Patent Application: US-99113801-A

Abstract:
the present invention relates to mutant 1 , 3 - propanediol dehydrogenase and a novel microorganism that is capable of growing in concentrations of at least 105 g / l 1 , 3 - propanediol , levels normally toxic to wild - type microorganisms . the present invention also provides expression vectors and host cells comprising the mutant 1 , 3 - propanediol dehydrogenase as well as methods for producing 1 , 3 - propanediol comprising the use of cells comprising the mutant 1 , 3 - propanediol dehydrogenase .

Description:
the terms “ 1 , 3 - propanediol dehydrogenase ” or “ pdd ” ( also known in the art as “ oxidoreductase ”) refer to the polypeptide ( s ) responsible for an enzyme activity that is capable of catalyzing the reduction of 3 - hydroxypropionaldehyde to 1 , 3 - propanediol . 1 , 3 - propanediol dehydrogenase includes , for example , the polypeptide encoded by the dhat gene . the present invention encompasses 3 - propanediol dehydrogenase from any source including , but not limited to e . blatte , k . pneumoniae , c . freundii , c . pasteurianum . as used herein , the term “ mutant ” or “ mutation ” refers to any genetic change that occurs in the nucleic acid of a microorganism and may or may not reflect a phenotypic change within the microorganism . a mutation may comprise a single base pair change , deletion or insertion ; a mutation may comprise a change , deletion or insertion in a large number of base pairs ; a mutation may also comprise a change in a large region of dna , such as through duplication or inversion . the amino acid sequence of a mutant 1 , 3 - propanediol dehydrogenase can be derived from a precursor 1 , 3 - propanediol dehydrogenase by the substitution , deletion or insertion of one or more amino acids of the naturally occurring 1 , 3 - propanediol dehydrogenase . methods for modifying genes ( e . g ., through site - directed oligonucleotide mutagenesis ) have been described in the art . the phrase “ corresponding to ” as used herein refers to the amino acid relatedness among 1 , 3 - propanediol dehydrogenases as exemplified by fig3 . specific residues discussed herein refer to an amino acid residue number which references the number assigned to the e . blatte geb pdd shown in fig3 . the mutation of his to leu is shown at residue 105 in fig3 . fig3 illustrates that 1 , 3 - propanediol dehydrogenases from a variety of microbial sources can be aligned using the algorithm clustalw . the invention is not limited to the mutation of the e . blattae pdd shown in fig1 and 2 , or the e . blattae deposited with the atcc and having accession number pta - 92 but encompasses all pdds containing amino acid residues at positions which are equivalent to the particular identified residue in e . blattae . a residue is equivalent if it is either homologous ( i . e ., corresponds in position for either the primary or tertiary structure ) or analogous to a specific residue or portion of that residue in e . blattae pdd ( i . e ., having the same or similar functional capacity to combine , react , or interact chemically or structurally ). in order to establish homology to primary structure , the amino acid sequence of a pdd is directly compared to the e . blattae pdd primary sequence ( shown in fig2 ) and particularly to a set of residues known to be invariant to all pdds for which sequences are known ( see , e . g ., fig3 ). the present invention encompasses the equivalent residue change in all sources of 1 , 3 - propanediol dehydrogenase as long as the mutant form is able to alter the km of the activity for 1 , 3 - propanediol . in a preferred embodiment , the km of the mutant form is increased for 1 , 3 - propanediol . the nucleic acid sequence of seq id no : 1 was obtained via pcr techniques . such techniques are often characterized by inadvertent pcr generated sequence error . therefore , the present invention also encompasses the 1 , 3 - propanediol dehydrogenase of e . blattae having atcc accession number pta - 92 and corresponding mutations in other microbial sources of the 1 , 3 - propanediol dehydrogenases . the term “ km ” refers to affinity of the enzyme for the substrate . a high km reflects