Article Cite This: J. Agric. Food Chem. 2019, 67, 6523−6531
pubs.acs.org/JAFC
Combinatorial Engineering of Mevalonate Pathway and Diterpenoid Synthases in Escherichia coli for cis-Abienol Production Lei Li,†,§,∥ Xun Wang,†,§,∥ Xinyang Li,†,§,∥ Hao Shi,‡ Fei Wang,†,§,∥ Yu Zhang,†,§,∥ and Xun Li*,†,§,∥
Downloaded via BUFFALO STATE on July 18, 2019 at 02:15:53 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
†
Jiangsu Provincial Key Lab for the Chemistry and Utilization of Agro-forest Biomass, Nanjing Forestry University, Nanjing 210037, China ‡ Jiangsu Provincial Engineering Laboratory for Biomass Conversion and Process Integration, Huaiyin Institute of Technology, Huaian 223003, China § Jiangsu Key Laboratory of Biomass-based Green Fuels and Chemicals, Nanjing Forestry University, Nanjing 210037, China ∥ College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China S Supporting Information *
ABSTRACT: Identification of diterpene synthase-encoding genes together with synthetic biology technology offers an opportunity for the biosynthesis of cis-abienol. The methylerythritol phosphate (MEP) and the mevalonate (MVA) pathways were both engineered for cis-abienol production in Escherichia coli, which improved the cis-abienol yield by approximately 7-fold and 31-fold, respectively, compared to the yield obtained by overexpression of the MEP pathway alone or the original MEP pathway. Furthermore, systematic optimization of cis-abienol biosynthesis was performed, such as diterpene synthase screening and two-phase cultivation. The combination of bifunctional class I/II cis-abienol synthase from Abies balsamea (AbCAS) and class II abienol synthase from Salvia sclarea (SsTPS2) was found to be the most effective. By using isopropyl myristate as a solvent in two-phase cultivation, cis-abienol production reached 634.7 mg/L in a fed-batch bioreactor. This work shows the possibility of E. coli utilizing glucose as a carbon source for cis-abienol biosynthesis through a modified pathway. KEYWORDS: cis-abienol, mevalonate pathway, diterpene synthase, two-phase fermentation, Escherichia coli 4-phosphate (MEP) pathway in prokaryotes.12,13 The theoretical maximum IPP yield of glucose via the MEP pathway is higher than that via the MVA pathway. However, the MEP pathway requires more reducing equivalents and energy than the MVA pathway.14 IPP is transformed by isopentenyl diphosphate isomerase genes (IDI) to its isomer DMAPP. DMAPP and IPP are converted to GGPP by farnesyl pyrophosphate synthase (ERG20) and geranylgeranylpyrophosphate synthase (GGPPs) and further converted to diterpenes by diterpene synthases (diTPS) through GGPP cyclization reactions.15−17 To date, the highest cis-abienol of 23.8 mg/L was reported in Yarrowia lipolytica.18 There is no report on the recombinant production of cis-abienol in E. coli, although E. coli is one of the most commonly used hosts for the heterologous production of isoprenoids. Typically, E. coli has only the MEP pathway. In recent years, metabolic engineering has been used as an efficient approach for producing diterpenoids by constructing MEP and/or MVA pathway in E. coli.12,19−22 Improving metabolite yield through metabolic engineering requires the reconstruction of optimal biosynthetic pathways in E. coli because different original genes, various enzyme activities, and special enzyme properties will affect the metabolic flux and yield.23,24 Sallaud et al. have identified and characterized two diTPS genes governing the biosynthesis of
1. INTRODUCTION Ambergris is used as a fixative in the perfume industry, allowing fragrances to last longer.1,2 However, ambergris is an expensive substance that is very difficult to obtain because it is only produced by an estimated one percent of sperm whales, which are classified as endangered species.3 In addition, it is illegal to sale ambergris in the US and Australia. Therefore, in order to balance the protection of the sperm whales and the increasing demand for ambergris, the artificial synthesis of ambergris compounds has become increasingly popular in the fragrance industry. cis-Abienol is a natural diterpenoid that can be used as a starting material for the semisynthesis of (−)-ambrox, an accepted substitute for ambergris.4,5 However, cis-abienol has been found only in a few plants, such as Abies balsamea and Nicotiana tabacum, and is present in small amounts usually in the form of complex mixtures.6,7 Climate and the environment also affect the acquisition of natural diterpenoids from plants. Furthermore, the purification process of cis-abienol is labor intensive and expensive.8 Advances in the synthetic engineering of diterpenoids provide an opportunity for the biosynthesis of cis-abienol to improve and extend the availability of this compound as starting material for the manufacture of ambroxide.9−11 Biosynthesis of cis-abienol in plants involves the sequential cyclization of (E,E,E)-geranylgeranyl diphosphate (GGPP; Figure 1), which is derived from two common building blocks, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) synthesized through the mevalonate (MVA) pathway in archaea/eukaryotes and the methyl-D-erythritol© 2019 American Chemical Society
Received: Revised: Accepted: Published: 6523
April 5, 2019 May 17, 2019 May 21, 2019 May 22, 2019 DOI: 10.1021/acs.jafc.9b02156 J. Agric. Food Chem. 2019, 67, 6523−6531
Article
Journal of Agricultural and Food Chemistry
Figure 1. Biochemical pathways to convert glucose to cis-abienol in recombinant E. coli. (a) Illustration of the MEP pathway and MVA pathway leading to the formation of GGPP. (b) Pathway to cis-abienol in tobacco involve the activity of two separate enzymes: a type II diTPS that convert GGPP to labda-13-en-8-ol diphosphate (LDPP) and a type I diTPS that converts LDPP to cis-abienol. Metabolites abbreviations: A-CoA, acetylCoA; GAP, glyceraldehyde 3-phosphate; PYR, pyruvate; DXP, 1-deoxy-D-xylulose 5-phosphate; A-CoA, acetyl-CoA; AA-CoA, acetoacetyl-CoA; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; MEP, 2C-methyl-D-erythritol 4-phosphate; CDP-ME, 4-diphosphocytidyl-2C-methyl-D-erythritol; MEcPP, 2C-methyl-D-erythritol 2,4-cyclodiphosphate; HMBPP, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; A-CoA, acetyl-CoA; AA-CoA, acetoacetyl-CoA; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; Mev-P, mevalonate 5-phosphate; Mev-PP, mevalonate 5-diphosphate.
