AU2001251397A1 - Method of site-specific insertion in zymomonas mobilis - Google Patents

Description

Method of Site-Specific Insertion in Zymomonas mobilis

Contractual Origin of the Invention:

The United States Government has rights in this invention pursuant to Contract No. DE- AC36-99GO 10337 between the United States Department of Energy and the Midwest Research Institute. Field Technical

The present invention relates to gene insertion in Zymomonas mobilis, and in particular to the insertion inactivation of specific gene products in recombinant Zymomonas mobilis strains which ferment xylose and arabinose, or both, into ethanol. Background Art 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. To overcome this disadvantage, 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 are based on the use of multicopy plasmids capable of expression of the desired enzyme product.

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. The Patent claims an integrated strain. US Pat. Nos. 5,712,133 and 5,726,053 disclose, inter alia, Z. mobilis arabinose fermenting transformants (CP4/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 transfoπnants for the fermentation of a glucose and arabinose containing substrate is also disclosed. The Patent claims integration of the exogenous genes into the host genome.

US Pat. No. 5,843,760 discloses a Z mobilis xylose and arabinose fermenting transformant (206C/pZB301) 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. The process of using the transformants together with the plasmid vectors (pZB301 , pZB401, pZB402, and pZB 403) is also disclosed. This Patent claims integration of the exogenous genes into the host genome.

Vital to the engineering of an economic fermentation process for the production of fuels and chemicals, from cellulose containing substrates, is to achieve a high rate of specific product formation and conversion efficiency. When using the pentose-fermenting recombinant strains of Z mobilis, where ethanol is the specific product, the formation of the by-products lactic acid, an end product of energy formation, and xylitol lowers conversion efficiency. Thus, it is desirable to develop new metabolic engineering methods for the site-specific insertion of genes in Z mobilis, such as for the insertion inactivation of lactate dehydrogenase, to enhance the conversion efficiency in a cellulose to ethanol process. In Esherichia coli, the classical method for generating chromosomal inserts of foreign genes involves the use of specialized λ phage cloning vectors that can exist stable in the lysogenic state. Alternatively, site specific genes can be inserted though homologous recombination, when bracketed with E. coli chromosomal sequences, or by random transposition if the genes can be cloned in the permissive sites of a transposon. While 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 (Tn or mini Mμ transposition of auxotrophy or antibiotic resistance for genetic analysis in Z mobilis) transposition is random and homologous recombination in Z mobilis has not been demonstrated. Further, no bacteriophage has ever been isolated from Zymomonas. In view of the foregoing, a need exists for a method of site specific insertion in Z mobilis. One practical application of the method would be for the elimination in the formation of by-products, in a Z mobilis fermentation, through the construction of stable recombinant strains, characterized by the insertion inactivation of genes encoding those enzymes in the specific by-product formation pathway, to be eliminated. Disclosure of Invention

It is therefore an object of the present invention to provide a method for site-specific insertion in Z. mobilis.

It is a further object of the present invention to provide a method of eliminating the formation of by-products in a Z mobilis fermentation through the construction of stable recombinant strains, characterized by insertion inactivation of genes encoding those enzymes in the specific by-product formation pathway, to be eliminated.

It is a further object of the invention to provide a method of insertion inactivation of the lactate dehydrogenase gene in Z mobilis to eliminate the formation of the by-product lactic acid.

To overcome the problems associated with the related art and in accordance with the purpose of the invention, as embodied and broadly described herein, the present invention briefly provides a method of site-specific insertion in Zymomonas, comprising, providing a Zymomonas gene fragment, interrupting a DNA sequence the fragment, and transforming the Zymomonas through homologous recombination with the interrupted fragment.

Additional advantages of the present invention will be set forth in part in the description that follows and in part will be obvious for that description or can be learned from practice of the invention. The advantages of the invention can be realized and obtained by the method particularly pointed out in the appended claims. Brief Description of Drawings

The accompanying drawings, which are incorporated in and which constitute a part of the specification, illustrate at least one embodiment of the invention and, together with the description, explain the principles of the invention. Figure 1 is a map of the shuttle vector pZB 101 and a chart showing the method of using the vector pZB 101 according to the present invention.

Figure 2 shows the Southern Analysis of the plasmids pZB102 and pZB121 using the Tc probe. Figure 3 is a map of the suicide vector pZB121 together with a chart showing the method of using the vector according to the present invention.

