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Article

Improving Ergometrine Production by easO and easP Knockout in Claviceps paspali

by
Yun-Ming Qiao
,
Yan-Hua Wen
,
Ting Gong
,
Jing-Jing Chen
,
Tian-Jiao Chen
,
Jin-Ling Yang
and
Ping Zhu
*
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, NHC Key Laboratory of Biosynthesis of Natural Products, CAMS Key Laboratory of Enzyme and Biocatalysis of Natural Drugs, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China
*
Author to whom correspondence should be addressed.
Fermentation 2022, 8(6), 263; https://doi.org/10.3390/fermentation8060263
Submission received: 28 April 2022 / Revised: 17 May 2022 / Accepted: 26 May 2022 / Published: 2 June 2022
(This article belongs to the Special Issue Fermentation and Bioactive Metabolites 3.0)
Browse Figures
Review Reports Versions Notes

Abstract

:
Ergometrine is widely used for the treatment of excessive postpartum uterine bleeding. Claviceps paspali is a common species for industrial production of ergometrine, which is often accompanied by lysergic acid α-hydroxyethylamide (LAH) and lysergic acid amide (LAA). Currently, direct evidence on the biosynthetic mechanism of LAH and LAA from lysergic acid in C. paspali is absent, except that LAH and LAA share the common precursor with ergometrine and LAA is spontaneously transformed from LAH. A comparison of the gene clusters between C. purpurea and C. paspali showed that the latter harbored the additional easO and easP genes. Thus, the knockout of easO and easP in the species should not only improve the ergometrine production but also elucidate the function. In this study, gene knockout of C. paspali by homologous recombination yielded two mutants ∆easOhetero-1 and ∆easPhetero-34 with ergometrine titers of 1559.36 mg∙L−1 and 837.57 mg∙L−1, which were four and two times higher than that of the wild-type control, respectively. While the total titer of LAH and LAA of ∆easOhetero-1 was lower than that of the wild-type control. The Aspergillus nidulans expression system was adopted to verify the function of easO and easP. Heterologous expression in A. nidulans further demonstrated that easO, but not easP, determines the formation of LAA.

1. Introduction

Claviceps species produce a lot of bioactive ergot alkaloids, some of which have been developed into clinically important drugs [1,2]. For example, ergometrine is used to cure postpartum uterus bleeding [3]; ergotamine is used for the treatment of migraine [4,5]; bromocriptine can be used for treating hyperprolactinemia and Parkinson’s disease [6,7]. On the other hand, ergot alkaloids are responsible for the toxicity of ergotism, a common cereal epidemics [8]. These alkaloids are generally biosynthesized from the common precursor D-lysergic acid. This step is catalyzed by the non-ribosomal peptide synthetases (NRPSs). From this reaction, a class of ergopeptides (also named ergopeptines), including ergotamine, ergocristine, ergocornine, α-ergocriptine and β-ergocriptine are synthesized [9,10]. Alternatively, D-lysergic acid can also be converted into simple lysergic acid amides, including ergometrine, lysergic acid α-hydroxyethylamide (LAH) and lysergic acid amide (LAA) [1,4,11,12].
C. purpurea produces not only ergopeptides but also ergometrine [13]. The ergot alkaloid synthesis (EAS) pathway of ergometrine in C. purpurea has been deciphered [4,9,14,15,16,17,18,19,20,21,22]. In comparison, C. paspali does not produce ergopeptides but produces ergometrine and other lysergic acid amides, including LAH and LAA [23]. A comparison of the EAS gene clusters of C. purpurea and C. paspali shows that both C. purpurea and C. paspali harbor lpsB (encoding NRPS) and lpsC (encoding NRPS), which guarantee the biosynthesis of ergometrine. However, lpsA1 and lpsA2 (encoding NRPSs) are missing from C. paspali, which results in the inability of C. paspali to produce ergopeptines (Figure 1a). Surprisingly, the EAS gene cluster of C. paspali additionally harbors easO and easP, which are also present in other non-Claviceps fungal species, such as Periglandula ipomoeae, Metarhizium robertsii and Metarhizium, which can produce the simpler lysergic acid amides [10]. The additional easO and easP are likely involved in the biosynthesis of LAH and LAA, in which LAH can spontaneously convert to LAA by a non-enzymatic process [10,24]. Recently, easO has been shown to control the biosynthesis of LAH in M. brunneum [25], suggesting the role of the enzyme in the biosynthesis of LAH in C. paspali.
Multiple sequence alignment displayed that the enzyme EasO of C. paspali harbors two ‘fingerprint’ motifs which are characteristic of type I Baeyer-Villiger monooxygenase (BVMO): (FXGXXXHXXXW[P/D]) and ([A/G]GXWXXXX[F/Y]P[G/M]XXXD), and the latter is located between the two Rossmann folds and contains the critically conserved active site asparagine (Figure S1). This type of BVMO acts on intermediates linked to an acyl carrier protein (ACP) and forms thiocarbonates through BV oxidation of the carbonyl group [26,27]. The proposed biosynthetic pathway of lysergic acid amides produced in C. paspali is shown in Figure 1b [9,24,28,29].
In addition to validating the function of the additional genes in the EAS gene cluster, down-regulating of easO and easP in C. paspali should be a good strategy for improving the ergometrine production by diverting the metabolic flux away from LAH and into ergometrine. In this study, a protoplast-mediated genetic transformation system for the C. paspali WL721 strain was established to disrupt easO and easP in the C. paspali genome, resulting in the mutants ∆easOhetero and ∆easPhetero. Fermentation products of the mutants were subsequently analyzed. Compared with the control, the titer of ergometrine in the mutant ∆easOhetero was significantly increased along with a sharp decline in the titers of LAH and LAA, demonstrating that easO plays a key role in the biosynthesis of LAH and LAA. The titer of ergometrine was also increased in the mutant ∆easPhetero, however, the titers of LAH and LAA did not decline compared with those of the wild-type strain. The role of easO in the biosynthesis of LAA was further demonstrated in the heterologous expression system of A. nidulans, by co-expressing easO with lpsB and lpsC from C. paspali.

