Engineering Saccharomyces cerevisiae for the production of the valuable monoterpene ester geranyl acetate

Engineering Saccharomyces cerevisiae for the production of the valuable monoterpene ester geranyl... Background: Geranyl acetate is widely used in the fragrance and cosmetic industries, and thus has great economic value. However, plants naturally produce a mixture of hundreds of esters, and geranyl acetate is usually only present in trace amounts, which makes its economical extraction from plant sources practically impossible. As an ideal host for heterologous production of fragrance compound, the Saccharomyces cerevisiae has never been engineered to produce the esters, such as geranyl acetate. Results: In this study, a heterologous geranyl acetate synthesis pathway was constructed in S. cerevisiae for the first time, and a titer of 0.63 mg/L geranyl acetate was achieved. By expressing an Erg20 mutant to divert carbon flux from FPP to GPP, the geranyl acetate production increased to 2.64 mg/L. However, the expression of heterologous GPP had limited effect. The highest production of 13.27 mg/L geranyl acetate was achieved by additional integration and expression of tHMG1, IDI1 and MAF1. Furthermore, through optimizing fermentation conditions, the geranyl acetate titer increased to 22.49 mg/L. Conclusions: We constructed a monoterpene ester producing cell factory in S. cerevisiae for the first time, and demonstrated the great potential of this system for the heterologous production of a large group of economically important fragrance compounds. Keywords: Monoterpene, Geranyl acetate, ERG20, tHMG1, IDI1, MAF1 Background which in turn is synthesized from isopentenyl diphos- Monoterpenes constitute a subclass of terpenoids [1] that phate (IPP) and its isomer dimethylallyl diphosphate are widely used as additives in the food, pharmaceutical, (DMAPP), derived from either the mevalonate (MVA) agrichemical and cosmetic industries, due to their strong pathway or the 2C-methyl-d-erythrtiol 4-phosphate flavor, fragrance and physiological activity [ 2, 3]. Moreo- (MEP) pathway [5, 6]. As the precursor of monoterpenes ver, some monoterpenes were shown to have great poten- such as geraniol and linalool, GPP reacts with one more tial as biofuels [4], which prompted increased attention IPP and gives rise to farnesyl diphosphate (FPP) [7, 8], from the research community in recent years. The basic which is the precursor of sesquiterpenes, squalene, diter- scaffold of monoterpenes contains two isoprene units penes, GGPP and so on. that are biosynthesized from geranyl diphosphate (GPP), Monoterpenes are mainly produced by plants, albeit at extremely low concentrations [9], and the traditional chemical synthesis and bio-extraction processes are both *Correspondence: Zhangbolin888@sina.com.cn; bi_ch@tib.cas.cn; costly and environmentally harmful. However, a num- zhang_xl@tib.cas.cn Tao Wu and Siwei Li have contributed equally to this work ber of research groups have been able to produce natu- College of Biological Sciences and Technology, Beijing Forestry ral products by metabolic engineering of microbial cell University, Beijing 100083, People’s Republic of China 2 factories [10, 11], most often derived from the model Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, People’s Republic of China organisms Escherichia coli and Saccharomyces cerevisiae Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wu et al. Microb Cell Fact (2018) 17:85 Page 2 of 10 [12–14]. The production of monoterpenes by metabolic production was achieved, which represents a 3.5-fold engineering has also been reported, but most of the cases increase compared with that achieved via the separate involved relative low production, which hindered their expression of Erg20 and SpSabS1 [20]. By the screen- industrial application [9]. Previous work has shown that ing of different sources of GESs and GPPSs, as well as engineered E. coli could produce 400  mg/L of limonene fusions of the two proteins, Hou’ group achieved the titer and approximately 100 mg/L of perillyl alcohol [13], and of 293  mg/L geraniol in S. cerevisiae [21]. Furthermore, engineered yeasts were able to produce 95  µg/L of lin- dynamic control of ERG20 expression combined with alool [15], as well as 36.04 mg/L–2.0 g/L of geraniol [16, minimized endogenous downstream metabolism led to 17]. impressive progress in the production of geraniol [22]. Saccharomyces cerevisiae possesses a native MVA On the other hand, HMG-CoA reductase was identi- pathway [6], which makes it suitable for the synthesis fied as a key rate-limiting enzyme in the MVA pathway of monoterpenes. Some reports also demonstrated the of S. cerevisiae [23, 24], and a truncated 3-hydroxyl- potential of metabolic engineering for monoterpene 3-methylglutaryl-CoA reductase gene (tHMGR) was production in S. cerevisiae [18]. GPP is synthesized by overexpressed to increase the supply of mevalonate. the bifunctional enzyme ERG20 (Fig.  1), which has both DMAPP is the common substrate for the biosynthesis GPP synthase (GPPS) and farnesyl pyrophosphate syn- of both GPP and tRNA, and MAF1 represses the tran- thase (FPPS) activities [19]. To decrease the metabolic scriptional activity of RNA polymerase III, serving as flux towards FPP, mutants with changes in the FPP syn - a negative regulator in the biosynthesis of tRNA [16]. thesis domain of Erg20p were screened, and mutations The isoprenoid diphosphate isomerase IDI1 catalyzes at position 197 (K197G, C, S, T, D, E) [18] and a double the interconversion between DMAPP and IPP [25], but mutant (N127W–F96W) [20] were proved to dramati- the formation of one molecule of GPP requires two cally increase the yield of monoterpenes. To increase the molecules of IPP and one molecule of DMAPP. Since production of sabinene, the sabinene synthase SpSabS1 the ratio of IPP to DMAPP is not optimal for GPP bio- was fused to the Erg20 variant, and 1.87  mg/L sabinene synthesis, Jingwen Zhou’s research indicated that the Glucose Geranylacetate Acetyl-CoA SAAT Geraniol HMG-CoA GES HMG1 IPP HMG2 ERG20 IDI1 ERG8,ERG12 ERG20 ERG19 IPP FPP MVA GPP ERG9 DMAPP MAF1 Squalene tRNA Fig. 1 Schematic diagram of the heterologous synthesis pathway for the production of geranyl acetate in the engineered S. cerevisiae. HMG-CoA 3-hydroxy-3-methylglutaryl coenzyme A, MVA mevalonate, IPP isopentenyl pyrophosphate, DMAPP dimethylallyl diphosphate, GPP geranyl pyrophosphate, FPP farnesyl pyrophosphate Wu et al. Microb Cell Fact (2018) 17:85 Page 3 of 10 isomerase IDI1 was the rate-limiting enzyme in geran- Methods iol production [16]. Thus, overexpression of truncated Media, strains and plasmids HMG-CoA (tHMG1), IDI1 and MAF1 could improve Escherichia coli Trans T1 (TransGen, China) was used as the production of monoterpenes. the host for plasmid construction and amplification. The Geranyl acetate, an acyclic monoterpene ester cells were grown at 37 °C in Luria–Bertani (LB) medium derived from geraniol, is widely used in the cosmet- (1% NaCl, 1% tryptone and 0.5% yeast extract with ics industry due to its pleasant scent, and it was also 100  mg/L of ampicillin (Solarbio, China). S. cerevisiae recently discovered to have an antinociceptive activ- BY4742 (MATα, his3Δ1, leu2Δ0, lys2Δ0, MET15, ura3Δ0; ity [26], making it a compound with great economic Euroscarf, Germany) was used as the host strain for DNA value. However, plants naturally produce a mixture assembly and integration. The cells were cultivated at of hundreds of esters, and geranyl acetate only makes 30  °C in yeast-extract peptone dextrose (YPD) medium up a small percentage of the total, which makes its (2% glucose, 2% peptone, and 1% yeast extract). Synthetic extraction and traditional plant-based production complete drop-out medium lacking leucine and/or his- uneconomical [27, 28]. Albeit the great commercial tidine (SC-LEU, SC-LEU-HIS, SC-LEU-HIS-TRP) was potential, as far as we know, no research