APPLIED MICROBIAL AND CELL PHYSIOLOGY Production and enhancement of the acetylcholinesterase inhibitor, huperzine A, from an endophytic Alternaria brassicae AGF041 Amira G. Zaki 1 & Einas H. El-Shatoury 2 & Ashraf S. Ahmed 1 & Ola E. A. Al-Hagar 1 Received: 27 March 2019 /Accepted: 13 May 2019 # Springer-Verlag GmbH Germany, part of Springer Nature 2019 Abstract Huperzine A (HupA) is a potent acetylcholinesterase (AChE) inhibitor of a great consideration as a prospective drug candidate for Alzheimer’ s disease treatment. Production of HupA by endophytes offers an alternative challenge to reduce the massive plant harvest needed to meet the increasing demand of HupA. In the current study, some endophytic fungal and actinobacterial isolates from the Chinese herb, Huperzia serrata, underwent liquid fermentation, alkaloid extraction, and screening for AChE inhibition and HupA production. Among these isolates, Alternaria brassicae AGF041 strain was the only positive strain for HupA production with the maximum AChE inhibition of 75.5%. Chromatographic analyses verified the identity of the produced HupA. The HupA production was efficiently maximized up to 42.89 μg/g of dry mycelia, after optimization of thirteen process parameters using multifactorial statistical approaches, Plackett–Burman and central composite designs. The statistical optimiza- tion resulted in a 40.8% increase in HupA production. This is the first report to isolate endophytic actinobacteria with anti-AChE activity from H. serrata, and to identify an endophytic fungus A. brassicae as a new promising start strain for a higher HupA yield. Keywords Huperzine A . Acetylcholinesterase inhibitor . Endophytes . Alternaria brassicae . Statistical optimization Introduction Alzheimer’ s disease (AD) is an elderly-targeting neurodegen- erative defect. It is characterized by a gradual memory loss and a cognitive function collapse (Zhu et al. 2004; Khairallah and Kassem 2011). There are about 36 million AD patients worldwide, and the number is expected to double in 2030 (Citron 2002). AChE inhibitors are considered as efficient therapeutic agents applied up to date for rebalancing the cholinergic neuro- transmission, and enhancing the memory and the cognitive functions of AD patients (Craig et al. 2011; Dey et al. 2017). Majority of the FDA-approved drugs for AD symptomatic ther- apy are inhibitors of AChE including tacrine, rivastigmine, donepezil, and galantamine (Colovic et al. 2013). The naturally occurring Lycopodium alkaloid, huperzine A (HupA), has received intensive attention due to its powerful AChE inhibitory activity besides its ability to improve memory of AD patients, with no toxicity and low side effects (Tang and Han 1999; Zangara 2003). HupA was firstly isolated in 1986 from the Chinese Club moss, Huperzia serrata (family: Huperziaceae) (Ishiuchi et al. 2013). In China, HupA is currently marketed as a drug for AD therapy. Additionally, it is available as a food supplement in the USA for minimizing the memory de- terioration (Zangara 2003). Also, ZT-1, the semi-synthesized de- rivative of HupA, is being improved in Europe and China, as a new therapeutic candidate for AD (Ma et al. 2007). However, H. serrata represents the main current source for obtaining HupA; the plant vegetative cycle is too long and the HupA content is extremely low (Ma and Gang 2008). Although other species of family Huperziaceae showed a higher HupA content, they rarely exist in nature (Zhao et al. 2013). Previous trials for HupA production by tissue culturing (Ma and Gang 2008; Ishiuchi et al. 2013) or chemical synthe- sis processes (Koshiba et al. 2009; Ding et al. 2012) showed some disadvantages and limitations. Endophytes are microorganisms (fungi, bacteria, or actinobacteria) colonizing the internal plant tissues, * Amira G. Zaki Amira_hegab39@yahoo.com 1 Plant Research Department, Nuclear Research Center, Atomic Energy Authority, Cairo, Egypt 2 Department of Microbiology, Faculty of Science, Ain Shams University, Cairo, Egypt Applied Microbiology and Biotechnology https://doi.org/10.1007/s00253-019-09897-7