Cloning of three Alnus sieboldiana type III polyketide synthases and formation of polyketides in recombinant Escherichia coli using cinnamic acid analogs as substrates

Konosuke Takemoto; Yuichi Mikota; Ryota Moriuchi; Yuko Yoneda; Shingo Kawai

doi:10.1016/j.heliyon.2024.e27698

Cloning of three Alnus sieboldiana type III polyketide synthases and formation of polyketides in recombinant Escherichia coli using cinnamic acid analogs as substrates

Heliyon. 2024 Mar 9;10(6):e27698. doi: 10.1016/j.heliyon.2024.e27698. eCollection 2024 Mar 30.

Authors

Konosuke Takemoto^{1

2}, Yuichi Mikota¹, Ryota Moriuchi³, Yuko Yoneda¹, Shingo Kawai¹

Affiliations

¹ Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan.
² The United Graduate School of Agricultural Science, Gifu University, 1-1, Yanagido, Gifu-shi, 501-1193, Japan.
³ Functional Genomics Section, Shizuoka Instrumental Analysis Center, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan.

Abstract

Alnus sieboldiana is an actinorhizal plant that coexists with the nitrogen-fixing actinomycete Frankia via nodules. It produces a variety of polyketides, including flavonoids, stilbenoids, and diarylheptanoids. These compounds have beneficial biological activities. Plant polyketides are produced by type III polyketide synthases (PKSIII). In this study, three A. sieboldiana PKSIIIs (AsPKSIII1, AsPKSIII2, and AsPKSIII3) predicted from next-generation sequencing analysis of A. sieboldiana seedling RNA were amplified and cloned. Phylogenetic tree analysis classified AsPKSIII2 and AsPKSIII3 into the chalcone synthase (CHS) group, whereas AsPKSIII1 was not classified into this group. We attempted to produce polyketides by adding cinnamic acid analogs to the culture medium of Escherichia coli, in which the respective PKSIII gene and the acetyl-CoA carboxylase (ACC) and 4-coumarate: CoA ligase (4CL) genes were simultaneously recombined. AsPKSIII1 is an enzyme that condensed only one molecule of malonyl-CoA to cinnamoyl-CoAs. In contrast, AsPKSIII2 and AsPKSIII3 produced chalcones as shown in a phylogenetic tree analysis, but also produced triketide pyrone. The ratio of these products differed between the two enzymes. We determined the gene and amino acid sequences as well as the substrate specificities of the two enzymes involved in flavonoid production and one enzyme potentially involved in diarylheptanoid production in A. sieboldiana.

Keywords: Actinorhizal symbiosis; Betulaceae; Diarylheptanoids; Diketide-CoA synthase; Flavonoids; Type III polyketide synthase.