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De novo biosynthesis of τ-cadinol in engineered Escherichia coli

De novo biosynthesis of τ-cadinol in engineered Escherichia coli b). Masao Takei et al. reported the effect of τ-cadinol on Introduction dendritic cells (immune response center), and the results Terpenoids are the largest class of secondary metabo- showed that τ-cadinol enhanced the differentiation and lites found in nature. So far, more than 50,000 terpenoids functional maturation of dendritic cells, which indicates have been identified. Due to the diversity of biological that τ-cadinol can be used for dendritic cell-based can- functions, many terpenoids are widely used in industries cer immunotherapy (Takei et al. 2006). In addition, it has such as chemistry, perfume, medicine and nutraceuti- been reported that τ-cadinol can effectively interact with cals (Li et  al. 2020). τ-Cadinol is a sesquiterpene widely the cell envelope of Staphylococcus aureus, resulting in distributed in plants, insects, and microorganisms with bacterial lysis and thus bactericidal effect (Claeson et  al. a wide range of application prospects (Pascal et al. 2003; 1992). Because of its effective fungicidal and insecticidal Yamada et al. 2015). The usual way to get it mainly comes action it could be used as a wood preservative in the from the heartwood of Cryptomeria japonica and Myrrh future (Wu et al. 2005). (Andersson et  al, 1997; Narita et  al. 2006). τ-Cadinol is Currently, two τ-cadinol synthases from Lavandula widely used in essential oils and spices because of its spe- angustifolia and maize have been identified (Fei et  al. cial fragrant odor, which makes it a highly sought-after 2016; Jullien et al. 2014). There are two pathways used for fragrances and scent compounds in the food and cosmet- terpenoid biosynthesis in plants: the mevalonate (MVA) ics industries. τ-Cadinol also displays extensive biologi- pathway in the cytosol and the methylerythritol phos- cal activity. As a pharmacological component of Myrrh in phate (MEP) pathway in the plastids. Therefore, MVA and Somali traditional medicine, it is used for the treatment MEP pathways may both contribute to τ-cadinol synthe- of diarrhea and wounds (Claeson et  al. 1991a, b). It has sis in plants through metabolite exchanges between cyto- been reported that τ-cadinol as a calcium antagonist can sol and plastid. The first τ-cadinol synthase, LaCADS, relax the smooth muscle in the rat aorta, which makes it was identified recently in L. angustifolia, which produced clinically potentially useful for the treatment of gastric τ-cadinol as the predominant product and r-cadinene as spasms and vasospasm diseases (Claeson et  al. 1991a, Sun  et al. Bioresources and Bioprocessing (2022) 9:29 Page 3 of 8 minor product (Jullien et  al. 2014). Subsequently, a ter- Farnesyl diphosphate synthase IspA gene from E. pene synthase ZmTPS7 from maize was characterized coli was amplified by PCR using primers IspA-XbaI/ as a τ-cadinol synthase (Fei et al. 2016). ZmTPS7 was co- IspA-SpeI-BamHI and cloned into pET28a (+) with expressed with farnesyl diphosphate synthase IspA in E. XbaI/BamHI restriction sites, creating plasmid pSY10. coli. GC–MS analysis showed that ZmTPS7 reacted with τ-Cadinol synthase CS from L. angustifolia was codon farnesyl diphosphate to form a blend of sesquiterpenoids. optimized and synthesized by Genscript (Jullien et  al. The predominant constituent was identified as τ-cadinol. 2014). CS was amplified by PCR using primers CS-XbaI/ So far there is no promising way to prepare τ-cadinol by CS-SpeI-BamHI and cloned into pET28a (+) with XbaI/ biological production method. Therefore, it is necessary BamHI restriction sites, creating plasmid pSY11. CS was to develop microbial cell factories to synthesize τ-cadinol. then excised from pSY11 with XbaI/XhoI and ligated In this study, we constructed a biosynthetic pathway for into SpeI and XhoI sites of pSY10 to create pSY12. The the production of τ-cadinol in E. coli with the heterolo- isopentenyl-diphosphate isomerase IdI gene from E. coli gous hybrid MVA pathway (Fig. 1). was amplified by PCR using primers IdI-XbaI/ IdI-XhoI and ligated into SpeI and XhoI sites of pSY12 to create Methods pSY13. The strains, primers and plasmids used in this Plasmid and strains study are summarized in Tables 1 and 2. The HMG-CoA synthase MvaS and bifunctional A110G acetyl-CoA acetyltransferase/HMG-CoA reductase Shake‑flask cultures MvaE genes from Enterococcus faecalis were synthe- Precultures of E. coli strains with recombinant plasmid sized by Genscript (Yoona et al. 2009; Wu et al. 2019) and were grown in LB at 37  °C. For production experiments amplified using primers MvaS-XbaI/MvaS-SpeI-BamHI the cells were transferred to 100 mL of YT medium con- and MvaE-XbaI/MvaE-SpeI-BamHI, and ligated into taining 20 g/L of glucose as previously described by Kong pET28a (+) via XbaI and BamHI to yield plasmid pSY06 (Kong et  al. 2020), cultivated at 30  °C and induced with and pSY07. The XbaI–XhoI fragment of MvaE from IPTG (0.1  mM). Chloromycetin (36  mg/L), ampicillin pSY07 was inserted into SpeI and XhoI sites of pSY06 to (100 mg/L) and kanamycin (50 mg/L) were added to the give pSY08. The XbaI–XhoI fragment of MvaS and