Engineered biosynthesis of indole alkaloids in Myxococcus xanthus Lea Winand 1 , Pascal Schneider 2 , Sebastian Kruth 1 , Nico-Joel Greven 1 , Wolf Hiller 3 , Marcel Kaiser 4 , Jörg Pietruszka 2 , Markus Nett 1 1 Laboratory of Technical Biology, TU Dortmund University, Germany, 2 Institute of Bioorganic Chemistry,Germany, 3 Department of Chemistry and Chemical Biology, TU Dortmund University, Germany, 4 Swiss Tropical and Public Health Institute, Basel, Switzerland The cholinesterase inhibitor physostigmine is an important lead structure for the development of new drugs against Alzheimer’s dementia. 1,2 To produce physostigmine analogues with improved bioactivity, selectivity and toxicity, we combined chemical synthesis with pathway refactoring. 3 In a first step, the late stages of the physostigmine pathway were reconstituted in the myxobacterial model organism Myxococcus xanthus . For this purpose, we made use of a myxobacterial expression plasmid, which had been endowed with the necessary biosynthesis genes. After reduction of the metabolic burden , implementation of a gene fusion strategy and selection of an appropriate host strain, a physostigmine titer of 72mg/L was obtained. Subsequently, the production strain was fed with a series of synthetically prepared precursors, which were successfully introduced into the heterologously expressed physostigmine pathway. The mutasynthetically produced physostigmine derivatives were tested for their inhibitory efficiency on acetyl- and butyrylcholinesterase. One analogue exhibits improved bioactivity and reduced toxicity compared to the parental natural product. 3 This derivative preferentially inhibits butyrylcholinesterase, which is a promising target in the treatment of Alzheimer’s dementia. 4 In addition, we present the first biocatalytic concept for the synthesis of the drug candidate phenserine. 3 New results further demonstrate the high substrate flexibility of the physostigmine biosynthetic enzymes, enabling the production of furoindole derived alkaloids. References 1. Andrade, O. A.; Zafar Gondal, A., Physostigmine, In : StatPearls, 2020 . Treasure Island (FL): StatPearls Publishing. www. ncbi.nlm.nih.gov/books/NBK545261 2. Greig, N. H.; Pei, X. F.; Soncrant, T. T.; Ingram, D. K.; Brossi, A., Med. Res. Rev. 1995 , 15 , 3-31. 3. Winand, L.; Schneider, P.; Kruth, S.; Greven, N.-J.; Hiller, W.; Kaiser, M.; Pietruszka, J.; Nett, M. Org. Lett. 2021 , 23 , 6563–6567 4. Greig, N. H.; Lahiri, D. K.; Sambamurti, K. Int. Psychogeriatr. 2002 , 14 , 77–91.
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