(+)-Borneol is a desirable monoterpenoid with effective anti-inflammatory and analgesic effects that is known as soft gold. (+)-bornyl diphosphate synthase is the key enzyme in the (+)-borneol biosynthesis pathway. Despite several reported (+)-bornyl diphosphate synthase genes, relatively low (+)-borneol production hinders the attempts to synthesize it using microbial fermentation. Here, we identified the highly specific (+)-bornyl diphosphate synthase CbTPS1 from Cinnamomum burmanni. An in vitro assay showed that (+)-borneol was the main product of CbTPS1 (88.70% of the total products), and the Km value was 5.11 ± 1.70 μM with a kcat value of 0.01 s–1. Further, we reconstituted the (+)-borneol biosynthetic pathway in Saccharomyces cerevisiae. After tailored truncation and adding Kozak sequences, the (+)-borneol yield was improved by 96.33-fold to 2.89 mg⋅L–1 compared with the initial strain in shake flasks. This work is the first reported attempt to produce (+)-borneol by microbial fermentation. It lays a foundation for further pathway reconstruction and metabolic engineering production of this valuable natural monoterpenoid.
Aconitum carmichaelii
is a high-value medicinal herb widely used across China, Japan, and other Asian countries. Aconitine-type diterpene alkaloids (DAs) are the characteristic compounds in
Aconitum
. Although six transcriptomes, based on short-read next generation sequencing technology, have been reported from the
Aconitum
species, the terpene synthase (TPS) corresponding to DAs biosynthesis remains unidentified. We apply a combination of Pacbio isoform sequencing and RNA sequencing to provide a comprehensive view of the
A. carmichaelii
transcriptome. Nineteen TPSs and five alternative splicing isoforms belonging to TPS-b, TPS-c, and TPS-e/f subfamilies were identified
. In vitro
enzyme reaction analysis functional identified two sesqui-TPSs and twelve diTPSs. Seven of the TPS-c subfamily genes reacted with GGPP to produce the intermediate
ent
-copalyl diphosphate. Five AcKSLs separately reacted with
ent
-CPP to produce
ent
-kaurene,
ent
-atiserene, and
ent
-13-
epi
-sandaracopimaradie: a new diterpene found in
Aconitum
. AcTPSs gene expression in conjunction DAs content analysis in different tissues validated that
ent
-CPP is the sole precursor to all DAs biosynthesis, with AcKSL1, AcKSL2s and AcKSL3-1 responsible for C
20
atisine and napelline type DAs biosynthesis, respectively. These data clarified the molecular basis for the C
20
-DAs biosynthetic pathway in
A. carmichaelii
and pave the way for further exploration of C
19
-DAs biosynthesis in the
Aconitum
species.
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