WO1992003546A1

WO1992003546A1 - Enhancement of production of native products in corynebacterium by expression of cloned bacterial hemoglobin

Info

Publication number: WO1992003546A1
Application number: PCT/US1991/005825
Authority: WO
Inventors: Fred C. Sander; Deborah L. Leenutaphong; Jorge L. Galazzo; Dallas E. Hughes; James E. Bailey; Chaitan S. Khosla
Original assignee: Exogene Corporation
Priority date: 1990-08-23
Filing date: 1991-08-22
Publication date: 1992-03-05
Also published as: ZA916702B; IL99272A0

Abstract

The invention relates to a method for expressing bacterial hemoglobin in Coryneform and, by this means, for enhancing production of amino acids or other metabolites by Coryneform. The invention also relates to expression vectors for expression of native and heterologous genes in Coryneform.

Description

ENHANCEMENT OF PRODUCTION OF NATIVE PRODUCTS

IN CORYNEBACTERIUM BY EXPRESSION OF CLONED

BACTERIAL HEMOGLOBIN

TECHNICAL FIELD

This invention relates to the expression of Vitreoscilla hemoglobin Coryneform bacteria to enhance amino acid production and growth characteristics.

This invention relates to all members of the Coryneform group of bacteria including the genera Corvnebacterium, revibacterium, Arthrobacter, Microbacterium and Cellulomonas.

BACKGROUND ART

The use of an intracellular globin to enhance growth and productivity in Corvnebacterium is based on several observations. First, oxygen requirements during Corvnebacterium fermentations are high, and increased oxygen uptake results in increased amino acid production (Yamada, et al . , "The Microbial Production of Amino Acids", Halstead Press, 1970). Also, altered redox state is known to influence amino acid production by Coryneform bacteria (Radjai, et al., Biotech, and Bioeng. Symposium No. 14, 1984). Second, we have observed that in individual cells there exists a possibly significant diffusional barrier between environmental oxygen and the cytochromes where the energy-producing reactions necessary for cell growth occur (Wittenburg, et al., J. Biol. Chem., 249:4057 (1974); Khosla and Bailey, J. Bol. Biol., 210: 79 (1989)). Third, the globins represent a family of heme-containing proteins that reversibly bind oxygen and are thus able to enhance the oxygen transfer rate to cells in multicellular organisms. Fourth, the expression of bacterial hemoglobin has been shown to enhance the growth properties of the bacterium Escherichia coli. especially under conditions of reduced oxygen (Khosla and Bailey, Nature, 331:633, 1986). Such enhancement may result in part from an altered intracellular redox state.

The bacteria of the Coryneform group are the single most important bacterial group used for the commercial production of amino acids (Atkinson and Mauituma, Biochemical Engineering and Biotechnology Handbook. MacMillan, England, 1987). Examples of widelyused amino acid products produced in Corvnebacterium fermentations include taste enhancers in human food, especially mono-sodium glutamate, and animal feed supplements, notably L-lysine. Also, production by Corvnebacterium of aromatic amino acids, for example, L-phenylalanine, L-tyrosine, and L-tryptophan, is of increasing industrial interest (Ito, et al. , Agricultural and Biological Chemistry 54 : 70 , 1990) . Phenylalanine is an important component of the new sweetener known as "Aspartame", and tryptophan, is used as an ingredient in animal feeds and in medicines. Corvnebacterium are also used in the production of nucleotides and related compounds (Shiio and Nakamoir, Fermentation Process - Development of Industrial Organisms, J. O. Newary, ed., Marcel Dekker, N.Y., 1989). Extensive "classical" mutagenesis of Corynebacterium has been used to develop strains which overproduce a variety of nucleotides, nucleosides, and amino acids. However, further increase in production by such traditional techniques is limited since the cumulative effect of many mutations results in almost complete physiological deregulation (Martin, et al., Bio\Technology, 5:137, 1987).

Corvnebacterium are facultative aerobes in which the accumulation of amino acids depends upon a metabolic balance that is greatly affected by environmental factors. Among these factors, the most important are the concentrations of oxygen, ammonium ion, phosphate, biotin, and the culture pH. A typical Corynebacterium qlutamicum fermentation begins with the growth of cells to high densities (growth phase). There is little glutamic acid production during growth phase. The final cell densities achieved are usually limited by the supply of the growth factor biotin, or by the addition of penicillin to the fermentation media late in the growth phase. Through an incompletely understood mechanism (s) the removal of biotin or the addition of penicillin causes changes in the cell wall synthesis, followed by induction of glutamic acid production and cessation of cell growth. After cessation of growth, amino acid production (production phase) is maintained as long as possible by supplying the appropriate nutrients. Eventually, acidic waste products accumulate and the cells die. Environmental factors which influence the production of specific amino acids in Corynebacterium have been investigated in detail. For example, oxygen deficiency in glutamic acid fermentations has been shown to be the dominant factor in switching the fermentation product from glutamate to succinate and lactate (Yamada, et al., "The Microbial Production of Amino Acids", Halsted Press, 1970). Both too little or too much dissolved oxygen in the fermentation media are associated with impaired cell growth and poor glutamic acid production. with sufficient, but not excessive, dissolved oxygen, glutamic acid production is high and is proportional to the rate of oxygen utilization by the cells.

