Luria-Bertani (LB) medium (1% Bacto Tryptone, 0.5% Bacto Yeast Extract, and 1% NaCl), M9 medium(1x M9 salts, 1 mM thiamine hydrochloride, 0.4% glucose, 0.2 casamino acids, 2mM magnesium sulfate, and 0.1 mM calcium chloride) was used for general cultivation. If necessary, chloramphenicol (Cm; 30 μg/ml), kanamycin (Kana; 50 μg/ml), ampicillin (Amp; 100 μg/ml) or/and tetracycline (Tet; 10 μg/ml) was added. For solid media, 1.5% (wt/vol) agar was added. As for the arginine synthesis experiment, 5 mM ammonia(aq) was added as an ammonia source; for the cysteine synthesis experiment, 30 mM ammonium sulfate was added as a sulfate source; and 1% glucose was added as an extra carbon source in myrcene synthesis experiment. For all experiments, EcN and DH10b grew at 37 degree Celsius and 200 rpm in Deep-well Multiwell Plate overnight and test tubes with 0.5 mM isopropyl-β-D-1-thiogalactopyr-anosid(IPTG), except gene knockout which was at 30 degree Celsius.
Strains and plasmids
Construction of arginine maximization plasmids
The arginine maximization plasmid was constructed by mutating argA gene(from E.coli strain DH10b, encoding for N-acetyl glutamate synthase) into argA^fbr which substituting the TAT (Tyr) in 19th position into TGT (Thr), and the GCG (Ala) in 389th position into GCT (Ala) through polymerase chain reaction (PCR)). Meanwhile, the original argA gene was inserted into the pTYT plasmid(pTYT-argA) as a control group. Besides, CRISPR-cas9 was used to knock out gene argR from the genome of DH10b. Therefore, DH10b with pTYT-argA, pTYT- argA^fbr, and pTYT-argA-∆argR, pTYT-argA^fbr -∆argR are constructed along with the wildtype.
Primers argA-F and argA-R (Table.5) were used to obtain the original argA gene from the genome by polymerase chain reaction(PCR). GoldenGate was used to connect the PCR product to the pET28B plasmid with promoter Ptac. A set of primers argA-Mut-F1, argA-Mut-R2, argA-Mut F2, and argA-Mut R1 (Table.5) were used to mutate the argA to argA^fbr gene, the PCR product was digested by DpnI Restriction Endonuclease and then connected by Gibson assembly. The connection was verified by primers rrnBT-F1 and Ptac-R1 (Table.5) to detect if the genes were successfully inserted.
The upstream and downstream fragments of argR gene were obtained by primer sets Up-arm F along with Up-arm R, and down-arm F along with down-arm R respectively (Table.5). The gRNA was designed with the website https://www.atum.bio/eCommerce/cas9/input and was obtained by gRNA-1-F1, gRNA-1-F2, and Scarfold-R (Table.5). The gRNA, upstream and downstream fragments were constructed into pTarget using Gibson assembly.
The constructed pTarget plasmid along with pCas plasmid was transformed into both E.coli EcN and E.coli DH10b to delete argR gene.
Table.4 The list of all stains and plasmid used. The relevant characteristics of plasmids are listed in the following order: type of replication origin, antibiotic resistance, type of promoter, standard plasmid name.
