict balance between product resistance, growth, and mycelial morphology.

Improving the production of FR901379 by overexpresing the transcriptional activator McfJ

Overexpression of transcriptional activators is an effective strategy to increase the production of secondary metabolites [32, 33]. Overexpressing the specific transcriptional regulator lovE significantly improved the production of monacolin J in the industrial strain Aspergillus terreus [33]. However, the biosynthetic genes of FR901379 were distributed in two separate BGCs, including the core biosynthetic gene cluster mcf and the O-sulfonation gene cluster. The regulation of FR901379 biosynthesis is more ambiguous and complicated. The gene deletion and reverse transcription-polymerase chain reaction (RT‒PCR) results showed that mcfJ might be a transcriptional activator for the biosynthesis of FR901379 (Additional file 1: Fig. S3). The production of FR901379 was abolished completely in the mcfJ deletion mutant. It is consistent with the phenomenon observed in C. empetri SIPI1284, deleting Cehyp (homologous gene of mcfJ) broke the production of FR901379 [26]. The transcriptional levels of the FR901379 biosynthetic pathway, including the genes in the large mcf cluster and the separate O-sulfonation cluster, were significantly downregulated following mcfJ disruption (Fig. 6A, Additional file 1: Table S3).

To further test the function of mcfJ and improve the production of FR901379, the expression cassette of mcfJ was constructed using the promoter PgpdAt and introduced into C. empetri MEFC09. The transformants harboring the expression cassette were confirmed by genomic PCR and designated MEFC09-J. The results of shake flask fermentation showed that all of the transformants produced significantly higher FR901379 titers than C. empetri MEFC09 (Fig. 6B, C). The highest FR901379 titer of 1.32 g/L was achieved in strain MEFC09-J-4, which was threefold higher than that in MEFC09 (Fig. 6C). In addition, the transcription levels of mcfJ and other biosynthetic genes of FR901379 were measured by RNA sequencing and were significantly increased in the mutant strain MEFC09-J (Fig. 6A, Additional file 1: Table S3). These results demonstrated that McfJ is indeed a transcriptional activator for the biosynthesis of FR901379. Although the biosynthetic genes were distributed in two separate BGCs, the production of FR901379 was coordinately regulated by McfJ. More importantly, because of this regulatory mechanism, overexpression of mcfJ was successfully developed as a very effective metabolic engineering strategy to increase the production capacity of FR901379.

Construction of a high-yield cell factory for FR901379 by combinatorial metabolic engineering

Overexpression of CYP enzymes mcfH and mcfF could significantly reduce the accumulation of byproducts, while overexpression of transcriptional activator mcfJ could remarkably increase the production of FR901379. Inspired by the performance of these two strategies, a mutant strain coexpressing mcfJ, mcfF and mcfH was constructed to achieve a superposition of beneficial effects. The expression cassettes of PgpdAt-mcfF-TtrpC and PgpdAt-mcfH-TtrpC were cointroduced into MEFC09-J-4 to generate the mutant strain MEFC09-JFH. The production of FR901379 was further improved in all engineered strains (Fig. 7A). The highest increase was observed in MEFC09-JFH-2, and the titer of FR901379 reached 2.03 g/L, which was 47% higher than that of MEFC09-J. In addition, the production of byproducts WF11899C was completely abolished in MEFC09-JFH-2, and the WF11899B ratio decreased from 14 to 4%. Therefore, a more efficient cell factory with a higher FR901379 titer and fewer byproducts was constructed by combinatorial metabolic engineering. This is the first transcriptional regulator identified in the biosynthesis of echinocandins, which will serve as a good reference for research on other echinocandins.