cis-abienol in N. tabacum.7 The class II abienol synthase from N. tabacum (NtTPS2) synthesizes labda-13-en-8-ol diphosphate from GGPP by binding the substrate’s diphosphate moiety and the class I diterpene synthase from N. tabacum (NtABS) that undergoes ionization of the allylic diphosphate at the class I active site and enzyme-specific rearrangement of intermediate carbocations to yield cis-abienol (Figure. 1).6,7 Furthermore, Zerbe et al. discovered a bifunctional class I/II cis-abienol synthase from A. balsamea (AbCAS) containing both class I and class II active sites and functionalities.6 AbCAS can convert GGPP into cis-abienol directly.6 Based on this, Royer produced cis-abienol by converting GGPP to cis-abienol in the presence of a combination of a bifunctional class I/II AbCAS and a class II diTPS in Y. lipolytica.18 Therefore, screening appropriate diTPS genes and reconstructing an efficient synthesis pathway are necessary to improve cis-abienol production. In this study, a series of systematic optimizations of cisabienol biosynthesis were described. To synthesize DMAPP and IPP in E. coli, the MEP pathway was strengthened and the MVA pathway was introduced into E. coli. Then, several cisabienol synthases from different sources were screened and
optimized in recombinant E. coli strains to obtain higher cisabienol production. Subsequently, the culture conditions for cis-abienol production in recombinant E. coli were optimized. Finally, to alleviate the low titer of cis-abeinol in single-phase cultures and spontaneous emulsification phenomenon in the two-phase bioprocess (TPB) fermentation, TPB with different solvents on engineered E. coli cultures was investigated.
■
MATERIAL AND METHODS
Bacterial Strains and Growth Conditions. E. coli strains were grown at 37 °C in Luria−Bertani (LB) medium or Terrific-Broth (TB) medium (containing 9.4 g of K2HPO4, 2.2 g of KH2PO4, 4 g of glucose, 24 g of yeast extract, and 12 g of tryptone per liter). If necessary, appropriate antibiotics were added aseptically to the culture medium at the following concentrations: ampicillin (Amp, 100 μg/ mL), streptomycin (Sm, 50 μg/mL), spectinomycin (Spec, 50 μg/ mL), and chloramphenicol (Cm, 34 μg/mL). Cell growth was determined by measuring the optical density at a wavelength of 600 nm (OD600) using Biophotometer Plus (Eppendorf, Hamburg, Germany). DNA Manipulation and Plasmid Construction. DNA manipulation was performed according to standard procedures.25 DNA polymerase, DNA markers, and restriction and modification enzymes were purchased from Takara (Dalian, China). DNA transformation 6524
DOI: 10.1021/acs.jafc.9b02156 J. Agric. Food Chem. 2019, 67, 6523−6531
Article
Journal of Agricultural and Food Chemistry
Methanosarcina mazei (MK MM ), phosphomevalonate kinase (PMKSC), and mevalonate pyrophosphate decarboxylase (PMDSC) from S. cerevisiae were amplified from the genomic DNA of CIBTS1758.21 The PCR product was then digested with BglII and XholI and subcloned into the same site of pCDFDuet-1 to create pML. The IDISC and ERG20SC genes were cloned into pML between SacI and PstI sites, resulting in plasmid pMLIE. The genes DXSEC and IDISC were integrated into pACYCDuet-1, forming plasmid pDI. The recombinant plasmid pGC was constructed to assemble the pETDuet-1 vector with two gene inserts encoding a truncated GGPP synthase from Taxus canadensis (TcGGPPs) and a truncated AbCAS, both of which were synthesized according to E. coli codon usage. Class II abienol synthase from Salvia sclarea (SsTPS2) was inserted into pGC between SacI and PstI sites, resulting in plasmid pGSC. Replacing SsTPS2 gene of pGSC with NtTPS2 or Class II abienol synthase gene from Coleus forskohlii (Cf TPS2) to obtain plasmid pGNC or pGFC, respectively. The plasmid pGNA was prepared by replacing AbCAS gene of pGNC with NtABS gene. Plasmid pGCLN was constructed by replacing the gene AbCAS with the gene AbCASL-NtTPS2, which is a fusion gene formed from NtTPS2 and AbCAS genes. The two diterpene synthase (NtABS and NtTPS2) genes were fused and inserted into pETDuet-1, resulting in plasmid pNLA/ pALN. The fusion enzymes were constructed by inserting a widely used (GGGGS)2 linker between the two corresponding genes. All modules were constructed using a one-step PCR strategy similar to overlap extension PCR. The TcGGPPs gene was then cloned into pNLA/pALN to obtain plasmid pGNLA/pGALN. Optimizing Bioconversion Conditions for cis-Abienol Production in Shake Flasks. The recombinant strain CA1168 was used in shake flasks to optimize the bioconversion conditions. CA1168 was inoculated into 50 mL of fresh TB medium in 250 mL shake flasks containing 100 μg/mL ampicillin, 50 μg/mL spectinomycin and 34 μg/mL chloramphenicol at 37 °C. The effects of the induction temperature (20∼35 °C), cell concentration (OD600 = 3, 4, 5, 6 or 7), IPTG concentration (0.1∼1.0 mM), and 10% (v/v) of the desired solvent (n-dodecane, oleyl alcohol, or isopropyl myristate) on cis-abienol production were determined. Preparation of cis-Abienol by the Recombinant Strains. The recombinant strain CA1168 was inoculated into 50 mL of fresh TB medium containing the appropriate antibiotics and grown at 37 °C until the OD600 reached 5. A total of 0.5 mM IPTG and 10% desired solvent were added to the recombinant strains, and the culture