a low affinity ; a low km reflects a high affinity . the terms “ carbon substrate ” and “ carbon source ” refer to a carbon source capable of being metabolized by host organisms of the present invention and particularly carbon sources selected from the group consisting of monosaccharides , oligosaccharides , polysaccharides , and one - carbon substrates or mixtures thereof . the terms “ host cell ” or “ host organism ” refer to a microorganism capable of receiving foreign or heterologous genes and of expressing those genes to produce an active gene product . as used herein , “ nucleic acid ” refers to a nucleotide or polynucleotide sequence , and fragments or portions thereof , and to dna or rna of genomic or synthetic origin which may be double - stranded or single - stranded , whether representing the sense or antisense strand . the terms “ native ” and “ wild - type ” refer to a gene as found in nature with its own regulatory sequences . as used herein “ amino acid ” refers to peptide or protein sequences or portions thereof . the term “ expression ” refers to the transcription and translation to gene product from a gene coding for the sequence of the gene product . the terms “ plasmid ”, “ vector ”, and “ cassefte ” refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell , and usually in the form of circular double - stranded dna molecules . such elements may be autonomously replicating sequences , genome integrating sequences , phage or nucleotide sequences , linear or circular , of a single - or double - stranded dna or rna , derived from any source , in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and dna sequence for a selected gene product along with appropriate 3 ′ untranslated sequence into a cell . “ transformation cassette ” refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitate transformation of a particular host cell . “ expression cassette ” refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host . the terms “ isolated ” or “ purified ” as used herein refer to a nucleic acid or amino acid that is removed from at least one component with which it is naturally associated . the present invention relates to mutant 1 , 3 - propanediol dehydrogenase ( pdd ) characterized by having an increased km for 1 , 3 propanediol . polynucleotide sequence as shown in seq id no : 1 encodes the 1 , 3 - propanediol dehydrogenase ( seq id no : 2 ) having the mutation of his to leu at residue 105 as shown in fig3 . as will be understood by the skilled artisan , due to the degeneracy of the genetic code , a variety of polynucleotides can encode seq id no : 2 . the present invention encompasses all such polynucleotides . the present invention encompasses nucleic acid encoding pdd comprising a mutation corresponding to e . blatte residue his 105 to leu as shown in fig3 . the nucleic acid and amino acid sequence for pdd from k . pneumoniae is given in genbank accession number u30903 ; pdd from c . freundii is given in genbank accession number u09771 ; for pdd from c . pasteurianum is given in genbank accession number af00034 . the present invention also encompasses mutant pdd obtainable from e . blattae having atcc accession number pta - 92 . methods of obtaining desired genes from a microbial genome are common and well known in the art of molecular biology . for example , if the sequence of the gene is known , suitable genomic libraries may be created by restriction endonuclease digestion and may be screened with probes complementary to the desired gene sequence . once the sequence is isolated , the dna may be amplified using standard primer directed amplification methods such as polymerase chain reaction ( pcr ) ( u . s . pat . no . 4 , 683 , 202 ) to obtain amounts of dna suitable for transformation using appropriate vectors . alternatively , methods of using cosmid vectors for the transformation of suitable bacterial hosts are well described in sambrook et al ., molecular cloning : a laboratory manual , second edition ( 1989 ) cold spring harbor laboratory press , cold spring harbon , n . y . ( 1989 ). methods of making mutations in pdd genes are known to the skilled artisan and include for example site - directed mutagenesis , procedures described in united states patent u . s . pat . no . 