CA1335576C - Methylotroph cloning vehicle - Google Patents

Abstract

A cloning vehicle comprising: a replication determinant effective for replicating the vehicle in a non-C1-utilizing host and in a C1-utilizing host; DNA
effective to allow the vehicle to be mobilized from the non-C1-utilizing host to the C1-utilizing host: DNA
providing resistance to two antibiotics to which the wild-type C1-utilizing host is susceptible, each of the antibiotic resistance markers having a recognition site for a restriction endonuclease; a cos site; and a means for preventing replication in the C1-utilizing host. The vehicle is used for complementation mapping as follows. DNA comprising a gene from the C1-utilizing organism is inserted at the restriction nuclease recognition site, inactivating the antibiotic resistance marker at that site. The vehicle can then be used to form a cosmid structure to infect the non-C1-utilizing (e.
g., E. coli) host, and then conjugated with a selected C1-utilizing mutant. Resistance to the other antibiotic by the mutant is a marker of the conjugation. Other phenotypical changes in the mutant, e.g., loss of an auxotrophic trait, is attributed to the C1 gene. The vector is also used to inactivate genes whose protein products catalyze side reactions that divert compounds from a biosynthetic pathway to a desired product, thereby producing an organism that makes the desired product in higher yields.

Description

Backq~ound of the Invention This invention relates to cloning vehicles and methods fo~ enginee~ing Cl-utilizing mic~oo~ganisms p~oducing a -desi~ed compound; it also celates to the ~esulting enginee~ed mic~oorganisms. t In theo~y, organisms that use Cl compounds as a sou~ce of carbon and ene~gy should be useful fo~ fe~mentation p~ocesses p~oducing de6ired compounds, because Cl compounds such as methanol a~e ~elatively cheap and available. The~e a~e various disclosu~es of fe~mentation processes based on Cl-utilizing organisms. For example, Kono U.S. Patent 3,663,370 discloses synthesizing glutamic acid using specific st~ains of Methanomonas, Protaminobacter, and Mic~ocyclus.
Nakayama U.S. Patent 3,907,637 discloses synthesizing L-lysine by fe~mentation of a mutant P~otaminobacte~ capable of utilizing methanol. Gatenbeck U.S. Patent 3,963,572 discloses synthesizing L-tryptophan f~om indole o~ derivatives thereof using Pseudomonas AM 1 o~ Methylomonas methanolica, in which methanol is the p~imary source of ca~bon. Kiyoshi et al.
(1975) Jap. Appl'n 29791/75 discloses synthesizing L-tryptophan using mutants de~ived from the gene~a Pseudomonas, Methanomonas, o~ P~otaminobacter.
It is desirable to enginee~ o~ganisms to imp~ove product yield; howeve~, engineering Cl-utilizing mic~oo~ganisms is generally difficult because theie genomes a~e ~elatively poo~ly characte~ized compa~ed to o~ganisms such as E. coli, and it is difficult to isolate stable mutants.
Moreover, enginee~ing Cl-utilizers is also difficult because of the lack of vehicles that di~ectly transfo~m C~-utilize~s;
thus it is difficult to develop efficient gene t~ansfe~ systems fo~ Cl-utilize~s. Finally, antibiotic resistance cha~acte~istics of Cl-utilizing mic~oo~ganisms may not be z - 1 3 3 5 5 7 6 compatible with many common marker genes used in engineering procedures, such as genes for resistance to trimethoprin, streptomycin, or ampicillin.
Various efforts have been made to study the genome of .-Cl-utilizing microorganisms using genetic engineering techniques. Haber et al. (1983) Science 221:1147-1153 reviews a number of articles including methods for transferring cloned DNA into methylotrophs using conjugative or mobilizable cloning vectors. The former are transferred between bacterial cells by simple mating techniques, and the latter are transferred only with the assistance of another mobilizing plasmid that codes for the gene products necessary for conjugal transfer. Those vectors include:
1) pRK290, a mobilizable plasmid derived ~from RK2 (a broad host range plasmid): pRK290 contains genes coding for resistance to three antibiotics including tetracycline: it is mobilized to transfer by conjugation between E. coli and various other strains in the presence of helper plasmid pRK2013 tDitta et al. (1980) PNAS USA 77:7347]

2) pLAFRl is a mobilizable cosmid derivative of pRK290 containing a gene for tetracycline resistance: pLAFRl cosmids are mobilized from Rhizobium meliloti into E. coli and back again in the presence of helper plasmid pRK2013 [Friedman et al. (1982) Gene 18:289]:

3) pVK100, pVK101, and pVK102 are mobilizable , derivatives of pRK290 containing a kanamycin resistance gene from plasmid R6-5: pVK102 is a cosmid vector having cloning sites in resistance gene~ that allow selection for inserts [Knauf et al. (1982) Plasmid 8:45]

4) R68.45 is a conjugative plasmid with a broad host range it is mobilizable to transfer between E. coli and Pseudomonas aeruqinosa, Pseudomonas AMl, Methylosinus trichosporium OB36, and Methylobacterium orqanoPhilum xx [Holloway (1981) Microbial Growth on C-l ComPounds, H. Dalton 35 EdLondon p. 317].

