Breeding high-yield pullulan engineering strains of Aureobasidium pullulans by ARTP mutagenesis and transcriptome analysis

doi:10.21203/rs.3.rs-3804345/v1

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Breeding high-yield pullulan engineering strains of Aureobasidium pullulans by ARTP mutagenesis and transcriptome analysis

https://doi.org/10.21203/rs.3.rs-3804345/v1

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published 14 Aug, 2024

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Pullulan is a microbial exopolysaccharide produced by Aureobasidium sp. with excellent physical and chemical properties, resulting in great application value. In this study, a novel strain of Aureobasidium pullulans with high pullulan yield up to 51.03 ± 1.03 g·L^− 1 isolated from the rhizosphere soil of plants in Taihu Lake in Wuxi, China was subjected to atmospheric and room temperature plasma (ARTP) mutagenesis, followed by selective and optimization to obtain high-yield and high-quality strains. Finally, two pullulan high-yielding and stable genetic mutants Mu0816 and Mu1519 were obtained, with polysaccharide yields after 72-hour fermentation were of 58.71 ± 0.78 and 60.03 ± 0.75 g·L^− 1, representing 15.05 and 17.64% increases compared with the original strain, respectively. Transcriptome sequencing was performed on the mutants and the original strain. Transcriptional analysis revealed that the high expression of abhd, gh18 and spMFS in the mutants may be related to the synthesis and secretion of pullulan. These results demonstrated the effectiveness of ARTP mutagenesis in A. pullulans, providing a basis for the investigation of genes related to pullulan production and metabolism.

Aureobasidium pullulans

Pullulan

ARTP mutagenesis

Transcriptome analysis

Pullulan is formed by polymerization of maltotriose units via α-1,6 glycosidic bonds (Shingel 2004; Agrawal et al. 2022). It is non-toxic, while also displaying good physicochemical properties such as film forming property, oxygen resistance and moisture retention (Yang et al. 2020; Li et al. 2020; Bulman et al. 2015; Emam et al. 2021). As an excellent biological material, pullulan is widely used in the fields of medicine, food, chemical industry and environmental protection (Hu et al. 2022; Singh et al. 2023; Singh et al. 2021). However, due to intrinsic differences in physiology, the production of exopolysaccharides varies greatly among different strains. Moreover, there is a lack of research on the underlying molecular and metabolic mechanisms, restricting further strain engineering and industrialization (Rai et al. 2021; Singh et al. 2022; Singh et al. 2019; Leathers et al. 2003). Therefore, it is crucial to select and breed excellent strains, as well as study the underlying metabolic mechanisms of increased pullulan production (Badhwar et al. 2020).

Aureobasidium pullulans, also known as black yeast (Wei et al. 2021), is widely found in water, soil, deep-sea sediments and plant surfaces (Prasongsuk et al. 2018; Gostinčar et al. 2019). It is a GRAS microbe certified by the United States FDA, and is the main producer of pullulan. In different stages of growth, A. pullulans can produce biomolecules such as melanin and pullulan, which has important economic value (Junior et al. 2022; Chi et al. 2009; Suzuki et al. 2021). The polysaccharide high-yielding strain RM1603 was isolated from plant rhizosphere soil. Morphological, phylogenetic and genome sequencing results showed that RM1603 is a strain of A. pullulans.

Atmospheric and room temperature plasma (ARTP) has emerged as a novel mutagenesis tool in recent years (Liu et al. 2020; Li et al. 2022). It uses a stream of plasma to cause gene damage, and has been widely used in biological breeding with excellent results (Zhu et al. 2022; Li et al. 2021).

In this study, the mutant strains Mu0816 and Mu1519 were obtained using ARTP mutagenesis technology based on the original strain RM1603, after which transcriptome sequencing was performed using the Illumina Novaseq 6000 sequencing platform (Jiang et al. 2015; Guo et al. 2021). The results were used as a basis to determine the genetic changes caused by ARTP treatment in A. pullulans, and thereby demonstrate the effectiveness of ARTP mutagenesis in this important pullulan producer (Xia et al. 2021; Wang et al. 2020; Suryadi et al. 2022). The identified differentially expressed genes (DEGs) can help us to understand the mechanism of pullulan synthesis and identify key genes related to polysaccharide metabolism, which can serve for further rational strain engineering in the future.

