Abstract
Huperzine A (HupA) isolated from Huperzia serrata is an important compound used to treat Alzheimer’s disease (AD). Recently, HupA was reported in various endophytic fungi, with Colletotrichum gloeosporioides ES026 previously isolated from H. serrata shown to produce HupA. In this study, we performed next-generation sequencing and de novo RNA sequencing of C. gloeosporioides ES026 to elucidate the molecular functions, biological processes, and biochemical pathways of these unique sequences. Gene ontology and Kyoto Encyclopedia of Genes and Genomes assignments allowed annotation of lysine decarboxylase (LDC) and copper amine oxidase (CAO) for their conversion of L-lysine to 5-aminopentanal during HupA biosynthesis. Additionally, we constructed a stable, high-yielding HupA-expression system resulting from the overexpression of CgLDC and CgCAO from the HupA-producing endophytic fungus C. gloeosporioides ES026 in Escherichia coli. Quantitative reverse transcription polymerase chain reaction analysis confirmed CgLDC and CgCAO expression, and quantitative determination of HupA levels was assessed by liquid chromatography high-resolution mass spectrometry, which revealed that elevated expression of CgLDC and CgCAO produced higher yields of HupA than those derived from C. gloeosporioides ES026. These results revealed CgLDC and CgCAO involvement in HupA biosynthesis and their key role in regulating HupA content in C. gloeosporioides ES026.
Highlights
Huperzine A (HupA) is a pyridine-type alkaloid derived from Huperzia serrata[1, 2] and constitutes a highly active acetylcholinesterase inhibitor, making it a valuable therapeutic option for the treatment of Alzheimer’s disease (AD)[3, 4]
lysine decarboxylase (LDC) and copper amine oxidase (CAO) were annotated and confirmed as the first enzymes known to participate in HupA biosynthesis in H. serrata[17], and de novo RNA sequencing of C. gloeosporioides ES026 performed by Zhang et al.[21] confirmed their roles in the HupA biosynthetic pathway
As mentioned in the introduction, very few biosynthetic studies have been performed with HupA, no investigations have been reported that have attempted to identify the biosynthetic pathway leading directly to HupA, two enzymes (LDC and CAO) have been proposed as the entry point enzymes into the pathway to the HupA22, 23
Summary
Huperzine A (HupA) is a pyridine-type alkaloid derived from Huperzia serrata[1, 2] and constitutes a highly active acetylcholinesterase inhibitor, making it a valuable therapeutic option for the treatment of Alzheimer’s disease (AD)[3, 4]. Some endophytic fungi associated with H. serrata are capable of producing HupA12–14 with Colletotrichum gloeosporioides ES026 yielding 45 μg/g dried mycelium according to our previous study[15]. HupA production by these endophytes is hindered by low yields and the loss of biosynthetic capability after several generations. Methods involving overexpression of the enzymes associated with HupA biosynthesis need to be developed in a heterologous host if stable and efficient production is to be achieved[15,16,17]. LDC and CAO were annotated and confirmed as the first enzymes known to participate in HupA biosynthesis in H. serrata[17], and de novo RNA sequencing of C. gloeosporioides ES026 performed by Zhang et al.[21] confirmed their roles in the HupA biosynthetic pathway. Our results provide valuable insights into genetic modification of strains for selective overexpression of biosynthetic enzymes
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