Patent Application
20130157319
The Sequence Listing ASCII text file, named as “KS-00011-Sequence-Listing.TXT”, sized as “5.41 Kbytes”, and created on Oct. 17, 2012 and submitted on Oct. 19, 2012 in the United States Patent and Trademark Office, is hereby incorporated by reference in this specification. Please attach the above mentioned ASCII text file of Sequence Listing named “KS-00011-Sequence-Listing.TXT” to the end of the specification as a separate part of the disclosure of Sequence Listing in the present application.
The attached ASCII text file of the disclosed “Sequence Listing” will serve as both the paper copy required by 37 C.F.R. §1.821(c) and the computer readable form (CRF) required by 37 C.F.R. §1.821(e). Thus, a statement under 37 C.F.R. §1.821(f) showing that the content of the sequence listing information recorded in the computer readable form is identical to a written copy on paper of “Sequence Listing” is no longer required pursuant to “Legal Framework for EFS-WEB, Section I”.
1. Field of the Invention
The present invention is related to a modified fermentation performance of a microorganism, more particularly to simultaneously utilizing pentose and hexose as the substrates for fermentation.
2. Description of Prior Art
The substitution of renewable resources for the petroleum-related chemicals is the main stream on the international market. Among renewable resources, plant-based biomass (e.g., lignocellulose) is the most abundant in nature. Lignocellulose contains cellulose, hemicellulose, and lignin. After cellulose and hemicellulose are hydrolyzed, the products of hydrolysis are mainly glucose and xylose. In the present invention, Escherichia coli (E. coli) is genetically re-constructed, which is able to metabolize glucose and xylose in a simultaneous and rapid way. The re-constructed E. coli can ferment glucose and xylose and convert them to bio-energy such as alcohol and other chemical such as lactate.
In the existed techniques, E. coli is commonly used to ferment monosaccharides. However, there are several problems to be solved. The advantages of E. coli are rapid growth, easy culturing with a simple medium, easy fermentation operation, and efficient utilization of various monosaccharides. When various monosaccharides are present, E. coli metabolizes glucose first. After glucose is totally consumed, other monosaccharides are utilized. Therefore, E. coli is unable to metabolize different monosaccharides at the same time in the presence of glucose. Therefore, the overall sugar metabolism rate of E. coli is inefficient. In the existing technology, some mutagens such as ultra-violate ray, gamma ray, and nitrosoguanidine are used to mutate bacterial strains. Through the screening process, a strain metabolizing pentose and hexose simultaneously is isolated. However, the mutation method requires repeated screening, which is not systematic and is laborious as well as complicated. The resulting mutant strains are usually inefficient in terms of co-utilization of pentose and hexose.
In
In the existing technique, two distinct strains able to metabolize glucose and xylose individually are adopted. One strain metabolizes glucose only and the other strain deficient in glucose metabolism utilizes xylose solely. The objective of co-utilization of pentose and hexose is then achieved. However, the process is not easy to operate and the efficiency of the two sugars co-fermentation needs to be optimized by the adjustment of fermentation conditions. In addition, the two strains are cultured, thus increasing the fermentation cost that is unfavorable for industrial applications.
The described drawbacks must be overcome. For example, the cost of fermentation is high, the rate of fermentation is not efficient, and the operation procedure of fermentation is complicated. It is necessary to develop a method to equip the strain with the ability to ferment pentose and xylose simultaneously, which can improve and simplify the procedure of and to increase the efficiency of fermentation. This developed technology is particularly important as long as the issue of production of value-added chemicals from renewable resources is concerned.
Bio-industry is a representative of the green industry that is recognized as the fourth industrial revolution. Bio-industry is founded on biotechnology. Comparing to the fossil fuels-based industry, biotechnology can reduce the energy consumption and the environmental pollution. In particular, biotechnology is a technology that can use the renewable resources to achieve the sustainable development and environmental progress. Biomass is the main renewable resources, comprising the wastes from agriculture, forestry, fishing, and animal husbandry and the organic waste released from industry and urban area. Through the process of biorefinery process, the biomass is transformed into the alternative energy for substitution of the petroleum-derived products. The biorefinery industries are growing at a roughly rate of 15% every year, and their market price of total global production will reach 1215 billion US dollars by 2012 (Gobina E, 2007, report code EGY054A, BCC Research publications). Among the renewable resources, lignocellulose is the most abundant and widespread. This biomass for current fermentation studies comes from (1) the agriculture residues from sugar cane residues, straw, chaff, corn straw, (2) non-crop plants such as sword grass, (3) woody plant biomass such as Physic Nut and (4) biowaste such as the residues of vegetable, fruit, pulp, and solid waste from the city (Dietmar P, 2006, Biotechnol J. 1:806-814). In general, lignocellulose comprises of 30-60% cellulose, 20-40% hemicellulose, 10-30% lignin. Cellulose is a polysaccharide in which glucose is linked by β-1,4 glycosidic linkage. Because of the hydrogen bonds between its molecules, they cause the formation of crystallinity and amorphous structure. The hemicellulose is a polysaccharide which is made of pentose and hexose with complicated side branches. The hemicellulose of soft wood is hexose like glucose and the hemicellulose of hard wood is pentose like xylose (Ganapathy S. et al., 2010, Eng. Life Sci. 10:8-18). The cellulose and hemicellulose are hydrolyzed mainly to glucose and xylose. Most microorganisms can metabolize glucose effectively; however, a few microorganisms can ferment xylose poorly. Therefore, the poor use of xylose by microorganisms affects the development of the biorefinery industry.
