WO2001083786A2 - Stable zymomonas mobilis xylose and arabinose fermenting strains - Google Patents
Stable zymomonas mobilis xylose and arabinose fermenting strains Download PDFInfo
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- WO2001083786A2 WO2001083786A2 PCT/US2001/011334 US0111334W WO0183786A2 WO 2001083786 A2 WO2001083786 A2 WO 2001083786A2 US 0111334 W US0111334 W US 0111334W WO 0183786 A2 WO0183786 A2 WO 0183786A2
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Definitions
- the present invention relates to the biological conversion of cellulosic substrates into fuels and chemicals, and in particular to recombinant Zymomonas mobilis strains which ferment xylose and arabinose, or both, into ethanol.
- Fermentation technology is useful for the conversion renewable biomass cellulose substrates into fuels and chemicals, such as ethanol.
- a typical substrate is comprised of 35-45% cellulose, 30-40% hemicellulose, and 15% lignin.
- the hydrolysis fraction contains glucose polymers, and the hemicellulose fraction contains mostly xylose.
- Arabinose is also a significant fermentable substrate found in biomass materials, such as switchgrass grass and corn fiber.
- Z. mobilis is widely reported for its ability to rapidly and efficiently convert glucose substrates into ethanol, at a low pH, in an anaerobic culture, and in a medium which contains the inhibitory compounds typically associated with a lignocellulose-hydrolysate.
- a distinct disadvantage in the use of Z. mobilis is, however, that it does not ferment pentose sugars.
- the prior art has focused on recombinant Z. mobilis strains which ferment a mixture of glucose, and xylose or arabinose, or both, using exogenous genes which catalyze the metabolism of xylose and arabinose. These strains, and the cloning vectors, are based on the use of multiple-copy plasmids having antibiotic resistance markers.
- US Pat. No. 5,514,583 discloses a transformed Z mobilis xylose fermenting strain (CP4/pZB4 and pZB5) having exogenous genes, and plasmid vectors (pZB4 and pZB5) encoding xylose isomerase, xylulokinase, transaldolase and transketolase, and further comprising at least one promoter (Pgap and Peno) recognized by Zymomonas which regulates the expression of at least one of said genes.
- the microorganism is capable of growing on xylose as a sole carbon source, and fermenting xylose to ethanol at about 88% of the maximum theoretic yield.
- US Pat. Nos. 5,712,133 and 5,726,053 disclose, inter alia, Z. mobilis arabinose fermenting transformants (39676/pZB 206), containing exogenous genes that encode L-arabinose isomerase, L-ribulokinase and L-ribulose-5-phosphate-4-epimerase, transaldolase and transketolase which impart arabinose to ethanol fermentation capability.
- the plasmid vector (pZB 206) and a process of using the transformants for the fermentation of a glucose and arabinose containing substrate is also disclosed.
- US Pat. No. 5,843,760 discloses a Z.
- mobilis xylose and arabinose fermenting transformant containing exogenous genes encoding xylose isomerase, xylulokinase, L-arabinose isomerase, L-ribulokinase, L-ribulose-5-phosphate 4-epimerase, transaldolase and transketolase, and further comprising at least one promoter recognized by Zymomonas which regulates the expression of at least one of said genes, wherein said microorganism is capable of growing on arabinose and/or xylose, alone or in combination, as the carbon source and fermenting said arabinose and xylose to ethanol.
- antibiotic resistance markers are generally perceived as undesirable for industrial application, such as the large-scale production of ethanol.
- the classical method for generating genomic inserts of foreign genes involves the use of specialized ⁇ phage cloning vectors that can exist stable in the lysogenic state.
- genes can be inserted though homologous recombination, when bracketed with E.
- transposition has been demonstrated in Z mobilis, Pappas, K. M., et al., (1997) Transposon mutagensesis and strain construction in Zymomonas mobilis, Journal of Applied Microbiology, Vol. 82, p.p. 379-388, it has been limited to mini M ⁇ or Tn5 multiple transposition of random auxotrophy or antibiotic resistance phenotypes for genetic analysis, and in the case of the Tn5 derivatives the insertions are reportedly stable for only 5-15 generations. Pappas, K. M., et seq. P. 383, FIG. 1. Moreover, site-specific insertion through homologous recombination in Z mobilis has not been demonstrated, and no bacteriophage has ever been isolated from Zymomonas.
- Transposons Tn5 and TniO are well known and have been widely used for mutagensis and insertion of cloned DNA into a variety of gram-negative bacteria.
- Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria J. Bacteriol.
- a procedure for cloning and stable insertion of foreign genes into the chromosome of gram-negative eubacteria by combining two sets of plasmids, (i) the transposition features of TnlO and Tn5, (ii) the resistance to certain compounds, and (iii) the suicide delivery properties of the R6K-based plasmid pGP704.