broths were incubated at 25 °C with shaking at 200 rpm for 48 h. For singlephase bioprocess, 10% desired solvent was added to the culture broth for extraction after cultivation. Bioreactor fermentation experiments were performed in a 1.3 L bioreactor (BioFlo/CelliGen 115, New Brunswick, U.S.A.) containing 0.5 L AM mineral medium (KH2PO4 4.2 g/L, K2HPO4·H2O 15.7 g/ L, (NH4)2SO4 2.0 g/L, citric acid 1.7 g/L, EDTA 8.4 mg/L, glucose 30 g/L, yeast extract 5 g/L, 1 M MgSO4·H2O 5 mL/L, 4.5 g/L thiamine•HCl 1 mL/L and 10 mL/L trace elements).26 Trace elements solution: 0.25 g of CoCl2·6H2O, 1.5 g of MnCl2·H2O, 0.15 g of CuCl2·2H2O, 0.3 g of H3BO3, 0.25 g of Na2MoO4·2H2O, 1.3 g of Zn(CH3COO)2·2H2O, 10 g of Fe(III) citrate per liter 1 M HCl. Strains CA1168 were inoculated into 50 mL of LB medium in a 250 mL shake flask containing the appropriate antibiotics and shaken at 200 rpm for 10 h at 37 °C. The seed liquid was transferred to fresh AM medium in fermenter and the inoculation amount was 5%. Fermentation was carried out at 37 °C and the pH maintained at 6.7 by automatic feeding of NH4OH 25% and HCl 15% v/v. The dissolved oxygen concentration (DO) was kept above 15% air saturation by adjusting the stirring speed from 200 rpm to a maximum of 700 rpm and by aeration with 1 vvm (air volume/working volume/ min). When the residual glucose in the medium dropped below 0.1 g/ L, the bioreactor was switched to fed-batch mode by feeding the addition medium (700 g/L glucose; 12 g/L, MgSO4·7H2O; 13 mg/L EDTA and 10 mL/L feed trace solution) to maintain the glucose concentrations between 0.1 and 2 g/L. The induction for expressing recombinant protein was started with an appropriate time point (OD600 nm of fermentation broth around 50−55). After addition of 0.5
was conducted via electroporation using the Gene Pulser Xcell system (Bio-Rad, Hercules, U.S.A.). The primers and optimized genes were synthesized by Springen (Nanjing, China). All plasmid construction procedures were carried out in E. coli TOP10 (Thermo Fisher Scientific, Shanghai, China). E. coli BL21 (DE3) was used for gene expression and cis-abienol production. The plasmids pACYCDuet-1, pCDFDuet-1, and pETDuet-1 (Novagen, Germany) were used as vectors for gene cloning and expression studies. E. coli B354 was used as the source for amplification of 1-deoxyxylulose-5-phosphate synthase (DXSEC) genes. Isopentenyl diphosphate isomerase (IDISC) genes and farnesyl pyrophosphate synthase (ERG20SC) genes were amplified from the genomic DNA of Saccharomyces cerevisiae. Plasmids and strains used in this study are listed in Table 1. All primers used are listed in Table
Table 1. Plasmids and Strains Used in This Study plasmids pACYCDuet-1 PCDFDuet-1 pETDuet-1 pMUD pMLIE pAGES pDI pGSC pGNC pGFC pGNA pGNLA pGALN pGCLN pGC strain CIBTS1758 BL21(DE3) CA0028 CA1028 CA1168 CA1128 CA1118 CA1126 CA11620 CA11260 CA11280 CA1108
genotype
ref
P15A origin, Cm CloDF13 origin, Smr ColE1 origin, Ampr T7 MvaE MvaS dxs, pACYCDuet-1 ori, Cmr T7 MK PMK PMD IDI Erg20, pCDFDuet-1 ori, Smr PGI* fldA ispG, Ptrc mvaEEFmvaSEF, pCL1920 ori, Specr T7 dxs, idi, pACYCDuet-1 ori, Cmr T7 GGPPs SsTPS2 AbCAS, pETDuet-1 ori, Ampr T7 GGPPs NtTPS2 AbCAS, pETDuet-1 ori, Ampr T7 GGPPs Cf TPS2 AbCAS, pETDuet-1 ori, Ampr T7 GGPPs NtTPS2 NtABS, pETDuet-1 ori, Ampr T7 GGPPs NtTPS2-L-NtABS, pETDuet-1 ori, Ampr T7 GGPPs NtABS-L-NtTPS2, pETDuet-1 ori, Ampr T7 GGPPs AbCAS-L-NtTPS2, pETDuet-1 ori, Ampr T7 GGPPs AbCAS, pETDuet-1 ori, Ampr BL21, glmS-pstS:: PL* MKMMPMKSCPMDSCidiSC,Δidi:: PGI* idiSC., PL** dxs, PGI* dxr F−, ompT, hsdSB(rB−mB−), gal, dcm (DE3) BL21(DE3)/ pGNC BL21(DE3)/ pDI + pGNC BL21(DE3)/ pMUD + pMLIE + pGSC BL21(DE3)/ pMUD + pMLIE + pGNC BL21(DE3)/ pMUD + pMLIE + pGFC BL21(DE3)/ pMUD + pMLIE + pGNA BL21(DE3)/ pMUD + pMLIE + pGNALN BL21(DE3)/ pMUD + pMLIE + pGNLA BL21(DE3)/ pMUD + pMLIE + pGCLN BL21(DE3)/ pMUD + pMLIE + pGC
Novagen Novagen Novagen this study this study
r
21 this study this study this study this study this study this study this study this study this study 21 Vazyme this study this study this study this study this study this study this study this study this study this study
S1. Six diTPS genes, which have been deposited in the Genbank database (Table S2), were codon optimized for E. coli expression and synthesized by Generay (Shanghai, China). To construct the top portion of the MVA pathway, the bifunctional acetoacetyl-CoA thiolase/3-hydroxy-3-methylglutaryl-CoA (HMGCoA) reductase (MvaE) and HMG-CoA synthase (MvaS) genes of Enterococcus faecalis were amplified from plasmid pAGES, which were generously donated by professor Yang.21 Polymerase chain reaction (PCR) products of MvaE and MvaS were cleaved by BamHI and AscI, while DXS, cleaved by BglII and XhoI, was ligated in the same site of pACYCDuet-1 resulting in pMUD. The bottom portion of the MVA pathway, including genes encoding mevalonate kinase from 6525
DOI: 10.1021/acs.jafc.9b02156 J. Agric. Food Chem. 2019, 67, 6523−6531
Article
Journal of Agricultural and Food Chemistry
Figure 2. Effect of constructed MEP and MVA pathways on cis-abienol production. Time courses of cis-abienol production (a), cell density (b), glucose consumption (c), cis-abienol yields and productivities (d). Data represent the mean/standard error of the mean (SEM) of three independent experiments.