4 , 760 , 025 issued jul . 26 , 1988 . the present invention provides a variety of vectors and transformation and expression cassettes suitable for the cloning , transformation and expression of mutant pdd as well as other proteins associated with 1 , 3 - propanediol production into a suitable host cell . suitable vectors will be those which are compatible with the bacterium employed . suitable vectors can be derived , for example , from a bacteria , a virus ( such as bacteriophage t7 or a m - 13 derived phage ), a cosmid , a yeast or a plant . protocols for obtaining and using such vectors are known to those in the art . ( sambrook et al ., molecular cloning : a laboratory manual — volumes 1 , 2 , 3 ( cold spring harbor laboratory , cold spring harbor , n . y ., ( 1989 )). typically , the vector or cassette contains sequences directing transcription and translation of the relevant gene , a selectable marker , and sequences allowing autonomous replication or chromosomal integration . suitable vectors comprise a region 5 ′ of the gene which harbors transcriptional initiation controls and a region 3 ′ of the dna fragment which controls transcriptional termination . it is most preferred when both control regions are derived from genes homologous to the transformed host cell although it is to be understood that such control regions need not be derived from the genes native to the specific species chosen as a production host . initiation control regions or promoters , which are useful to drive expression of pdd in the desired host cell , are numerous and familiar to those skilled in the art . virtually any promoter capable of driving these genes is suitable for the present invention including but not limited to cyc1 , his3 , gal1 , gal10 , adh1 , pgk , pho5 , gapdh , adc1 , trp1 , ura3 , leu2 , eno , tpi ( useful for expression in saccharomyces ); aox1 ( useful for expression in pichia ); and lac , trp , ip l , ip r , t7 , tac , and trc ( useful for expression in e . coli ). termination control regions may also be derived from various genes native to the preferred hosts . optionally , a termination site may be unnecessary , however , it is most preferred if included . for effective expression of the instant enzymes , dna encoding the enzymes are linked operably through initiation codons to selected expression control regions such that expression results in the formation of the appropriate messenger rna . once suitable cassettes are constructed they are used to transform appropriate host cells . introduction of the cassette containing mutant 1 , 3 - propanediol dehydrogenase , either separately or together with other proteins necessary for the production of 1 , 3 - propanediol , into the host cell may be accomplished by known procedures such as by transformation ( e . g ., using calcium - permeabilized cells , electroporation ) or by transfection using a recombinant phage virus . ( sambrook et al ., supra .). suitable host cells for the recombinant production of 1 , 3 - propanediol may be either prokaryotic or eukaryotic and will be limited only by the host cell ability to express active enzymes . preferred hosts will be those typically useful for production of glycerol or 1 , 3 - propanediol such as citrobacter , enterobacter , clostridium , klebsiella , aerobacter , lactobacillus , aspergillus , saccharomyces , schizosaccharomyces , zygosaccharomyces , pichia , kluyveromyces , candida , hansenula , debaryomyces , mucor , torulopsis , methylobacter , escherichia , salmonella , bacillus , streptomyces and pseudomonas . most preferred in the present invention are e . coli , klebsiella species and saccharomyces species . fermentation media in the present invention must contain suitable carbon substrates . suitable substrates may include but are not limited to monosaccharides such as glucose and fructose , oligosaccharides such as lactose or sucrose , polysaccharides such as starch or cellulose , or mixtures thereof , and unpurified mixtures from renewable feedstocks such as cheese whey permeate , cornsteep liquor , sugar beet molasses , and barley malt . additionally , the carbon substrate may also