The mechanism by which glutamic acid production in Corynebacterium is regulated is most influenced by two major factors: oxygen demand and biotin concentration (Hirose, "The Microbial Production of Amino Acids", Halsted Press, 1970) . Oxygen demand is closely correlated with cell growth, glutamic acid formation, and utilization of substrate. The biotin concentration in the medium controls the rate of substrate utilization and hence the cell oxygen demand. Higher concentrations of biotin result in increased rate of substrate utilization and oxygen demand.

The effect of bacterial hemoglobin expression on growth of a unicellular organism was investigated by Khosla and

Bailey (Khosla and Bailey, ibidl. The bacterial hemoglobin was originally discovered in the obligate aerobic bacterium, Vitreoscilla (Tyres and Webster, J.

Biol. Chem., 253:6988, 1978). The hemoglobin is a soluble, dimeric protein that combines with oxygen and displays a spectral response to carbon monoxide binding characteristic of eukaryotic hemoglobins (Wakabayashi, et al., Nature. 332:481, 1986). It was conjectured by

Wakabayashi (ibid) that the hemoglobin protein functioned as an "oxygen storage trap" for Vitreoscilla and thus allowed it to propagate under oxygen-poor conditions.

The gene for the Vitreoscilla hemoglobin has been isolated along with its native transcriptional regulatory sequences (Khosla and Bailey, Mol. Gen. Genet..214:158, 1988). Interestingly, this gene was expressed from its native promoter when introduced into E. coli. Of particular interest was the observation that expression of hemoglobin was regulated by the culture oxygen content such that maximal induction occurred under microaerobic conditions. Under fed-batch fermentation conditions, E. coli cells expressing hemoglobin displayed significantly higher specific growth rates and achieved final cell densities 2-3-fold those attained by non- expression strains (Khosla and Bailey, Nature. 331:633, 1988).

SUMMARY OF THE INVENTION

The present invention relates to oxygen-binding proteins, particularly hemoglobins, a recombinant-DNA method of producing same, and to portable DNA sequences capable of directing intracellular production of these oxygenbinding proteins in Coryneform. The present invention also relate vectors containing these portable DNA sequences. One object of the present invention is to provide a recombinant-DNA method for the production of these oxygen-binding proteins. To facilitate the recombinantDNA synthesis of these oxygen-binding proteins, it is a further object of the present invention to provide portable DNA sequences capable of directing intracellular production of oxygen-binding proteins in Coryneform. It is also an object of the present invention to provide cloning vectors containing these portable sequences. These vectors are capable of being used in recombinant Coryneform to enhance the growth characteristics of organisms, and to produce useful quantities of oxygenbinding proteins. In cells augmented by intracellular synthesis of oxygen-binding proteins, the production of amino acids, nucleotides and other products can also be enhanced. The present invention also provides novel methods and materials for expression of cloned genes in Coryneform. A preferred expression vector, a recombinant-DNA method of producing same, and a preferred portable DNA sequence capable of directing the translation and transcription initiation and control of the expression of desired gene products are disclosed.

Additionally, in accordance with the purposes of the present invention, a preferred recombinant-DNA method is disclosed which results in manufacture by cells of the genus Coryneform of the oxygen-binding proteins using the preferred portable DNA sequences.

The accompanying drawing, which is incorporated in and constitutes a part of this specification, illustrates ne embodiment of the invention and, together with the description, serves to explain the principles of the invention.

BRIEF DESCRIPTION OF THE DRAWING

Figure 1 is a partial restriction map of plasmid pBHb3. BEST MODES FOR CARRYING OUT THE INVENTION

Reference will now be made in detail to the present preferred embodiments of the invention, which, together with the drawing and the following examples, serve to explain the principles of the invention. The present invention provides methods for expression of hemoglobin in Coryneform bacteria to enhance aerobic growth, respiration, and/or the productivity of amino acids, nucleotides or other πietabolites. As used herein, the phrase "portable DNA sequence" is intended to refer either to a synthetically produced nucleotide sequence or to a restriction fragment of a naturally-occurring DNA sequence. The phrase "oxygen-binding protein" is intended to mean a protein having oxygen-binding activity, with a primary structure as defined by the codons present in the deoxyribonucleic acid sequence which directs intracellular translation and which may or may not include post-translational modifications. Such post-translational modifications may include, for example, association with a heme prosthetic group. It is further intended that the term "oxygen-binding protein" refers to its intracellular forms which are excretable or non-excretable.

In a particularly preferred embodiment, portable DNA sequences are provided which are capable of directing intracellular production of a hemoglobin biologically equivalent to that isolated from the filamentous bacterium, Vitreoscilla. By "biologically equivalent", as used herein, it is meant that a protein, produced using a portable DNA sequence of the present invention, is capable of binding oxygen in the same fashion, but not necessarily to the same degree, as the homodimeric soluble heme protein (subunit MW 15,775) isolated from Vitreoscilla.

The portable DNA sequences of the present invention may also include DNA sequences downstream from a promoter/regulator which code for at least one foreign protein. For purposes of this specification, "foreign protein" is intended to mean a protein with a primary structure as defined by the codons present in the deoxyribonucleic acid sequence which directs translation to the corresponding amino acid sequence, and which may or may not include post-translational modifications. It is further intended that the term "foreign protein" refers to its intracellular forms which are excretable or non-excretable. It is intended that promoter/regulator sequences of the present invention may control and initiate transcription and translation of many other endogenous and/or exogenous foreign proteins, such as, proteins involved in amino acid synthesis.