has focused used for transformant selection. For solid media, 2% agar to heterologous produce ester fragrance compounds. was added. And as an ideal host for heterologous production of The codon-optimized genes encoding geraniol syn - fragrance compound, the S. cerevisiae has never been thase (GES) from Ocimum basilicum without the engineered to produce the esters, such as geranyl N-terminal transit peptide (1–34aa), and alcohol acyl- acetate. transferases (AATs) (SAAT) from cultivated strawberry Thus, in this study, we intended to construct a S. cer- (Fragaria × ananassa) were synthesized by GENEWIZ evisiae cell factory for production of geranyl acetate to (Suzhou, China) and embedded in plasmid pUC57- study and demonstrate the capacity of this system for Amp. The sequences of the two genes are provided heterologous production of this group of economically in Additional file  1. The SAAT and GES genes were important fragrance compounds, and explore the fer- excised from pUC57-SAAT/GES with SexAI and AscI. mentation methods and conditions for the production SAAT was ligated into the vector pM2 under the con- process (Fig.  1). Geraniol synthase (GES) [16, 22, 29] trol of the PGK1 promoter and ADH1 terminator, and from Ocimum basilicum and alcohol acyltransferases pM3-GES was constructed with GES under the control (AATs) [29] from strawberry (Fragaria × anana ssa) of the TEF2 promoter and CYC1 terminator. tHMG1 (SAAT) were introduced into the yeast chromosome. containing the catalytic domain of HMG1 was ampli- Several groups have used the GES from O. basilicum fied from the genome of S. cerevisiae using the primer for the formation of geraniol, and SAAT showed a high pair SexA-tHMG1/AscI-tHMG1 and cloned into vector affinity and efficiency for the biosynthesis of geranyl pM4 using T4 ligase (Thermo, USA). The plasmids pM4- acetate [29]. ERG20(F96W–N127W), pM2-MAF1 and pM3-IDI1 were constructed in the same way (Table 1). Table 1 Strains and plasmids used in this study Name Description Source pUC57-GES Cloning vector with a synthetic version of the GES gene from O. basilicum GENEWIZ pUC57-SAAT Cloning vector with a synthetic version of the SAAT gene from cultivated strawberry (Fra- GENEWIZ garia × ananassa) pEASY-Blunt Cloning vector for blunt ligation This study pM3-GES pEASY-Blunt vector with pTEF2-GES-tCYC1cassette This study pM2-SAAT pEASY-Blunt vector with pPGK1-SAAT-tADH1 cassette This study pM4-ERG20(F96W–N127W ) pEASY-Blunt vector with pTDH3-ERG20(F96W–N127W )-tTPI1 cassette This study pM2-MAF1 pEASY-Blunt vector with pPGK1-MAF1-tADH1 cassette This study pM3-IDI1 pEASY-Blunt vector with pTEF2-IDI1-tCYC1cassette This study pM4-tHMG1 pEASY-Blunt vector with pTDH3-tHMG1-tTPI1 cassette This study pRS313-TRP pRS313 vector: HIS selection marker was replased withTRP This study pRS313-GPPS pRS313-TRP vector with pPGK-GPPS -tCYC1cassette This study At At pRS313-GPPS pRS313-TRP vector with pPGK-GPPS -tCYC1cassette This study Mp Mp Wu et al. Microb Cell Fact (2018) 17:85 Page 4 of 10 Genetic manipulation of S. cerevisiae The plant GPP synthase genes were amplified from The construction and assembly of functional transcrip - cDNA of Arabidopsis thaliana and Mentha piperita, tional units on the chromosome of S. cerevisiae was and cloned into the expression vector pRS313-TRP performed by the DNA assembler method [30, 31]. The using T4 ligase. The maps of the corresponding expres - transcription unit encoding GES (pTEF2-GES-tCYC1) sion vectors pRS313-GPPS and pRS313-GPPS are At Mp was amplified by PCR from plasmid pM2-GES, that of shown in Additional file  1: Fig. S1. All plasmids were SAAT (pPGK1-GES-tADH1) from pM3-SAAT, and that verified by PCR and DNA sequencing, and the relevant of ERG20 (pTDH3-ERG20(F96W–N127W)-tTPI1) from primers are listed in Additional file 1 : Table S1. pM4-ERG20. The selection marker and integration locus fragments were amplified by PCR. All primers used for amplification and integration into the S. cerevisiae chro - Site‑directed Mutagenesis of the farnesyl diphosphate mosome are listed in Additional file  1: Table  S1. Strain synthase ERG20 GA01 was constructed by integrating the GES and SAAT Two point-mutations (F96W and N127W) were intro- cassettes into the gal 80 site along with the LEU selection duced into ERG20 by overlap-extension PCR. Yeast marker, the strain GA02 was constructed by integrating genomic DNA was used as the template, and the ERG20 the GES, SAAT and ERG20(F96W–N127W) cassettes gene was divided into three parts, 1–96aa, 94–127aa into the gal 80 site along with the LEU selection maker and 127–353aa. The three parts of the gene were ampli - (Fig.  2), and strain GA03 was constructed by integrating fied separately using primers with embedded muta - the tHMG1, MAF1 and IDI1 cassettes into the NDT80 tions, and then fused into a complete mutated gene by site of strain GA02 along with the HIS selection maker. overlap-extension PCR. The corresponding primers are The DNA fragments, GPP synthase expression plas - listed in Additional file 1 : Table S1. mid pRS313-GPPSAt and pRS313-GPPSMp were all L1 L1 pPGK1 SAAT tADH1 R Gal80 site-1 L1 Leu2 F GES pTEF2 tCYC1 L3R L3-2 L3R Gal80 site-1 chromosome Gal80 DNA site b L1 L1 pPGK1 SAAT tADH1 F R Gal80 site-1 L1 Leu2 F ERG20m L2 pTDH3 tTPI1 L2 F R pTEF2t GES CYC1 L3 L3 F R L3 R Gal80 site-1 chromosome Gal80 DNA site Fig. 2 Schematic diagram of the integrated expression cassettes of the heterologous geranyl acetate synthesis pathway (a) the yeast genome integration of geraniol synthase gene (GES) and alcohol acyltransferases gene (SAAT ) (b) the yeast genome integration of the geraniol synthase gene (GES), strawberry acyltransferases gene (SAAT ) and Erg20 mutant (F96W–N127W ) gene Wu et al. Microb Cell Fact (2018) 17:85 Page 5 of 10 introduced into S. cerevisiae BY4742 by conventional Results electroporation method. When four or five fragments Construction of a microbial cell factory by integrating (each fragment was 100  ng) were used for homologous the geranyl acetate biosynthetic pathway recombination (HR) in S. cerevisiae, about 200–400 colo- into the chromosome of S. cerevisiae nies could be achieved. As volatile esters, geranyl acetate is the essential com- ponents of fruit characteristic aroma and presents in the essential oils of various plant species. It serves as respon- Yeast cultivation and PCR confirmation ser to stress or insect infestation. It has been reported For PCR confirmation of transformants, single colonies that truncated O. basilicum geraniol synthase is very were used to inoculate 4  mL of SC-LEU/SC-LEU-HIS/ efficient in geraniol synthesis with geranyl diphosphate SC–LEU-HIS-TRP medium, and grown at 30°C and (GPP) as the substrate [3, 16]. We found that alcohol 250  rpm overnight. Cells were harvested by centrifuga- acyltransferase gene from cultivated strawberry (SAAT) tion, and the genomic DNA was extracted using the Yeast was a highly active enzyme capable of transferring the Gene DNA Kit (CW Biotech, China). 2 μL of total DNA acetyl group from acetyl-CoA to various substrates, and was used as template for PCR using the 2 × Taq Mas- possibly to geraniol to form geranyl acetate [29]. In order ter Mix (CW Biotech). 