Figure 4 shows the production of lactic acid in fermentations using strains 2-1 ,2-2 and #30, created according to the method of the invention, compared to the wild-type strain 39676. The graph shows that lactic acid production was eliminated using homologous recombination in the Idh gene.

Figure 5 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 pZB1862 of Z mobilis. Plasmid pZB1862 is a derivative of pZB186, a plasmid disclosed in US Pat. Nos. 5,514,583 and 5,712,133, incorporated herein by reference. Figure 6 shows the Southern analysis of the chromosomal integrated xylose/arabinose- fermenting Z mobilis strains from homologous recombination using DIG-αrα and DIG-/Jb probes. AX1, 13, 23, 101 , 104, and 107 are araBAD integrants. C25 is the host control pZB1862-/JbL- r is the plasmid control isolated form DH5α. λ/H is a molecular weight marker: 23, 9.4, 6.6 4.3, 2.3 and 2.0 kb. Figure 7 represents bar graph results of the enzymatic activities of L-arabinose isomerase, L-ribulokinase and L-ribulose-5-phosphate-4 epimerase of chromosomal integrated strains. 206C/pZB301 is a plasmid control. 206C is a host control. C25 is the xylose- fermenting integrant. AX1 and AX101 are the lactate dehydrogenase inactivated xylose/arabinose-fermenting integrants from homologous recombination. Figure 8 represents a bar graph result of the ethanol process yields of the chromosomal integrated xylose and arabinose-fermenting Zymomonas strains on RMGXA (1 :2:2%) at T=30° C, without pH control. These strains were inoculated form cultures at various generations on non-selective media.

Figure 9 represents a bar graph results of the xylose and arabinose utilization of the chromosomal 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 carious generations on non-selective media.

Figure 10 is a line graphic representation of the fermentation performance of the chromosomal integrated xylose and arabinose-fermenting Zymomonas strains in RM containing 4% glucose, 4% xylose and 2% arabinose at pH 5.5 and 30° C.

Detailed Description of Drawings

Unless 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 this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All U.S. patents are incorporated by reference as though fully set forth herein.

Reference now will be made in detail to the presently preferred embodiments of the invention, examples of which are illustrated in the accompanying drawings. For the examples described below, "plasmid-bearing strains" refers or relates to those strains and vectors described in the US Patents identified in the Description of the Related Art. "Z mobilis genome, or genomic" means the genes which, in toto, specify all the expressed and potentially expressible with a given Z. mobilis, including native plasmid and chromosomal. Examples The following examples illustrate a homologous recombination method for site specific insertion in Z. mobilis and the practical use of the method for insertion inactivation of an unwanted gene, such as a gene associated with a by-product formation. In the following examples, The putative lactate dehydrogenase (Idh) gene was the target region for illustration of the principles of the present invention. Idh converts pyruvate to lactic acid, a by-product in Zymomonas an ethanol product specific fermentation. Integration of the pentose metabolism genes (Example 2) into the Z mobilis Idh genome using homologous recombination stabilizes the phenotypic response without the use of antibiotics, or other selective pressure.

E. coli DH5 was used as a host for the construction of the plasmids. Strains of Z mobilis ATCC 39676 and its derivative 206C (US Pat. No. 5,843,760) were used as recipients in the construction of C25. For Example 2, the construction of strain C25 is disclosed in the co- pending U.S. Patent Application Serial No.09/565,233, filed May 1 , 2000. 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 KH₂P0₄) supplemented with 20g/L glucose, D- xylose or L-arabinose, unless otherwise specified. All strains containing plasmids were grown in the presence of tetracycline (Tc) ((10μg/ml in liquid Z mobilis, and E. coli; 20 μg/ml in agar for Z mobilis and 15 μg/ml in agar, or ampicillin (Ap), 100 μg/ml for E. coli)).

Chloramphenicol was used at 50 μg/ml, for both liquid and solid media, for Ε. coil, and at a concentration of 100 μg/ml, for both liquid and solid media, for Z. mobilis.

For regeneration and selection of Z. mobilis transformants, mating plates ((10 g/L yeast extract, 5 g/L tryptone, 2.5 g/L (NH₄)₂S0₄, 0.2 g/L K₂HP0₄ and 50 g/L sugar)) supplemented with tetracycline or nalidixic acid (20 μg/ml) were used. All agar plates were made with 15 g/L agar.