Comparing to other bacteria, Escherichia coli (E. coli) is a bioprocess-friendly strain. It is characterized as rapid growth, being cultured by simple media formula and easy fermentation operation. Moreover, this bacterium is able to metabolize an array of monosaccharides including pentose (including xylose). However, if there is sufficient glucose in the surrounding, it utilizes glucose first. The metabolism of other monosaccharides is inhibited. After glucose is totally consumed, other monosaccharides will be used sequentially. This slows down the rate of monosaccharide metabolism. Even, it makes the other metabolism uncompleted and ineffective.
Because of aforementioned reasons, the present invention is aimed at metabolic engineering of E. coli. In the step (a) of
) lactate production.
The technologies in the present invention refer to the content in the textbook, such as Sambrook J, Russell D W, 2001, Molecular Cloning: a Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press, New York. The technologies comprise cleavage reaction by restriction enzyme, DNA ligation with T4 ligase, polymerase chain reaction (PCR), agarose gel electropohoresis, sodium dodecyl sulfate-polyacrylamide electrophoresis, and plasmid transformation. All the technologies can be conducted by experienced people who are well acquainted with those. The density of bacteria in the cultured media is measured by spectrophotometer (V530, Hasco) with the wave length at 550 nm; the bacterial density is recorded as OD550. The protein assay Reagent (BioRad Co.) is used to measure the concentration of proteins for the total protein quantification. Individually marked protein is analyzed by Alphalmager EP (Alphalnnotech) to quantify the protein resolved by the electrophoresis.
The purification of the chromosome and plasmid of bacteria and phage is carried out by the commercial kit from Wizard® Genomic DNA purification kit (Promega Co.), High-Speed Plasmid Mini Kit (Geneaid Co.) and Gel/PCR DNA Fragments Extraction Kit (Geneaid Co.). The DNA point mutation is carried out by the QuickChange® Sit-Directed Mutagenesis Kit (Stratagene Co.). The restriction enzyme is purchased from New England Boplabs and Fermentas Life Science. The T4 ligase and Pfu DNA polymerase is purchased from the Promega Co. All the primers are synthesized by Mission biotech and Tri-I biotech, Inc. (Taipei, Taiwan).
In the DNA cloning procedure, the bacterial cells used are DH5α (Stratagene Co), BW25142 (Haldimann and Wanner, 2001, J. Bacterior., 183: 6384-93) and BL21 (DE3) (Invitrogen Co.). Bacteria are cultured in LB media (Miller J H, 1972, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, New York). The transformed bacteria are cultured in the media with antibiotics such as: ampicillin (50 μg/mL), kanamycin (50 μg/mL)
The present invention is aimed at developing a process for a microorganism to acquire the ability to simultaneously utilize pentose and hexose as the carbon sources for fermentation. Escherichia coli (E. coli) is used as the main host, because it possesses a lot of advantages and is widely used in industry. Several steps are conducted to achieve the objective. The present invention is detailed by the following descriptions in conjunction with drawings therein.
