Claims
- 1. A cloning vehicle selected from the group consisting of the following plasmids: pLA 2917 as deposited in E. coli strain HB101 as ATCC 39840; pLA 2920 as deposited in E. coli strain HB101 as ATCC 39841; pLA 2901; pLA 2905; pLA 2910; and derivatives engineered therefrom.
- 2. A C.sub.1 -utilizing microorganism of the species Methylobacterium organophilium, the species Pseudomonas aeruginosa, or the strain Pseudomonas AM 1, and wherein said microorganism comprising a cloning vehicle selected from the group consisting of the following plasmids; pLA 2917 as deposited in E. coli strain HB101 as ATCC 39840; pLA 2920 as (deposited in E. coli strain HB101 as ATCC 39841; pLA 2901; pLA 2905; pLA 2910; and derivatives engineered therefrom.
- 3. The microorganism of claim 2 wherein said microorganism is Methylobacterium organophilum deposited as ATCC 39866 or a derivative thereof.
BACKGROUND OF THE INVENTION
This is a continuation of co-pending application Ser. No. 650,825 filed on 9/14/84 abandoned.
This invention relates to cloning vehicles and methods for engineering C.sub.1 -utilizing microorganisms producing a desired compound; it also relates to the resulting engineered microorganisms.
In theory, organisms that use C.sub.1 compounds as a source of carbon and energy should be useful for fermentation processes producing desired compounds, because C.sub.1 compounds such as methanol are relatively cheap and available. There are various disclosures of fermentation processes based on C.sub.1 -utilizing organisms. For example, Kono U.S. Pat. No. 3,663,370 discloses synthesizing glutamic acid using specific strains of Methanomonas, Protaminobacter, and Microcyclus. Nakayama U.S. Pat. No. 3,907,637 discloses synthesizing L-lysine by fermentation of a mutant Protaminobacter capable of utilizing methanol. Gatenback U.S. Pat. No. 3,963,572 discloses synthesizing L-tryptophan from indole or derivatives thereof using Pseudomonas AM 1 or Methylomonas methanolica, in which methanol is the primary source of carbon. Kiyoshi et al. (1975) Jap. Appl'n No. 29721/75 discloses synthesizing L-tryptophan using mutants derived from the genera Pseudomonas, Methanomonas, or Protaminobacter.
It is desirable to engineer organisms to improve product yield; however, engineering C.sub.1 -utilizing microorganisms is generally difficult because their genomes are relatively poorly characterized compared to organisms such as E. coli, and it is difficult to isolate stable mutants. Moreover, engineering C.sub.1 -utilizers is also difficult because of the lack of vehicles that directly transform C.sub.1 -utilizers; thus it is difficult to develop efficient gene transfer systems for C.sub.1 -utilizers. Finally, antibiotic resistance characteristics of C.sub.1 -utilizing microorganisms may not be 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 C.sub.1 -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 [Ditta et al. (1980) PNAS USA 77:7347];
(2) pLAFR1 is a mobilizable cosmid derivative of pRK290 containing a gene for tetracycline resistance; pLAFR1 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 genes 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 aeruginosa, Pseudomonas AM1, Methylosinus trichosporium OB36, and Methylobacterium organophilum xx [Holloway (1981) Microbial Growth on C-1 Compounds, H. Dalton Ed., London p. 317].
(5) pM061 is a conjugative plasmid derived from R68.45 with enhanced chromosomal mobilization host range and [stability of R' plasmid derivatives - ? in article]. Transfer frequencies reportedly are similar 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 AM1 [Bagdasarian et al. (1981) Gene 16:237; Gautier et al. (1980) Mol. Gen. Genet. 178:375]; and
(7) pKT230 and pKT231 are mobilizable derivatives of RSF1010 containing cloning sites in resistance genes to allow selection for inserts [Bagdasarian (1981), cited above]>
Moore et al. (9183) J. Gen. Microbiol. 129:785-799 disclose a method of complementation mapping of Methylophilus methylotrophus using a plasmid that is an R.sup.1 derivative of plasmid pM0172.
Gautier et al. (1980) Mol. and Gen. Genetics 144:243-251 disclose cloning the wild-type methanol dehydrogenase gene of Psuedomonas AM 1 in E. coli using plasmid R1162. The methanol dehydrogenase gene is then transferred to a methanol dehydrogenase mutant of Pseudomonas AM 1 using RP4 to mobilize the hybrid plasmid.
