Combination of genetic engineering and random mutagenesis for improving production of raw-starch-degrading enzymes in Penicillium oxalicum

Microbial Cell Factories

Research

Jia-Xun Feng¹  Shuai Zhao¹  Fa-Ting Ye¹  Xue-Mei Luo¹  Yuan-Peng Chen¹  Rui-Xian Wang¹  Ming-Zhu Tan¹ 

[1] State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi Research Centre for Microbial and Enzyme Engineering Technology, College of Life Science and Technology, Guangxi University, Nanning, China;

关键词: Raw starch-degrading enzymes;    Genetic engineering;    Random mutagenesis;    Penicillium oxalicum;    Biorefining;   

DOI  :  10.1186/s12934-022-01997-w

 received in 2022-09-01, accepted in 2022-12-17,  发布年份 2022

来源: Springer

【 摘 要 】

BackgroundRaw starch-degrading enzyme (RSDE) is applied in biorefining of starch to produce biofuels efficiently and economically. At present, RSDE is obtained via secretion by filamentous fungi such as Penicillium oxalicum. However, high production cost is a barrier to large-scale industrial application. Genetic engineering is a potentially efficient approach for improving production of RSDE. In this study, we combined genetic engineering and random mutagenesis of P. oxalicum to enhance RSDE production.ResultsA total of 3619 mutated P. oxalicum colonies were isolated after six rounds of ethyl methanesulfonate and Co60-γ-ray mutagenesis with the strain A2-13 as the parent strain. Mutant TE4-10 achieved the highest RSDE production of 218.6 ± 3.8 U/mL with raw cassava flour as substrate, a 23.2% compared with A2-13. Simultaneous deletion of transcription repressor gene PoxCxrC and overexpression of activator gene PoxAmyR in TE4-10 resulted in engineered strain GXUR001 with an RSDE yield of 252.6 U/mL, an increase of 15.6% relative to TE4-10. Comparative transcriptomics and real-time quantitative reverse transcription PCR revealed that transcriptional levels of major amylase genes, including raw starch-degrading glucoamylase gene PoxGA15A, were markedly increased in GXUR001. The hydrolysis efficiency of raw flour from cassava and corn by crude RSDE of GXUR001 reached 93.0% and 100%, respectively, after 120 h and 84 h with loading of 150 g/L of corresponding substrate.ConclusionsCombining genetic engineering and random mutagenesis efficiently enhanced production of RSDE by P. oxalicum. The RSDE-hyperproducing mutant GXUR001 was generated, and its crude RSDE could efficiently degrade raw starch. This strain has great potential for enzyme preparation and further genetic engineering.

【 授权许可】

CC BY   
© The Author(s) 2022

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