Figure 3. cis-Abienol production by recombinant E. coli harboring modulars overproducing various diterpene enzymes. The strains were cultivated for 48 h in TB medium, and cis-abienol was extracted with n-dodecane. The data represent the means of three replicates and error bars represent standard deviations. mM IPTG, the fermentation temperature was decreased to 25 °C, meanwhile 10% (v/v) of biocompatible organic solvent was added to the culture broth as an in situ extractant of cis-abienol. Identification and Quantification of cis-Abienol. The mixture of culture and solvent was centrifuged at 10 000g for 10 min. The organic layer was collected followed by filtrated with 0.22 μm nylon-6 membrane (Aesculap Corp.). The titer of cis-abienol was analyzed by high performance liquid chromatography (HPLC, Agilent 1260, Agilent, Santa Clara, CA, U.S.A.) equipped with an ultraviolet (UV, 237 nm) detector and an Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 μm, Agilent, Saμnta Clara, U.S.A.). The column temperature was 25 °C, the mobile phase was 71% methanol, 18% ethanol, and 11% aqueous acetic acid solution (0.1%, v/v), and the flow rate was 0.8 mL/min. The product was characterized by direct comparison with standard cis-abienol (Sigma-Aldrich, St. Louis, U.S.A.). Liquid chromatography−mass spectrometry (LC-MS) analysis of cis-abienol was performed using a hybrid quadrupole time-of-flight mass spectrometry (Q-TOF MS) system (AB SCIEX, Concord, U.SA.) and coupled to a TripleTOF 4600 system (AB SCIEX,
Concord, U.S.A.), which equipped with atmospheric pressure chemical ionization (APCI) source in positive mode. The ion spray was operated at 3 kV and 350 °C. Separation of the different compounds was achieved in a Welch Ultimate XB-C18 column (2.1 mm × 100 mm, 3 μm, Welch Materials, Egerkingen, Switzerland). Solvents were composed of water/acetonitrile/methanoic acid (A: 0/ 100/0%, B: 99.9/0/0.1%). The gradient program was as follows: start with 70% A and hold 1 min, increase to 100% A within 1 min and also hold 1 min, then decrease to 70% A within 6 s and hold 2 min for stabilization. The flow rate was 0.4 mL/min and the injection volume was 1 μL. The identities of cis-abienol were confirmed based on the concordance of the retention index and mass spectra of authentic standard. Analysis of the Concentration of Glucose and Acetate. The amount of glucose in the culture was determined by the glucose oxidase-peroxidase method following the protocol of the glucose assay kit (Rsbio, Shanghai, China).27 The concentration of acetic acid was determined by gas chromatography (GC) analysis. A total of 1 mL supernatant of fermentation broth was added to a 5 mL tube, followed 6526
DOI: 10.1021/acs.jafc.9b02156 J. Agric. Food Chem. 2019, 67, 6523−6531
Article
Journal of Agricultural and Food Chemistry
Figure 4. Effect of culture conditions on cis-abienol production in the strain CA1168. (a) Effect of induction temperature on cis-abienol production. (b) Effect of IPTG concentration on cis-abienol production. (c) Effect of induction time on the cis-abienol production. (d) Effect of initial medium pH on cis-abienol production. (e) Cultivation profiles of the recombinant strain CA1168 in biphasic culture. Real-time pH (■), the concentration of acetic acid (●), the titer of cis-abienol (▲), OD600 (★), and glucose consumption (▼). Error bars represent one standard deviation from triplicate experiments.
of NAD(P)H and ATP in the cells.28 In this study, three E. coli strains associated with the MEP and MVA pathway were first studied, and their products were analyzed by LC-MS consistent with the cis-abienol standard (Figure S1). The time course of their cis-abienol production is shown in Figure 2a. The cis-abienol titer of strain CA0028 with original MEP pathway was 3.5 mg/L after 24 h, while the corresponding titer of engineered strain CA1028 with strengthened MEP pathway increased to 15.4 mg/L. Subsequently, strain CA1128 harboring plasmids pMUD, pMLIE, and pGNC produced 109.2 mg/L of cis-abienol, which was a 7-fold increase compared to that from strain CA1028. Additionally, cis-abienol production might affect the growth phenotype and glucose
by the addition of 0.2 mL of 50% sulfuric acid and 1 mL of ether and then centrifuged at 10 000g for 1 min. GC analysis was performed on an Agilent 7890A equipped with a flame ionization detector (FID) and a HP-INNOWAX column (30 m × 0.25 mm, 0.25 μm, Agilent, Santa Clara, U.S.A.). The column temperature profile was initiated at 70 °C for 3 min and then increased to 230 °C with an incremental rate of 8 °C per minute and held for 3 min. Nitrogen (N2) was used as carrier gas with a linear velocity of 2 mL/min; the temperature of FID was 300 °C.
