Chemicals and materials
Restriction enzymes, T4 DNA ligase, T4 polynucleotide kinase, PrimeSTAR Max DNA polymerase, and PCR reagents were purchased from Takara (Dalian, China). The primers were synthesized by Sangon (Shanghai, China). The corresponding codon-optimized genes were chemically synthesized by Generay (Shanghai, China). The gel extraction, PCR purification, and plasmid mini kits were purchased from Axygen Scientific (Suzhou, China). All other chemicals used were of the highest available purity.
Plasmid construction
The primers used for the genetic modifications are listed in Additional file 1: Table S4. The plasmids pHGFH and pAGES containing expression cassettes for the partial DXP and full-length MVA pathway genes were gifts from Prof. Sheng Yang. The codon-optimized LIS genes from Lavandula angustifolia (Genbank No. ABB73045.1), Osmanthus fragrans var. thunbergii (Genbank No. ABB73045.1), and Streptomyces clavuligerus (Genbank No. D5SL78.1) were chemically synthesized with codons optimized for Escherichia coli expression. The LIS gene was cloned between the NcoI and BamHI sites of the pETDuet-tac vector. The plasmid products were pETDuet-laLIS, pETDuet-ofLIS, and pETDuet-bLIS. The gene encoding MK was cloned downstream of bLIS on pETDuet-bLIS and yielded the pETDuet-bLIS-MK. The RBS sequences with various TIRs were calculated by Sails Lab [29]. The RBS sequences with TIRs of 100, 200, 500, 5,000, 50,000, and 176,000 au were added to the 5’-end of bLIS in pETDuet-bLIS. The plasmid products were pETDuet-bLIS (0.1 k), pETDuet-bLIS (0.2 k), pETDuet-bLIS (0.5 k), pETDuet-bLIS (5 k), pETDuet-bLIS (50 k), and pETDuet-bLIS (max). The MBP, NusA, CmR29, and GST fusion tag genes were chemically synthesized and subcloned into the upstream region of bLIS in pETDuet-bLIS with a ClonExpress II one-step cloning kit (Vazyme Biotech Co. Ltd., Nanjing, China). The plasmid products were pETDuet-MBP*bLIS, pETDuet-NusA*bLIS, pETDuet-CmR29*bLIS, and pETDuet-GST*bLIS. The optimized RBS sequence was ligated into the 5’-end of fused CmR29*bLIS and generated pETDuet-CmR29*bLIS (maximum). The gene encoding GPP synthase from Abies grandis (Genbank No. AAN01134.1) was truncated by 86 aa from its N-terminus, codon-optimized for E. coli, and inserted behind the second tac promoter of pETDuet-CmR29*bLIS (max). The plasmid product was pETDuet-CmR29*bLIS (max)-AgGPPS. The plasmids pETDuet-CmR29*bLIS (max)-IspA and pETDuet-CmR29*bLIS (max)-Erg20 were constructed based on pETDuet-Cm29R*bLIS (max)-AgGPPS by replacing AgGPPS with IspA from E. coli and Erg20 from Saccharomyces cerevisiae. The mutants of IspA (S80F), Erg20 (F96W), Erg20 (N127W), and Erg20 (F96W/N127W) were obtained by inverse PCR and construction of pETDuet-CmR29*bLIS (max)-IspA (S80F), pETDuet-CmR29*bLIS (max)-Erg20 (F96W), pETDuet-CmR29*bLIS (max)-Erg20 (N127W), and pETDuet-CmR29*bLIS (max)-Erg20 (F96W/N127W). The DNA sequences of all constructs were verified by full re-sequencing.
Strains and culture conditions
All plasmids and strains constructed here are listed in Additional file 1: Table S1. For R-(-)-linalool production, the seed culture was incubated at 37 °C overnight in Luria-Bertani (LB) medium (10 g L-1 tryptone, 5 g L-1 yeast extract, and 10 g L-1 sodium chloride) with antibiotics (100 mg L-1 ampicillin, 50 mg L-1 spectinomycin, and 34 mg L-1 chloramphenicol). Shake flask cultures were initiated by inoculating 0.5 mL seed culture into 50 mL TB medium (12 g L-1 tryptone, 24 g L-1 yeast extract, 9.4 g L-1 K2HPO4, and 2.2 g L-1 KH2PO4) containing 0.5% (v/v) glycerol as the main carbon source. When OD600 ~0.8–1, 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added and 10% (v/v) isopropyl myristate was overlaid on the medium to prevent the loss of volatile products. After induction, the temperature was decreased to 25 °C. The experiments were run in duplicate. The R-(-)-linalool concentration and OD600 were quantified every 24 h.
Product analysis by GC-MS and GC-FID
One milliliter of uniformly mixed culture broth with the isopropyl myristate phase was sampled and concentrated by centrifuging at 13,500 ×g for 5 min. The isopropyl myristate layer was analyzed by GC-FID and GC-MS. R-(-)-linalool, geraniol, and nerolidol were identified by a Trace GC 2000 Ultra (Thermo Fisher Scientific, Waltham, MA, USA) fitted with a DB-5 column (30 m × 0.25 mm × 0.25 μm) and a Thermo Finnigan Trace DSQ mass spectrometer operating with electron ionization (ion source temperature: 200 °C; electron energy: 70 eV). The scan rate was set to 500 amu s-1. The operating parameters were as follows: injector: 220 °C; detector: 250 °C; oven temperature: start at 40 °C, hold for 1 min, programmed to rise from 40 °C to 240 °C at 10 °C min-1, hold for 5 min; helium carrier gas flow: 1.0 mL min-1; injection mode: 1 mL sample with splitless injection. Monoterpene and sesquiterpene identities were confirmed by comparing mass spectra and retention times against authentic standards.