- the resulting constructions contain unique N ⁇ tl or Stzl sites internal to either the TnlO or the Tn5 inverted repeats. These sites are used for cloning D ⁇ A fragments with the help of two additional specialized cloning plasmids, pUC18 ⁇ ot and pUC18Sf ⁇ .
- Donor plasmids containing hybrid transposons are transformed into a specialized ⁇ pri lysogenic E. coli strain, such as E. c /7 SmlO( ⁇ pz ' r), with a chromosomally integrated RP4 that provided broad-host range conjugal transfer functions.
- Delivery of the donor plasmids into selected host bacteria is accomplished through mating with the target strain.
- Transposition of the hybrid transposon from the delivered suicide plasmid to a replicon in the target is mediated by the cognate transposase encoded on the plasmid at a site external to the transposon. Since the transposase function is not maintained in target cell, such cells are immune to further transposition rounds. Multiple insertions in the same strain are therefore only limited by the availability of distinct selection markers. Further disclosed in Herrero, M.
- Tn5-derived minitransposons such as Tn5Tc
- the minitransposons consist of genes specifying the resistance to kanamycin, and tetracycline as selection markers and a unique Notl cloning site flanked by 19-base-pair terminal repeat sequences of TnJ.
- the transposons are located on a R6K-based suicide delivery plasmid that provides the ISJ0 R transposase tnp gene in cis but external to the mobile element and whose conjugal transfer to recipients is mediated by RP4 mobilization functions in the donor.
- insertions produced by these elements are generally more stable because of the absence of transposase-mediated secondary transpositions, deletions, and inversions.
- Berg et al (1989) Transposable elements and the genetic engineering of bacteria, p.p. 879-926, in D.E. Berg, Mobile DNA, American Society for Microbiology, Washington, DC.
- Very stable insertions can in this way be obtained with elements derived, for instance also from TnlO. Way,
- mini-TnJTc Herrero, et seq., p. 6569
- the structure of mini-TnJTc, Herrero, et seq., p. 6569, is disclosed for use for insertion mutagenesis or as a transposon vector for the cloning of DNA fragments flanked by Notl sites (readily isolated by cloning D ⁇ A fragments first into the pUCl 8 derivatives pUCl 8 ⁇ ot and pUC18Not).
- the Mini-TnJTc element is constructed, in vitro, using standard recombinant DNA techniques. Maniatis, T., et al., (1989) Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
- Tc tetracycline resistance
- This fragments is subsequently cloned into the single EcoRI site of pUC18Sfi, excised as an Sfil fragment, and inserted between the TnJ 19-base pair termini in pUT so that the mobile unit is present in all cases as an Xbal-EcoK (partial) portion of the delivery plasmid.
- the resulting element is mini- TnJ7c.
- TnlO-based transposon vector delivery systems are described in Herrero. M., et seq. 172:6557-6567.
- Phage ⁇ a derivative ⁇ RP167, carries a 5.1-kb EcoRI insert containing the mini-Tn/OKm element and the transposase gene of IS70 R is located outside the inverted repeats of the mobile element and downstream of the pTac promoter.
- the EcoRI insert fragment is ligated to pBOR8, a derivative of pGP704 containing the lacF gene from plasmid pMJR1560.
- pGP704 contains the conjugal transfer origin (oriT sequence) of the RP4 plasmid and can therefore be transferred to gram-negative bacteria when provided in trans with mobilization (Mob) functions.
- the Mlul fragment internal to the inverted repeats containing the original-specified ⁇ protein product of the original kanamycin resistance gene of the mini-TniO is replaced by a fragment containing a S/7I-Ptt cassette, appropriately modified by the addition of the Notl site and Mlul adapters, which produced the pLODPtt.
- This construction has unique Sfil, Notl, and ⁇ b ⁇ l cloning sites between the mini-TniO inverted repeats.
- the Ptt resistance marker (P t r ) of pLOFPtt is exchanged by kanamycin resistance to produce plasmid pLOFKm.
- the strains should be free of antibiotic resistance and stable for more than 40 generations in a non-selection media. It is also desirable for the strains to demonstrate a high specific rate of product formation at close to maximum theoretical product yield.
- the present invention briefly includes: a transposon for stable insertion of foreign genes into a bacterial genome, including: at least one operon having structural genes encoding enzymes selected from the group consisting of xylAxylB, araBAD and tal/tkt, and at least one promoter for expression of the structural genes in the bacterium, a pair of inverted insertion sequences, the operons contained inside the insertion sequences, and a transposase gene located outside of the insertion sequences; a plasmid shuttle vector for transformation of foreign genes into a bacterial genome, including: at least one operon having structural genes encoding enzymes selected from the group consisting of xylAxylB, araBAD and tal/tkt, at least one promoter for expression of the structural genes in the bacterium, and at least two DNA fragments having homology with a gene in the
- transposon and shuttle vectors are useful in constructing significantly different Zymomonas mobilis strains, according to the present invention, which are useful in the conversion of the cellulose derived pentose sugars into fuels and chemicals, using traditional fermentation technology, because they are stable for expression in a non-selection medium.