|strain or plasmid||relevant characteristics||reference|
|E.coli DH5a||F-80d lacZ∆M15 ∆(lacZYA-argF) U169 end A1 recA1 hsdR17 (rk-, mk+) supE44– thi-1 gtrA96 relA1 phoA||Ref. Trans5α Chemically Competent Cell, n.d.|
|E.coli EcN||E.coli Nissle 1917|
|E.coli DH10b||F- mcrA ∆(mrr-hsdRMS-mcrBC) 80 lacZ∆M15∆lacX74 recA1 ara∆139∆(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG||Ref. Trans10 Chemically Competent Cell, n.d.|
|E.coli DH10b ∆argR||F- mcrA ∆(mrr-hsdRMS-mcrBC) 80 lacZ∆M15∆lacX74 recA1 ara∆139∆(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG ∆argR||this work|
|plasmid for arginine synthesis|
|pTYT-argA||pBR322, Kanar, Ptac, pET28B-argA||this work|
|pTYT-argAfbr||pBR322, Kanar, Ptac, pET28B-argA^fbr||this work|
|plasmid for cysteine synthesis|
|pTYT-cysE||pBR322, Kanar, Ptac, pET28B-cysE||this work|
|pTYT-cysE-256||pBR322, Kanar, Ptac, pET28B-mut cysE 256||this work|
|pTYT-cysE-5||pBR322, Kanar, Ptac, pET28B-mut cysE 5||this work|
|pTYT-cysE-11-2||pBR322, Kanar, Ptac, pET28B-mut cysE 11-2||this work|
|pTYT-cysE-5-11-2||pBR322, Kanar, Ptac, pET28B-mut cysE 5&11-2||this work|
|pTYT-cysE-256-5||pBR322, Kanar, Ptac, pET28B-mut cysE 256&5||this work|
|pTYT-cysE-256-11-2||pBR322, Kanar, Ptac, pET28B-mut cysE 256&11-2||this work|
|pTYT-cysE-256-5-11-2||pBR322, Kanar, Ptac, pET28B-mut cysE 256&5&11-2||this work|
|plasmid for myrcene synthesis|
|pTYT-GPPS-MS||pBR322, Kanar, Ptac, pET28B-tGPPS-MS_Qi||this work|
|pMevT||p15A, Cmr, Plac, pMBIS-atoB-HMGS-tHMGR||Jay Keasling's engineered Saccharomyces cerevisiae|
|pMBIS||pBBR1, Tetr, Plac , pMBIS-ERG12-ERG8-MVD1-idi-ispa||Jay Keasling’s engineered Saccharomyces cerevisiae|
|pTarget||pBR322, Ampr, J23119, pTarget-upArm-gRNA-downarm||this work|
|pCas9||pSC101, Kanar, Pcas, pCas-cas9-gam-bet||Ref. Jiang, 2015|
Construction of cysteine maximization plasmids
Three mutants in cysE gene were utilized and inserted into the following plasmids: pTYT-cysE-256 to express the mutant cysE-256(substitute the ATG (Met) on the 256th position into TGG (Trp)), pTYT-cysE-5 to express the mutant cysE-5 (mutate the GTC (Val), GAT (Asp) on the 95th and 96th position into AGA (Arg), CCC (Pro)), and pTYT-cysE-11-2 to express the mutant cysE-11-2(substitutes CGT (Arg), ACC (Thr) in 89th, 90th positions into CAT (His), GTA (Val)), and substitutes CCG (Pro), GCA (Ala) in 93th and 94th position into GCT (Ala), ACA (Thr)). By combining these mutants, more mutants were created: plasmid pTYT-cysE-256-5(combine the
positions of mutations in mutants cysE-256 and cysE-5), pTYT-cysE-256-11-2(combine the positions of mutations in mutants cysE-256 and cysE-11-2), pTYT-cysE-256-5-11-2(combine the positions of mutations in mutants cysE-256, cysE-5, and cysE-11-2), and pTYT-cysE-5-11-2 (combine the positions of mutations in mutants cysE-5 and cysE-11-2) were constructed on the pET28B plasmids. Meanwhile, original cysE was constructed on the same plasmid as pTYT-cysE with the same promoter served as the control along with wildtype (Table.4).
