ARTP/EMS-combined multiple mutagenesis efficiently improved production of raw starch-degrading enzymes in Penicillium ox
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Biotechnology for Biofuels Open Access
RESEARCH
ARTP/EMS‑combined multiple mutagenesis efficiently improved production of raw starch‑degrading enzymes in Penicillium oxalicum and characterization of the enzyme‑hyperproducing mutant Li‑Sha Gu, Ming‑Zhu Tan, Shi‑Huan Li, Ting Zhang, Qi‑Qiang Zhang, Cheng‑Xi Li, Xue‑Mei Luo, Jia‑Xun Feng and Shuai Zhao*
Abstract Background: Application 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 process‑ ing. Breeding assisted by random mutagenesis is an efficient way to improve fungal enzyme production. Results: A 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. Conclusions: Overall, ARTP/EMS-combined mutagenesis effectively improved fungal RSDE yield. An RSDE-hyperpro‑ ducing mutant, A2-13, was obtained, and its RSDEs could efficiently hydrolyze raw starch, in combination with com‑ mercial α-amylase at low temperature, which provides a useful RSDE resource for future starch processing. Keywords: Raw starch-degrading enzymes, ARTP/EMS-combined mutagenesis, Penicillium oxalicum
*Correspondence: [email protected] 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, Nanning 530004, Guangxi, People’s Republic of China
Background Plant biomass biorefineries use renewable and relatively inexpensive raw materials as feedstocks for processing into value-added biofuels and chemicals, with potential benefits for industry and the environment. Biorefinery processes can help alleviate problems associated with industrial chemicals and fossil fuels, such as increasing
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