Strain mutagenesis

Activation and Cultivation of the strain

The strain was transferred to PDB medium, and cultured at 28℃ and 200 rpm for 24 to 48 h. Then, the activated seed culture was used to inoculate the seed medium at a 2% (v/v, OD₆₀₀ = 1) ratio, followed by incubation at 28℃ and 200 rpm before mutagenesis (Monjazeb et al. 2018; Liu et al. 2021).

ARTP mutagenesis

The optimal mutagenesis time was determined in preliminary experiments based on the viability time profile at different treatment times. The treatment time with a fatality rate between 80% and 99% was selected the highest mutation rate was determined as the best mutagenesis time through the preliminary screening microtiter plates and re-screening in shake flasks, resulting in a large number of mutants library obtained by repeated mutagenesis (Gu et al. 2020; Dong et al. 2022). Strains with polysaccharide yields more than 10% higher than that of the original strain were selected, and subjected to 15 rounds of subculture. The polysaccharide yield of the original strain and the mutant strains was determined in triplicate fermentations (Wei et al. 2022). If the polysaccharide yield of each generation was not significantly different and was significantly higher than that of the original strain, the mutant strain was considered genetically stable.

Characteristics of the mutant strains

The genetically stable strains were verified by repeated shake flask fermentations. Polysaccharide yield, biomass, OD₆₀₀ value, pH and other indexes were determined, after which the polysaccharide yield and sucrose conversion rate were calculated. The mutant strains and original strain were streaked in lines on plates of fermentation medium, and cultured at 28℃ for 3 ~ 5 d to observe the colony morphology (Wu et al. 2022). Finally, the two genetically stable high-yield mutant strains obtained through ARTP mutagenesis were named Mu0816 and Mu1519.

Transcriptome sequencing

Strain culture and sample preparation

A. pullulans RM1603 (CK), Mu0816 and Mu1519 were streaked on pullulan fermentation medium plate for activation for 48 h, and then grown in liquid pullulan fermentation medium at 28℃ and 200 rpm for 60 h. The resulting seed broth was used to inoculate pullulan fermentation medium at a ratio of 2% (v/v, OD₆₀₀ = 1) and cultured at 28 ℃ and 200 rpm for 72 h (Lu et al. 2022).

The cells were collected by centrifugation and cryo-preserved at -80℃. The samples were sent to the headquarters of Marjor Biological Co, Ltd. in Shanghai for transcriptome sequencing.

Library sequencing and functional analysis of DEGs

Illumina Novaseq 6000 equencing platform was used to complete the transcriptome sequencing of A. pullulans. The Illumina PE library was constructed for 2×150 bp sequencing. The clean reads obtained after quality control were compared and analyzed, including sequence analysis and expression level analysis (Guo et al. 2021).

DESeq2 was used to quantify gene expression, and the expression level of the identified genes was analyzed for different genes among samples. Only the genes meeting the criteria of |log2 fold change (FC)|>2 and P < 0.05 were identified as being differentially expressed genes. Functional enrichment analysis of DEGs using software Goatools (https://github.com/tanghaibao/GOatools), only the P < 0.05 were considered significant.

Validation of expression using qPCR

The expression of differentially expressed genes in the mutants was confirmed by qPCR. The Trizol method (Schmittgen et al. 2008; Islas-Flores et al. 2006) was used to extract total RNA of the mutants and RM1603, after which the concentration and purity were assessed by denaturing RNA gel electrophoresis. The cDNA was obtained by reverse transcription PCR using ReverTra Ace™ qPCR RT Master Mix with gDNA Remover (TOYOBO. Co., LTD), and the real-time fluorescence quantitative PCR was carried out based on this template with ACT1 as internal standard.

ARTP mutagenesis

The morphology of the regenerated strains obtained by ARTP was observed on plates, and the large and viscous colonies were selected for primary screening in 24 deep well plates, followed by rescreening in shake flasks. From a total of 1988 regenerated strains from the ARTP treatment, 26 strains with more than 10% increases of extracellular polysaccharide yield were obtained.