10 colonies from SC-Leu agar to construct a metabolic pathway for the production of plates were randomly picked and inoculated in SC-Leu geranyl acetate, the geraniol synthase (GES) gene from O. medium. After that, genomic DNA was extracted, and basilicum and SAAT were integrated into the chromo- PCR determination was performed respectively. The some of S. cerevisiae BY4742 at the gal80 site (Fig.  2a). ratio of positive clones to all the colonies was calculated The expression of the synthetic cassette was controlled to be above 90%. by a constitutive strong promoter and the resulting strain was designated as GA01. The production of geranyl acetate was measured by Cell‑culture, extraction and quantification of geranyl GC–MS (Fig.  3a), and the titer ranged from 0.25 to acetate 0.63 mg/L during the fermentation process (Fig. 3b). The The correct colonies were picked and grown in the cor - titer reached its maximum value after 48  h of cultiva- responding synthetic complete drop-out medium or YPD tion, at an OD of 5.23. However, while the density of medium overnight, transferred into a flask with fresh the yeast culture increased persistently for 5  days, gera- medium to yield an initial OD of 0.05–0.10, and cul- nyl acetate production did not improve accordingly, tured for 6 days at 30 °C and 250 rpm. The increase of cell which may be due to gaseous escape of the volatile gera- biomass during the fermentation process was detected by nyl acetate during the aerobic fermentation process [32]. measuring the OD value using a UV-2550 spectropho- Besides, another four alcohol acyltransferase genes from tometer (Shimadzu, Japan). plants were used for geranyl acetate production, but the To quantify the titer of geranyl acetate in the yeast titer was very low or no geranyl acetate was detected cultures, aliquots comprising 1  mL of the fermentation compared with SAAT (Additional file 1: Table S2). broth were concentrated by centrifugation at 16,200×g for 2  min, after which 1  mL of n-hexane was added to extract the products that were secreted into the medium. The expression of Erg20 mutants for improved geranyl The cell pellet was extracted with another 1 mL of n-hex - acetate production ane under ultrasonic agitation for 30 min, and the n-hex- GPP is the starting substrate of monoterpene produc- ane phase was collected by centrifugation at 16,200×g tion pathways [19]. However, in yeast cells GPP can be for 2  min. The two extraction liquids were mixed, and converted into FPP by the bifunctional synthase Erg20. 1  µL of the combined extract was analyzed using a Agi- In addition, since FPP is the precursor of ergosterol, lent 5975C GC–MS system equipped with a HP-5 ms GC the outright deletion of Erg20 is potentially lethal [5]. column (30  m × 0.25  mm × 0.5  μm; Agilent, USA) and a Accordingly, the Erg20 F96W–N127W mutant, which triple-Axis detector. The GC–MS temperature program has a decreased FPP formation efficiency but consistent encompassed an initial temperature of 45°C for 1  min GPP production, was constructed according to an earlier and a ramp of 10°C/min to 220°C, which was maintained report [20]. The mutated Erg20, GES and SAAT genes for 5 min. Helium was used as the mobile phase at a flow were integrated into the chromosome of S. cerevisiae rate of 1.0 mL/min. The injection port, interface, and MS BY4742 at the gal80 site (Fig.  2b). The resulting strain source temperatures were 250, 300, and 180  °C, respec- GA02 produced 2.64  mg/L of geranyl acetate in 48  h, tively [29]. A reference standard comprising authentic which represented a 419% increase of titer over GA01 geranyl acetate purchased from Sigma Aldrich, USA, was (Fig. 4a). used for quantification. Wu et al. Microb Cell Fact (2018) 17:85 Page 6 of 10 Standard Sample geranyl acetate Time (min) 0.7 0.6 0.5 0.4 0.3 0.2 OD 0.1 Geranyl acetate 024487296120 144 Time (h) Fig. 3 Identification of fermentation products of strain GA01. a The time-course of OD and geranyl acetate in aerobic fermentation of strain GA01. 38. b GC–MS analysis of a cell extract of strain GA01. The mass spectrum of geranyl acetate is shown in the top right corner and the red line indicates the authentic geranyl acetate standard Overexpression of tHMG1, IDI1 and MAF1 for the enhanced biosynthesis of geranyl acetate, IDI1, MAF1 and tHMG1 production of geranyl acetate were integrated into the chromosome of GA02, resulting Isoprenoid diphosphate isomerase (IDI1) catalyzes the in strain GA03. In this best strain, the titer of geranyl ace- isomeric interconversion between IPP and DMAPP [33], tate reached 13.27  mg/L, representing a 400% increase and its overexpression can therefore potentially enhance compared with GA02, and a remarkable 2100% increase the synthesis of GPP and benefit the production of over the starting strain GA01 (Fig. 4a). monoterpenes. MAF1 represses the transcriptional activ- Since S. cerevisiae does not have a specific GPP syn - ity of RNA polymerase III, serving as a negative regula- thase (GPPS, EC 2.5.1.1), which belongs to the short- tor of the biosynthesis of tRNAs [16, 18]. Since DMAPP chain prenyltransferase family [34]. To supply more is a common substrate of both tRNA and GPP synthe- precursor for GPP synthesis, The homomeric GPPS sis, overexpression of MAF1 can divert the carbon flux from Arabidopsis thaliana (GPPS ) and the heteromeric At toward GPP formation [18]. Thus, to further improve the GPPS from Mentha piperita (GPPS ) were separately Mp Abundance OD 600 geranyl acetate(mg/L) Wu et al. Microb Cell Fact (2018) 17:85 Page 7 of 10 GPPS protein, we constructed another plasmid pRS313- pPGK-LSU-tCYC-pTEF-SSU-tADH,the two subunit were expressed with strong promoter respectively, but 12 6 the geranyl acetate production was not increased either. 10 5 Improving geranyl acetate production by optimizing 8 4 the fermentation conditions 6 3 The highest geranyl acetate production of strain GA3 reached to 13.27  mg/L in SC-LEU-HIS medium after cultivated for 48  h, and the OD reached about 5.23 (Fig.  4a). To further improve geranyl acetate produc- 2 1 tion, we adjusted the initial OD of fermentation to 0.1, which made the final OD increased to about 7.2 in 0 0 GA01 GA02 GA03 synthetic complete drop-out medium, and to about 15.6 geranyl acetate OD in YPD medium after incubated at 30 °C for 48 h. When YPD medium was used to fermentation, the titer of gera- 16 7 nyl acetate increased to 1.98, 6.07 and 20.48 mg/L respec- tively for strain GA01, GA02 and GA03 after cultivating for 48  h respectively, as shown in Fig.  5a. Furthermore, in order to prevent volatilization of geranyl acetate, 10% 4 isopropyl myristate was added to the culture medium after 24  h. The production of geranyl acetate increased to 22.49  mg/L in strain GA03 as shown in Fig.  5b, and the organic layer was easily harvested by centrifugation of the fermentation medium. However, we found geranyl acetate production did not improve continually when the strains were inoculated for 5  days. The geranyl acetate 0 0 GA03- GA03- production was improved by 1.69-fold through optimiza- GA03 pRS313-GPPSAt pRS313-GPPSMp tion of the fermentation conditions. OD geranyl acetate Fig. 4 Fermentation and quantification of geranyl acetate. a OD Discussion and geranyl acetate titer of the engineered strains GA01, GA02 and GA03. b Geranyl acetate titers of strains with plant GPP synthases In the past decades, more and more attention has arisen (GPPS : Arabidopsis thaliana GPP synthase, GPPS : Mentha piperita At Mp on heterologous production of the monoterpene geran- GPP synthase) iol. The highest titer of geraniol is about 2.0 g/L in engi - neered E. coli and 1.68  g/L in engineered S. cerevisiae respectively [17, 22]. However, there are rare reports for metabolic engineering of heterologous production of introduced into GA03 on the plasmid pRS313-TRP, monoterpene esters, such as geranyl acetate. Monoter- resulting in the strains GA03-pRS313-GPPS and GA03- At penoids such as geranyl acetate are active compounds pRS313-GPPS respectively. The relevant primers are Mp derived from many plants, which play important roles in listed in Additional file  1: Table S1, and plasmid diagrams protection against pathogens and attraction of animals, are shown in Additional file  1: Fig. S1. Structurally, the also traditionally used as additive of medicines, essential heteromeric GPPS is composed of a large subunit (LSU) oils and perfume [26]. and a small subunit (SSU) [19]. The LSU is inactive alone, The truncated O. basilicum geraniol synthase (GES) and the non-catalytic SSU acts as a modulator of the and alcohol acyltransferase from strawberry (SAAT) interaction between the two inactive