2. Materials and Methods

2.1. Strains

C. paspali WL721 (GenBank No. OK326692) was preserved at 4 °C in our laboratory and was used throughout this study. A. nidulans A1145 was used for heterologous expression. Saccharomyces cerevisiae BJ5464 was used for in vivo yeast DNA recombination cloning. The nucleotide sequences of C. paspali RRC-1481(GenBank No. AFRC01000001.1) and C. purpurea (GenBank No. CAGA01000001.1) were used.

2.2. Establishment of the Protoplast-Mediated Transformation System

Fungal genomic DNA isolation was performed according to the previous method [30,31]. Preparation and transformation of protoplast were performed according to a modified version of the method previously used in other filamentous fungi [32]. Protoplasts were successively diluted to three concentration gradients: 5 × 107, 5 × 106 and 5 × 105 protoplasts/mL with STC buffer. Two different protoplast regeneration methods were used and tested, namely the monolayer medium culture method and the layered medium culture method (Figure S2). Follow-up experiments for protoplast regeneration were carried out on the basis of the most suitable protoplast concentration and regeneration mode. The minimal inhibitory concentrations (MICs) of 5 antibiotics were tested on C. paspali WL721 for screening the proper antibiotic as a dominant selection marker. Specific operation methods and details are provided in the supporting information.

2.3. Construction of Deletion Cassettes and Characterization of ΔeasO and ΔeasP Mutants

The nucleotide sequence of the EAS gene cluster of C. paspali RRC-1481 was obtained from the National Center for Biotechnology Information (NCBI), with the GenBank accession number JN186800.1, and was used to design primers for the PCR amplification of gene sequences from the genome of C. paspali WL721. The deletion cassettes for easO and easP knockout were constructed as described by Szewczyk et al. [33] DNA fragments comprising the 5′ and 3′ DNA sequences flanking the target gene and the hygromycin B phosphotransferase gene (hph) cassette was assembled by the fusion PCR method to construct the fusion fragments. The size of 5′- and 3′-flanking fragments were 1152 bp and 1657 bp for easO, and 2055 bp and 2077 bp for easP, respectively. The hph cassette was 2.6 kb. The fusion fragments and pEasy-Blunt vectors were combined to form the gene disruption plasmids, designated as pEasy-Blunt-fusion-easO-hph and pEasy-Blunt-fusion-easP-hph, respectively. Then, the deletion cassettes were generated by PCR based on the above disruption plasmids. These two deletion cassettes were separately transformed into protoplast of C. paspali WL721. The genes of easO and easP were knocked out by homologous recombination. The primers used for the amplification of DNA fragments and PCR verification of transformants are shown in Table S1.