Scale-up production of FR901379 in the 5 L bioreactor

To evaluate the scale-up potential of FR901379 production, batch experiments were carried out in a 5 L bioreactor using engineered strains MEFC09-HF and MEFC09-JFH, with MEFC09 as a control. The titers of FR901379 reached 0.9 g/L, 1.4 g/L, and 2.4 g/L in the strains MEFC09, MEFC09-HF, and MEFC09-JFH, respectively (Fig. 7B). This is significantly higher than those in the shake flask fermentation, which demonstrated the potential for production improvement in the stirred-tank bioreactor. The FR901379 productivity of MEFC09-JFH was much higher than that of MEFC09, MEFC09-HF in the batch fermentation (Additional file 1: Fig. S4). In addition, there was almost no increase in FR901379 production of MEFC09-JFH from Day 6, which was observed from Day 8 in other strains (Fig. 7B). In the fermentation medium MKF for the production of FR901379, D-sorbitol is the main carbon source. Further examination of the D-sorbitol concentration in the fermentation culture showed that D-sorbitol was completely depleted from Day 5 (Additional file 1: Fig. S5A). Therefore, fed-batch fermentation was carried out to achieve higher production of FR901379 using strain MEFC09-JFH. The downward trend in productivity has been significantly slowed. And 4.0 g/L of FR901379 was produced on Day 11 after feeding D-sorbitol three times (Fig. 7C), which was the highest production reported to date. To the best of our knowledge, it is substantially higher than the current titer achieved in industrial production .

Materials and methods

Strains and cultural conditions

All fungal strains used or constructed in this study are listed in Table 1. These strains were cultivated on PDA medium (pH 5.6, purchased from BD company) at 25 ℃ for 7 days and used for multiplication of the inoculum. The transformants were selected on PDAS (PDA with 0.8 M D-sorbitol, pH 5.6) supplemented with appropriate antibiotics as needed, such as 100 mg/L of hygromycin B or geneticin, and cultured for 5–7 days at 30 ℃.

Table 1 Strains used in this study

Full size table

Overexpression of target genes in C. empetri MEFC09

All plasmids used here are listed in Additional file 1: Table S1. All primers used in this study are listed in Additional file 1: Table S2. The DNA fragments of mcfF, mcfH, mcfP, and mcfS were amplified from genomic DNA of C. empetri MEFC09 using respective primer pairs and cloned into vector pXH2-1 at the restriction sites of Pci I and Hind III, resulting in the plasmids pPM-mcfF, pPM-mcfH, pPM-mcfP and pPM-mcfS, respectively (Additional file 1: Fig. S1) [35]. To construct the mcfJ-overexpressing mutant strain, mcfJ was amplified from the genomic DNA of C. empetri MEFC09 using the primers mcfJ-F1/mcfJ-R and cloned into the PU-ZX vector digested with Xba I, resulting in the PU-mcfJ plasmid (Additional file 1: Fig. S1). The mcfJ expression cassette PgpdAt-mcfJ-Tpgk-hph was amplified from plasmid PU-mcfJ using primers PgpdAt-F/hph-R.

All of the expression cassettes were individually introduced into C. empetri MEFC09 through (PEG)-CaCl₂-mediated protoplast transformation [34]. Transformants were selected on PDAS plates amended with 100 mg/L hygromycin B and verified by genomic PCR. To construct the mutant strain MEFC09-HF, the expression cassette PgpdAt-mcfF-TtrpC was fused with marker neo from pPM-4 by fusion PCR and introduced into MEFC09-H. Similarly, the expression cassettes PgpdAt-mcfF-TtrpC-neo and PgpdAt-mcfH-TtrpC-neo were cointroduced into MEFC09-J, resulting in the engineered strain MEFC09-JFH.

Identification of β-1,3-glucan synthase

The β-1,3-glucan synthase coding genes (CEfks1 and CEfks2) were searched in the genome of C. empetri MEFC09 with the sequences of prfks1n and prfks1a from Pezicula radicicola NRRL 12192 as probes [31]. The putative function of the predicted enzymes was confirmed with the online NCBI BLASTP programmer (http://blast.ncbi.nlm.nih.gov). Gene disruption was carried out via homologous recombination as described previously [34]. To knock out the CEfks1 gene, approximately 1.2 kb of 5' and 3' DNA of the CEfks1 gene were amplified by PCR from the genome of C. empetri MEFC09 using the primer pairs UCEfks1-F/UCEfks1-R and DCEfks1-F/DCEfks1-R and were fused with the marker hph by fusion PCR. The gene-targeting cassette was amplified using nest primers and introduced into MEFC09-∆ku80 by (PEG)-CaCl₂-mediated protoplast transformation. The mutant strains were verified using the primers UCEfks1-F/DCEfks1-R. A similar strategy was used for the deletion of CEfks2. The CEfks2-overexpressing strain was constructed as described above.

