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[Abstract]This study constructed a suicide plasmid named pPICPJ-mazF that uses the mazF gene of Escherichia coli as a counterselectable marker for the markerless editing of C.
[Key words]Candida tropicalis 1798； Glycerol； Suicide plasmid； Long-chain dicarboxylic acid
中圖分类号：Q933 文献标识码：A 文章编号：1009-914X（2018）29-0251-01
1 Materials and Methods
1.1 Strains and plasmids
Escherichia coli DH5α was used as the preliminary host for all genetic modifications. C. tropicalis 1798 and C. lipolytica 1457 were purchased from the CICC. The plasmids pPICZαA and pPIC9K with a G418 resistance marker were purchased from Hangzhou Biosci Biotech Co.， Ltd.
1.2 Construction of markerless genome editing vector
For the construction of a counterselectable marker for C. tropicalis 1798， we spliced three segments into a counterselectable cassette as follows： the pGAL heterologous promoter from C. lipolytica， the mazF toxin gene from E. coli， and the aox1 terminal sequence from pPICZαA. With the use of the pGAL and mazF gene fragments as templates， overlap PCR， the pGAL-mazF recombination fragment was amplified. and the pGAL-mazF recombination fragment was then ligated to pPICZαA-Kanr with a One Step Cloning Kit to produce the suicide plasmid pPICPJ-mazF.
1.3 Construction of a vector for the replacement of the GK gene promoter
The homology arm of the glycerol kinase promoter named gkpF and gkpR from C. tropicalis 1798 and the pGAP promoter gene from C. tropicalis 1798 were obtained used for PCR amplification. The recombinant fragment gkpF-gkpR was obtained used for overlap PCR. The gkpF-gkpR fragment was ligated to the SacI site of the suicide plasmid to produce the recombinant vector pPICPJm-gkpFR， and the pGAP fragment was ligated to the AvrII site of the recombinant vector to produce the gene replacement vector pPICPJm-gkp
1.4 Preparation，transformation，and selection of competent cells
The abovementioned vectors were transfected into C. tropicalis 1798 with a Gene Pulser Apparatus. Then， C. tropicalis transformants were selected on the basis of their ability to grow on YPD plates containing G418.
Colonies of the abovementioned cells that exhibited G418 resistance were inoculated into liquid YPD medium and cultured overnight at 30 °C， and cells were collected by centrifugation at 8000 rpm. The genome was isolated from recombinant yeast using an Ezup Column Bacteria Genomic DNA Purification Kit （Sangon Biotech）， The amplification products were verified by 1% agarose gel electrophoresis. Finally， we obtained recombinant strains that were positive for gene knockout and the promoter. The three positive recombinant strains that were obtained were inoculated into 50 mL liquid YPD medium and cultured for 12 h. Then， transferred to YPGs medium for 24 h； this step was repeated， and selected microorganisms were streaked across solid YPGs medium and cultured for 36 h. Single colonies were transferred to liquid-phase YPD culture medium. By validation of the absence of the purpose band， we obtained engineered strains with the following features： the pGAP promoter without marker replacement （C. tropicalis-gkPr）；
1.5 Analysis of glycerol utilization
Samples of 500 μL C. tropicalis 1798 and C. tropicalis gkPr that had been preserved in a glycerol tube were inoculated into 50 mL liquid sugar-free YPD medium containing 2% glycerol， and the culture was shaken at 200 rpm at 30 °C. The optical density at 600 nm （OD600） of samples was measured at intervals of 2 h. Determination of glycerol was performed using the potassium permanganate oxidation method.
1.6 Fermentative activity of recombinant strains and product analysis
Samples of 500 μL C. tropicalis 1798， C. tropicalis-gkPr， which had been preserved in glycerol tubes， were inoculated into 50 mL liquid YPD medium and cultured at 200 rpm for 30 h at 30 °C. The seed culture medium was transferred to 50 mL fermentation medium， and a 10% inoculum was cultured at 200 rpm with shaking at 30 °C.
