BR0110987B1 - Transposon for stable insertion of foreign genes into a bacterial genome, plasmid vector, bacterium, and reaction mixture comprising a fermentable medium. - Google Patents

Description

"TRANSPOSON FOR STABLE INSERTION OF ALIEN GENES WITHIN A BACTERIAL GENOME, PLASMID VECTOR, BACTERIA, AND REACTION MIX UNDERSTANDING A FERRELABLE MEANS." Contractual Origin of the Invention; The United States Government has rights with respect to the present invention by virtue of Contract No. DE-AC36-99GO10337 between the United States Department of Energy and the Midwest Research Institute.

Technical scope The present invention relates to the biological conversion of cellulose substrates into fuels and chemicals, and in particular to recombinant Zymomonas mobilis strains fermenting xylose and arabinose, or both, in ethanol.

State of the art Fermentation technology is useful for converting renewable biomass cellulose substrates into fuels and chemicals such as ethanol. A typical substrate is 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. Thus, achieving a high specific product formation rate and conversion efficiency in the fermentation of pentose sugars is vital for commercial production of fuels and chemicals from renewable substrates. Z. mobitis is widely known for its ability to rapidly and efficiently convert glucose substrates in ethanol at low pH into an anaerobic culture and within a medium containing the inhibitor compounds typically associated with a hydrolyzate. lignocellulose. A distinct disadvantage of using Z. mobilis is, however, that it does not ferment pentose sugars. To overcome this disadvantage, prior art has focused on recombinant Z mobilis strains that ferment a mixture of glucose, and xylose or arabinose, or both, using exogenous genes that catalyze xylose and arabinose metabolism. These strains, and the cloning vectors, are based on a use of multiple copy plasmids bearing antibiotic resistance markers. United States Patent No. 5,514,583 discloses a transformed Z. mobilis xylose fermentor strain (CP4 / pZB4 and pZB5) featuring exogenous genes, and plasmid vectors (pZB4 and pZB5) encoding xylose isomerase, xylulokinase, iransaldolase and transquetolase, and further comprising at least one Zymomonas recognized promoter (Pgap and Peno) that regulates the expression of at least one of said genes. The microorganism is capable of growing with xylose as the sole carbon source, and fermenting xylose in ethanol at about 88% of the theoretical maximum yield. The patent claims an integrated strain.

U.S. Patent Nos. 5,712,133 and 5,726,053 disclose, among other things, arabinose fermenting Z mobilis transformants (39676 / pZB 206), containing exogenous genes encoding L-arabinose isomerase, L-ribulokinase and L-ribulose -5-phosphate-4-epimerase, Iransaldolase and transquetolase which confer a capacity for arabinose fermentation in ethanol. Also disclosed is the plasmid vector (pZB 206) and a process of using the transformants for the fermentation of a substrate-containing glucose and arabinose. The patent claims the integration of exogenous genes into a host genome. U.S. Patent No. 5,843,760 discloses an xylose and arabinose fermenter Z mobilis transformant (206C / pZB301) containing exogenous genes encoding xylose isomerase, xylulokinase, L-arabinose isomerase, L-ribulokinase, L-ribulose- 5-phosphate 4-epimerase, transaldolase and transquetoiasis, and further comprising at least one Zymomonas recognized promoter that regulates the expression of at least one of said genes, wherein said microorganism is capable of growing having arabinose and / or xylose, alone. or in combination as the carbon source and fermenting said arabinose and xylose in ethanol. The process of using the transformants along with the plasmid vectors (pZB301, pZB401, pZB402, and pZB 403) is also disclosed. This patent claims the integration of exogenous genes into the host genome.

Although hybrid plasmids can be readily maintained in Z. mobilis when cultured in mono-culture under controlled conditions, they often become unstable when the host organism is developed in the absence of selection pressure for plasmid maintenance, ie in the presence of antibiotics. For example, exogenous genes in the strains referenced above are capable of stable expression for about forty generations. Instability can be exacerbated when Z mobilis has to compete with other organisms in a mixed culture, such as a simultaneous cellulose fermentation-saccharification process. In addition, antibiotic resistance markers are generally perceived as undesirable for industrial application, such as large scale ethanol production. Thus, it is preferable to insert the cloned genes into the Z. mobilis genome, where they are kept in a low natural copy number, and thus not overexpressed, and where, at least theoretically, they should be as stable as genomic DNA. .

In Esherichia coli, the classical method for generating alien gene genomic inserts involves the use of specialized cloning λ-phage vectors that may exist stable in the lysogenic state. Alternatively, genes may be inserted by homologous recombination when grouped with E. coli chromosome sequences, or by transposition if genes can be cloned at the permissive sites of a transposon. Although transposition has been demonstrated in Z. mobilis, Pappas, KM, et al., (1997) Transposon mutagensesis and strain construction in Zymomonas mobilis (Transposon mutagenesis and strain construction in Zymomonas mobilis), Journal of Applied Microbiology, Vol. 82 , ρ.ρ. 379-388, this has been limited to mini posiçãoμ or Tn5 multiple transposition of phenotypes with auxotrophy or random antibiotic resistance for genetic analysis, and in the case of TnJ derivatives the inserts are described as stable for only 5-15 generations. Pappas, K.M., et seq. P. 383, see Figure 1. Furthermore, insertion at a specific site by homologous recombination in Z. mobilh has not been demonstrated, and no bacteriophage has yet been isolated from Zymomonas.

Tn5 and Tn 10 transposons are well known and have been widely used for mutagenesis and insertion of cloned DNA into a wide variety of gram-negative bacteria. In Herrero, M. et al., (1990) Transposon vectors containing non-resistance antibiotic selection markers for cloning and stable chromosomic insertion of foreign genes in gram-negative bacterium (Transposon vectors not containing antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes into gram-negative bacteria), J. Bacteriol. 172: 6557-6567, the procedure is disclosed for the stable cloning and insertion of extraneous genes into the gram-negative eubacterial chromosome by combining two sets of plasmids, (i) the transposition characteristics of Tn / 0 and Tn5, ( ii) resistance to certain compounds; and (iii) the incidence of suicidal properties of the R6K-based plasmid pGP704. The resulting constructs contain unique Notl or SfiI sites internal to both TnJO and Tn5 inverted repeaters. These sites are used for cloning DNA fragments with the help of two additional specialized cloning plasmids, pUCISNot and pUC18Sfi. The novel derived constructs are maintained only in donor host strains that produce the R6K-specified π protein, which is an essential replication protein for R6K and plasmids derived therefrom. Donor plasmids containing hybrid transposons are transformed into a specialized strain of E. coli lysogenic λρή, such as E. coii. 8ιη10 (λρ;>), with a chromosomally integrated RP4 that ensures broad-band marital transfer functions. The distribution of donor plasmids to selected host bacteria is accomplished by mating with the target strain. Transposition of the hybrid transposon from the distributed suicide plasmid to a target replicon is mediated through the cognate transposase encoded in the plasmid at a site external to the transposon. Since transposase function is not maintained in the target cell, such cells are immune to further transposition attempts. Multiple insertions in the same strain are therefore only limited by the availability of different selection markers.