medium, when needed. In a two-phase organic overlay- A110G MvaE from pDG08 was inserted into XbaI and XhoI sites culture system, 10  ml of n-dodecane was added as the of pDG30 to give pSY09. upper covering extraction system. Fig. 1 Engineered pathway for the production of τ-cadinol from glucose Sun et al. Bioresources and Bioprocessing (2022) 9:29 Page 4 of 8 Table 1 Primers used in this study Primer name Sequence (5′–3′) MvaS-XbaICTA TCT AGA AAG AGG AGA TAT AAT GAC CAT TGG TAT TGA TAA AAT CAG CT MvaS-SpeI-BamHITAC GGA TCC ACT AGT TTA GTT GCG ATA GCT GCG CAC G MvaE-XbaIGTA TCT AGA AAG AGG AGA TAT AAT GAA GAC CGT TGT GAT TAT TGACG MvaE-SpeI-BamHITAT GGA TCC ACT AGT TTA CTG TTT ACG CAG GTC GTT CAGG IspA-XbaIGTA TCT AGA AAG AGG AGA TAT AAT GGA CTT TCC GCA GCA ACT CG IspA-SpeI-BamHITAT GGA TCC ACT AGT TTA TTT ATT ACG CTG GAT GAT GTA GTC CG CS-XbaIGTA TCT AGA AAG AGG AGA TAT AAT GGC GAC GAG CGC GGT CS-SpeI-BamHITAT GGA TCC ACT AGT TTA AAA CAC CAG CGG ATC TAA AAACA IdI-XbaIATC TCT AGA AAG AAG GAG ATA TAA TGC AAA CGG AAC ACG TCA TTT TAT IdI-XhoIACA TCT CGA GTT ATT TAA GCT GGG TAA ATG CAG ATA ATC Table 2 Plasmids and strains used in this study Plasmids Properties Copy number Source pDG30 pSC101 origin, Amp , pT7: AtoB, ERG13 and tHMG1 1 Guo et al. 2018 pDG31 pBBRMCS1 origin, Chl , pT7: IdI, ERG8, MVD1 and ERG12 10–20 Guo et al. 2018 pSY09 pSC101 origin, Amp , pT7: Mva and MvaE 1 This study SA110G pSY12 pBR322 origin, Kan , pT7: IspA and CS 100–200 This study pSY13 pBR322 origin, Kan , pT7: IspA, CS and IdI 100–200 This study Strains SY01 BL21(DE3) derivative: pSY09 carrying Mva and MvaE, pDG31 carrying IdI, ERG8, MVD1 and ERG12, This study SA110G and pSY12 carrying IspA and CS SY02 BL21(DE3) derivative: pDG30 carrying AtoB, ERG13 and tHMG1, pDG31 carrying IdI, ERG8, MVD1 and This study ERG12, and pSY12 carrying IspA and CS SY03 BL21(DE3) derivative: pDG30 carrying AtoB, ERG13 and tHMG1, pDG31 carrying IdI, ERG8, MVD1 and This study ERG12, and pSY13 carrying IspA, CS and IdI Glucose assays pentadecanoate was used as internal standard for quan- Residual glucose concentrations were measured with titative τ-cadinol. Compound identification was per - a Glucose Assay Kit (Cat. MAK263, Merck). A glu- formed using NIST and Adams mass spectra databases. cose standard solution was used to create a calibration curve. Fermentation broth samples were thawed and Results centrifuged at 6000  g for 5  min to isolate supernatants Construction of τ‑cadinol biosynthetic pathway in E. coli for glucose content analysis. Dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP) are two precursor units of terpe- noids. E. coli synthesizes DMAPP and IPP through the GC/MS analysis of τ‑cadinol MEP pathway. However, due to the regulatory mecha- Cells from 5  mL culture broth were lysed with glass nism in E. coli, the production of target products by beads (0.1  mm) by vigorous vortexing for 5  min. Equal the MEP pathway is restricted (Martin et al. 2003). To volume of ethyl acetate is used to extract τ-cadinol. get rid of regulatory constraints, we design a heterolo- In a two-phase organic overlay-culture system, the gous hybrid MVA pathway to produce τ-cadinol in E. n-dodecane phase was diluted in ethyl acetate with coli. The heterologous hybrid MVA pathway is mainly 10:1 prior to GC/MS analysis. The ethyl acetate phase composed of nine genes: mutated HMG-CoA syn- was analyzed using an Agilent 7890A GC/MS equipped thase MvaS and bifunctional acetyltransferase/ A110G with a HP–5MS column. After splitless injection, the HMG-CoA reductase MvaE from E. faecalis for the initial temperature of 80  °C was increased for 20  °C/ conversion of acetoacetyl-CoA to mevalonate. IPP min to 260  °C, maintain 260  °C for 8  min. Methyl isomerase IdI and farnesyl diphosphate synthase IspA Sun  et al. Bioresources and Bioprocessing (2022) 9:29 Page 5 of 8 from E. coli, phosphomevalonate kinase ERG8, meva- Table 3 τ-Cadinol production in engineered E. coli strains in shake flasks for 36 h lonate pyrophosphate decarboxylase MVD1, meva- lonate kinase ERG12 from Saccharomyces cerevisiae Strains τ‑ Cadinol (mg/L) Yield (mg/g) for the conversion of mevalonate to farnesyl diphos- SY01 7.2 ± 0.8 0.45 ± 0.05 phate, τ-cadinol synthase CS from L. angustifolia for SY02 24.4 ± 2.6 1.51 ± 0.16 the conversion of farnesyl diphosphate to τ-cadinol. SY03 35.9 ± 4.3 2.20 ± 0.26 Plasmid pSY09 with co-expression of m vaS and A110G SY03 with overlay- 133.5 ± 11.2 8.04 ± 0.67 mvaE, pDG31 with co-expression of IdI, ERG8, MVD1 solvent and ERG12 (Guo et  al. 2018), and pSY12 with co- Yield calculated by milligrams τ-cadinol produced divided by grams glucose expression of IspA and CS were introduced into BL21 consumed (DE3), generating strain SY01. Strain with empty plas- All experiments were performed in triplicate and SD is indicated mid without the gene of interest is used as a negative control. Strain SY01 was cultured in YT medium with 20  g/L glucose. When the OD600 reaches about 0.8, (Additional file  1: Fig. S2). The time course of cell IPTG at a final concentration of 0.1  mM was added OD600 and τ-cadinol concentration of recombinant E. to induce gene expression. τ-Cadinol was extracted coli in shake flasks are shown in Fig.  2. τ-Cadinol can from recombinant