METHOD TO CONTROL AND PRODUCE NOVEL BIOPOLYMERS

Abstract: A method for identifying, characterizing, utilizing and modifying a set of genes which interact to produce a specific polymer. The method is demonstrated for production of an exocellular polysaccharide produced by the bacteria Zoogloea ramigera. The isolated polysaccharide is useful as a viscosity modifier, oil field chemical, drag reducing agent, dispersant or flocculant. Modification of the isolated genes, for example, by insertion of a regulatable promoter, provides a means for alteration of the enzymes responsible for synthesis of the polysaccharide and its resulting structure.

Description: "METHOD TO CONTROL AND PRODUCE NOVEL BIOPOLYMERS" BACKGROUND OF THE INVENTION The present invention is in the field of biotechnology and in particular the area of genetic manipulation of production and structure of biopolymers. Biopolymers , especially polysaccharide polymers, produced in biological systems, have found applications in many industries, including the food, cosmetic, chemical, biomedical, waste treatment and oil industries. However, the potential for new biopolymers with unique properties is still enormous. Biotechnology can help develop this potential and substantially increase the applicability and usage of biologically synthesized polymers. Flocculation is an important commercial use of biopolymers. Flocculation involves polymeric substances of bacterial origin, particularly extracellular polysaccharides. Entanglement and adsorption of microorganisms by exocellular polysaccharide fibrils and zoogloeal matrices are major causes of flocculation in aerobic waste treatment facilities. Understanding the development of microbial floes and the structure-function relationships of the polysaccharides causing them will aid in the engineering of more efficient flocculants and floc-forming bacterial systems. Several types of floc-forming bacteria have been identified. The most efficient are the cellulose (or cellulose-li e) produci- bacteria such as certain species of Pseudomonas , Aerobacte , Agrobacterium, Azotobacter and Zooqloea. With these bacteria, flocculation appears to occur when cells become embedded in a network of polysaccharide fibrils. Other floc-forming bacteria produce capsular polysaccharides enclosing large packets of cells which lead to floe formation. An example of this phenomenon occurs with Zoogloea ramigera 115. Still others produce " water soluble ionic exopolysaccharides that cause flocculation in a manner analogous to synthetic polyelectrolyte flocc'-iants. That is, the bridging of cells by the adsorption of polymers to their surfaces. This adsorption is usually attributed to ionic carboxyl groups or, in the case of neutral polysaccharides, to non-ionic hydroxyl groups. 2. ramigera is a gram-negative, rod-shaped, floc-forming, single polar flagellated, obligate aerobe found in aerobic waste treatment facilities and natural aquatic habitats, capable of growing on a variety of carbon and nitrogen sources. Zooqloea is distinguished from other gram-negative pseudomonads by the production of several distinct exocellular polysaccharides. These vary according to strain and are thought to function to concentrate nutrients around the cell floes enabling them to grow in nutrient deficient environments. Heavy metal ions, including cobalt, copper, iron, nickel, cadmium, and uranium, are also adsorbed by this matrix in an amount up to 40% of their total cell floe weight. " " Z. rami era isolate 115, available from the American Type Culture Collection, Rockville, D, is a zoogloeal matrix forming strain which, when grown in a nitrogen limiting medium, converts 60% (w/w) of the available glucose substrate into a water soluble capsular branched heteropolysaccharide composed of glucose and galactose in a molar ratio of 2:1 and approximately 3% to 5% pyruvate. The negatively charged carboxyl groups of the pyruvate are thought to be primarily responsible for the biopoly er's high affinity for heavy metal ions. Due to the unique rheological and metal binding properties of the Zooqloea ramigera exocellular polysaccharide, it is desirable to have a method to isolate, characterize, express and modify the genes for the polysaccharide. It is also desirable to develop a method for isolation, characterization, expression and modification of genes for a variety of polysaccharides for application to other biological systems so that the genes can be arranged in a system for the production of enhanced or novel polymers with multiple uses. It is therefore an object of the present invention to provide a method for the isolation, characterization, production and modification of biopolymers, especially biopolysaccharides. It is a further object of the present invention to provide the genes or their resulting nucleotide sequences required for production of an exocellular polysaccharide produced by Zoogloea ramigera. „ It is another object of the present invention to provide a method and system for the design and synthesis of novel biopolymers. It is a' further object of the present invention to provide unique, well-defined polymers for specific applications. Summary of the Invention A method for identifying, characterizing, modifying and utilizing genes for biopolymers, particularly an exocellular polypjccharide produced by Zooqloea ramigera. The