Strains and reagents
DNA polymerases, Taq Mix, T4 DNA ligase, Gibson Assembly® One Step Cloning Kit were purchased from Vazyme Biotech Co. Ltd. (Nanjing, China). Restriction enzymes were purchased from New England Biolabs (USA). Plasmid Mini Kits, PCR Clean-Up Kits, and Gel Extraction kits were all obtained from Omega Bio-Tek Co. Ltd. (USA). P-coumaric acid and raspberry ketone was purchased from Solarbio Biochemical Co. Ltd. (Shanghai, China). All other regular chemicals were purchased from ShengGong Biochemical Co. Ltd. (Shanghai, China). The primer synthesis and gene sequencing were done in GENEWIZ, Inc. (Beijing, China).
The E. coli strain DH5α was used for plasmids construction, BW25113/F′ were used for protein expression and raspberry ketone production, respectively. Luria-Bertani (LB) medium was used for all molecular construction experiments and strain culture. Strains containing temperature-sensitive plasmids pKD46, pCP20, and pSB1s-cre were cultured at 30 °C, and the remaining strains were cultured at 37 °C without special instructions.
Luria-Bertani (LB) medium containing 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl was used to grow E. coli cells unless otherwise stated. 5 × M9 salts containing 12.8 g/L Na2HPO4•7H2O, 3 g/L KH2PO4, 0.5 g/L NaCl, 1 g/L NH4Cl was used to bioconversion. M9-FAs medium containing 1 × M9 salts, 2 mM MgSO4, 10 g/L palmitic acid, M9-FAs + YE(supplemented with YE), M9-glucose(10/L glucose instead of palmitic acid) were used to screen FA response promoters. CM medium containing 10 g/L tryptone, 5 g/L yeast extract, 5 g/L glycerol, 0.5 g/L glucose, 25 mM Na2HPO4, 25 mM KH2PO4, 50 mM NH4Cl, 5 mM Na2SO4, 2 mM MgSO4 and trace elements was used for one-step fermentation and cell preparation for bioconversion. Modified CM medium containing 1 × M9 salts, 5 g/L tryptone, 2 mM MgSO4 and trace elements was used to one-step fermentation and bioconversion. When necessary, the antibiotics were used (ampicillin, 100 µg/mL; streptomycin, 50 µg/mL; and kanamycin, 50 µg/mL.).
Construction of plasmids and strains
All strains and plasmids used in this study are shown in Table 1. All primers are listed in Supplementary Table S1. At4CL1 (GenBank ID: AAA82888.1) from Arabidopsis thaliana, RpBAS (GenBank ID: AAK82824.1) from Rheum palmatum, and RiRZS1 (GenBank ID: JN166691) from Rubus idaeus were codon-optimized for E. coli expression[17, 38–40].
Molecular cloning and genetic editing were performed using standard protocols. For knockout genes, a single knockout library stored in the laboratory was used to achieve integration through P1 phage infection[41]. pCP20 eliminated the resistance marker by identifying the FRT sites at both ends of the resistance marker gene. The plasmids pYB1s and pLB1a were previously constructed in our laboratory; the specific features were as follows: streptomycin and kanamycin resistance genes, araBAD promoter, multiple cloning sites, rrnB terminator, p15A, and R6k. Promoter replacement and gene insertion replacement used the gene-editing tool plasmids constructed in our laboratory as templates for amplification to obtain homologous recombination fragments. pSB1s-cre eliminated the resistance marker by identifying the lox66 and lox71 sites at both ends of the resistance marker gene.
Table 2
Strains and plasmids used in this study.