Deletion of a ptsG Gene Sequence
In step (a) of
The PCR reaction is carried out using the purified chromosome of E. coli strain CGSC 9031(E. coli Genetic Stock Center, USA) as the template and with primer 1 and primer 2. A DNA cassette (2.8 kb) is amplified, and it contained the FRT sites-surrounded anti-kanamycin gene (FRT-kan-FRT) that is flanked by two homologous regions of the ptsG gene sequence. E. coli strain BL21 is transformed with plasmid pKD46 (Datsenko K. A. and Wanner B. L., 2000, Proc. Natl. Aca. Sci. USA, 97:6640-6645) to obtain strain BL21/pKD46. This linear PCR DNA fragment is then transformed into the competent strain BL21/pKD46 by electroporation. The competent cell with linear DNA is cultured in SOC media with 1 mM arabinose at 30° C. to induce the expression of the λ-Red gene sequence on the plasmid. The λ-Red gene sequence product facilitates the homologous recombination between the genomic ptsG gene and the homologous sequences that flank the FRT-kan-FRT of the DNA cassette. After 2-hour incubation, the culture temperature is raised to 42° C. for another 2 hours. Bacterial cells are collected by centrifugation and cultured on LB media with kanamycin. The in situ PCR reaction is carried out with primer 3 and 4 to confirm that bacterial cells carried the inserted copy of the anti-kanamycin gene within the genomic ptsG gene. In
Construction of a Recombinant E. coli Strain with Introducing the glf Gene
Construction of Integration Plasmid pHK-glf
In the former study, the glucose consumption rate of E. coli strain lacking the ptsG was decreased significantly. Meanwhile, a previous study reported that introduction of the glf gene encoding the glucose facilitator from Zymomonas mobilis (Z. mobilis) could restore the glucose metabolism of E. coli that lost the ability of transporting glucose (Parker C et al., 1995, Mol Microbiol. 15:795-802). In step (b) of
Primers 5 and 6 are synthesized according to the glf gene sequence in the NCBI database. The forward primer 5 contained the XbaI site (underline) while the reverse one carried the XhoI site (underline). The PCR reaction is carried out with aforementioned primers using the Z. mobilis genome as the template. One DNA fragment containing the glf gene sequence is amplified (1.4 kb). After purifying the amplified DNA fragment by Gel/PCR DNA Fragments Extraction Kit, it is cleaved with the restriction enzyme XbaI and XhoI. Plasmid pND707 (Love C A et al., 1996, Gene, 176:49-53) purified by High-Speed Plasmid Mini kit is also cleaved with the XbaI and XhoI. The cleaved DNA fragment is purified and recovered by Gel/PCR DNA Fragments Extraction Kit. T4 ligase is used to incorporate the linearized plasmid pND707 DNA fragment with the glf gene—containing DNA. As a result, plasmid pND-glf is obtained from E. coli strain DH5α as shown in
Primers land 8 are synthesized according to the pND-glf; the forward one containing the BamHI site (underline). The PCR is carried out with the primer 7 and primer 8 from plasmid pND-glf. An amplified DNA fragment (1.8 kb) is obtained, and it contained the λPRPL promoter-driven glf gene. The amplified DNA fragment purified by Gel/PCR DNA Fragments Extraction Kit is cleaved with the restriction enzyme BamHI and SmaI. Integration plasmid pHK-Km (Chiang C J et al., 2008, Biotechnol. Bioeng. 101:985-995) purified by High-Speed Plasmid Mini kit is cleaved by BamHI and SmaI. The cleaved DNA fragment is recovered by Gel/PCR DNA Fragments Extraction Kit. The glf gene-containing DNA and the linearized plasmid pHK-Km are spliced together to obtain plasmid pHK-glf from E. coli strain BW25142 as shown in
Transformation of Plasmid pHK-glf into Strain BL-G
Helper plasmid pAH69 (Haldimann A and Wanner B L., 2001, J Bacteriol., 183:6384-6393) is transformed into strain BL-G by the chemical transformation method to obtain strain BL-G/pAH69. The pHK-glf is then transformed into BL-G/pAH69 to facilitate integration of plasmid pHK-glf. Cells are selected in LB media containing kanamycin and the inserted glf gene is verified by in situ PCR with the primers 7 and 8 as shown in lane 3 of
Introducing at Least One Gene in the Pentose Phosphate Pathway
The expression of rpe, tktA, rpiA, and talB genes or the combination thereof is enhanced to increase the metabolic rate of xylose in the pentose phosphate pathway. As shown in step (c) and (d) of
Enhanced Expression of the rpe and tktA Genes
Primers 9 and 10 are synthesized according to the rpe gene sequence in the database of NCBI; the forward primer containing the NdeI site (underline), the reverse primer containing the EcoRI site (underline). The PCR reaction is carried out with the primers 9 and 10 and the chromosome of BL21 as the template. One DNA fragment (0.7 kb) including the rpe gene is amplified. The amplified DNA fragment is purified by Gel/PCR DNA Fragments Extraction Kit and cleaved with the restriction enzyme NdeI and EcoRI. The cleaved DNA fragment is purified and recovered by the Gel/PCR DNA Fragments Extraction Kit.