2.4. Detection and Analysis of Ergot Alkaloid Production for C. paspali Mutants

The culture method and fermentation conditions of the mutants are described in the supporting information. For ergot alkaloid extraction, the fermentation broth was adjusted to pH 8–9 by using NH4OH, and 5 mL of cultures were extracted with 20 mL of ethyl acetate and concentrated with a rotary evaporator followed by dissolving with 1 mL of methanol. The solution was filtered through a 0.22 μm MultiScreen filter plate (Merck Millipore, Burlington, MA, USA). Then, the Agilent 1200 high-performance liquid chromatography (HPLC) system was used, and the separation was carried out on a C18 column (Capcel pak C18 MG II, 4.6 mm I.D. × 150 mm, 5 μm) at the flow rate of 1 mL∙min−1, operated at the temperature of 28 °C. The detection UV wavelength was 314 nm. Solvents were: (A) HPLC grade H2O containing 20 mM of ammonium formate; (B) HPLC grade CH3CN. Gradients were as follows: 0–15 min, 10–18% B; 15–20 min, 18–42% B; 20–25 min, 42–100% B; 25–26 min, 100–10% B; 26–30 min, 10% B. LC-MS analysis was conducted on an LCMS-2020 system (Shimadzu Corporation, Japan, Kyoto) with LC-20AT pumps, a PDA detector, an electrospray ionization (ESI) source interface, and an SQ mass detector (Shimadzu, 5 µm, 2.1 × 100 mm, C18 column) using positive and negative mode electrospray ionization. Water (solvent A) and acetonitrile (solvent B) were used at a flow rate of 0.3 mL∙min−1. Samples were analyzed with the following gradient: 0–20 min, 10–42% B; 20–25 min, 42% B; 25–30 min, 42–100% B; 30–31 min, 100–10% B; 31–36 min, 10% B. Ergometrine, LAH, LAA and D-lysergic acid prepared by our lab were used as standards.
The titer of ergometrine was calculated by the standard curve of ergometrine, which was plotted based on the different concentrations of standard sample ergometrine (0.1625, 0.325, 0.65, 0.8125 and 0.975 mg∙L−1). The regression equation was y = 19304x + 385.55 (R2 = 0.9953). In addition, this regression equation was utilized to roughly estimate the titers of LAH, LAA and the isomers.

2.5. Heterologous Expression in A. nidulans A1145

The plasmids pYTU-lpsB, pYTP-lpsC, pYTR-easO, pYTR-easP and pYTR-easO-glaA-easP for A. nidulans expression were assembled by yeast homologous recombination [34]. The genes of lpsB, lpsC, easO and easP carrying 200 bp terminators were amplified from the genomic DNA of C. paspali WL721 by Q5 high fidelity DNA polymerase (NEB, Cat# M0491L) using primers containing 30 bp overlapping regions with the A. nidulans vectors. The PCR products were co-transformed into S. cerevisiae BJ5464 with Pac I digested pYTU and pYTP and BamH I digested pYTR, respectively. The plasmid was extracted from yeast using the Zymoprep Yeast Miniprep Kit (Zymo Research, Cat# D2001) and transformed into E. coli T1 for sequencing. The combinations of pYTU-lpsB/pYTP-lpsC, pYTU-lpsB/pYTP-lpsC/pYTR-easO, pYTU-lpsB/pYTP-lpsC/pYTR-easP and pYTU-lpsB/pYTP-lpsC/pYTR-easO-glaA-easP were, respectively, co-transformed into the protoplast of A. nidulans A1145. Preparation of the protoplast and transformation of A. nidulans A1145 were performed as previously described [35]. Spores of each transformant were collected separately and suspended in sterile distilled water before use.

2.6. Product Analysis for Transformants of A. nidulans

For product analysis, the transformants were cultured on solid and in liquid CD-ST medium (GMM liquid medium containing 20 g∙L−1 starch without glucose), respectively. For solid cultivation, 2 mg of D-lysergic acid was dissolved in 100 μL of DMSO and mixed with 20 mL of CD-ST for each plate. The spore solution of each transformant of A. nidulans was spread on the plate containing the substrate D-lysergic acid and cultivated for 5 days at 30 °C, then, the culture was soaked in methanol overnight and extracted with methanol. For liquid cultivation, each transformant was incubated in 50 mL of liquid CD-ST medium at 25 °C for 2 days, and 5 mg of D-lysergic acid dissolved in 250 μL of DMSO was added to the medium and cultivation was continued for another 3 days. The culture broth was extracted with ethyl acetate. The extracts were concentrated by rotary evaporator and subjected to the liquid chromatograph-mass spectrometer (LC-MS) analysis. The equipment and conditions used in LC-MS analysis were the same as those in the previous steps.

2.7. Statistical Analysis

Data are means ± standard deviation (SD) of three biological repeats. One-way ANOVA was used to analyze the statistical significance of the differences between means of the yield of ergometrine, total ergot alkaloids, the total of LAA, LAH and their isomers for wild-type and ΔeasOhetero-1, wild-type and ΔeasPhetero-34. * p < 0.05 and ** p < 0.01 are relative to the wild-type. Differences with * p < 0.05 were considered statically significant.

3. Results

3.1. The Protoplast-Mediated Transformation of C. paspali

Since C. paspali could not generate spores under laboratory conditions, protoplasts were used for genetic manipulation. The first step of our study was to investigate the conditions for the protoplast preparation and regeneration system for C. paspali WL721.
The commercial lywallzyme worked well in releasing the protoplast from the mycelia of C. paspali WL721. The number of protoplasts released from mycelium increased obviously with the prolonged incubation and reached its maximum value at 90 min. Beyond 90 min, the number of protoplasts remained basically unchanged (Figure 2a). Based on the regenerated colony dispersion, the layered medium culture method was chosen for further study and the optimal protoplast concentration was 105 protoplasts/mL (Figure S2). After preliminary screening, hygromycin B resistance was chosen as the dominant selectable marker. Hygromycin B could inhibit hyphal growth at the suitable concentration of 0.3 mg∙L−1 after cultivation for 7 days (Figure 2b). The PEG-mediated transformation method was used and the protoplasts could be successfully transformed via pAN7-1 [36] which harbored the hph cassette containing the hygromycin B-resistance gene (hygromycin phosphotransferase gene, hph) (Figure 2c). The selected transformants could still grow normally on the solid medium containing 0.6 mg∙L−1 of hygromycin B (Figure 2d). The hph cassette (2.6 kb in length) could be amplified from 14 of the 19 transformants (transformation frequency: ~74%), indicating that the genetic transformation system was successfully established (Figure 2e).