Further disclosed in Herrero, M. et cil., 1990, Mini-Tn5 Transposon Derivatives for Insertion Mutagenesis Promoter Probing, and Genomic Insertion of Cloned DNA in Gram-Negative Bubacteria (Transposon Mini-Tn5 Derivatives for Mutagenesis Promoter Probing) by insertion, and genomic insertion of cloned DNA into Gram-negative eubacteria), Journal de Bacteriol. Vol 172, No. 11, p. 6568-6572, the construction of a collection of Tn5-derived minitransposons, such as Tn5Tc, that allows the introduction of extraneous DNA fragments into the chromosome of a series of gram-negative bacteria. Minitransposons consist of genes specifying quenamycin resistance, and tetracycline as selection markers and a unique Notl cloning site flanked by base pair 19 repeater Tn5 sequences. The transposons are located on a R6K-based suicide distribution plasmid which provides the ISJOu tnp transposase gene in eis but external to the mobile element and whose marital transfer to recipients is mediated through donor RP4 mobilization functions. Therefore, the insertions produced by these elements are generally more stable due to the absence of transposase-mediated transpositions, deletions and secondary inversions.

See also, Berg et al., (1989) Transposable elements and the genetic engineering of bacteria (Transposable elements and the genetic engineering of bacteria), pp. 879-926, in D.E. Berg, Mobile DNA, American Society for Microbiology, Washington, DC. Several insertion stable can be obtained in this way with derived elements, for example also from Tnl6. Way, JC et al., (1984) New Tn / 0 derivatives for transposon mutagenesis and for construction of lacZ operon fusions by transposition (New Tn / 0 derivatives for transposon mutagenesis and for construction of lacZ operon fusions transposition). Gene 32: 369-379. The structure of the mini-Tn5Tc, Herrero, et seq., P. 6569, is disclosed for use for insertion mutagenesis or as a transposon vector for cloning DNA fragments flanked by Notl sites (readily isolated by cloning DNA fragments primarily into pUC18 derivatives called pUC18Not and pUC18Not). The Mini-Tn5Tc element is constructed in vitro using standard DNA recombination techniques.

Maniatis, T., et al (1989) Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor-, NY The determining factor for (Tc) resistance is obtained. as a fragment of R a RI from plasmids carrying these as an interposon. Fellay, R., et al (1987) Interposon mutagensesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertion mutagenesis of Gram-negative bacteria (Interposon mutagenesis of soil and water bacteria: a family of fragments of DNA designed for in vitro insertion mutagenesis of Gram-negative bacteria), Gene 52: 147-154. This fragment is subsequently cloned into the single pUCISSfi EcoR1 site, excised as an Sfd fragment, and inserted between the Tn5 19 base pair terminals in the pUT so that the mobile unit is present in all cases as an Xbal-EcoRl moiety. (partial) part of the distribution plasmid. The resulting element is the mini Tn57c.

Tn10-based transposon vector delivery systems are described in Herrero, M., et seq. 172: 6557-6567. The λ-phage, a derivative of XRP167, carries a 5.1-kb insert containing the mini-Tn / OKm element and the IS / (¾ transposase gene) is located outside the inverted repeaters of the mobile element and downstream of the pTdc promoter To obtain a transposon delivery plasmid with host-independent transposition regulation, the EcoR1 insert fragment is ligated to pBOR8, a derivative of pGP704 containing the lacE gene from plasmid pMJR1560. unable to replicate in host strains devoid of the pir gene RKK-specified π protein product pGP704 contains the marital transfer origin (oriT sequence) of plasmid RP4 and can therefore be transferred to a gregaregative bacterium when disposed. in trans with mobilization functions (Mob) The Mlu.1 fragment internal to inverted repeaters containing the specified original π protein product of the original resistance gene kanamycin mini-Tn / 0 is replaced with a fragment containing an S / II-Ptt cassette, appropriately modified by the addition of the NotI site and Mtul adapters, which produced the pLODPtt. This construct features unique cloning sites Sftl, Notl, and Xbal among mini-Tn / 0 inverted repeaters. PLOFPtt resistance marker Ptt (Ptt1) is exchanged for quenamycin resistance to produce plasmid pLOFKm.

In view of the foregoing, there is a need for the construction of stable recombinant Z. mobilis strains capable of fermenting xylose and arabinose, both of which in ethanol through the generation of stable genomic inserts encoding enzymes which are necessary for the development of the enzyme. xylose and arabinose catabolism. The strains should be free of antibiotic resistance and stable for more than 40 generations in a non-selective medium. It is also desirable that the strains demonstrate a high specific rate of product formation close to the theoretical maximum product yield.

Disclosure of the Invention It is an object of the present invention to provide transposable elements and plasmid vectors useful for stable integration of foreign gene structures, encoding enzymes selected from the group consisting of xylAxylB, tal / tkt, and araBAD and at least one regulatory gene for induction. of gene structures in a genome of Z. mobilis.

It is a further object of the present invention to provide significantly different Z. mobilis strains capable of stable expression of gene structures in a non-selection medium.

It is yet another object of the present invention to provide significantly different Z. mobilis strains showing stable expression of gene structures, but which are characterized by a high rate of specific product formation and conversion efficiency when used as bio-catalysts in a reaction mixture of cellulose hydrosalysate.