E. coli SY01 and analyzed by GC– reach 7.2 ± 0.8  mg/L after 36  h of shake-flask fermen- MS (Additional file  1: Fig. S1). The biosynthesis of tation (Table  3). The results indicate that the designed τ-cadinol was confirmed through comparison with τ-cadinol biosynthetic pathway from glucose was the standard mass fractionation in GC–MS database effective in E. coli. Fig. 2 The time course of cell OD600 and τ-cadinol concentration of engineered E. coli strains in shake flasks. A SY01 strain; B SY02 strain; C SY03 strain; D SY03 strain with overlay-solvent Sun et al. Bioresources and Bioprocessing (2022) 9:29 Page 6 of 8 Improving τ‑cadinol biosynthesis by replacing mvaS a two-phase organic overlay-culture system with A110G and mvaE with AtoB, ERG13 and tHMG1 n-dodecane overlay. After 36  h of shake-flask fermenta - Mevalonate is a precursor substrate for the biosynthesis tion, the τ-cadinol titer of E. coli strain SY03 obtained of τ-cadinol. Therefore, we hypothesized that increasing by two-phase organic overlay-culture system reached mevalonate availability would improve τ-cadinol produc- 133.5 ± 11.2  mg/L (Table  3), representing 3.7-fold tion. For biosynthesis of mevalonate from acetyl-CoA, improvements, when compared with that achieved in no- an earlier study showed that a hybrid pathway consists solvent cultures. The time course of glucose concentra - of E. coli acetoacetyl-CoA thiolase AtoB, S. cerevisiae tion of recombinant E. coli in shake flasks are shown in 3-hydroxy-3-methylglutaryl-CoA synthase ERG13 and Fig. 3. τ-Cadinol yield was calculated to be 8.04 mg/g glu- truncated HMG-CoA reductase tHMGR can enhance the cose and 2.9% of the theoretical yield (Table 3). metabolic flux of mevalonate in E. coli (Zhu et al. 2014). Therefore, we evaluated the effect of replacing MvaS Discussion A110G and MvaE with AtoB, ERG13 and tHMG1 on τ-cadinol Terpenoids, including monoterpenes (C10), sesquit- synthesis. Plasmid pDG30 with co-expression of AtoB, erpenes (C15), diterpenes (C20) and triterpenoids ERG13 and tHMG1 (Guo et al. 2018), pDG31and pSY12 (C30), are synthesized by terpene synthases with IPP were introduced into BL21 (DE3), generating strain and DMAPP as substrates (Yang et  al. 2013; Zhang SY02. The strain SY02 produced up to 24.4 ± 2.6  mg/L et  al. 2013; Zong et  al. 2019). Traditionally, terpenoids τ-cadinol in shake flasks for 36  h (Table  3), which indi- are synthesized by plants in low amounts. The pro - cated that the replacement of MvaS and MvaE duction of terpenoids by physical extraction is a time- A110G with AtoB, ERG13 and tHMG1 can effectively improve consuming and laborious process. In addition, the long τ-cadinol production. growing seasons of such plants is subject to high lev- els of variation due to differences in soil, climate and geography, which creates difficulties for the company’s Overexpression of IdI gene promotes the production supply chain. Synthetic biology applies genetic engi- of τ‑cadinol neering tools to build synthetic pathways in microbial The farnesyl diphosphate synthase IspA catalyzes the for - cell factories to overproduce chemicals with high titers mation of farnesyl diphosphate through the head-to-tail and yields. Thus, it is of great significance to develop condensation of one DMAPP and two IPP (Fujisaki et al. microbial cell factories for achieving terpenoids bio- 1990). The IPP isomerase IdI catalyzes the isomeriza - synthesis. Sesquiterpene synthases catalyze the con- tion between DMAPP and IPP. Previous reports indicate version from farnesyl diphosphate to a large variety that IdI is a rate-limiting enzyme and its overexpression of sesquiterpenes. To date, various sesquiterpenes can improve the biosynthesis of farnesyl diphosphate were successfully produced by recombinant micro- (Guo et  al. 2018). Farnesyl diphosphate is the precur- organism, such as β-copaene (Mischko et  al. 2018), sor substrate for the biosynthesis of τ-cadinol. Thus, nerolidol (Qu et  al. 2019), farnesol (Chonglong et  al. it is expected to increase the production of τ-cadinol by enhancing the synthesis ability of farnesyl diphos- phate. In this study, IdI was placed on the high-copy plasmid pSY12 and overexpressed together with CS and IspA. The resulting E. coli strain SY03 produced up to 35.9 ± 4.3  mg/L τ-cadinol in shake flasks for 36  h (Table  3), which indicated that the overexpression of IdI can effectively improve τ-cadinol production. Establishment of a two‑phase fermentation system The efficient production of terpenoids by microorgan - isms may be limited by end product feedback inhibi- tion. Two-phase organic overlay-culture system has been shown to work with a large number of different recom - binant strains and to improve the production capacity of the host strain by relieving the end product feedback inhibition (Li et al. 2019; Sun et al. 2021). Dodecane was Fig. 3 The time course of glucose concentration of engineered E. coli considered as an ideal overlay-solvent (Frohwitter et  al. strains in shake flasks 2014; Nadja et al. 2018). In this study, we have established Sun  et al. Bioresources and Bioprocessing (2022) 9:29 Page 7 of 8 2010), patchoulol (Liu et  al. 2021), humulene (Harada link the upstream MVA pathway with the downstream et  al. 2009) and