3.2. Knockout of easO and easP and Characterization of ΔeasO and ΔeasP Mutants

The established genetic transformation system was used to knock out the genes easO and easP from the genome of C. paspali WL721. The gene knockout efficiency (per regenerated colony) was ~17%. The diagnostic lengths and sequences of the PCR products for ∆easO and ∆easP are summarized in Figure 3 and Tables S2–S8, respectively.
For analyzing the easO deletion, the two overlapped fragments with 3.5 kb and 3.3 kb in lengths were yielded when the primer pairs P1/P8 and P7/P6 were used, respectively, which harbored the 5′ flank and 3′ flank regions of the easO gene and the whole hph expression cassette (Figure 3b). This result, together with the 2.6 kb-hph amplified fragment, demonstrated that the deletion cassette has replaced the corresponding part of easO and led to the knockout of easO from the genome of C. paspali WL721 (Figure 3b). However, the 1.9 kb-easO gene could still be amplified from both the wild-type control and each of the six recombinants (Figure 3b), indicating that all the recombinants were heterokaryons. In other words, the easO-deleted nucleus (ΔeasO) and the easO-preserved nucleus were simultaneously present in each cell. These recombinants are designated as ΔeasOhetero mutants.
Similarly, the ΔeasPhetero mutants were obtained in which the fragments of 3.9 kb (5′ flank regions of easP gene), 3.8 kb (3′ flank regions of easP gene), the hph cassette (2.6 kb), and the 1.1 kb-easP gene could be simultaneously amplified (Figure 3c).

3.3. Analysis of Ergot Alkaloid Production

The alkaloid-producing capabilities of these partial gene knockout mutants were analyzed in which the mutants ∆easOhetero-1 and ∆easPhetero-34 were chosen as the representatives for further investigation. The HPLC profiles of the product yields were shown in Figure 4, which also included the MS spectra data of LAA, LAH and ergometrine, as well as their isomers.
Compared with the wild-type strain, ∆easOhetero-1 accumulated more ergometrine, however, the production of LAA and LAH decreased significantly, indicating that the partial deletion of easO has redirected the lysergic acid flux away from LAH/LAA to ergometrine. More ergometrine was also accumulated in ∆easPhetero-34, although the yields of LAH and LAA seemed unchanged compared with those of the wild-type strain. This result implied that easP may play a different role in the production of ergometrine.
The titer of ergometrine in ∆easOhetero-1 reached 1559.36 mg∙L−1 after 14 days of fermentation, which is nearly 4 times higher than that of the wild-type (400.84 mg∙L−1) (Figure 5). The proportion of ergometrine in the total ergot alkaloid fermentation products of the ∆easOhetero-1 mutant was more than 80%. On the other hand, the total titer of LAH, LAA and their isomers of the ∆easOhetero-1 mutant was lower than that of the wild-type control (459.13 mg∙L−1 vs. 521.03 mg∙L−1), and the significant decreases in the LAH and LAA titers suggested that easO is indispensable for the biosynthesis of these two alkaloids. The mutant could maintain the productivity and quantity of the alkaloids for at least three generations. The ergometrine titer of the ∆easPhetero-34 reached 837.57 mg∙L−1 after 14 days of fermentation, which was 2.1 times that of the wild-type. Additionally, the ergometrine content was more than 50% of the total alkaloids produced by the mutant. However, the total titer of LAH, LAA and their isomers of ∆easPhetero-34 mutant was even higher than that of the wild-type control (641.12 mg∙L−1 vs. 521.03 mg∙L−1) (Figure 5), suggesting that easP may not be involved in the biosynthesis of LAH and LAA.