In order to overcome the problems associated with the related state of the art and according to the purpose of the present invention, as incorporated and broadly described herein, the present invention briefly includes: a transposon for stable insertion of foreign genes within a bacterial genome, including: at least one operon displaying enzyme encoding gene structures selected from the group consisting of xylAxilB, araBAD and tal / tkt, and at least one promoter for the expression of gene structures in the bacterium, a pair of inverted insertion sequences, the operons contained within the insertion sequences, and a transposase gene located outside the insertion sequences; a plasmid launcher vector for transforming foreign genes within a bacterial genome, including: at least one operon displaying gene structures encoding enzymes selected from the group consisting of xiAxylB, araBAD and tal / tkt, at least one promoter for expression of gene structures in the bacterium, and at least two DNA fragments showing homology to a gene in the genome of the bacterium to be transformed. The transposon and the launcher vector are useful in constructing significantly different Zymomonas mobilis strains according to the present invention which are useful in converting cellulose-derived pentose sugars into fuels and chemicals using traditional fermentation technology because are stable for expression in a non-selection medium.

Additional advantages of the present invention will be defined in part in the following description and in part will be obvious from that description or may be deduced from the practice of the present invention. The advantages of the present invention may be realized and obtained by the method particularly indicated in the appended claims.

Brief Description of the Figures The accompanying figures, which are incorporated and form an integral part of the present specification, illustrate at least one embodiment of the present invention and together with said description explain the principles of the present invention. 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-Tn5 series constructs according to the present invention. Figure 3 is an illustration of the series of constructs resulting in the pVCtaltktSfi plasmid map. Figure 4 shows the results of the Southern Analysis of taUtkt-xylAxilB Z. mobilis transconjugates with the Tal probe, C is the mini Tn5 tal / tkt-xylAxHB of the control plasmid. λΗ is the DNA size marker. Figure 5 shows the results of Southern Analysis of tal / tkt-xylAxylB Z. mobilis transconjugates with Tnp probe. C is the tcJ / tkt-xylAxilB mini-TnJ of the control plasmid. λΗ is the DNA size marker. Figure 6 is a graph of the enzymatic activities for various isolates of the stable xylose fermenting Z. mobilis strain according to the present invention. Figure 7 illustrates the graphs of fermentation profiles for four of the isolates (Nos. 21, 22 and 5) of Figure 6 and their performance relative to plasmid carrying strains 39673 / pZB4 and BCl / pZB4L. Figure 8 is a graphical representation illustrating the stability of stable xylose fermenting Z25 mobilis strains C25 and D95 according to the present invention. The graph illustrates the stability of plasmid-bearing and genome-integrated xylose fermentor strains using the ethanol process yield and xylose utilization parameters as indicators. The C25 and D95 strains of the present invention have remained stable for more than 90 generations. The fermentation medium comprised 1% glucose and 5% xylose RM and the temperature was constant at 30 ° C. Figure 9 illustrates the optical density biomass concentration (ODSoo), the xylose utilization as a function of time, the ethanol production as a function of time, and the process yield compared to the xylose percentage utilization for batch fermentation of genome-integrated xylose fermentor strains C25 and D95 in RM medium containing 4.5% glucose and 4.5% xylose under pH 5.0 and pH 5.5 at 30 ° C ° C according to the present invention.

Figure 10 is a map of integrative plasmid pZB 1862-ldhL-ara. AmBAD is inserted into the klL Notl site, interrupting the wool. The construct is based on the Z. mobilis replicating plasmid pZB 1862. Figure 1! is the Tn / OG plasmid map. IS 10r is the transposase gene. IR is the sequence of the inverted repeating transposon. ArciBAD was inserted between the two inverted repeaters at NotI sites. Figure 12 illustrates Southern analysis of genome-integrated xylose / arabinose fermenting Z. mobilis strains from homologous recombination using DIG-ara and DIG-1dh probes. AX1, 13, 23, 101, 104, and 107 are members of araBAD. C25 is the host control. 1% pZB 62-ldhL-ara is an isolated form of DH5a plasmid control. λ / Η is a molecular weight marker: 23, 9.4, 6.6 4.3, 2.3 and 2.0 kb. Figure 13 represents the Southern analysis of genome-integrated xylose / arabinose fermenting Z. mobilis strains from transposon integration using DIG-tnp and DIG-ara probes. G5,6, 1,15,17,14 and 19 are members of araBAD. C25 is the host control. Tn / OG is the plasmid control. λΗ is a molecular weight marker: 23,9,4,6,6,4,3,2,3 and 2,0 kb. Figure 13 (a) is the Pstl tnp digestion and Figure 13 (b) is the Pstl digestion probe. Figure 14 represents bar graphs of the enzymatic activities of transquetolase, transaldolase, xylose isomerase and xylulokinase of genomic integrated strains. 206C / pZB301 is a plasmid control. 206C is a host control. C25 is the integral xylose fermenter. G8 is the xylose / arabinose fermenting member of the Tn / 0 transposition. AX1 and AX101 are the members of xylose / arabinose fermentation 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 from genomic integrated strains. 206C / pZB30! It is a plasmid control. 206C is a host control. C25 is the integral xylose fermenter. G8 is the integrating xylose / arabinose fermenting form of the Tn / 0 transposition. AXI and AXI01 are the members of xylose / arabinose fermentation from homologous recombination. Figure 16 represents the bar graph result of ethanol processing yields of genomic integrated strains of xylose and arabinose fermenting Zymomonas in RMGXA (1: 2: 2%) at a temperature T = 30 ° C, without pH control. These strains were cultures of forms inoculated over generations in non-selective media. Figure 17 represents the bar graph result of the use of xylose and arabinose from genomic integrated strains of xylose and arabinose fermenter Zymomonas in RM containing 1% glucose, 2% xylose and 2% arabinose at a temperature of 30 ° C. with pH control. These strains were cultures of forms inoculated over generations in non-selective media. Figure 18 is a graphical representation of the fermentation performance of the genomic integrated strains of xylose and arabinose fermenter Zymomonas in RM containing 4% glucose, 4% xylose and 2% arabinose at a pH of 5.5 and temperature of 30 ° C.

Detailed Description of the Figures Except as specifically defined otherwise, all technical or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. While any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, preferred methods and materials will now be described. All United States Patents are incorporated herein by reference as fully defined herein.

