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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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• • C—CHEMISTRY; METALLURGY
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  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds

Definitions

• 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.
• 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.
• floc-forming bacteria Several types have been identified. The most efficient are the cellulose (or cellulose-like) producing bacteria such as certain species of Pseudomonas, Aerobacter, 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 floccul ants. 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.
• z. 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 .
• ramigera isolate 115 available from the American Type Culture Collection, Rockville, MD, 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 biopolymer's high affinity for heavy metal ions.
• 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 strai ns us ing the present invention, as follows.
• 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-M is referred to as Zooglan I-16-M.
• Complementation of these mutations was achieved with a Z . rarigera I-16-M gene library constructed in the broad host range cosmid vector pLAFR3 and introduced into the 1-16-M mutants by conjugation. Transformed colonies were identified as having restored Zooglan I-16-M production by fluorescence on Cellufluor and flocculation.
• a gene bank of Z. 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. ramigera strains and identification of their functions with respect to their 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 conrcruction 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.
• Fig. 1 is a schematic of the cloning of Z. ramigera polysaccharide genes in non-polysaccharide producing Z. ramigera.
• Fig. 2 is a schematic of the method for clone bank construction in pLAFR3 of B. Staskawicz, U.C. Berkeley.
• Fig. 3 is an autoradiogram of 32 p-Iabeled Tn5 hybridized to a Southern blot of DNA from mutants T18, T25, T27, T30, T48, and T49 cut with BamHI (lanes 2-7), Hindlll (lanes 8-13) and PstI (lanes 15-20). Lanes 1 and 14 contain size markers.
• 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 Zoogloea raqimera.
• Strategies for genetic manipulations include both classical mutagenesis and recombinant DNA technology.
• Two methods were used to transferZ. ramigera DNA into a second organism, either a second distinct strain of Z. ramigera as determined from its DNA composition, or E. coli.
• 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 in 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.
• 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 scheme is the introduction of the plasmid DNA into Z. ramigera.
• the strategy for cloning in Z. 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.
• the broad host range cloning vector if transferred to Z. ramigera, can be selected for by growth on appropriate medium.
• 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 transcon jugants 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.
• Zooglan I-16-M 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-M gene library constructed in the broad host range cosmid vector pLAFR3 and introduced into the I-16-M mutants by conjugation.
• 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.
• Z . ramigera cultures are stored frozen at -70oC in trypticase soy broth (TSB) medium containing 7% DMSO and 15% glycerol.
• TTB trypticase soy broth
• the various Z. ramigera strains were routinely cultured in either a defined medium, described by Norberg and Enfors in Appl. Env. Microbiol. 44, 1231-1237 (1982) having the following composition in (g/liter): 25g glucose, 2 g K 2 HPO 4 , 1 g
• KH 2 PO 4 1 g NH 4 CI 0.2 g MgSO 4 .7H 2 O; 0.01 g yeast extract (Difco Laboratories) in one liter distilled water where the glucose, MgSO 4 7H 2 O, yeast extract and salts were autoclaved separately, or the TSB medium.
• 100 ml cultures of Z. ramigera were grown on a rotary shaker (200 rpm) at 30oC in 500 ml baffled shake flasks for periods up to two weeks.
• E. coli 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).
• 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 byextraction with ether to remove excess phenol or by ultraf iltration. 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.
• Cellufluor (Polysciences Chemicals, Warrington, PA) is a fluorescent dye, disodium salt of
• Purified polysaccharide was hydrolyzed in 1 M trifluoroacetic acid at 120oC for times varying between 1 ⁇ 2 . 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 85oC, 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 2 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 mg dry KBr and pressed into a disk) using a Perkin Elmer Model 283B Infrared Spectrophotometer.
• the polymer produced by Zooqloea ramigera 115 consists of glucose and galactose in a ratio of approximately 2:1.
• 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.
• Plasmid DNA from E. coli 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). Where necessary, the plasmid copy number per cell was increased by chloramphenicol amplification as described by Curtiss et al., Molecular Cloning of Recombinant DNA, Soft and Werner, eds., pages 99-114 (Academic Press, NY, 1977).
• Restriction endonucleases and T4 DNA ligase were purchased from IBI (New Haven, CT). Calf intestinal alkaline phosphatase (CIP) was obtained from Boehringer Mannhe im (Indianapolis, IN). DNA polymerase I was obtained from Amersham (Arlington Heights, IL.). All enzymes were used according to the manufacturers' recommended conditions.
• DNA was digested for 1 h, followed by the addition of EDTA to 25 mM and incubation at 68oC for 10 min.
• DNA was precipitated with 2 volumes of 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 (0.01 units/ microy DNA) was added and the mixture incubated at 37°c for 30 min.
• the enzyme was inactivated at 68oC for 10 min and partially digested DNA was electrophoresed on a 0.75% (w/v) agarose gel. 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 BamHI 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 14oC.
• Ligated DNA was packaged using in vitro packaging extracts prepared from E. coli BHB2688 and BHB2690 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.
• Transfer of pLAFR3 and pLAFR3 recombinant DNA molecules i nto 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 30oC. Cells were resuspended in a 1 ml LB and dilutions were plated on LB agar containing Carbenicillin (Cb) (100 microg/ml and Tc (10 microg/ml).
• 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 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-32P] 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 65oC i sealed polyethylene bags.
• the pre-hybridization/hybridization solution contained 5 x SSCP (1 x SSCP contains 0.15 M NaCl, 0.15 M Na Citrate, 10 mM Na 2 HPO 4 10 mM NaH 2 PO 4 ), 5 X Denhardts solution (0.5 g Ficoll, 0.5 g polyvinyl pyrrolidone, 0.5 g BSA (Pentax Fraction V) H 2 O up to 500 ml), 0.1% (w/v) SDS, 10 mM EDT and 100 microg sonicated denatured salmon DNA.
• Filters were pre-hybridized for 8-18 h and hybridized for 16-18 h using 10 7 Cpm 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 65oC.
• Chromosomal DNA was isolated from Cel- Tn5 mutants and used in the following hybridization analyses.
• 32P-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, T30, T48 and T49.
• the pLAFR3/I-16-M gene library described previously was mated en masse from E. coli DH5 into the four I-16-M Celmutants. 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 Zooglan I-16-M was deposited in E. coli HB101 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.
• Piamid 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
• Z . ramigera gene 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 in vitro and transduced into E. coli DH5. This procedu re was carried out for both I-16-M and 115 and yielded approximately 105 recombinants/micro g of insert DNA in each case.
• 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 i include placing the polysaccharide biosynthetic genes under the control of regulatable promoters. Isolation of the prlysaccharide biosynthetic genes from Z. 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.
• polysaccharide structure examples 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. pyruryl, 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.
• ionic groups e.g.
• Identification of the gene products encoded by the polysaccharide yenes help to determine the enzymology of the biosynthetic pathway and 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.
• 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.
• 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.
• 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.

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