Chiral gas chromatography was performed to determine enantiomeric linalool distribution. The isopropyl myristate phase was analyzed on a Cyclosil-B β-cyclodextrin chiral capillary column (30 m × 0.25 mm × 0.25 μm). Samples were injected at 200 °C into a GC (No. 7890A; Agilent Technologies, Santa Clara, CA, USA) fitted with a flame ionization detector (FID). The oven temperature was first held at 80 °C for 5 min and gradually increased at a rate of 5 °C min-1 to 200 °C where it was held for another 5 min. The detector temperature was maintained at 260 °C. Product identity was confirmed by comparison against authentic R-(-)-linalool standards (Additional file 1: Fig. S4).
Protein analysis
To establish the expression levels and solubilities of the fused bLISs, pETDuet-bLIS, pETDuet-MBP*bLIS, pETDuet-NusA*bLIS, pETDuet-Cm29*bLIS, and pETDuet-GST*bLIS were transformed into E. coli BL21 to yield the recombinant strains WX6009, WX6019, WX6029, WX6039, and WX6049, respectively. Heterologous expression of the fused bLISs in E. coli BL21 was compared by 12% SDS-PAGE analysis of the crude protein extracts.
Fed-batch fermentation in a 1.3-L bioreactor
The process used for R-(-)-linalool production was adapted from a previously published method [18]. A single colony of strain WX6637 was incubated overnight in 5 mL LB medium at 37 °C and rotated at 200 rpm. Then 40 mL LB medium (10% v/v) was inoculated with this pre-culture. After 6–8 h cultivation (37 °C; 200 rpm) and at OD600 = 5, the pre-culture was then used to inoculate the main culture. Fed-batch fermentation experiments were conducted in a 1.3-L bioreactor (New Brunswick Scientific BioFlo/CelliGen 115, Edison, NJ, USA). At the start of the batch phase, the bioreactor contained 0.4 L AM mineral medium (4.2 g L-1 KH2PO4, 15.7 g L-1 K2HPO4·3H2O, 2 g L-1 (NH4)2SO4, 1.7 g L-1 citric acid, 8.4 mg L-1 EDTA, 15 g L-1 glycerol, 1.2 g L-1 MgSO4·7H2O, 4.5 mg L-1 thiamine HCl, and 10 mL L-1 trace metal solution), 100 mg L-1 ampicillin, 50 mg L-1 spectinomycin, and 34 mg L-1 chloramphenicol. The trace metal solution consisted of 0.25 g L-1 CoCl2·6H2O, 1.5 g L-1 MnCl2·4H2O, 0.15 g L-1 CuCl2·2H2O, 0.3 g L-1 H3BO4, 0.25 g L-1 Na2MoO4·2H2O, 1.3 g L-1 Zn(CH3COO)2·2H2O, and 10 g L-1 Fe(III) citrate hydrate. Fermentation was run at 37 °C and 25% (v/v) ammonia or 10% (v/v) HCl was used to maintain the pH at 7.0. The former also served as a nitrogen source during fermentation. The dissolved oxygen (DO) concentration was maintained at > 15% saturation by increasing the stirring speed from 200 rpm to a maximum of 700 rpm and by aeration with 3 vvm air. Antifoam 204 (Sigma-Aldrich Corp., St. Louis, MO, USA) was added to prevent foaming. When the initial glycerol in the medium was nearly depleted (< 0.1 g L-1) and the DO spiked, a glycerol concentration-based feed was activated by adding a solution at an appropriate rate to maintain the glycerol concentration < 5 g L-1. The feed solution consisted of 650 g L-1 glycerol, 12 g L-1 MgSO4·7H2O, 10.7 g L-1 (NH4)2SO4, 13 mg L-1 EDTA, and 10 mL L-1 feed trace metal solution (0.4 g L-1 CoCl2·6H2O, 2.35 g L-1 MnCl2·4H2O, 0.25 g L-1 CuCl2·2H2O, 0.5 g L-1 H3BO4, 0.4 g L-1 Na2MoO4·2H2O, 1.6 g L-1 Zn(CH3COO)2·2H2O, and 4 g L-1 Fe(III) citrate hydrate). R-(-)-linalool production was induced with 0.5 mM IPTG at OD600 = 35. Then 10% (v/v) isopropyl myristate was added to the medium to extract the product in situ with a biocompatible organic phase. The post-induction culture temperature was held at 30 °C. Samples were drawn throughout fermentation to measure cell density and the glycerol and R-(-)-linalool concentrations. Glycerol was analyzed with a glycerol assay kit (Jiancheng, Nanjing, China) according to the manufacturer’s instructions.
R-(-)-linalool toxicity assay
To evaluate the toxicity of R-(-)-linalool to E. coli CIBTS1758, a 12-h seed culture was diluted with 1 mL fresh LB medium (OD600 = 0.2) containing 100, 200, 500, 1,000, or 2,000 mg L-1 R-(-)-linalool. Two hundred-microliter samples were transferred to 96-well plates and incubated with continuous shaking at 37 °C in a Biotek Cytation 3 imaging plate reader (Biotek Instruments Inc., Winooski, VT, USA). OD600 was automatically measured every 0.5 h. To determine the minimum inhibitory concentrations of R-(-)-linalool, equal amounts of cells at OD600 = 1 were subjected to serial 10-fold dilutions with fresh LB medium. Then 100 μL of a 10-5 dilution was spread onto inhibitory LB plates containing 0, 1, 1.1, 1.2, 1.3, 1.4, or 1.5 g L-1 R-(-)-linalool and incubated for 12 h at 37 °C. The viable cells on each plate were then counted. Three independent biological / experimental replicates were prepared. The minimum inhibitory concentration of R-(-)-linalool was defined as that which reduced colony formation by ≤ 2× on inhibitory LB plates.