subunits, resulting were found with high activity in our lab [22, 29], the in an active GPPS [19]. Consequently, the flexible fusion two genes were integrated into the chromosome of S. protein linker GGGS and (GGGS) were introduced to cerevisiae BY4742 at the gal80 site (Fig.  2a) and a titer construct a heteromeric Mentha piperita GPPS (LSU- of 0.63 mg/L was achieved. According to the method of GGGS/(GGGS) -SSU). Unfortunately, the introduction Huizhou Liu’s group [17], the titer of geraniol in cul- of plant GPPS did not effectively improve the produc - ture medium and in yeast cells after fermentation was tion of geranyl acetate (Fig.  4b). Since fusion of the two analyzed, which was measured to be zero. This result subunits might affect the expression and function of indicated geraniol was entirely converted to geranyl geranyl acetate (mg/L) geranyl acetate (mg/L) OD 600 OD 600 Wu et al. Microb Cell Fact (2018) 17:85 Page 8 of 10 higher production of 13.27 mg/L was achieved by addi- 25 18 tional integration and expression of tHMG1, IDI1 and MAF1, Overexpression of which with strong promoters increased the supplement of precursors. In the fermentation optimization process, when we used YPD media instead of the synthetic complete drop-out medium for fermentation, the OD value of 6 the fermentation broth and the yield of geranyl acetate were both increased. The OD value could reach up 4 600 to about 15.6, presenting a 198% increase compared with that in SC-LEU-HIS medium. The titer of geranyl 0 0 GA01 GA02 GA03 acetate reached 20.48  mg/L, showing a 54% increase compared with the production in SC-LEU-HIS medium geranyl acetate OD (13.27 mg/L). So YPD was more suitable for cell growth and fermentation of the engineered strains. Besides,due 25 20 to the possible gaseous escape of the volatile geranyl acetate, 10% isopropyl myristate was added to the cul- ture after 24 h fermentation. TThe production of gera - nyl acetate increased to 22.49 mg/L in strain GA03 with YPD medium as shown in Fig.  5b. However, geranyl acetate production did not improve continually in the 5-days fermentation process. We thought during the time, the accumulation of the product might affect the 0 0 growth condition and inhibit further improvement of 48 h 120 h geranyl acetate production as described by Zhao’s arti- geranyl acetate OD cle [21]. To sum up, optimization of the fermentation condi- Fig. 5 Fermentation optimization and titer of geranyl acetate. a OD and geranyl acetate titer of the engineered strains GA01, GA02 tions led to a 1.69-fold improvement of geranyl acetate and GA03 in YPD medium. b Geranyl acetate titer of strains in YPD production. And with 10% isopropyl myristate added, medium with 10% isopropyl myristate added to the culture after 24 h it might be able to prevent the volatilization of geranyl acetate and relieve the cell toxicity of geranyl acetate by extracting it from the fermentation broth [21]. acetate by SAAT by the strains we constructed, and suggested the SAAT we used was very efficient and not Conclusion the rate-limiting step of the synthesis pathway. Besides, In this research, a heterologous geranyl acetate synthe- a report just published described truncated GES from sis pathway was constructed in S. cerevisiae for the first Catharanthus roseus with site-directed mutation (Y436 time, and a product titer of 0.63  mg/L was achieved in and D501) was found to has high catalytic activity the starting strain. By expressing an Erg20 mutant to [35]. Thus, for the further improvement of the gera - divert carbon flux from FPP to GPP, the geranyl ace - nyl acetate production, selection and optimization of tate production was increased to 2.64  mg/L, although GESs might be the key focus. In engineered S. cerevi- expression of heterologous GPP did not have a signifi - siae producing monoterpene, the farnesyl diphosphate cant effect. The highest production of 13.27  mg/L was synthase Erg20p was found to be the key enzyme that achieved by additionally expressing tHMG1, IDI1 and limiting monoterpene formation [20]. GPP and FPP MAF1, which represents a remarkable 2100% increase formation are both catalyzed by Erg20p, towards either over the starting strain. Furthermore, optimization of geraniol or downstream squalene synthesis. For more the fermentation conditions led to 22.49  mg/L gera- GPP synthesis, Erg20 F96W–N127W mutant was inte- nyl acetate production, which exhibited a 35.69-fold grated into the genome, which had decreased farnesyl increase over the parent strain GA01 (Fig.  6). To our diphosphate synthase function without interference best knowledge, this work offers the first microbial cell of the growth of S. cerevisiae. And the production of factory for specific production of a monoterpene ester, geranyl acetate increased to 2.64 mg/L. Furthermore, a geranyl acetate (mg/L) geranyl acetate (mg/L) OD OD 600 Wu et al. Microb Cell Fact (2018) 17:85 Page 9 of 10 Geranyl acetate Increase Figure 8 production value 0.63 mg/L 1 GA01 OverexpressionofErg20 mutant 2.64 mg/L 4.19 GA02 Overexpression of tHMG1, IDI1 andMAF1 13.27 mg/L 21.06 GA03 GA03 in YPD medium 20.48 mg/L 32.50 GA03 in YPD medim with 35.69 22.49 mg/L 10 % isopropyl myristate Fig. 6 Diagram summarizing the increase of geranyl acetate Specifically, plasmid maps and DNA sequence data can be found in Additional demonstrating great potential for the heterologous file 1. production of many more economically important fra- grance compounds. Consent for publication I hereby give the Journal of Microbial Cell Factories the right and permission Additional file to publish this article. Ethical approval and consent to participate Additional file 1: Table S1. Primers used in this study. Table S2. Intro - Not applicable. duction of alcohol acyltransferases from plants and the titer of geranyl acetate. Fig. S1. Plasmid maps and DNA sequences. Funding This research was supported by grants from the National Natural Science Foundation of China (31522002), Natural Science Foundation of Tianjin (15JCY- BJC49400), and the Tianjin Key Technology R&D program of Tianjin Municipal Authors’ contributions Science and Technology Commission (11ZCZDSY08600). TW and SWL planned and performed the experiments, analyzed and inter- preted the data. TW wrote the manuscript, and SWL checked the manu- script. BZ, CB and XZ supervised the study, designed the experiments and Publisher’s Note analyzed and interpreted the results. All authors read and approved the final Springer Nature remains neutral with regard to jurisdictional claims in pub- manuscript. lished maps and institutional affiliations. Author details Received: 22 December 2017 Accepted: 11 May 2018 College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, People’s Republic of China. Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, People’s Repub- lic of China. Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, People’s Republic of China. References 1. Mcgarvey DJ, Croteau R. Terpenoid metabolism. Plant Cell. Acknowledgements 1995;7:1015–26. Not applicable. 2. Lapczynski A, Bhatia SP, Foxenberg RJ, Letizia CS, Api AM. Fragrance mate- rial review on geraniol. Food Chem Toxicol. 2008;46:S160. Competing interests 3. Chen W, Viljoen AM. Geraniol—a review of a commercially important The authors declare that they have no competing interests. fragrance material. S Afr J Bot. 2010;76:796–807. 4. Renninger NS, Ryder JA, Fisher KJ: Jet fuel compositions and methods of Availability of data and materials making and using same. US; 2011. We provide all necessary data for the publication of the article. All additional data is present in the article and the supplemental material documents. 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Engineering Saccharomyces cerevisiae for the production of the valuable monoterpene ester geranyl acetate

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Chemistry; Applied Microbiology; Biotechnology; Microbiology; Microbial Genetics and Genomics; Enzymology; Genetic Engineering