5 ) pM061 is a conjugative plasmid derived frcrn R68.45 with enhanced chr~nos~nal ITK)bilization host range, Transfer frequencies ~epoctedly a~e similac to R68.45. [Reiss et al.
(1980) Genet. Res. 36:99]:

6) RSF1010 is a mobilizable plasmid having a broad host range that transfers between E. coli and Pseudomonas AMl [~agdasa~ian et al. (1981) Gene 16:237 Gautier et al. (1980) Uol. Gen. Genet. 178:375]; and

7) pKT230 and pKT231 a~e mobilizable derivatives of RSF1010 containinq cloning sites in cesistance genes to allow 6election fo~ insects tBagdasa~ian (19~1), cited above].
Moore et al. (1983) J. Gen. Mic~obiol. 129:785-799 disclose a method o complementation mapping of Methylophilus methylotrophus using a plasmid that is an R derivative of plasmid ~M0172.
Gautier et al. (1980) Mol. and Gen. Genetics 144:243-251 disclose cloning the wild-type methanol dehydrogenase gene of Pseudomonas AM 1 in E. coli using plasmid R116Z. The methanol dehyd~ogenase gene is then t~ans~e~ed to a methanol dehyd~ogenase mutant o~ Pseudomonas AM 1 using RP4 to mobilize the hybcid plasmid.
O'Conno~ et al. (1978) J. Gen. Microbiol. 104:105-111 disclose transfo~ming Methylobacte~ium o~qanophilum in o~de~ to ~tudy the linkage of Cl-utilizing genes in that o~ganism.
Wa~ner et al. (1980) FEMS Microbiol. Letters 7:181-lB5 and Jeyaseelan et al. (1979) FEMS Mic~obiol. Lettecs 6:87-89 disclose an attempt to use a b~oad host-~ange plasmid, R68.45, to mae chromosomes of 6eve~al geneca of Cl-utilizing ocganisms.
Tat~a et al. (1983) J. Gen Mic~obiol. lZ9:Z6Z9-263Z
disclose that R68.45, ~efe~red to above, can mobilize the chcomo60me of Pseudomonas AM 1. Ma~ke~s a~e linked to genes to demonstrate their location.

-We have discoveced vecsatile cosmid cloning vehi~les that can be used genecally to chacactecize and engineer the genome o Cl-utilizing miccoocganisms. The vehicles have a bcoad host cange and can tcan6~ec by conjugation between a non-Cl-utilizing host and a Cl-utilizing host so that complementation mapping can be used to chacactecize the genome of the Cl-utilizing organism, and having done so, to enginee~
selected genes to inccease pcoduction of desiced compounds by the Cl-utilizing ocganism.
~y the tecm C1-utilizing miccoocganisms, we mean to include all ocganisms that can use as a cacbon/enecgy sou~ce Cl comeounds 6uch a~ methane oc methanol. We specifically mean to include facultative and obligate Cl-utilizing miccoocganisms; methane and methanol-utilizing miccoocganisms:
and type I (i.e. those using the cibulose monophosphase pathway) and type 2 (i.e. those using the secine pathwayJ
miccoocganisms. The tecm also include6 bactecia as well as yeast oc othec Cl-utilizing miccoocganisms. For a general discussion of classification of Cl-utilizing miccoocganisms, see Habec et al. (19B~) Science 221:1147-1153 and ceecences cited thecein. Reecences hecein to genus and species cefe~ to ocganisms as classi~ied in ~uchanan et al., The Shoctec ~ecgey's Manual Foc Detecminative Bacteciology (William Wilkins, l9a2).
In a fi~st aspect, the invention genecally ~eatuces a cloning vehicle compcising: a ceplication detecminant efective foc ceplicating the vehicle in a non-Cl-utilizing host and in a Cl-utilizing host: DNA efective to allow the vectoc to be mobilized fcom the non-C -utilizing host to the Cl-utili~ing host in the presence of a mobilizing pl~mi~; -DNA
providing resistance to at least two antibiotics to which the wild-type non-Cl -utilizing host is susceptible, at least one of the antibiotic resistance markers having a recognition site for a restriction endonuclease- that , generates f~agments that are ligatable to DNA digested by a cestriction endonuclease that recognizes sites consisting of fou~ nucleotides or less; and a cos site.
The vehicle is capable of~ eceiving, at the cestriction nuclease recognition site, an insertion of DNA
comprising a gene-containing fragment of the digested genome of the Cl-utilizing o~ganism, the~eby inactivating resistance to one of the antibiotics as a marker of the inse~tion: 2) forming a cosmid structu~e: 3) infecting the non-Cl-utilizing host:
and 4) conjugating with a mutant of the Cl-utilizing host that is mutated in the gene, whe~eby the gene-containing fragment vehicle complements the mutant, thus signaling the function of the gene.
In ereferred embodiments, the replication determinant is a ~eplicon such as the one reported in pRK290; the antibiotic ~esistance is to kanamycin or tetracycline the cos site is from phage lambda; the DNA effective to allow the vehicle to be mobilized is de~ived from pRK290: the mobilizing plasmid is pRK2913: the Cl-genome-digesting restriction endonuclease is Sau3A the vehicle is selected feom pLA2901, pLA2905, pLA2910, and pLA2917 and the internal recognition 6ite of the first antibiotic resistance gene is B~lII or Sau3A.
In a second aspect, the invention features using the above-described vehicle fo~ complementation mapping of a Cl-utilizing o~ganism by: digesting the genome of the Cl-utilizing organism with a ~estriction endonuclease that recognizes a 4-nucleotide base-pai~ sequence to generate gene-containing fragments: inserting one of the gene-containing fragments into the recognition site of the internal antibiotic resistance gene of the vehicle to inactivate the resistance, packaging the gene fragment-containing vehicle into a cosmid st~uctu~e infecting a non-Cl-utilizing host selecting the infected non-Cl-utilizers from a heterogeneous population that a~e resistant to the second but not the first antibiotic ..