The practice of the present invention will employ, unless otherwise indicated, conventional molecular biology, microbiology, and recombinant DNA techniques with the skill of the art. Such techniques are explained fully in the literature. See, e.g., Maniatis, Fritsch & Sambrook, "Molecular Cloning: A Labortory Manual" (1982); "DNA Cloning: A Practical Approach", Volumes I and II (D.N. Glover ed. 1985); "Oligonucleotide Synthesis" (M.J. Gait ed. 1984); "Nucleic Acid Hybridization" (B.D. Hames & S.J. Higgins eds. 1985); "Transcription and Translation" (B.D. Hames & S. J. Higgins eds. 1984) ; "Animal Cell Culture" (R.I. Freshney ed. 1986); "Immobilized Cells and Enzymes" (IRL Press, 1986); B. Perbal, "A Practical Guide to Molecular Cloning" 1984).

In a preferred embodiment, a functional promoter/regulator in Coryneform bacteria is operatively fused with at least a major portion of the following nucleotide sequence which reads 5 to 3 and includes the translation initiation sequence ATG (double underlined) and the nucleotide sequence of the Vitreoscilla structural gene (also underlined): AAGCTTAACG GACCCTGGGG TTAAAAGTAT TTGAGTTTTG

ATGTGGATTA AGTTTTAAGA 60

GGCAATAAAG ATTATAATAA GTGCTGCTAC ACCATACTGA TGTATGGC AA AACCATAATA 120

ATGAACTTAA GGAAGACCCT CATGTTAGAC CAGCAAACCA TTAACATCAT CAAAGCCACT 180

GTTCCT GTATTGAAGGAGCA TGGCGTTACC ATTACCACGA CTTTTTATAA AAACTTGTTT 240

GCC AAACACC CTGAAGTACG TCCTTTGTTT GATATGGGTC

GCCAAGAATC TTTGGAGCAG 300

CCTAAGGCTTTGGQGATGAC GGTATTGGCG GCAGCGCAAA ACATTGAAAA TTTGCCAGCT 360

ATTTTGCCTG CGGTCAAAAA AATTGCAGTC AAACATTGTC AAGCAGGCGT GGCAGCAGCG 420

CATTATCCGA TTGTCGGTCA AGAATTGTTG GGTGHGATTA AAGAAGTATT GGGCGATGCC 480

GCAACCGATG ACATTTTGGA CGCGTGGGGC AAGGCTTATG GCGTGATTGC AGATGTGTTT 540

ATTCAAGTGG AAGCAGATTT GTACGCTCAA GCGGTTGAAT AAAGTTTCAG GCCGCTTTCA 600

GGACATAAAA AACGCACCAT AAGGTGGTCT TTTTACGTCT GATATTTACA CAGCAGCAGT 660

TTGGCTGTTG GCCAAAACTT GGGACAAATA TTGCCCTGTG TAAGAGCCCG CCGTTGCTGC 720

GACGTCTTCA GGTGTGCCTT GGCAT 745

The nucleotide coding sequence is operatively fused to the promoter/regulator sequences in a cell when RNA polymerase transcribes the nucleotide coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence. The promoter\regulator thus comprise operational elements for expression.

5 The protein so expressed in this preferred embodiment

should contain at least a major portion of the following amino acid sequence of Vitreoscilla hemoglobin, which portion is capable of binding oxygen.

5 10 15

Met-Leu-Asp-Gln-Gln-Thr-lle-Asn-lIe-lle-Lys-Ala-T-h-r-Val-Pro-Val-Leu

20 25 30

Lys-G!u-His-Gly-Val-Thr-lle-Thr-eThr-Thr-Ph -Tyr-Lys-Asn-Leu-Phe-

35 40 45

Ala-Lys-His-Pro-Glu-Val-Arg-Pro-Leu-Phe-Asp-Met-Gly-Arg-Gln-Glu-

50 55 60 65

Ser-Leu-GIu-Gln-Pro-Lys-Ala-Leu-Ala-Met-Thr-Val-Lθu-Ala-Ala-Aia-

70 75 80

Gln-Asn-lle-Glu-Asn-Leu-Pro-Ala-Ile-Leu-Pro-Ala-Val-Lys-Lys-lle-

85 90 95

Ala-Val-Lys-His-Cys-Gln-Ala-Gly-Val-Ala-Ala-Ala-Hts-Tyr-Pro-lle-

100 . 105 110

Val-Gly-Gln-Glu-Leu-Leu-Gly-Ala-lle-Lys-Glu-Val-Leu-Gly-Asp-Ala-

115 120 125

Ala-Thr-Asp-Asp-lie-Leu-Asp-AIa-Trp-Gly-Lys-Ala-Tyr-Gly-Val-lle-

130 135 ^* 140 145

Ala-Asp-Val-Phe-Ile-Gln-Val-Glu-Ala-Asp-Leu-Tyr-Ala-Gln-Ala-Val-

145

G!u This amino acid sequence is disclosed in Wakabayashi, et al., supra, Nature, 322:483, 1986. It is presently believed that the protein purified and prepared through the practice of this invention will exhibit a homology of over 80% with this sequence. The present invention encompasses proteins made in Coryneform which are substantially homologous with the protein with the above- described amino acid sequence. Two DNA sequences are "substantially homologous" when at least about 80% (preferably. at least about 90%, and most preferably at least about 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis, et al., supra; DNA Cloning, Vols. I & II, supra ; Nucleic Acid Hybridization, supra. The amino acids represented by the foregoing abbreviations are as follows:

Amino Acid 3-Letter Symbol

Glycine Gly

Alanine Ala

Valine Val

Leucine Leu

Isoleucine He

Arginine Arg

Lysine Lys

Glutamic acid Glu

Aspartic acid Asp

Glutamine Gin

Asparagine Asn

Threonine Thr

Serine Ser

Cysteine Cys

Methionine Met

Phenylalanine Phe

Tyrosine Tyr

Tryptophan Trp

Proline Pro

Histidine His

It must be borne in mind in the practice of the present invention that the alteration of some amino acids in a protein sequence may not affect the fundamental properties of the protein. Therefore, it is also contemplated that other portable DNA sequences, both those capable of directing intracellular production of identical amino acid sequences and those capable of directing intracellular production of analogous amino acid sequences which also possess oxygen-binding activity, are included within the ambit of the present invention.

It must also be borne in mind in the practice of the present invention that the alteration of some nucleotide bases in a DNA sequence may not affect the fundamental properties of the coding sequence. Therefore, it is also contemplated that other analogous portable DNA promoter\regulator sequences are included within the ambit of the present invention. It is contemplated that some of these analogous amino acid sequences will be substantially homologous to native Vitreoscilla hemoglobin while other amino acid sequences, capable of functioning as oxygen-binding proteins, will not exhibit substantial homology to native Vitreoscilla hemoglobin. By "substantial homology" as used herein, is meant a degree of homology to native Vitreoscilla hemoglobin in excess of 50%, preferably in excess of 80%.

The portable DNA sequences of the present invention may be synthetically created, by hand or with automated apparatus. The methods used for synthetic creation of these polynucleotide sequences are generally known to one of ordinary skill in the art, particularly in light of the teachings contained herein. As examples of the current state of the art relating to polynucleotide synthesis, one if directed to Maniatis, et al. , Molecular Cloning - A Laboratory Manual. Cold Spring Harbor Laboratory (1984), and Horvath, et aJL. , "An Automated DNA Synthesizer Employing Deoxynucleoside 3¹Phosphoramidites, Methods in Enzymology 154:313-326, 1987, hereby incorporated by reference.

Additionally, a portable DNA sequence may be a fragment of a natural sequence, i. e., a fragment of a polynucleotide which occurred in nature. In one embodiment, a portable DNA sequence may be a restriction fragment isolated from a genomic library. In a preferred embodiment, the genomic library is created from the bacterium Vitreoscilla. In other alternative embodiments, a portable DNA sequence may be isolated from other genomic and cDNA libraries.

While it is envisioned that the portable DNA sequences of this invention may desirably be inserted directly into the host Coryneform chromosome, the present invention also provides a series of vectors, each containing at least one portable DNA sequence as described herein. It is contemplated that additional copies of a portable DNA sequence may be included in a single vector to increase a host cell's ability to produce large quantities of the desired oxygen-binding protein. It is also envisioned that other desirable DNA sequences may also be included in the vectors of this invention. Further, the invention may be practiced through the use of multiple vectors, with additional copies of at least one portable DNA sequences and, optionally, other desirable DNA sequences. Other desirable sequences may encode metabolites normally made by Coryneform. or of natural or unnatural metabolites and proteins expressed in Coryneform via genetic manipulation. This would include heterologous proteins — either intracellular or extracellular — as well as biopolymers such as polysaccharide materials,. simpler metabolites such as amino acids and nucleotides, antibiotics and other chemicals.

In addition, cloning vectors within the scope of the present invention may contain supplemental nucleotide sequences preceding or subsequent to the portable promoter/regulator and/or DNA sequence. These supplemental sequences are those that will not adversely interfere with transcription of the portable promoter/regulator and/or any fused DNA sequence and will, in some instances, enhance transcription, translation, post-translational processing, or the ability of the primary amino acid structure of the resultant gene product to assume an active form.

A preferred vector of the present invention is set forth in Figure 1. This vector, pBHb3, contains a nucleotide sequence which codes for an oxygen-binding protein derived from Vitreoscilla hemoglobin gene. Plasmid pBHb3 may also contain supplemental nucleotide sequences such as terminators, enhancers, attenuators and the like. For proteins to be exported form the intracellular space, at least one leader sequence and any other DNA sequences necessary or preferred for appropriate transcription and subsequent translation of the vector DNA may be included within the scope of this invention.

In a preferred embodiment, cloning vectors containing and capable of expressing the portable DNA sequence of the present invention in Coryneform contain various operational elements in addition to or instead of the promotor/regulator disclosed and claimed herein. These "operational elements" may include at least one promoter, at least one sequence that acts as expression regulator, and at least one terminator codon, at least one leader sequence, and any other DNA sequences necessary or preferred for appropriate transcription and subsequent translation of the vector DNA. Additional embodiments of the present invention include modifications employing other vectors modified to contain one or more of the preferred portable DNA sequences described herein. In particular, it is preferred that such vectors have some or all of the following characteristics: (1) possess a minimal number of host- organisms sequences; (2) be stable in the desired host; (3) be capable of being present in a high copy number in the desired host; (4) possess a regulatable promoter; and (5) have at least one DNA sequence coding for a selectable trait present on a portion of the plasmid separate from that where the portable DNA sequence will be inserted. Alteration of vectors to meet the above criteria are readily performed by those of ordinary skill in the art in light of the available literature and the teachings herein.