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Abstract

Background: Geranyl acetate is widely used in the fragrance and cosmetic industries, and thus has great economic value. However, plants naturally produce a mixture of hundreds of esters, and geranyl acetate is usually only present in trace amounts, which makes its economical extraction from plant sources practically impossible. As an ideal host for heterologous production of fragrance compound, the Saccharomyces cerevisiae has never been engineered to produce the esters, such as geranyl acetate. Results: In this study, a heterologous geranyl acetate synthesis pathway was constructed in S. cerevisiae for the first time, and a titer of 0.63 mg/L geranyl acetate was achieved. By expressing an Erg20 mutant to divert carbon flux from FPP to GPP, the geranyl acetate production increased to 2.64 mg/L. However, the expression of heterologous GPP had limited effect. The highest production of 13.27 mg/L geranyl acetate was achieved by additional integration and expression of tHMG1, IDI1 and MAF1. Furthermore, through optimizing fermentation conditions, the geranyl acetate titer increased to 22.49 mg/L. Conclusions: We constructed a monoterpene ester producing cell factory in S. cerevisiae for the first time, and demonstrated the great potential of this system for the heterologous production of a large group of economically important fragrance compounds. Keywords: Monoterpene, Geranyl acetate, ERG20, tHMG1, IDI1, MAF1 Background which in turn is synthesized from isopentenyl diphos- Monoterpenes constitute a subclass of terpenoids [1] that phate (IPP) and its isomer dimethylallyl diphosphate are widely used as additives in the food, pharmaceutical, (DMAPP), derived from either the mevalonate (MVA) agrichemical and cosmetic industries, due to their strong pathway or the 2C-methyl-d-erythrtiol 4-phosphate flavor, fragrance and physiological activity [ 2, 3]. Moreo- (MEP) pathway [5, 6]. As the precursor of monoterpenes ver, some monoterpenes were shown to have great poten- such as geraniol and linalool, GPP reacts with one more tial as biofuels [4], which prompted increased attention IPP and gives rise to farnesyl diphosphate (FPP) [7, 8], from the research community in recent years. The basic which is the precursor of sesquiterpenes, squalene, diter- scaffold of monoterpenes contains two isoprene units penes, GGPP and so on. that are biosynthesized from geranyl diphosphate (GPP), Monoterpenes are mainly produced by plants, albeit at extremely low concentrations [9], and the traditional chemical synthesis and bio-extraction processes are both *Correspondence: Zhangbolin888@sina.com.cn; bi_ch@tib.cas.cn; costly and environmentally harmful. However, a num- zhang_xl@tib.cas.cn Tao Wu and Siwei Li have contributed equally to this work ber of research groups have been able to produce natu- College of Biological Sciences and Technology, Beijing Forestry ral products by metabolic engineering of microbial cell University, Beijing 100083, People’s Republic of China 2 factories [10, 11], most often derived from the model Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, People’s Republic of China organisms Escherichia coli and Saccharomyces cerevisiae Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wu et al. Microb Cell Fact (2018) 17:85 Page 2 of 10 [12–14]. The production of monoterpenes by metabolic production was achieved, which represents a 3.5-fold engineering has also been reported, but most of the cases increase compared with that achieved via the separate involved relative low production, which hindered their expression of Erg20 and SpSabS1 [20]. By the screen- industrial application [9]. Previous work has shown that ing of different sources of GESs and GPPSs, as well as engineered E. coli could produce 400  mg/L of limonene fusions of the two proteins, Hou’ group achieved the titer and approximately 100 mg/L of perillyl alcohol [13], and of 293  mg/L geraniol in S. cerevisiae [21]. Furthermore, engineered yeasts were able to produce 95  µg/L of lin- dynamic control of ERG20 expression combined with alool [15], as well as 36.04 mg/L–2.0 g/L of geraniol [16, minimized endogenous downstream metabolism led to 17]. impressive progress in the production of geraniol [22]. Saccharomyces cerevisiae possesses a native MVA On the other hand, HMG-CoA reductase was identi- pathway [6], which makes it suitable for the synthesis fied as a key rate-limiting enzyme in the MVA pathway of monoterpenes. Some reports also demonstrated the of S. cerevisiae [23, 24], and a truncated 3-hydroxyl- potential of metabolic engineering for monoterpene 3-methylglutaryl-CoA reductase gene (tHMGR) was production in S. cerevisiae [18]. GPP is synthesized by overexpressed to increase the supply of mevalonate. the bifunctional enzyme ERG20 (Fig.  1), which has both DMAPP is the common substrate for the biosynthesis GPP synthase (GPPS) and farnesyl pyrophosphate syn- of both GPP and tRNA, and MAF1 represses the tran- thase (FPPS) activities [19]. To decrease the metabolic scriptional activity of RNA polymerase III, serving as flux towards FPP, mutants with changes in the FPP syn - a negative regulator in the biosynthesis of tRNA [16]. thesis domain of Erg20p were screened, and mutations The isoprenoid diphosphate isomerase IDI1 catalyzes at position 197 (K197G, C, S, T, D, E) [18] and a double the interconversion between DMAPP and IPP [25], but mutant (N127W–F96W) [20] were proved to dramati- the formation of one molecule of GPP requires two cally increase the yield of monoterpenes. To increase the molecules of IPP and one molecule of DMAPP. Since production of sabinene, the sabinene synthase SpSabS1 the ratio of IPP to DMAPP is not optimal for GPP bio- was fused to the Erg20 variant, and 1.87  mg/L sabinene synthesis, Jingwen Zhou’s research indicated that the Glucose Geranylacetate Acetyl-CoA SAAT Geraniol HMG-CoA GES HMG1 IPP HMG2 ERG20 IDI1 ERG8,ERG12 ERG20 ERG19 IPP FPP MVA GPP ERG9 DMAPP MAF1 Squalene tRNA Fig. 1 Schematic diagram of the heterologous synthesis pathway for the production of geranyl acetate in the engineered S. cerevisiae. HMG-CoA 3-hydroxy-3-methylglutaryl coenzyme A, MVA mevalonate, IPP isopentenyl pyrophosphate, DMAPP dimethylallyl diphosphate, GPP geranyl pyrophosphate, FPP farnesyl pyrophosphate Wu et al. Microb Cell Fact (2018) 17:85 Page 3 of 10 isomerase IDI1 was the rate-limiting enzyme in geran- Methods iol production [16]. Thus, overexpression of truncated Media, strains and plasmids HMG-CoA (tHMG1), IDI1 and MAF1 could improve Escherichia coli Trans T1 (TransGen, China) was used as the production of monoterpenes. the host for plasmid construction and amplification. The Geranyl acetate, an acyclic monoterpene ester cells were grown at 37 °C in Luria–Bertani (LB) medium derived from geraniol, is widely used in the cosmet- (1% NaCl, 1% tryptone and 0.5% yeast extract with ics industry due to its pleasant scent, and it was also 100  mg/L of ampicillin (Solarbio, China). S. cerevisiae recently discovered to have an antinociceptive activ- BY4742 (MATα, his3Δ1, leu2Δ0, lys2Δ0, MET15, ura3Δ0; ity [26], making it a compound with great economic Euroscarf, Germany) was used as the host strain for DNA value. However, plants naturally produce a mixture assembly and integration. The cells were cultivated at of hundreds of esters, and geranyl acetate only makes 30  °C in yeast-extract peptone dextrose (YPD) medium up a small percentage of the total, which makes its (2% glucose, 2% peptone, and 1% yeast extract). Synthetic extraction and traditional plant-based production complete drop-out medium lacking leucine and/or his- uneconomical [27, 28]. Albeit the great commercial tidine (SC-LEU, SC-LEU-HIS, SC-LEU-HIS-TRP) was potential, as far as we know, no research has focused used for transformant selection. For solid media, 2% agar to heterologous