The original cysE gene was obtained from the genome in DH5a by PCR with primers cysE-F and cysE-R (Table.5). GoldenGate was used to connect gene cysE to plasmid pET28B to construct pTYT-cysE. Primers cysE-mut-F1, cysE-Mut-R2, cysE-Mut R1, and cysE-Mut-F2 were used to mutate cysE to cysE-256 with templated pTYT-cysE, which was digest by DpnI Restriction Endonuclease (Table.5), then connected by overlapped PCR to construct the plasmid pTYT-cysE-256. Similarly, primers cysE5&11-Mut-F1, cysE5&11-Mut-R2, cysE5-mut-F1, cysE5-mut-R2, cysE11-mut-F1, and cysE11-mut-R2 were used to obtain genes cysE-5, cysE-11-2, cysE-5-11-2, cysE-256-5, cysE-256-11-2, and cysE-256-5-11-2 by PCR with templates pTYT-cysE and pTYT-cysE-256 (Table.6), which were digested by DpnI, and connected by overlap PCR to pET28B plasmid. These were verified by PCR with primers cysE-JC-F and cysE-JC-R if the mutation was gained, and rrnBT-F1 and Ptac-R1 if the genes were inserted into the pET28B plasmid (Table.5).
Table. 5 Information on primers used in this research
|argA-F||Obtain the argA gene.||5'-TGGAATTCGCGGCCGCTTCTAGAGGTGGTAAAGGAACGTAAAACCG-3'|
|argA-R||Obtain the argA gene.||5'-GGACTGCAGCGGCCGCTACTAGTATTACCCTAAATCCGCCATCAA-3'|
|argA-Mut-F1||Mutate the argA to argAfbr gene||5'-TTCCCTGTATCAATACCCACCG-3'|
|argA-Mut-R2||Mutate the argA to argAfbr gene||5'-CGGTGGGTATTGATACAGGGAA-3'|
|argA-Mut-F2||Mutate the argA to argAfbr gene||5'-TCAGGCTAAGCAGAGC-3'|
|argA-Mut-R1||Mutate the argA to argAfbr gene||5'-GCTCTGCTTAGCCTGA-3'|
|cysE-F||Obtain the cysE gene.||5'-TGGAATTCGCGGCCGCTTCTAGAGATGTCGTGTGAAGAACTGGAA-3'|
|cysE-R||Obtain the cysE gene.||5'-CGCTACTAGTATTAGATCCCATCCCCATACTC-3'|
|pTarget-JC-F||Verify the connection of up-Arm down-Arm and gRNA||5'-TTGCTGGCCTTTTGCTCACATG-3'|
|pTarget-JC-R||Verify the connection of up-Arm down-Arm and gRNA||5'-TCGATCATAGCACGATCAACGGC-3'|
|gRNA-1-F2||Construct gRNA sequence||5'-CAGTCCTAGGTATAATACTAGTAGAAGAGAAATTTAGCTCCCGTTTTAG-3'|
|gRNA-1-F1||Construct gRNA sequence on pTarget||5'-TAATACTAGTAGAAGAGAAATTTAGCTCCCGTTTTAGAGCTAGAAATAGCAAGTTAAAA-3'|
|Scarfold-R||Construct and connect gRNA sequence on pTarget||5'-CTGCAGGTCGACTCTAGAGA-3'|
|pTarget-VR||Construct pTarget plasmid vector||5'-ACTAGTATTATACCTAGGACTGAGCT-3'|
|pTarget-VF||Construct pTarget plasmid vector||5'-AAGCTTAGATCTATTACCCTGTTATCC-3'|