2 Results and Discussion
2.1 Construction of pPICPJ-mazF traceless knockout vector
In order to replace the Zeocin resistance marker in pPICZαA with the G418 resistance gene using pPIC9k as a template， the fragment of the G418 resistance gene Kanr was obtained by PCR and ligated to pPICZαA to obtain the recombinant vector pPICZαA-Kanr. In addition， fragment of the pGAL promoter of the GAL gene from C. lipolytica 1457， which can be induced by galactose， and a fragment of the toxin gene mazF from E. coli DH5α were amplified. After connection via overlap PCR， the overlapped fragment was ligated to the SalI site upstream of the AOX1 transcription termination region in pPICZαA-Kanr with a One Step Cloning Kit. Finally， the plasmid-free knockout vector pPICPJm was constructed
2.2 Construction of pPICPJm-gkp traceless knockout vector
The gkpF-gkpR fragment was obtained by overlap PCR and ligated to the SalI restriction site in pPICPJ-mazF to obtain pPICPJm-gkpFR. Next， the pGAP fragment derived from C. tropicalis 1798 was ligated to the AvrII site in pPICPJm-gkpFR， and pPICPJm-gkp was finally obtained. After electrotransfection and G418 selection， the first single exchange was achieved to produce recombinant C. tropicalis-gkpA. To confirm the expression of mazF in C. tropicalis 1798. Then， screening with 10 g/L galactose was carried out for the subsequent production of the second single-exchange recombinant. C. tropicalis-gkpA was cultured in 100 mL liquid YPD medium and then transferred to galactose-containing YPGs screening medium as a 1.0% inoculum. After cultivation for 24 h， galactose-resistant strains were isolated using a plate streaking method， and the genome was isolated for PCR validation to produce the second single-exchange recombinant C. tropicalis-gkPr. 2.3 Enhancement of production of long-chain dicarboxylic acids by C. tropicalis
The replacement of the GK gene promoter in the strain was investigated and the results showed that when glucose was the only carbon source， the growth rate of the two bacteria were basically the same， and the growth of both bacteria entered the stationary phase after 10 h. when glycerol was the only carbon source， the growth rate of the engineered strains was clearly higher than that of the original strain. the OD600 values reached a maximum， and the maximum value for the engineered strain C.
3 Conclusions
In this study， the mazF gene of E. coli was used as a reverse screening marker to construct the suicide vector pPICPJ-mazF. The results showed that mazF from E. coli can be induced by galactose in C. tropicalis under the control of the pGAL promoter from C. lipolytica. In addition， we used the suicide vector pPICPJ-mazF to edit the genome of C. tropicalis， replace the promoters of the GK gene reductase. The yield of long-chain dibasic acids from the engineered strain C. The results showed that a construct containing mazF from E. coli can be used as a traceless editing system in C. tropicalis to complete the gene-free editing of the genome of C. tropicalis， which lays the foundation for further metabolic engineering transformations of C. tropicalis.
Supported by the Independent Innovation and Achievement Transformation Project in Shandong Province （grant number 201422CX02602）， Major Program of National Natural Science Foundation of Shandong （grant number ZR2017ZB0208）， Shandong Provincial Natural Science Foundation （grant number ZR2016CB04）
Corresponding author： WANG Jun-qing. Tel： +86-0531-8963-1138； E-mail： wjqtt.6082@163.com；
[Key words]Candida tropicalis 1798； Glycerol； Suicide plasmid； Long-chain dicarboxylic acid
中圖分类号：Q933 文献标识码：A 文章编号：1009-914X（2018）29-0251-01
1 Materials and Methods
1.1 Strains and plasmids
Escherichia coli DH5α was used as the preliminary host for all genetic modifications. C. tropicalis 1798 and C. lipolytica 1457 were purchased from the CICC. The plasmids pPICZαA and pPIC9K with a G418 resistance marker were purchased from Hangzhou Biosci Biotech Co.， Ltd.
1.2 Construction of markerless genome editing vector
For the construction of a counterselectable marker for C. tropicalis 1798， we spliced three segments into a counterselectable cassette as follows： the pGAL heterologous promoter from C. lipolytica， the mazF toxin gene from E. coli， and the aox1 terminal sequence from pPICZαA. With the use of the pGAL and mazF gene fragments as templates， overlap PCR， the pGAL-mazF recombination fragment was amplified. and the pGAL-mazF recombination fragment was then ligated to pPICZαA-Kanr with a One Step Cloning Kit to produce the suicide plasmid pPICPJ-mazF.
1.3 Construction of a vector for the replacement of the GK gene promoter
The homology arm of the glycerol kinase promoter named gkpF and gkpR from C. tropicalis 1798 and the pGAP promoter gene from C. tropicalis 1798 were obtained used for PCR amplification. The recombinant fragment gkpF-gkpR was obtained used for overlap PCR. The gkpF-gkpR fragment was ligated to the SacI site of the suicide plasmid to produce the recombinant vector pPICPJm-gkpFR， and the pGAP fragment was ligated to the AvrII site of the recombinant vector to produce the gene replacement vector pPICPJm-gkp
1.4 Preparation，transformation，and selection of competent cells
The abovementioned vectors were transfected into C. tropicalis 1798 with a Gene Pulser Apparatus. Then， C. tropicalis transformants were selected on the basis of their ability to grow on YPD plates containing G418.