- Figure 1 is a plasmid map of Mini-Tn5 Tc in pGP704 containing the xylose assimilation operon according to the present invention.
- Figure 2 is a series of plasmid maps illustrating the mini-TnJ series constructs, according to the present invention.
- Figure 3 is an illustration of the series of constructs resulting in the plasmid map of p CtaltktSfi.
- Figure 4 is the results of the Southern analysis tal/tkt-xylAxylB Z. mobilis transconjugates with Tal probe.
- C is the control plasmid mini TnJ tal/tkt-xylAxylB.
- ⁇ H is the DNA size marker.
- Figure 5 is the results of the Southern analysis of tal/tkt-xy I AxylB Z. mobilis transconjugates with Tnp probe.
- C is the control plasmid mini-TnJ tal/tkt-xylAxylB .
- ⁇ H is the DNA size marker.
- Figure 6 is a graph of the enzymatic activities for several isolates of the stable xylose fermenting Z mobilis strain according to the present invention.
- Figure 7 illustrates graphs of the fermentation profiles for four of the isolates (nos. 21, 22 and 5) of Figure 6 and their performance in relation to the plasmid bearing strains 39673/pZB4 and BCl/pZB4L.
- Figure 8 is a graphic representation which illustrates the stability of the stable xylose fermenting Z mobilis strains C25 and D95 according to the present invention.
- the graph shows the stability of the plasmid-bearing and genomic integrated xylose-fermenting strains, using the ethanol process yield and xylose utilization parameters as indicators.
- Strains C25 and D95 of the present invention remained stable for more than 90 generations.
- the fermentation medium comprised RM with 1% glucose and 5% xylose and the temperature was constant at 30°C.
- Figure 9 illustrates the biomass concentration at optical density (OD 600 ), the xylose utilization as a function of time, ethanol production as a function of time, and process yield compared to the percent xylose utilization for batch fermentation of the genomic integrated xylose-fermenting strains, C25 and D95, in a RM medium containing 4.5% glucose, and 4.5% xylose at pH 5.0 and pH 5.5 at 30°C, according to the present invention.
- Figure 10 is a map of the integrative plasmid pZB 1862-ldhL-ara.
- the araBAD is inserted in the Notl site of ldhL disrupting Idh.
- the construction is based on the replicative plasmid pZB 1862 of Z mobilis.
- Figure 1 1 is the plasmid map of TnlOG.
- IS10 R is the transposase gene.
- IR is the inverted repeat sequence of the transposon.
- AraBAD was inserted between two the two inverted repeats at the Notl sites.
- Figure 12 shows the Southern analysis of the genomic integrated xylose/arabinose- fermenting Z mobilis strains from homologous recombination using DIG- ⁇ ra and DlG-ldh probes.
- AX1, 13, 23, 101, 104, and 107 are araBAD integrants.
- C25 is the host control.
- ⁇ pZBlS62-ldhL-ara is a plasmid control isolated form DH5 ⁇ .
- ⁇ /H is a molecular weight marker: 23, 9.4, 6.6 4.3, 2.3 and 2.0 kb.
- Figure 13 represents a Southern analysis of the genomic integrated xylose/arabinose- fermenting Zymomonas strains from transposon integration using DIG-tnp and DIG-ara probes. G5,6,l 1,15,17,14 and 19 are araBAD integrants. C25 is the host control. TniOG is the plasmid control. ⁇ H is a molecular weight marker: 23,9.4,6.6,4.3,2.3 and 2.0 kb.
- Figure 13 (a) is the tnp Pstl digestion and Figure 13 (b) is the probe are Pstl digestion.
- Figure 14 represents bar graphs of the enzymatic activities of the transketolase, transaldolase, xylose isomerase and xylulokinase of the genomic integrates strains.
- 206C/pZB301 is a plasmid control.
- 206C is a host control.
- C25 is the xylose-fermenting integrant.
- G8 is the xylose/arabinose fermenting integrant from Tn/0 transposition.
- AX 1 and AX101 are the xylose/arabinose fermenting integrants from homologous recombination.
- Figure 15 represents bar graph results of the enzymatic activities of L-arabinose isomerase, L-ribulokinase and L-ribulose-5-phosphate-4 epimerase of genomic integrated strains.
- 206C/pZB301 is a plasmid control.
- 206C is a host control.
- C25 is the xylose-fermenting integrant.
- G8 is the xylose/arabinose-fermenting integrant form TnlO transposition.
- AX1 and AX101 are the xylose/arabinose-fermenting integrants from homologous recombination.
- Figure 17 represents a bar graph results of the xylose and arabinose utilization of the genomic integrated xylose and arabinose-fermenting Zymomonas strains on RM containing 1% glucose, 2% xylose and 2% arabinose at 30°C with pH control. These strains were inoculated form cultures at various generations on non-selective media.