O'Connor et al. (1978) J. Gen. Microbiol. 104:105-111 disclose transforming Methylobacterium organophilum in order to study the linkage of C.sub.1 -utilizing genes in that organism.
Warner et al. (1980) FEMS Microbiol. Letters 7:181-185 and Jeyaseelan et al. (1979) FEMS Microbiol. Letters 6:87-89 disclose an attempt to use a broad host-range plasmid, R68.45, to map chromosomes of several genera of C.sub.1 -utilizing organisms.
Tatra et al. (1983) J. Gen Microbiol. 129:2629-2632 disclose that R68.45, referred to above, can mobilize the chromosome of Pseudomonas AM 1. Markers are linked to genes to demonstrate their location.
We have discovered versatile cosmid cloning vehicles that can be used generally to characterize and engineer the genome of C.sub.1 -utilizing microorganisms. The vehicles have a broad host range and can transfer by conjugation between a non-C.sub.1 -utilizing host and a C.sub.1 -utilizing host so that complementation mapping can be used to characterize the genome of the C.sub.1 -utilizing organism, and having done so, to engineer selected genes to increase production of desired compounds by the C.sub.1 -utilizing organism.
By the term C.sub.1 -utilizing microorganisms, we mean to include all organisms that can use as a carbon/energy source C.sub.1 compounds such as methane or methanol. We specifically mean to include facultative and obligate C.sub.1 -utilizing microorganisms; methane and methanol-utilizing microorganisms; and type I (i.e. those using the ribulose monophosphase pathway) and type 2 (i.e. those using the serine pathway) microorganisms. The term also includes bacteria as well as yeast or other C.sub.1 -utilizing microorganisms. For a general discussion of classification of C.sub.1 -utilizing microorganisms, see Haber et al. (1983) Science 221:1147-1153 and references cited therein. References herein to genus and species refer to organisms as classified in Buchanan et al., The Shorter Bergey's Manual For Determinative Bacteriology (Williams & Wilkins, 1982).
In a first aspect, the invention generally features a cloning vehicle comprising: a replication determinant effective for replications the vehicle in a non-C.sub.1 -utilizing host and in a C.sub.1 -utilizing host; DNA effective to allow the vector to be mobilized from the non-C.sub.1 utilizing host to the C.sub.1 utilizing host; DNA providing resistance to at least two antibiotics to which the wild-type non-C.sub.1 -utilizing host is susceptible, at least one of the antibiotic resistance markers having a recognition site for a restriction endonuclease that generates fragments that are ligatable to DNA digested by a restriction endonuclease that recognizes sites consisting of four nucleotides or less; and a cos site.
The vehicle is capable of: (1) receiving, at the restriction nuclease recognition site, an insertion of DNA comprising a gene-containing fragment of the digested genome of the C.sub.1 -utilizing organism, thereby inactivating resistance to one of the antibiotics as a marker of the insertion; (2) forming a cosmid structure; (3) infecting the non-C.sub.1 -utilizing host; and (4) conjugating with a mutant of the C.sub.1 -utilizing host that is mutated in the gene, whereby the gene-containing fragment vehicle complements the mutant, thus signaling the function of the gene.
In preferred embodiments, the replication determinant is a replicon such as the one reported in pRK290; the antibiotic resistance is to kanamycin or tetracycline; the cos site is from phage lambda; the DNA effective to allow the vehicle to be mobilized is derived from pRK290; the mobilizing plasmid is pRK2913; the C.sub.1 -genome-digesting restriction endonuclease is Sau3A; the vehicle is selected from pLA2901, pLA2905, pLA2910, and pLA2917; and the internal recognition site of the first antibiotic resistance gene is BglII or Sau3A.
In a second aspect, the invention features using the above-described vehicle for complementation mapping of a C.sub.1 -utilizing organism by: digesting the genome of the C.sub.1 -utilizing organism with a restriction endonuclease that recognizes a 4-nucleotide base-pair 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 structure; infecting a non-C.sub.1 -utilizing host; selecting the infected non-C.sub.1 -utilizers from a heterorgeneous population that are resistant to the second but not the first antibiotic; mobilizing the gene fragment-containing vehicle from the selected non-C.sub.1 -utilizing host into a C.sub.1 -utilizing host that is mutated in the gene of interest; and determining whether the gene fragment-containing vehicle complements the mutant and therefore whether the vehicle contains the gene of interest.