Any strain of Coryneform which admits stable insertion of cloned DNA may serve as a host for the practice of this invention. Examples of Coryneform strains which can be transformed or transduced are: Corynebacterium glutamicum - Thierbach, et al., Applied Microbiology and Biotechnology. 29:356, 1988.

Corvnebacterium callunae - Dunican and Shivnan, Bio/Technology. 7:1067, 1989.

Brevibacterium lactofermentum - Sano and Matsui, Gene. 53:191, 1987.

Brevibacterium ammoniagenes - Dunican and Shivana, loc. cit.

Brevibacterium flavum - Sugimoto and Shilo, Agric. Biol. Chemistry. 47:2295, 1983.

Cellulomonas flavigena - Alemohammad and Pembroke, Biotech. Reviews. 4:147, 1990.

Microbacterium ammoniaphilum - Katsumata, et al., J. of Bacteriology. 159:306, 1984. Various vector systems will be suitable for Coryneform species, including plasmids and bacteriophages. The following, noninclusive list of cloning vectors is believed to set forth vectors which can readily be altered using the teachings herein to meet the above criteria. Such alterations are readily performed by those of ordinary skill in the art in light of the available literature and the teaching herein.

For example, the following Coryneform plasmids are useful vectors:

pGX1415 - Smith, et al., Applied Environm. Microbiology. 51:636, 1986.

pAJ1844 - Miwa, et al., Gene. 39:281, 1985.

pUL330 - Santamaria, et al., Gen. Microbiology. 130:2, 1985.

pJA85 - Alemohammad and Pembroke, Biotech. Technigues.

4:147, 1990.

pHY416 - Yoshihama, et al., J. Bacteriology. 162:591, 1985.

pBK10 - Paradis, et al., Gene, 61:199, 1987. pEB003 - Morinaga, et al., J. of Biotechnology, 5:305, 1987.

Phages used as Coryneform vectors include derivatives of f1A (Miwa, et al., Gene. 39:281, 1985). Synthesis and/or isolation of necessary and desired component parts of such cloning vectors, and their assembly is believed to be within the capability of those of ordinary skill in the art and, such , are capable of being performed without undue experimentation. In construction of the cloning vectors of the present invention, it should additionally be noted that multiple copies of a portable DNA sequence coding for an oxygen- binding protein and its attendant operational elements as necessary may be inserted into each vector. In such an embodiment, the Coryneform host may produce greater amounts per vector of the cloned protein. The number of multiple copies of the DNA sequence which may be inserted into the vector is limited by the ability of the resultant vector, due to its size, to be transferred into and replicated and expressed in the Coryneform host.

Additionally, it is preferred that a cloning vector contain a selectable marker, such as a drug resistance marker or other marker which causes expression of a selectable trait by the host. In a particularly preferred embodiment of the present invention, the gene for kanamycin resistance is preferred and is thus included in the preferred vector pBHb3. Such a drug resistance or other selectable marker is intended in part to facilitate in the selection of transformants. Additionally, the presence of such a selectable marker on the cloning vector may be of use in keeping contaminating microorganisms from multiplying in the culture medium. A biologically pure culture of the transformed Coryneform host would be obtained by culturing under conditions which require the induced phenotype for survival.

It is noted that a portable DNA sequence of the present invention may be used as a selectable marker, in that it provides enhanced growth characteristics in low oxygen circumstances.

It is noted that promoter/regulators useful in the practice of this invention are not restricted to the tac promoter, but may also include other promoter/regulations used in Coryneform vectors, for example: trp - Morinaga, et al., J. of Biotechnology.

5:305,1987.

lac - Morinaga, et al., ibid.

The present invention also provides a recombinant-DNA method for the production of oxygen-binding proteins comprising the steps of:

(a) preparing a portable DNA sequence capable of directing a Coryneform host cell to produce a protein having oxygen-binding activity;

(b) transferring the portable DNA sequence directly into the host, or

(c) cloning the portable DNA sequence into a vector capable of being transferred into and replicating in the host cell, such vector containing operational elements for the portable DNA sequence;

(d) transferring the vector from (c) containing the portable DNA sequence and operational elements into the host cell capable of expressing the oxygen-binding protein;

(e) culturing the host cell from (b) or (d) under conditions appropriate for replication and propagation of the vector and/or expressing of the protein; and

(f) in either order:

(i) harvesting the protein, if desired; and

(ii) maturing the protein to an active structure whereby it possesses oxygen-binding activity.

Maturation of the protein to its oxygen-binding active structure occurs by folding into an active configuration and incorporation of heme.