- Figure 18 is a line graph representation of the fermentation performance of the genomic integrated xylose and arabinose-fermenting Zymomonas strains in RM containing 4% glucose,
- Z mobilis genome, or genomic means the genes which, in toto, specify all the expressed and potentially expressible features associated with a given Z. mobilis.
- C25 refers to Zymomonas mobilis Patent Deposit Designation PTA-1799;
- AX refers to Zymomonas mobilis Patent Deposit Designatin PTA-1797;
- G8 refers to Zymomonas mobilis Patent Deposit Designation PTA-1796 xylAxylB rfers to Escherichia coli: DH5 ⁇ (pZb 1862- zZ-ara) Patent Deposit Designation PTA- 1798; and
- C25 refers to Zymomonas mobilis Patent Deposit Designation PTA-1799.
- E. coli bacterial strains CC1 18 ⁇ (pir), CC1 18 ⁇ (/?z ' r) (mini-tn5Tc), SM10 ⁇ (pir), and plasmids pUC 19, pLOF/Km, pUC 18sfi, pUT/Tc containing mini transposon Tn5, Tn 10 and pUC18Not were obtained from Dr. K. Timmis, GBF - Deutschen Fur Biotechnololgische Anlagen mbH, Mascheroder weg 1 D - 38124 Braunschweig, Federal Republic of Germany.
- E. coli DH5 ⁇ was used as a host for the construction of the plasmids.
- coli SMIO ⁇ pir was used as donor strain in mating experiments.
- Strains of Z mobilis ATCC 39676 and its derivative, 206C (US Pat. No. 5,843,760) were used as recipients in accordance with the invention.
- TniO-based plasmids were constructed and maintained in E. coli CC118.
- E. coli strains were cultured in LB medium at 37° C.
- Z. mobilis strains were maintained anaerobically in RM (10 g/L yeast extract, 2 g/L KH 2 PO 4 ) supplemented with 20g/L glucose, D- xylose or L-arabinose, unless otherwise specified.
- Tc tetracycline
- mating plates ((10 g/L yeast extract, 5 g/L tryptone, 2.5 g/L (NH 4 ) 2 SO 4 , 0.2 g/L K 2 HPO 4 and 50 g/L sugar)) supplemented with tetracycline or nalidixic acid (20 ⁇ g/ml) were used. All agar plates were made with 15 g/L agar.
- Genomic DNA of Z. mobilis was extracted using three-milliliters of overnight cells resuspended in 250 ml of 50 mM Tris-50 mM ⁇ DTA buffer.
- the cells were treated with lysozyme at 37°C for 30 min and 100 ml of 5% SDS solution and RNAase (final concentration equal to 20 ng/ml) were then added and the mixture was incubated for an additional 30 min. A phenol/chloroform extraction was performed twice, to remove the proteins. Genomic DNA was recovered by ethanol precipitation.
- Plasmids were transferred from the donor strains E. coli SMIO ⁇ pir or SI 7-1 into Z. mobilis strains by conjugation with a filter mating technique (Conway et al., 1987). Plasmid DNAs were transformed into either Z mobilis or E. co// cells by electroporation (Zhang et al.,
- DNA probes Tc, xylB, Tnp, and Tal were digoxigenin-UTP labeled by Polymerase
- EXAMPLE I The following example illustrates the construction of Mini-TnJTc containing genes encoding the xylose assimilation enzymes and the conjugal transfer of this construct into Z. mobilis 206C and ATCC 39676.
- Mini-TnJTc containing genes encoding the xylose assimilation enzymes was constructed by inserting a Vgap-xylAxylB operon into the unique Notl site of Mini- TnJTc contained in plasmid pGP704. See. Figure 1. The Vgap-xylAxylB operons, were taken from plasmid pZB4 or pZB5, US Pat. ⁇ os. 5,712,133 and 5,726,053.
- Mini-Tn5Tc xylA/xylB(X4) and Mini-Tn5Tc xylAxylB (X5) were transformed into the donor strain E. coli SMIO ⁇ pir by electroporation and mated with either Z. mobilis ATCC 39676 and 206C strains.
- Z. mobilis 206C is disclosed in US Pat. No. 5,843,760 which is incorporated herein by reference.
- Tc r transconjugates were obtained from both SMIO ⁇ pir donors containing Mini-Tn5Tc y//4 y/.S (X4) and Mini-Tn5 TcxylAxylB (X5) by selection on media containing Tc and nalidixic acid. See, Figure 2(b).
- Genomic and plasmid DNA from the nine Z mobilis Tc r transconjugates was then subjected to Southern blot analyses. Genomic and plasmid DNA from the transconjugates was digested with Sphl, which cuts in the hybrid transposon and in the xylose operon, and the blots were then hybridized to either a Tc probe, a XylB probe, or a transposase Tnp probe.