Fermentation and HPLC analysis

For the fermentation of all C. empetri strains, the fresh mycelia were crushed and inoculated in 50 mL of seed medium MKS (soluble starch 15 g/L, sucrose 10 g/L, cottonseed meal 5 g/L, peptone 10 g/L, KH₂PO₄ 1 g/L, and CaCO₃ 2 g/L; pH 6.5) in 250 mL shake flasks for 2 days at 25 °C and 220 rpm [34]. Then, 5 mL of seed culture was inoculated into 50 mL of fermentation medium MKF (glucose 10 g/L, corn starch 30 g/L, peptone 10 g/L, D-sorbitol 160 g/L, (NH₄)₂SO₄ 6 g/L, KH₂PO₄ 1 g/L, FeSO₄·7H₂O 0.3 g/L, ZnSO₄·7H₂O 0.01 g/L, and CaCO₃ 2 g/L; pH 6.5) and cultivated at 25 °C and 220 rpm for 8 days. The cultivation broth of C. empetri MEFC09 and the derived engineered strains was analyzed after cultivated for 8 days. Four-fold volume of methanol was added in each sample and shaken on the vortex oscillator at 2600 rpm for 1 h at room temperature. Three independent experiments were performed for each transformant.

The samples were analyzed by HPLC equipped with a reverse-phase C₁₈ column (Agilent, 4.6 × 150 mm, 5 µm) monitored at 210 nm. For HPLC analysis, solvent A was deionized water with 0.05% trifluoroacetic acid and solvent B was acetonitrile with 0.05% trifluoroacetic acid. The following gradient was used at a flow rate of 1 mL/min: 5%-40% solvent B for 3 min, 40%-60% solvent B for 15 min, 100% solvent B for 5 min, and 5% solvent B for 3 min [34]. The amount of FR901379 was quantified based on the peak area.

Sample preparation for scanning electron microscopy

Sample preparation for SEM was performed according to the method reported previously with modification [36]. The fresh mycelia were collected by centrifugation and washed with phosphate buffer solution (PBS, pH 7.4) three times. The samples were fixed in 2.5% glutaraldehyde fixative for at least 1 h and washed with PBS three times for 10 min each time. Osmium tetroxide was added to the samples for 10 min, then they were washed in PBS three times. The samples were dehydrated with 30%, 50%, 70%, 90%, and 100% ethanol for 5 min each. Next, tertiary butanol was added to the samples for 15 min, and the samples were dried in a freezer dryer and loaded onto specimen stubs. They were coated with gold–palladium before observation under a Hitachi S-4800 SEM Cold Field Emission Microscope (Japan).

RNA isolation and transcript quantification

Mycelia were collected from the MKF cultures for 2 days and immediately frozen in liquid nitrogen. Total RNA was isolated using a Takara MiniBEST Universal RNA Extraction Kit (Takara, Japan) according to the manufacturer’s protocol. RNA samples were treated with RNase-free DNase I (Takara, Japan) for 15 min to eliminate the genomic DNA. First strand cDNA was synthesized with the PrimeScript™ RT Reagent Kit with gRNA Eraser (Takara, Japan). All of the primers used for RT‒PCR are listed in Additional file 1: Table S2, with the actin gene used as a reference.

The expression profiles of target genes responsible for the biosynthesis of FR901379 of C. empetri MEFC09, MEFC09-∆mcfJ, and MEFC09-J were compared by RNA sequencing performed by GENEWIZ (Suzhou, China). Then, the corresponding expression levels were obtained by calculating FPKM. The results were illustrated using TBtools based on Euclidean distance calculation [37].

Cultivation in a stirred-tank bioreactor

For batch fermentation, strains MEFC09, MEFC09-HF, and MEFC09-JFH were cultivated in a 5 L stirred-tank bioreactor containing 3 L of MKF medium. Seed cultures of engineered strains were prepared as described above, then 10% (v/v) seed culture was inoculated into the bioreactor and cultivated at 25 °C for 9 days. The aeration was kept at 1.0 vvm (volume of air under standard conditions/volume of liquid/minute) and the dissolved oxygen (DO) level was controlled at 10% air saturation with an automated cascade to control the stirring rate within a 400–600 rpm range. For fed-batch cultivation, the engineered strain MEFC09-JFH was cultivated for 12 days. After 5 days of incubation, an additional 180 g of D-sorbitol was added to the bioreactor on Day 5, Day 6, and Day 8, respectively. Samples were taken every day for HPLC analysis at the same time each day.