be one - carbon substrates such as carbon dioxide , or methanol for which metabolic conversion into key biochemical intermediates has been demonstrated . preferred carbon substrates are monosaccharides , oligosaccharides , polysaccharides , and one - carbon substrates . more preferred are sugars such as glucose , fructose , sucrose and single carbon substrates such as methanol and carbon dioxide . most preferred is glucose . in addition to an appropriate carbon source , fermentation media must contain suitable minerals , salts , cofactors , buffers and other components , known to those skilled in the art , suitable for the growth of the cultures and promotion of the enzymatic pathway necessary for glycerol production . particular attention is given to co ( ii ) salts and / or vitamin b 12 or precursors thereof . typically , cells are grown at 30 ° c . in appropriate media . preferred growth media in the present invention are common commercially prepared media such as luria bertani ( lb ) broth , sabouraud dextrose ( sd ) broth or yeast malt extract ( ym ) broth . other defined or synthetic growth media may also be used and the appropriate medium for growth of the particular microorganism will be known by someone skilled in the art of microbiology or fermentation science . the use of agents known to modulate catabolite repression directly or indirectly , e . g ., cyclic adenosine 2 ′: 3 ′- monophosphate or cyclic adenosine 2 ′: 5 ′- monophosphate , may also be incorporated into the reaction media . similarly , the use of agents known to modulate enzymatic activities ( e . g ., sulphites , bisulphites and alkalis ) that lead to enhancement of glycerol production may be used in conjunction with or as an alternative to genetic manipulations . suitable ph ranges for the fermentation are between ph 5 . 0 to ph 9 . 0 , where ph 6 . 0 to ph 8 . 0 is preferred as range for the initial condition . reactions may be performed under aerobic or anaerobic conditions where anaerobic or microaerobic conditions are preferred . the manner and method of carrying out the present invention may be more fully understood by those of skill in the art by reference to the following examples , which examples are not intended in any manner to limit the scope of the present invention or of the claims directed thereto . example 1 describes the kinetic changes associated with the mutant pdd shown in seq id no : 2 . strains — wild type atcc 33429 , e . blattae comprising the mutant pdd atcc accession number pta - 92 . growth — cells were grown in a complex medium at 30c 500 ml in a 2800 ml fernbach with shaking at 225 rpm for 20 hr . the medium consists of kh2po4 , 5 . 4 g / l ; ( nh4 ) 2so4 , 1 . 2 g / l ; mgso47h2o , 0 . 4 g / l ; yeast extract , 2 . 0 g / l ; tryptone , 2 . 0 g / l ; and glycerol , 9 . 2 g / l in tap water . the ph was adjusted to 7 . 1 with koh before autoclaving ( honda , et al ., 1980 , j . bacteriol , 143 : 1458 - 1465 ). extract prep — cells were harvested by centrifugation with care to avoid anaerobic conditions . pellets were resuspended in 100 mm tricine ph 8 . 2 containing 50 mm kcl and 1 mm dtt . cells were disrupted by passage through a french pressure cell . crude extracts were clarified by centrifugation at 20k × g for 20 min followed by 100k × g for 1 hr to yield the high speed supernatant ( hss ) fraction . assays — the assay for pdd was performed as described by johnson , e . a . et al ., 1987 , j . bacteriol . 169 : 2050 - 2054 . partial purification of pdd — hss was separated on a 16 × 100 poros 20hq column . the buffers were a , 50 mm hepes , ph 7 . 4 containing 100 um mncl and b , a buffer containing 500 mm kcl . the column was loaded and developed at 10 ml / min . the gradient was 10 cv wash , a linear gradient to 70 % b in 10 cv , and 1 cv to 100 % b . the activity was detected in the very early fractions of the gradient . pooled column fractions of the 33429 strain were used as collected for assays after the addition of additional of dtt to 1 mm . the active fractions from strain geb031 were pooled and concentrated on a pm30 membrane and used as concentrated after the addition of additional 1 mm dtt . strain gd ( u / mg ) pdd ( u / mg ) ratio gd / pdd 33429 0 . 