3.4. Heterologous Expression of the Ergot Alkaloid Biosynthetic Genes in A. nidulans

To examine the function of easO and easP in the biosynthesis of LAA based on the proposed biosynthetic pathway, different combinations of lpsB, lpsC, easO and easP were expressed in the heterologous host A. nidulans, and the recombinants were cultured on the solid medium fed with the substrate lysergic acid during cultivation. The metabolic products from A. nidulans were analyzed by LC-MS.
As shown in Figure 6, co-expression of lpsB and lpsC in A. nidulans led to the production of ergometrine, confirming the roles of the two genes in the biosynthesis of ergometrine. The product LAA, along with the major products ergometrine, were produced when lpsB, lpsC and easO were heterologously co-expressed in A. nidulans, demonstrating the key function of easO in the formation of LAA. LAH was not detected in the lpsB/lpsC/easO recombinant, and the most possible reason is that an unknown factor in A. nidulans triggered the rapid conversion from LAH to LAA; and/or after a long time soaking in methanol, nearly all the remaining LAH was spontaneously converted into LAA [10,24]. However, when lpsB, lpsC and easP were co-expressed in A. nidulans, only ergometrine could be detected, confirming that easP is not responsible for the biosynthesis of LAH/LAA. Specifically, when easO was co-expressed with lpsB, lpsC and easP genes, the production of additional minor LAA was restored, which again demonstrated that easO catalyzed the formation of LAA. Interestingly, the recombinant A. nidulans with the lpsB/lpsC/easO combination grew very poorly in the medium while the addition of easP in the combination (lpsB/lpsC/easO/easP) could restore growth (Figure 6), which deserves further analysis. In fact, the recombinant with the lpsB/lpsC/easO combination almost stopped growth in the liquid medium, and the experiment could not be completed for this mutant.

4. Discussion

Ergometrine is clinically used for the treatment of postpartum uterus bleeding. The valuable commodity is mainly produced by C. paspali, which also produces other simple lysergic acid amides, including LAH and LAA. Both ergometrine and LAH/LAA are derived from D-lysergic acid, given that LAA is spontaneously converted from LAH. The presence of additional genes (easO and easP) in the EAS gene cluster of C. paspali suggests their functions in the biosynthesis of LAH/LAA. Thus, the proposed biosynthetic pathway suggested that easO and easP may be involved in the biosynthesis of LAH/LAA. Moreover, down-regulation of easO and easP should be a good strategy to improve the productivity of the ergometrine of C. paspali. On the other hand, although the function of easO in M. brunneum has recently been illustrated to control the biosynthesis of LAH by the gene knockout test, additional evidence is required to demonstrate the roles of easO and easP in the biosynthesis of LAH/LAA in C. paspali.
Currently, there is no universal genetic transformation method that can be applied to every fungal species. Moreover, the current protoplast-mediated transformation protocols of C. paspali are undermined by its inefficiencies in protoplast regeneration, low frequency of DNA integration and low mitotic stability of the nascent transformants [31]. Therefore, in the present study, a protoplast-mediated genetic transformation system for the C. paspali WL721 strain was first established to enable subsequent gene manipulation.
C. paspali mutants were produced using homologous fragment recombination, and they were screened with a high concentration of hygromycin B. Figure 3 showed that the event of gene replacement between the deletion cassette and easO (Figure 3b) or easP (Figure 3c) has taken place in every tested gene knockout mutant, indicating the high efficacy of the homologous recombination approach. On the other hand, due to the multinucleate property of the strain, the nucleus harboring the easO or easP gene was hardly eliminated from the mutants. However, ergometrine production of the heterokaryon ∆easOhetero-1 was significantly increased compared with that of the wild-type control (1559.36 mg∙L−1 vs. 400.84 mg∙L−1), accompanied by a significant decrease in the production of LAH and LAA (Figure 5), which supported the key role of the enzyme in the formation of LAH/LAA (Figure 1b). Furthermore, the down-expression of the gene could attenuate the lysergic acid (the common precursor of ergometrine and LAH/LAA) flux towards LAH/LAA but enhance it towards ergometrine biosynthesis. Typically, the function of easO has been demonstrated in this study by heterologous expression of it in the A. nidulans system (Figure 6). To the best of our knowledge, this study is the first to demonstrate the role of the easO in the biosynthesis of LAA of C. paspali. Although the mutant of C. paspali was a heterokaryon to the gene easO, the increase in ergometrine productivity was maintained for at least four generations, suggesting that the mutant can be utilized for industrial production if the strain improvement is accompanied during the industrial process. On the other hand, the great increase in the titer of ergometrine and the significant decrease in the impurities of the fermentation products may further benefit the pharmaceutical industry.
The productivity of LAH and LAA was slightly enhanced in the ∆easPhetero-34 strain compared with that in the wild-type control (641.12 mg∙L−1 vs. 521.03 mg∙L−1 in total), suggesting that easP was not involved in the biosynthesis of LAH/LAA. The non-involvement of easP in the biosynthesis of LAH/LAA was further supported by the heterologous expression analysis in the A. nidulans system (Figure 6). However, the titer of ergometrine in the mutant was surely increased. Taken together, this enzyme does not participate in the formation of LAH/LAA, but knocking it out indirectly improved the biosynthesis of ergometrine.
In summary, a protoplast-mediated genetic transformation system for the C. paspali WL721 strain was established to delete easO and easP in the C. paspali genome. A. nidulans system was used to further investigate the function of the additional genes of easO and easP in the EAS gene cluster of C. paspali. The two ergot mutants, ΔeasOhetero-1 and ΔeasPhetero-34, were obtained. The ergometrine yields of ΔeasOhetero-1 and ΔeasPhetero-34 at the flask fermentation level reached 1559.36 mg∙L−1 and 837.57 mg∙L−1, which were 4 and 2 times higher than that of the wild-type control, respectively. Meanwhile, the yields of LAH and LAA in ΔeasOhetero-1 were significantly decreased, which strongly supported that the gene of easO was involved in the branch pathway of the biosynthesis of ergot alkaloids. The function of easO was further demonstrated by heterologous expression of it in the A. nidulans system. In addition, the increase in the ergometrine yield in ΔeasPhetero-34 suggests that although easP is not involved in the biosynthetic pathway of LAH/LAA, knocking it out can indirectly improve the formation of ergometrine.

