Chemicals, enzymes, plasmids and strains
The standards α,β-unsaturated alcohols and aldehydes were obtained from Sigma-Aldrich (Shanghai) Trading Co., Ltd. (Shanghai, China). Other chemicals of analytical grade were purchased from Sangon Biotech Co. Ltd (Shanghai, China) or Shanghai Jingchun Reagent Co., Ltd (Shanghai, China). The kits and the enzymes for gene manipulation were obtained from Takara Biomedical Technology Co., Ltd. (Beijing, China). The vectors pET28a and pACYC Duet-1 were used for the over-expression of the enzymes, and the E. coli strain BL21(DE3) was used as the host. E. coli cultures were grown routinely in Luria Bertani (LB) medium at 37 °C for 12 h.
Cloning and over-expression of YsADH, TkNOX and VsHGB in E. coli
The genes encoding YsADH (GenBank accession number: KF887947), TkNOX (BAD85488) and VsHGB (AAA27584) were colon-optimized and synthesized at Vazyme Biotech Co.,Ltd (Nanjing, China). The gene encoding YsADH was introduced into the sites Nco I/Hind III of the vector pACYC Duet-1, yielding the recombinant plasmid pACYC Duet-1-YsADH. Moreover, the gene encoding TkNOX was further inserted into the sites Nde I/Xho I of the plasmid pACYC Duet-1-YsADH, yielding the recombinant plasmid pACYC Duet-1-YsADH-TkNOX. In addition, the gene encoding VsHGB was introduced into the sites EcoR I and Hind III of the vector pET28a, offering the recombinant plasmid pET28a-VsHGB.
The recombinant plasmids pACYC Duet-1-YsADH and pACYC Duet-1-YsADH-TkNOX were transformed into the host strain E. coli BL21(DE3), giving the recombiant strains E. coli BL21(DE3)/pACYC Duet-1-YsADH and E. coli BL21(DE3)/pACYC Duet-1-YsADH-TkNOX, respectively. Furthermore, the recombinant plasmid pET28a-VsHGB was transformed into the strain E. coli BL21(DE3)/pACYC Duet-1-YsADH-TkNOX, leading to the strain E. coli BL21(DE3)/pACYC Duet-1-YsADH-TkNOX/pET28a-VsHGB.
The recombinant E. coli strains were routinely grown in LB medium containing 50 µg/ml chloramphenicol and/or 50 µg/ml kanamycin at 37 °C until the OD600 of 0.6–0.8. Specifically, chloramphenicol was used for the cells containing the vector pACYC Duet-1, kanamycin was used for the cells containing the vector pET28a, meanwhile both chloramphenicol and kanamycin were supplemented for the cells containing the vectors pACYC Duet-1 and pET28A. The strains were induced by adding 0.3 mM IPTG and cultured at 20 ℃ for 12 h. The cells were washed twice using 50 mM Tris-HCl buffer (pH 8.0) and then harvested by 8000 g centrifugation at 4 °C for 10 min. Finally, lyophilized cells were obtained by freeze-drying and stored at -20 °C for further use.
Construction and over-expression of the fusion enzymes YsADH-(linker)-TkNOX and YsADH-(linker)-TkNOX-(linker)-VsHGB in E. coli
To construct four YsADH–(linker)–TkNOX fusion genes, the stop codon of the YsADH gene was removed, and the linkers of different lengths, (GSG)n (n = 1, 2) or (GGGGS)n (n = 1, 2), were introduced between the open reading frames of the YsADH and TkNOX genes via two rounds of PCR. The first round of the PCR introduced the linkers (GSG)n (n = 1, 2) and (GGGGS)n (n = 1, 2) into the YsADH gene using four pairs of primers. Simultaneously, the complementary linkers (GSG)n (n = 1, 2) and (GGGGS)n (n = 1, 2) were introduced into the TkNOX gene using four other pairs of primers (Table 3). Each PCR product was purified and served as a template in the second round of PCR. The PCR program included a 4 min period at 98 °C, 32 cycles at 98 °C (10 s), 58 °C (10 s) and 72 °C (30 s), and a final 5 min extension at 72 °C. The gel purified PCR products were ligated into a pACY CDuet-1 vector. Next, the PCR products of the YsADH and TkNOX genes were joined by overlapping extension PCR. The PCR program included a 4 min period at 98 °C, 32 cycles at 98 °C (10 s), 58 °C (10 s) and 72 °C (30 s), and a final 5 min extension at 72 °C. The gel purified PCR products were ligated into a pACYC DUet-1 vector. The four fusion genes were confirmed by sequencing. Finally, the four fusion genes were ligated to pACYC Duet-1 between Nco I and Hind III sites, yielding pACYC Duet-1-YsADH-(GGGGS)-TkNOX, pACYC Duet-1-YsADH-(GGGGS)2-TkNOX, pACYC Duet-1-YsADH-(GSG)-TkNOX and pACYC Duet-1-YsADH-(GSG)2-TkNOX. Each expression construct pACYC DUet-1-YsADH-(GGGGS)-TkNOX, pACYC DUet-1-YsADH-(GGGGS)2-TkNOX, pACYC Duet-1-YsADH-(GSG)-TkNOX or pACYC Duet-1-YsADH-(GSG)2-TkNOX, was transformed into E. coli BL21(DE3) grown in LB medium containing 50 µg/ml chloramphenicol.