Preparing a DNA Fragment Including the tktA Gene
Primers 11 and 12 are synthesized according to the tktA gene sequence in the database of NCBI; the forward primer 11 containing the EcoRI site (underline); the reverse primer 12 containing the XhoI site (underline). The PCR reaction is carried out with the primers 11 and 12 and the chromosome of BL21 as the template. One DNA fragment (2.01 kb) including the tktA gene is amplified. The amplified DNA fragment is purified by Gel/PCR DNA Fragments Extraction Kit and cleaved with the restriction enzyme EcoRI and XhoI. The cleaved DNA fragment is purified and recovered by the Gel/PCR DNA Fragments Extraction Kit. Plasmid pND707 (Love C A et al., 1996, Gene, 176:49-53) purified by the High-Speed Plasmid Mini kit is digested with restriction enzyme NdeI and EcoRI and then purified by the Gel/PCR DNA Fragments Extraction Kit. DNA fragments containing the rpe and tktA genes and linearized plasmid pND707 are spliced together to obtain plasmid pND-rTA.
Integration of the rpe and tktA Genes into Strain BL-Gf
According to plasmid pND-rTA, the primers 13 and 14 are designed: the forward primer 13 containing the BamHI site (underline). A DNA fragment (2.7 kb) containing the λPRPL promoter-driven rpe and tktA genes is amplified by PCR with the primer 13, 14 and the pND-rTA as the template. The PCR DNA fragment is purified by the Gel/PCR DNA Fragments Extraction Kit and cleaved with the restriction enzyme BamHI. Plasmid pPhi80-km (Chiang C J et al., 2008, Biotechnol. Bioeng. 101:985-995) purified by High-speed Plasmid Mini kit is cleaved by the restriction enzyme BamHI and SmaI. The cleaved fragment is purified by the Gel/PCR DNA Fragments Extraction Kit. The DNA fragment containing the λPRPL promoter-driven rpe and tktA genes and linearized plasmid pPhi80-km are spliced together to obtain plasmid pPhi80-rTA from strain BW25142 as shown in
Helper plasmid pAH123 (Haldimann A and Wanner B L., 2001, J Bacteriol., 183:6384-6393) is transformed into strain BL-Gf to obtain strain BL-Gf/pHA123. Followed by transformation of plasmid pPhi80-rTA into the BL-Gf/pHA123, the DNA containing the rpe and tktA genes controlled by the λPRPL promoter is incorporated in to the bacterial chromosome. Cell colonies grown on LB media with kanamycin are picked up and the inserted rpe and tktA genes are verified by in situ PCR based on the primer13 and 14 as shown in lane 3 of
Enhanced Expression of the rpiA and talB Genes
Preparing a DNA Fragment Including the rpiA Gene
Primers 15 and16 are synthesized according to the rpiA gene sequence in the database in NCBI; the forward primer 15 containing the NdeI site (underline), the reverse primer 16 containing the EcoRI site (underline). The PCR reaction is carried out with primers 15 and 16 and the chromosome of BL21 as the template. One DNA fragment (0.7 kb) including the rpiA gene is amplified. The amplified DNA fragment is purified by Gel/PCR DNA Fragments Extraction Kit and cleaved with the restriction enzyme NdeI and EcoRI. The cleaved DNA fragment is purified and recovered by the Gel/PCR DNA Fragments Extraction Kit. Preparing a DNA fragment including the talB gene
Primers 17 and 18 are synthesized according to the talB gene sequence in the database in NCBI; the forward primer 17 containing the EcoRI site (underline); the reverse primer 18 containing the XhoI site (underline). The PCR reaction is carried out with the primers 17 and 18 and the chromosome of BL21 as the template. One DNA fragment (1.0 kb) including the talB gene is amplified. The amplified DNA fragment is purified by Gel/PCR DNA Fragments Extraction Kit and cleaved with the restriction enzyme EcoRI and XhoI. The cleaved DNA fragment is purified and recovered by the Gel/PCR DNA Fragments Extraction Kit. Plasmid pND707 (Love C A et al., 1996, Gene, 176:49-53) purified by the High-Speed Plasmid Mini kit is digested with the restriction enzyme NdeI and EcoRI and then purified by the Gel/PCR DNA Fragments Extraction Kit. DNA fragments containing the rpiA and talB genes and linearized plasmid pND707 are spliced together to obtain plasmid pND-rTB.