mobilizing the gene fragment-containing vehicle from the selected non-Cl-utilizing host into a Cl-utilizing host that is mutated in the gene of interest: and determining whethec the gene fragment-containing vehicle complements the mutant and therefore whethee the vehicle contains the gene of interest.
In prefecred embodiments of the second aspect of the invention, the gene of interest is a gene expressing an enzyme of an aromatic amino acid synthesis pathway; the vehicle is pLA2901, pLA2905, pLA2910, or pLA2917; and the Cl-utilizing organism is Methylobactecium orqanophilum.
The above-described vehicle is particularly useful for characterizing the genome of the Cl-utilizer by tcansferring those genes to a mutant Cl-utilizer or to a non-Cl-utilizer and perfotming complementation mapping. 5pecifically, the cosmid will harbor coding capacity sufficient such that after digesting the Cl-utilizer chromosome and packaging the segments, the phage particles used to infect the non-Cl-utilizing host, specifically E. coli, include a complete representation of the host chromosome. Inseets are identified by loss of resistance, and complementation mapping allows pairing of specific changes in genome to specific phenotypical traits.
Having located and cloned the gene coding foe a target enzyme, it is possible to inactivate that enzyme by engineering the gene in the non-Cl-utilizing host. Specifically, a transposon i6 inserted in the gene, and the engineered gene is eeturned to the Cl-utilizing host where it replaces the wild-type gene on the chromosome by homologous recombination.
The Cl-utilizer gene peoduct is thus inactivated. By inactivating enzymes catalyzing undesired side reactions, yield of the desired compound is increased.
Thus, a third aspect of the invention features a vehicle for integrating DNA in the chromosome of a Cl-utilizer, which includes a homologous chromoscmal fragment containing the transposon, as well as the above-described replication and mobilization enabling elements of the cosmid vehicle. In preferred embodiments, the vehicle further includes a means of S preventing replication in the Cl-utilizing host including a temperature sensitive repressor; and the transposon is Tn5.
In a fourth aspect, the invention features a Cl-utilizing microorganism having such an engineered gene integrated in its chromosame. In preferred embodiments, the organism is a facultative methanol utilizer such as a Methylobacterium organophilum. The desired compound is an aromatic amino acid, e.g., phenylalanine, tyrosine, or tryptophan, and the undesired side pathways that are blocked involve systhesis of other aromatic amino acids. For example, the desired product is L-phenylalanine and the blocked side pathway steps include conversion of prephenate to p-hydroxyphenylpyruvate and conversion of chorismate to anthranilate.
In a fifth aspect, the invention features producing a desired compound by culturing a Cl-utilizing microorganism as described above.
Finally, the invention features a method of engineering a Cl-utilizing microorganism by identifying the gene to be engineered, transferring the gene to be engineered to a non-Cl-utilizer, altering the gene by inserting a site-directed transposon therein, and transferring the altered gene to a Cl-utilizing microorganism where it integrates in the chramosame.
Thus, according to a further aspect of the invention a Cl-utilizing microorganism is provided that is capable of synthesizing a desired compound by a first bioconversion pathway, said pathway comprising a first reaction in which a first intermediate compound is converted to a second intermediate compound, said microorganism comprising genetic material engineered from naturally occurring genetic material coding for an enzyme catalyzing a second .../7a - 7a -reaction in which said first intermediate is converted to a thirdintermediate compound, not in said synthesis pathway, said derived material being chromosomally located and comprising a DNA insert rendering the protein product of said derived genetic rnaterial ineffective to catalyze said second bioconversion, said insert coding for resistance to an antibiotic to which said Clutilizing microorganisrn is otherwise susceptible; wherein said derived genetic material is engineered by cloning said naturally occurring sequence, inserting said gene into a non-Cl-utilizing microorganisrn~
exposing said naturally occurring sequence to a transposon comprising said insert sequence, thereby creating a second sequence, and inserting said derived material into a Cl-utilizing microorganism, and causing it to integrate in said Cl-utilizer's chromosome; and whereby said DNA insert blocks said second reaction to prevent diversion of said first intermediate fr~n the synthesis of said desired compound, thereby increasing the level of said desired compound produced by said microorganism.
Other features and advantages will appear fr~n the following description of the preferred embodiment.