Strain | Genotype | Source |
E. coli BW25113/F׳ | rrnBT14 ΔlacZWJ16 hsdR514 ΔaraBADAH33 ΔrhaBADLD78 [F׳ proAB lacIqZΔM15 Tn10 (Tetr)] | CGSC |
FA09 | E. coli BW25113/F׳, ΔfadR, PCPA1-fadD, P119-fadL, ΔsthA, PCPA1-pntAB | [28] |
CC1 | FA09, ΔpoxB:: Pfrd3-AT4CL1 | This study |
CC2 | CC1, ΔfabB:: P119−BT-CA | This study |
CC3 | CC2, Pfrd3-pntAB | This study |
CR1 | E. coli BW25113/F׳ carrying pLB1a-RB and pYB1s- 4CL | This study |
CR2 | E. coli BW25113/F carrying pYB1s-RB and pLB1a-4CL | This study |
CR3 | E. coli BW25113/F׳ carrying pLB1a-RB4 | This study |
CR4 | E. coli BW25113/F׳ carrying pYB1s-RB4 | This study |
CR5 | FA09 carrying pYB1s-RB4 | This study |
CR6 | CC1 carrying pYB1s-RB4 | This study |
CR7 | CC2 carrying pYB1s-RB4 | This study |
CR8 | CC3 carrying pYB1s-RB4 | This study |
Plasmid | Description | Source |
pLB1a | araBAD promoter, R6k ori, Ampr | Our laboratory |
pYB1s | araBAD promoter, p15A ori, Strr | Our laboratory |
pKD46 | Temperature-sensitive vector carrying Red recombinase, Ampr | [42] |
pSB1s-Cre | Temperature-sensitive vector carrying Cre recombinase, Strr | Our laboratory |
pCP20 | Temperature-sensitive vector carrying FLP recombinase, Ampr | [42] |
pLB1a-RB | pLB1a containing Rubus idaeus RZS1 gene and Rheum palmatum BAS gene | This study |
pLB1a-4CL | pLB1a containing Arabidopsis thaliana 4CL1 gene | This study |
pYB1s-RB | pYB1s containing Rubus idaeus RZS1 gene and Rheum palmatum BAS gene | This study |
pYB1s- 4CL | pYB1s containing Arabidopsis thaliana 4CL1 gene | This study |
pLB1a-RB4 | pLB1a containing Rubus idaeus RZS1 gene, Rheum palmatum BAS gene and Arabidopsis thaliana 4CL1 gene | This study |
pYB1s-RB4 | pYB1s containing Rubus idaeus RZS1 gene, Rheum palmatum BAS gene and Arabidopsis thaliana 4CL1 gene | This study |
pYfrd11s-GFP | frd1 promoter, p15A ori, Strr, GFP gene | This study |
pYfrd21s-GFP | frd2 promoter, p15A ori, Strr, GFP gene | This study |
pYfrd31s-GFP | frd3 promoter, p15A ori, Strr, GFP gene | This study |
pYfrd41s-GFP | frd4 promoter, p15A ori, Strr, GFP gene | This study |
One-step fermentation conditions
When used CM medium for one-step fermentation, the recombinant strains were grown in CM medium to an OD600 of 0.4–0.6 at 30 °C, added 5 mM p-coumaric acid, and 2 g/L L-arabinose, then cultured for a given time. When used glucose for one-step fermentation, the recombinant strains were grown in M9 modified medium to an OD600 of 0.4–0.6 at 30 °C, added 2 g/L L-arabinose and induced overnight, supplemented with 10 g/L glucose, 5 mM p-coumaric acid, then cultured for a given time. When used fatty acids for one-step fermentation, the process was the same as the glucose for one-step fermentation process, except for supplementing 10 g/L fatty acids.
For condition optimization, the following methods were used to optimize the substrate: (1) different concentrations of fatty acids were added (2) 1% fatty acids and different concentrations of glycerol were used together with different concentrations of fatty acids (2) 1% fatty acids and 0.5% glucose.
Bioconversion conditions
The induction conditions we changed according to the experiment in the study, cells were collected after induction by centrifugation at 6000 × g for 10 min, washed twice with 0.85% NaCl solution, suspended in 3 mL bioconversion medium in a test tube containing 5 mM p-coumaric acid and different feedstocks, starting biomass of OD600 = 30(unless otherwise specified), growing at 30 °C, 200 rpm for 24 h. The bioconversion medium changed based on experimental conditions. The carbon sources glucose, glycerol, or fatty acids was also added based on the experimental conditions. In the process of optimizing the bioconversion conditions, the bioconversion starting biomass of OD600 was investigated at 10,20,30 and 40.
Cell growth and Fluorescence analysis
To compare the strength of FA response promoters, the GFP gene was placed under different promoters' control and introduced into a FA utilization chassis cell FA09. After overnight cultured, inoculated to M9-FAs medium, M9-glucose medium, and M9-FAs + YE medium, starting biomass of OD600 = 0.1, 37 °C, and 200 rpm culture for 36 h. GFP intensity was measured using the BioTek Synergy Mx enzyme marker (BioTek, Winooski, VT, USA). Excitation at 468 nm, emission at 512 nm, automatic gain.
Fatty acids emulsified in the medium were opaque emulsion, so OD600 could not be used directly to detect biomass. We mixed an equal volume of ethyl acetate with the bacterial solution, centrifugation at 6000 × g for 1 min, the fatty acid was sandwiched between the ethyl acetate and the medium, discarded all the supernatant, resuspended the cells with an appropriate amount of 0.85% NaCl solution, and monitored the biomass in OD600.
Analytical methods
HPLC determined product concentration. A 500 µL sample was taken and mixed thoroughly with the same amount of absolute ethanol by vortexing 30 s. The sample was centrifuged at 8,000 × g for 10 min and filtered with a 0.2 µm PES membrane filter (Jinteng, Tianjin, China).
The concentrations of raspberry ketone and p-coumaric acid were measured by high-performance liquid chromatography (HPLC, LC 20A LabSolutions, Shimadzu Corp., Kyoto, Japan) with an Agilent Extend-C18 3.5 µm column (4.6 × 250 mm). The column was at 35 °C, and a flow rate of 0.5 ml/min was used. Mobile phase A (65%) was water with 0.1% (v/v) formic acid; mobile phase B (35%) was acetonitrile. Raspberry ketone and p-coumaric acid were detected via DAD detection. The raspberry ketone was detected at 222 nm, p-coumaric acid was detected at 305 nm.