|UP-Arm-F||Obtain and construct up-Arm gene from genome||5'-TCTCTAGAGTCGACCTGCAGGGTTTTTAACAGTAGTGCAAGCGC-3'|
|UP-Arm-R||Obtain and construct up-Arm gene from genome||5'-AAGTCACCCGATATGGTGGTTG-3'|
|DOWN-Arm-F||Obtain and construct up-Arm gene from genome||5'-ACCACCATATCGGGTGACTTTCTCTGCCCCGTCGCTTCTG-3'|
|DOWN-Arm-R||Obtain and construct up-Arm gene from genome||5'-CAGGGTAATAGATCTAAGCTTGCCACACCACTTACGGATACG-3'|
|rrnBT1-R1||Verify gene on pTYT||5'-TGCGCCGCTACAGGGCGCGTGAGAGCGTTCACCGACAAACAA-3'|
|Ptac-F1||Verify gene on pTYT||5'-AGATCTCGATCCCGCGAAATTTCGTCAGGCCACATAGCTT-3'|
|MS-F||Obtain the MS gene and connect to GPPS||5'-TACTAGAGAAAGAGGAGAAATACTAGATGCGAAGAAGCGCGAATTATCA-3'|
|MS-R||Obtain the MS and connect to pTYT||5'-TCGTTTTATTTGATGCCTGGACTAGTATTAGTCCTTGTTCAGCGGGA-3'|
|GPPS-F||Obtain the GPPS and connect to pTYT||5'-AAACAGCCTCTACAAATAATTTTGTTTAAATACCCGTTTTTTGGGCTAA-3'|
|GPPS-R||Obtain the GPPS gene and connect to MS||5'-CTAGTATTTCTCCTCTTTCTCTAGTATTATTTGCTGCGTTTGTAAACCT-3'|
|cysE-Mut-F1||Mutate cysE to cysE-256||5'-CGCTAAGGATGATTTCTGGAATTCGC-3'|
|cysE-Mut-R2||Mutate cysE to cysE-256||5'-GCGAATTCCAGAAATCATCCTTAGCGAAAG-3'|
|cysE-Mut-R1||Mutate cysE to cysE-256||5'-GTTGAAATGCTGGTCCCAATCCATTGATGG-3'|
|cysE-Mut-F2||Mutate cysE to cysE-256||5'-CATCAATGGATTGGGACCAGCATTTCAAC-3'|
|cysE5&11-Mut-F1||Mutate cysE to cysE-5-11-2 & cysE-256-5-11-2||5'-CATGTACGCGACGCTACAAGACCCAAATACTCAACCCCGTTG-3'|
|cysE5&11-Mut-R2||Mutate cysE to cysE-5-11-2 & cysE-256-5-11-2||5'-CTTGTAGCGTCGCGTACATGCACCGCCTGAATATCACAGG-3'|
|cysE5-mut-F1||Mutate cysE to cysE-5 & cysE-256-5||5'-CCCGCGACCCGGCAAGACCCAAATACTCAACCCCGTTGTTAT-3'|
|cysE5-mut-R2||Mutate cysE to cysE-5 & cysE-256-5||5'-GGGTCTTGCCGGGTCGCGGGTACGCAC-3'|
|cysE11-mut-F1||Mutate cysE to cysE-11-2 & cysE-256-11-2||5'-TGCATGTACGCGACGCTACAGTCGATAAATACTCAACCCCGTTGT-3'|
|cysE11-mut-R2||Mutate cysE to cysE-11-2 & cysE-256-11-2||5'-TGTAGCGTCGCGTACATGCACCGCCTGAATATCACAGG-3'|
|cysE-JC-F||Verify the mutation of cysE||5'-TGCTGGCGAACAAGCTGTCATC-3'|
|cysE-JC-R||Verify the mutation of cysE||5'-GGAACGTCACAGAAACCTGGTTTTG-3'|
|genone-F||Verify the gene knockout||5'-CTGGAGCGATATCATACAGAGAGAGTTC-3'|
|genone-R||Verify the gene knockout||5'-GTTCAGCATTTCACGCATATCCATTGGC-3'|
Table.6 Designed mutations on plasmids used in this research.