produce ester fragrance compounds. was added. And as an ideal host for heterologous production of The codon-optimized genes encoding geraniol syn - fragrance compound, the S. cerevisiae has never been thase (GES) from Ocimum basilicum without the engineered to produce the esters, such as geranyl N-terminal transit peptide (1–34aa), and alcohol acyl- acetate. transferases (AATs) (SAAT) from cultivated strawberry Thus, in this study, we intended to construct a S. cer- (Fragaria × ananassa) were synthesized by GENEWIZ evisiae cell factory for production of geranyl acetate to (Suzhou, China) and embedded in plasmid pUC57- study and demonstrate the capacity of this system for Amp. The sequences of the two genes are provided heterologous production of this group of economically in Additional file  1. The SAAT and GES genes were important fragrance compounds, and explore the fer- excised from pUC57-SAAT/GES with SexAI and AscI. mentation methods and conditions for the production SAAT was ligated into the vector pM2 under the con- process (Fig.  1). Geraniol synthase (GES) [16, 22, 29] trol of the PGK1 promoter and ADH1 terminator, and from Ocimum basilicum and alcohol acyltransferases pM3-GES was constructed with GES under the control (AATs) [29] from strawberry (Fragaria × anana ssa) of the TEF2 promoter and CYC1 terminator. tHMG1 (SAAT) were introduced into the yeast chromosome. containing the catalytic domain of HMG1 was ampli- Several groups have used the GES from O. basilicum fied from the genome of S. cerevisiae using the primer for the formation of geraniol, and SAAT showed a high pair SexA-tHMG1/AscI-tHMG1 and cloned into vector affinity and efficiency for the biosynthesis of geranyl pM4 using T4 ligase (Thermo, USA). The plasmids pM4- acetate [29]. ERG20(F96W–N127W), pM2-MAF1 and pM3-IDI1 were constructed in the same way (Table 1). Table 1 Strains and plasmids used in this study Name Description Source pUC57-GES Cloning vector with a synthetic version of the GES gene from O. basilicum GENEWIZ pUC57-SAAT Cloning vector with a synthetic version of the SAAT gene from cultivated strawberry (Fra- GENEWIZ garia × ananassa) pEASY-Blunt Cloning vector for blunt ligation This study pM3-GES pEASY-Blunt vector with pTEF2-GES-tCYC1cassette This study pM2-SAAT pEASY-Blunt vector with pPGK1-SAAT-tADH1 cassette This study pM4-ERG20(F96W–N127W ) pEASY-Blunt vector with pTDH3-ERG20(F96W–N127W )-tTPI1 cassette This study pM2-MAF1 pEASY-Blunt vector with pPGK1-MAF1-tADH1 cassette This study pM3-IDI1 pEASY-Blunt vector with pTEF2-IDI1-tCYC1cassette This study pM4-tHMG1 pEASY-Blunt vector with pTDH3-tHMG1-tTPI1 cassette This study pRS313-TRP pRS313 vector: HIS selection marker was replased withTRP This study pRS313-GPPS pRS313-TRP vector with pPGK-GPPS -tCYC1cassette This study At At pRS313-GPPS pRS313-TRP vector with pPGK-GPPS -tCYC1cassette This study Mp Mp Wu et al. Microb Cell Fact (2018) 17:85 Page 4 of 10 Genetic manipulation of S. cerevisiae The plant GPP synthase genes were amplified from The construction and assembly of functional transcrip - cDNA of Arabidopsis thaliana and Mentha piperita, tional units on the chromosome of S. cerevisiae was and cloned into the expression vector pRS313-TRP performed by the DNA assembler method [30, 31]. The using T4 ligase. The maps of the corresponding expres - transcription unit encoding GES (pTEF2-GES-tCYC1) sion vectors pRS313-GPPS and pRS313-GPPS are At Mp was amplified by PCR from plasmid pM2-GES, that of shown in Additional file  1: Fig. S1. All plasmids were SAAT (pPGK1-GES-tADH1) from pM3-SAAT, and that verified by PCR and DNA sequencing, and the relevant of ERG20 (pTDH3-ERG20(F96W–N127W)-tTPI1) from primers are listed in Additional file 1 : Table S1. pM4-ERG20. The selection marker and integration locus fragments were amplified by PCR. All primers used for amplification and integration into the S. cerevisiae chro - Site‑directed Mutagenesis of the farnesyl diphosphate mosome are listed in Additional file  1: Table  S1. Strain synthase ERG20 GA01 was constructed by integrating the GES and SAAT Two point-mutations (F96W and N127W) were intro- cassettes into the gal 80 site along with the LEU selection duced into ERG20 by overlap-extension PCR. Yeast marker, the strain GA02 was constructed by integrating genomic DNA was used as the template, and the ERG20 the GES, SAAT and ERG20(F96W–N127W) cassettes gene was divided into three parts, 1–96aa, 94–127aa into the gal 80 site along with the LEU selection maker and 127–353aa. The three parts of the gene were ampli - (Fig.  2), and strain GA03 was constructed by integrating fied separately using primers with embedded muta - the tHMG1, MAF1 and IDI1 cassettes into the NDT80 tions, and then fused into a complete mutated gene by site of strain GA02 along with the HIS selection maker. overlap-extension PCR. The corresponding primers are The DNA fragments, GPP synthase expression plas - listed in Additional file 1 : Table S1. mid pRS313-GPPSAt and pRS313-GPPSMp were all L1 L1 pPGK1 SAAT tADH1 R Gal80 site-1 L1 Leu2 F GES pTEF2 tCYC1 L3R L3-2 L3R Gal80 site-1 chromosome Gal80 DNA site b L1 L1 pPGK1 SAAT tADH1 F R Gal80 site-1 L1 Leu2 F ERG20m L2 pTDH3 tTPI1 L2 F R pTEF2t GES CYC1 L3 L3 F R L3 R Gal80 site-1 chromosome Gal80 DNA site Fig. 2 Schematic diagram of the integrated expression cassettes of the heterologous geranyl acetate synthesis pathway (a) the yeast genome integration of geraniol synthase gene (GES) and alcohol acyltransferases gene (SAAT ) (b) the yeast genome integration of the geraniol synthase gene (GES), strawberry acyltransferases gene (SAAT ) and Erg20 mutant (F96W–N127W ) gene Wu et al. Microb Cell Fact (2018) 17:85 Page 5 of 10 introduced into S. cerevisiae BY4742 by conventional Results electroporation method. When four or five fragments Construction of a microbial cell factory by integrating (each fragment was 100  ng) were used for homologous the geranyl acetate biosynthetic pathway recombination (HR) in S. cerevisiae, about 200–400 colo- into the chromosome of S. cerevisiae nies could be achieved. As volatile esters, geranyl acetate is the essential com- ponents of fruit characteristic aroma and presents in the essential oils of various plant species. It serves as respon- Yeast cultivation and PCR confirmation ser to stress or insect infestation. It has been reported For PCR confirmation of transformants, single colonies that truncated O. basilicum geraniol synthase is very were used to inoculate 4  mL of SC-LEU/SC-LEU-HIS/ efficient in geraniol synthesis with geranyl diphosphate SC–LEU-HIS-TRP medium, and grown at 30°C and (GPP) as the substrate [3, 16]. We found that alcohol 250  rpm overnight. Cells were harvested by centrifuga- acyltransferase gene from cultivated strawberry (SAAT) tion, and the genomic DNA was extracted using the Yeast was a highly active enzyme capable of transferring the Gene DNA Kit (CW Biotech, China). 2 μL of total DNA acetyl group from acetyl-CoA to various substrates, and was used as template for PCR using the 2 × Taq Mas- possibly to geraniol to form geranyl acetate [29]. In order ter Mix (CW Biotech). 