other important sesquiterpenes. Yang carotenogenic pathway increased carotenoid production et  al. reported that the engineered E. coli synthesized by 5.7-fold (Kang et  al. 2019). Therefore, future efforts 220 ± 6  mg/L of β-caryophyllene from glucose by co- (for example, reduce the accumulation of intermediates expression of geranyl diphosphate synthase, glucose- by the optimization of each biosynthetic component 6-phosphate dehydrogenase and β-caryophyllene and identification of feedback-resistant enzyme, increas - synthase genes (Du et  al. 2016). Liu et  al. reported ing the supply of redox cofactors, discovery of novel that patchoulol titer in the recombinant S. cerevisiae τ-cadinol synthase, and prevent the diffusion of inter - reached 141.5  mg/L in a shake flask (Liu et  al. 2021). mediate by assembling multienzyme complexes) will be Han et  al. reported that engineered E. coli synthesized needed to improve the production of τ-cadinol. 80 mg/L of (−)-α-bisabolol in the shake-flask culture by combinatorial expression of the exogenous MVA path- Conclusions way, farnesyl diphosphate synthase and (−)-α-bisabolol τ-Cadinol is present in minute quantities in plants, so synthase (Han et al. 2016). physical extracts of τ-cadinol is limited by the low yields. τ-Cadinol is a sesquiterpene that is widely used in Production of τ-cadinol by microbial cell factories may be perfume, fine chemicals and medicines industry. In this an alternative and promising approach. This study shows study, we constructed a biosynthetic pathway from glu- an efficient method for the biosynthesis of τ-cadinol in E. cose to τ-cadinol in E. coli by combinatorial expression coli with the heterologous hybrid MVA pathway. of the exogenous MVA pathway, farnesyl diphosphate synthase IspA and τ-cadinol synthase CS. The biosyn - Supplementary Information thesis of τ-cadinol was further improved by optimizing The online version contains supplementary material available at https:// doi. biosynthetic pathway and overexpression of rate-limit-org/ 10. 1186/ s40643- 022- 00521-7. ing enzyme IdI. Finally, we increased the production of Additional file 1: Figure S1. GC/MS analyses of τ-cadinol in engineered τ-cadinol to 133.5 ± 11.2  mg/L by relieving end product E. coli strains. Identified substances: 1, τ-cadinol; 2, methyl pentade - feedback repression with a two-phase organic overlay- canoate (internal standard). Figure S2. Identification of τ-cadinol by culture system. Zhu et al. established an in vitro reconsti- Mass fractionation comparison. Mass fractionation of peaks identified in GC–MS (peak 1, RT = 7.7 min) of samples from engineered E. coli strains tuted terpenoid pathway system that allows monitoring (red) match that obtained from τ-cadinol standard with database searches of the steady-state kinetic and biochemical parameters (blue). Figure S3. The schematic diagram of the two-phase organic and the accumulation of intermediates (Zhu et al. 2014). overlay-culture system. Figure S4. Diagram of plasmids pSY 09. Figure S5. Diagram of plasmid pSY 13. The information thus gained could be used to guide the optimization of each biosynthetic component in E. coli for improvement of the production of terpenoid-derived Acknowledgements Not applicable. compounds in vivo. Based on this information, we placed AtoB, ERG13 and tHMG1 genes on low-copy plasmids, Authors’ contributions IdI, ERG8, MVD1 and ERG12 genes on medium-copy DG and CL conceived and designed the research. YS, SW and XF performed the experiments. DG analyzed the data and drafted the manuscript. All plasmids, and IspA and CS genes on high-copy plasmids. authors read and approved the final manuscript. Recruiting better performing enzymes is often a good strategy to break bottlenecks in engineered MVA path- Funding This work was financially supported by the National Natural Science Founda- ways. S. cerevisiae ERG12 is inhibited by substrate MVA. tion of China (31960216 and 31860019) and National Science Foundation of Therefore, the use of feedback-resistant ERG12 can Jiangxi Province (20192BBG70007, 20192BCBL23012 and 2019BBG70070). reduce MVA accumulation and promote the biosyn- Availability of data and materials thesis of terpenoids. Chen et  al. reported that lycopene All data generated or analyzed in this study are included in this published titer was increased 2.4 folds by replacing the wild-type article and its Additional file 1. ERG12 from S. cerevisiae with the feedback-resistant ERG12 (Chen et  al. 2018). Rad et  al. reported that use Declarations of B. licheniformis IdI in place of E. coli IdI increased Ethics approval and consent to participate lycopene production 1.4 folds in engineered E. coli (Rad Not applicable. et  al. 2012). HMGR consumes 2 molecules of NADPH to reduce HMG-CoA to mevalonate. Therefore, increas - Consent for publication Not applicable. ing the supply of NADPH will be a potentially important strategy to improve the yield of terpenoids. Recently, Competing interests assembling multienzyme complexes to prevent interme- The authors declare that they have no competing interests. diate diffusion through RIAD–RIDD interaction so as to Sun et al. 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De novo biosynthesis of τ-cadinol in engineered Escherichia coli