The mutants from rounds 1, 3, 5, 10 and 15 were selected for shake flask fermentation to compare the differences in polysaccharide yield and verify the stability of mutation inheritance. Among them, the 120s and 140s mutants showed no significant differences in polysaccharide yield from the original strain in F3 generation, and no further verification was conducted. After 160s fermentation, the polysaccharide yield of strains 0816 and 1519 was increased by more than 10% compared with that of the original strain, and there was no significant difference between the groups of each generation. When 180s strains were transferred to the F2 generation, the polysaccharide yield of each strain had no significant difference from that of the original strain. The two genetically stable high-yield mutants obtained through mutagenesis, screening and passage verification were named Mu0816 and Mu1519 (Fig. 1). The least significant difference (LSD) test confirmed that there were no significant differences in polysaccharide yield between the samples of Mu0816 and Mu1519, which had increases of 13% and 15.62%, respectively, compared to the original strain.

Differential expression analysis

A visual analysis of differentially expressed genes in samples of each group was performed (Schwartz et al. 2015). As shown in Fig. 2. A total of 88 genes were significantly changed in Mu0816, 50 of which were significantly upregulated, while 38 genes were significantly downregulated. A total of 419 genes of Mu1519 had significant changes, including 289 significantly upregulated and 193 significantly down-regulated genes. In addition, the transcriptome of mutant strain Mu1519 was significantly different from that of strain Mu0816. Differential expression analysis showed that among the 230 significantly upregulated genes, except for one apolipoprotein (c21771_g1) gene, most were upregulated, including tubulin related genes, glutathione S-transferase genes, and NAD(P)-binding protein genes. However, no direct correlation between these genes and pullulan synthesis has been reported, while the role of apolipoprotein in pullulan biosynthesis has been confirmed. Glutathione S-transferases (GSTs) are a group of multifunctional isoenzymes widely distributed in various organisms, which participate in the tricarboxylic acid cycle and sugar metabolism in vivo, and can affect cellular metabolism. As important intracellular metabolic regulators, GSTs have been widely studied in animals and plants, but there are relatively few related studies in fungi (Xu et al. 2022; Yoshimi et al. 2017). As an NADP-dependent enzyme, glyeraldehyde-3-phosphate dehydrogenase was reported to be associated with pullulan anabolism. In addition, 18.7% of upregulated genes were not annotated, including c20004_g1 and c20151_g1 (hypothetical protein GLE_3544 [Lysobacter enzymogenes] and hypothetical protein GLE_4140 [Lysobacter enzymogenes]), the two transcripts with the highest expression in Mu1519, which were not annotated in each pathway, NCBI analysis showed that the sequence similarity of these two genes with lysogens was 99% and 97% respectively, which may be related to the synthesis of melanin.

A Venn diagram was used to more intuitively show the number and distribution of differential genes in each group of samples (Coelho et al. 2002). As shown in Fig. 3, 455 genes were significantly differentially expressed between CK and Mu1519, while only 61 genes were significantly differentially expressed between CK and Mu0816, and 27 genes were significantly differentially expressed between Mu1519 and Mu0816, including ABC transporter transmembrane region (c12767_g1), Translation factor, c15086_g4), Phosphate transporter family (c7769_g2), Clock controlled protein 6 (c3697_g1), Protein TAR1 (c17182_g1), Putative polyketide synthase (c21929_g1), unknown protein (c7547_g1, c11223_g1, c13144_g1, c9492_g2), and unannotated pathway gene (c10141_g2). In addition, the polyketide synthase encoded by PKSIII has been reported to be involved in the synthesis of melanin by A. pullulans, which may explain the phenotypic changes of increased melanin production in the mutant strains. TAR1 protein has been reported to be localized to the mitochondria, and overexpression of this protein can affect mitochondrial gene expression and mitochondrial DNA (mtDNA) stability. The genes that are upregulated together are generally related to the biological process of the strain and may lead to phenotypic changes during growth. In addition, some of the upregulated genes also encoded proteins of unknown function. These changes may be associated with the increase of pullulan production, and provide a new entry point for the exploration of glucose metabolism and related genes of A. pullulans.