It is believed that some percentage of the oxygen-binding proteins will assume their proper, active structure upon expression in the host Coryneform. If desired, the oxygen-binding protein may be transported across a cell membrane. This will generally occur if DNA coding for an appropriate leader sequence has been linked to the DNA coding for the recombinant protein. The structures of numerous signal peptides have been published. It is envisioned that these leader sequences, included in or added to at least some portion of the portable DNA as necessary, will direct intracellular production of a fusion protein which will be transported through the cell membrane and will have the leader sequence cleaved upon release from the cell. It is envisioned that the portable DNA sequences may be inserted directly into the Coryneform host chromosome, or alternatively may utilize a plasmid cloning system. In a preferred embodiment, the cloning vector pBHb3 is used in the disclosed method. A vector thus obtained may be transferred into the appropriate Coryneform species. It is believed that any Coryneform species having the ability to take up exogenous DNA and express genes and attendant operational elements thereof may be used. Particular hosts which may be preferable for use in this invention include those described above. Methods for transfer of vectors into hosts are within the ordinary skill in the art. For advantageous expression in Coryneform. it may be desirable that the cloning vector be first transferred into another microorganism for amplification such as E. coli, where the vector would be allowed to replicate and, from which the vector would be obtained and purified after amplification, and then transferred into the Coryneform for ultimate expression of the oxygen-binding protein.

The Coryneform host cells are cultured under conditions appropriate for the expression of the oxygen-binding protein. These conditions are generally specific for the host organism, and are readily determined by one of ordinary skill in the art.

This invention also provides a recombinant-DNA method for the use in Coryneform of the Viteoscilla hemoglobin promoter/regulator. Generally, a selected DNA sequence (for example, a structural gene or a sequence in which a foreign protein is encoded) is subjected to external control in Coryneform under given environmental conditions which comprises the steps of:

(a) providing a SNA expression vector comprising at least one selected isolated structural gene or foreign DNA that is transcriptionally and/or translationally responsive to a Vitreoscilla hemoglobin promoter/regulator DNA sequence in Coryneform under the given environmental conditions, which is operatively fused with that promoter/regulator DNA sequence;

(b) introducing the vector into a Coryneform host and culturing in an appropriate medium and environment for expressing a protein encoded in the structural gene or foreign DNA. The host Coryneform cells are cultured under conditions similar to those appropriate for the expression of oxygen-binding proteins, described above. If harvesting of the protein products of the present invention is desired, it may be done prior or subsequent to purification and prior or subsequent to assumption of an active structure.

It is understood that the application of the teachings of the present invention to a specific problem or environment will be within the capabilities of one having ordinary skill in the art in light of teachings contained herein. Examples of the products of the present invention and representative processes for their isolation, use and manufacture appear below. INDUSTRIAL APPLICABILITY

The products and processes of the present invention find usefulness in the production of amino acids and other metabolites using Coryneform in labortory and industrial applications. The invention provides metabolically engineered cells with enhanced growth characteristics for increasing production of proteins, amino acids or other metabolites in Coryneform.

EXAMPLE 1

A plasmid was constructed for the expression of a bacterial hemoglobin in Corynebacterium. This plasmid, pBHb3, contains the Vitreoscilla hemoglobin gene (Khosla and Bailey, Mol. Gen. Genet..214:158, 1988) cloned into a common Corvnebacterium plasmid pBK10. Specifically, a 5.5 kilobase plasmid pINTl (Khosla and Bailey, J. Mol. Biol., 210:79, 1989) which consists of the E. coli plasmid pBR322 with a 1.2 kilobase insert consisting of the Vitreoscilla hemoglobin gene and the 122 base pair tac promoter (P.L. Biochemicals), was digested with Sal 1 and EcoR1. The plasmid ends were then made blunt by filling in with DNA polymerase 1 (Klenow fragment), and the 1.5 kilobase fragment containing the Vitreoscilla hemoglobin gene, tac promoter and flanking pBR322 sequences was isolated. This fragment was ligated to EcoR1 linearized plasmid pBK10 (Paradis, et al., Gene, 61:199, 1987), the ends of which had also been made blunt. The resulting fragment was made circular with T4 DNA ligase. This plasmid, pBHb3, was transformed into E. coli, and was stably maintained by selection with the antibiotic kanamycin. Corynebacterium glutamicum strain ATTC 39022, a variant of the wild type C. glutamicum strain ATTC 13032, was transformed with pBHb3 DNA out of E. coli. A single kanamycin resistant colony was isolated. This clone was designated 39022 :pBHb3-7. The wild-type C. glutamicum strain ATTC13032 was then transformed with pBHb3 DNA isolated out of clone 39022 :pBHb3-7 and a kanamycin resistant colony, designated 13032 :pBHb3 15 was selected for further experiments. Hemoglobin expression in 13032 :pBHb3-15 was confirmed by Western analysis of total cell protein. A crude cell extract was generated by sonication and the proteins separated by SDS-polyacrylamide gel electrophoresis. The proteins were then electrotransferred to a nitrocellulose membrane and screened with polyclonal antiserum generated against Vitreoscilla hemoglobin purified from E. coli. harboring plasmid pRed 2 (Khosla and Bailey, Mol. Gen. Genet., 1988). A band of identical molecular weight as pure hemoglobin was detected in the cell extracts.