Availability of data and materials

All data generated and analyzed during this study were included in this manuscript and the additional files.

Abbreviations

BGCs:: Biosynthetic gene clusters
CYP:: Cytochrome P450
HPLC:: High performance liquid chromatography
PgpdAt :: Promoter of glyceraldehyde-3-phosphate dehydrogenase gene
PDA:: Potato dextrose agar
SEM:: Sample preparation for scanning electron microscopyreverse
RT‒PCR:: Transcription-polymerase chain reaction
PBS:: Phosphate buffer solution
FPKM:: Fragments Per Kilobase of exon model per Million mapped fragments

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Acknowledgements

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Funding

This research was supported by National Key R&D Program of China (No. 2021YFC2102600), the National Natural Science Foundation of China (U2032139, 32170098), Key R&D Program of Shandong Province (No. 2021ZDSYS02). X.L. and X.H. are supported by the Shandong Taishan Scholarship.

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Authors and Affiliations

Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, 266101, China
** Men, Yu Zhou, Li **e, Xuan Zhang, Wei Zhang, Xuenian Huang & Xuefeng Lu
Shandong Energy Institute, Qingdao, 266101, China
** Men, Yu Zhou, Li **e, Xuan Zhang, Wei Zhang, Xuenian Huang & Xuefeng Lu
Qingdao New Energy Shandong Laboratory, Qingdao, 266101, China
** Men, Yu Zhou, Li **e, Xuan Zhang, Wei Zhang, Xuenian Huang & Xuefeng Lu
University of Chinese Academy of Sciences, Bei**g, 100049, China
** Men, Wei Zhang & Xuefeng Lu
Institute for Smart Materials & Engineering, University of **an, **an, 250022, China
Yu Zhou
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330096, China
Li **e
Marine Biology and Biotechnology Laboratory, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266237, China
Xuefeng Lu

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** Men
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Yu Zhou
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Contributions

XL and XH conceived the project; XL, XH, and PM designed the research; The fungal genetic experiments were carried out by PM, LX, XZ, and WZ; The fermentation experiments were performed by PM and YZ; PM, XH, and XL wrote the manuscript. All authors read and approved the manuscript.

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Correspondence to Xuenian Huang or Xuefeng Lu.

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Supplementary Information

Additional file 1: Table S1.

Plasmids used in this study. Table S2. Primers used in this study. Table S3. The expression profiles of C. empetri MEFC09 and mutant strains. Figure S1. Plasmid maps and cassettes of mcfF (A, G), mcfH (B, G), mcfP (C), mcfS (D), and mcfJ (E), and CEfks2 (F). Figure S2. Titers of FR901379 were quantified in the mutant strains MEFC09-P and MEFC09-S. Figure S3. Functional identification of gene mcfJ. Figure S4. The concentration of D-sorbitol of batch fermentation (A) and fed-batch fermentation (B) in a 5 L bioreactor.

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Men, P., Zhou, Y., **e, L. et al. Improving the production of the micafungin precursor FR901379 in an industrial production strain. Microb Cell Fact 22, 44 (2023). https://doi.org/10.1186/s12934-023-02050-0

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Received: 21 December 2022
Accepted: 25 February 2023
Published: 06 March 2023
DOI: https://doi.org/10.1186/s12934-023-02050-0

Improving the production of the micafungin precursor FR901379 in an industrial production strain

Abstract

Background

Results

Conclusions

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Background

Improving the production of FR901379 by overexpresing the transcriptional activator McfJ

Construction of a high-yield cell factory for FR901379 by combinatorial metabolic engineering

Scale-up production of FR901379 in the 5 L bioreactor

Materials and methods

Strains and cultural conditions

Overexpression of target genes in C. empetri MEFC09

Identification of β-1,3-glucan synthase

Fermentation and HPLC analysis

Sample preparation for scanning electron microscopy

RNA isolation and transcript quantification

Cultivation in a stirred-tank bioreactor

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Supplementary Information

Additional file 1: Table S1.

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