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Identification of (-)-bornyl diphosphate synthase from Blumea balsamifera and its application for (-)-borneol biosynthesis in Saccharomyces cerevisiae.

ABSTRACT: Borneol is a precious monoterpenoid with two chiral structures, (-)-borneol and (+)-borneol. Bornyl diphosphate synthase is the key enzyme in the borneol biosynthesis pathway. Many (+)-bornyl diphosphate synthases have been reported, but no (-)-bornyl diphosphate synthases have been identified. Blumea balsamifera leaves are rich in borneol, almost all of which is (-)-borneol. In this study, we identified a high-efficiency (-)-bornyl diphosphate synthase (BbTPS3) from B. balsamifera that converts geranyl diphosphate (GPP) to (-)-bornyl diphosphate, which is then converted to (-)-borneol after dephosphorylation in vitro. BbTPS3 exhibited a K _m value of 4.93 ± 1.38 μM for GPP, and the corresponding k _cat value was 1.49 s^-1. Multiple strategies were applied to obtain a high-yielding (-)-borneol producing yeast strain. A codon-optimized BbTPS3 protein was introduced into the GPP high-yield strain MD, and the resulting MD-B1 strain produced 1.24 mg·L^-1 (-)-borneol. After truncating the N-terminus of BbTPS3 and adding a Kozak sequence, the (-)-borneol yield was further improved by 4-fold to 4.87 mg·L^-1. Moreover, the (-)-borneol yield was improved by expressing the fusion protein module of ERG20^F96W-N127W-YRSQI-t14-BbTPS3K2, resulting in a final yield of 12.68 mg·L^-1 in shake flasks and 148.59 mg·L^-1 in a 5-L bioreactor. This work is the first reported attempt to produce (-)-borneol by microbial fermentation.

SUBMITTER: Ma R

PROVIDER: S-EPMC8671873 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:IntroductionBlumea balsamifera L. (Ainaxiang) DC. is a perennial herb of the compositae family. It is also the primary source of natural borneol. Endo-borneol, the principal medical active element in B. balsamifera, is anti-inflammatory, antioxidant, and analgesic; enhances medicine absorption; refreshes; and is used as a spice and in cosmetic. Industrialization of B. balsamifera is limited by its low L-borneol concentration. Thus, understanding the accumulation pattern of the secondary metabolite endo-borneol and its synthesis process in secondary metabolism is critical for increasing B. balsamifera active ingredient content and cultivation efficiency.MethodsIn this work, B. balsamifera was treated with varying concentrations (1.00 and 10.00 mmol/L) of methyl jasmonate (MeJA) as an exogenous foliar activator. The physiological parameters and L-borneol concentration were then assessed. Transcriptome sequencing of B. balsamifera-induced leaves was used to identify key genes for monoterpene synthesis.ResultsThe treatment effect of 1 mmol/L MeJA was the best, and the leaves of all three leaf positions accumulated the highest L-borneol after 120 h, correspondingly 3.043 mg·g-1 FW, 3.346 mg·g-1 FW, and 2.044 mg·g-1 FW, with significant differences from the control. The main assembly produced 509,285 transcripts with min and max lengths of 201 and 23,172, respectively. DEG analysis employing volcano blots revealed 593, 224, 612, 2,405, 1,353, and 921 upregulated genes and 4, 123, 573, 1,745, 766, and 763 downregulated genes in the treatments D1_1vsCK, D1_10vsCK, D2_1vsCK, D2_10vsCK, D5_1vsCK, and D5_10vsCK. Interestingly, when exposed to MeJA treatments, the MEP pathway's unigenes express themselves more than those of the MVA route. Finally, when treated with 1 mmol/L, the genes DXR, DXS, and GPS showed increased expression over time. At the same time, a 10 mmol/L therapy resulted in elevated levels of ispH and GGPS.DiscussionOur preliminary research indicates that exogenous phytohormones can raise the level of L borneol in B. balsamifera (L.) DC when given in the appropriate amounts. The most significant discovery made while analyzing the effects of different hormones and concentrations on B. balsamifera (L.) DC was the effect of 1 mmol/L MeJA treatment.

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Identification of (-)-bornyl diphosphate synthase from Blumea balsamifera and its application for (-)-borneol biosynthesis in Saccharomyces cerevisiae.

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