disclosed recombinant DNA technology to control and produce novel biopolymers is applicable to the bacterium Zooqloea ramigera. This technique is also applicable to other Gram-negative, exopolysaccharide producing bacteria. Several genes involved in exopolysaccharide production were isolated from Z. ramigera strains using the present invention, as follows. In the preferred technique, transposon insertion mutants, negative for exopolysaccharide production-, were isolated by screening for non-fluorescence on plates containing the dye Cellufluor. These mutants do not flocculate during growth, a phenomenon linked to the presence of extracellular polysaccharide in wild type Z^ ramigera strains. The exocellular polysaccharide normally produced by strain I-16-H is referred to as Zooglan I-16-M. Complementation of these mutations was achieved with a Z. rarigera I-16-.'i gene library constructed in the broad host range cosπid vector pLAFR3 and introduced into the 1-lfi-M mutant? by conjugation. Transformed colonies were identified as having restored Zooglan I-16-M production by fluorescence on Cellufluor and flocculation. Genes involved in exopolysaccharide synthesis in Z. ramigera strain 115 were isolated by introducing a similarly constructed 2^ ramigera 115/pLAFR3 gene library into the I—16—M exopolysaccharide negative mutants. In this case, transformants were screened for a morphology characteristic of strain 115 due to the presence of the exopolysaccharide, Zooglan 115. Several transformed Z ramigera I—16—l mutant colonies were found to possess this unique morphology and also to flocculate when grown in liquid media. The ability of the 115 plasmids to produce this effect in the I-16-M mutants provides evidence that these plasmids encode the entire exopolysaccharide biosynthetic pathway of strain 115 for Zooglan 115. In a second technique, a gene bank of 2_^ ramigera I-16-M DNA was made by ligating partially digested I-16-M DNA into the cosmid vector pHC79, packaging the recombinant molecule into lambda phage and transducing the phage into E_ ; _ coli. The transductants were then screened for polysaccharide production. Isolation of the polysaccharide genes from these Z ra icera strains and identification of their functions with -6- respect to thei- respective biosynthetic pathways enable development of strategies for the manipulation and control of the pathways at the genetic level. Strategies for controlling polymer production and structure include: placing the polysaccharide biosynthetic genes under the control of regulatable promoters; the introduction of these genes into new host strains to enable the development of more economic processes for polysaccharide production; mutagenesis of the genes to alter the enzyme activities and therefore polymer structure; and the conr ruction of novel pathways for polysaccharide synthesis by "mixing" genes from different strains of Z^ ramigera and other organisms. The system is useful in designing novel polymer structures for specific functional applications. A major application of the 'method of the present invention with respect to Z^ ramigera exopolysaccharides is to be able to control the time, rate and/or level of flocculation and chelation achieved with the polysaccharide. Brief Description of the Drawings Fig. 1 is a schematic of the cloning of 2_^ ramigera polysaccharide genes in non-polysaccharide producing Z^ ramigera. Fig. 2 is a schematic of the method for clone bank construction in p AFR3 of B. Staskawicz, U.C. Berkeley. Fig. 3 is an autoradiogram of 32 p-iabe ed Tn5 hybridized to a Southern blot of DNA from mutants T18, T25, T2 t T30, T48, and T ? cut with Bamril (lar.^s 2-7), Hind ll (lanes 8-13) and PstI (lanes 15-20). Lanes 1 and 14 contain size markers. Detailed Description of the Invention The present invention is the alteration of polymer structure and function through genetic manipulation as demonstrated by identifying, characterizing and altering the genes for polysaccharide synthesis by strains of Zooqloea raqinera . Strategies for genetic manipulations include both classical mutagenesis and recombinant DNA technology. Two methods were used to transfer l _ ramigera DNA into a second organism, either a second distinct strain of 2^ ramigera as determined from its DNA composition, or E^ coli. In an example of the first method, a gene bank of Z ramigera I-16-M was made by ligating partially digested I-16-M DNA into the cosmid vector pHC79. The recombinant molecules were then packaged _i_n vitro into lambda phage and transduced into E_^ coli . The transductants were plated on medium containing Cellufluor and screened for fluorescent colonies. Although exopolysaccharide producing colonies were present, the overall rate of success was not as high as in the second method. However, expression of the genes for the polysaccharide in E^ coli results in polysaccharide being produced and can produce morphological changes under optimal conditions. In theory this production can be enhanced by- genetic manipulation of the transduced recombinant molecules. SUBSTITUTESHEET In an example of the second, preferred method, the I-16-M DNA was introduced into Z_^ ramigera I-16-M which did not produce the exopolysaccharides. A new technique was developed for introducing the DNA into the host organism or a host-related organism for the cloning of the polysaccharide