Plasmid DNA isolation, restriction endonuclease digestion, ligation and transformation, agarose electrophoresis and other recombinant DNA techniques were carried out in accordance with published protocols, Sambrook et al., (1989) Molecular cloning: a laboratory manual, Cold Spring Harbor laboratory press, Cold Spring Harbor, NY, or the respective reagent manufacture's instructions, were specified, and are well known in the art. Genomic DNA of Z. mobilis was extracted using three-milliliters of overnight cells resuspended in 250 ml of 50 mM Tris-50 mM ΕDTA buffer. The cells were treated with lysozyme at 37^° C for 30 min 100 μl of 5% SDS solution and RNAase (final concentration equal to 20 ng/ml) were then added and incubated for an additional 30 min. A phenol/chloroform extraction was performed twice, to remove the proteins. Genomic DNA was recovered by ethanol precipitation. Example 1

The following example demonstrates gene insertion and inactivation into the Zymomonas genome via a homologous recombination This system is based on t e(ldh gene of Zymomonas having a tetracycline (Tc) resistant gene insert (Idhy.Tc). Plasmids containing the

Idhr.Tc cassette were used to transform Z mobilis, and the resultant Tc resistance transformants were analyzed by Southern hybridization. The results showed that the Idhr.Tc cassette had been inserted into the Idh region of the Zymomonas genome. Thus, the invention of gene integration based on homologous recombination, in Zymomonas, together with targeted integration resulting in an inactivated Idh gene, eliminated lactic acid by-product formation in an ethanol fermentation. A 1-kb Idh fragment was made by PCR using total DNA of Z mobilis ATCC39676, as a template. PCR was performed using the PCR kit from Perkin Elmer, and is well known. The primers were designed based on a DNA sequence of Z mobilis CP4, as published in Yamano I., (1993) Journal of Bacteriology, Vol. 175, 3926-3933. The primers for Idh were: 5'-TCGCGGATCCGTCTATGCGCGTCGCAATATTCAGTTCC-3'

5'-TCGCGGATCCGTCGCTTGTCTATTAAACAAGCGCATCCGGC-3' in which a BamHl site (underlined) was incorporated at the 5 '-ends of each primer. The PCR product (Idh) was approximately 995 bp in length covering the structural gene of Idh. The PCR product was digested using BamHl and ligated with a pUC19, made linear by digestion with BamHl and dephosphorylated by treatment with calf intestinal phosphatase. The ligated DNA was used to transform E. coli DH5α and restriction analyses of the plasmid DNA from ampicillin-resistant transformants confirmed the presence of the expected plasmid, which is designated as pUC19-LDH. A Tcr gene was then inserted in the Ncol site of Idh in pUC19- LDH. The primers used for Tc^r gene were: 5'-CTAGGCGTATCACGAGGCCCTTT-3'

5'-CTAGGCGGACGCGATGGATATGT-3'

The template DΝA used for synthesizing Tc^r was pBR332. The PCR product of the Tc^r gene was approximately 1400 bp. The PCR product of the Tc^r gene and pUC19-LDH were made linear by digestion with Ncol were both subjected to a treatment with Klenow fragment and ligated, The ligation mixture was transformed to E. coil DH5α and restriction analyses of the plasmid DΝA from ampicillin and tetracycline-resistant transformants confirmed the presence of the expected plasmid, designated as pUC 10-KDH-Tc^r.

A. Idh inactivation using a replicative (shuttle) vector.

The Tc^r gene, bracketed by the Idh sequences, was then cloned into are plicative vector pZB1861 (Cm^r a pZB186 derivative). pZB1861 was constructed by digesting pZB186 (U.S.

Patent No. 5, 514,583) using Cla I and BssH II to remove the Tc^r gene and ligated with a adapter containing a BamH l site. The sequence of the adapter is:

5 '-CGATGATATCGGATCCG-3 '