- EXAMPLE II (C25) This example shows that to enhance the genetic stability of the recombinant Z. Mobilis, in the absence of antibiotic selection, the xylose assimilation genes xylA and xylB, encoding xylose isomerase and xylulokinase, and pentose phosphate pathway genes, talB and tktA, encoding transaldolase and transketolase, were introduced into the Z. mobilis genome, using mini-TnJ. Two operons containing Vgap-xylA/xylB and Veno-talB/tktA were assembled in mini-TnJ and the resulting plasmid was conjugated into Z mobilis.
- the BgFll fragment containing the operon Veno-talB/tktA was inserted into the BamHI site of a newly constructed auxiliary plasmid, pUSCfiMCS as shown in Figure 3.
- the auxiliary plasmid, pUSCfiMCS was constructed by inserting a EcoR I- Hind III fragment containing the multicloning sites from pUC19 into pUCSfi.
- ⁇ UCtaltkt was then constructed from pUCpfiMCS and pUCtaltktSfi, as shown in the Figure.
- the Feno-talB/tktA was then excised, from pUCtaltktSfi, as a Sfil fragment and was used to clone into mini-tn5-Tc- y/-4 y/i?.
- the Tc r gene is flanked by Sfil sites on the Tn5-Tc-xylAxylB cassette.
- Mini-tn5-Tc-xylAxylB was partially and completely digested with Sfi I and ligated to the Veno-talB/tktASfi fragment, as shown in Figure 2(c).
- the partial digestion yielded a plasmid containing the Tc r gene, designated as mini-tn5- Tc tal/tkt-xylAxylB ((Fig. 2(c)), while complete digestion yielded a plasmid, according to the invention herein, without the Tc r gene, designated as miniTn5-to//tA:t- xylAxylB. See Figure 2(d).
- Both plasmids were transformed into the donor strain E. coli, SI 7-1 and mated with Z mobilis 206C.
- the resulting transconjugates were selected on mating media containing glucose,
- Tc, and nalidixic acid for mmiTn5-tal/tkt-xylAxy ⁇ B-Tc.
- the transconjugates were directly selected on mating media, containing xylose and nalidixic acid.
- a number of Tc r transconjugates (glucose-grown) were obtained for mini-tn5 tal/tkt-xylAxylB-Tc.
- Several xylose-grown transconjugates were obtained for mini-tn5-tal/tkt-xylAxylB.
- Xylose and ethanol were analyzed using a Hewlett-Packard 1090L HPLC equipped with an HP 1047 A refractive index detector and a Biorad HPX-87H organic acid analysis column operating at 65° C, with a 0.01 N sulfuric acid mobile phase flow rate of 0.6 ml/min.
- the ethanol yield was calculated using either the weight of sugar fermented or the total available sugar in the medium. The maximum theoretical yield was based on 0.51 g ethanol/ g xylose.
- Southern bolt analysis of genomic and plasmid DNA samples from the Z. mobilis transconjugates was made in order to investigate whether transposition of the mini-tn5 tal/tkt- xylAxylB cassette had occurred. Genomic and plasmid DNAs prepared from four transconjugates of mini-tn5 tal/tkt-xylAxylB were digested with Sphl, which cuts twice within the cassette yielding one fragment with Tal probe homology.
- Fig. 4 shows that one unique band greater than 4kb (the size of f 'eno-talB/tki A), which is adjacent to Z mobilis DNA , was detected from all the genomic DNA preparations when hybridized with Tal probe.
- Three of the samples nos. 22,23, and 24 possibly siblings also showed a band in the plasmid fraction, suggesting that the integration had occurred in the native plasmid.
- Clearly integration had occurred in the Z. mobilis genome for sample no. 21, and only one copy was inserted. Hybridization of genomic and plasmid DNA samples form these transconjugates with a Tnp probe (Fig.
- Zymomonas genome were considerably higher than was expected when compared to that of the 10 copies per cell found on a plasmid- bearing strain.
- the cells were used to inoculate a flask of RM media containing 1% glucose,and 5% xylose in order to measure their ability to ferment xylose to ethanol. Ethanol process yields and xylose utilization rates were used as the milestones for stability.
- Two of the genomic integrated strains demonstrated stability for more than 90 (C25 and D95) generations, while the plasmid-bearing strains (206/pZB4,
- strains C25 and D95 were analyzed in an RM medium containing a different concentrations of total glucose and xylose (1% glucose and 5% xylose; 3% glucose and 3% xylose; and 4.5% glucose and 4.5% xylose) under pH controlled conditions.
- strain C25 demonstrated much better xylose utilization and ethanol process yields at both pH 5, and pH 5.5 in RM containing 4.5% glucose and 4.5% xylose than D95. Consequently, in the subsequent examples the three arabinose assimilating genes (araBAD) were integrated into the C25 genome.
