Abstract
Background
The lipopeptide herbicolin A (HA) secreted by the biocontrol agent Pantoea agglomerans ZJU23 is a promising antifungal drug to combat fungal pathogens by targeting lipid rafts, both in agricultural and clinical settings. Improvement of HA production would be of great significance in promoting its commercialization. This study aims to enhance the HA production in ZJU23 by combining fermentation optimization and strain engineering.
Results
Based on the results in the single-factor experiments, corn steep liquor, temperature and initial pH were identified as the significant affecting factors by the Plackett–Burman design. The fermentation medium and conditions were further optimized using the Box-Behnken response surface method, and the HA production of the wild type strain ZJU23 was improved from ~ 87 mg/mL in King’s B medium to ~ 211 mg/mL in HA induction (HAI) medium. A transposon library was constructed in ZJU23 to screen for mutants with higher HA production, and two transcriptional repressors for HA biosynthesis, LrhA and PurR, were identified. Disruption of the LrhA gene led to increased mRNA expression of HA biosynthetic genes, and subsequently improved about twofold HA production. Finally, the HA production reached ~ 471 mg/mL in the ΔLrhA mutant under optimized fermentation conditions, which is about 5.4 times higher than before (~ 87 mg/mL). The bacterial suspension of the ΔLrhA mutant fermented in HAI medium significantly enhanced its biocontrol efficacy against gray mold disease and Fusarium crown rot of wheat, showing equivalent control efficacies as the chemical fungicides used in this study. Furthermore, HA was effective against fungicide resistant Botrytis cinerea. Increased HA production substantially improved the control efficacy against gray mold disease caused by a pyrimethanil resistant strain.
Conclusions
This study reveals that the transcriptional repressor LrhA negatively regulates HA biosynthesis and the defined HAI medium is suitable for HA production. These findings provide an extended basis for large-scale production of HA and promote biofungicide development based on ZJU23 and HA in the future.
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Background
Food production needs to increase by 60% to meet the demands of approximately 10 billion people around the world by 2050 [1]. The fast-growing human population requires an increased crop yield, along with a reduction in crop loss caused by crop pathogens and pests. An expert-based assessment of crop losses on an individual pathogen and pest basis for wheat, rice, maize, potato and soybean, as five major crops, globally indicated that the average of crop losses caused by pathogens and pests are about 22.5% for all five crops [2]. Wheat is the staple crop for an estimated 35% of the world population [3]. A critical approach to meeting the increased demand is better management of fungal diseases, which can be responsible for 15%–20% yield losses per annum, such as rusts, blotches and Fusarium head blight (FHB) [4]. For example, the annual average occurrence of FHB, which is mainly caused by Fusarium graminearum in China, affects more than 4.5 million hectares, approximately 20% of the total planted area of wheat, and has caused serious yield losses [5]. Furthermore, FHB pathogens contaminate grains with various mycotoxins, especially deoxynivalenol (DON), which poses a health threat to humans and livestock. Currently, chemical fungicides are still the most effective approach to control FHB. However, fungicide resistant F. graminearum isolates have been detected in fields after long-term intensive use of fungicides [6, 7]. Moreover, treatments with some fungicides at sub-lethal concentrations stimulate mycotoxin production [8,9,10]. There is an urgent need to develop and apply new approaches to control FHB and mycotoxin contamination.
Biological control by living microorganisms is considered to be a suitable, alternative strategy for the control of plant diseases. Several antagonistic microorganisms were identified as biocontrol agents (BCAs) to combat plant diseases and achieved comparatively good efficiency [11]. Moreover, more than 30 species of microbes have been reported to specifically inhibit the mycelial growth of F. graminearum, or/and provided successful control efficiencies against FHB and mycotoxin reduction under greenhouse and/or field conditions [12,4: Fig. S3a). The ORF2643 gene encodes a putative LysR-type DNA-binding transcriptional repressor, LrhA (Additional file 4: Fig. S3b). Its homologues were previously reported to repress biosynthesis of secondary metabolisms in Photorhabdus and Xenorhabdu [43,44,45]. The two identified genes were named purR and lrhA for further investigation.