3 '-TACTATAGCCTAGGCGCGC-5 ' The ligation mixture was transformed to E. Coli DH5α and the Cm' Tc^s transformants were selected. DNA prepared from the transformants was analyzed and the plasmid, designated pZB1861, lacking the Tcr gene and having a new BamHl site was obtained. The Idh-Tc fragment was excised as a BamHl fragment from pUC 19-ldh-Tc¹, ligated to pZBl 861 linearized by digestion with BamHl, and dephosphorylated by treatment with calf intestinal phosphatase. The ligated DNA was used to transform E. coli DH5α and restriction analyses of the plasmid DNA from chloramphenicol and tetracycline-resistant transformants confirmed the presence of the plasmid, designated as pZBlOl (or pZB1861-/Jb-Tc). The resulting plasmid, pZBlOl (CπvTc') (Figure 1) was transformed into Z mobilis 39676. To cure the plasmid, Tcr transformants were inoculated in RMG-Tc tubes (30^° C) followed by a transfer in RMG tubes (37° C), where the integrates can be selected at elevated temperature which promotes the loss of the plasmid. After approximately 10 transfers (90-100 generations), the cultures were transferred into RMG-Tcl O (30° C) to enrich the growth of potential Tc' integrants. The cells were diluted and plated on RMG-Tc20 plates. Tc' colonies were picked on both RMG-Tc20 and RMG-CmlOO plates. Several Tc' Cm^s colonies were analyzed for integration of the Tc' marker by Southern hybridization of the total and plasmid DNA. Probes including DIG-Tc' and DIG- Idh were used (the probes are shown in Example 2 below). The results showed that all the Tc'

Cms colonies contained the Tc' marker integrated in Idh gene in the chromosome. See, Figure 2. B. Idh inactivation using a non-replicative (suicide) vector. pZBlOl was digested using Ava I and BssHϊl to remove the Z mobilis origin of replication, from the plasmid, and then an intra-molecular ligation on the E. coli origin of replication containing backbone was made. In that procedure, the Ava I and BssH 11 fragment of pZBlOl, containing the E. coli origin of replication, was gel-purified and then filled in by a treatment using Klenow fragment. Intramolecular ligation then resulted in a suicide plasmid, designated as pZB121. See, Figure 3. Z mobilis 39676 was transformed by electroporation using either supercoiled pZB121 or pZB121 linearized by digestion using Ncol. Recombinant cells were selected from mating plates containing 20 μg/ml of Tc. The resulting colonies were replica picked onto RMG-Tc and RMG-Cm plates. Only one colony out of 250 analyzed was Tc' Cm^s. Southern hybridization confirmed a double-crossover event in isolate #30.

D- or L-lactic acid production was measured in ethanol fermentations using the above shuttle and suicide vector integrants. These tests were conducted in RM containing 10% glucose at 30° C, pΗ6.0, in 500-ml chemostats for Z. mobilis wild type strain 39676 and 4 of the integrants ((including 2-1 , 2-2 (from shuttle vector integration) and #30 (from suicidal vector integration)). Samples were taken periodically for HPLC analysis and Boehringer Mannheim Kit analysis (more completely described in Example 2 below). The results demonstrated that 39676 produced D-lactic acid and the production of this by-product was eliminated through integration of Tc' marker in the Idh gene in the integrants (Figure 4). In the foregoing example, construction of the Idh integrative cassette, by insertion of a

Tc resistance gene into a convenient Ncol site, is shown. However, it is within the scope of the invention to construct an Idh integrative cassette without a restriction site. In this manner the cassette could be made using either PCR fusing methodology, such as the PCR mediated overlap extension technique described in US Pat No. 5,514,583 to insert gene of interest into the Idh gene, or to create a deleted/inactivated gene by PCR. These products would then be used to obtain Z. mobilis strains having an inactivated Idh, and no lactic acid formation. Example 2

The following example demonstrates the introduction of the arabinose assimilation enzymes in the genome of C25 through homologous recombination via Idh resulting in the inactivation of the lactate dehydrogenase gene of the lactic acid by-product energy metabolism pathway.

Plasmid DNAs were transformed into either Z mobilis or E. coli cells by electroporation (Zhang et al., 1995)) Plasmid pZBl 862-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' colonies were further confirmed to be

Ara⁺Xyl⁺ by growth on RM supplemented with xylose or arabinose (RMS and RMA).

Previous attempts to integrate araBAD in the Zymomonas chromosome, using the above 1-kb Idh fragment as the homologous region, did not succeed. In order to increase the recombination frequency, a larger homology region was used. A 2.5.-kb DNA fragment, which includes Idh and the flanking region was amplified using Pfu PCR. The primers were designed based on the DNA sequence of Z mobilis CP4, published in Yomano et seq. Although a 2.5-kb fragment was expected from PCR, according to the published sequence, a 3.4-kb fragment was obtained instead. After digesting the 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 anneal to only the 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 former contained the Idh sequence. Therefore, the 2.5- kb BamHl fragment (designated ldhL) was cloned and used as the homologous region for gene integration into C25.

