ARTP/EMS-combined multiple mutagenesis efficiently improved production of raw starch-degrading enzymes in Penicillium oxalicum and characterization of the enzyme-hyperproducing mutant

Biotechnology for Biofuels

Xue-Mei Luo¹  Qi-Qiang Zhang¹  Shuai Zhao¹  Jia-Xun Feng¹  Shi-Huan Li¹  Cheng-Xi Li¹  Ming-Zhu Tan¹  Ting Zhang¹  Li-Sha Gu¹ 

[1] State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi Research Center for Microbial and Enzyme Engineering Technology, College of Life Science and Technology, Guangxi University, 100 Daxue Road, 530004, Nanning, Guangxi, People’s Republic of China;

关键词: Raw starch-degrading enzymes;    ARTP/EMS-combined mutagenesis;    Penicillium oxalicum;   

DOI  :  10.1186/s13068-020-01826-5

来源: Springer

【 摘 要 】

BackgroundApplication of raw starch-degrading enzymes (RSDEs) in starch processing for biofuel production can effectively reduce energy consumption and processing costs. RSDEs are generally produced by filamentous fungi, such as Penicillium oxalicum, but with very low yields, which seriously hampers industrialization of raw starch processing. Breeding assisted by random mutagenesis is an efficient way to improve fungal enzyme production.ResultsA total of 3532 P. oxalicum colonies were generated after multiple rounds of mutagenesis, by atmospheric and room-temperature plasma (ARTP) and/or ethyl methanesulfonate (EMS). Of these, one mutant A2-13 had the highest RSDE activity of 162.7 U/mL, using raw cassava flour as substrate, a yield increase of 61.1%, compared with that of the starting strain, OXPoxGA15A. RSDE activity of A2-13 further increased to 191.0 U/mL, through optimization of culture conditions. Increased expression of major amylase genes, including the raw starch-degrading glucoamylase gene, PoxGA15A, and its regulatory gene, PoxAmyR, as well as several single-nucleotide polymorphisms in the A2-13 genome, were detected by real-time reverse transcription quantitative PCR and genomic re-sequencing, respectively. In addition, crude RSDEs produced by A2-13, combined with commercial α-amylase, could efficiently digest raw corn flour and cassava flour at 40 °C.ConclusionsOverall, ARTP/EMS-combined mutagenesis effectively improved fungal RSDE yield. An RSDE-hyperproducing mutant, A2-13, was obtained, and its RSDEs could efficiently hydrolyze raw starch, in combination with commercial α-amylase at low temperature, which provides a useful RSDE resource for future starch processing.

【 授权许可】

CC BY