10 colonies from SC-Leu agar to construct a metabolic pathway for the production of plates were randomly picked and inoculated in SC-Leu geranyl acetate, the geraniol synthase (GES) gene from O. medium. After that, genomic DNA was extracted, and basilicum and SAAT were integrated into the chromo- PCR determination was performed respectively. The some of S. cerevisiae BY4742 at the gal80 site (Fig.  2a). ratio of positive clones to all the colonies was calculated The expression of the synthetic cassette was controlled to be above 90%. by a constitutive strong promoter and the resulting strain was designated as GA01. The production of geranyl acetate was measured by Cell‑culture, extraction and quantification of geranyl GC–MS (Fig.  3a), and the titer ranged from 0.25 to acetate 0.63 mg/L during the fermentation process (Fig. 3b). The The correct colonies were picked and grown in the cor - titer reached its maximum value after 48  h of cultiva- responding synthetic complete drop-out medium or YPD tion, at an OD of 5.23. However, while the density of medium overnight, transferred into a flask with fresh the yeast culture increased persistently for 5  days, gera- medium to yield an initial OD of 0.05–0.10, and cul- nyl acetate production did not improve accordingly, tured for 6 days at 30 °C and 250 rpm. The increase of cell which may be due to gaseous escape of the volatile gera- biomass during the fermentation process was detected by nyl acetate during the aerobic fermentation process [32]. measuring the OD value using a UV-2550 spectropho- Besides, another four alcohol acyltransferase genes from tometer (Shimadzu, Japan). plants were used for geranyl acetate production, but the To quantify the titer of geranyl acetate in the yeast titer was very low or no geranyl acetate was detected cultures, aliquots comprising 1  mL of the fermentation compared with SAAT (Additional file 1: Table S2). broth were concentrated by centrifugation at 16,200×g for 2  min, after which 1  mL of n-hexane was added to extract the products that were secreted into the medium. The expression of Erg20 mutants for improved geranyl The cell pellet was extracted with another 1 mL of n-hex - acetate production ane under ultrasonic agitation for 30 min, and the n-hex- GPP is the starting substrate of monoterpene produc- ane phase was collected by centrifugation at 16,200×g tion pathways [19]. However, in yeast cells GPP can be for 2  min. The two extraction liquids were mixed, and converted into FPP by the bifunctional synthase Erg20. 1  µL of the combined extract was analyzed using a Agi- In addition, since FPP is the precursor of ergosterol, lent 5975C GC–MS system equipped with a HP-5 ms GC the outright deletion of Erg20 is potentially lethal [5]. column (30  m × 0.25  mm × 0.5  μm; Agilent, USA) and a Accordingly, the Erg20 F96W–N127W mutant, which triple-Axis detector. The GC–MS temperature program has a decreased FPP formation efficiency but consistent encompassed an initial temperature of 45°C for 1  min GPP production, was constructed according to an earlier and a ramp of 10°C/min to 220°C, which was maintained report [20]. The mutated Erg20, GES and SAAT genes for 5 min. Helium was used as the mobile phase at a flow were integrated into the chromosome of S. cerevisiae rate of 1.0 mL/min. The injection port, interface, and MS BY4742 at the gal80 site (Fig.  2b). The resulting strain source temperatures were 250, 300, and 180  °C, respec- GA02 produced 2.64  mg/L of geranyl acetate in 48  h, tively [29]. A reference standard comprising authentic which represented a 419% increase of titer over GA01 geranyl acetate purchased from Sigma Aldrich, USA, was (Fig. 4a). used for quantification. Wu et al. Microb Cell Fact (2018) 17:85 Page 6 of 10 Standard Sample geranyl acetate Time (min) 0.7 0.6 0.5 0.4 0.3 0.2 OD 0.1 Geranyl acetate 024487296120 144 Time (h) Fig. 3 Identification of fermentation products of strain GA01. a The time-course of OD and geranyl acetate in aerobic fermentation of strain GA01. 38. b GC–MS analysis of a cell extract of strain GA01. The mass spectrum of geranyl acetate is shown in the top right corner and the red line indicates the authentic geranyl acetate standard Overexpression of tHMG1, IDI1 and MAF1 for the enhanced biosynthesis of geranyl acetate, IDI1, MAF1 and tHMG1 production of geranyl acetate were integrated into the chromosome of GA02, resulting Isoprenoid diphosphate isomerase (IDI1) catalyzes the in strain GA03. In this best strain, the titer of geranyl ace- isomeric interconversion between IPP and DMAPP [33], tate reached 13.27  mg/L, representing a 400% increase and its overexpression can therefore potentially enhance compared with GA02, and a remarkable 2100% increase the synthesis of GPP and benefit the production of over the starting strain GA01 (Fig. 4a). monoterpenes. MAF1 represses the transcriptional activ- Since S. cerevisiae does not have a specific GPP syn - ity of RNA polymerase III, serving as a negative regula- thase (GPPS, EC 2.5.1.1), which belongs to the short- tor of the biosynthesis of tRNAs [16, 18]. Since DMAPP chain prenyltransferase family [34]. To supply more is a common substrate of both tRNA and GPP synthe- precursor for GPP synthesis, The homomeric GPPS sis, overexpression of MAF1 can divert the carbon flux from Arabidopsis thaliana (GPPS ) and the heteromeric At toward GPP formation [18]. Thus, to further improve the GPPS from Mentha piperita (GPPS ) were separately Mp Abundance OD 600 geranyl acetate(mg/L) Wu et al. Microb Cell Fact (2018) 17:85 Page 7 of 10 GPPS protein, we constructed another plasmid pRS313- pPGK-LSU-tCYC-pTEF-SSU-tADH,the two subunit were expressed with strong promoter respectively, but 12 6 the geranyl acetate production was not increased either. 10 5 Improving geranyl acetate production by optimizing 8 4 the fermentation conditions 6 3 The highest geranyl acetate production of strain GA3 reached to 13.27  mg/L in SC-LEU-HIS medium after cultivated for 48  h, and the OD reached about 5.23 (Fig.  4a). To further improve geranyl acetate produc- 2 1 tion, we adjusted the initial OD of fermentation to 0.1, which made the final OD increased to about 7.2 in 0 0 GA01 GA02 GA03 synthetic complete drop-out medium, and to about 15.6 geranyl acetate OD in YPD medium after incubated at 30 °C for 48 h. When YPD medium was used to fermentation, the titer of gera- 16 7 nyl acetate increased to 1.98, 6.07 and 20.48 mg/L respec- tively for strain GA01, GA02 and GA03 after cultivating for 48  h respectively, as shown in Fig.  5a. Furthermore, in order to prevent volatilization of geranyl acetate, 10% 4 isopropyl myristate was added to the culture medium after 24  h. The production of geranyl acetate increased to 22.49  mg/L in strain GA03 as shown in Fig.  5b, and the organic layer was easily harvested by centrifugation of the fermentation medium. However, we found geranyl acetate production did not improve continually when the strains were inoculated for 5  days. The geranyl acetate 0 0 GA03- GA03- production was improved by 1.69-fold through optimiza- GA03 pRS313-GPPSAt pRS313-GPPSMp tion of the fermentation conditions. OD geranyl acetate Fig. 4 Fermentation and quantification of geranyl acetate. a OD Discussion and geranyl acetate titer of the engineered strains GA01, GA02 and GA03. b Geranyl acetate titers of strains with plant GPP synthases In the past decades, more and more attention has arisen (GPPS : Arabidopsis thaliana GPP synthase, GPPS : Mentha piperita At Mp on heterologous production of the monoterpene geran- GPP synthase) iol. The highest titer of geraniol is about 2.0 g/L in engi - neered E. coli and 1.68  g/L in engineered S. cerevisiae respectively [17, 22]. However, there are rare reports for metabolic engineering of heterologous production of introduced into GA03 on the plasmid pRS313-TRP, monoterpene esters, such as geranyl acetate. Monoter- resulting in the strains GA03-pRS313-GPPS and GA03- At penoids such as geranyl acetate are active compounds pRS313-GPPS respectively. The relevant primers are Mp derived from many plants, which play important roles in listed in Additional file  1: Table S1, and plasmid diagrams protection against pathogens and attraction of animals, are shown in Additional file  1: Fig. S1. Structurally, the also traditionally used as additive of medicines, essential heteromeric GPPS is composed of a large subunit (LSU) oils and perfume [26]. and a small subunit (SSU) [19]. The LSU is inactive alone, The truncated O. basilicum geraniol synthase (GES) and the non-catalytic SSU acts as a modulator of the and alcohol acyltransferase from strawberry (SAAT) interaction between the two inactive subunits, resulting were found with high activity in our lab [22, 29], the in an active