Integration of the rpiA and talB Genes into Strain BL21e
According to plasmid pND-rTB, primers 19 and 20 are designed: the reverse primer containing the SalI site (underline). A DNA fragment (1.7 kb) containing the λPRPL promoter-driven rpiA and talB genes is amplified by PCR with the primers 19, 20 and pND-rTB as the template. The PCR DNA fragment is purified by the Gel/PCR DNA Fragments Extraction Kit and cleaved with the restriction enzyme BamHI. Plasmid pLambda-km (Chiang C J et al., 2008, Biotechnol. Bioeng. 101:985-995) purified by High-speed Plasmid Mini kit is cleaved by the restriction enzyme SalI and SmaI. The cleaved fragment is purified by the Gel/PCR DNA Fragments Extraction Kit. The DNA fragment containing the λPRPL promoter-driven rpiA and talB genes and linearized plasmid pLambda-km are spliced together to obtain plasmid pLam-rTB from strain BW25142 as shown in
Helper plasmid pAH121 (Haldimann A and Wanner B L., 2001, J Bacteriol., 183:6384-6393) is transformed into strain BL21e to obtain strain BL21e/pHA121. Followed by transformation of plasmid pLam-rTB into the BL21e/pHA121, the DNA containing the rpiA and talB genes controlled by the λPRPL promoter is incorporated in to the bacterial chromosome. Cell colonies grown on LB media with kanamycin are picked up and the inserted rpiA and talB genes are verified by in situ PCR based on primer 19 and 20 as shown as lane 3 of
E. coli is able to produce various organic acids under the fermentative condition, known as the mixed acid fermentation. These organic acids are indeed wastes and may exhibit an inhibitory effect on the pentose phosphate pathway. As shown in the step (e), (f), (g), (h) of
Deletion at Least One Gene Sequence or the Combination Thereof which is Responsible for Synthesis of Organic Acid
Deletion the poxB Gene
Primers 21 and 22 are synthesized according to the poxB gene sequence in the database of NCBI; the forward primer 21 containing the HindIII site (underline) and the reverse primer 22 containing the SpeI site (underline). The DNA fragment containing the poxB gene sequence (0.84 kb) is amplified from strain BL21 genome by PCR with the primer 21 and 22. After purifying with the Gel/PCR DNA Fragments Extraction Kit, the PCR DNA is cleaved by the restriction enzyme HindIII and SpeI. The cleaved fragment is recovered by the Gel/PCR DNA Fragments Extraction Kit. Plasmid pMCS-5 (Mo Bi Tec, Germany) purified with the High-speed Plasmid Mini kit is cleaved by HindIII and SpeI and is recovered using the Gel/PCR DNA Fragments Extraction Kit. The poxB gene sequence-containing DNA fragment and linearized plasmid pMCS-5 are ligated together to obtain plasmid pMC-pox from strain DH5α. Primer 23 and 24 are synthesized based on the poxB gene sequence in the database of NCBI; the forward primer 23 containing the EcoRI site (underline) and the reverse primer 24 containing the SalI site (underline). The PCR is carried out with primers 23,-24 and pMC-pox as the template. A DNA fragment (3.5 kb) is amplified. After purification with the Gel/PCR DNA Fragments Extraction Kit, the DNA fragment is cleaved with the restriction enzyme EcoRI and SalI and recovered by the Gel/PCR DNA Fragments Extraction Kit. Moreover, primer 25 and 26 are synthesized according to the sequence of plasmid pKD13 (Datsendo K. A. and Wanner B. L., 2000, Proc. Natl. Aca. Sci. USA, 97:6640-6645) in the database of NCBI ; the forward primer 25 containing the EcoRI site (underline) and the reverse primer 26 containing the SalI site (underline).The PCR is carried out with plasmid pKD13 as the template and with the primers 25 and 26. A DNA fragment (1.3 kb) containing an anti-kanamycin gene sequence flanked by two FRT sites (FRT-kan-FRT) is amplified. After purifying with the Gel/PCR DNA Fragments Extraction Kit, the amplified fragment is cleaved with the restriction enzyme EcoRI and SalI and recovered by the Gel/PCR DNA Fragments Extraction Kit. The FRT-kan-FRT DNA fragment is incorporated into linearized plasmid pMC-pox to obtain plasmid pMC-poxKm as shown in
