Abstract: The present invention provides a synthetic mRNA leader for enhancing the expression of a gene encoding a desired protein, vectors comprising said synthetic mRNA leader and methods of producing a desired gene product using said synthetic mRNA leader and vector, said method comprising expressing said gene using a synthetic mRNA leader which comprises from 5? to 3?: (i) a first mRNA leader sequence element; (ii) a spacer region; and (iii) a second mRNA leader sequence element; wherein said first mRNA leader sequence element is a modified transcription-stimulating mRNA leader capable of enhancing transcription of a gene relative to an unmodified reference mRNA leader sequence and/or said second mRNA leader element is a modified translation-stimulating mRNA leader capable of enhancing the translation of a gene transcript relative to an unmodified reference mRNA leader sequence.

Abstract: The present invention concerns a method of producing a desired gene product in a recombinant gene expression system, said method comprising expressing said gene from a Pm promoter-based expression system using at least two mutant elements selected from: (i) a mutant Pm promoter; (ii) a mutant mRNA leader; and (iii) a mutant XyIS; wherein said mutant elements each comprise one or more mutations which enhance expression of said desired gene. Particularly combinations of a mutant Pm promoter and a mutant mRNA leader are concerned. Isolated nucleic acid molecules, vectors, host cells, libraries, expression systems, methods of enhancing expression, obtaining nucleic acid molecules and identifying combination mutants which enhance expression, artificially constructed operons and their uses are also encompassed.

Abstract: The present invention provides a synthetic mRNA leader for enhancing the expression of a gene encoding a desired protein, vectors comprising said synthetic mRNA leader and methods of producing a desired gene product using said synthetic mRNA leader and vector, said method comprising expressing said gene using a synthetic mRNA leader which comprises from 5? to 3?: (i) a first mRNA leader sequence element; (ii) a spacer region; and (iii) a second mRNA leader sequence element; wherein said first mRNA leader sequence element is a modified transcription-stimulating mRNA leader capable of enhancing transcription of a gene relative to an unmodified reference mRNA leader sequence and/or said second mRNA leader element is a modified translation-stimulating mRNA leader capable of enhancing the translation of a gene transcript relative to an unmodified reference mRNA leader sequence.

Abstract: The present invention concerns a method of producing a desired heterologous gene product wherein said heterologous gene product is expressed from a strong promoter, said method comprising expressing said gene using a mutant mRNA leader which comprises one or more mutations which enhance transcription of the gene. The invention also provides a mutant Pm mRNA leader sequence, and a vector and a library comprising the leader sequence. Methods of obtaining an mRNA mutant leader and identifying an mRNA mutant leader are encompassed, along with a vector for selection or identification of an mRNA leader mutant and a use thereof for screening.

Abstract: It is described biologically pure bacterial cultures of mutant strains of Pseudomonas fluorescens, which produces large amounts of alginate. The alginate may contain a certain determined content of mannuronate and guluronate residues, possible presence and determined level of acetyl groups in the alginate, and a desired molecular weight of the alginate. Also high yielding mutants with regulation of alginate production, is described. The invention further provides methods for producing new mutant strains of Pseudomonas fluorescens and variants thereof, and use of the resulting strains in alginate production.

Abstract: The invention provides a nucleic acid molecule comprising: (a) a nucleotide sequence as shown in SEQ ID No. 35; or (b) a nucleotide sequence which is the complement of SEQ ID No. 35; or (c) a nucleotide sequence which is degenerate with SEQ ID No. 35; or (d) a nucleotide sequence hybridising under conditions of high stringency to SEQ ID No. 35, to the complement of SEQ ID No. 35, or to a hybridisation probe derived from SEQ ID No. 35 or the complement thereof; or (e) a nucleotide sequence having at least 80% sequence identity with SEQ ID No. 35; or (f) a nucleotide sequence having at least 65% sequence identity with SEQ ID No. 35 wherein said sequence preferably encodes or is complementary to a sequence encoding a nystatin PKS enzyme or a part thereof.

Abstract: The present invention concerns a method of producing a desired heterologous gene product wherein said heterologous gene product is expressed from a strong promoter, said method comprising expressing said gene using a mutant mRNA leader which comprises one or more mutations which enhance transcription of the gene. The invention also provides a mutant Pm mRNA leader sequence, and a vector and a library comprising the leader sequence. Methods of obtaining an mRNA mutant leader and identifying an mRNA mutant leader are encompassed, along with a vector for selection or identification of an mRNA leader mutant and a use thereof for screening.