Colonies of the abovementioned cells that exhibited G418 resistance were inoculated into liquid YPD medium and cultured overnight at 30 °C， and cells were collected by centrifugation at 8000 rpm. The genome was isolated from recombinant yeast using an Ezup Column Bacteria Genomic DNA Purification Kit （Sangon Biotech）， The amplification products were verified by 1% agarose gel electrophoresis. Finally， we obtained recombinant strains that were positive for gene knockout and the promoter. The three positive recombinant strains that were obtained were inoculated into 50 mL liquid YPD medium and cultured for 12 h. Then， transferred to YPGs medium for 24 h； this step was repeated， and selected microorganisms were streaked across solid YPGs medium and cultured for 36 h. Single colonies were transferred to liquid-phase YPD culture medium. By validation of the absence of the purpose band， we obtained engineered strains with the following features： the pGAP promoter without marker replacement （C. tropicalis-gkPr）；
1.5 Analysis of glycerol utilization
Samples of 500 μL C. tropicalis 1798 and C. tropicalis gkPr that had been preserved in a glycerol tube were inoculated into 50 mL liquid sugar-free YPD medium containing 2% glycerol， and the culture was shaken at 200 rpm at 30 °C. The optical density at 600 nm （OD600） of samples was measured at intervals of 2 h. Determination of glycerol was performed using the potassium permanganate oxidation method.
1.6 Fermentative activity of recombinant strains and product analysis
Samples of 500 μL C. tropicalis 1798， C. tropicalis-gkPr， which had been preserved in glycerol tubes， were inoculated into 50 mL liquid YPD medium and cultured at 200 rpm for 30 h at 30 °C. The seed culture medium was transferred to 50 mL fermentation medium， and a 10% inoculum was cultured at 200 rpm with shaking at 30 °C.
2 Results and Discussion
2.1 Construction of pPICPJ-mazF traceless knockout vector
In order to replace the Zeocin resistance marker in pPICZαA with the G418 resistance gene using pPIC9k as a template， the fragment of the G418 resistance gene Kanr was obtained by PCR and ligated to pPICZαA to obtain the recombinant vector pPICZαA-Kanr. In addition， fragment of the pGAL promoter of the GAL gene from C. lipolytica 1457， which can be induced by galactose， and a fragment of the toxin gene mazF from E. coli DH5α were amplified. After connection via overlap PCR， the overlapped fragment was ligated to the SalI site upstream of the AOX1 transcription termination region in pPICZαA-Kanr with a One Step Cloning Kit. Finally， the plasmid-free knockout vector pPICPJm was constructed
2.2 Construction of pPICPJm-gkp traceless knockout vector
The gkpF-gkpR fragment was obtained by overlap PCR and ligated to the SalI restriction site in pPICPJ-mazF to obtain pPICPJm-gkpFR. Next， the pGAP fragment derived from C. tropicalis 1798 was ligated to the AvrII site in pPICPJm-gkpFR， and pPICPJm-gkp was finally obtained. After electrotransfection and G418 selection， the first single exchange was achieved to produce recombinant C. tropicalis-gkpA. To confirm the expression of mazF in C. tropicalis 1798. Then， screening with 10 g/L galactose was carried out for the subsequent production of the second single-exchange recombinant. C. tropicalis-gkpA was cultured in 100 mL liquid YPD medium and then transferred to galactose-containing YPGs screening medium as a 1.0% inoculum. After cultivation for 24 h， galactose-resistant strains were isolated using a plate streaking method， and the genome was isolated for PCR validation to produce the second single-exchange recombinant C. tropicalis-gkPr. 2.3 Enhancement of production of long-chain dicarboxylic acids by C. tropicalis
The replacement of the GK gene promoter in the strain was investigated and the results showed that when glucose was the only carbon source， the growth rate of the two bacteria were basically the same， and the growth of both bacteria entered the stationary phase after 10 h. when glycerol was the only carbon source， the growth rate of the engineered strains was clearly higher than that of the original strain. the OD600 values reached a maximum， and the maximum value for the engineered strain C.
3 Conclusions
In this study， the mazF gene of E. coli was used as a reverse screening marker to construct the suicide vector pPICPJ-mazF. The results showed that mazF from E. coli can be induced by galactose in C. tropicalis under the control of the pGAL promoter from C. lipolytica. In addition， we used the suicide vector pPICPJ-mazF to edit the genome of C. tropicalis， replace the promoters of the GK gene reductase. The yield of long-chain dibasic acids from the engineered strain C. The results showed that a construct containing mazF from E. coli can be used as a traceless editing system in C. tropicalis to complete the gene-free editing of the genome of C. tropicalis， which lays the foundation for further metabolic engineering transformations of C. tropicalis.
Supported by the Independent Innovation and Achievement Transformation Project in Shandong Province （grant number 201422CX02602）， Major Program of National Natural Science Foundation of Shandong （grant number ZR2017ZB0208）， Shandong Provincial Natural Science Foundation （grant number ZR2016CB04）
Corresponding author： WANG Jun-qing. Tel： +86-0531-8963-1138； E-mail： wjqtt.6082@163.com；