■
RESULTS AND DISCUSSION Engineering both MEP and MVA Pathways for cisAbienol Production. Coexpression of the MVA and MEP pathways can further increase products yield due to the balance 6527
DOI: 10.1021/acs.jafc.9b02156 J. Agric. Food Chem. 2019, 67, 6523−6531
Article
Journal of Agricultural and Food Chemistry
encoding high efficient enzymes is a key step in reconstructing an effective metabolic pathway in microbes to obtain natural plant derived products.29 In this study, three diTPS (from N. tabacum,7 C. forskohlii,17 and S. sclarea.16), AbCAS,6 and NtABS7 were collected, optimized, and synthesized based on the preferred codon usage of E. coli. Since the previous studies suggested the presence of molecular interactions among CAS, diTPS, and ABS,6,7,16 a series of combination variants having these proteins fused or expressed singly or multiply were obtained and named: CA1168, CA1128, CA1118, CA1126, CA11260, CA11620, CA11280, and CA1108 (Table 1). As shown in Figure 3, the strain CA1126 carrying both NtTPS2 and NtABS modules produced 4.1 mg/L cis-abienol. While the strain CA1108 carrying a bifunctional abienol synthase AbCAS module produced 9.0 mg/L cis-abienol, which is over 2-fold compared to the production of CA1126. We speculate that the inability of NtABS to function properly in E. coli could be one of the reasons for the low yield of cis-abienol in CA1126. Therefore, more variants using the AbCAS were constructed and each variant was designed to have AbCAS and diTPS derived from different plants.7,16,17 The obtained strains CA1118, CA1128, and CA1168 could produce 91.9, 110.7, and 123.7 mg/L of cis-abienol, respectively. The titer of cisabienol from CA1168 harboring AbCAS and SsTPS2 represents a 13.7-fold and 30-fold improvement compared to that from CA1126 and CA1108 strains, respectively. These results indicated that the combination of AbCAS with SsTPS2 was more effective than with other diTPS. Meanwhile, the strains (CA1118, CA1128, and CA1168) had both AbCAS and diTPS2 modules improved the cis-abienol titers over 10-fold, compared to strains with AbCAS alone, which is consistent with Royer’s result.18 Zhou et al. showed that the fusion of different diTPS might be beneficial to miltiradiene production because it brings active sites into a closer proximity to each other.30 Thus, we made three modules that produced fusion proteins AbCAS-LNtTPS2, NtTPS2-L-NtABS, and NtABS-L-NtTPS2, respectively. While the addition of the AbCAS-L-NtTPS2 module (strain CA11280) gave a sharply reduced cis-abienol
Figure 5. Effect of solvent on cis-abienol production and cell growth. Error bars represent one standard deviation from triplicate experiments.
consumption. As shown in Figure 2b, the introduction of MVA pathway had little effect on cell growth during the process of cultivation, with OD600 of all strains were around 10.0. The glucose consumption rates of strains CA0028 and CA1028 were faster than CA1128, but CA1128 produced the highest cis-abienol with a steady increase in titer up to 40 h (Figure 2c). The yield of cis-abienol from CA1128 carrying the MEP and MVA pathway reached to 0.027 g/g glucose, which was much higher than that from CA0028 and CA1028 (Figure 2d). Thus, overexpression of both the MEP and MVA pathways improved the cis-abienol synthesis significantly in E. coli. The strains with original MEP pathway expression or MEP pathway overexpression reached a productivity of 0.083 and 0.36 mg/ L/h, respectively, whereas overexpression of both pathways increased the cis-abienol productivity to 2.68 mg/L/h (Figure 2d). Overall, the results demonstrated that production improvement of cis-abienol had been achieved using the hybrid MVA and MEP pathway. Optimization of the Combination of Different cisAbienol Synthases for cis-Abienol Production. Generally, screening and obtaining the appropriate exogenous genes
Figure 6. Fed-batch fermentation of CA1168 strains in a 1.3-L bioreactor. Time profiles of OD600 (●), glucose consumption (■), the concentration of acetic acid (▲), and the titer of cis-abienol (★). 6528
DOI: 10.1021/acs.jafc.9b02156 J. Agric. Food Chem. 2019, 67, 6523−6531
Article
Journal of Agricultural and Food Chemistry
was comparable at 0 mg/L and up to 700 mg/L of added cisabienol in vitro (data not shown). Analysis of the results showed that cis-abienol exhibits low or no toxicity to E. coli when it is added exogenously to the growth medium. This is consistent with a previous finding that cis-abienol had little or no antibacterial activity against Ralstonia solanacearum.35,36 Although cis-abienol had little or no antibacterial activity on E. coli, the titer of cis-abeinol (41.1 mg/L) was low in single-phase cultures. We hypothesized that the solubility of cis-abeinol in water is easy to reach the threshold in the broth of single-phase culture. It is difficult to export out of the cell for the intracellular cis-abeinol, which caused negative feedback inhibition of the cis-abeinol biosynthesis. TPB has already proven to be applicable for a large number of different recombinant strains and increases production capacity of host strains.29,37,38 Dodecane was considered as a reasonable choice of solvent for the TPB;37 however, the results of our cis-abienol biosynthesis experiment showed a spontaneous emulsification phenomenon in TPB fermentation. In order to get a successful application of TPB, it is important to select a suitable solvent. An ideal solvent should be biocompatible yet nonbioavailable and have low aqueous solubility, high selectivity and a low emulsion-forming tendency.39 Based on this, the effect of n-dodecane, oleyl alcohol, and isopropyl myristate on cell growth, and cis-abienol production of the engineered strain CA1168 were investigated, with a no-solvent culture as a control. After 48 h of shake-flask fermentation, in the presence of n-dodecane, oleyl alcohol, and isopropyl myristate, the cis-abienol titers reached 165.8, 134.8, and 143.9 mg/L, representing 4-fold, 3.2-fold, and 3.5-fold improvements, respectively, when compared with that achieved in no-solvent cultures (41.1 mg/L; Figure 5). Additionally, the results showed that cell growth was not significantly decreased in biphasic cultures when compared with that in no-solvent cultures (Figure 5). It is verified that the endogenously synthesized cis-abienol and these solvents exhibit no or low inhibitory effects on cell growth, and TPB is useful for improving the yield of cis-abienol by relieving end product feedback repression. Although oleyl alcohol also has the lower emulsion-forming tendency than n-dodecane (Figure S3), the viscosity of oleyl alcohol is high and the cis-abienol formation/ DCW (0.052 g/g) were lower than the other two (0.076 and 0.066 g/g). Meanwhile, emulsification was also significantly alleviated using isopropyl myristate as the solvent (Figure S3). Furthermore, the cis-abienol formation/DCW of CA1168 using isopropyl myristate (0.076 g/g) as the solvent was the highest among these three solvents. In order to achieve the delicate balance between the yield of products and emulsionforming tendency, we selected isopropyl myristate as the TPB solvent. In order to improve cis-abienol production, the recombinant strain CA1168 was cultured in a 1.3 L fermenter using fedbatch fermentation mode under control condition. The fedbatch cultivation studies were carried out using isopropyl myristate overlay. The carbon source (glucose) concentration was controlled below 2 g/L in fed-batch process, and the acetate concentration was kept below 3 g/L throughout the cultivation. Under these fermentation conditions, the titer of cis-abienol reached a maximum of 634.7 mg/L with a corresponding specific productivity was 6.6 mg/L/h in almost 96 h after the induction of cis-abienol production (Figure 6), which was 27 times of the highest reported titer.18 Maximum cell densities were 18 g/L dry cell weight (OD600 = 83) during
