The disruption of the MAPKK gene triggering the synthesis of flavonoids in endophytic fungus Phomopsis liquidambaris
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ORIGINAL RESEARCH PAPER
The disruption of the MAPKK gene triggering the synthesis of flavonoids in endophytic fungus Phomopsis liquidambaris Qian Yang . Mei Wu . Ya-Li Zhu . Ya-Qiong Yang . Yan-Zhen Mei Chuan-Chao Dai
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Received: 1 September 2020 / Accepted: 27 October 2020 Ó Springer Nature B.V. 2020
Abstract Flavonoids, which are mainly extracted from plants, are important antioxidants and play an important role in human diseases. However, the growing market demand is limited by low productivity and complex production processes. Herein, the flavonoids biosynthesis pathway of the endophytic fungus Phomopsis liquidambaris was revealed. The mitogen-activated protein kinase kinase (MAPKK) of the strain was disrupted using a newly constructed CRISPR-Cas9 system mediated by two gRNAs which was conducive to cause plasmid loss. The disruption of the MAPKK gene triggered the biosynthesis of flavonoids against stress and resulted in the precipitation of flavonoids from fermentation broth. Naringenin, kaempferol and quercetin were detected in fedbatch fermentation with yields of 5.65 mg/L, 1.96 mg/ L and 2.37 mg/L from P. liquidambaris for dry cell weigh using the mixture of glucose and xylose and corn steep powder as carbon source and nitrogen source for 72 h, respectively. The biosynthesis of flavonoids was triggered by disruption of MAPKK Q. Yang M. Wu Y.-L. Zhu Y.-Q. Yang Y.-Z. Mei (&) C.-C. Dai (&) Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering and Technology Research Center for Industrialization of Microbial Resources, College of Life Sciences, Nanjing Normal University, Nanjing 210023, Jiangsu, China e-mail: [email protected] C.-C. Dai e-mail: [email protected]
gene in P. liquidambaris and the mutant could utilize xylose. Keywords Phomopsis liquidambaris CRISPRCas9 Mitogen-activated protein kinase kinase Flavonoids Xylose
Introduction Flavonoids, such as naringenin (40 ,5,7-trihydroxydihydroflavone) and its derivatives, are ubiquitous in higher plants such as tomatoes and citrus fruits (Tomas et al. 2017). They play an important role in plant growth, development, flowering, and fruiting and exhibit antibacterial and disease prevention properties (Winkel-Shirley 2002). Some studies have shown that different flavonoids have various activities, such as anti-inflammatory, antioxidant, atherosclerosis prevention, and anticancer effects (Iwashina 2003; Khan et al., 2019). At present, the strategies for the production of flavonoids mainly include four approaches: (1) Chemical synthesis was involved in toxic chemicals and extreme reaction conditions and usually limited by low chiral purity, inability and high cost, although the yield was relatively high (Xia et al. 2007). (2) Extraction for most flavonoids from plants was hindered by the long growth cycle and the growing region of plants, production capacity from raw materials, complex metabolic products and high
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cost (Wang et al. 2011)
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