genes. In this technique, the genes for polysaccharide synthesis are ligated onto a plasmid which is then introduced into a _Z_^ ramigera non-producing strain (or related organism) , expressed and identified by a screening technique. The crucial part of this sche.ie is the introduction of the plasmid DNA into Z_^ ramigera. The strategy for cloning in ^_ ramigera involves the conjugal transfer of a broad host range cloning vector from E^ coli to Z. ramigera. The conjugation procedure is a triparental mating in which two E_^ coli donors and the Z_^ ramigera recipient participate. The broad host range vector includes the mobilization yenes and is contained in one of the E^ coli strains. The other E^ coli strain contains a "helper" plasmid which carries the transfer genes. When the three strains are mated, the broad host range cloning vector, if transferred to Z. ramigera, can be selected for by growth on appropriate medium. Once the conjugation had been successfully accomplished and the conditions optimized, a Z_^ ramigera gene library was constructed in the broad host range vector and the transfer procedure repeated into a polysaccharide non-producing strain or non-producing mutants of the original polysaccharide producing strain, with the transcoπjugants being screened for polysaccharide production on Cellufluor plates. Any candidates that have regained polysaccharide production will presumably contain a gene or genes responsible for production of the exopolysaccharide on the plasmid, which can then be studied and manipulated in a variety of ways. Several genes involved in production of Zooglan I-16-M were isolated from strain I-16-M. At least five different transposon insertion mutants, negative for exopolysaccharide production, were isolated by screening for non-fluorescence on plates containing the dye Cellufluor. These mutants do not flocculate during growth, a phenomenon linked to the presence of extracellular polysaccharide in wild type Z ramigera strains. Complementation of these mutations was achieved with a Z. ramigera I-16- gene library constructed in the broad host range cosmid vector pLAFR3 and introduced into the I-16-M mutants by conjugation. Transformed colonies identified as having restored exopolysaccharide production as indicated by fluorescence on Cellufluor and flocculation were identified for four of the five mutant strains. The plasmids having the genes for synthesis of Zooglan I-16- have been partially characterized. Genes involved in expolysaccharide synthesis in Z . ramigera strain 115 were isolated by introducing a similarly constructed Z^ ramigera 115/pLAFR3 gene library into the I-16-M SUBSTITUTE SHEET exopolysacchaπde negative mutants. In this case, transformants were screened for a morphology due to the production of Zooglan 115. Several transformed Z ramigera I-16- mutant colonies were found to possess the 115 morphology and to flocculate when grown in liquid media. The ability of the 115 plasmids to produce this effect in all five I-16- mutants provides evidence that these plasmids encode the entire exopolysaccharide biosynthetic pathway of strain 115 for Zooglan 115. Materials and Methods used in the Isolation, Characterization and Genetic Manipulation of the Exopolysaccharides Produced by Zooqloea ramigera strains. The Zoogloea ramigera strains were characterized by microscopy, morphological characterization and determination of the ability to yrow and produce polysaccharide on different mediums. Strains I-16-M, 106 and 115 all flocculate and produce polysaccharide. Strain 115 is the only strain of the three to have a discernable polysaccharide capsule layer surrounding the cell floes and has a very unique colonial morphology. A complex medium and a defined medium were selected that contain all the requirements for growth and polysaccharide production. Zoogloea ramigera strains I-16-M and 106 were provided by Dr. P.R. Dugan, Ohio State university, Columbus, Ohio. Z. ramigera 115 was obtained from the American Type Culture Collection (ATCC) , Rockville, Maryland. SUBSTITUTE SHEET " e following strains of E. coli and plasmids were used, TABLE I E. col i Strains and Plasmids Strain/Plasmid Genotype Source or Reference E. coli Strains HB101 F~, hsdS20(r" B m _ B ) , T. Maniatis et al. recAl3, ara-14, Molecular Cloning proA2, lacYl, galK2, A Laboratory rpsL20, (Sm^) χyl-5, Manual, p. 504 mtl-1, supE44, (Cold Spring lambda" Harbor Laboratory, Cold Spring Harbor, Nϊ, 1982) BHB2688 (N205 recA" [lambda Hohn and Murray, imm434 f cits, b2,red~, Proc. Natl . Acad. Earn, Sam/lambda] ) Sci. 74, 3259- 3263 (1977) BHB2690 (N205 recA-[lambda Hohn and Murray, lmm 434 , cits, b2, red" 1977 Dam, Sam/lambda) ) MM294A pro-82 thi-1 Leigh, J.A. , . endAl hsdR!7 supE44 Signer, E.R. and Walker, G.C. Proc. Natl. Acad. Sci . USA 82, 6231-6235 (1985) DH5 p-, end Al , hsdRl7 Bethesda Research (r~ , m + k) , supE44 ( Laboratories thi-1, lambda-K Maryland recAl, gyrA96, relAl SUBSTITUTE SHEET Strain/Piasmiα Genotype Source or Reference Plasmids: pUC8 Ara r Maniatis et al. PRK2013 K r D.H. Figurski and D.R. Helinski, Proc. Natl. Acad. Sci. USA, 76, 1648-1652 (1979) PLAFR3 Tc^, cos B. Staskawicz University of California at Berkeley y RK602 Kmr, N m r T.M. Finan e_t a_l. , Cell, 40, 869- 877 (1985; Media and Culture Conditions Z. ramigera cultures are stored frozen at -70 β C in trypticase soy broth (TSB) medium containing 7% DMSO and 15% glycerol. The various Z^ ramigera strains were routinely cultured in either a defined medium, described by Norberg and Enfors ih Appl. Env. Microbiol. 