GO classification statistics and enrichment analysis of DEGs

The GO (Gene Ontology) database can be used to define and describe the functions of genes and proteins, as well as classify genes according to their participation in biological processes, cellular components and molecular functions. Through the significant enrichment analysis of GO functions, the functional enrichment of differential genes can be explained, after which the differences between samples can be clarified at the level of gene function (Gostinčar et al. 2014). As shown in Fig. 4, A total of 37 differentially expressed genes of CK vs. Mu0816 were annotated into the GO classification, among which 31 differentially expressed genes were upregulated, mainly focusing on metabolic and cellular processes under biological processes, while 6 differentially expressed genes were downregulated, mainly focusing on catalytic activities under molecular functions and metabolic processes. In CK vs. Mu1519, a total of 760 differentially expressed genes were annotated into the GO classification, among which 396 differentially expressed genes were upregulated, focusing on metabolic processes, cellular processes, single-organism process, cells, cell part, catalytic activity and binding. In addition, 364 differentially expressed genes were downregulated, focusing on metabolic processes, cellular processes, single-organism processes, cells, cell parts, organelles, catalytic activity and binding.

The significant enrichment of GO functions among differentially expressed genes in Mu0816 is shown in Fig. 5. There were 12 significantly enriched GO pathways among the differentially expressed genes of Mu0816, with 3 highly significant pathways including primary amine oxidase activity, inorganic phosphor transmembrane transporter activity, and phosphate ion transport. A total of 366 differentially expressed genes in Mu1519 were significantly enriched in GO pathways, among which 148 were extremely significantly enriched, mainly including Translational termination, Protein complex subunit organization, Plasma membrane, Protein complex disassembly, Cellular protein complex disassembly, Carbohydrate biosynthetic process, Macromolecular complex disassembly, Macromolecular complex subunit organization, Cellular component disassembly, Endonuclease activity, Cell part, Cytoskeletal part and Protein polymerization. Among the differentially expressed genes of the two mutant strains, the upregulated PKSIII gene encodes a polyketide synthase, which was reported to be associated with the synthesis of melanin in A. pullulans. This may explain the more intense colony pigmentation of mutant strains compared with the original strain. However, there may also be a relationship between the secretion of melanin and pullulan synthesis, which may indirectly lead to the increase of pullulan yield. Based on these changes and analysis, we concluded that ARTP caused membrane damage (manifested by significant upregulation of the microporin gene) and altered the internal metabolic network of A. pullulans strains.

KEGG enrichment pathway analysis of DEGs

KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis was performed using KOBAS and calculated based on Fisher’s exact test. Multiple tests were performed using the BH (FDR) method to reduce the false positive rate, and the corrected P-value was set to 0.05. The KEGG pathways consistent with the corrected conditions were selected as the KEGG pathways with significant enrichment among the differentially expressed genes, as shown in Fig. 6. The DEGs of Mu0816 were significantly enriched in 5 pathways, including Chlorocyclohexane and chlorobenzene degradation, Chloroalkane and chloroalkene degradation, Tropane, piperidine and pyridine alkaloid biosynthesis, Isoquinoline alkaloid biosynthesis, and Bile secretion. The significantly enriched pathways of strain Mu1519 included Gap junction, Glycolysis/Gluconeogenesis, Biosynthesis of amino acids, Carbon metabolism, Carbon fixation in photosynthetic organisms, Glycerolipid metabolism, and Methane metabolism.

Screening of genes related to pullulan synthesis

According to current studies, enzymes related the polysaccharide production and metabolism in A. pullulans mainly include α-phosphate mutase, glucose-transferase, UDPG-pyrophosphorylase and pullulanase, which are directly related to pullulan synthesis, as well as apolipoprotein and glucose repressor protein, which are involved in the control of pullulan synthesis. Based on the comparison and annotation of the original fungal transcripts, some reported genes related to pullulan synthesis were found (Table.S1). The main components included UDPG pyrophosphorylase base factor and phosphoglucomutase genes, glycosyltransferase genes, apolipoprotein genes, and DNA-binding protein creA gene. At the same time, the enrichment analysis of the GO and KEGG pathways of the differentially expressed genes of the two mutant strains with increased yield was conducted to explore the internal correlation between the significantly enriched pathway-related genes and the polysaccharide production and metabolism of the strain.