We will now refer in detail to the presently preferred embodiments of the present invention, examples of which are illustrated in the accompanying Figures. For the examples described below, "plasmid-bearing strains" refers to or relates to those strains and vectors described in United States Patents identified in the State of the Art Description. The “genome of Z. mobilis, ου Z. mobiiis genomic” means genes that foolishly specify all the expressed and potentially expressive characteristics associated with a given Z. mobilis. C25 refers to Zymomonas mobilis' Patent Application PTA-1799 Designation, AX refers to Zymomonas mobilis PTA-1797 Patent Application Designation; G8 refers to Zymomonas mobilis PTA-1796 Patent Application Designation, mim-TnS-tal / tkt-xylAxilB refers to PTA-1798 Escherichia coli Patent Application Designation: DH5 · (pZb 1862 ~ / (// iL-ara); and C25 refers to Zymomonas mobilis Patent Application PTA-1799 Designation. All of the above are deposited with the American Type Culture Collection, 10801 University Blvd., Manassas, VA 201 10-2209, received on May 2, 2000, in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Patent Procedure Effects and have been duly accepted.

Examples Strains, Plasmids, and Media.

The bacterial strains of E. coli CC118Xipir), CC118λ (/? Ί>) (mini-tn5Tc), SM10Xipir), and plasmids pUC19, pLOF / Km, pUC18sfi, pUT / Tc containing Tn5, Tn 10 and pUC18Not minitransposons were obtained from Dr. K. Timmis, GBF - Gesellschaft Fur Biotechnololgische Forschung mbH, Mascheroder weg 1 D - 38124 Braunschweig, Federal Republic of Germany. E. coli DH5a was used as a host for plasmid construction. E. coli SMIOLpir was used as a donor strain in mating experiments. Strains of Z. mobilis ATCC 39676 and its derivative, 206C (United States Patent No. 5,843,760) were used as recipients according to the present invention. Tn / 0-based plasmids were constructed and maintained in E. coli CC1 18.

E. coli strains were cultured in LB medium at a temperature of 37 ° C. Z. mobilis strains were anaerobically maintained on MRI (10 g / L yeast extract, 2g / L KH2PO4) supplemented with glucose, D -xylose or L-arabinose at 20g / L, except where otherwise specified. All strains containing plasmids were developed in the presence of tetracycline (Tc) ((10pg / ml in liquid Z. mobilis, and E. coli; 20 pg / ml in agar for Z. mobilis; and 15 pg / ml in agar, or ampicillin (Ap), 100 pg / ml for E. coli)). For regeneration and selection of Z. mobilis transformants or transconjugates, mating plates were used ((10 g / L yeast extract, 5 g / L tryptone, 2.5 g / L (NH4) 2SO4, K2HPO4 to 0.2 g / l and sugar at 50 g / l)) supplemented with tetracycline or nalidixic acid (20 pg / ml). All agar plates were made with 15 g / L agar. Recombinant DNA techniques. Isolation of plasmid DNA, restriction endonuclease digestion, ligation and transformation, agarose electrophoresis and other recombinant DNA techniques were performed according to published protocols, Sambrook et al. (1989) Molecular cloning: a laboratory manual (Molecular Cloning: A Laboratory Manual), Cold Spring Harbor laboratory press, Cold Spring Harbor, NY, or the reagent manufacturer's instructions, which have been specified, and are well known in the art. Z. mobilis genomic DNA was extracted using three milliliters of cells overnight resuspended in 250 ml of 50 mM Tris-50 mM EDTA buffer. Cells were treated with lysozyme at 37 ° C for 30 min and 100 ml of 5% SDS and RNAase solution (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.

Conjugation and transformation.

Plasmids were transferred from E. coli SM1OZpir or SI7-1 donor strains into Z. mobilis strains by conjugation with a filter mating technique (Conway et al., 1987). Plasmid DNAs were transformed into Z. mobilis or E. coli cells by electroporation (Zhang et al., 1995)).

Southern Blot Analysis, DNA was transferred to a nylon membrane using a Stratagene Posi Blot pressure blotter. Tc, xilB, Tnp, and Tal DNA probes were labeled with digoxigenin-UTP by polymerase chain reactions (PCR). The following primers were used for DNA labeling: XilB 5'-TATGGGTTCAGCGGCATGAG-3 'SEQ ID NO. 3. 5'-ATGGGCATGAGATCCATAGCC-3 'SEQ ID NO. 4. Τηρ 5 '-TCCT A AC AT GGT AACGTTC-3' SEQ ID NO. 5. 5'-CCAACCTTACCAGAGGGC-3 'SEQ ID NO. 6

Such: 5 '-CGTCT A A AG ATTTT A AG A AGGTTTCG AT ATG ACGG AG A A ATTGACC-3' SEQ ID NO. 7

5 '-CATTTTGACTCC AGATCTAG ATTACAGCAGATCGCCG ATC ATTTTTTCC-3' SEQ ID NO. 8. Prehybridization and hybridization were performed according to established protocols described in the Boehringer Mannheim Hybridization Kit.

EXAMPLE 10 The following example illustrates the construction of Mini-Tn5Tc containing genes encoding xylose assimilation enzymes and the marital transfer of this construct into Z. mobilis 206C and ATCC 39676. The Mini-TnJTc containing genes encoding xylose assimilation enzymes was constructed by inserting a Pgap-xilAxitB operon into the unique Notl site of the Mini-Tn5Tc contained in plasmid pGP704. See Figure 1. Pgap-xAXylB operons were taken from plasmid pZB4 or pZB5, see United States Patents No. 5,712,133 and 5,726,053. The resulting plasmids, designated Mini-Tn5Tc xylA / xilB (X4) and Mini-Tn5Tc xylAxilB (X5), were transformed into the donor E. coli SM 10Xpir strain by electroporation and mated as Z. mobilis ATCC 39676 or 206C strains. Z. mobilis 206C is disclosed in United States Patent No. 5,843,760 which is incorporated herein by reference. Nine Zyntomonas Tcr transconjugates were obtained from both 8Μ! 0λρό 'donors containing Mini-Tn5Tcxi7Ar // ZJ (X4) and Mini-Tn5 Tc xylAxylB (X5) by selection on media containing Tc and nalidixic acid. See Figure 2 (b). The genomic DNA and plasmid DNA of the nine Z transconjugates.