44, 1231-1237 (1982) having the following composition in ( /liter) : 25g glucose, 2 g K2HPO4, 1 g KH 2 P04, 1 g NH4CI ; 0.2 g MgSθ4.7H 2 0; 0.01 g yeast extract (Difco Laboratories) in one liter distilled water where the glucose, MgSθ4 7H2ϋ, yeast extract and salts were autoclaved separately, or the TSB medium. 100 ml cultures of Z_ j _ ramigera were yrown on a rotary shaker (200 rpm) at 30 β C in 500 ml baffled shake flasks for periods up to two weeks. E. ccli strains were grown in Luria-Bertani (LB) medium, 1% (w/v) NaCl, 1% (w/v) peptone (Difco) and 0.5% (w/v) yeast extract (Difco). Purification of the Polymer The polymer is purified by the addition of concentrated NaOH to the cell culture to a final concentration of 0.2 M, followed by the addition of 3 volumes of ethanol to precipitate the polymer and other materials. The precipitate is collected and redissolved in half the original volume of water. Protein is removed by either extracting twice with phenol, followed by ■ extraction with ether to remove excess phenol or by ultrafiltration. The aqueous phase is dialyzed, lyophilized and ground to yield a fine white powder. The product is 96% pure and recovered at a yield of approximately 1 g/liter. Characterization of the Polymers Total carbohydrate concentration in culture broths and polymer solutions was determined by the Phenol reaction, described by Gerhardt in Manual of Methods for General Bacteriol. (Washington Amer. Soc. Microbiol. 1981). Glucose, galactose and Xanthan gum (Sigma Chemical Co., St. Louis, Mo.) were used as standards. Total protein concentration in culture broths and polymer solutions was determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Richmond, CA, 1979). Lysozyme was used as the standard. cellular protein was released by boiling in U.2 N NaOH. Cellufluor (Polysciences Chemicals, Warrington, PA) is a fluorescent dye, disodium salt of 4 ,4'- is- [4-anilino-bis-diethyl-amino-S-triazin-2-ylamino]- 2,2'-stilbene-disulfonic acid, that binds specifically to beta (1-3) and beta (1-4) glycosyl linkages and fluoresces when exposed to UV light. Cellufluor was added to agar plates, pH 7.4, at a concentration of 200 micro y/ml and used to determine polysaccharide production in _^ ramigera and E^ coli. Purified polysaccharide was hydrolyzed in 1 M trifluoroacetic acid at 120 β C for times varying between 1ft. hour and 2 hours. Monosaccharides in the polysaccharide hydrolysate were separated using a Waters HPLC equipped with a Brownlee Polypore PB, lead loaded cation exchange column, operated at 85 β C, with water as the eluent. Detection was by refractive index using a Waters Model 401 Differential Refractometer. The polysaccharide was further characterized by proton NMR spectroscopy and infared spectroscopy. The polysaccharide hydrolysate (10 mg) was dissolved in D O and analyzed using a 500 MHz proton NMR spectrometer. Testing was performed at the NMR Facility for Biomolecular Research located at the Francis Bitter National Magnet Laboratory, Massachusetts Insitute of Technology, Cambridge, Massachusetts. Infrared spectra were obtained on purified polysaccnaride (1 to 5 mg ground with 100 g dry KBr an pressed into a disk) using a Perkin Elmer Model 283B Infrared Spectrophotome er. As characterized by HPLC and proton-NMR, the polymer produced by Zooqloea ramigera 115 consists of ylucose and galactose in a ratio of approximately 2:1. Functional group assignments were made from an infrared scan of the 115 polysaccharide as follows: OH, 2.93 microns, C-H, 3.43 microns; C=0 of an ionized carboxyl, 6.15 microns and 7.15 microns; tertiary CH-OH, 8.65 microns, saccharide ring, 9.5 microns, and the C=0 of the ionized carboxyl group of pyruvic acid, 6.15 microns and 7.15 microns. Infrared analysis indicating that the 115 polysaccharide contains ionized carboxyl groups results in a "fingerprint" scan which along with the monosaccharide composition data is compared to the composition and IR scans of polysaccharides from mutant or genetically manipulated strains to detect changes in structure. The composition of the I-16-M polysaccharide is not known but it is believed to consist predominantly of 8(1-4) linked glucose and to have properties similar to those of cellulose. Isolation of Plasmid DNA from E. coli Plasmid DNA from E^ coli (large scale and mini-preparations) was prepared using the methods of Birnboim and Doly, Nucleic Acids Res. 7, 1513-1523 (1979) as modified by Ish-Horowicz and Burke Nucleic Acids Res. 9, 2989-2998 (1981). here necessary, the plasmid copy number per cell was increased by chlora phenicol amplification as described by Curtiss et al., Molecular Cloning of Recombinant DNA, Soft and Werner, eds. , pages 99-114 (Academic Press, NY, 1977). Enzymatic Treatement of DNA Restriction endonucleases and T4 DNA ligase were purchased from IBI (New Haven, CT) . Calf intestinal alkaline phosphatase (CIP) was obtained from Boehringer Mannheim (Indianapolis, IN). DNA polymerase I was obtained from A ersham (Arlington Heights, IL. ) . All enzymes were used according to the manufacturers' recommended conditions. Construction of Z. rameriga Gene Libraries Mbol partial digestion conditions for Z^ ramigera DNA were determined as required to yield fragments in the size range of 15-28 kb, as described by Maniatis, Molecular Cloning (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982). 