- Plasmid pZB1862-ldhL-ara described below, was used to transform Z mobilis or E. coli by electroporation. Transformants were selected on mating plates supplemented with glucose and tetracycline. Tc r colonies were further confirmed to be Ara + Xyf by growth on RM supplemented with xylose or arabinose (RMS and RMA). Plasmid TniOG, also described below, was transferred from an E.
- the ldhL fragment and digoxygenin (DIG)-labeled Idh, ara, and tnp probes were amplified by PCR using either Pfu (Stratagene, La Jolla, C A) or Taq DNA polymerase (Qiagen,
- ldhL 5'-TCGCGGATCCTCTATCCCTTTATTTTTCTATCCCCATCACCTCGG-3' SEQ. ID NO. 9.
- DIG-ara 5'-CTAACATGTTGACTCCTTCTCTAGACTTAGCG-3' SEQ. ID NO. 13.
- a Not I site was introduced in ldhL by insertion of an oligonucleotide 5'-CATGCGCGGCCGCC-3' at Ncol site, which is located in the middle of the Idh gene.
- the new Notl site was approximately 1.4 and 1.1 kb from either end of ldhL.
- a BamHl fragment of ldhL (2.5 kb) containing the Notl site was ligated into pZBl 862 at a BcR site.
- a 4.4-kb Vgap-araBAD isolated from pZB206 (US Pat. ⁇ os. 5,712.133 and 5,726,053), was cloned into the Notl site, of ldhL, to form the integrative plasmid, pZB1862- ldhL-ara. See. Figure 10.
- Vgap-araBAD operon containing the three arabinose-assimilating genes, was integrated into the Idh site in the C25 genome through homologous recombination.
- pZB1862-ldhL-ara was constructed in E. coli DH5 ⁇ .
- the plasmid pZB1862-ldhL-ara was transferred into C25 by electroporation.
- the Tc resistant transformants were selected and tested for growth on arabinose.
- Vgap-ara-BAD could be integrated in the genome of C25 by the replacement of ldhL with the ldhL" -araBAD-ldhL ' cassette (from the plasmid) through homologous recombination.
- Plasmid pZB1862-ldhL-ara will replicate in Z. mobilis. However, Z mobilis tends to lose foreign plasmids at sub-optimal growth conditions (e.g. 37° C). Using this characteristic, curing of pZB1862-ldhL-ara was achieved by subculturing C25 transformants at 37° C in the absence of Tc for several transfers. Cultures form each transfer were constantly monitored of for the loss of the plasmid. By the third transfer, 100% of the cells became Tc s , indicating a loss of the plasmid.
- Mini-TnJ was used for constructing C25 with Veno-tal/tkt and Vgap-xylAB operons. Although the transposase gene did not exist in C25, mini-TniO was used for the subsequent integration of Vgap-araBAD to avoid any possible incompatibility between the same transposons.
- Plasmid TnlOG (Fig.l 1) was constructed based on the Tn70-based delivery plasmid, pLOFKm. The Km r gene was replaced by a Notl fragment of Vgap-araBAD. isolated from pZB206. TnlOG was constructed and maintained in E. coli CI 18. The plasmid was then transferred into the mating donor, E. coli SMIO ⁇ pir for conjugation with Z mobilis. Since
- TnlOG is a suicide plasmid in Z. mobilis, only transconjugates with araBAD integration were able to grow on mating plates, supplemented with arabinose.
- the E. coil SMIO ⁇ pir donor was inhibited, in the plates, by the presence of nalidixic acid.
- Transconjugates appeared on mating/ara plates in 7 days. Colonies were replica-picked onto RMA and RMX to confirm their phenotypes. Eighty-six percent of the colonies picked were Xyl + Ara + . Twenty colonies from the pick-plates were cross-transferred to different plates (from xylose plates to arabinose plates or vice versa). Sixty percent of those colonies remained Xyl + Ara + .
- Pstl cuts TnlOG into two fragments (7.7 and 5.9 kb) and only the 5.9-kb fragment carries the transposase gene (IS10 R ). Both fragments hybridized with an ara probe. According to the blots, only G8. Gl 1, G15 and GH17 were ⁇ ro-positive and t/?/?-negative. Different band patterns indicated that Vgap-araBAD was integrated at different loci in the genome. Although the transposase gene was not expected to remain in the genome of the integrants, four strains (G5, G6, G14 and G19), out of the eight, contained the transposase gene. Furthermore, G14, and G19 contained the transposase gene on the plasmid.
- Xylose isomerase (XI), xylulokinase (XK), L-arabinose isomerase (L-AI), L-ribulokinase (L-RK), L-ribulose-5-P-4-epimerase (L-Repi), transketolase(TKT) and transaldolase (TAL) were assayed, using cell-free extracts of the Z. mobilis integrants and control strains, according to Zhang, et al, 1995; and Deanda et al, 1996, with minor modifications.
