Materials and methods for the production of phenylalanine
AssigneePurdue Research Foundation
InventorsJohn A. Morgan, Arnav Deshpande, Melissa Marsing
Life of this patent
Estimating the term…
Abstract
Polynucleotides, constructs/vectors, host cells, polypeptides, and methods of producing phenylalanine in a prokaryotic host, such as for subsequent isolation.
Claims29
An isolated or purified polynucleotide encoding a prephenate dehydratase having an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:
- 1:
- 1 MAALRFQAWL TDQDQQPSHL LACRSIPATL QTLADGTVDY AVVPVENSVE GSVAATLDSL
- 61 WQLPQLSIQR ALILPIAHAL ISFEDDRTAI RQVLSHPQAL AQCQQWLQRQ LPQAELIPTN
- 121 STTEALQDLE RHPQRAVIAS TRAAELYQMP IQSFPINDSP DNRTRFWVVS RNLTPGGACT
- 181 SLSFSLDANV PGALVKPLQI LAERQINLSR IESRPTKRSL EYLFFLDLE ADLREPAIAQ
- 241 AVQAVADCTE QLRVLGSYDS LDFTQVVQPS [SEQ ID NO:
- 1], in which the glycine at amino acid position 221 (G221) is substituted with an amino acid having an aromatic R group.
An isolated or purified polynucleotide encoding a TolC family protein having an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:
- 2:
- 1 MAAFLYRLSL LSALAIAAHG MEPPAAIADI VDPQATGPSP TIAQNSPPPA ATTPAPTTPP
- 61 SSPVKEVVPD ANLLKELQAN PNPFQLPNQP NQVQTESLQP LTLEQALNLA RLNNPQVQIR
- 121 QLQVEQRRAA LRGTEAALYP TLGLQGNAGY QQSGNRLNVT EGSTVQPTGS SLFTTLGQSS
- 181 IGATLNLNYT IFDFVRGAQL AASRDQVTQA ELDLEATLED LQLTVSEAYY QLQNADQLVR
- 241 IARESVVASE RSLKDAEALF RAGVGTQFDV LRQQVQLAQD QQNLVDSIGN QDKARRSLVQ
- 301 ALNLPQNVNV LTADPVELAA PWNLSLDESI VLAFQNRPEL EREVLQRNIS YNQAQAARGQ
- 361 ILPQLGLQAS YGVTGSINSN LRSGSQALTF PSPTLTNNSN YSYSIGLVLN VPLFDGGLAN
- 421 ANAQQQELNG QIAEQNFVLT RNQIRTDVET AFYDLQTNLA NIGTTRKAVE QAREALRLAR
- 481 LRFQAGVGTQ TEVIDSQRDL TRAEANALNA ITAYNLALAR IKRAVSNVSN ARVGS [SEQ ID NO:
- 2], in which the leucine at amino acid position 531 (L531) is substituted with an amino acid having an aromatic R group.
An isolated or purified polynucleotide encoding a circadian clock protein having an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:
- 3:
- 1 MLSQIAICIW VESTAILQDC QQALAGDRYQ LQICSSGDLL LDYAQTHRDQ IDCLLLVATN
- 61 PGCKTVIQQL CFQGIVVPAI VVGDRDTDDV TDTQKDWVYH SAELHLGIHQ LEQLPYQVDA
- 121 ALAEFLRQAP VETIADQVML MAATHDPELA SHQRDLAQRL QERLGYLGVY YKRDPDRFLR
- 181 NLPAEGQKL LEAMQTSYRE IVLSYFSPNS NLNQSLDNFV NMAFFADVPV TQVVEIHMEL
- 241 MDEFAKKLRV EGRSEDILLD YRLTLIDVIA HLCEMYRRSI PRET [SEQ ID NO:
- 3], in which there is a frameshift mutation at tyrosine at amino acid position 185 (Y185) resulting in the substitution of Y185 with an amino acid having a nonpolar, aliphatic R group.
A host cell comprising and expressing an isolated or purified polynucleotide encoding a prephenate dehydratase having an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:
- 4, an isolated or purified polynucleotide encoding a TolC family protein having an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:
- 5.
An isolated or purified polypeptide having the amino acid sequence of SEQ ID NO:
- 4.
An isolated or purified polypeptide having the amino acid sequence of SEQ ID NO:
- 5.
An isolated or purified polypeptide having the amino acid sequence of SEQ ID NO:
- 6.
A method of producing phenylalanine in a prokaryotic host, which method comprises culturing a host cell in medium under culture conditions that support the production of phenylalanine, wherein the host cell comprises and expresses a construct or vector comprising:
- (a) an isolated or purified polynucleotide encoding a prephenate dehydratase having an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:
- 1, in which the glycine at amino acid position 221 (G221) is substituted with an amino acid having an aromatic R group, operably linked to a promoter;
- (b) an isolated or purified polynucleotide encoding a TolC family protein having an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:
- 2, in which the leucine at amino acid position 531 (L531) is substituted with an amino acid having an aromatic R group, operably linked to a promoter;
- (c) an isolated or purified polynucleotide encoding a circadian clock protein having an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:
- 3, in which there is a frameshift mutation at tyrosine at amino acid position 185 (Y185) resulting in the substitution of Y185 with an amino acid having a nonpolar, aliphatic R group, operably linked to a promoter;
- (d) an isolated or purified polynucleotide encoding a prephenate dehydratase having an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:
- 4, an isolated or purified polynucleotide encoding a TolC family protein having an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:
- 5, operably linked to a promoter;
- or
- (e) an isolated or purified polynucleotide encoding a prephenate dehydratase having an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:
- 4, an isolated or purified polynucleotide encoding a TolC family protein having an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:
- 5, and an isolated or purified polynucleotide encoding an isolated or purified polynucleotide encoding a circadian clock protein having an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:
- 6, operably linked to a promoter;
- whereupon phenylalanine in the prokaryotic host is produced.
Dependencies are inferred from the claim text.