EXAMPLE 2

Expression of a Bacterial Hemoglobin in Coryneforms Enhances Amino Acid Yield and Productivity

in Shake Flask Cultures Lysine production in Corvnebacterium glutamicum ATCC 13287 and in similar cells transformed with the plasmid BHb3 was compared in a shake flask culture experiment. Equal amounts of exponential phase cells were inoculated into 75. mL (250 mL flasks) of the following medium: glucose, 175 g/L; yeast extract, 2 g/L; ammonium sulphate, 55 g/L; magnesium sulfate (heptahydrate), 0.8 g/L; potassium phosphate, 1 g/L; manganese sulfate (tetrahydrate), 0.01 g/L; ferrous sulfate (heptahydrate), 0.01 g/L; biotin, 100 mg/L; thiamine-HCl, 200 mg/L; L- leucine, L-methionine and L-threonine, 0.001 mM each. The pH of the culture was maintained at neutrality by adding 50 g/L of calcium carbonate to each flask. The cells were grown at 250 rpm shaking speed and 30 C. Samples were taken at different times, and the culture optical density was measured at 600 nm in a spectrophotometer. An OD 600 of 1.0 was determined to correspond to a dry cell weight of 0.35g/Liter. Glucose and lysine concentrations in the sample supernatants were measured by high performance liquid chromatography. Cell extracts were prepared from samples taken from each time point throughout the experiment. Proteins were separated by SDS-PAGE and screened with anti-Vitreoscilla hemoglobin antisera. Western analysis confirmed that hemoglobin was being expressed throughout the experiment. This hemoglobin was demonstrated to be functional by a carbon monoxide difference spectrum technique (Webster and Liu, J. Biol. Chem. , 1974).

Table 2-1 shows the results obtained 48 hours after inoculation.

Table 2-1: Effect of hemoglobin expression on the production of L-lysine in C. glutamicum ATCC 13287

Strain 13287 :pBHb3 13287

glucose (g/L) 159 155

lysine (g/L) 1.60 1.45

OD (600 nm) 5.14 5.67

cell mass (gdw/L) 1.8 2.0 Yields :

g cells/kg glucose 110 100

g lysine/kg glucose 100 70

g lysine/kg cells 910 730

Productivity:

g lysine/ (kg cells.h) 19 15

Although the cell yield per glucose consumed is similar in both strains (110 g/kg vs 100 g/kg), the lysine yield per glucose consumed is 43% higher in the hemoglobin containing cells (100 g/kg va 0.07 g/kg). Also, the lysine produced per cell mass is 25% higher in the hemoglobin containing cells (910 g/kg vs 730 g/kg).

This experiment indicates that hemoglobin expression in Coryneform increases lysine yield and productivity. EXAMPLE 3

The Presence of Active Vitreoscilla Hemoglobin in

Corynebacterium glutamicum Enhances Lysine

Production, Yield, and Oxygen Uptake Rate

in Batch Fermentation

In this example, lysine production by C. glutamicum ATCC 13287 cells transformed with the plasmid pBHb3 and plasmid-free cells was studied in batch fermentation experiments.

C. glutamicum ATCC 13287 :pBHb3 and C. glutamicum ATCC 13287 :no-plasmid cells were grown in 250-mL shake flasks at 30'C and 250 rpm in the following synthetic medium: glucose, 75 g/L; yeast extract, 2 g/L; ammonium sulfate, 55 g/L; magnesium sulfate (heptahydrate), 0.8 g/L; potassium phosphate, 1 g/L; manganese sulfate (tetrahydrate), 0.01 g/L; ferrous sulfate (heptahydrate), 0.01 g/L; biotin, 100 mg/L; thiamine-HCl, 200 mg/L; L- leucine, L-methionine and L-threonine, 200 mg/L each; p 7.0. These cells were used to seed the fermentors. Fermentations were conducted in 3-L B. Braum MD fermentors in the medium described above, at 30 ° C and under constant air sparging (0.5 L/min). Initially, the impeller agitation rate was constant. During that time, the dissolved oxygen concentration gradually decreased. When the dissolved oxygen concentration reached 5% of air saturation, the agitation rate was adjusted by the fermentor controller in order to maintain the dissolved oxygen concentration at 5% of air saturation until the end of the fermentations. The culture Ph was maintained at Ph 7.0 by the fermentor Ph controller by periodic additions of 4 N sodium hydroxide.

Samples were taken at different times throughout the fermentations. Glucose and lysine concentrations were determined HPLC. Optical density was measured at 600 nm using a Beckman spectrometer. An OD600 of 1.0 corresponds to a cell mass of 0.35 g dw/L. Analysis of the off-gas oxygen and carbon dioxide content were done using a Perkin-Elmer MGA 1200 mass spectrometer. Oxygen uptake rates (OUR) and respiration coefficients were calculated using the off-gas data. Hemoglobin expression was stable throughout the fermentation as demonstrated by Western electroblotting of the samples taken. The hemoglobin produced by 13287 :pBHb3 was biologically active as demonstrated by carbon monoxide binding assay.