|Allele of the cysE gene||sequence of proteins and cysE gene at position 89-96||SAT protein and cysE gene at position 254-256|
|cysE wild-type||Arg Thr Arg Asp Pro Ala Val AspCGT ACC CGC GAC CCG GCA GTC GAT||Met Asp Met Asp GlnATG GAT ATG GAC CAG|
|cysE 256||Arg Thr Arg Asp Pro Ala Val AspCGT ACC CGC GAC CCG GCA GTC GAT||Met Asp Trp Trp GlnATG GAT TGG GAC CAG|
|cysE 5||Arg Thr Arg Asp Pro Ala Arg ProCGT ACC CGC GAC CCG GCA AGA CCC||Met Asp Met Asp GlnATG GAT ATG GAC CAG|
|cysE 11-2||His Val Arg Asp Ala Thr Val AspCAT GTA CGC GAC GCT ACA GTA GAT||Met Asp Met Asp GlnATG GAT ATG GAC CAG|
|cysE 5-11-2||His Val Arg Asp Ala Thr Arg ProCAT GTA CGC GAC GCT ACA AGA CCC||Met Asp Met Asp GlnATG GAT ATG GAT CAG|
|cysE 256-5||Arg Thr Arg Asp Pro Ala Arg ProCGT ACC CGC GAC CCG GCA AGA CCC||Met Asp Trp Trp GlnATG GAT TGG GAC CAG|
|cysE 256-11-2||His Val Arg Asp Ala Thr Val AspCAT GTA CGC GAC GCT ACA GTA GAT||Met Asp Trp Trp GlnATG GAT TGG GAC CAG|
|cysE 256-5-11-2||His Val Arg Asp Ala Thr Arg ProCAT GTA CGC GAC GCT ACA AGA CCC||Met Asp Trp Trp GlnATG GAT TGG GAC CAG|
Construction of myrcene production plasmids
Two engineered Saccharomyces cerevisiae plasmids pMevT and pMBIS constructed by Jay Keasling (originally used to produce arteannuic acid) were provided. The pMevT plasmid contains three genes AtoB(from E.coli), HMGS(from Saccharomyces cerevisiae), and tHMGR(from Saccharomyces cerevisiae) with promoter Plac (Table.4). The pMBIS plasmid contains five genes, which are ERG12(from Saccharomyces cerevisiae), ERG8(from Saccharomyces cerevisiae), MVD1(from Saccharomyces cerevisiae), idi(from Saccharomyces cerevisiae), and ispA(from Saccharomyces cerevisiae), with promoter Plac. The third plasmid pTYT-GPPS-MS was constructed and it contains genes GPPS(from Abies Grandis, position 1-84) and MS(from Quercus ilex L, position 1-56). They were constructed on the pET28B substituting promoter PT7 to Ptac.
pMevT and pMBIS were obtained from Jay Keasling's laboratory. GPPS was obtained from plasmid pLB1s-Erg20(M) provided by biotech company Bluepha with primers GPPS-F and GPPS-R, and MS was obtained from synthesis plasmid with primer MS-F and MS-R provided by biotech company GenScript. The pET28B vector was linearized with BsaI. The GPPS fragment, MS fragment, and the linearized vector were connected by overlap PCR with primer GPPS-F and MS-R to construct pTYT-GPPS-MS. Primers rrnBT-F1 and Ptac-R1 were designed to detect if the genes are inserted into the pET28B plasmid(table.5).
Measurement of ammonia
Nessler's reagent was prepared by weighing 16 g sodium hydroxide (NaOH), dissolving with 50 ml distilled water, and waiting until it is cold as the surrounding. 7 g potassium iodide and 10 g mercury iodide are dissolved consecutively into the solution. The solution then is poured into the aqua of sodium hydroxide slowly while stirring and diluted into 100 ml.
Sodium potassium tartrate tetrahydrate reagent is prepared by weighing 50 g sodium potassium tartrate tetrahydrate dissolving with 100 ml distilled water.
Besides, the molarities of standard samples which mixed ammonium sulfate with distilled water are 0.025mM, 0.05mM, 0.1mM, 0.2mM, 0.4mM, 0.5mM.
Determination of Calibration line
96-well plates were used added with 150 μL samples, 30 μL sodium potassium tartrate tetrahydrate reagent, and 20 μL Nessler's reagent in turn. After 10 minutes of waiting, infinite 200Pro spectrophotometer was used to test the light absorption at 420 nm. A reagent blank without ammonium sulfate was prepared under the same condition. The least-squares regression line was drawn according to data collected. The x-axis refers to the absorbance of light at a certain wavelength, and the y-axis refers to the concentration of NH4+ in solution.