production (4.7 mg/L) as compared to that of the strain CA1128, suggesting that the hybrid enzyme could not be expressed properly in the host. As shown in Figure S2, the expression quantity of fusion protein gene was limited (lanes 2, 5, and 8) and most of the recombinant protein was insoluble (lane 8). In comparison, the strain CA1128, which expressing the AbCAS and NtTPS2 module separately, produced much more amount of recombinant protein than the strain CA11280, and few inclusion bodies were detected in the cell extracts (lane 7). The limited soluble expression in the strain CA11280 might be the main reason for a reduced cis-abienol production. Similarly, strains CA11260 and CA11620 expressing the NtTPS2-L-NtABS and NtABS-L-NtTPS2 module produced only 3.6 and 3.9 mg/L cis-abienol, respectively. These results indicated that the fusion proteins were disadvantageous to the production of cis-abienol. Effect of Induction Conditions on the cis-Abienol Production. Induction temperature, inducer concentration, and its addition time can affect the growth of strains and the expression of recombinant proteins.31 cis-Abienol production in CA1168 improved substantially when the induction temperature was decreased from 35 °C and reached 141 ± 0.3 mg/L at 25 °C after 48 h (Figure 4a). High (30 and 35 °C) or low (20 °C) induction temperatures decreased cis-abienol production. Different IPTG concentrations revealed that cisabienol production reached 141.8 mg/L when IPTG was 0.5 mM, whereas high (1.0 mM) or low (0.1 mM) IPTG decreased cis-abienol production (Figure 4b). The optimal cell concentration when adding IPTG was at OD600 of 5, and the titer of cis-abienol production reached 148 ± 0.4 mg/L (Figure 4c). There was no significant difference in cis-abienol production when CA1168 was induced at the early log phase; however, cis-abienol production decreased significantly when induced at OD600 of 7. Too late induction time point leads to excessive cells and insufficient carbon source and thus the subsequent low cis-abienol production. These results indicated that early induction in the log phase was more effective than induction in the later log phase. Terpene cis-abienol cyclases from N. tabacum are reported to have an optimum pH near 7.0.32 To investigate the effect of pH on cis-abienol production, different initial medium pHs (5.5−7.0) were tested and the optimal initial pH is 6.5 (Figure 4d). when the initial pH was 7.0, the pH of culture broth was around 6.5 for the first 36 h and then increased, and the titer of cis-abienol did not increase after 40 h. Meanwhile, when the initial pH was 6.5 in this study, the culture broth remained at a pH around 6.0 for most of the time, and the titer of cis-abienol showed an upward trend (Figure 4e). Glucose was almost depleted after 24 h and acetate only accumulated to below 1.5 g/L throughout the cultivation. The final titer of cis-abienol reached 158 mg/L after 48 h using the engineered strain CA1168. These results are consistent with that of sclareol production reported by Schalk,26 who found that sclareol was formed at an optimal pH of 6.0 and only trace sclareol was formed at pH 7.0. These results verified that acetic acid will be produced when the content of glucose is sufficient and the pH will decrease, but when the glucose is completely consumed, acetic acid will be used as a carbon source, thus the pH will rise.33,34 Given this data, we conclude that pH 6.0 is the optimal reaction pH of cis-abienol synthase. cis-Abienol Production in Two-Phase Bioprocess. Microbial production of terpenoids in high titers may be restricted by its potential toxicity to the host. E. coli cell growth 6529
DOI: 10.1021/acs.jafc.9b02156 J. Agric. Food Chem. 2019, 67, 6523−6531
Article
Journal of Agricultural and Food Chemistry
of China (No. 21878160), and the Topnotch Academic Programs Project (TAPP) Education Institutions.
the high cell density fermentations (Figure 6). Correspondingly, the study about sclareol titers of about 1.5 g/L in highcell-density fermentation.26 Tracing it to its cause, the main reason is that different terpenoid synthases and different upper MVA pathway. Although sclareol and cis-abienol has the similar mechanisms of biosynthesis, there is a definitely difference between sclareol synthase (SsScS) and cis-abienol synthase (AbCAS and SsTPS2), which have different expression level and activity in E. coli. Furthermore, differences in upper MVA pathway and selected expression plasmids have also played an important role in yield gap between sclareol and cis-abienol. In conclusion, the MEP pathway and the MVA pathway have been engineered in E. coli. Heterologous expression of optimized versions of cis-abienol synthases in E. coli strain led to a dramatic increase in cis-abienol titers reaching 634.7 mg/L in high cell density fermentation. To the best of our knowledge, this is the highest titer reported so far for cis-abienol in E. coli. Our work provides an alternative route to the conventional plant extraction process (labor-intensive and expensive) or using ambergris (very difficult to obtain/ illegal). Despite the extensive progress made on cis-abienol production in E. coli, many possible improvements can be achieved in enhancing cis-abienol production. The conversion efficiency of glucose to cis-abienol (gram to gram) in the metabolically engineered strain is only 1.3% in the high celldensity fermentation and the highest cell density was only 83 OD600. One approach is to optimize the high cell-density fermentation process to elevate the conversion efficiency and the cell densities. Furthermore, we transformed three recombinant plasmids into E. coli, which resulted in slow growth of the recombinant strains. So, another possibility is employing a chromosome integration technique to decrease the cell growth burden on the host. Based on the above reason, our subsequent work would focus on alternating and integrating MVA pathways genes or other key heterologous genes into the E. coli genome to overcome cell growth burden would lay the foundation for industrialized production of cisabienol.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
REFERENCES
We thank Prof. Sheng Yang at Chinese Academy of Sciences for the MVA gene (CIBTS1758 and pAGES).