Table 3.1 shows the optical density, glucose and lysine concentration as a function of time for 13287 :pBHb3 and 13287:np. Table 3.1

Time OD600 glucose lysine

(h) (g/L) (g/L)

10 13287 :pBHb3 3.5 64 0.1

13287:np 3.7 74 0.1

20 13287 :pBHb3 23 47 4.4

13287:np 24 47 4.6

35 13287 :pBHb3 35 20 15

13287:np 36 27 12

45 13287 :pBHb3 44 5 22

13287:np 44 11 16

56 13287 :pBHb3 42 0.1 27

13287:np 43 0.2 21

The cell yield per glucose consumed is similar in both strains: 196 g cells/kg glucose for 13287:pBHb3 and 201 g cells/kg glucose for 13287 :np. The lysine yield per glucose consumed is 360 g/kg glucose for 13287:pBHb3 and

289 g/kg for 13287 :np. The hemoglobin containing cells have a lysine yield 25% higher than the no-plasmid cells. Also, the lysine produced per cell mass is 31% higher in the hemoglobin-producing cells (1,840 g/kg cells for

13287:pBhb3 vs 1,400 g/kg cells for 13287:np).

Accordingly, lysine productivity per cell mass is 31% higher in the hemoglobin containing cells (32.9 g/kg cells/h for 13287 :pBHb3 and 25.0 g/kg cells/h for

13287:np).

Table 3.2 shows the results from the off-gas analysis during the lysine production period for the 13287 :pBHb3 and 13287 :np fermentations.

Table 3.2:

Time OUR PQ

(h) (mmol/L/h)

35 13287 :pBHb3 11.2 0.98

I3287:np 7.60 1.05

45 13287 :pBHb3 10.2 1.06

13287:np 6.40 0.92

56 13287 :pBHb3 11.1 1.06

13287:np 6.60 0.94

During the lysine production phase (35, 45 and 58 h), the oxygen uptake rate (OUR) of 13287:pBHb3 averages 10.8 mmol/L/h, a 57% higher than the average OUR of 13287 :np during the same time period (6.87 mmol/L/h).

These results indicate that expression of active hemoglobin in Corvneforms increases lysine yield and productivity, and oxygen uptake rate.

Claims

WHAT IS CLAIMED IS:

1. A recombinant-DNA vector capable of directing intracellular production in Coryneform of a major portion of Vitreoscilla hemoglobin protein.

2. A vector according to Claim l wherein said Coryneform comprises Corynebacterium glutamicum.

3. A recombinant-DNA method for production of at least a major portion of the Vitreoscilla hemoglobin protein, or oxygen-binding analogs of said protein, in a Coryneform host grown in the presence of oxygen, comprising:

(a) introducing a vector capable of directing intracellular production in Coryneform. of at least a major portion of Vitreoscilla hemoglobin protein or a protein substantially homologous thereto into said Coryneform host; and

(b) culturing said host under conditions appropriate for expression of said protein or homolog thereof.

4. A method according to Claim 3 wherein said host comprises Corynebacterium glutamicum.

5. A protein prepared by the method of Claims 3 or 4.

6. Recombinantly modified Coryneform containing a vector according to Claim 1 and capable of intracellular production of at least a major portion of Vitreoscilla hemoglobin protein or a protein substantially homologous thereto.

7. Recombinantly modified Corvnebacterium glutamicum according to Claim 6.

8. A method for expressing in Coryneform a selected chromosomal or extrachromosomal gene or DNA sequence comprising the steps of:

(a) introducing into a Coryneform host cell capable of expressing said selected gene or said sequence a vector capable of directing intracellular production of Corynebacterium of at least a major portion of Vitreoscilla hemoglobin protein, or a protein substantially homologous thereto, into said host cell;

(b) culturing said host under conditions appropriate for expression of said selected gene or DNA sequence and for production of said protein or homolog thereof.

9. A method according- to Claim 8 wherein said host comprises Corynebacterium glutamicum.

10. A method according to Claim 9 wherein said selected gene is a chromosomal gene and said expression produces an amino acid, nucleotide, or other metabolite.

11. A method according to Claim 10 wherein said amino acid comprises glutamic acid.

12. A method according to Claim 10 wherein said amino acid comprises L-lysine.

13. A method according to Claim 8 further comprising, prior to culturing said host in said step (b), the step of introducing into said host cell a second vector capable of directing intracellular expression of said selected gene or said selected DNA sequence in said host cell.

14. A method for increasing amino acid productivity of an amino acid producing Coryneform host culture comprising the steps of: (a) introducing into said host a vector capable of directing intracellular production in Coryneform of at least a portion of Vitreoscilla hemoglobin protein, or a protein substantially homologous thereto;

15. A method according to Claim 14 wherein said Coryneform comprises Corynebacterium glutamicum.

16. A method according to Claim 14 wherein said amino acid comprises glutamic acid.

17. A method according to Claim 14 wherein said amino acid comprises L-lysine.

18. In a genetic engineering process for securing expressin of at least one selected isolated structural gene sequence in a Coryneform host, wherein said selected structural gene is stably incorporated as a chromosomal or extrachromosomal constituent of said host, the improvement comprising the step of : operatively fusing with said selected structural gene sequence a Vitreoscilla hemoglobin promoter/regulator DNA sequence which is responsive to environmental variations of the concentration of oxygen within said Coryneform host.

19. A method for expressing any foreign protein in a host Coryneform cultured cell, comprising:

(a) introducing into said host cultured cell an expression vector comprising a Vitreoscilla hemoglobin promoter/regulator operatively fused to foreign DNA sequences coding for said foreign protein; and

(b) growing said host cultured cell in an appropriate medium and environment and isolating the protein expressed by said foreign DNA.

20. The method of Claim 19, wherein said vector is a portable DNA sequence, and is introduced directly and integrated into the chromosome of said Coryneform host cultured cell.