40 microg lots of DNA were digested for 1 h, followed by the addition of EDTA to 25 mM and incubation at 68*C for 10 in. DNA was precipitated with 2 volumes o€ ethanol and resuspended in 185 microl TE, then 10 microl of 1 M Tris HCl pH 9.5 was added followed by 5 microl of 10 mg/ml spermidine. CIP (ϋ.01 units/ microy DNA. was added and the mixture incubated at 37°c for 30 min. The enzyme was inactivated at 68 β C for 10 in and partially digested DNA was electrophoresed on a 0.75% (w/v) agarose yel. DNA in the range of 15-28 kb was cut out of the gel, electroeluted, ethanol precipitated and resuspended in TE. Vector DNA was prepared as follows. Two aliquots (10 microg each) were digested to completion, one with Hindlll and one with EcoRI, followed by CIP treatment. Samples were purified by phenol extration, ethanol precipitation and resuspended in TE. Both aliquots were then completely cleaved with Ba HI and purified by phenol extraction. The desired fragments were precipitated with 0.7 volumes isopropanol in the presence of 0.2 M sodium acetate and resuspended in TE at a concentration of 1 microy/microl . Ligation reactions contained 1 microg of Hindlll/BamHI cut vector and 1 microy of EcoRI/BamHI cut vector and 2 microg of target DNA in a total volume of 10 microl for 12-16 hours at 14"C. Ligated DNA was packaged using in vitro packaging extracts prepared from E^ coli BHB2688 and BHri2690 using the method of Ish-Horowicz and Burke, Nucleic Acids Res. 9, 2989-2998 (1981). Recombinant phage particles were transduced into E. coli HB101 as described by Maniatis et al.. Molecular Cloning (1982) and plated on LB agar containing 10 microy Tc/ml and 200 microg Cellufluor/ml. SUBSTITUTE SHEET Table II: Z_^ ramigera Mutant Strains Strain Mutaqen Phenotype 106-C1 (and C2) NTG Cel", Flc", Gal I-16M-C4 (and C5) UV Cel " , Flc", Gal", Cb r NmS, sms I-16M-P1 (and P2-P12) UV c", Gal + , Cb r I-16M-T1 (T7, T9, T12, Tn5 Cel", Flc", Gal + , Cb r T18, T24, T25, T27 Nπ.r, sm∑- T28, 130, T32, T33 T44, T48, T49) Cel- non-fluorescent on Cellufluor Flc" non-flocculating . Gal" unable to utilize galactose Cb r carbenicillin resistant " NmS neomycin sensitive Sm s streptomycin sensitive Conjugation in Z. ramigera Transfer of pLAFR3 and pLAFR3 recombinant DNA molecules into Z ramigera I-16-M was done using the conjugative plasmid pRK2013 as follows: E^ coli MM294A (pRK2013) and E^ coli DH5 containing pLAFR3 or one of the pLAFR3/Z. ramigera gene libraries were each grown up in LB broth containing Kanamycin (Km) (50 microy/microl for pRK2013) or Tetracycline (Tc) (10 microy/microl for pLAFR3) to a density of approximately 2 X 10 9 . Equal amounts (0.5 ml) of each were mixed, after washing, with 0.5 ml of I-16-M parent or mutant strains. The mixture was plated on a single 100 mm LB ayar plate and incubated overnight at 30 β C. Cells were SUBSTITUTE SHEET resuspended in a 1 ml LB and dilutions were plated on LB agar containing Carbenici11in (Cb) (10U microg/ml and Tc (10 icrog/ml) . Transposon Mutagenesis Transfer of pRK602 (a derivative of pRK2013 containing Tn5) into Z ramigera was carried out as described above using E_. coli MM294A (pRK602) as the only donor. Tn5 insertions into the Z. ramigera chromosome were selected for by growth on streptomycin (100 microy/ml). DNA Blotting and Hybridization Analysis DNA blots were prepared usiny DNA frayments separated on ayarose yels by the sandwich blot method described by G.E. Smith and M.D. Summers in Anal. Biochem. 1-09, 123-129 (1980), based on the technique developed by Southern, in J_ Mol. Biol . 113, 503-517 (1975). Filters were hybridized with DNA probes labelled to a high specific activity (0.1-1 x 10 8 cpm/microg of DNA) with [alpha- 32 P] dATP, by nick translation described by P.W.J. Rigby et al., in J^ Mol. Biol. 113, 237-251 (1977). Pre-hybridizations and hybridizations were carried out at 65 β C i sealed polyethylene bags. The pre-hybridization/hybridization solution contained 5 x SSCP (1 x SSCP contains 0.15 M NaCl, 0.1Ξ M Na Citrate, 10 mM Na 2 HP0 4f 10 m M NaH 2 P0 4 ), 5 X Denhardts solution (0.5 g Ficoll, 0.5 g polyvinyl pyrrolidone, 0.5 g BSA (Pentax Fraction V) H 2 0 up to 500 ml), 0.1% (w/v) SDS, 10 M Em SUBSTITUTE SHEET and 100 microg sonicated denatured salmon DNA. Filters were pre-hybridized for 8-18 h and hybridized for 16-18 h using lϋ 7 C m of labelled DNA probe per filter. Final wash conditions were 2 x SSC (20 x SSC: 3.0 M NaCl, 0.3 M sodium citrate), 0.1% (w/v) SDS at 65 β C. Characterization of I-16-M (Cel") Tn5 Mutants Chromosomal DNA was isolated from Cel" Tn5 mutants and used in the following hybridization analyses. First, to confirm the presence of and identify the fragment containing Tn5, 32 P-labeled Tn5 DNA was hybridized to a Southern blot of EcoRI digested DNA from each of the Cel" mutants and pRK602. From this autoradiogram six mutants were selected that had Tn5 insertions in apparently different EcoRI fragments. They are strains T18, T25, T27 r T30, T48 and T49. These six mutants were further characterized by hybridizing 32 P-labeled Tn5 DNA to a Southern blot of BamHI, Hindlll and PstI digests of chromosomal DNA from each of the six strains 