During the construction of YsADH-(linker)-TkNOX-(linker)-VsHGB fusion gene, the linker was chosen set as GSG. Similar to the construction of YsADH–(linker)–TkNOX fusion genes, the YsADH-(GSGr)-TkNOX-(GSG)-VsHGB fusion gene was obtained via two rounds of PCR and confirmed by sequencing. The fusion gene was ligated to pACYC Duet-1 between Nco I and Hind III sites and the resulting recombinant plasmid pACYC Duet-1-YsADH-(GSGr)-TkNOX-(GSG)-VsHGB. was transformed into E. coli BL21(DE3) grown in LB medium containing 50 µg/ml chloramphenicol. Following the same procedure for the induction mentioned above, the cells expressing the fusion enzymes YsADH-(linker)-TkNOX or YsADH-(GSG)-TkNOX-(GSG)-VsHGB were induced, harvested and lyophilized.
Table 3
The primers used for the construction of the fusion enzymes
Linker | Gene | | Primers |
GGGGS | ADH | F | 5-TAACTTTAATAAGGAGATATACCATGGGCATGTCTATTATAAAAAGCTATGCC-3 |
R | 5-ACCACGGTTTTACGTTCCATGCTGCCGCCGCCGCCAAAGTCGGCTTGCAGTACCAC-3 |
TkNOX | F | 5-TGGTACTGCAAGCCGACTTTGGCGGCGGCGGCAGCATGGAACGTAAAACCGTGGTG-3 |
R | 5-ACTTAAGCATTATGCGGCCGCAAGCTTTCAAAATTTCAGAACACGTGCC-3 |
GSG | ADH | F | 5-TAACTTTAATAAGGAGATATACCATGGGCATGTCTATTATAAAAAGCTATGCC-3 |
R | 5-CACCACGGTTTTACGTTCCATGCCGCTGCCAAAGTCGGCTTGCAGTACCAC-3 |
TkNOX | F | 5-GTGGTACTGCAAGCCGACTTTGGCAGCGGCATGGAACGTAAAACCGTGGTG-3 |
R | 5-ACTTAAGCATTATGCGGCCGCAAGCTTTCAAAATTTCAGAACACGTGCC-3 |
(GGGGS)2 | ADH | F | 5-TAACTTTAATAAGGAGATATACCATGGGCATGTCTATTATAAAAAGCTATGCC-3 |
R | 5-CACCACGGTTTTACGTTCCATGCTGCCGCCGCCGCCGCTGCCGCCGCCGCCAAAGTCGGCTTGCAGTACCAC-3 |
TkNOX | F | 5-GTGGTACTGCAAGCCGACTTTGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCATGGAACGTAAAACCGTGGTG-3 |
R | 5-ACTTAAGCATTATGCGGCCGCAAGCTTTCAAAATTTCAGAACACGTGCC-3 |
(GSG)2 | ADH | F | 5-TAACTTTAATAAGGAGATATACCATGGGCATGTCTATTATAAAAAGCTATGCC-3 |
R | 5-CACCACGGTTTTACGTTCCATGCCGCTGCCGCCGCTGCCAAAGTCGGCTTGCAGTACCAC-3 |
TkNOX | F | 5-GTGGTACTGCAAGCCGACTTTGGCAGCGGCGGCAGCGGCATGGAACGTAAAACCGTGGTG-3 |
R | 5-ACTTAAGCATTATGCGGCCGCAAGCTTTCAAAATTTCAGAACACGTGCC-3 |
GSG | YsADH-(GSG)-TkNOX | F | 5-TAACTTTAATAAGGAGATATACCATGGGCATGTCTATTATAAAAAGCTATGCC-3 |
R | 5-GGTCTGCTGGTCCAGCATGCCGCTGCCAAATTTCAGAACACGTGCC-3 |
VsHGB | F | 5-GGCACGTGTTCTGAAATTTGGCAGCGGCATGCTGGACCAGCAGACC-3 |
R | 5-ACTTAAGCATTATGCGGCCGCAAGCTTTTATTCAACTGCCTGAGCG-3 |
Enzyme assays
The lyophilized cells were re-suspended in the 50 mM Tris-HCl buffer (pH 8.0) and disrupted through ultrasonication for 10 min. After that, the cell debris and cell lysate were removed by centrifugation to result in a clear cell extract.