Tc ‘mobilis were then subjected to Southern blot analysis. The genomic DNA and plasmid DNA from the nine transconjugates were digested with Sphl, which sectioned on the hybrid transposon and xylose operon, and the blots were then hybridized to a Tc probe, an XilB probe or a Τηρ transposase probe. From the autoradiograph, it was determined that: (1) the Pgap-xylAxilB xylose operon had been inserted into the Zymomonas genome, along with the Tc1 gene; (2) only a single insertion had occurred for each transconjugate; (3) the inserts were each at a distinct locus in the genome; (4) the gene Tc1 and ο XilB were not present in the plasmid fraction; and (5) the transposase tnp gene was not present in the transconjugates. Enzymatic analysis of Zymomonas Tc1 Pgap-xylAxIIB transconjugates was performed to confirm the expression of xylose isomerase (XI) and xylulokinase (XK) Zymomonas Tc1 Pgap-xylAxilB transconjugate nodes. This analysis revealed that xylose isomerase enzyme levels for all transconjugates were about half that of their equivalent plasmids. Similarly, about half of the xylulokinase activity of plasmid-bearing strains was observed in the members. Both XI and XK activities, expressed from a single copy of the genes in the Zymomonas genome, were considerably higher than expected when compared to those of the 10 copies per cell detected in plasmid-bearing strains. EXAMPLE II (C2S) This example illustrates that to increase the genetic stability of Z.

Recombinant Mobilis, in the absence of antibiotic selection, the xylose and xylB assimilation genes encoding xylose isomerase and xylulokinase, and pentose, talB and tktA phosphate pathway genes encoding transaldolase and transquetolase were introduced into the Z mobilis genome. using mini Tn5. Two operons containing Pgap-xylA / xilB and Peno-talB / tktA were pooled into mini-Tn.5 and the resulting plasmid was conjugated to one Z. mobilis. With the aid of transposase located outside the mini-Tnó cassette, individual copies of Pgap-xylAxilB and Peno-talB / tktA were inserted into the Z mobilis genome, as illustrated by Southern hybridization. Enzymatic analysis of xylose isomerase, xylulokinase, transaldolase and transaldolase indicated that all genes were expressed in a coordinated manner and that the integrated strains produced about 30-70% of the enzymatic activities of the plasmid-bearing strains. These enzyme levels were sufficient for the body to develop and ferment xylose in ethanol.

To facilitate the cloning process, the Zig / ΓΙ fragment containing the Peno-talB / tktA operon was inserted into the BamHI site of a newly constructed helper plasmid pUSCfiMCS as shown in Figure 3. The helper plasmid pUSCfiMCS was constructed by insertion. of an EcoB fragment. 1-Hind III containing pUCI9 multicloning sites in pUCSi. YUCtalíkt was then constructed from pUCpfiMCS and pUCtaltktSfi, as shown in Figure.

Referring now to Figure 2, Peno-taiB / tktA was then excised from pUCtaltktSfi as an Sfil fragment and was used to clone into mini-tn5-Tc-xylAxUB. As shown in the Figure, the Tcr gene is flanked by Sfil sites in the Tn5-Tc-xylAxylB cassette. The mini-tnS-Tc-xMmax / f was partially and completely digested with Sfi I and ligated to the Peno-talB / tktASfi fragment as shown in Figure 2 (c). partial digestion produced a plasmid containing the Tcr gene, designated mini-tn5-Tc tal / tkt-xylAxylB ((Figure 2 (c)), while complete digestion produced a plasmid according to the present invention without the Tcr gene , designated min \ Tf \ 5-tal / tkt-xylAxyl B. See Figure 2 (d).

Both plasmids were transformed into donor E. coli strain, SI7-1 and mated with Z. mobilis 206C. The resulting transconjugates were selected on mating media containing glucose, Tc and nalidixic acid for mm 2 Pn5-tal / tkt-xylAxylB-Tc.

For mini-tn5-tal / tkt-xylAxilB, the transconjugates were selected directly in mating media containing xylose and nalidixic acid. A number of Tcr (glucose-developed) transconjugates were obtained for mini-tn5 tal / tkt-xylAxylB-Tc. Several xylose-developed transconjugates were obtained for mm \ -tn5-tal / tkt-xylAxylB.

Preliminary batch cultures were statistically tested at a temperature of 30 ° C without pH control in 80 ml RM bottles containing 2% xylose for their fermentation dynamics. Colonies taken from RM + 2% xylose plaques were grown in a 2% xylose RM medium overnight at a temperature of 20 ° C until the culture had reached the stationary development phase (optical density600). = 0.1 at 500 nm) were used as inoculum. Xylose and ethanol were analyzed using an HPLC.

Hewlett-Packard 1090L equipped with an HP 1047 A refractive index detector and Biorad HPX-87H organic acid analysis column operating at a temperature of 65 ° C with a 0.01 N sulfuric acid mobile phase flow rate 0.6 ml / min. The ethanol yield was calculated using either the weight of fermented sugar or the total sugar available in the medium. The theoretical maximum yield was based on 0.51 g ethanol / g xylose.

Southern blot analysis of genomic DNA and plasmid DNA samples of the Z. furniture transconjugates was performed to investigate whether or not transposition of the mini-tn5 tal / tkt-xylAxilB cassette had occurred. Genomic and plasmid DNAs prepared from the four min-tn5 tal / tkt-xylAxyl B transconjugates were digested with SphI, which cuts twice inside the cassette producing a fragment with Tal probe homology. The blots were then hybridized to either the Tal probe or the Tnp probe. The autoradiograph in Figure 4 illustrates that an exclusive band greater than 4kb (the size of Veno-talB / tktA), which is adjacent to Z. mobilis DNA, was detected in all genomic DNA preparations when hybridized to the. Tal probe. Three of the samples (nos. 22, 23 and 24 possibly sisters) also had a band in the plasmid fraction, suggesting that integration occurred in the native plasmid. It is clear that integration occurred in the Z genome.

mobilis for sample no. 21, and only one copy was inserted. Hybridization of genomic and plasmid DNA samples from these transconjugates with a Tnp probe (Figure 5) revealed an absence of homology between the sample DNA and the Tnp probe. These results suggested that xylose assimilation and the pentose phosphate pathway genes, together with the Tc 'gene, were transposed into the Z. mobilis genome, and that only a single insertion occurred in each of the transconjugates, and the insertions. they occurred in different places of the genome.