5. Conclusions

By means of protoplast-mediated genetic transformation and homologous recombination, we obtained two mutants, ∆easOhetero-1 and ∆easPhetero-34, from the C. paspali WL721 strain, with ergometrine titers of 1559.36 mg∙L−1 and 837.57 mg∙L−1, which were 4 and 2 times higher than that of the wild-type control, respectively, and have practical implication for improving ergometrine production of C. paspali. Meanwhile, the yields of LAH and LAA in ΔeasOhetero-1 were significantly decreased, supporting that at least the gene of easO was involved in the branch pathway of the biosynthesis of ergot alkaloids. Heterologous expression of easO or easP, together with lpsB and lpsC from C. paspali in the A. nidulans system, further demonstrated that easO, but not easP, determines the formation of LAA. To the best of our knowledge, this is the first evidence of the biosynthetic mechanism of LAH and LAA from lysergic acid in C. paspali and paves the way for the improvement of ergometrine production of C. paspali.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation8060263/s1. Table S1. Primers used in this study. Table S2. The sequence of hph cassette. Table S3. The sequence of 5′ homologous integration of the replacement construct in the location. Table S4. The sequence of 3′ homologous integration of the replacement construct in the location of target gene in ΔeasO strain when primers P6 and P7 were used. Table S5. The sequence of 5′ homologous integration of the replacement construct in the location of target gene in ΔeasP strain when primers P1′ and P8 were used. Table S6. The sequence of 3′ homologous integration of the replacement construct in the location of target gene in ΔeasP strain when primers P6′ and P7 were used. Table S7. The sequence of easO. Table S8. The sequence of easP. Table S9. ITS sequence of Claviceps paspali WL721 strain. Figure S1. Sequence alignment of representative Baeyer-Villiger monooxygenase and EasO. Figure S2. (a) Protoplast regeneration by two different methods. (1) Layered medium culture method. (2) Monolayer medium culture method. (b) Determination of minimal inhibitory concentrations (MICs) of 5 antibiotics. The concentrations of antibiotics are measured in mg·mL−1.

Author Contributions

Y.-M.Q. designed and conducted the experiments, collected data, created the graphs and wrote the manuscript; Y.-H.W. performed parts of the experiments; T.G. analyzed the data of mass spectra and helped in taking the photographs; J.-J.C., T.-J.C. and J.-L.Y. helped to analyze the data; P.Z. conceived the experiments and revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the CAMS Innovation Fund for Medical Sciences (No. CIFMS-2021-I2M-1-029), the National Key Research and Development Program of China (grant No. 2018YFA0901900), and the PUMC Disciplinary Development of Synthetic Biology (201920100801, China).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

The authors have agreed upon the publication of this manuscript.

Data Availability Statement

The datasets generated during and/or analyzed during the current study are available from the author Y.-M.Q. on reasonable request.

