Abstract
To improve β-1,3–1,6-D-glucan (β-glucan) production by Aureobasidium pullulans, an Agrobacterium tumefaciens–mediated transformation method was developed to screen a mutant A. pullulans CGMCC 19650. Based on thermal asymmetric–interlaced PCR detection, DNA sequencing, BLAST analysis, and quantitative real-time PCR assay, the T-DNA was identified to be inserted in the coding region of mal31 gene, which encodes a sugar transporter involved in pullulan biosynthesis in the mutant. The maximal biomass and β-glucan production under batch fermentation were significantly increased by 47.6% and 78.6%, respectively, while pullulan production was decreased by 41.7% in the mutant, as compared to the parental strain A. pullulans CCTCC M 2012259. Analysis of the physiological mechanism of these changes revealed that mal31 gene disruption increased the transcriptional levels of pgm2, ugp, fks1, and kre6 genes; increased the amounts of key enzymes associated with UDPG and β-glucan biosynthesis; and improved intracellular UDPG contents and energy supply, all of which favored β-glucan production. However, the T-DNA insertion decreased the transcriptional levels of ags2 genes, and reduced the biosynthetic capability to form pullulan, resulting in the decrease in pullulan production. This study not only provides an effective approach for improved β-glucan production by A. pullulans, but also presents an accurate and useful gene for metabolic engineering of the producer for efficient polysaccharide production.
Key points
• A mutant A. pullulans CGMCC 19650 was screened by using the ATMT method.
• The mal31 gene encoding a sugar transporter was disrupted in the mutant.
• β-Glucan produced by the mutant was significantly improved.
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Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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This work was supported by the National Natural Science Foundation of China (21776189), the Natural Science Foundation of Jiangsu Province (BK20181440), and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.
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XZ and GYW conceived and designed research. XC, YW, and CYH conducted experiments. GCZ and CLW analyzed the data. XC and DHW wrote the manuscript. XZ and GYW reviewed and edited the manuscript. DHW and GYW administrated project and acquired funding. All authors read and approved the final version of manuscript.
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Chen, X., Wang, Y., He, CY. et al. Improved production of β-glucan by a T-DNA–based mutant of Aureobasidium pullulans. Appl Microbiol Biotechnol 105, 6887–6898 (2021). https://doi.org/10.1007/s00253-021-11538-x
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DOI: https://doi.org/10.1007/s00253-021-11538-x