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Abstract

b). Masao Takei et al. reported the effect of τ-cadinol on Introduction dendritic cells (immune response center), and the results Terpenoids are the largest class of secondary metabo- showed that τ-cadinol enhanced the differentiation and lites found in nature. So far, more than 50,000 terpenoids functional maturation of dendritic cells, which indicates have been identified. Due to the diversity of biological that τ-cadinol can be used for dendritic cell-based can- functions, many terpenoids are widely used in industries cer immunotherapy (Takei et al. 2006). In addition, it has such as chemistry, perfume, medicine and nutraceuti- been reported that τ-cadinol can effectively interact with cals (Li et  al. 2020). τ-Cadinol is a sesquiterpene widely the cell envelope of Staphylococcus aureus, resulting in distributed in plants, insects, and microorganisms with bacterial lysis and thus bactericidal effect (Claeson et  al. a wide range of application prospects (Pascal et al. 2003; 1992). Because of its effective fungicidal and insecticidal Yamada et al. 2015). The usual way to get it mainly comes action it could be used as a wood preservative in the from the heartwood of Cryptomeria japonica and Myrrh future (Wu et al. 2005). (Andersson et  al, 1997; Narita et  al. 2006). τ-Cadinol is Currently, two τ-cadinol synthases from Lavandula widely used in essential oils and spices because of its spe- angustifolia and maize have been identified (Fei et  al. cial fragrant odor, which makes it a highly sought-after 2016; Jullien et al. 2014). There are two pathways used for fragrances and scent compounds in the food and cosmet- terpenoid biosynthesis in plants: the mevalonate (MVA) ics industries. τ-Cadinol also displays extensive biologi- pathway in the cytosol and the methylerythritol phos- cal activity. As a pharmacological component of Myrrh in phate (MEP) pathway in the plastids. Therefore, MVA and Somali traditional medicine, it is used for the treatment MEP pathways may both contribute to τ-cadinol synthe- of diarrhea and wounds (Claeson et  al. 1991a, b). It has sis in plants through metabolite exchanges between cyto- been reported that τ-cadinol as a calcium antagonist can sol and plastid. The first τ-cadinol synthase, LaCADS, relax the smooth muscle in the rat aorta, which makes it was identified recently in L. angustifolia, which produced clinically potentially useful for the treatment of gastric τ-cadinol as the predominant product and r-cadinene as spasms and vasospasm diseases (Claeson et  al. 1991a, Sun  et al. Bioresources and Bioprocessing (2022) 9:29 Page 3 of 8 minor product (Jullien et  al. 2014). Subsequently, a ter- Farnesyl diphosphate synthase IspA gene from E. pene synthase ZmTPS7 from maize was characterized coli was amplified by PCR using primers IspA-XbaI/ as a τ-cadinol synthase (Fei et al. 2016). ZmTPS7 was co- IspA-SpeI-BamHI and cloned into pET28a (+) with expressed with farnesyl diphosphate synthase IspA in E. XbaI/BamHI restriction sites, creating plasmid pSY10. coli. GC–MS analysis showed that ZmTPS7 reacted with τ-Cadinol synthase CS from L. angustifolia was codon farnesyl diphosphate to form a blend of sesquiterpenoids. optimized and synthesized by Genscript (Jullien et  al. The predominant constituent was identified as τ-cadinol. 2014). CS was amplified by PCR using primers CS-XbaI/ So far there is no promising way to prepare τ-cadinol by CS-SpeI-BamHI and cloned into pET28a (+) with XbaI/ biological production method. Therefore, it is necessary BamHI restriction sites, creating plasmid pSY11. CS was to develop microbial cell factories to synthesize τ-cadinol. then excised from pSY11 with XbaI/XhoI and ligated In this study, we constructed a biosynthetic pathway for into SpeI and XhoI sites of pSY10 to create pSY12. The the production of τ-cadinol in E. coli with the heterolo- isopentenyl-diphosphate isomerase IdI gene from E. coli gous hybrid MVA pathway (Fig. 1). was amplified by PCR using primers IdI-XbaI/ IdI-XhoI and ligated into SpeI and XhoI sites of pSY12 to create Methods pSY13. The strains, primers and plasmids used in this Plasmid and strains study are summarized in Tables 1 and 2. The HMG-CoA synthase MvaS and bifunctional A110G acetyl-CoA acetyltransferase/HMG-CoA reductase Shake‑flask cultures MvaE genes from Enterococcus faecalis were synthe- Precultures of E. coli strains with recombinant plasmid sized by Genscript (Yoona et al. 2009; Wu et al. 2019) and were grown in LB at 37  °C. For production experiments amplified using primers MvaS-XbaI/MvaS-SpeI-BamHI the cells were transferred to 100 mL of YT medium con- and MvaE-XbaI/MvaE-SpeI-BamHI, and ligated into taining 20 g/L of glucose as previously described by Kong pET28a (+) via XbaI and BamHI to yield plasmid pSY06 (Kong et  al. 2020), cultivated at 30  °C and induced with and pSY07. The XbaI–XhoI fragment of MvaE from IPTG (0.1  mM). Chloromycetin (36  mg/L), ampicillin pSY07 was inserted into SpeI and XhoI sites of pSY06 to (100 mg/L) and kanamycin (50 mg/L) were added to the give pSY08. The XbaI–XhoI fragment of MvaS and medium, when needed. In a two-phase organic overlay- A110G MvaE from pDG08 was inserted into XbaI and XhoI