The PCR is carried out with primers 21 and 22 and using plasmid pMC-poxKm as template. The PCR resulted in a DNA cassette (1.9 kb) that contained the FRT-kan-FRT DNA fragment flanked by the homologous regions of the poxB gene sequence, which is purified by the Gel/PCR DNA Fragments Extraction Kit. Helper plasmid pKD46 (Datsenko K. A. and Wanner B. L., 2000, Proc. Natl. Aca. Sci. USA, 97:6640-6645) is transformed into strain BL21e-RB, resulting in strain BL21e-RB/pKD46. The obtained DNA cassette is then transformed into competent strain BL21e-RB/pKD46 by electroporation. The competent cell with linear DNA is cultured in SOC media with 1 mM arabinose at 30° C. to induce the expression of λ-Red gene sequence on the plasmid. The λ-Red gene sequence product facilitates the homologous recombination between the genomic poxB gene sequence and the homologous sequences that flank the FRT-kan-FRT of the DNA cassette. After 2-hour incubation, the culture temperature is raised to 42° C. for another 2 hours. Bacterial cells are collected by centrifugation and cultured on LB media with kanamycin. The in situ PCR reaction is carried out with the primer 21 and 22 to confirm that bacterial cells carried the inserted copy of the anti-kanamycin gene sequence within the genomic poxB gene sequence. To remove the integrated anti-kanamycin gene sequence, plasmid pCP20 (Datsenko K. A. and Wanner B. L., 2000, Proc. Natl. Aca. Sci. USA, 97:6640-6645) is transformed into the bacterial strains and induced by shifting the culture temperature from 30° C. to 40° C. to express the FLP protein whose function is to recombine two FRT sites while leaving a single FRT site behind As depicted in
Primers 27 and 28 are synthesized according to the pta gene sequence in the database of NCBI; the forward primer 27 containing the HindIII site (underline) and the reverse primer 28 containing the SpeI site (underline). The DNA fragment containing the pta gene sequence (0.95 kb) is amplified from strain BL21 genome by PCR with the primer 27 and 28. After purifying with the Gel/PCR DNA Fragments Extraction Kit, the PCR DNA is cleaved by the restriction enzyme HindIII and SpeI. The cleaved fragment is recovered by the Gel/PCR DNA Fragments Extraction Kit. Plasmid pMCS-5 (Mo Bi Tec, Germany) purified with the High-speed Plasmid Mini kit is cleaved by HindIII and SpeI and is recovered using the Gel/PCR DNA Fragments Extraction Kit. The pta gene sequence-containing DNA fragment and linearized plasmid pMCS-5 are ligated together to obtain plasmid pMC-pta from strain DH5α. Primers 29 and 30 are synthesized based on the pta gene sequence in the database of NCBI; the forward primer containing the EcoRI site (underline) and the reverse primer containing the SalI site (underline). The PCR is carried out with the primers 29, 30 and pMC-pox as the template. A DNA fragment (3.5 kb) is amplified. After purification with the Gel/PCR DNA Fragments Extraction Kit, the DNA fragment is cleaved with the restriction enzyme EcoRI and SalI and recovered by the Gel/PCR DNA Fragments Extraction Kit. Moreover, the primer 25 and 26 are synthesized according to the sequence of plasmid pKD13 (Datsendo K. A. and Wanner B. L., 2000, Proc. Natl. Aca. Sci. USA, 97:6640-6645) in the database of NCBI ; the forward primer 25 containing the EcoRI site (underline) and the reverse primer 26 containing the SalI site (underline).The PCR is carried out with plasmid pKD13 as the template and with the primers 25 and 26. A DNA fragment (1.3 kb) containing an anti-kanamycin gene sequence flanked by two FRT sites (FRT-kan-FRT) is amplified. After purifying with the Gel/PCR DNA Fragments Extraction Kit, the amplified fragment is cleaved with the restriction enzyme EcoRI and SalI and recovered by the Gel/PCR DNA Fragments Extraction Kit. The FRT-kan-FRT DNA fragment is incorporated into linearized plasmid pMC-pta to obtain plasmid pMC-ptaKm as shown in
The PCR is carried out with primers 27 and 28 and using plasmid pMC-ptaKm as template. The PCR resulted in a DNA cassette (1.9 kb) that contained the FRT-kan-FRT DNA fragment flanked by the homologous regions of the pta gene sequence, which is purified by the Gel/PCR DNA Fragments Extraction Kit. Helper plasmid pKD46 (Datsenko K. A. and Wanner B. L., 2000, Proc. Natl. Aca. Sci. USA, 97:6640-6645) is transformed into strain BLA1, resulting in strain BLA1/pKD46. The obtained DNA cassette is then transformed into competent strain BLA1/pKD46 by electroporation. The competent cell with linear DNA is cultured in SOC media with 1 mM arabinose at 30° C. to induce the expression of the λ-Red gene sequence on the plasmid. The λ-Red gene sequence product facilitates the homologous recombination between the genomic pta gene sequence and the homologous sequences that flank the FRT-kan-FRT of the DNA cassette. After 2-hour incubation, the culture temperature is raised to 42° C. for another 2 hours. Bacterial cells are collected by centrifugation and cultured on LB media with kanamycin. The in situ PCR reaction is carried out with the primers 27 and 28 to confirm that bacterial cells carried the inserted copy of the anti-kanamycin gene sequence within the genomic pta gene sequence. To remove the integrated anti-kanamycin gene sequence, plasmid pCP20 (Datsenko K. A. and Wanner B. L., 2000, Proc. Natl. Aca. Sci. USA, 97:6640-6645) is transformed into the bacterial strains and induced by shifting the culture temperature from 30° C. to 40° C. to express the FLP protein whose function is to recombine two FRT sites while leaving a single FRT site behind As depicted in
