Benzoate-and anthranilate-inducible promoters

• US 20050202544A1
• Filed: 01/03/2005
• Published: 09/15/2005
• Est. Priority Date: 07/03/2002
• Status: Active Grant

• Current Assignee: Pfenex Inc.
• Original Assignee: Dow Global Technologies, Inc. (Dow, Inc.)
• Inventors: Subramanian, Venkiteswaran, Retallack, Diane M.
• Granted Patent: US 7,794,972 B2
• Time in Patent Office: Days
• US Class Current: 435/108
• CPC Class Codes: C07K 14/21 from Pseudomonadaceae (F)
• CPC Class Codes: C12N 15/78 for Pseudomonas

Abstract

Novel benzoate- or anthranilate-inducible promoters, and novel tandem promoters, and variants and improved mutants thereof, useful for commercial prokaryotic fermentation systems, nucleic acid constructs containing the promoters, expression systems using them, methods for expressing proteins by use thereof, and proteins expressed thereby.

Claims

1-21. -21. (canceled)

22. (canceled)

23. A nucleic acid construct comprising a non-catabolite-repressed promoter operably linked upstream to a natively catabolite-repressed promoter.

73. The construct of claim 23 wherein at least one of the non-catabolite-repressed or natively catabolite-repressed promoters is obtained from a Pseudomonad.

74. The construct of claim 73 wherein the Pseudomonad is a Pseudomonas fluorescens.

75. The construct of claim 23 wherein the non-catabolite-repressed and the natively catabolite-repressed promoters are both obtained from at least one gene encoding an alternative carbon source utilization enzyme or pathway.

76. The construct of claim 75 wherein the non-catabolite-repressed promoter is a promoter from a gene encoding an anthranilate degradation pathway protein.

77. The construct of claim 76 wherein the anthranilate promoter is selected from the group consisting of nucleotides 1221-1359, 1221-1371, 1328-1359, and 1328-1371 OF SEQ ID NO:

13.

78. The construct of claim 75 wherein the natively catabolite-repressed promoter is a promoter from a gene encoding a benzoate degradation pathway protein.

79. The construct of claim 78 wherein the benzoate promoter is selected from the group consisting of nucleotides 1430-1503, 1430-1509, 1477-1503, and 1477-1509 of SEQ ID NO:

13.

80. The construct of claim 23 further comprising a linker sequence.

81. The construct of claim 80 wherein the linker sequence comprises nucleotide sequence 1396-1429 of SEQ ID NO:

13.

82. The construct of claim 23 further comprising at least one activator protein binding site.

83. The construct of claim 82 wherein at least one activator protein binding site is a benzoate activator protein binding site or an anthranilate activator protein binding site.

84. The construct of claim 23 comprising comprises nucleotide sequence 1121 -1503 of SEQ ID NO:

13.

85. The construct of claim 23 wherein induction of the non-catabolite repressed promoter can regulate induction of the catabolite repressed promoter.

86. The construct of claim 23 further comprising an exogenous polypeptide of interest.

87. The construct of claim 86 wherein the polypeptide of interest is operably linked to the expression of the catabolite-repressed promoter.

24-72. -72. (canceled)

88. A host cell comprising a nucleic acid construct comprising a non-catabolite-repressed promoter operably linked upstream to a natively catabolite-repressed promoter.

89. The host cell of claim 88 wherein at least one of the non-catabolite-repressed or natively catabolite-repressed promoters is obtained from a Pseudomonad.

90. The host cell of claim 88 wherein the non-catabolite-repressed promoter is a promoter from a gene encoding an anthranilate degradation pathway protein.

91. The host cell of claim 88 wherein the natively catabolite-repressed promoter is a promoter from a gene encoding a benzoate degradation pathway protein.

92. The host cell of claim 88 further comprising an exogenous polypeptide of interest.

93. The host cell of claim 88 further comprising at least one benzoate activator protein binding site and at least one anthranilate activator protein binding site.

94. A process for protein expression comprising growing a cell comprising a nucleic acid construct comprising a non-catabolite-repressed promoter operably linked upstream to a natively catabolite-repressed promoter and a recombinant protein expression cassette in the presence of an inducing condition.

95. The process of claim 94 wherein the presence of an inducing condition produces production of a recombinant protein or peptide.

96. The process of claim 94 wherein the inducing condition comprises the presence of anthranilate.

97. The process of claim 96 wherein the inducing condition further comprises the presence of benzoate.

98. The process of claim 94 wherein induction of the non-catabolite repressed promoter changes induction of the catabolite repressed promoter.