To confirm the roles of PurR and LrhA on HA production, we constructed single deletion mutants, double deletion mutants, and corresponding complementation strains of the two genes. The mRNA expression levels of genes in the biosynthetic gene cluster AcbA-AcbJ [21]. Lipopeptides constitue a specific class of microbial secondary metabolites and harbor diverse biological functions, especially antimicrobial and anticancer activities [19, 20, 57,58,59,60,61,62]. Lipopeptides can be employed for pharmaceutical, food-related, agricultural, and environmental protection applications [63, 64]. In the present study, we demonstrated that increased HA yields in the ZJU23 deletion mutant result in control effects of fungal pathogens that are comparable to synthetic fungicides. This highlights its applicability for crop protection. HA belongs to the cyclic lipopeptides and its structure consists of a peptide ring of eight amino acids where a fatty acid chain, a dehydrobutyric acid, and a sugar moiety are attached [Quantification of HA via HPLC analysis Quantitative analysis of HA was performed as previously described [13]. Briefly, 1 mL of the fermentation supernatant was collected and freeze-dried. The precipitate formed was resuspended with 1 mL methanol for HA extraction and the crude samples were subjected to HPLC analysis (Agilent Technologies 1100 Infinity) under the following conditions: C18 reversed-phase column [Agilent ZORBAX RX-C18 column (250 × 4.6 mm)] eluted with methanol/H2O (A/B) (1 mL/min, 30–90% A in 30 min, followed by 90–100% A in 10 min). The peak areas were used to quantify the production of HA according to the standard sample. To quantify HA more precisely, the extracted samples were analyzed by liquid chromatography-mass spectrometry (LC–MS). A transposon mutagenesis library and gene deletion mutants of ZJU23 were constructed as described previously [13]. Briefly, transposon mutants were generated by conjugation of recipient ZJU23 resistant to rifampicin and donor E. coli SM10λ-pir. Each transposon was screened in an inhibition zone assay against F. graminearum. The selected transposons were re-sequenced by Bei**g Novogene Bioinformatics Technology Co., Ltd. to localize the insertion by comparing it with the wild type strain ZJU23. Gene deletion mutants were generated by using the λ-red recombinase method. The transformants were confirmed by PCR. Double mutant strains were generated using single mutants as a background strain as indicated. Complementation constructructions were generated as described previously [68]. The primers designed in this study for mutant strains and complementation constructions are listed in the Additional file 1: Table S4. The primer pair M13-F and M13-R were used for identification of pBBR-LrhA and pBBR-PurR plasmid construction [69]. For real-time quantitative PCR (RT-qPCR), all strains were cultured with HI medium at 25 °C and 180 rpm for 18 h. Total RNA purification was performed by using RNAprep Pure Cell/ Bacteria Kit (TIANGEN, DP430) and reverse transcription was done using HiScript II Q RT SuperMix for qPCR (+ gDNA wiper) (Vazyme, R223-01) according to the manufacturer's instructions. RT-qPCR was performed via qRT-PCR using ChamQ SYBR qPCR Master Mix (Vazyme, Q311-02). The experiments were performed in independent biological triplicates and 16S rRNA of ZJU23 was used as the internal control. The mRNA fold change was estimated by the threshold cycle (Ct) values of 2−(ΔΔCt). The primers used for qRT-PCR assays are listed in Additional file 1: Table S4. Biocontrol experiments with a fermentation suspension produced by ZJU23 in KB and ΔLrhA in HAI against gray mold caused by B. cinerea and Fusarium crown rot caused by F. pseudograminearum were conducted. The gray mold assay was performed as previously described [70]. The corresponding fermentation broth, pyrimethanil (50 mg/L) or water were sprayed on the tomato or apple surface. After 1 h air-drying, fresh mycelial plugs (6 mm in diameter) were inoculated. Pyrimethanil (50 mg/L) and clean water were used as fungicide treatment and negative control. Disease lesions were observed and measured 72 h after inoculation with the pathogenic fungus. The Fusarium crown rot assay was performed as described previously but with specific modifications [71]. F. pseudograminearum F303 conidia (1 × 106 CFU/mL) were mixed with soil at 1:10 (W/W) for three days. Then wheat seeds (cultivar: Jimai 22) were planted into conidia-inoculated soil. Non-inoculated soil was used as a control. At the seventh day, 50 mL of the fermentation suspension were sprinkled on the wheat root. Tebuconazole (150 mg/L) and clean water were used as fungicide treatment and negative control. After 21 days, the roots were washed and the browning areas of the wheat stem base were observed. The disease index (DI) was calculated as previously described [72] but with modifications. Five evaluation classes ranging from 0 to 4, were applied for the disease index. The disease index corresponds to the percentage of the browning length of the first stem node (0 = 0, 1 = 1–25%, 2 = 26–50%, 3 = 51–75%, and 4 ≥ 75%). The disease index was calculated as follows in Eq. (2): The control efficacy was calculated as follows in Eq. (3): All experiments in this study were repeated three times. Data presented are the mean ± standard errors. Differences between two groups were analyzed by Student’s t-test. Multiple comparisons were analyzed by one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) multiple-range test.Construction of transposon mutants and gene deletion mutants
RNA preparation and quantitative reverse transcription PCR (qRT-PCR)
Evaluation of biocontrol efficacy of the fermentation suspension
Statistical analysis
Availability of data and materials
All data generated or analyzed during this study are included in this published article [and the additional files].
Abbreviations
- PBD:
-
Plackett–Burman design
- BBD:
-
Box-Behnken design
- RSM:
-
Response surface methodology
- HA:
-
Herbicolin A
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This research was supported by National Key Research and Development Program of China (2022YFD1400100), the National Natural Science Foundation (31922074), the Natural Science Foundation of Zhejiang Province (Z23C140008), China Agriculture Research System (CARS-3-1-15) and the Fundamental Research Funds for the Central Universities (2021FZZX001-31).
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YC initiated, coordinated and supervised the project. HW, YZ, SX, BZ performed the experiments. H.W and YC collected and analyzed the data. HW and YC wrote the manuscript. YC, ZM and TC revised the manuscript. All authors read and approved the final manuscript.
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Additional file 1: Table S1.
The component of different media used for basic medium screening. Table S2. List of levels and factors in the Plackett–Burman design experiments. Table S3. Strains and plasmids used in this study. Table S4. PCR primers used in this study.
Additional file 2: Fig. S1.
Selection of a base medium for HA production.
Additional file 3: Fig. S2.
Effects of different components in the medium and various fermentation parameters on the HA production.
Additional file 4: Fig. S3.
Phylogenetic analysis based on the amino acid sequences of proteins encoded by ORF22 a and ORF2643 b in P. agglomerans ZJU23.
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Wang, H., Zhou, Y., Xu, S. et al. Enhancement of herbicolin A production by integrated fermentation optimization and strain engineering in Pantoea agglomerans ZJU23. Microb Cell Fact 22, 50 (2023). https://doi.org/10.1186/s12934-023-02051-z
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DOI: https://doi.org/10.1186/s12934-023-02051-z