2-7 (BamHI cut), 8-13 (Hindlll cut) and 15-20 (PstI cut); lanes 1 and 14 contain size markers, as shown in Fig. 3. The 3.4 kb fragment in lanes 8-13 and the 2.5, 1.1 and 0.9 Kb fragments in lanes 15-20 are internal Tn5 fragments. It is clear that five of the mutants have Tn5 inserted into different EcoRI fragments while T48 and T49 have Tn5 inserts in the same frayment. Subsequent work was done using only the five strains, T18 r T25, T27, T30 and T48. The EcoRI fragments containing Tn5 were isolated by completely digesting the chromosomal DNA from each of five mutants with EcoRI which was ligated into pUC8, transformed into E. coli HBlOl and plated on LB-ayar with neomycin (50 ng/ml) and a picillin (50 ng/ml). Colonies resistant to both antibiotics should contain pUC8 and Tn5 as a recombinant plasmid. This procedure was successful in isolating EcoRI fragments that contain Tn5 from strains T25, T27, T30 and T48 ligated into pUC8 which are designated pCLT25, pCLT27, pCLT30 and pCLT48, resp ctively. Complementation of Cel~ Mutants The pLAFR3/I-16-M gene library described previously was mated en masse from E^ coli DH5 into the four I-16-M Cel- mutants. More than 5000 transconjugants for each mutant strain were screened for fluorescence on Cellufluor plates. Fluorescent candidates were found at a frequency of approximately 1 in 100-200 colonies. Pictures of cultures of wild-type I-16-M, mutant T27 and complemented mutant T27 containing plasmid pPS27 showing the tubes just after shaking to disperse the floes, as well as after the floes were allowed to settle, clearly demonstrate that the mutant strain forms a turbid culture while both the wild-type and complemented mutant form cell floes. Plasmid pPS27 was able to complement all five Tn5 mutations indicating that several linked genes for exopolysaccharide synthesis are encoded on this plasmid. This plasmid encoding SUBSTSTUT SHE! :T Zooglan I-16-M was deposited in E_^ coii HBlOl with the American Type Culture Collection, Rockvilie, Maryland on July 28, 1986 and assigned ATCC designation The pLAFR3/115 gene library was used in a similar manner to complement the I-16-M Cel- mutants. Again, over 5000 transconjuyants for each mutant strain were screened for fluorescence. -Mildly fluorescent candidates were found but at a much lower frequency (less than 1 in 1000). These transronjugants were also screened for changes in colony morphology since the two Z_^ ramigera strains are easily distinguishable using this characteristic. Several candidates were isolated that have a morphology similar to that of strain 115, indicating that these transformed strains are producing Zooglan 115. These candidates along with the fluorescent candidates are currently being analyzed. Pia id pHP30 was able to complement all the mutations deficient for exopolysaccharide synthesis, indicating that the plasmid encodes for all of the genes involved in Zooglan 115 synthesis. Plasmid pHP30 was deposited in E_^ coli DH5 with the American Type Culture Collection, Rockvilie, Maryland on July 28, 1986 and assigned ATCC designation Control of a polysaccharide's structure by manipulation of the genes that code for its biosynthesis first requires that those genes be isolated and characterized. The strategy developed for isolation and characterization of these genes, as demonstrated for the exocellular polysaccharides produced by Zooqloea ramigera strains involves cloning the polysaccharide genes directly in Z _ ramigera, as shown schematically in Fig. 2. The requirements for cloning the genes are 1) a method for introducing foreign DNA into Z^ ramigera, 2) the construction of a Z. ramigera gene library, 3) the isolation of polysaccharide mutant strains, and 4) the subsequent complementation of those mutants using the Z ramigera gene library. To meet the first requirement, a conjugation system was developed for Z _ ramigera strain I-16-M using a broad host range vector. The frequency of transfer for this system is approximately 10~ 3 transconjugants/recipient. To meet the second requirement, construction of Z. ramigera yene libraries, the cosmid pLAFR3, derived from RK2 via pRK290, was used to construct Z ramigera I-16-M and 115 gene libraries, as shown in Fig. 2, using the method of B. Staskawicz, University of California at Berkeley. This procedure increases the cloning efficiency by ensuring that only recombinant molecules can be packaged. A "right" and "left" arm are created which can only be packaged if they ligate to an insert molecule that is between 15 and 28 kb, thus a high frequency of recombinants are obtained. The recombinant molecules are packaged _i_n vitro and transduced into E_ j _ coli DH5. This procedure was carried out for both I-1S-M and 115 and yielded approximately 105 recombinants/micro y of insert DNA in each case. For the third requirement, isolation of mutant strains from Z ramigera I-1.6-M, mutants devoid of exopolysaccharide production, were selected. Since non-production of Zooglan I-16-ff has no significant effect on the growth of the organism, a technique using the fluorescent dye Cellufluor is used for screeniny. Mutants that do not fluoresce on Cellufluor (Cel") were obtained using UV and transposon mutagenesis, as described in Table I. For the fourth requirement, complementation of the different mutants using the Z ramigera gene library, a genetic transfer