The activities of xylose isomerase, xylulokinase, transaldolase and transquetolase were measured as previously described (Zhang et ah, 1995) for Z mobilis transconjugates. As defined above, it was observed that the expression of a single Pgap-xylAxilB copy in the genome was about half that of the enzymes XI and XK produced for the plasmid-bearing strains. However, when enzymatic analysis was performed to confirm whether transaldolase (TAL) and transquetolase (TKT) had been expressed in the Zymomonas tal / tkt-xylAxilB-Tc and tal / tkt-xylAxilB transconjugates, as shown in Figure 6, that the enzyme levels of TAL for all transconjugates (nos. 4, 5, 17, 18, 21, 23 and 24) was about 50-70% of that of their equivalent plasmids. About 30-70% of TKT activities of strains bearing piasmid were observed in the members. Although the members of the piasmid exhibit significantly elevated TAL and TKT activities, both TAL and TKT activity, expressed from a single copy of the genes in the Zymomonas genome, were considerably higher than expected when compared to those of 10 copies per found in a piasmid carrier strain.

All four members of tal / tkt-xylAxilB Zymomonas (nos. 21,22, 5 and 1 I) were able to develop into xylose. Preliminary fermentation studies for these members were then performed using a 2% xylose substrate at a temperature of 30 ° C. The fermentation profiles for these members are shown in Figure 7. In this Figure, the development rates. sugar elitilization of member no. 21 of Z. mobtlis were slower than those of the piasmid carrier strain 39676 / pZB4. However, most members of Z. mobilis (nos. 22, 5 and 1 1 have read comparable to strains carrying piasmid.

All members produced ethanol from xylose, with 92% of the maximum theoretical product yield. The stability of these genomic integrated strains and three strains carrying piasmid was measured in a non-selective medium. All strains were grown in RMG medium, and serially transferred daily for about 10 generations. Every 40 generations, cells were used to inoculate a RM medium vial containing 1% glucose and 5% xylose to measure their ability to ferment xylose in ethanol. Ethanol processing yields and xylose utilization rates were used as a stability reference. Two of the genomic integrated strains demonstrated stability for more than 90 (C25 and D95) generations, whereas strains carrying plasmids (206 / pZB4, 206 / pZB4L and BC 1 pZB4L) were stable for about 40 generations. See Figure 8. The fermentation performance of C25 and D95 strains were analyzed in an RM medium containing 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. As shown in the Figure, strain C25 demonstrated much better xylose utilization and ethanol processing yields at both pH 5 and pH 5.5 in an RM containing 4.5% glucose and 4.5% xylose. that D95. Consequently, in the following examples the three arabinose assimilating genes (araBAD) were integrated into the C25 genome. EXAMPLE III (AX) The following example demonstrates the introduction of arabinose assimilation enzymes into the C25 genome by homologous recombination via Idh and transposition using Tn10 mini-transposon. Plasmid pZB 1862-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. The Tc1 colonies were further confirmed to be Ara + Xii + by development on MRI supplemented with xylose or arabinose (RMS and RMA). Plasmid Tn / OG, also described below, was transferred from an E. coli 3Μ10λ donor to Z. mobilis C25 by conjugation to a filter mating technique (Conway et al., 1987). The resulting transconjugates were selected on mating plates containing arabinose. A. Construction of pZB1862-IdhL-ara and C25 integration using homologous recombination.

Earlier attempts to integrate araBAD into the Zymomonas genome using an! -Kb Idh fragment as the homologous region were unsuccessful.

In order to increase the recombination frequency, a larger homology region was used. A 2.5-kb DNA fragment including Idh and the flank region was amplified using Pfu PCR and Z. mobilis ATCC 39676 DNA, see U.S. Patent No. 5,726,053 as a template. Primers were designated based on the Z. mobilis CP4 DNA sequence, published in Yamano I (1993) Journal of Bacteriology, Vol. 175, 3926-3933. Although a 2.5-kb fragment was expected from PCR, according to the published sequence, a 3,4-kb fragment was obtained. After digestion of a 3,4-kb fragment with BamHl, two fragments (2.5 and 0.9 kb) were obtained. Both fragments were tested by PCR using primers designed to ring only with Idh. The 2.5-kb fragment produced a PCR product of the correct size, whereas the 0.9-kb fragment did not, indicating that the first one contained the klh sequence.

Therefore, the 2.5-kb BamR1 fragment (called ldhL) was cloned and used as the homologous region for gene integration in C25. The ldhL fragment and digoxigenin-labeled (DIG) -idh, ara and Tnps probes were amplified by PCR using Pfu (Stratagene, La Jolla, CA) or Taq DNA polymerase (Qiagen, Valencia, CA). DIG-UTP was purchased from Boehringer Mannheim, Indianapolis, IN. Probe I was used as a probe for IS / Or, the Tn 10 transposase gene. The PCR products for ldhL, tdh, ara, and tnp were 2.5, 1, 1.4, and 0.8 kb, respectively.

The following primer sequences were used: 1dhL: 5'-TCGCGGATCÇTCT ATCCCTTTATTTTCT ATCCCC ATC ACCTCGG-3 'SEQ ID NO. 9. 5'-TCGCGGATÇGCGGCTG AC AT AC ATCTTGCG AAT ATAGGG-3 'SEQ ID NO. 10. DIG-1dh: 5'-TCGCGGATCCGTCTATGCGCGTCGCAATATTCAGTTCC-3 'SEQ ID NO. 11. 5'-TCGCGG ATCCGTCGCTTGTCT ATT A TO AC AGCGC AT CCGGC-3 'SEQ ID NO. 12. DIG-ara: 5'-CTAACATGTTGACTCCTTCTCTAGACTTAGCG-3 'SEQ ID NO. 13. 5'-GTTGAAACCGCTGGGCACCACGC-3 'SEQ ID NO. 14. DIG-tnp: 5'-CGCACT AC AC GGTCGTTCT GTT AC-3 'SEQ ID NO. 15. 5'-GGTTGCAGCCACGAGTAAGTCTTC-3 'SEQ ID NO. 16

For cloning purposes, a Not I site was introduced into ldhL by insertion of a 5'-CATGCGCGGCCGCC-3 'oligonucleotide at the NcoI site, which is located in the middle of the Idh gene. The new Not \ site was approximately 1.4 and 1.1 kb from either end of the HDL. A 1dHL (2.5 kb) BamHI fragment containing the Notl site was ligated to pZBI862 at a Bell site. Finally, a 4,4-kb Pgap-araBAD isolated from pZB206 (U.S. Patent Nos. 5,712,133 and 5,726,053) was cloned into the IclhL Notl site to form the integrating plasmid, pZB 1862-ldhL-ara. See Figure 10. The Pgap-araBAD operon containing the three arabinose assimilating genes was integrated into the idh site in the C25 genome by homologous recombination. To integrate the amBAD genes into the C25 genome, pZB 1862-ldhL-ara was constructed in E. coli DH5a. Plasmid pZB 1862-ldhL-ara was transferred to C25 by electroporation. Tc resistant transformants were selected and tested for arabinose development. During propagation of transformants, Pgap-ara-BAD could be integrated into the C25 genome by replacing ldhL with the \ u \ ciraBAD-ldhL (plasmid) cassette by homologous recombination.