In preferred 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 C.sub.1 -utilizing organism is Methylobacterium organophilum.
The above-described vehicle is particularly useful for characterizing the genome of the C.sub.1 -utilizer by transferring those genes to a mutant C.sub.1 -utilizer or to a non-C.sub.1 -utilizer and performing complementation mapping. Specifically, the cosmid will harbor coding capacity sufficient such that after digesting the C.sub.1 -utilizer chromosome and packaging the segments, the phage particles used to infect the non-C.sub.1 -utilizing host, specifically E. coli, include a complete representation of the host chromosome. Inserts are identified by loss of resistance, and complementation mapping allows pairing of specific changes in genome to specific phenotypical traits.
Having locating and cloned the gene coding for a target enzyme, it is possible to inactivate that enzyme by engineering the gene in the non-C.sub.1 -utilizing host. Specifically, a transposon is inserted in the gene, and the engineered gene is returned to the C.sub.1 -utilizing host where it replaces the wild-type gene on the chromosome by homologous recombination. The C.sub.1 -utilizer gene product 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 C.sub.1 -utilizer, which includes a homologous chromosomal 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 preventing replication in the C.sub.1 -utilizing host including a temperature sensitive repressor; and the transposon is Tn5.
In a fourth aspect, the invention features a C.sub.1 -utilizing microorganism having such an engineered gene integrated in its chromosome. 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 synthesis 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 C.sub.1 -utilizing microorganism as described above.
Finally, the invention features a method of engineering a C.sub.1 -utilizing microorganism by identifying the gene to be engineered, transferring the gene to be engineered to a non-C.sub.1 -utilizer altering the gene by inserting a site-directed transposon therein, and transferring the altered gene to a C.sub.1 -utilizing microorganism where it integrates in the chromosome.
Other features and advantages of the invention will appear from the following description of the preferred embodiment and from the claims.
Government Interests
This invention was made with Government support under Contract No. (DE-AC02-82ER12029) awarded by the Department of energy. The Government has certain rights in this invention.
US Referenced Citations (3)
Foreign Referenced Citations (1)
| Number |
Date |
Country |
| 2979175 |
Nov 1980 |
JPX |
Non-Patent Literature Citations (18)
| Entry |
| Haber et al. (1983), Science, 221:1147-1153. |
| Ditta et al. (1980), PNAS USA, 77:7347. |
| Friedman et al. (1982), Gene, 18:289. |
| Knauf et al. (1982), Plasmid, 8:45. |
| Holloway (1981), Microbial Growth on C--1 Compounds, H. Dalton Ed., London, pp. 317-324. |
| Rieb et al. (1980), Genet. Res., 36:99. |
| Bagdasarian et al. (1981), Gene, 16:237. |
| Gautier et al. (1980), Mol. Gen. Genet., 178:375. |
| Moore et al. (1983), J. Gen. Microbiol., 129:785-799. |
| Tatra et al. (1983), J. Gen. Microbiol., 129:2629-2632. |
| O'Connor et al. (1978), J. Gen. Microbiol., 104:105-111. |
| Warner et al. (1980), FEMS Microbiol. Letters, 7:181-185. |
| Jeyaseelan et al. (1979), FEMS Microbiol. Letters, 6:87-89. |
| Haber, 1984, "Plasmid DNA from an Obligate Methanetroph: Physical and Conjugative Characteristics", Ph.D. Dissertation, Univ. Wisc. Madison, pp. 128-156. |
| Gowrishankar et al. 1982, "Regulation of Phenylalanine Biosynthesis in E. Coli K--12 . . . ", J. Bact., v 150(3), pp. 1130-1137. |
| Toukdarian et al. (1984), Journal of Bacteriology, 157(3):979-983. |
| Suzuki et al. (1977), J. Ferment. Technol., 55(s):466-475. |
| Allen et al. (1984), Microbial Growth on C1 Compounds, Proc. 4th Int'l. Symp. Crawford, Ed. |
Continuations (1)
|
Number |
Date |
Country |
| Parent |
650825 |
Sep 1984 |
|
Patent Grant
4824786