- Cell-free extracts were prepared by collecting the cultures at late-log phase (30°C, OD 600 approximately 1.2), washing once with sonication buffer (10 mM Tris-HCl, pH 7.6 10 mM MgCl 2 ) and sonicating. The cell debris was removed by centrifugation (14,000 rpm, 45 min 4°C). In the L-AI assay, the volumes of timed samples were scaled down by half (50 ⁇ l), 70% H 2 SO 4 (1.5 ml) and 0.12% carbazole (50 ⁇ l). All of the tubes were maintained in a 25 °C water bath, both before and after the addition of 70% H 2 SO 4 , until reading the absorbency. The samples were taken at 0,5,10, and 20 min during the reaction.
- the integrants showed lower activities than the plasmid- bearing strain (206C/pZB301). This is presumable related to the copy number of the genes.
- the cultures were inoculated into test tubes containing RMG, incubated overnight at 30°C. and transferred daily to RMG tubes. The inoculum was controlled to allow transfer every 10 generations. At every 40 generations, the cells were used to inoculate flasks, containing a mixture of sugars, to test the fermentation capabilities on the sugars without pH control at 30°C.
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Application Number | Priority Date | Filing Date | Title |
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AU4992601A AU4992601A (en) | 2000-05-01 | 2001-04-06 | Stable zymomonas mobilis xylose and arabinose fermenting strains |
JP2001580393A JP4510355B2 (en) | 2000-05-01 | 2001-04-06 | A stable xylose and arabinose fermentation strain of Thymomonas mobilis |
AU2001249926A AU2001249926B2 (en) | 2000-05-01 | 2001-04-06 | Stable zymomonas mobilis xylose and arabinose fermenting strains |
BRPI0110987-1A BR0110987B1 (en) | 2000-05-01 | 2001-04-06 | Transposon for stable insertion of foreign genes into a bacterial genome, plasmid vector, bacterium, and reaction mixture comprising a fermentable medium. |
EP01923213A EP1278876A2 (en) | 2000-05-01 | 2001-04-06 | STABLE i ZYMOMONAS MOBILIS /i XYLOSE AND ARABINOSE FERMENTING STRAINS |
US10/134,655 US7223575B2 (en) | 2000-05-01 | 2002-04-27 | Zymomonas pentose-sugar fermenting strains and uses thereof |
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US09/565,233 US7354755B2 (en) | 2000-05-01 | 2000-05-01 | Stable zymomonas mobilis xylose and arabinose fermenting strains |
US09/565,233 | 2000-05-01 | ||
CA2,304,929 | 2000-05-02 | ||
CA002304929A CA2304929C (en) | 2000-05-02 | 2000-05-02 | Stable zymomonas mobilis xylose and arabinose fermenting strains |
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US09/565,233 Continuation-In-Part US7354755B2 (en) | 2000-05-01 | 2000-05-01 | Stable zymomonas mobilis xylose and arabinose fermenting strains |
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US10/134,655 Continuation-In-Part US7223575B2 (en) | 2000-05-01 | 2002-04-27 | Zymomonas pentose-sugar fermenting strains and uses thereof |
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EP (1) | EP1278876A2 (en) |
JP (1) | JP4510355B2 (en) |
AU (2) | AU2001249926B2 (en) |
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Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2001083784A2 (en) * | 2000-05-01 | 2001-11-08 | Midwest Research Institute | Method of site-specific insertion in zymomonas mobilis |
AU2003299464B8 (en) * | 2000-05-01 | 2004-05-13 | Alliance For Sustainable Energy, Llc | Zymomonas pentose-sugar fermentating strains and uses thereof |
EP1539785A2 (en) * | 2002-06-26 | 2005-06-15 | Transgenrx, Inc. | Gene regulation in transgenic animals using a transposon-based vector |
EP1549736A2 (en) * | 2002-04-27 | 2005-07-06 | Midwest Research Institute | Zymomonas pentose-sugar fermentating strains and uses thereof |
CN100374570C (en) * | 2004-07-07 | 2008-03-12 | 新疆农业科学院微生物应用研究所 | Electric conversion method of making shuttle plasmid guide into yellow brevibacterium |
DE102007048134A1 (en) * | 2007-10-05 | 2009-04-09 | Evocatal Gmbh | Cloning, integration and expression of a gene cluster comprises using gene cassettes comprising sequences that mediate the transfer, integration and expression of a flanked nucleic acid |
WO2013096366A1 (en) * | 2011-12-20 | 2013-06-27 | E. I. Du Pont De Nemours And Company | Gene inactivation allowing immediate growth on xylose medium by engineered zymomonas |