_ - 8 - 1 335~76 Description of the Preferred Embodiment We turn now to a description of the preferred embodiment of the invention, first briefly describing the drawings. -Fig. 1 is a flow diagram of a process for producing 5 the cloning vector pLA2901.
Fig. 2 is a flow diagram of a process for producing the cloning vectors pLA2905, pLA2910, and pLA2917.
Fig. 3 is a flow diagram of a process for producing the cloning vector pLA2920.
The preferred cloning vehicles are illustrated by the following descriptions of specific vehicles denoted pLA2920, pLA2905, pLA2910, and pLA2917; the two most preferred vehicles are pLA2917 and pLA2920. The latter two vehicles have been deposited with the American Type Culture Collection and given 15 accession nos. 39840 and 39841, respectively. Applicants recognize their obligation to notify the ATCC of the issuance of a patent on this application, to make the above deposits publicly available thereafter, and to replace the deposit should it die during the effectiveness of any patent issuing on this application.
We will describe first the construction of pLA2917 and the derivation of pLA2920 from it, thereby providing a detailed description of the elements of those plasmids.
We will then describe complementation analysis using those plasmids to characterize the genome of a Cl-utilizing microorganism, exemplified by a strain of Methylobacterium organophilum; we then will describe engineering selected genes of the Cl-utilizing microorganism and integration of those genes in the chromosome of that or another Cl-utilizing microorganism.
Finally, we will describe production of a desired product, exemplified by production of L-phenylalanine, by culturing the engineered Cl-utilizing microorganism on a C
carbon source.

, _ - 9 - 1 335576 Genecal Techniques Except as noted below or as noted elsewheee in this application, the experimental techniques that can be used to accomplish the engineecing described in this application are desccibed in Maniatis Molecular Cloninq, Cold Spcing Harbor Laboratocy, ~983.
Escherichia coli is coutinely cultuced on LB medium as desc~ibed in Miller (1972) Experiments in Moleculac Genetics, P. 433 (Cold Spcing Hacbo~ Laboratory, Cold Spcing Hacbor~
N.Y.). Unless othecwise specified, M. orqanophilum ATCC 27886, M. o~qanophilum DSM 760, and Ps. AM 1 strains ace grown on MacLennan salts (see Maclennan et al. (1971) J. Gen. Mic~obiol.
69:395-404) supplemented with sodium succinate (0.1%
weight/vol.) oc methanol (0.5% vol/vol).
Antibiotics, when added, are used at final concentrations of 25 ~g/ml (kanamycin) and 20 ~g~ml (tetracycline) for E. coli and 10 ~g/ml each foc methylotcophs.
Pcepacative amounts of plasmid DNA ace isolated fcom E. coli HB101 essentially by the method of Birnboim (1983) Methods Enzymol. 100:243-255. Cells ace lysed in alkaline SDS
followed by high salt pcecipitation. This matecial is centrifuged (8,000 cpm, 30 minutes) and the plasmid DNA is precipitated by the addition of 0.55 vol of cold isopropanol.
Plasmid DNA is then further purified by banding twice in ethidium beomide/CsCl dye bouyant density gcadients.
Plasmid DNA from recombinant clones is scceened by the minipcep pcocedure of Crosa et. al. [~Plasmids" (pp. 266-282) Manual Of Methods fo~ General Biochemistry, Gechardt ed., Am.
Soc. Miccobiol. Wash, D.C., L981] except that following isopropanol pcecipitation, cestrictable plasmid preps ace obtained by two successive precipitations with ethanol.
Chcomosomal DNA from wild-type strain xx is pucified fcom cells lysed in the following manner. A late log phase culture o M.
orqanophilum grown on Penassay broth is hacvested by centcifugation (8,000 cpm, 10 minutes) and washed once by centcifugation with 1 M NaCl. The pellet is cesuspended in 1 M
NaCl (20 ml/gm wet weight) and incubated at 55C foc 30 minutes. EDTA (ethylenediaminetetraacetate) is added to 0.1 M
and the incubation at 55C is continued for 15 minutes. Cells ace collected by centcifugation (8,000 cpm, 10 minutes) and cesuspended in TES (10 mM TRIS(hydcoxymethyl-amino methane)-HCl pH 8.0, 1 mM EDTA, 0.15 M NaCl) plus 10 mg/ml lysozyme. The cell suspensions ace incubated at 37C foc 45 minutes followed by the addition of sodium dodecyl sulfate (SDS) to 1%
(wt/vol). Aftec addition of SDS, the cells ace incubated foc 45 minutes at 65C. Chcomosomal DNA fcom cells lysed in the mannec desccibed above is pucified by the method of Maemuc ~1961) J. Mol. Biol. 3:208-218.
Where indicated, DNA is pucified fcom agacose gels by electcoelution onto dialysis membcanes accocding to the method of Yang et al. (1979) Methods Enzymol. 68:176-182.
Restciction endonucleases ace used acco~ding to manufactucecs' cecommendations (Bethesda Reseacch Labocatocies (BRL), Rockville, MD and Peomega Biotech Inc., Madison, Wisc.).
Nuclease Bal31 (BRL) is incubated with DNA at 0.09 units/~g DNA (500 ~1) at 32C in 20 mM TRIS-HCl pH 8.0, lZ mM
CaClz, 12 mM MgC12, 1 mM EDTA, 200 mM NaCl and 250 ~g/ml BSA. Samples (22 ~1) ace taken at cegular intecvals and the 25 ceaction stopped by the addition of 2 ~1 of 200 mM ethylene glycol-bis(B-amino ethyl ether)-N,N'-tetcacetate(EGTA).
T4 DNA polymecase (BRL) and deoxynucleotide tciphosphates ace used to genecate blunt ends in Bal31 tceated molecules. T4 DNA ligase (Pcomega Biotech, Inc.) and Bactecial Alkaline Phosphatase (BRL) ace used accocding to manufactucecs' cecommendations.
Bacteciophage lambda packaging extcacts and in vitco packaging is pecfocmed by the method of Hohns (1979) Methods Enzymol. 68:Z99-309 as modified by Ostcow et al. (1983) J. of Invest. Decmatol. 80:436-440.