Acknowledgments

We thank Rui-Lin Yu at Purdue University for proofreading the manuscript and English editing, and for assistance in the screening of mutant strains during her internship in our lab.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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Figure 1. (a) Comparison of the two EAS gene clusters from C. purpurea and C. paspali. (b) The proposed biosynthetic pathway of lysergic acid amides in C. paspali.
Figure 1. (a) Comparison of the two EAS gene clusters from C. purpurea and C. paspali. (b) The proposed biosynthetic pathway of lysergic acid amides in C. paspali.
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Figure 2. Establishment of the protoplast-mediated transformation system for C. paspali. (a) Protoplast formation at different time of enzymatic hydrolysis. The bar is 10 μm. (b) Determination of minimal inhibitory concentrations (MICs) of 5 antibiotics. The concentrations of antibiotics are measured in mg∙mL−1. (c) Protoplast regeneration on the regeneration plates with different concentrations of hygromycin B. −pAN7-1: protoplasts transformed with mock plasmid (negative control). +pAN7-1: protoplasts transformed with dominant pAN7-1 harboring hph gene. The layered medium culture method was used and the upper layer medium (5 mL) contained 0, 0.1, 0.2, and 0.3 mg∙mL−1 hygromycin B, respectively. (d) Growth of the transformants on fresh PGA medium containing 0.6 mg∙mL−1 hygromycin B. (e) Identification of positive transformants via PCR. Lanes 1–19, transformants; Lane 20, pAN7-1 (positive control); Lane 21, the wild-type strain (negative control). M: DNA marker.
Figure 2. Establishment of the protoplast-mediated transformation system for C. paspali. (a) Protoplast formation at different time of enzymatic hydrolysis. The bar is 10 μm. (b) Determination of minimal inhibitory concentrations (MICs) of 5 antibiotics. The concentrations of antibiotics are measured in mg∙mL−1. (c) Protoplast regeneration on the regeneration plates with different concentrations of hygromycin B. −pAN7-1: protoplasts transformed with mock plasmid (negative control). +pAN7-1: protoplasts transformed with dominant pAN7-1 harboring hph gene. The layered medium culture method was used and the upper layer medium (5 mL) contained 0, 0.1, 0.2, and 0.3 mg∙mL−1 hygromycin B, respectively. (d) Growth of the transformants on fresh PGA medium containing 0.6 mg∙mL−1 hygromycin B. (e) Identification of positive transformants via PCR. Lanes 1–19, transformants; Lane 20, pAN7-1 (positive control); Lane 21, the wild-type strain (negative control). M: DNA marker.
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Figure 3. Schematic diagram of the progress of constructing mutant strains and screening of gene knockout mutants. (a) Knockout of easO and easP in C. paspali WL721. 5′ and 3′ flank fragments are amplified separately from genomic DNA with primers P1/P3 and P4/P6 for easO or P1′/P3′ and P4′/P6′ for easP, respectively. Primers P3 (or P3′) and P4 (or P4′) have 5′ tails homologous to the hph cassette. The two flanks and the hph cassette are assembled by overlapping PCR method and the fusion fragment containing the deletion cassette is obtained by amplification with primers P2 (or P2′ for easP) and P5 (or P5′ for easP). Homologous recombination creates the circular construct. The deletion cassette harboring the 5′ and 3′ flank regions of easO or easP was obtained by PCR amplification with primers P2 and P5 for easO or primers P2′ and P5′ for easP. The fragment harboring the easO or easP in the genomic DNA of C. paspali W721 can be replaced by the deletion cassette containing the hph gene through the double crossover between the deletion cassette and the genomic DNA. The hph is transcribed in the antisense direction relative to the target gene. Transformants carrying the homologous integration of the replacement construct were identified via PCR with primers P1 (or P1′) and P8, P7 and P6 (or P6′), respectively. Other pairs of primers were used to conduct PCR verification at the same time: easO-F and easO-R (1900 bp of amplified fragment), hph-F and hph-R (2660 bp of amplified fragment), easP-F and easP-R (1100 bp of amplified fragment). The easP gene was knocked out in the same way as the easO gene. Transformants carrying the homologous integration of the replacement construct were identified via PCR with two different pairs of primers P1′ and P8, P6′ and P7, respectively. Two additional pairs of primers, easP-F and easP-R (1100 bp of amplified fragment), hph-F and hph-R (2660 bp of amplified fragment), were used to conduct PCR verification. (b) Screening of ΔeasO transformants via PCR. M: DNA marker. 1–6: ΔeasO transformants. (c) Screening of ΔeasP transformants via PCR. M: DNA marker. 1–4: ΔeasP transformants. W: C. paspali WL721 wild strain. P: pAN7-1 plasmid.
