RSC Advances View Article Online PAPER View Journal | View Issue Stepwise engineering of Saccharomyces cerevisiae to produce (+)-valencene and its related Cite this: RSC Adv.,2019,9,30171 sesquiterpenes† Xiaodan Ouyang, a Yaping Cha, a Wen Li, a Chaoyi Zhu, a Muzi Zhu, b Shuang Li, *a Min Zhuo, a Shaobin Huang c and Jianjun Li b (+)-Valencene and (+)-nootkatone are high value-added sesquiterpenoids found in grapefruit. The synthesis of (+)-nootkatone by chemical oxidation from (+)-valencene cannot meet the increasing demand in natural aromatics markets. Development of a viable bioprocess using microorganisms is attractive. According to the yields of b-nootkatol and (+)-nootkatone by strains harboring different expression cassettes in the resting cell assay, premnaspirodiene oxygenase from Hyoscyamus muticus (HPO), cytochrome P450 reductase from Arabidopsis thaliana (AtCPR) and alcohol dehydrogenase (ADH1) from Saccharomyces cerevisiae were finally selected and overexpressed in CEN$PK2-1Ca, À1 Creative Commons Attribution-NonCommercial 3.0 Unported Licence. yielding b-nootkatol and (+)-nootkatone with 170.5 and 45.6 mg L ethyl acetate, respectively. A combinational engineering strategy including promoter change, regulator ROX1 knockout, squalene pathway inhibition, and tHMGR overexpression was performed to achieve de novo (+)-valencene production. Subsequent culture investigations found that galactose as the induced carbon source and a lower temperature (25 C) were beneficial to target accumulation. Also, replacing the inducible promoters (GAL1) of HPO and AtCPR with constitutive promoters (HXT7 and CYC1) dramatically increased the b-nootkatol accumulation from 108.2 to 327.8 mg LÀ1 ethyl acetate in resting-cell Received 18th July 2019 experiments using (+)-valencene as a substrate. Finally, the total terpenoid titer of the engineered strain Accepted 9th September 2019 of PK2-25 using glucose as a carbon source was improved to 157.8 mg LÀ1 cell culture, which was 56 This article is licensed under a DOI: 10.1039/c9ra05558d times the initial value. We present a new candidate for production of (+)-valencene and its related rsc.li/rsc-advances sesquiterpenoids with attraction for industry. Open Access Article. Published on 24 September 2019. Downloaded 9/26/2021 6:58:52 AM. home products. It is on the authorized avor list with a 0.5 mg 1. Introduction À kg 1 concentration level for food in the European Regulations Sesquiterpenes are a class of abundant natural products derived by the European Food Safety Authority.3 (+)-Nootkatone is re- from the 15-carbon linear precursor, farnesyl pyrophosphate ported to have several pharmacological properties, such as (FPP). (+)-Valencene is an important sesquiterpene that is an antioxidant, antiseptic and antiallergic activities, and is also an aroma ingredient in orange peel oil. Its odor characteristics effective repellent against such various insects as mosquitoes, include fresh citrus notes with sweet, orange and woody bed bugs, termites and ticks.4 Recently, (+)-nootkatone was balsamic proles. (+)-Nootkatone is an oxidized product of considered as protective agent against air pollution-induced (+)-valencene. It is the main contributor to the aroma compo- respiratory effects.5 nent of grapefruit and citrus-derived odorants with relatively Employing conventional extraction processes from low perception threshold.1,2 Applications of these compounds natural sources, it takes 2.5 million kilograms of oranges to include food and beverages, fragrances, personal care and obtain a single kilogram of (+)-valencene. The trace amount of (+)-valencene and (+)-nootkatone in natural plants leads to high price of these fragrance compounds.6 The high cost, aSchool of Biology and Biological Engineering, South China University of Technology, Higher Education Mega Center, Guangzhou 510006, China. E-mail: shuangli@scut. limited supply and inconsistent product quality have edu.cn; Fax: +86 20 3938 0601; Tel: +86 20 3938 0601 restricted their applications. Several chemical synthetic bState Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial strategies have been developed via the Robison annulation, Key Laboratory of Microbial Culture Collection and Application, Guangdong Institute intramolecular Sakurai reaction, and Diels–Alder/Aldol of Microbiology, Guangzhou 510070, China tandem reaction, etc.7 However, the mentioned above cSchool of Environment and Energy, South China University of Technology, Higher generally provide racemic (Æ)-nootkatone. One-pot synthesis Education Mega Center, Guangzhou 510006, China † Electronic supplementary information (ESI) available. See DOI: of (+)-nootkatone through singlet oxygenation of (+)-valen- 10.1039/c9ra05558d cene has been realized with carcinogenic