-The Vehicles Fig. 1 outlines the steps in the construction of pLA2901 fcom pRK290, which is then modified as shown in Fig. Z
to yield pLA2905, pLA2910, and pLA2917.
Fig. 3 outlines the steps in forming pLA2920 from pLA2917.
Plasmid pLA2917 is based on the broad host cange mobile cloning vector pRK290 ~eported by Ditta et al. PNAS USA
77:7347-7351. pRK290 is a 20 kbp plasmid that can be mobilized fcom E. coli to several gram negative bacteria by RK2-decived plasmid pRK2013. Specifically, pLA2917 and pLA2920 include the kanamycin resistance gene of the TnS transposon, described below.
In order to incorporate the kanamycin resistance element of Tn5 into pRK290, pBR322 is digested with HindIII, and the HindIII fragment of t~ansposon Tn5 is cloned into pBR322.
Next, the kanamycin resistance element is excised from the resulting plasmid with BamHl. This element is inselted into pRK290 [Ditta et al. (1980) PNAS USA 77:7347-7351] by a BamHl/BqlII fusion to yield a 22.4 kbp plasmid, pLA2901.
Plasmid pLA2901 harbors genes coding for resistance to kanamycin and tetracycline. It contains 5 unique restriction sites: the EcoRl site of pRK290, the HindIII site of pBR322, and the BqlII, BstEII, and XhoI sites of Tn5. DNA insertions at the unique BqlII site can be detected by inactivation of Km ~esistance.
More versatile cosmid vectors, pLA2910 and pLA2917, are then constructed from pLA2901 in the following manne~. In order to generate unique restriction sites fo~ PstI and SalI, pLA2901 is linearized with XhoI and treated with Bal31 Nuclease. The extent of Bal31 digestion is followed by subcutting samples at 2 minute intervals with PvuII and monito~ing the PvuII fragment (originally 730 bp) of Tn5 on 4%

polyacrylamide gels according to the method of Rothstein et al.
(1980) Cell 19:795-805. An inc~ease in the mobility (dec~ease in size) of this f~agment indicates that Bal31 activity has p~oceeded past the SalI and adjacent PstI sites and into the ~egion of the PvuII flagment.
The cohesive ends (cos site) of bacte~iophage lambda a~e then int~oduced in ocder to geneeate a vecto~ with inc~eased DNA insect capacity. The o~igin of the lambda cos site in pLA2910 and pLA2917 is pMF517 disclosed by Feiss et al.
(1972) Gene 17:123-130. This plasmid is linea~ized with HindIII, treated with Nuclease Bal31 to ~educe the size of the cesulting cos f~agment by app~oximately 800 bp, and blunt ends a~e gene~ated with T4 DNA polyme~ase plus dNTP's. The ~esulting DNA is subcut with PvuII, sepalated on a 0.7% agaeose gel and the gel-pucified cos f~agment is ligated to pLA2901 which i8 t~eated as desc~ibed above.
Cosmid pLA2917 is shown in Fig. 2: cosmid pLA29L0 is the same as pLA2917, except that the lambda cos f~agment is in the opposite orientation. The cos site allows in vitro packaging into bacteeiophage lambda of inse~ts up to 30 kbp in length. Cosmid pLA2917 (21 kbp) contains 6 unique rest~iction enzyme cleavage sites: BqlII, (Km), BstEII, HindIII, HPaI~
Pstl, (Km), and SalI (Tc). Seve~al possible cas6ette6 exist fo~ the Km ~esistance gene. Fo~ example, a HPaI, EcoRV
f~agment of 1.4 kbp contains unique rest~iction sites for B~lII, HindIII and Pstl.
Plasmid pLA2920 is c~eated from pLA2917 by deleting the kanamycin ~esistance segment to avoid inte~nal ~ea~angement when the t~ansposon segment moves and to pe~mit selection of conjugants that have a Tn5 insertion. To const~uct pLA2920 from pLA2917, the latte~ was t~eated with HindIII and HpaI, and then with phage T4 DNA polymerase and the ~esulting st~and was ligated as shown in Fig. 3.