NH4+ analysis and quantification
Before the experiment, DH10b was cultivated in M9 medium with 5mM ammonia and 0.1 mM IPTG in Deep-well Multiwell Plate at 37 degree Celsius overnight. 10 μL supernatant of each medium was taken from Deep-well Multiwell Plate and diluted with 990 μL distilled water in 1.5 mL centrifuge tubes respectively and mixed thoroughly. The reagent blank with only pure M9 medium was prepared under the same condition. The same operation was repeated to test standard samples, and then used spectrophotometer with light absorption at 420 nm to test the concentration of ammonia in the solutions.
Measurement of cysteine
The acid ninhydrin reagent contained 250mg of ninhydrin in a mixture of 6ml of acetic acid and 4 ml of conc. HCl, which was mixed repeatedly at room temperature, yielded a solution in 20 - 30 min. This reagent was prepared before use. Besides, the molarities of standard samples which mixed solid L-cysteine with distilled H2O are 0.1mM, 0.2mM, 0.4mM, 0.8mM and 1mM respectively (Gaitonde, 1967).
Determination of Calibration Line
Centrifuge tubes are added 0.1mM, 0.2mM, 0.4mM, 0.8mM, and 1mM to 1.5 ml standard samples of L-cysteine respectively with 0.1 ml acid ninhydrin reagent and 0.1 ml acetic acid, mixed thoroughly at room temperature. The centrifuge tubes are heated in the metal bath with 100 degrees Celsius for 10 minutes, and then rapidly cool in ice. Then 0.7 ml 95% ethanol is added in each centrifuge tubes and mixed thoroughly. A reagent blank without cysteine is prepared under the same condition. The pipette is used to shift 200 microliter solutions of each mixture to 96-well plates so that solution can be tested by infinite 200Pro spectrophotometer, with light absorption at 560 nm. The least-squares regression line is created according to the data collected by the spectrophotometer. The x-axis refers to the absorbance of light at a certain wavelength, and the y-axis refers to the concentration of cysteine in solution.
Cysteine analysis and quantification
Before the test, EcN was cultivated in M9 medium with 0.5 mM IPTG in Deep-well Multiwell Plate at 37 degree Celsius overnight. 0.1 ml supernatant of each EcN solution is taken from Deep-well Multiwell Plate and added to 1.5 ml centrifuge tubes respectively. The same operation is repeated as testing standard samples, and then use spectrophotometer with light absorption at 560 nm to test the concentration of cysteine in the solutions.
Measurement of myrcene
Before the experiment, DH10b is introduced in 2 ml M9 medium with 0.5 mM IPTG and 1% glucose, in 37 degree Celsius overnight. 20% dodecane (v/v) was added to collect the myrcene produced by DH10b. After the centrifugation (12000rpm for a minute), the dodecane layer was collected to test the production of myrcene (Kim, E. et al, 2015).
Myrcene analysis and quantification
After the dodecane layer was collected, the collected sample was analyzed by gas chromatography−mass spectrometry [GC−MS, Agilent 6890N series GC/TOF-MS(LECO), under these conditions: injector temperature, 250°C; flowrate, 1.2 mL/min; split ratio, 2:1; oven initially at 60°C for 5 min, followed by 4°C/min, and increase to 240°C; carrier gas, N2; and HP-Ultra2 column (25 m long, 0.2 mm diameter, and 0.11μm film thick)]. 95% myrcene sample was used as a standard for quantitative analysis, and the concentrations were normalized using an internal standard.
Gaitonde MK. A spectrophotometric method for the direct determination of cysteine in the presence of other naturally occurring amino acids. Biochem J. 1967;104(2):627-633.
Kim, E. et al. " Microbial Synthesis of Myrcene by Metabolically Engineered Escherichia coli.pdf", Journal of Agricultural and Food Chemistry, 2015.