(1) Brito, C.; Jordão, V. L.; Pierce, G. J. Ambergris as an overlooked historical marine resource: its biology and role as a global economic commodity. J. Mar. Biol. Assoc. U. K. 2015, 96 (3), 585−596. (2) Rowland, S. J.; Sutton, P. A. Chromatographic and spectral studies of jetsam and archived ambergris. Nat. Prod. Res. 2017, 31 (15), 1752−1757. (3) Christopher, K. On Long Beach. In Floating Gold: A Natural (and Unnatural) History of Ambergris, 1st ed.; Christopher, K., Eds.; University of Chicago Press: Chicago, IL, 2012; pp 5−18. (4) Zerbe, P.; Bohlmann, J. Enzymes for synthetic biology of ambroxide-related diterpenoid fragrance compounds. Adv. Biochem. Eng./Biotechnol. 2015, 148, 427−447. (5) Barrero, A. F.; Alvarez-Manzaneda, E. J.; Altarejos, J.; Salido, S.; Ramos, J. M. Synthesis of Ambrox® from (−)-sclareol and (+)-cisabienol. Tetrahedron 1993, 49 (45), 10405−10412. (6) Zerbe, P.; Chiang, A.; Yuen, M.; Hamberger, B.; Hamberger, B.; Draper, J. A.; Britton, R.; Bohlmann, J. Bifunctional cis-abienol synthase from Abies balsamea discovered by transcriptome sequencing and its implications for diterpenoid fragrance production. J. Biol. Chem. 2012, 287 (15), 12121−12131. (7) Sallaud, C.; Giacalone, C.; Topfer, R.; Goepfert, S.; Bakaher, N.; Rosti, S.; Tissier, A. Characterization of two genes for the biosynthesis of the labdane diterpene Z-abienol in tobacco (Nicotiana tabacum) glandular trichomes. Plant J. 2012, 72 (1), 1−17. (8) Jablonsky, M.; Nosalova, J.; Sladkova, A.; Haz, A.; Kreps, F.; Valka, J.; Miertus, S.; Frecer, V.; Ondrejovic, M.; Sima, J.; Surina, I. Valorisation of softwood bark through extraction of utilizable chemicals. A review. Biotechnol. Adv. 2017, 35 (6), 726−750. (9) Ignea, C.; Trikka, F. A.; Nikolaidis, A. K.; Georgantea, P.; Ioannou, E.; Loupassaki, S.; Kefalas, P.; Kanellis, A. K.; Roussis, V.; Makris, A. M.; Kampranis, S. C. Efficient diterpene production in yeast by engineering Erg20p into a geranylgeranyl diphosphate synthase. Metab. Eng. 2015, 27, 65−75. (10) Zerbe, P.; Bohlmann, J. Plant diterpene synthases: exploring modularity and metabolic diversity for bioengineering. Trends Biotechnol. 2015, 33 (7), 419−428. (11) Ignea, C.; Ioannou, E.; Georgantea, P.; Loupassaki, S.; Trikka, F. A.; Kanellis, A. K.; Makris, A. M.; Roussis, V.; Kampranis, S. C. Reconstructing the chemical diversity of labdane-type diterpene biosynthesis in yeast. Metab. Eng. 2015, 28, 91−103. (12) Miziorko, H. M. Enzymes of the mevalonate pathway of isoprenoid biosynthesis. Arch. Biochem. Biophys. 2011, 505 (2), 131− 43. (13) Ajikumar, P. K.; Xiao, W. H.; Tyo, K. E.; Wang, Y.; Simeon, F.; Leonard, E.; Mucha, O.; Phon, T. H.; Pfeifer, B.; Stephanopoulos, G. Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli. Science 2010, 330 (6000), 70−74. (14) Whited, G. M.; Feher, F. J.; Benko, D. A.; Cervin, M. A.; Chotani, G. K.; McAuliffe, J. C.; LaDuca, R. J.; Ben-Shoshan, E. A.; Sanford, K. J. Technology update: Development of a gas-phase bioprocess for isoprene-monomer production using metabolic pathway engineering. Ind. Biotechnol. 2010, 6 (3), 152−163. (15) Falara, V.; Pichersky, E.; Kanellis, A. K. A copal-8-ol diphosphate synthase from the angiosperm Cistus creticus subsp. creticus is a putative key enzyme for the formation of pharmacologically active, oxygen-containing labdane-type diterpenes. Plant Physiol. 2010, 154 (1), 301−310.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.9b02156.
■
■
Primers used in this study, Genebank ID and sequences of diterpene synthase used in this study, HPLC and HPLC-MS analysis of cis-abienol product, SDS-PAGE analysis of the recombinant diterpene synthase, and effect of solvent on emulsification in biphasic culture (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Phone: (+86)-025-85427635. Fax: (+86)-025-85427635. ORCID
Xun Li: 0000-0002-1267-9263 Funding
This work was financially supported by the National Key Research & Development Program of China (2017YFD0600205), the National Natural Science Foundation 6530
DOI: 10.1021/acs.jafc.9b02156 J. Agric. Food Chem. 2019, 67, 6523−6531
Article
Journal of Agricultural and Food Chemistry
catabolite repression of acetyl-CoA synthetase. BMC Syst. Biol. 2010, 4, 166. (34) Pei, J.; Sun, Q.; Zhao, L.; Shi, H.; Tang, F.; Cao, F. Efficient biotransformation of luteolin to isoorientin through adjusting induction strategy, controlling acetic acid and increasing UDP-glucose supply in Escherichia coli. J. Agric. Food Chem. 2019, 67 (1), 331−340. (35) Kerstiens, G. Plant cuticles-an integrated functional approach. J. Exp. Bot. 1996, 47 (supp1), 50−60. (36) Seo, S.; Gomi, K.; Kaku, H.; Abe, H.; Seto, H.; Nakatsu, S.; Neya, M.; Kobayashi, M.; Nakaho, K.; Ichinose, Y.; Mitsuhara, I.; Ohashi, Y. Identification of natural diterpenes that inhibit bacterial wilt disease in tobacco, tomato and Arabidopsis. Plant Cell Physiol. 2012, 53 (8), 1432−1444. (37) Etschmann, M. M.; Schrader, J. An aqueous-organic two-phase bioprocess for efficient production of the natural aroma chemicals 2phenylethanol and 2-phenylethylacetate with yeast. Appl. Microbiol. Biotechnol. 2006, 71 (4), 440−443. (38) Jeschek, M.; Gerngross, D.; Panke, S. Combinatorial pathway optimization for streamlined metabolic engineering. Curr. Opin. Biotechnol. 2017, 47, 142−151. (39) Bruce, L. J.; Daugulis, A. J. Solvent selection strategies for extractive biocatalysis. Biotechnol. Prog. 1991, 7 (2), 116−124.