_ L3 - -The above-described vectors carcy ~esistance detecminants for kanamycin and/or tetracycline and have multiple unique ~est~iction endonuclease cleavage sites.
Insertional inactivation of kanamycin (BqlII and Pstl) and tetracycline (SalI) can be used to detect inseets. These vecto~s can t~ansfer efficiently to a broad host range including E. coli HBlOl, M. organophilum strain xx, M.
o~qanophilum DSM 761, Pseudomonas AM 1, Pseudomonas putida and Pseudomonas ae~uqinosa. The p~eviously desc~ibed pVK100 se~ies cosmids also have multiple drug resistance maekers; howeve~, the cosmids desc~ibed here have the additional advantage of utilizing Sau3A gene~ated partials of insert DNA to clone into the BqlII site. The tet~anucleotide ~ecognition sequence of Sau3A allows the cloning of a mo~e complete random set of partial digestion p~oducts than does a rest~iction endonuclease with a hexanucleotide recognition sequence. Indeed, examples of BqlII or Sau3A inse~ts greate~ than 25 kbps with no internal sites for BqlII andtor HindIII have been found in this laboratory. These libra~ies would not contain the entire genome.
Cha~acterization of the Cl-Utilizec's Genome A Cl-utilizing organism is selected to be transformed to the ultimate fe~mentation host. For example, a strain of Methylobacte~ium orqanophilum (ATCC 27886), a facultative methylotroph that p~efe~s methanol as a carbon and energy source, is a suitable sta~ting strain. Other suitable organisms include Methylophilus methylot~ophus, Pseudomonas aeruqinosa mutants, Pseudomonas AM 1, Methylobacte~ium orqanophilum DSM761, and Pseudomonas putida.
Once the ultimate fe~mentation host has been selected, its genome can be mapped and cha~acterized to enable engineering that will enhance fu~ther the yield of the desi~ed compound. Such complementation mapping is illust~ated by the following description of mapping M. orqanophilum.

M. orqanophilum is naturally resistant to trimethoprin (100 ~g/ml), mutates spontaneously at high frequency to streptomycin resistance, and due to the extended period necessary to select transconjugants (10-lZ days), ampicillin is not sufficiently stable to allow selection of resistant colonies. As a result, these thcee antibiotics, frequently used foe selection in genetic studies of other gram negative bacteria, cannot be used with this organism. M. orqanophilum strain xx is very sensitive ( <10 ~g/ml) to kanamycin and a convenient source of this antibiotic cesistance determinant is the neomycin phosphotransferase gene of TnS as described by Beck et al. (198Z) Gene 19:3Z7-336. These factoes, in addition to convenient restciction enzyme cleavage sites available for the isolation of this gene, are desirable factors.
A genomic lib~ary of M. orqanophilum strain xx DNA is constructed in the BqlII site of pLAZ917. A Sau3A partial digest of strain xx chromosomal DNA is size fractionated on a 0.5% agarose gel (500 ml) and partial digestion peoducts ranging f~om ZZ to 30 kbp are isolated. pLA2917 is linearized with BalII, tceated with bacterial alkaline phosphatase, mixed with the M. orqanophilum strain xx DNA fragments, and the DNA
mixture is treated with DNA ligase. Vector and chromosomal DNA
are ligated at a vector-to-insert ratio of 2:1 and a total DNA
concenteation of 300 ~g/ml. The ligation mixture is packaged into bacteciophage lambda and the resulting phaqe particles are used to infect E. coli HB101 (ATCC No. 33694 ). Several thousand transfectants can be obtained, and 2,000 are picked for further study. Tc clones contain insert DNA with an average size of 28 kbp.
Complementation assays can be performed by patch plate matings. Fifty recombinant E. coli HB101 donors containing pLA2917 with inserts, arranged in a grid pattern on a master plate, can be replica plated onto a lawn of E. coli HB101 (pRKZ013) and a Ps. aeruqinosa auxotroph. Matings are !