GPPS [19]. Consequently, the flexible fusion two genes were integrated into the chromosome of S. protein linker GGGS and (GGGS) were introduced to cerevisiae BY4742 at the gal80 site (Fig.  2a) and a titer construct a heteromeric Mentha piperita GPPS (LSU- of 0.63 mg/L was achieved. According to the method of GGGS/(GGGS) -SSU). Unfortunately, the introduction Huizhou Liu’s group [17], the titer of geraniol in cul- of plant GPPS did not effectively improve the produc - ture medium and in yeast cells after fermentation was tion of geranyl acetate (Fig.  4b). Since fusion of the two analyzed, which was measured to be zero. This result subunits might affect the expression and function of indicated geraniol was entirely converted to geranyl geranyl acetate (mg/L) geranyl acetate (mg/L) OD 600 OD 600 Wu et al. Microb Cell Fact (2018) 17:85 Page 8 of 10 higher production of 13.27 mg/L was achieved by addi- 25 18 tional integration and expression of tHMG1, IDI1 and MAF1, Overexpression of which with strong promoters increased the supplement of precursors. In the fermentation optimization process, when we used YPD media instead of the synthetic complete drop-out medium for fermentation, the OD value of 6 the fermentation broth and the yield of geranyl acetate were both increased. The OD value could reach up 4 600 to about 15.6, presenting a 198% increase compared with that in SC-LEU-HIS medium. The titer of geranyl 0 0 GA01 GA02 GA03 acetate reached 20.48  mg/L, showing a 54% increase compared with the production in SC-LEU-HIS medium geranyl acetate OD (13.27 mg/L). So YPD was more suitable for cell growth and fermentation of the engineered strains. Besides,due 25 20 to the possible gaseous escape of the volatile geranyl acetate, 10% isopropyl myristate was added to the cul- ture after 24 h fermentation. TThe production of gera - nyl acetate increased to 22.49 mg/L in strain GA03 with YPD medium as shown in Fig.  5b. However, geranyl acetate production did not improve continually in the 5-days fermentation process. We thought during the time, the accumulation of the product might affect the 0 0 growth condition and inhibit further improvement of 48 h 120 h geranyl acetate production as described by Zhao’s arti- geranyl acetate OD cle [21]. To sum up, optimization of the fermentation condi- Fig. 5 Fermentation optimization and titer of geranyl acetate. a OD and geranyl acetate titer of the engineered strains GA01, GA02 tions led to a 1.69-fold improvement of geranyl acetate and GA03 in YPD medium. b Geranyl acetate titer of strains in YPD production. And with 10% isopropyl myristate added, medium with 10% isopropyl myristate added to the culture after 24 h it might be able to prevent the volatilization of geranyl acetate and relieve the cell toxicity of geranyl acetate by extracting it from the fermentation broth [21]. acetate by SAAT by the strains we constructed, and suggested the SAAT we used was very efficient and not Conclusion the rate-limiting step of the synthesis pathway. Besides, In this research, a heterologous geranyl acetate synthe- a report just published described truncated GES from sis pathway was constructed in S. cerevisiae for the first Catharanthus roseus with site-directed mutation (Y436 time, and a product titer of 0.63  mg/L was achieved in and D501) was found to has high catalytic activity the starting strain. By expressing an Erg20 mutant to [35]. Thus, for the further improvement of the gera - divert carbon flux from FPP to GPP, the geranyl ace - nyl acetate production, selection and optimization of tate production was increased to 2.64  mg/L, although GESs might be the key focus. In engineered S. cerevi- expression of heterologous GPP did not have a signifi - siae producing monoterpene, the farnesyl diphosphate cant effect. The highest production of 13.27  mg/L was synthase Erg20p was found to be the key enzyme that achieved by additionally expressing tHMG1, IDI1 and limiting monoterpene formation [20]. GPP and FPP MAF1, which represents a remarkable 2100% increase formation are both catalyzed by Erg20p, towards either over the starting strain. Furthermore, optimization of geraniol or downstream squalene synthesis. For more the fermentation conditions led to 22.49  mg/L gera- GPP synthesis, Erg20 F96W–N127W mutant was inte- nyl acetate production, which exhibited a 35.69-fold grated into the genome, which had decreased farnesyl increase over the parent strain GA01 (Fig.  6). To our diphosphate synthase function without interference best knowledge, this work offers the first microbial cell of the growth of S. cerevisiae. And the production of factory for specific production of a monoterpene ester, geranyl acetate increased to 2.64 mg/L. Furthermore, a geranyl acetate (mg/L) geranyl acetate (mg/L) OD OD 600 Wu et al. Microb Cell Fact (2018) 17:85 Page 9 of 10 Geranyl acetate Increase Figure 8 production value 0.63 mg/L 1 GA01 OverexpressionofErg20 mutant 2.64 mg/L 4.19 GA02 Overexpression of tHMG1, IDI1 andMAF1 13.27 mg/L 21.06 GA03 GA03 in YPD medium 20.48 mg/L 32.50 GA03 in YPD medim with 35.69 22.49 mg/L 10 % isopropyl myristate Fig. 6 Diagram summarizing the increase of geranyl acetate Specifically, plasmid maps and DNA sequence data can be found in Additional demonstrating great potential for the heterologous file 1. production of many more economically important fra- grance compounds. Consent for publication I hereby give the Journal of Microbial Cell Factories the right and permission Additional file to publish this article. Ethical approval and consent to participate Additional file 1: Table S1. Primers used in this study. Table S2. Intro - Not applicable. duction of alcohol acyltransferases from plants and the titer of geranyl acetate. Fig. S1. Plasmid maps and DNA sequences. Funding This research was supported by grants from the National Natural Science Foundation of China (31522002), Natural Science Foundation of Tianjin (15JCY- BJC49400), and the Tianjin Key Technology R&D program of Tianjin Municipal Authors’ contributions Science and Technology Commission (11ZCZDSY08600). TW and SWL planned and performed the experiments, analyzed and inter- preted the data. TW wrote the manuscript, and SWL checked the manu- script. BZ, CB and XZ supervised the study, designed the experiments and Publisher’s Note analyzed and interpreted the results. All authors read and approved the final Springer Nature remains neutral with regard to jurisdictional claims in pub- manuscript. lished maps and institutional affiliations. Author details Received: 22 December 2017 Accepted: 11 May 2018 College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, People’s Republic of China. Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, People’s Repub- lic of China. Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, People’s Republic of China. References 1. Mcgarvey DJ, Croteau R. Terpenoid metabolism. Plant Cell. Acknowledgements 1995;7:1015–26. Not applicable. 2. Lapczynski A, Bhatia SP, Foxenberg RJ, Letizia CS, Api AM. Fragrance mate- rial review on geraniol. Food Chem Toxicol. 2008;46:S160. Competing interests 3. Chen W, Viljoen AM. Geraniol—a review of a commercially important The authors declare that they have no competing interests. fragrance material. S Afr J Bot. 2010;76:796–807. 4. Renninger NS, Ryder JA, Fisher KJ: Jet fuel compositions and methods of Availability of data and materials making and using same. US; 2011. We provide all necessary data for the publication of the article. All additional data is present in the article and the supplemental material documents. 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Appl Microbiol Biotechnol. 2016;100:4561–71. Ready to submit your research ? Choose BMC and benefit from: fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions

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Microbial Cell FactoriesSpringer Journals

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