Figure 3. Schematic diagram of the progress of constructing mutant strains and screening of gene knockout mutants. (a) Knockout of easO and easP in C. paspali WL721. 5′ and 3′ flank fragments are amplified separately from genomic DNA with primers P1/P3 and P4/P6 for easO or P1′/P3′ and P4′/P6′ for easP, respectively. Primers P3 (or P3′) and P4 (or P4′) have 5′ tails homologous to the hph cassette. The two flanks and the hph cassette are assembled by overlapping PCR method and the fusion fragment containing the deletion cassette is obtained by amplification with primers P2 (or P2′ for easP) and P5 (or P5′ for easP). Homologous recombination creates the circular construct. The deletion cassette harboring the 5′ and 3′ flank regions of easO or easP was obtained by PCR amplification with primers P2 and P5 for easO or primers P2′ and P5′ for easP. The fragment harboring the easO or easP in the genomic DNA of C. paspali W721 can be replaced by the deletion cassette containing the hph gene through the double crossover between the deletion cassette and the genomic DNA. The hph is transcribed in the antisense direction relative to the target gene. Transformants carrying the homologous integration of the replacement construct were identified via PCR with primers P1 (or P1′) and P8, P7 and P6 (or P6′), respectively. Other pairs of primers were used to conduct PCR verification at the same time: easO-F and easO-R (1900 bp of amplified fragment), hph-F and hph-R (2660 bp of amplified fragment), easP-F and easP-R (1100 bp of amplified fragment). The easP gene was knocked out in the same way as the easO gene. Transformants carrying the homologous integration of the replacement construct were identified via PCR with two different pairs of primers P1′ and P8, P6′ and P7, respectively. Two additional pairs of primers, easP-F and easP-R (1100 bp of amplified fragment), hph-F and hph-R (2660 bp of amplified fragment), were used to conduct PCR verification. (b) Screening of ΔeasO transformants via PCR. M: DNA marker. 1–6: ΔeasO transformants. (c) Screening of ΔeasP transformants via PCR. M: DNA marker. 1–4: ΔeasP transformants. W: C. paspali WL721 wild strain. P: pAN7-1 plasmid.
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Figure 4. LC-MS analysis of fermentation products of ΔeasOhetero-1, ΔeasPhetero-34 and the wild-type strain. Notes: Ergot alkaloids might convert into their isomers. 1, LAA; 2, LAH; 3, ergometrine; 4, 6 and 7, isomers of LAH; 5, isomer of ergometrine. The standards of ergometrine, LAH and LAA were used, respectively.
Figure 4. LC-MS analysis of fermentation products of ΔeasOhetero-1, ΔeasPhetero-34 and the wild-type strain. Notes: Ergot alkaloids might convert into their isomers. 1, LAA; 2, LAH; 3, ergometrine; 4, 6 and 7, isomers of LAH; 5, isomer of ergometrine. The standards of ergometrine, LAH and LAA were used, respectively.
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Figure 5. The quantitative analysis of ergot alkaloids production (mg∙L−1) from different strains after 14 days of fermentation. One-way ANOVA was used to analyze the significance between means. * p < 0.05, ** p < 0.01 relative to the wild-type. Notes: 1, LAA; 2, LAH; 3, ergometrine; 4, 6 and 7, isomers of LAH; 5, isomer of ergometrine; 8, total ergot alkaloids; 9, total of LAA, LAH & isomers.
Figure 5. The quantitative analysis of ergot alkaloids production (mg∙L−1) from different strains after 14 days of fermentation. One-way ANOVA was used to analyze the significance between means. * p < 0.05, ** p < 0.01 relative to the wild-type. Notes: 1, LAA; 2, LAH; 3, ergometrine; 4, 6 and 7, isomers of LAH; 5, isomer of ergometrine; 8, total ergot alkaloids; 9, total of LAA, LAH & isomers.
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Figure 6. LC-MS analysis and morphologies of the corresponding strains. Note: Ergot alkaloids might convert into their isomers. a and b, D-lysergic acid and isomer; c and d, ergometrine and isomer; e and f, LAA and isomer. a, c and e were the standards of D-lysergic acid, ergometrine and LAA, respectively. AN-lpsB + lpsC: co-expression of lpsB and lpsC in A. nidulans host. AN-lpsB + lpsC + easO: co-expression of lpsB, lpsC and easO in A. nidulans host. AN-lpsB + lpsC + easP: co-expression of lpsB, lpsC and easP in A. nidulans host. AN-lpsB + lpsC + easO + easP: co-expression of lpsB, lpsC, easO and easP in A. nidulans host. The morphologies of different recombinants on the solid medium show the sporulation (left) and the color patterns (right) after incubation at 30 °C for 5 days.
Figure 6. LC-MS analysis and morphologies of the corresponding strains. Note: Ergot alkaloids might convert into their isomers. a and b, D-lysergic acid and isomer; c and d, ergometrine and isomer; e and f, LAA and isomer. a, c and e were the standards of D-lysergic acid, ergometrine and LAA, respectively. AN-lpsB + lpsC: co-expression of lpsB and lpsC in A. nidulans host. AN-lpsB + lpsC + easO: co-expression of lpsB, lpsC and easO in A. nidulans host. AN-lpsB + lpsC + easP: co-expression of lpsB, lpsC and easP in A. nidulans host. AN-lpsB + lpsC + easO + easP: co-expression of lpsB, lpsC, easO and easP in A. nidulans host. The morphologies of different recombinants on the solid medium show the sporulation (left) and the color patterns (right) after incubation at 30 °C for 5 days.
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Qiao, Y.-M.; Wen, Y.-H.; Gong, T.; Chen, J.-J.; Chen, T.-J.; Yang, J.-L.; Zhu, P. Improving Ergometrine Production by easO and easP Knockout in Claviceps paspali. Fermentation 2022, 8, 263. https://doi.org/10.3390/fermentation8060263

AMA Style

Qiao Y-M, Wen Y-H, Gong T, Chen J-J, Chen T-J, Yang J-L, Zhu P. Improving Ergometrine Production by easO and easP Knockout in Claviceps paspali. Fermentation. 2022; 8(6):263. https://doi.org/10.3390/fermentation8060263

Chicago/Turabian Style

Qiao, Yun-Ming, Yan-Hua Wen, Ting Gong, Jing-Jing Chen, Tian-Jiao Chen, Jin-Ling Yang, and Ping Zhu. 2022. "Improving Ergometrine Production by easO and easP Knockout in Claviceps paspali" Fermentation 8, no. 6: 263. https://doi.org/10.3390/fermentation8060263

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