sites culture system, 10  ml of n-dodecane was added as the of pDG30 to give pSY09. upper covering extraction system. Fig. 1 Engineered pathway for the production of τ-cadinol from glucose Sun et al. Bioresources and Bioprocessing (2022) 9:29 Page 4 of 8 Table 1 Primers used in this study Primer name Sequence (5′–3′) MvaS-XbaICTA TCT AGA AAG AGG AGA TAT AAT GAC CAT TGG TAT TGA TAA AAT CAG CT MvaS-SpeI-BamHITAC GGA TCC ACT AGT TTA GTT GCG ATA GCT GCG CAC G MvaE-XbaIGTA TCT AGA AAG AGG AGA TAT AAT GAA GAC CGT TGT GAT TAT TGACG MvaE-SpeI-BamHITAT GGA TCC ACT AGT TTA CTG TTT ACG CAG GTC GTT CAGG IspA-XbaIGTA TCT AGA AAG AGG AGA TAT AAT GGA CTT TCC GCA GCA ACT CG IspA-SpeI-BamHITAT GGA TCC ACT AGT TTA TTT ATT ACG CTG GAT GAT GTA GTC CG CS-XbaIGTA TCT AGA AAG AGG AGA TAT AAT GGC GAC GAG CGC GGT CS-SpeI-BamHITAT GGA TCC ACT AGT TTA AAA CAC CAG CGG ATC TAA AAACA IdI-XbaIATC TCT AGA AAG AAG GAG ATA TAA TGC AAA CGG AAC ACG TCA TTT TAT IdI-XhoIACA TCT CGA GTT ATT TAA GCT GGG TAA ATG CAG ATA ATC Table 2 Plasmids and strains used in this study Plasmids Properties Copy number Source pDG30 pSC101 origin, Amp , pT7: AtoB, ERG13 and tHMG1 1 Guo et al. 2018 pDG31 pBBRMCS1 origin, Chl , pT7: IdI, ERG8, MVD1 and ERG12 10–20 Guo et al. 2018 pSY09 pSC101 origin, Amp , pT7: Mva and MvaE 1 This study SA110G pSY12 pBR322 origin, Kan , pT7: IspA and CS 100–200 This study pSY13 pBR322 origin, Kan , pT7: IspA, CS and IdI 100–200 This study Strains SY01 BL21(DE3) derivative: pSY09 carrying Mva and MvaE, pDG31 carrying IdI, ERG8, MVD1 and ERG12, This study SA110G and pSY12 carrying IspA and CS SY02 BL21(DE3) derivative: pDG30 carrying AtoB, ERG13 and tHMG1, pDG31 carrying IdI, ERG8, MVD1 and This study ERG12, and pSY12 carrying IspA and CS SY03 BL21(DE3) derivative: pDG30 carrying AtoB, ERG13 and tHMG1, pDG31 carrying IdI, ERG8, MVD1 and This study ERG12, and pSY13 carrying IspA, CS and IdI Glucose assays pentadecanoate was used as internal standard for quan- Residual glucose concentrations were measured with titative τ-cadinol. Compound identification was per - a Glucose Assay Kit (Cat. MAK263, Merck). A glu- formed using NIST and Adams mass spectra databases. cose standard solution was used to create a calibration curve. Fermentation broth samples were thawed and Results centrifuged at 6000  g for 5  min to isolate supernatants Construction of τ‑cadinol biosynthetic pathway in E. coli for glucose content analysis. Dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP) are two precursor units of terpe- noids. E. coli synthesizes DMAPP and IPP through the GC/MS analysis of τ‑cadinol MEP pathway. However, due to the regulatory mecha- Cells from 5  mL culture broth were lysed with glass nism in E. coli, the production of target products by beads (0.1  mm) by vigorous vortexing for 5  min. Equal the MEP pathway is restricted (Martin et al. 2003). To volume of ethyl acetate is used to extract τ-cadinol. get rid of regulatory constraints, we design a heterolo- In a two-phase organic overlay-culture system, the gous hybrid MVA pathway to produce τ-cadinol in E. n-dodecane phase was diluted in ethyl acetate with coli. The heterologous hybrid MVA pathway is mainly 10:1 prior to GC/MS analysis. The ethyl acetate phase composed of nine genes: mutated HMG-CoA syn- was analyzed using an Agilent 7890A GC/MS equipped thase MvaS and bifunctional acetyltransferase/ A110G with a HP–5MS column. After splitless injection, the HMG-CoA reductase MvaE from E. faecalis for the initial temperature of 80  °C was increased for 20  °C/ conversion of acetoacetyl-CoA to mevalonate. IPP min to 260  °C, maintain 260  °C for 8  min. Methyl isomerase IdI and farnesyl diphosphate synthase IspA Sun  et al. Bioresources and Bioprocessing (2022) 9:29 Page 5 of 8 from E. coli, phosphomevalonate kinase ERG8, meva- Table 3 τ-Cadinol production in engineered E. coli strains in shake flasks for 36 h lonate pyrophosphate decarboxylase MVD1, meva- lonate kinase ERG12 from Saccharomyces cerevisiae Strains τ‑ Cadinol (mg/L) Yield (mg/g) for the conversion of mevalonate to farnesyl diphos- SY01 7.2 ± 0.8 0.45 ± 0.05 phate, τ-cadinol synthase CS from L. angustifolia for SY02 24.4 ± 2.6 1.51 ± 0.16 the conversion of farnesyl diphosphate to τ-cadinol. SY03 35.9 ± 4.3 2.20 ± 0.26 Plasmid pSY09 with co-expression of m vaS and A110G SY03 with overlay- 133.5 ± 11.2 8.04 ± 0.67 mvaE, pDG31 with co-expression of IdI, ERG8, MVD1 solvent and ERG12 (Guo et  al. 2018), and pSY12 with co- Yield calculated by milligrams τ-cadinol produced divided by grams glucose expression of IspA and CS were introduced into BL21 consumed (DE3), generating strain SY01. Strain with empty plas- All experiments were performed in triplicate and SD is indicated mid without the gene of interest is used as a negative control. Strain SY01 was cultured in YT medium with 20  g/L glucose. When the OD600 reaches about 0.8, (Additional file  1: Fig. S2). The time course of cell IPTG at a final concentration of 0.1  mM was added OD600 and τ-cadinol concentration of recombinant E. to induce gene expression. τ-Cadinol was extracted coli in shake flasks are shown in Fig.  2. τ-Cadinol can from recombinant E. coli SY01 and analyzed by GC– reach 7.2 ± 0.8  mg/L after 36  h of shake-flask fermen- MS (Additional file  