US8623632B2 (en) | 2005-08-09 | 2014-01-07 | Helmholtz-Zentrum Fur Infektionsforschung Gmbh | Extracellular polyhydroxyalkanoates produced by genetically engineered microorganisms |
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US7741119B2 (en) * | 2006-09-28 | 2010-06-22 | E. I. Du Pont De Nemours And Company | Xylitol synthesis mutant of xylose-utilizing zymomonas for ethanol production |
ES2385857T3 (en) * | 2007-10-30 | 2012-08-01 | E. I. Du Pont De Nemours And Company | Zymomonas with increased ethanol production in medium containing concentrated sugars and acetate |
JP2011515067A (en) * | 2007-10-30 | 2011-05-19 | イー・アイ・デュポン・ドウ・ヌムール・アンド・カンパニー | Method for producing ethanol from a medium containing xylose using a recombinant zymomonas strain with reduced expression of himA |
EP2271743A2 (en) * | 2008-03-27 | 2011-01-12 | E. I. du Pont de Nemours and Company | Zymomonas with improved xylose utilization |
WO2010036978A2 (en) | 2008-09-25 | 2010-04-01 | Transgenrx, Inc. | Novel vectors for production of growth hormone |
WO2010036976A2 (en) | 2008-09-25 | 2010-04-01 | Transgenrx, Inc. | Novel vectors for production of antibodies |
EP2417263B1 (en) | 2009-04-09 | 2015-09-23 | ProteoVec Holding L.L.C. | Production of proteins using transposon-based vectors |
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- 2001-04-06 WO PCT/US2001/011334 patent/WO2001083786A2/en active IP Right Grant
- 2001-04-06 AU AU2001249926A patent/AU2001249926B2/en not_active Ceased
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WO1997042307A1 (en) * | 1996-05-06 | 1997-11-13 | Purdue Research Foundation | Stable recombinant yeasts for fermenting xylose to ethanol |
WO1998050524A1 (en) * | 1997-05-06 | 1998-11-12 | Midwest Research Institute | Single zymomonas mobilis strain for xylose and arabinose fermentation |
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Cited By (14)
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WO2001083784A2 (en) * | 2000-05-01 | 2001-11-08 | Midwest Research Institute | Method of site-specific insertion in zymomonas mobilis |
WO2001083784A3 (en) * | 2000-05-01 | 2003-10-02 | Midwest Research Inst | Method of site-specific insertion in zymomonas mobilis |
AU2003299464B8 (en) * | 2000-05-01 | 2004-05-13 | Alliance For Sustainable Energy, Llc | Zymomonas pentose-sugar fermentating strains and uses thereof |
US7374939B1 (en) | 2000-05-01 | 2008-05-20 | Midwest Research Institute | Method of inactivation of an end product of energy metabolism in Zymomonas mobilis |
AU2003299464B2 (en) * | 2000-05-01 | 2007-09-06 | Alliance For Sustainable Energy, Llc | Zymomonas pentose-sugar fermentating strains and uses thereof |
EP1549736A4 (en) * | 2002-04-27 | 2006-12-13 | Midwest Research Inst | Zymomonas pentose-sugar fermentating strains and uses thereof |
EP1549736A2 (en) * | 2002-04-27 | 2005-07-06 | Midwest Research Institute | Zymomonas pentose-sugar fermentating strains and uses thereof |
EP1539785A4 (en) * | 2002-06-26 | 2006-03-29 | Transgenrx Inc | Gene regulation in transgenic animals using a transposon-based vector |
EP1539785A2 (en) * | 2002-06-26 | 2005-06-15 | Transgenrx, Inc. | Gene regulation in transgenic animals using a transposon-based vector |
CN100374570C (en) * | 2004-07-07 | 2008-03-12 | 新疆农业科学院微生物应用研究所 | Electric conversion method of making shuttle plasmid guide into yellow brevibacterium |
US8623632B2 (en) | 2005-08-09 | 2014-01-07 | Helmholtz-Zentrum Fur Infektionsforschung Gmbh | Extracellular polyhydroxyalkanoates produced by genetically engineered microorganisms |
DE102007048134A1 (en) * | 2007-10-05 | 2009-04-09 | Evocatal Gmbh | Cloning, integration and expression of a gene cluster comprises using gene cassettes comprising sequences that mediate the transfer, integration and expression of a flanked nucleic acid |
WO2013096366A1 (en) * | 2011-12-20 | 2013-06-27 | E. I. Du Pont De Nemours And Company | Gene inactivation allowing immediate growth on xylose medium by engineered zymomonas |
CN104220590A (en) * | 2011-12-20 | 2014-12-17 | 纳幕尔杜邦公司 | Gene inactivation allowing immediate growth on xylose medium by engineered zymomonas |
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BR0110987B1 (en) | 2014-07-15 |
WO2001083786A3 (en) | 2002-06-20 |
WO2001083786B1 (en) | 2002-07-18 |
AU2001249926B2 (en) | 2006-07-20 |
AU4992601A (en) | 2001-11-12 |
BR0110987A (en) | 2006-02-07 |
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