To enrich and isolate the members, the plasmid was cured for the transformants. Plasmid pZB 1862-ldhL-ara will replicate in Z. mobilis. However, Z. mobilis tends to lose foreign plasmids under suboptimal development conditions (for example at a temperature of 37 ° C). Using this feature, the cure of pZB 1862-ldhL-ara was accomplished by subculturing C25 transformants at a temperature of 37 ° C in the absence of Tc for various transfers. Cultures from each transfer were constantly monitored for plasmid loss. By the third transfer, 100% of the cells became Tcs, indicating a plasmid loss. 3rd, 4th, 5th, and 6th-transfer cultures were inoculated into MRI containing arabinose (RMA) at a temperature of 30 ° C to enrich the development of potential Pgap-araBAD integrants. The enriched cells were transferred to RMG plates and replicated on RMA, RMX, and RMGTc plates. Several members (AX) with the Xil + Ara + Tcs phenotype were submitted for further analysis as described below. These members were able to use xylose or arabinose as the sole carbon source. B. Tn / 0G Construction and C25 Conjugation, The Mini-Tnó was used to construct C25 with Peno-tcd / tkt and Pgap-xilAB operons. Although the transposase gene does not exist in C25, the minimum Tn / 0 was used for subsequent integration of Pgap-araBAD to avoid any possible incompatibility between the same transposons. The Tn / OG plasmid (Figure 11) was constructed based on the Tn 10 based distribution plasmid, pLOFKm. The Km "gene was replaced by a Pgap-araBAD Notl fragment isolated from pZB206. Tn 10G was constructed and maintained in E. coli Cl 18. The plasmid was then transferred to the mating donor, E. coli SMKW. Since Tn / riG is a suicide plasmid in Z. mobilis, only araBAD-integrating transconjugates were able to grow on mating plates supplemented with arabinose Donor E. coli SMlOZ / ur was inhibited in the plates by the presence of naidic acid Transconjugates appeared in the mating / ara plates within 7 days The colonies were replicated on RMA and RMX to confirm their phenotypes Eighty-six percent of colonies taken were Xil + Ara + Twenty colonies of the taking plates were cross-transferred to different plates (from xylose plates to arabinose plates or vice versa) Sixty percent of these colonies remained Xil + Ara +.

Twenty colonies were analyzed on preliminary Southern hybridization (data not shown).

Using the Tnp probe, about 50% of the strains contained the transposase gene in the genome. Eight members were then subjected to further analysis by Southern hybridization. The integration of P gap-araBAD in C25 Idh was confirmed by Southern hybridization to the DNA of pZB 1862-ldhL-ara integrants using the DIG ara and Idh labeled probes. See Figure 12 (a) and (b). there is only one Pstl site on pZB 1862-ldhL-ara and it is located in Pgap-araBAD. Therefore a hybridization band (12.9 kb) of the Pst1 digested plasmid was expected using the ara probe. With Pgap-arciBAD integrated into the genome, two bands generated by the Pstl site in Pgap-araBAD and the adjunct Pstl sites in the genome outside Pgap-araBAD were expected. The results from Figure 13 (a) clearly showed that two bands form the total DNA preparation hybridized with the ara probe and demonstrated Pgap-araBAD integration. The absence of member plasmid DNA hybridization bands indicated that integration occurred in the genome rather than in native plasmids. To demonstrate that Idh was disrupted by Pgap-araBAD integration, the same DNA was transferred and hybridized to the Idh probe. As expected, the hybridization patterns of the members were exactly the same in both blots except for C25, as shown in Figure 12 (b). The total DNA of the host strain, C25, used for Pgap-araBAD integration, which has an intact Idh, demonstrated only one band. The results confirmed that ciraBAD had integrated with the C25 Idh.

Figures 13 (a) and (b) illustrate Southern hybridization results using DIG-labeled Tnp and ara probes, respectively, for the eight integrating Pgap-araBAD transposons generated by the posiçãoη / (λ transposition. DNA was digested with an enzyme PstI restriction The PstI cuts Tn / OG into two fragments (7.7 and 5.9 kb) and only the 5.9-kb fragment carries the transposase gene (IS / Or). According to the blots, only G8, G11, G15, and GH17 were both positive and tap-negative, and different band patterns indicated that Pgap-araBAD was integrated at different sites in the genome. transposase gene remained in the integrant genome, four strains (G5, G6, G14 and G19) of eight contained the transposase gene, and G14 and G19 contained the plasmid transposase gene. two bands of the members were expected, but only simple bands were observed. To address this ambiguity, a PCR was performed for the members using ara initializers and it was confirmed that all eight members contained Pgap-araBAD in the genome (data not shown).

Xylose isomerase (XI), xylulokinase (XK), L-arabinose isomerase (L-AI), L-ribulokinase (L-RK), L-ribulose-5-P-4-epimerase (L-Repi), transquetolase (TKT) and transaldolase (TAL) using cell-free extracts of the 2. mobilis members and control strains, according to Zhang, et al, 1995; and Deanda et al, 1996, with slight modifications. Cell-free extracts were prepared by harvesting late log cultures (30 ° C, approximately 1.2 OD), once washing with sonication buffer (10 mM Tris-HCl, pH 7.6 10 mM MgCL) and sonication. Cell debris was removed by centrifugation (14,000 rpm for 45 min at 4 ° C). In the L-AI assay, the timed sample volumes were reduced by half (50μΙ), 70% H2SO4 (1.5 mL) and 0.12% carbazole (50μΙ). All tubes were maintained in a 25 ° C water bath both before and after the addition of 70% H2SO4 until absorbance reading. Samples were taken at 0, 5, 10 and 20 min during the reaction.

Although the integrants of both homologous recombination and transposon integration were able to develop into D-xylose and L-arabinose, the expression level of the integrated genes was determined by measuring enzymatic activity. C25 / AXI, C25 / AX10I, and C25 / G8 isolates were chosen for the enzyme assays because they were the most stable integrants as determined in the stability studies described below. The results of the enzymatic assays are summarized in Figures 14 and 15. For all assays (except for xylulokinase), the members had positive activities compared to controls (C25 and / or 206C). It is believed at the present time that the low activity of XK for members may be due to an experimental error, the nature of the assay or even both. In most assays (excluding L-ribulokinase and xylose isomerase), the members had lower activities than the plasmid carrier strain (206C / pZB301). This is presumably related to the copy number of the genes.