system was developed using the conjugal properties of pRK2013 and a broad host range cloning vector derived from RK2, yielding a frequency of transfer of approximately 10~ 3 transconjuyants/recipient. Conjugation frequencies from other Gram-negative organisms using vectors also derived from RK2 are similar. The transfer of genetic material by conjugation enabled the development of a transposon mutagenesis procedure using Tn5. Cel" mutants obtained by Tn5 insertion into the I-16-M genome were complemented with a pLAFR3/ I-16-M gene library. The insertion of Tn5 into the polysaccharide biosynthetic genes or regulatory genes pinpoints the exact location of the mutation which simplifies sub-cloning of the DNA fragments containing these genes. Identification of the functions of these genes helps solve the biosynthetic pathway and enables genetic manipulation to control structure and increase production of novel polymers. Proposed strategies for controlling polymer production and structure .include placing the polysaccharide biosynthetic genes under the control of regulatable promoters. Isolation of the prlysaccharide biosynthetic genes from Z_ j _ ramigera provides a means for the determination of the relative positions of the various polysaccharide genes in the Z _ ramigera chromosome to show if the genes are linked or unlinked and whether or not it is possible that these .yenes exist in the format of an ' operon. Further, the cloning of the entire biosynthetic pathway as an operon under the control of an inducible promoter enables production to be turned on and -off. For example, in a large scale process, a two stage system can be developed in which cells are first grown to a high density with the polymer genes off, then in the second stage the genes are induced and large amounts of polysaccharide are produced by the high density culture. Other possibilities for alteration and control of polysaccharide structure include the subcloning of the genes coding for the pathway enzymes onto a single plasmid with different inducible promoters for key genes. This enables the manipulation of polysaccharide composition and structure by controlling the levels of expression of these yenes and thus the relative amounts of each enzyme. For example, if one desires a highly branched polysaccharide, the yene coding for a branching enzyme could be overexpressed resulting in higher levels of this particular enzyme and a higher degree of branching in the polymer. Additionally, the potential exists for controlling the charge density of a polysaccharide by regulating the genes that code for enzymes responsible for transferring ionic groups (e.g. pyru' * yl, acetyl, succinyl, amino moieties, etc.) to the polysaccharide. Using such a system, polysaccharides that have optimal charge distributions for improved flocculating properties can be designed and produced. Other strategies for controlling polymer production and structure include the introduction of these genes into new host strains to enable .the development of more economic processes for polysaccharide production; mutagenesis of the genes to alter the enzyme activities and therefore polymer structure; and the construction of novel pathways for polysaccharide synthesis by "mixing" genes from different strains of _ j _ ramigera and other organisms. The system is useful in designing novel polymer structures for specific functional applications. Identification of the gene products encoded by the polysaccharide yenes help to determine the enzymology of the biosynthetic pathway ano any control mechanisms it might be subject to, and therefore facilitate development of these strategies for controlling polymer production and structure. A detailed enzymology study is required to completely characterize the pathway. The development of assays for each enzyme is necessary to determine the enzyme levels _in vivo, the kinetics of the reactions they carry out, and their substrate specificity. This information is further used to develop strategies for the manipulation and control of the pathway at the genetic level. Inserting the yenes for flocculation into different host strains can lead to improved purification procedures for bacterial products. For example, bacteria that produce intracellular products can be flocculated by induction of the polysaccharide yenes at the end of their production cycle providing an easier separation of the cell floes from the culture broth. Similarly, if the desired product is excreted into the broth, cells induced to flocculate at the end of the production staye can then be removed from the supernatant by sedimentation or in a settler as opposed to costly centrifugation or filtration. If the product of interest is the polysaccharide itself then it is possible to transfer and express the genes for the biosynthetic pathway into a more desirable strain. For instance, a polysaccharide producing bacterium that has a high growth rate and is able to grow on low cost substrates would be beneficial. This would result in higher polysaccharide yields and a more economical product. Althnugh ti.is invention _ιas been described with reference to specific embodiments, it is understood that modifications and variations may occur to those skilled in the art. It is intended that all such modifications and variations be included within the scope of the appended claims.