- 1 33-~576 _ - - 15 -incubated at 37C for nine hours and then replica plated onto selective media. In this way, mutant clones exhibiting a particular phenotypical trait (such as auxotrophes for some, but not all, aromatic amino acids) are complemented by specific ~
clones containing specific recombinant plasmids, thus enabling identification and mapping of the gene responsible for that trait.
Thus, the ability of recombinant molecules to complement mutants unable to grow without an amino acid is determined by patch plate matings. Plates are examined and donors are scored for the ability to complement markers in the recipients i.e., to restore ability to grow without the amino acid.
DNA from complementing clones is subcloned into pLA2920 and mutagenized in vivo with Tn5 in E. coli HslOl as described below. Since Tn5 does not appear to transpose in M.
organophilum, these can be mobilized into strain xx wild type to create mutants by marker exchange. This establishes which functions are coded for by each cloned fragment and enables determination of linkages between other C-l markers in _. organophilum strain xx .
Engineering the Cl-Utilizing Host Where production of a spçcific compound is desired, mutants of the organism that are resistant to analogs of the product can be selected. For example, where phenylalanine production is sought, the original Methylotrophs are mutated chemically (for example, with N-nitrosoguanidine) and the mutants are exposed to 5-fluorophenylalanine. Resistant mutants such as ATCC No. ~ ~P~ ~ are selected.
Having identified and cloned a gene for a side reaction which lessens product yield as described above, that gene can be rendered inactive as follows.
A plasmid such as pLA2920 containing the gene of _ - 16 - 1 3 3 5 5 7 6 interest is inserted into E. coli and is mutagenized by t~ansposition of Tn5, a discrete 5.7 kilobase segment of bactecial DNA which can inseet at high frequency into numecous sites in gram negative bacteria. It encodes resistance to kanamycin and neomycin in bacteria. Tn5 is disclosed in Berg et al. Bio/Technology July, 1983, pp. 417-435. A suitable soucce of TnS is plasmid pJBJI eeported by Becinger et al.
(1978) Natu~e 276:633-634. The transposon can move to new locations in DNA molecules without ~elying on extensive DNA
sequence homology between the insected element and the insertion site, and without relying on ec genes needed for classical homologous crossing over. That transposition is marked by an antibiotic resistance marker on TnS transposon plasmid. By restriction mapping techniques, it is possible to select events in which the transposon has inserted into the gene of interest.
The selected E. coli organisms are then conjugated with the ultimate Cl-utilizing host, and the gene o~ interest containing the inactivating insert integ~ates into the chromosome to ~eplace the wild-type active gene.
In this way, genes whose protein products catalyze undesired side reactions are inactivated. The resulting Cl-utilizing organism provides enhanced yields of desired compounds.
Fermentation The enginee~ed CL-utilizing microorganism may be cultured according to known techniques using a Cl compound, p~eferably methanol as the primary source of carbon and energy. The desired compound is harvested according to known techniques. For example, the fecmentation conditions described at p. 1089 in Demain at al. (1981) Appl. and Env. Microbiol 41:1088-1096 may be used, and the product recovery technique described at p. 777 in Yamada et al. (1981) Appl. and Env.
Microbiol 42:773-778 may be used.

Othec Embodiments Othe~ embodiments a~e within the following claims.
Foc example, othec tcansposons, othec antibiotic cesistance tcaits, and othec ceplicons may be used. Similacly, othe~
Cl-utilizing o~ganisms may be used, and other desi~ed compounds may be pcoduced.

Claims (27)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A cloning vehicle comprising:
a replication determinant effective for replicating said vehicle in a non-C1-utilizing host and in a C1-utilizing host;
DNA effective to allow said vehicle to be mobilized from said non-C1-utilizing host to said C1-utilizing host in the presence of a mobilizing plasmid;
DNA providing resistance to a first antibiotic and comprising an internal recognition site for a restriction endonuclease, said C1-utilizing host being naturally susceptible to said first antibiotic;
said restriction recognition site generating fragments that are ligatable to DNA digested by a restriction endonuclease that recognizes sites consisting of four nucleotides or less;
DNA providing resistance to a second antibiotic, said non-C1-utilizing host being susceptible to said first and to said second antibiotic; and a cos site;
whereby said vehicle is capable of:
1 ) receiving, at said restriction nuclease recognition site, an insertion of DNA comprising a gene-containing fragment of the digested genome of said C1-utilizing organism which encodes an enzyme necessary for synthesis of an aromatic amino acid, thereby inactivating resistance to one of said antibiotics as a marker of said insertion;
2) forming a cosmid structure;
3) infecting said non-C1-utilizing host; and 4) mobilizing into a mutant of said C1-utilizing host that is mutated in said gene, whereby said gene-fragment containing vehicle complements said mutant, thus signaling the function of said gene.