The ldhL fragment and digoxygenin (DIG)-labeled Idh and ara probes were amplified by PCR using either Pfu (Stratagene, La Jolla, CA) or Taq DNA polymerase (Qiagen, Valencia, CA). DIG-UTP was purchased from Boehringer Mannheim, Indianapolis, IN. PCR products for ldhL, Idh, and ara are 2.5, 1 , and 1.4 kb, respectively. The following primer sequences were used: ldhL: 5'-TCGCGGATCCTCTATCCCTTTATTTTTCTATCCCCATCACCTCGG-3' 5 '-TCGCGGATCCGCGGCTGACATACATCTTGCGAATATAGGG-3 ' DIG-ldh:5'-TCGCGGATCCGTCTATGCGCGTCGCAATATTCAGTTCC-3'

5'-TCGCGGATCCGTCGCTTGTCTATTAAACAAGCGCATCCGGC-3' DIG-ara: 5 ' -CTAAC ATGTTGACTCCTTCTCTAGACTT AGCG-3 '

5 '-GTTGAAACCGCTGGGCACCACGC-3 ' For cloning purposes, 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 pZB1862 at a BcR site. Finally, a 4.4-kb Ygap-araBAD, isolated from pZB206 (US Pat. Νos. 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 5.

The Ygap-araBAD operon, containing the three arabinose-assimilating genes, was integrated into the Idh site in the C25 chromosome through homologous recombination. To integrate the araBAD genes into the genome of C25, pZB1862-ldhL-ara was constructed in E. coli DH5α. The plasmid pZB1862-ldhL-ara was transferred into C25 by electroporation. The Tc resistant transformants were selected and tested for growth on arabinose. During propagation of the transfoπnants, Pgap-ara-BAD could be integrated in the chromosome of C25 by the replacement of ldhL with the ldhL' -araBAD-ldhL ' cassette (from the plasmid) through homologous recombination.

To enrich and isolate the integrants, plasmid curing was conducted for the transformants. Plasmid pZB 1862-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. Cultures from the 3^rd, 4^th, 5^th, and 6^th transfers were inoculated in RM containing arabinose (RMA), at 30° C, to enrich the growth of potential Ygap-araBAD integrants. The enriched cells were transfeπed to RMG plates and replica-picked onto RMA, RMX, and RMGTc plates. Several integrants (AX) with the phenotype of Xyl⁺Ara⁺Tc^s were subjected to further analysis, as described below. These integrants were able to use either xylose or arabinose as a sole carbon source. Integration of Ygap-araBAD in Idh of C25 was confirmed by Southern hybridization, for the integrants DNA using the DIG-labeled ara and Idh probes. See, Figure 6(a) and (b). There is only one Pstl site on pZB1862-ldhL-ara and it is located in Ygap-araBAD. Therefore, one hybridizaion band (12.9 kb) from the Pstl-digested plasmid was expected, using the ara probe. With Ygap-araBAD integrated in the genome two bands generated by the Pstl site in Ygap- araBAD and the adjunct Pstl sites on the chromosome located outside the Ygap-araBAD were expected. The results from Figure 7(a) clearly showed that two bands form the total DNA preparation hybridized with the ara probe and demonstrated integration of Ygap-araBAD. The lack of hybridization bands from plasmid DNA of integrants indicated that integration had occurred on the chromosome, rather than on native plasmids. To show that the Idh was disrupted by the Ygap-araBAD integration, the same DNA was transferred and hybridized with the Idh probe. As expected, the hybridization patterns for the integrants were exactly the same on both blots, except for C25, as shown in Figure 7(b). The total DNA from the host strain, C25, used for Ygap-araBAD integration, which has an intact Idh, showed only one band. The results confirmed that araBAD was integrated in Idh of C25. 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, OD₆₀₀ approximately 1.2), washing once with sonication buffer (10 mM Tris-HCl, pH 7.6 . 10 mM MgCl₂) 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 (50μl), 70% H₂S0₄ (1.5 ml) and 0.12% carbazole (50μl). All of the tubes were maintained in a 25° C water bath, both before and after the addition of 70% H₂S0₄, until reading the absorbency. The samples were taken at 0,5,10, and 20 min during the reaction. Although the integrants, from homologous recombination were able to grow on D-xylose and L-arabinose, the expression level of the integrated genes it was determined by measuring enzymatic activity. Isolates C25/AX1, C25/AX101, and C25/G8 were chosen for the enzymatic assays because they were the most stable integrants, as determined in the stability studies described below. The results of the enzymatic assays for L arabinose Isomerase,. L- Ribulokinase, -and L Ribulose-5-Phosphate-4-Epimerase are summarized in Figure 8 (other enzymes not shown). For all assays (with and exception of xylulokinase), integrants showed positive activities as compared to the controls (C25 and/or 206C). It is believed, at this moment, that the low activity of XK for the integrants, might be due to experimental eπor, the nature of the assay, or both. In most assays, (excluding L-ribulokinase and xylose isomerase), the integrants showed lower activities than the plasmid-bearing strain (206C/pZB301). This is presumable related to the copy number of the genes.