Deletion of the ldhA Gene
Primers 3 1 and 32 are synthesized according to the adjacent sequence of the ldhA gene sequence in EcoCye database. The chromosome of CGSC 9216 strain (E. coli Genetic Stock Center, USA) is purified by Wizard Genomic DNA purification kit (Promega Co.). With the primers 31 and 32, the PCR is conducted using the purified chromosome of CGSC 9216 as the template. A DNA cassette (2.8 kb) comprising the FRT site-surrounded anti-kanamycin gene sequence (FRT-kan-FRT) that is flanked by two homologous regions of the ldhA gene sequence is amplified and then purified by the Gel/PCR DNA Fragments Extraction Kit. Helper plasmid pKD46 (Datsenko K. A. and Wanner B. L., 2000, Proc. Natl. Aca. Sci. USA, 97:6640-6645) is transformed into strain BL-A2 to obtain strain BL-A2/pKD46. This linear PCR DNA fragment is then transformed into the competent strain BL-A2/pKD46 by electroporation. The competent cell with linear DNA is cultured in SOC media with 1 mM arabinose at 30° C. to induce the expression of the β-Red gene sequence on the plasmid. The β-Red gene sequence product facilitates the homologous recombination between the genomic ldhA gene sequence and the homologous sequences that flank the FRT-kan-FRT of the DNA cassette. After 2-hour incubation, the culture temperature is raised to 42° C. for another 2 hours. Bacterial cells are collected by centrifugation and cultured on LB media with kanamycin. The in situ PCR reaction is carried out with the primers 31 and 32 to confirm that bacterial cells carried the inserted copy of the anti-kanamycin gene sequence within the genomic ldhA gene sequence. To remove the integrated anti-kanamycin gene sequence, plasmid pCP20 (Datsenko K. A. and Wanner B. L., 2000, Proc. Natl. Aca. Sci. USA, 97:6640-6645) is transformed into the bacterial strains and induced by shifting the culture temperature from 30° C. to 40° C. to express the FLP protein whose function is to recombine two FRT sites while leaving a single FRT site behind Finally, cells that are unable to grow on the LB media with kanamycin are chosen, and one of them is picked up and re-named BL-A3.
Deletion of the frdA Gene
Primers 33 and 34 are synthesized according to the adjacent sequence of the frdA gene sequence in EcoCye database. The chromosome of CGSC 10964 strain (E. coli Genetic Stock Center, USA) is purified by Wizard Genomic DNA purification kit (Promega Co.). With the primers 33 and 34, the PCR is conducted using the purified chromosome of CGSC 9216 as the template. A DNA cassette (3.0 kb) comprising the FRT site-surrounded anti-kanamycin gene sequence (FRT-kan-FRT) that is flanked by two homologous regions of the frdA gene sequence is amplified and then purified by the Gel/PCR DNA Fragments Extraction Kit. Helper plasmid pKD46 (Datsenko K. A. and Wanner B. L., 2000, Proc. Natl. Aca. Sci. USA, 97:6640-6645) is transformed into strain BL-A3 to obtain strain BL-A3/pKD46. This linear PCR DNA fragment is then transformed into the competent strain BL-A3/pKD46 by electroporation. The competent cell with linear DNA is cultured in SOC media with 1 mM arabinose at 30° C. to induce the expression of the λ-Red gene sequence on the plasmid. The λ-Red sequence gene product facilitates the homologous recombination between the genomic frdA gene sequence and the homologous sequences that flank the FRT-kan-FRT of the DNA cassette. After 2-hour incubation, the culture temperature is raised to 42° C. for another 2 hours. Bacterial cells are collected by centrifugation and on cultured on the LB media with kanamycin. The in situ PCR reaction is carried out with the primers 33 and 34 to confirm that bacterial cells carried the inserted copy of the anti-kanamycin gene sequence within the genomic frdA gene sequence. To remove the integrated anti-kanamycin gene sequence, plasmid pCP20 (Datsenko K. A. and Wanner B. L., 2000, Proc. Natl. Aca. Sci. USA, 97:6640-6645) is transformed into the bacterial strains and induced by shifting the culture temperature from 30° C. to 40° C. to express the FLP protein whose function is to recombine two FRT sites while leaving a single FRT site behind Finally, cells that are unable to grow on the LB media with kanamycin are chosen, and one of them is picked up and re-named BL-A4.