1: Fig. S1). The biosynthesis of tation (Table  3). The results indicate that the designed τ-cadinol was confirmed through comparison with τ-cadinol biosynthetic pathway from glucose was the standard mass fractionation in GC–MS database effective in E. coli. Fig. 2 The time course of cell OD600 and τ-cadinol concentration of engineered E. coli strains in shake flasks. A SY01 strain; B SY02 strain; C SY03 strain; D SY03 strain with overlay-solvent Sun et al. Bioresources and Bioprocessing (2022) 9:29 Page 6 of 8 Improving τ‑cadinol biosynthesis by replacing mvaS a two-phase organic overlay-culture system with A110G and mvaE with AtoB, ERG13 and tHMG1 n-dodecane overlay. After 36  h of shake-flask fermenta - Mevalonate is a precursor substrate for the biosynthesis tion, the τ-cadinol titer of E. coli strain SY03 obtained of τ-cadinol. Therefore, we hypothesized that increasing by two-phase organic overlay-culture system reached mevalonate availability would improve τ-cadinol produc- 133.5 ± 11.2  mg/L (Table  3), representing 3.7-fold tion. For biosynthesis of mevalonate from acetyl-CoA, improvements, when compared with that achieved in no- an earlier study showed that a hybrid pathway consists solvent cultures. The time course of glucose concentra - of E. coli acetoacetyl-CoA thiolase AtoB, S. cerevisiae tion of recombinant E. coli in shake flasks are shown in 3-hydroxy-3-methylglutaryl-CoA synthase ERG13 and Fig. 3. τ-Cadinol yield was calculated to be 8.04 mg/g glu- truncated HMG-CoA reductase tHMGR can enhance the cose and 2.9% of the theoretical yield (Table 3). metabolic flux of mevalonate in E. coli (Zhu et al. 2014). Therefore, we evaluated the effect of replacing MvaS Discussion A110G and MvaE with AtoB, ERG13 and tHMG1 on τ-cadinol Terpenoids, including monoterpenes (C10), sesquit- synthesis. Plasmid pDG30 with co-expression of AtoB, erpenes (C15), diterpenes (C20) and triterpenoids ERG13 and tHMG1 (Guo et al. 2018), pDG31and pSY12 (C30), are synthesized by terpene synthases with IPP were introduced into BL21 (DE3), generating strain and DMAPP as substrates (Yang et  al. 2013; Zhang SY02. The strain SY02 produced up to 24.4 ± 2.6  mg/L et  al. 2013; Zong et  al. 2019). Traditionally, terpenoids τ-cadinol in shake flasks for 36  h (Table  3), which indi- are synthesized by plants in low amounts. The pro - cated that the replacement of MvaS and MvaE duction of terpenoids by physical extraction is a time- A110G with AtoB, ERG13 and tHMG1 can effectively improve consuming and laborious process. In addition, the long τ-cadinol production. growing seasons of such plants is subject to high lev- els of variation due to differences in soil, climate and geography, which creates difficulties for the company’s Overexpression of IdI gene promotes the production supply chain. Synthetic biology applies genetic engi- of τ‑cadinol neering tools to build synthetic pathways in microbial The farnesyl diphosphate synthase IspA catalyzes the for - cell factories to overproduce chemicals with high titers mation of farnesyl diphosphate through the head-to-tail and yields. Thus, it is of great significance to develop condensation of one DMAPP and two IPP (Fujisaki et al. microbial cell factories for achieving terpenoids bio- 1990). The IPP isomerase IdI catalyzes the isomeriza - synthesis. Sesquiterpene synthases catalyze the con- tion between DMAPP and IPP. Previous reports indicate version from farnesyl diphosphate to a large variety that IdI is a rate-limiting enzyme and its overexpression of sesquiterpenes. To date, various sesquiterpenes can improve the biosynthesis of farnesyl diphosphate were successfully produced by recombinant micro- (Guo et  al. 2018). Farnesyl diphosphate is the precur- organism, such as β-copaene (Mischko et  al. 2018), sor substrate for the biosynthesis of τ-cadinol. Thus, nerolidol (Qu et  al. 2019), farnesol (Chonglong et  al. it is expected to increase the production of τ-cadinol by enhancing the synthesis ability of farnesyl diphos- phate. In this study, IdI was placed on the high-copy plasmid pSY12 and overexpressed together with CS and IspA. The resulting E. coli strain SY03 produced up to 35.9 ± 4.3  mg/L τ-cadinol in shake flasks for 36  h (Table  3), which indicated that the overexpression of IdI can effectively improve τ-cadinol production. Establishment of a two‑phase fermentation system The efficient production of terpenoids by microorgan - isms may be limited by end product feedback inhibi- tion. Two-phase organic overlay-culture system has been shown to work with a large number of different recom - binant strains and to improve the production capacity of the host strain by relieving the end product feedback inhibition (Li et al. 2019; Sun et al. 2021). Dodecane was Fig. 3 The time course of glucose concentration of engineered E. coli considered as an ideal overlay-solvent (Frohwitter et  al. strains in shake flasks 2014; Nadja et al. 2018). In this study, we have established Sun  et al. Bioresources and Bioprocessing (2022) 9:29 Page 7 of 8 2010), patchoulol (Liu et  al. 2021), humulene (Harada link the upstream MVA pathway with the downstream et  al. 2009) and other important sesquiterpenes. Yang carotenogenic pathway increased carotenoid production et  al. reported