tert-butyl This journal is © The Royal Society of Chemistry 2019 RSC Adv.,2019,9,30171–30181 | 30171 View Article Online RSC Advances Paper hydroperoxide in combination with Co(OAc)2,Cu(OAc)2 or Ltd. (Osaka, Japan). Restriction enzymes and T4 DNA ligase 7 V(OAc)2 catalysts. The explosive, corrosive, and toxic heavy were bought from Fermentas of Thermo Fisher Scienti c metal chemocatalyst requirements of the chemical (Pittsburgh, PA, USA). DNA preparations kits for plasmid approaches call for greener and more effective methods for extraction and DNA purication were purchased from Tiangen (+)-valencene and (+)-nootkatone preparation. Previously, (Beijing, China). (+)-Valencene, b-nootkatol, (+)-nootkatone and (+)-valencene extracted from Valencia orange was converted isolongifolene were provided by Tokyo Chemical Industry Co. into (+)-nootkatone by biotransformation using Aspergillus, Ltd. (TCI, Tokyo, Japan). For all other procedures, the highest Rhodococcus, Chlorella, Mucor species Botryodiplodia theo- purity grade chemicals were used. bromae 1368, Yarrowia lipolytica 2.2ab, and Phanerochaete chrysosporium.1,8 These reported biotransformation 2.2 Strains, media and genetic materials approaches suffered frequently from long reaction time, E. coli DH5a was used for gene cloning and plasmid mainte- relatively low yields and troublesome purication nance. All recombinant E. coli cells were cultured in Luria- procedures.9 Bertani (LB) medium with appropriate antibiotics (100 mg Recent rapid developments in metabolic engineering by À mL 1 ampicillin). All recombinant S. cerevisiae strains in this rewiring cellular metabolism provide an attractive alternative study were constructed from BY4741, BJ5464 or CEN$PK2-1Ca. approach for producing plant terpenoids.6,10,11 Saccharomyces Complete medium of yeast extract peptone dextrose (YPD, cerevisiae has been widely used in terpenoid biosynthesis containing 1% (w/v) yeast extract, 2% (w/v) peptone and 2% (w/ because it is generally regarded as safe (GRAS),10 industrially v) D-glucose) was used for yeast cultivation. Engineered yeast robust and able to functionally express eukaryotic cytochrome strains were grown in minimal synthetic dened (SD) medium P450 enzymes.12 There are many discussions about terpenoid (Clontech, Mountain View, USA), in which leucine and/or uracil production through metabolic engineering of S. cerevisiae at were omitted for auxotrophic selection.21,22 For the expression of hierarchical levels of genetic manipulation tools, biological 10,12–15 galactose-inducible genes, SG, SGR and SGG medium were Creative Commons Attribution-NonCommercial 3.0 Unported Licence. parts, pathways, organelles and systems. Although used, in which the SD medium was changed as the medium signicant progresses have been achieved in the past decades, containing 2% (w/v) galactose instead of glucose, or 2% (w/v) there are still a lot of efforts to be made. galactose supplemented with 0.7% (w/v) raffinose, or 2% (w/v) In S. cerevisiae, the mevalonate (MVA) pathway is naturally galactose combined with 10% (w/v) glycerol, respectively. tightly regulated at transcriptional and post-transcriptional All heterologous genes used in this study were synthesized by levels, limiting the overall metabolic ux for sesquiterpene Sangon Biotech (Shanghai, China) with codon optimization for production. To increase the target sesquiterpene accumulation, expression in S. cerevisiae. The truncated version of HMG1 genetic modications that block the competition branch ux (tHMGR), alcohol dehydrogenase isozymes (ADH1 and ADH3) could be considered. The squalene synthase (ERG9) is a branch- were amplied from CEN$PK2-1Ca genomic DNA. Genes used This article is licensed under a point enzyme in the mevalonate (MVA) pathway.16–18 As sterols are in this study are listed in Table S1.† Plasmids and strains used essential for yeast cells, so it is not feasible to thoroughly knock in this work are listed in Tables S2 and S3,† respectively. out squalene synthase (ERG9). Down-regulating the expression of ERG9 by weakening its promoter is an alternative way. In addi- Open Access Article. Published on 24 September 2019. Downloaded 9/26/2021 6:58:52 AM. S. cerevisiae tion, the ROX1 is a transcription factor that inhibits expression of 2.3 Cloning and integration of pathway in hypoxia-induced genes in the presence of oxygen and also The assembly of synthetic operon in YEp352 was performed represses target genes during osmotic stress.19 Previous study according to the BioBrick principles23 described by Zelcbuch showed that transcriptional regulation of ROX1 mediates the et al.24 To further simplify the cloning process, a seamless, transcriptional regulation of ERG genes. Especially, ERG9 could recombination-based