99. The process of claim 94 wherein at least one of the non-catabolite-repressed or natively catabolite-repressed promoters is obtained from a Pseudomonad.

100. The process of claim 94 wherein the cell is derived from a Pseudomonad.

101. The process of claim 94 further comprising isolating the recombinant protein or peptide.

102. An isolated nucleic acid comprising a −

35 region of a Pseudomonas fluorescens native benzoate promoter.

103. The nucleic acid of claim 102 further comprising a −

10 region of the benzoate promoter.

104. The nucleic acid of claim 103 further comprising a nucleotide linker.

105. The nucleic acid of claim 102 wherein the nucleic acid further comprises a benzoate promoter activator protein binding site.

106. The nucleic acid of claim 105 wherein the activator protein comprises a coding sequence for amino acids selected from the group consisting of an amino acid sequence at least 90% homologous to residues 1-335 OF SEQ ID NO:

2;

residues 1-335 OF SEQ ID NO;

2 containing Asn152;

residues 21-335 of SEQ ID NO;

2;

residues 21-335 of SEQ ID NO;

2 containing Asn152, and at least one of the mutations listed therein.

107. The nucleic acid of claim 102 wherein the nucleic acid further comprises a benzoate promoter activator protein coding sequence.

108. The nucleic acid of claim 102 wherein the nucleic acid comprises a sequence of nucleotides 1275-1280 of SEQ ID NO:

1.

109. The nucleic acid of claim 102 wherein the nucleic acid further comprises a sequence of nucleotides 1296-1301 OF SEQ ID NO:

1.

110. The nucleic acid of claim 102 wherein the nucleic acid comprises a sequence of a mutation listed in SEQ ID NO:

1.

111. A nucleic acid construct comprising a nucleotide sequence comprising a −

35 region of a Pseudomonas fluorescens native benzoate promoter.

112. The construct of claim 111 further comprising a nucleotide sequence comprising a −

10 region of the benzoate promoter.

113. The construct of claim 112 further comprising a nucleotide linker sequence.

114. The construct of claim 113 further comprising at least one coding sequence for an exogenous polynucleotide of interest operably linked to the benzoate promoter.

115. The construct of claim 111 comprising a nucleotide sequence of nucleotides 1275-1280 of SEQ ID NO:

1.

116. The construct of claim 111 comprising a nucleotide sequence of nucleotides 1296-1301 OF SEQ ID NO:

1.

117. An isolated nucleic acid comprising a −

35 region of a Pseudomonas fluorescens native anthranilate promoter.

118. The nucleic acid of claim 117 further comprising a −

10 region of the anthranilate promoter.

119. The nucleic acid of claim 118 further comprising a nucleotide linker.

120. The nucleic acid of claim 117 wherein the nucleic acid further comprises an anthranilate promoter activator protein binding site.

121. The nucleic acid of claim 120 wherein the activator protein comprises a coding sequence for amino acids consisting of an amino acid sequence at least 90% homologous to residues 1-330 of SEQ ID NO:

9 or residues 1-330 of SEQ ID NO;

9 containing Ala268.

122. The nucleic acid of claim 117 wherein the nucleic acid further comprises an anthranilate promoter activator protein coding sequence.

123. The nucleic acid of claim 117 wherein the nucleic acid comprises a sequence of nucleotides 1239-1244 of SEQ ID NO:

7.

124. The nucleic acid of claim 117 wherein the nucleic acid further comprises a sequence of nucleotides 1264-1268 of SEQ ID NO:

7.

125. The nucleic acid of claim 117 wherein the nucleic acid comprises a sequence of a mutation listed in SEQ ID NO:

7.

126. A nucleic acid construct comprising a nucleotide sequence comprising a −

35 region of a Pseudomonas fluorescens native anthranilate promoter.

127. The construct of claim 126 further comprising a nucleotide sequence comprising a −

10 region of the anthranilate promoter.

128. The construct of claim 127 further comprising a nucleotide linker sequence.

129. The construct of claim 126 further comprising at least one coding sequence for an exogenous polynucleotide of interest operably linked to the anthranilate promoter.

130. The construct of claim 126 comprising a nucleotide sequence of nucleotides 1239-1244 of SEQ ID NO:

7.

131. The construct of claim 126 comprising a nucleotide sequence of nucleotides 1264-1268 of SEQ ID NO:

7.