that the engineered E. coli synthesized by 5.7-fold (Kang et  al. 2019). Therefore, future efforts 220 ± 6  mg/L of β-caryophyllene from glucose by co- (for example, reduce the accumulation of intermediates expression of geranyl diphosphate synthase, glucose- by the optimization of each biosynthetic component 6-phosphate dehydrogenase and β-caryophyllene and identification of feedback-resistant enzyme, increas - synthase genes (Du et  al. 2016). Liu et  al. reported ing the supply of redox cofactors, discovery of novel that patchoulol titer in the recombinant S. cerevisiae τ-cadinol synthase, and prevent the diffusion of inter - reached 141.5  mg/L in a shake flask (Liu et  al. 2021). mediate by assembling multienzyme complexes) will be Han et  al. reported that engineered E. coli synthesized needed to improve the production of τ-cadinol. 80 mg/L of (−)-α-bisabolol in the shake-flask culture by combinatorial expression of the exogenous MVA path- Conclusions way, farnesyl diphosphate synthase and (−)-α-bisabolol τ-Cadinol is present in minute quantities in plants, so synthase (Han et al. 2016). physical extracts of τ-cadinol is limited by the low yields. τ-Cadinol is a sesquiterpene that is widely used in Production of τ-cadinol by microbial cell factories may be perfume, fine chemicals and medicines industry. In this an alternative and promising approach. This study shows study, we constructed a biosynthetic pathway from glu- an efficient method for the biosynthesis of τ-cadinol in E. cose to τ-cadinol in E. coli by combinatorial expression coli with the heterologous hybrid MVA pathway. of the exogenous MVA pathway, farnesyl diphosphate synthase IspA and τ-cadinol synthase CS. The biosyn - Supplementary Information thesis of τ-cadinol was further improved by optimizing The online version contains supplementary material available at https:// doi. biosynthetic pathway and overexpression of rate-limit-org/ 10. 1186/ s40643- 022- 00521-7. ing enzyme IdI. Finally, we increased the production of Additional file 1: Figure S1. GC/MS analyses of τ-cadinol in engineered τ-cadinol to 133.5 ± 11.2  mg/L by relieving end product E. coli strains. Identified substances: 1, τ-cadinol; 2, methyl pentade - feedback repression with a two-phase organic overlay- canoate (internal standard). Figure S2. Identification of τ-cadinol by culture system. Zhu et al. established an in vitro reconsti- Mass fractionation comparison. Mass fractionation of peaks identified in GC–MS (peak 1, RT = 7.7 min) of samples from engineered E. coli strains tuted terpenoid pathway system that allows monitoring (red) match that obtained from τ-cadinol standard with database searches of the steady-state kinetic and biochemical parameters (blue). Figure S3. The schematic diagram of the two-phase organic and the accumulation of intermediates (Zhu et al. 2014). overlay-culture system. Figure S4. Diagram of plasmids pSY 09. Figure S5. Diagram of plasmid pSY 13. The information thus gained could be used to guide the optimization of each biosynthetic component in E. coli for improvement of the production of terpenoid-derived Acknowledgements Not applicable. compounds in vivo. Based on this information, we placed AtoB, ERG13 and tHMG1 genes on low-copy plasmids, Authors’ contributions IdI, ERG8, MVD1 and ERG12 genes on medium-copy DG and CL conceived and designed the research. YS, SW and XF performed the experiments. DG analyzed the data and drafted the manuscript. All plasmids, and IspA and CS genes on high-copy plasmids. authors read and approved the final manuscript. Recruiting better performing enzymes is often a good strategy to break bottlenecks in engineered MVA path- Funding This work was financially supported by the National Natural Science Founda- ways. S. cerevisiae ERG12 is inhibited by substrate MVA. tion of China (31960216 and 31860019) and National Science Foundation of Therefore, the use of feedback-resistant ERG12 can Jiangxi Province (20192BBG70007, 20192BCBL23012 and 2019BBG70070). reduce MVA accumulation and promote the biosyn- Availability of data and materials thesis of terpenoids. Chen et  al. reported that lycopene All data generated or analyzed in this study are included in this published titer was increased 2.4 folds by replacing the wild-type article and its Additional file 1. ERG12 from S. cerevisiae with the feedback-resistant ERG12 (Chen et  al. 2018). Rad et  al. reported that use Declarations of B. licheniformis IdI in place of E. coli IdI increased Ethics approval and consent to participate lycopene production 1.4 folds in engineered E. coli (Rad Not applicable. et  al. 2012). HMGR consumes 2 molecules of NADPH to reduce HMG-CoA to mevalonate. Therefore, increas - Consent for publication Not applicable. ing the supply of NADPH will be a potentially important strategy to improve the yield of terpenoids. Recently, Competing interests assembling multienzyme complexes to prevent interme- The authors declare that they have no competing interests. diate diffusion through RIAD–RIDD interaction so as to Sun et al. 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Journal

Bioresources and BioprocessingSpringer Journals

Published: Mar 21, 2022

Keywords: Metabolic engineering; Escherichia coli; τ-Cadinol; MVA

References