School of Biology and Basic Medical Sciences, Soochow University, 199# Ren'ai Road, Suzhou, 215123, People's Republic of China.
College of Pharmaceutical Sciences, Southwest University, 2# TianSheng Road, Beibei, Chongqing, 400715, People's Republic of China.
Appl Microbiol Biotechnol. 2021 Sep;105(18):6887-6898. doi: 10.1007/s00253-021-11538-x. Epub 2021 Aug 27.
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.
为提高出芽短梗霉（Aureobasidium pullulans）β-1,3-1,6-D-葡聚糖（β-glucan）的产量，采用根癌农杆菌介导的转化方法，对出芽短梗霉 CGMCC 19650 进行了筛选。基于热不对称交错 PCR 检测、DNA 测序、BLAST 分析和定量实时 PCR 分析，鉴定 T-DNA 插入突变体 mal31 基因的编码区，该基因编码参与合成普鲁兰的糖转运蛋白。与出发菌株 A. pullulans CCTCC M 2012259 相比，突变体在分批发酵中的最大生物量和β-glucan 产量分别显著提高了 47.6%和 78.6%，而普鲁兰产量下降了 41.7%。这些变化的生理机制分析表明，mal31 基因缺失增加了 pgm2、ugp、fks1 和 kre6 基因的转录水平；增加了与 UDPG 和β-glucan 生物合成相关的关键酶的含量；并提高了细胞内 UDPG 含量和能量供应，所有这些都有利于β-glucan 的生产。然而，T-DNA 插入降低了 ags2 基因的转录水平，降低了形成普鲁兰的生物合成能力，导致普鲁兰产量下降。本研究不仅为出芽短梗霉提高β-glucan 产量提供了一种有效方法，而且为该生产菌的代谢工程提供了一个准确而有用的基因，以实现高效多糖生产。关键点：• 采用 ATMT 法筛选出芽短梗霉突变体 CGMCC 19650。• 突变体中编码糖转运蛋白的 mal31 基因被破坏。• 突变体产生的β-glucan 显著提高。