For stability studies, the cultures were inoculated into test tubes containing RMG, incubated overnight at 30° C, and transfeπed 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. Batch fermentation studies were performed at 30° C with pH control in Bio-StatQ chemostats, a trademark of B. Braun, Allentown, PA., using 500 ml as the working volume. The pH was automatically controlled with 2N KOH. Initial sugar concentration and pH varied between each batch, depending on the culture conditions. All the sugars used were reagent grade. Samples were taken periodically throughout the fermentation, and analyzed for sugars, ethanol and by-products with HPLC, as described previously (Zhang 1995). Optical density, at 600 ran (OD600), was measured in order to monitor cell growth. Ethanol yield was based on the amount of total available sugar.

Several chromosomal integrated xylose and arabinose fermenting Z. mobilis strains developed through both homologous recombination and transposition were studied for their stability in a non-selective medium (RMG). These strains were cultured in RMG medium and 13

serially transfeπed, daily, after about 10 generations. After every 40 generations, the cells were used to inoculate a flask containing 1% glucose and 2% xylose and 2% arabinose for examination of their ability to ferment xylose and arabinose to ethanol. Ethanol process yields, and xylose and arabinose utilization rates, were used as the stability trait. Two of the isolates remained stable for 160 generations. Tliree integrated strains and a plasmid-bearing strain were further tested for fermentation performance, in a media containing a mixture of 4% glucose, 4% xylose, and 2% arabinose at pH 5.5 and 30° C. As shown in Figure 10, all three strains utilized glucose, xylose and arabinose in 72 hours, while the plasmid-bearing strains still had 6 g/L residual arabinose. However, the integrated strains produced more xylitol (4 g/1) than the plasmid bearing strain (1 g/L). The two homologous recombination AXl and AXl 01 strains did not produce lactate because the lactate dehydrogenase gene was inactivated through the gene integration. The process yields (about 83% of theoretical) of the integrated strains were very similar to the plasmid bearing strain. Moreover, the integrated strains grew to a greater cell densities, which is probably due to a the lesser metabolic burden associated with having only singly 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 invention.

Claims (12)

Claims

1. A method of site-specific insertion in Zymomonas, comprising: (a) providing a Zymomonas DNA fragment; (b) interrupting a sequence in the DNA fragment; and

(c) transforming the Zymomonas through homologous recombination with the inteπupted fragment.

2. The method of claim 1 wherein the DNA fragment is encoding a structural protein in a metabolic pathway of the by-product to be eliminated.

3. The method of claim 1 wherein interrupting is by inserting a DNA sequence inside the

DNA fragment;

4. The method of claim 1 wherein interrupting is by deleting a DNA sequence inside the DNA fragment.

5. The method of claim 1 further comprising ligating the inteπupted DNA fragment with a plasmid vector; wherein transforming the Z mobilis organism is through homologous recombination with the inteπupted fragment of the plasmid vector.

6. The method of claim 2 wherein the gene fragment is Idh and the product to be eliminated is lactic acid.

7. The method of claim 3 wherein the insertion is ldhL.

8. The method of claim 3 wherein the insertion is a selection marker.

9. The method of claim 3 wherein the insertion is an operon encoding at least one structural gene selected from the group consisting of xylose isomerase, xylulokinase, L-arabinose isomerase, L-ribulokinase, L-ribulose-5-phosphate 4-epimerase, transaldolase or transketolase, and a promoter for expression of the structural gene in Z. mobilis.

10. The method of claim 5 wherein the plasmid is pZB 101 , pZB 102, or pZB 121.

1 1. The method of claim 5 further comprising curing the Z. mobilis of the plasmid.

12. The method of claim 1 1 wherein the plasmid is pZ l962-ldhL-ara.