For stability studies, cultures were inoculated into RMG-containing test tubes, incubated overnight at 30 ° C, and transferred daily to RMG tubes. The inoculum was controlled to allow transfer every 10 generations. Every 40 generations, cells were used to inoculate flasks containing a sugar mixture to test fermentation capacities on uncontrolled sugars at a temperature of 30 ° C. Batch fermentation studies were performed at a temperature of 30 ° C. C with pH control on Bio-StatQ chemostats, a registered trademark of B, Braun, Allentown, PA., Using 500 ml as the workload. The pH was automatically controlled with 2N KOH. initial sugar concentration and pH varied between lots depending on the growing conditions. All sugars used were of the reagent standard. Samples were taken periodically by fermentation, and analyzed for sugars, ethanol, and HPLC by-products as previously described (Zhang 1995). Optical density at 600 nm (OD600) was measured to monitor cell development. Ethanol yield was based on the amount of total sugar available.

Several strains of genome-integrated xylose and arabinose fermenter Z. mobtlis developed through both homologous recombination and transposition were studied for their stability in a non-selective medium (RMG). These strains were grown in a RMG medium and serially transferred daily after about 10 generations. After every 40 generations, cells were used to inoculate a flask containing 1% glucose and 2% xylose and 2% arabinose to examine their ability to ferment xylose and arabinose in ethanol. Ethanol processing yields and xylose and arabinose utilization rates were used as the stability characteristic. Two of the isolates remained stable for 160 generations. See Figures 16 and 17. Three integrated strains and plasmid-bearing strains were further tested for fermentation performance, on an average containing a mixture of 4% glucose, 4% xylose, and 2% arabinose at a pH of 5. , 5 and temperature of 30 ° C. As shown in Figure 18, all three strains used glucose, xylose and arabinose within 72 hours, while the plasmid-bearing strains still had 6 g / L residual arabinose. However, the integrated strains produced more xylitol (4 g / l) than the plasmid carrier strain (1 g / l). the two homologous recombination strains AX1 and AX101 did not produce lactate because the lactated dehydrogenase gene was deactivated by gene integration. The process yield (about 83% of theory) of the integrated strains was very similar to that of the plasmid carrier strain. Moreover, the integrated strains developed to higher cell densities, which is probably due to the lower metabolic load associated with the presentation of only a single copy of the seven genes.

While the present invention has been described in connection with the illustrated embodiments, it will be appreciated and understood that modifications may be made without departing from the true spirit and scope of the present invention.

SEQUENCE LIST

<110> MIDWEST RESEARCH INSTITUTE

<I20> ZYMOMONAS MOBILIS STABLES FIXED XYLOSIS AND

ARABINOSIS <I30> PCT / 00-14 <I60> 16 <I70> Patentln See, 2.1 <210> 1 <2II> 18 <212> DNA <213> Artificial Sequence <220>

<223> Artificial Sequence Description: Tc primer for DNA labeling <400> 1 ttgctaacgc agtcaggc 18 <210> 2 <21l> 18 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Tc primer for DNA labeling <400> 2 gaatccgtta gcgaggtg 18 <2I0> 3 <211> 20 <2I2> DNA <213> Artificial Sequence <220>

<223> Artificial Sequence Description: xilB primer used for DNA labeling <400> 3 tatgggttca gcggcatgag 20 <210> 4 <211> 21 <2I2> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: xilB primer used for DNA labeling <400> 4 atgggcatga gatccatagc c 21 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Tnp primer for DNA labeling <400> 5 tcctaacatg gtaacgttc 19 <210> 6 <211> 18 <2I2> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Tnp primer for DNA labeling <400> 6 ccaaccttac cagagggc 18 ------- <210> 7 <211> 48 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Primer Such for DNA labeling <400> 7 cgtctaaaag attttaagaa aggtttcgat atgacggaca aattgacc 48 <210> 8 <211> 49 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Primer Such for DNA labeling <400> 8 cattttgact ccagatctag attacagcag atcgccgatc attttttcc 49 <210> 9 <211> 45 <2I2> DNA <213> Artificial Sequence <220> <223> Sequence Description Artificial: Initiator for ldhL gene fragment amplification using PCR <400> 9 tcgcggatcc tctatccctt tatttttcta tccccatcac ctcgg 45 <2I0> 10 <21l> 40 <212> DNA ---------------- ------------------ <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer for ldhL gene fragment amplification using PCR <400> 10 tcgcggatcc gcggctgaca tacatcttgc gaatataggg 40 <210> 11 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer for DIG-ldh gene fragment amplification using PCR <400> 11 tcgcggatcc gtctatgcgc gtcgcaat gtcgcaat tcagttcc 38 <210> 12 <211> 4I

Claims (10)

1. Transposon for stable insertion of foreign genes into a bacterial genome, characterized in that it comprises mini-Tn5-Tcital / tkt-xylA xilB. 2. Transposon for stable insertion of foreign genes into a bacterial genome, characterized in that it comprises mini Tx \ 5-tal / tkt-xylA xilB, ATCC Accession No. PTA-1795. Plasmid vector comprising transposon as defined in claim 1 or 2. Plasmid vector comprising the mini-Tn5 transposon as defined in claim 1 and the mini-Tn5 transposon as defined in claim 2. 5. Plasmid vector, characterized in that it is vector pZB 1862-ldhL-ara, ATCC Accession No. PTA-1798. Zymomonas mobilis bacterium, characterized in that it is transformed with the plasmid vector as defined in any one of claims 3 to 5, wherein the expression of a plasmid product is stable for at least 45 generations. A bacterium according to claim 6, characterized in that it is Z. mobilis strain G8, ATCC Accession No. PTA-1796. A bacterium according to claim 6, characterized in that it is Z. mobilis strain C25, ATCC Accession No. PTA-1799. A bacterium according to claim 6, characterized in that it is Z. mobilis strain AX, ATCC Accession No. PTA-1797. A reaction mixture comprising a fermentable medium, characterized in that the medium includes xylose or arabinose, and a bacterium as defined in any one of claims 6 to 9.