Activities of TkNOX in crude cell extracts were determined according to the previously-reported procedure [20]. Activities of YsADH in crude cell extracts were measured at 45 °C by monitoring the change of the absorbance at 340 nm. The enzyme assay for alcohol oxidation was carried out 45 °C in triplicate in a reaction mixture (2.5 ml) composed of 20 mM crotyl alcohol and 1 mM NADP+ in 50 mM Tris-HCl (pH 8.0) buffer. The reaction was started by the addition of the enzyme. One unit of the activity is defifined as formation or oxidation of 1 µmol NADPH per min. The protein concentrations of all samples were determined using the Bradford reagent with bovine serum albumin as the standard protein. In addition, the determination of H2O2 was conducted according to the previously-reported procedure [33].
The reaction mixture of α,β-unsaturated alcohol oxidation and its optimization
The standard reaction mixture (5 ml) contained 100 mM α,β-unsaturated alcohols, 0.3 g lyophilized cells, 0.4 mM NADP+, 0.2 mM FAD, 50 mM Tris-HCl buffer (pH 8.0). The reaction was carried out at 45 °C and 600 rpm for 2 h. The optimal temperature of alcohol oxidation was determined at a series of temperatures ranging from 40 to 65 °C. The optimal pH was determined over a range of pH 6.0 to 8.5 at 45 °C. The buffers used were piperazine-1,4-bisethanesulfonic acid (PIPES, pH 6.1–7.5), Tris-HCl (pH 7.5-9.0). The optimal rotation was determined over a range of 400 to 900 rpm. The concentrations of FAD and NADP+ were explored within the range of 0 to 1 mM. After alcohol oxidation, the reaction mixture was extracted by 5 ml of ethyl acetate under strong vibration. The organic phase in samples was separated by centrifugation and dehydrated by anhydrous sodium sulfate, and then 1 µl dehydrated sample was applied for GC analysis.
Determination of substrates, products and possible by-products by gas chromatograph
α,β-unsaturated alcohols/aldehydes and possible by-products were determined by GC (Agilent 6890N) equipped with an FID detector and chiral capillary BGB-174 column (BGB Analytik, Böckten, Switzerland, 30 m × 250 µm × 0.25 µm). The flow rate and split ratio of N2 as the carrier gas were set as 1.38 ml/min and 1:100, respectively. Both injector and detector were kept at 250◦C. The injection volume was 1 µl.
For crotyl alcohol/crotonaldehyde, 3-methyl-2-buten-1-ol/3-methyl-2-butenal, trans-2-hexenol/trans-2-Hexenal and cinnamyl alcohol/cinnamaldehyde, the column temperature program was listed as follows; initial temperature of 75 °C for 3 min, 10 °C/min ramp to 120 ◦C for 3 min, and 30 °C/min ramp to 180 °C for 3 min. For geraniol/geranal and nerol/neral, the column temperature program was listed as follows; initial temperature of 75◦C for 3 min, 4 °C/min ramp to 120 °C for 3 min, and 30 °C/min ramp to 180 °C for 3 min. The retention times of the above-mentioned substrates and products were summarized in Table 4.
Table 4
The retention time of substrates and products in GC analyses
Entry | Alcohols | Time (min) | Aldehydes | Time (min) |
1 | Crotyl alcohol | 3.509 | Crotonaldehyde | 3.777 |
2 | 3-Methyl-2-buten-1-ol | 5.320 | 3-Methyl-2-butenal | 7.752 |
3 | trans-2-Hexenol | 6.115 | trans-2-Hexenal | 13.151 |
4 | Nerol | 17.619 | Neral | 18.858 |
5 | Geraniol | 18.562 | Geranial | 19.504 |
6 | Cinnamyl alcohol | 14.851 | Cinnamaldehyde | 14.117 |
7 | Farnesol | 19.662 | Farnesal | 19.471 |
For possible by-products isovaleraldehyde, isovaleric acid and 3,3-dimethylacrylic acid, the column temperature program was listed as follows; initial temperature of 60 °C for 5 min, 10 °C/min ramp to 120 °C for 3 min, and 30 ◦C/min ramp to 180 °C for 3 min. The retention times for isovaleraldehyde, 3,3-dimethylacrylic acid and isovaleric acid were 6.987 min, 7.839 min and 9.612 min, respectively.
HPLC-based determination of retinol and retinal
Retinol and retinal were determined by HPLC (Waters 2010) equipped with an UV detector and C-18 column (Welwich, 30 m × 250 µm × 0.25 µm). The HPLC conditions were listed as follows: Temperature, 40 °C; the wavelength of UV detector, 340 nm; flow rate, 1 ml/min; mobile phase, methanol: acetonitrile = 95: 5. The retention time for retinol and retinal were 5.237 min and 5.618 min, respectively.