Production of Ethanol in the Constructed Strain by Fermentation of Glucose and Xylose
Construction of Plasmid pND-Pet
The pdc gene encoding pyruvate decarboxylase and the adhII gene encoding alcohol dehydrogenas from Z. mobilis have been studied previously (Ingram Lo et al., 1987, Appl. Environ. Microbiol. 53:2420-2425). The two genes mediate a two-step reaction by conversion of pyruvate to ethanol. In the step (i) of
Primers 35 and 36 are synthesized according to the pdc gene sequence in NCBI database; the forward primer 35 containing the NdeI site (underline) and the reverse primer 36 containing the BamHI site (underline). With the primers 35 and 36, the PCR is carried out using the chromosome of Z. mobilis as the template. A DNA fragment (1.7 kb) containing the pdc gene is amplified and purified by the Gel/PCR DNA Fragments Extraction Kit. Followed by digestion with BamHI and NdeI, the pdc gene-containing DNA fragment is purified by the Gel/PCR DNA Fragments Extraction Kit. Primers 37 and 38 are synthesized according to the adhII gene sequence in NCBI database; the forward primer 37 containing the BamHI site (underline) and the reverse primer 38 containing the XhoI site (underline). With the primers 37 and 38, the PCR is carried out using the chromosome of Z. mobilis as the template. A DNA fragment (1.15 kb) containing the adhII gene is amplified and then purified by the Gel/PCR DNA Fragments Extraction Kit. Followed by digestion with BamHI and XhoI, the adhII gene-containing DNA fragment is recovered by the Gel/PCR DNA Fragments Extraction Kit. Plasmid pND707 purified with the High-Speed Plasmid Mini kit is cleaved by restriction enzyme NdeI and XhoI and followed by purification with the Gel/PCR DNA Fragments Extraction Kit. The linearized plasmid pND707 and the DNA fragments containing the pdc and adhII genes are spliced together to obtain plasmid pND-pet from E. coli strain DH5α as shown in
Finally, plasmid pND-pet is transformed into wild-type strain BL21 and genetically constructed strain BL-G, BL-Gf, BL21e-RB and BL-A4 to obtain recombinant strains BL21/pND-pet, BL-G/pND-pet, BL-Gf/pND-pet, BL21e-RB/pND-pet, and BL-A4/pND-pet, respectively.
The fermentation performance of the 5 recombinant strains is investigated by determining the ethanol production and the sugar consumption rate in the presence of mixed sugars (i.e., glucose and xylose). The results are shown as follows:
A single colony of each recombinant strain is picked up and cultured in the 5 mL LB broth with ampicillin at 30° C. and 200 rpm overnight. Each of described strains is seeded respectively in the 25 mL fresh LB broth with ampicillin plus 3% glucose and 3% xylose. The initial optical density (550 nm) of cells reached 2.0. The cell culture is then carried out at 37° C. and 150 rpm. The concentration of glucose, xylose, and ethanol are measured along the time course.
In
The main objective of the present invention is to construct a strain of E. coli capable of co-utilizing glucose and xylose and producing ethanol in an efficient way. For this purpose, the producer strain is constructed in a systematic manner by deletion of the ptsG gene sequence (giving strain BL-G), introduction of the glf gene sequence (giving strain BL-Gf), and enhanced expression of the rpiA, tktA, rpe, and talB genes (giving BL21e-RB). In addition, the ldhA, poxB, pta, and frdA genes of strain BL21e-RB are deleted, thus producing strain BL-A4, to curtail the waste production and to ease the inhibitory effect on the pentose phosphate pathway. In a similar culture condition, strain BL-A4/pND-pet enabled to consume both glucose and xylose simultaneously and rapidly. As shown in
Production of Lactate by Simultaneous Fermentation of Glucose and Xylose
The ability of the genetically constructed strain to co-ferment glucose and xylose for lactate production, but not limited, is illustrated within following embodiment.
Construction of Plasmid pTrc-H/D-Ldh
Primers 39 and 40 are synthesized based on the ldhA gene sequence in the database of NCBI; the forward primer 39 containing NcoI site (underline) and the reverse primer 40 containing the HindIII site (underline). Using the chromosome of E. coli BL21 as the template, the PCR is carried out with the primers 39 and 40. A DNA fragment (1 kb) containing the ldhA gene is amplified and purified by the Gel/PCR DNA Fragments Extraction Kit. The amplified DNA fragment is cleaved by NcoI and HindIII and recovered by Gel/PCR DNA Fragments Extraction Kit. Plasmid pTrc99A (National Institute of Genetics, Japan) purified with High-Speed Plasmid Mini kit is cleaved by NcoI and HindIII and recovered by Gel/PCR DNA Fragments Extraction Kit. The DNA fragment containing the ldhA gene and linearized plasmid pTrc99A are ligated together to obtain plasmid pTrc-H/D-Ldh from strain DH5α as shown in
Lactate Production by Simultaneous Fermentation of Xylose and Glucose
Another example is shown in step (i) of ) is produced after 48-hour fermentation and no other organic acids are detected.
As illustrated in this embodiment, the genetically re-constructed strain BL-A4 based on the technology developed in this present invention is able to ferment glucose and xylose simultaneously and rapidly.
| Number | Date | Country | Kind |
|---|---|---|---|
| 100146856 | Dec 2011 | TW | national |