(16) Caniard, A.; Zerbe, P.; Legrand, S.; Cohade, A.; Valot, N.; Magnard, J. L.; Bohlmann, J.; Legendre, L. Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture. BMC Plant Biol. 2012, 12, 119. (17) Pateraki, I.; Andersen-Ranberg, J.; Hamberger, B.; Heskes, A. M.; Martens, H. J.; Zerbe, P.; Bach, S. S.; Moller, B. L.; Bohlmann, J.; Hamberger, B. Manoyl oxide (13R), the biosynthetic precursor of forskolin, is synthesized in specialized root cork cells in Coleus forskohlii. Plant Physiol. 2014, 164 (3), 1222−1236. (18) Royer, J. Methods for producing abienol. International Patent WO 2016/094178 A1, 2016. (19) Yang, L.; Wang, C.; Zhou, J.; Kim, S.-W. Combinatorial engineering of hybrid mevalonate pathways in Escherichia coli for protoilludene production. Microb. Cell Fact. 2016, 15 (1), 409. (20) Yoon, S. H.; Lee, S. H.; Das, A.; Ryu, H. K.; Jang, H. J.; Kim, J. Y.; Oh, D. K.; Keasling, J. D.; Kim, S. W. Combinatorial expression of bacterial whole mevalonate pathway for the production of betacarotene in E. coli. J. Biotechnol. 2009, 140 (3−4), 218−226. (21) Yang, C.; Gao, X.; Jiang, Y.; Sun, B.; Gao, F.; Yang, S. Synergy between methylerythritol phosphate pathway and mevalonate pathway for isoprene production in Escherichia coli. Metab. Eng. 2016, 37, 79−91. (22) Coussement, P.; Bauwens, D.; Maertens, J.; De Mey, M. Direct combinatorial pathway optimization. ACS Synth. Biol. 2017, 6 (2), 224−232. (23) Pei, J.; Chen, A.; Zhao, L.; Cao, F.; Ding, G.; Xiao, W. One-pot synthesis of hyperoside by a three-enzyme cascade using a UDPgalactose regeneration system. J. Agric. Food Chem. 2017, 65 (29), 6042−6048. (24) Alonso-Gutierrez, J.; Kim, E. M.; Batth, T. S.; Cho, N.; Hu, Q.; Chan, L. J. G.; Petzold, C. J.; Hillson, N. J.; Adams, P. D.; Keasling, J. D.; Garcia Martin, H.; Lee, T. S. Principal component analysis of proteomics (PCAP) as a tool to direct metabolic engineering. Metab. Eng. 2015, 28, 123−133. (25) Randall, A. H.; Andrew, D. E. Synthetic DNA synthesis and assembly: Putting the synthetic in synthetic biology. In Synthetic biology: Tools for engineering biological systems, 1st ed.; Daniel, G. G., Clyde, A. H., Hamilton, O. S., John, C. V., Eds.; Cold Spring Harbor Laboratory: New York, 2017; pp 10−40. (26) Schalk, M.; Pastore, L.; Mirata, M. A.; Khim, S.; Schouwey, M.; Deguerry, F.; Pineda, V.; Rocci, L.; Daviet, L. Toward a biosynthetic route to sclareol and amber odorants. J. Am. Chem. Soc. 2012, 134 (46), 18900−18903. (27) Tiffany, T. O.; Jansen, J. M.; Burtis, C. A.; Overton, J. B.; Scott, C. D. Enzymatic kinetic rate and end-point analyses of substrate, by use of a gemsaec fast analyzer. Clin. Chem. 1972, 18 (8), 829−840. (28) Gruchattka, E.; Hädicke, O.; Klamt, S.; Schütz, V.; Kayser, O. In silico profiling of Escherichia coli and Saccharomyces cerevisiae as terpenoid factories. Microb. Cell Fact. 2013, 12, 84−84. (29) Peralta-Yahya, P. P.; Ouellet, M.; Chan, R.; Mukhopadhyay, A.; Keasling, J. D.; Lee, T. S. Identification and microbial production of a terpene-based advanced biofuel. Nat. Commun. 2011, 2, 483−491. (30) Zhou, Y. J.; Gao, W.; Rong, Q.; Jin, G.; Chu, H.; Liu, W.; Yang, W.; Zhu, Z.; Li, G.; Zhu, G.; Huang, L.; Zhao, Z. K. Modular pathway engineering of diterpenoid synthases and the mevalonic acid pathway for miltiradiene production. J. Am. Chem. Soc. 2012, 134 (6), 3234− 3241. (31) Donovan, R. S.; Robinson, C. W.; Glick, B. R. Review: optimizing inducer and culture conditions for expression of foreign proteins under the control of the lac promoter. J. Ind. Microbiol. 1996, 16 (3), 145−154. (32) Guo, Z.; Severson, R. F.; Wagner, G. J. Biosynthesis of the diterpene cis-abienol in cell-free extracts of tobacco trichomes. Arch. Biochem. Biophys. 1994, 308 (1), 103−108. (33) Valgepea, K.; Adamberg, K.; Nahku, R.; Lahtvee, P. J.; Arike, L.; Vilu, R. Systems biology approach reveals that overflow metabolism of acetate in Escherichia coli is triggered by carbon 6531
DOI: 10.1021/acs.jafc.9b02156 J. Agric. Food Chem. 2019, 67, 6523−6531