Claims: CLAIMS : 1. A method for identifying, expressing and modifying the yenes for biopolymers produced by a cell strain comprising: a) isolating mutants of the cell strain or a closely related cell strain wherein said isolated mutants do not produce a specific polymer and the original cell strain produces the polymer; b) constructing a library of the genes from the original cell strain; c) introducing genes from said library into said isol?-.ed mutants; d) screening said mutants for polymer production; and e) isolating and characterizing said introduced genes until the genes encoding the entire polymer biosynthetic pathway of the specific polymer of the cell strain are isolated and characterized. 2. The method of claim 1 further comprising selecting said cell strain from strains of Zoogloea ramigera. 3. The method of claim 1 further comprising mutating said original cell strain by introducing a transposon-containing vector into said original cell strain. 4. The method of claim 1 wherein the library is constructed in a vector. 5. The method of claim 4 wherein step b further comprises selecting a broad host range cosmid vector for said vector. 6. The method of claim 5 wherein the cell strain is a strain of Zoogloea ramigera and said broad host ranye cosmid vector is pLAFR3. 7. The method of claim 4 for constructing the library comprising: isolating DNA from the original cell strain; partially digesting said isolated DNA with restriction enzymes to produce approximately 15-28 kb DNA fragments; and inserting said DNA fragments into the vector to form a recombinant molecule. 8. The method of claim 7 tor constructing the library further comprising: packaging said recombinant molecules; and transducing said packaged recombinant molecules into an appropriate host bacteria. 9. The method of claim 8 wherein said cell strain is a strain of Z-oogloea ramigera, said broad host range cosmid vector is pLAFR3, and said host bacteria is E^ coli DH5. 10. The method of claim 1 wherein step c comprises introducing the yenes from the library into said mutants by conjugation. 11. The method of claim 7 wherein step c comprises introducing said recombinant molecules into said mutants by conjugation. 12. The method of claim 11 wherein step c comprises introducing said recombinant molecules into said mutants by conjugation using the conjugation plasmid pRK2013. 13. The method of claim 11 further comprising mutating said original cell strain by introducing a transposon delivery vector by conjugation. 14. The method of claim 13 further comprising selecting pRK602 as said transposon delivery vector and E^ coli MM294 as the host bacteria. 15. The method of claim 1 wherein the polymer confers an identifiable morphological characteristic on the bacterial strain and said mutants are screened for acquired morphological characteristics. 16. The method of claim 1 further comprising modifying said isolated and characterized genes for the specific polymer. 17. The method of claim 16 wherein said isolated and characterized polymer genes are placed under the control of regulatable promoters. 18. The method of claim 1 further comprising introducing said isolated and characterized polymer genes into a second host strain. 19. The method of claim 18 further comprising selecting said introduced polymer genes conferring enhanced polymer production on said host strain. 20. The method of claim 16 wherein said polymer genes having enzymatic activity are modified. 21. e methoα of claim 16 wnerein said polymer genes responsible for polymer structure are modified. 22. The method of claim 16 further comprising selecting and combining the isolated and characterized genes for more than one specific polymer; placing said selected and combined genes in a host wherein said genes are translated; and selecting and modifying the host conditions, wherein novel polymers are produced from said selected and combined genes. 23. A gene for an exopolysaccharide produced by Zoogloea ramigera, wherein the encoded exopolysaccharide causes flocculation of the produciny organism when the organism is grown in a liquid media and confers a distinct morphology on said organism. 24. The gene of claim 23 wherein said gene is inserted into a broad host range cosmid vector. 25. The gene of claim 24 wherein tne vector is pLAFR3. 26. e yene of claim 23 v erein said yene is isolated from Zoogloea raπiqera strain I-lfi-M. 27. The yene of claim 26 as deposited with the ATCC in host E^ coli HB101 on July 28, 1986 and designated ATCC 28. The gene of claim 23 further comprising a regulatable promoter. 29. The gene of claim 23 wherein said gene is isolated for Zooqloea ramigera strain 115. 30. The gene of claim 29 as deposited with the ATCC in host E, coli DH5 on July 28, 1986 and designated ATCC 31. Λ 'ene foi a polymer, wherein said gene is isolated and identified by a) isolating mutants of the cell strain or a closely related cell strain wherein said isolated mutants do not produce a specific polymer and the original cell strain produces the polymer; b) constructing a library of the genes from the original cell strain; c) introducing yenes from said library into said isola'.ed mutants; d) screening said mutants for polymer production; and e) isolating and characterizing said introduced genes until the yenes encodiny the entire polymer biosynthetic pathway of the specific polymer of the cell strain are isolated and characterized. 32. A polymer synthesized by introducing the genes of claim 31 for the entire polymer biosynthetic pathway of said polymer into an appropriate host and inducing said yenes. 33. The polymer synthesized from plasmid pPS27, deposited with the ATCC on July 28, 1986 in host E_^ coli H6101 and designated ATCC 34. The polymer synthesized from plasmid deposited with the ATCC on July 28, 1986 in host E. coli HBlOl and designated ATCC

Inventors: EASSON DONALD DAVIDSON JR, RHA CHOKYUN, SINSKEY ANTHONY J, EASSON, DONALD, DAVIDSON, JR., RHA, CHOKYUN, SINSKEY, ANTHONY, J.
Applicants: MASSACHUSETTS INST TECHNOLOGY, MASSACHUSETTS INSTITUTE OF TECHNOLOGY
Application year: 1987
Main classification: C07H15
IPC codes: C07H21, C09K8, C12N15, C12P19, C12R1