2. The vehicle of claim 1 wherein said replication determinant comprises the replicon of pRK290.

3. The vehicle of claim 1 wherein said first antibiotic is kanamycin or tetracycline.

4. The vehicle of claim 1 wherein said cos site comprises the cos gene from bacteriophage lambda.

5. The vehicle of claim 1 wherein said DNA effective to allow said vehicle to be mobilized is derived from pRK290.

6. The vehicle of claim 5 wherein said mobilizing plasmid is pRK2913.

7. The vehicle of claim 1 wherein said C1-genome-digesting restriction endonuclease is Sau3A.

8. The vehicle of claim 7 wherein said recognition site of said first antibiotic resistance gene is Bg1II or Sau3A.

9. The vehicle of claim 1 wherein said vehicle is selected from pLA2901, pLA2905, pLA2910, and pLA2917.

10. The vehicle of claim 9 wherein said vehicle is pLA2917 having ATCC accession no. 39866.

11. A method of mapping a gene of interest of a C1-utilizing organism using the vehicle of claim 1 comprising:
digesting the genome of said C1-utilizing organism with said restriction endonuclease that recognizes a 4-nucleotide base-pair sequence, to generate gene-containing fragments, the gene being one which encodes an enzyme necessary for synthesis of an aromatic amino acid;
inserting one of said gene-containing fragments into said recognition site of said first antibiotic resistance gene of a said vehicle;
packaging said gene fragment-containing vehicle into a cosmid structure;
infecting a said non-C1-utilizing host with said structure;
selecting said infected non-C1-utilizing host from a heterogeneous population by selecting organisms that are resistant to said second but not said first antibiotic; and mobilizing said gene fragment-containing vehicle from said selected non-C1-utilizing host into a C1-utilizing host that is mutated in said gene of interest; and determining whether said vehicle complements said mutated C1-utilizer and therefore contains said gene of interest.

12. The method of claim 11 wherein said vehicle is pLA2901, pLA2905, pLA2910, or pLA2917.

13. The method of claim 11 wherein said C1-utilizing organism is Methylobacterium organophilum.

14. A cloning vehicle comprising:
a replication determinant effective for replicating said vehicle in a non-C1-utilizing host and in a C1-utilizing host;
DNA effective to allow said vehicle to be mobilized from said non-C1-utilizing host to said C1-utilizing host in the presence of a mobilizing plasmid;
DNA providing resistance to a first antibiotic;
a C1-utilizer gene, which encodes an enzyme necessary for synthesis of an aromatic amino acid, inactivated by a transposon containing a second antibiotic to which said C1-utilizing host is susceptible; and a cos site.

15. The vehicle of claim 14 wherein said vehicle further comprises a means of preventing replication in said C1-utilizing host comprising a temperature sensitive repressor.

16. The vehicle of claim 14 wherein said transposon is Tn5.

17, A C1-utilizing microorganism capable of synthesizing a desired compound by a first bioconversion pathway, said pathway comprising a first reaction in which a first intermediate compound is converted to a second intermediate compound, said microorganism comprising genetic material engineered from naturally occuring genetic material coding for an enzyme catalyzing a second reaction in which said first intermediate is converted to a third intermediate compound, not in said synthesis pathway, said derived material being chromosomally located and comprising a DNA insert rendering the protein product of said derived genetic material ineffective to catalyze said second bioconversion, said insert coding for resistance to an antibiotic to which said C1-utilizing microorganism is otherwise susceptible;
wherein said derived genetic material is engineered by cloning said naturally occurring sequence, inserting said gene into a non-C1-utilizing microorganism, exposing said naturally occurring sequence to a transposon comprising said insert sequence, thereby creating a second sequence, and inserting said derived material into a C1-utilizing microorganism, and causing it to integrate in said C1-utilizer's chromosome; and whereby said DNA insert blocks said second reaction to prevent diversion of said first intermediate from the synthesis of said desired compound, thereby increasing the level of said desired compound produced by said microorganism.

18. The microorganism of claim 17 wherein said microorganism is a methanol-utilizing organism.

19. The microorganism of claim 18 wherein said microorganism is a facultative methanol-utilizing organism.

The microorganism of claim 19 wherein said microorganism is a member of the species Methylobacterium organophilum.

21. The microorganism of claim 20 wherein said microorganism comprises a cloning vehicle comprising:
a replication determinant effective for replicating said vehicle in a non-C1-utilizing host and in a C1-utilizing host;
DNA effective to allow said vehicle to be mobilized from said non-C1-utilizing host to said C1-utilizing host in the presence of a mobilizing plasmid;
DNA providing resistance to a first antibiotic;
a C1-Utilizer gene inactivated by a transposon containing a second antibiotic to which said C1-utilizing host is susceptible; and a cos site.

22. The microorganism of claim 21 wherein said microorganism comprises the vehicle pLA2920 having a transposon-inactivated gene inserted therein.

23. The microorganism of claim 17 wherein said desired compound is an aromatic amino acid.

24. The microorganism of claim 23 wherein said non-pathway enzyme is selected from enzymes catalyzing the following:

1) formation of prephenate from chorismate;
2) formation of anthranilate from chorismate;
3) formation of phenylpyruvate from prephenate;
and 4) formation of p-hydroxyphenylpyruvate from prephenate.

25. The microorganism of claim 24 wherein said desired compound is phenylalanine and said microorganism comprises genetic material engineered from naturally occurring genetic material coding for enzymes catalyzing conversion of prephenate to p-hydroxyphenylpyruvate, and for bioconversion of chorismate to anthranilate, and said derived material comprises at least one DNA
insert rendering the protein product of said derived genetic material inneffective to catalyze at least one said conversion.

26. A method of making a desired compound by culturing the microorganism of claim 17.

27. A method of making a desired amino acid selected from phenylalanine, tyrosine, and tryptophan, comprising culturing the microorganism of claim 17 in a medium that includes a sufficient amount of said non-selected aromatic acids to support said microorganism.