Validation of candidate genes by qRT-PCR

As the member of the hydrolase family, α/β-hydrolase (ABHD) has a wide range of functions. α-amylase (AMY1) has been reported to be involved in pullulan synthesis (Liu et al. 2018), but its specific role is not clear. It has been reported that during the synthesis of pullulan, α-1,4-linked glucooligosaccharide chains need to be synthesized first, and then cut into trisaccharide units by a specific hydrolase (Du et al. 2018; Peng et al. 2021). Sugar porter family MFS transporters (SPF-MFS) are a special class of transmembrane transporters that participate in polysaccharide secretion in several reported strains (Ma et al. 2016). The transcriptome data showed that the high expression of these three protein-coding genes may be related to the synthesis and secretion of pullulan, which was verified by the qPCR results. The process of polysaccharide synthesis in fungi is thought to start from the primer of glycogen synthesis. In addition, sterol glycosyltransferase (SGT) has been reported to be one of the key enzymes in pullulan synthesis. When Chen (Chen et al. 2020) silenced the sgt gene, the production of pullulan decreased, indicating the active role of this enzyme in pullulan synthesis. Our transcriptome data showed that in the two mutant strains with high pullulan production, both sgt1 and sgt2 genes had lower expression levels compared with the original strain, and the qPCR data also supported this result. This indicates that other synthetases may be able to provide sufficient precursors for pullulan synthesis. In addition, two hypothetical proteins (HP1 and HP2) also showed extremely significant differential expression in the mutant strains, and the reliability of the transcriptomic data was also verified by the qPCR results (Figure.7). Subsequent knockout and overexpression of these genes should be conducted in future studies to further verify their roles in the synthesis and secretion of pullulan.

In this study, genetically stable and high-yielding pullulan mutants Mu0816 and Mu1519 were obtained by ARTP mutagenesis. Transcriptome analysis of the two mutant strains revealed that genes related to pullulan metabolism reported in the literature were corroborated by the transcriptomic data, including the genes of UDP-glucose pyrophosphorylase and phosphoglucomutase, apolipoprotein, and glucose repressor (DNA-binding protein CreA). There were significant changes in the transcriptomes of the mutant strains. A total of 419 genes of Mu1519 had significant changes, including 230 significantly upregulated and 189 significantly downregulated genes. A total of 366 significantly enriched GO pathways were enriched among the differentially expressed genes in strain Mu1519, which included some pathways directly related to pullulan synthesis, such as UDP-glucosyltransferase activity, glucosyltransferase activity, UDPG metabolic process, polysaccharide biosynthetic process and polysaccharide metabolic process. In addition, there were seven significantly enriched KEGG pathways. The most significant change was found in gap junctions, where the annotation indicated that the microporin related unigenes were significantly upregulated. It was concluded that ARTP caused membrane damage and altered the internal metabolic network. Significantly upregulated genes associated with increasing pullulan yield and phenotypic traits included tubulin related genes, glutathione S-transferase genes, NAD(P)-binding protein genes, and polyketide synthase genes. Their roles in the metabolism and pullulan biosynthesis process of A. pullulans should be verified through knockout and overexpression in future studies.

Author contribution HZ and FX conceived and designed the experimental plan. YH, RB and JC conducted an investigation and further optimized the plan. RB, JC ,YH, YD, TG and KR, conducted experiments. RB, SZ and JC analyzed the experimental results and wrote the original manuscript. All authors conducted scientific discussions and commented on the manuscript. HZ, RB and FX revised the manuscript. RB, YH and JC supervised the research and reviewed and edited the manuscript. All authors read and approved the manuscript.

Funding This work was financially supported by the National Science Foundation of China (Grant No. 31970038), and Shenyang Rural Science and Technology Special Mission (Grant No. 20-207-3-47).

Data availability The datasets generated or analyzed and other findings of this study are available from the corresponding author upon reasonable request.

Ethics approval This article does not contain any studies on human participants or animals performed by any of the authors.

Conflict of interest The authors declare no competing interests.

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Breeding high-yield pullulan engineering strains of Aureobasidium pullulans by ARTP mutagenesis and transcriptome analysis

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Journal Publication

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Abstract

Figures

Introduction

Materials and methods

Strain mutagenesis

Activation and Cultivation of the strain

ARTP mutagenesis

Characteristics of the mutant strains

Transcriptome sequencing

Library sequencing and functional analysis of DEGs

Validation of expression using qPCR

Results and discussion

ARTP mutagenesis

Differential expression analysis

GO classification statistics and enrichment analysis of DEGs

KEGG enrichment pathway analysis of DEGs

Screening of genes related to pullulan synthesis

Validation of candidate genes by qRT-PCR

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1