64 0 . 22 2 . 9 geb031 0 . 79 0 . 08 9 . 9 example 2 : cloning and sequencing the 1 , 3 - propanediol dehydrogenase genes ( dhat ) from e . blattae . the dhat genes were amplified by pcr from genomic dna from e . blattae as template dna using synthetic primers ( primer 1 and primer 2 ) based on the k . pneumoniae dhat sequence and incorporating an xbai site at the 5 ′ end and a bamhi site at the 3 ′ end . the product was subcloned into pcr - blunt ii - topo ( invitrogen ). the cloning dhat were then sequenced was standard techniques . the results of the dna sequencing are given in seq id no : 1 and seq id no : 2 . as will be readily understood by the skilled artisan , nucleic acid sequence generated via pcr methods may comprise inadvertent errors . the present invention also encompasses nucleic acid encoding pdd obtainable from e . blattae having atcc accession number pta - 92 . all references cited herein , including patents , patent applications , sequences and publications are hereby incorporated in their entirety by reference . val leu asp gly leu ala met phe arg lys glu gln cys asp met ile ile ala ala thr his pro gly asp leu tyr ser tyr ala gly ile glu gly thr ala ser glu val thr arg his cys val leu thr asn thr lys thr lys val lys phe val ile val ser trp arg asn leu pro ser val ser ile asn asp pro leu leu met ile gly lys pro ala gly leu thr ala ala thr gly met asp ala leu thr his ala val glu ala tyr ile leu pro his val cys arg tyr asn leu ile ala 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ala met phe arg lys glu gln cys asp met ile ile ala ala thr his pro gly asp leu tyr ser tyr ala gly ile glu gly thr ala ser glu val thr arg his cys val leu thr asn thr lys thr lys val lys phe val ile val ser trp arg asn leu pro ser val ser ile asn asp pro leu leu met ile gly lys pro ala gly leu thr ala ala thr gly met asp ala leu thr his ala val glu ala tyr ile leu pro his val cys arg tyr asn leu ile ala asn pro glu lys phe ala asp ile ala thr phe met gly glu asn thr thr gly leu ser thr asp val gly ile pro gln his leu arg glu leu gly val lys glu ala asp phe pro tyr met ala glu met ala leu lys asp gly asn ala phe ser asn pro arg lys gly asn glu lys glu ile ala asp ile phe arg val leu asp gly leu ala met phe arg lys glu gln cys asp met ile ile ala ala thr his pro gly asp leu tyr ser tyr ala gly ile glu gly thr ala ser glu val thr arg his cys val leu thr asn thr lys thr lys val lys phe val ile val ser trp arg asn leu pro ser val ser ile asn asp pro leu leu met ile ser lys pro ala gly leu 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ile ala asn pro glu lys phe asp ile gly ile pro gln his leu arg asp leu gly val lys glu ala asp phe pro tyr met ala glu met ala leu lys asp gly asn ala phe ser asn pro arg lys gly asn glu gln glu ile ala ala ile phe arg gly pro asn ala ile ser val val gly glu arg cys lys leu leu gly asp gly ala val asp lys thr leu thr his leu arg glu ala gly ile val arg asp gly leu glu val phe arg lys glu his cys asp ile ile gly thr ala ser glu val thr arg his cys val leu thr asn thr lys thr lys val lys phe val ile val ser trp arg asn leu pro ser val ser ile asn asp pro leu leu met leu gly lys pro ala pro leu thr ala ala thr gly met asp ala leu thr his ala val glu ala tyr ile leu pro his val ala arg tyr asn leu ile ala asn pro glu lys phe ala asp ile ala glu phe met gly glu asn thr asp gly leu ser thr asp ile gly ile pro gln his leu arg asp leu gly val lys glu ala asp phe pro tyr met ala glu met ala leu lys asp gly asn ala phe met arg met tyr asp phe leu ala pro asn val asn phe met gly ala lys ala leu ile val thr asp lys phe leu arg asn met glu asp gly ala phe tyr asp asp val glu pro asn pro lys asp thr asn val arg asp gly leu lys val tyr arg lys glu asn cys asp leu ile val thr gly ser glu val thr arg his cys val ile thr asn thr lys thr lys thr gly met asp ala leu thr his ala ile glu ser tyr val ser lys his val glu arg tyr asn leu ile ser asn pro lys lys phe ala asp ala ala glu lys ala ile asp ala met phe arg leu ser lys asp val gly ile pro ala ser leu lys glu met gly val asn glu gly asp phe glu tyr met ala lys met ala leu lys asp gly asn ala phe ser asn pro arg lys gly asn glu lys asp ile val lys ile phe arg glu ala