Abstract: The present invention relates to a cloning vector for cloning of DNA in a broad host range of bacteria, the vector being an autonomously replicating artificial chromosome comprising: (i) the RK2 origin of replication oriV; (ii) the RK2 origin of conjugate transfer oriT; (iii) par DE from RK2; (iv) a cloning region; (v) a further origin of replication which permits replication of said vector at a copy number of no more than 1 or 2; wherein the vector is no more than 15 kb in size, does not contain trfA of RK2, and is capable of cloning inserts of at least 12 kb and wherein the content of RK2 DNA in the vector is no more than 10% of RK2. The invention also relates to host cells and a vector system having the cloning vector. Methods of cloning DNA and preparing a library and uses of the vector and the RK2 replicon in metagenomic cloning are provided.

Abstract: The invention provides a nucleic acid molecule comprising: (a) a nucleotide sequence as shown in SEQ ID No. 35; or (b) a nucleotide sequence which is the complement of SEQ ID No. 35; or (c) a nucleotide sequence which is degenerate with SEQ ID No. 35; or (d) a nucleotide sequence hybridising under conditions of high stringency to SEQ ID No. 35, to the complement of SEQ ID No. 35, or to a hybridisation probe derived from SEQ ID No. 35 or the complement thereof; or (e) a nucleotide sequence having at least 80% sequence identity with SEQ ID No. 35; or (f) a nucleotide sequence having at least 65% sequence identity with SEQ ID No. 35 wherein said sequence preferably encodes or is complementary to a sequence encoding a nystatin PKS enzyme or a part thereof.

Abstract: The invention provides a nucleic acid molecule comprising: (a) a nucleotide sequence as shown in SEQ ID No. 35; or (b) a nucleotide sequence which is the complement of SEQ ID No. 35; or (c) a nucleotide sequence which is degenerate with SEQ ID No. 35; or (d) a nucleotide sequence hybridising under conditions of high stringency to SEQ ID No. 35, to the complement of SEQ ID No. 35, or to a hybridisation probe derived from SEQ ID No. 35 or the complement thereof; or (e) a nucleotide sequence having at least 80% sequence identity with SEQ ID No. 35; or (f) a nucleotide sequence having at least 65% sequence identity with SEQ ID No. 35 wherein said sequence preferably encodes or is complementary to a sequence encoding a nystatin PKS enzyme or a part thereof.

Abstract: The present invention concerns a method of producing a desired gene product in a recombinant gene expression system, said method comprising expressing said gene from a Pm promoter-based expression system using at least two mutant elements selected from: (i) a mutant Pm promoter; (ii) a mutant mRNA leader; and (iii) a mutant XyIS; wherein said mutant elements each comprise one or more mutations which enhance expression of said desired gene. Particularly combinations of a mutant Pm promoter and a mutant mRNA leader are concerned. Isolated nucleic acid molecules, vectors, host cells, libraries, expression systems, methods of enhancing expression, obtaining nucleic acid molecules and identifying combination mutants which enhance expression, artificially constructed operons and their uses are also encompassed.

Abstract: It is described biologically pure bacterial cultures of mutant strains of Pseudomonas fluorescens, which produces large amounts of alginate. The alginate may contain a certain determined content of mannuronate and guluronate residues, possible presence and determined level of acetyl groups in the alginate, and a desired molecular weight of the alginate. Also high yielding mutants with regulation of alginate production, is described. The invention further provides methods for producing new mutant strains of Pseudomonas fluorescens and variants thereof, and use of the resulting strains in alginate production.

Abstract: It is described biologically pure bacterial cultures of mutant strains of Pseudomonas fluorescens, which produces large amounts of alginate. The alginate may contain a certain determined content of mannuronate and guluronate residues, possible presence and determined level of acetyl groups in the alginate, and a desired molecular weight of the alginate. Also high yielding mutants with regulation of alginate production, is described. The invention further provides methods for producing new mutant strains of Pseudomonas fluorescens and variants thereof, and use the resulting strains in alginate production.

Abstract: The invention provides a nucleic acid molecule comprising: (a) a nucleotide sequence as shown in SEQ ID No. 35; or (b) a nucleotide sequence which is the complement of SEQ ID No. 35; or (c) a nucleotide sequence which is degenerate with SEQ ID No. 35; or (d) a nucleotide sequence hybridising under conditions of high stringency to SEQ ID No. 35, to the complement of SEQ ID No. 35, or to a hybridisation probe derived from SEQ ID No. 35 or the complement thereof; or (e) a nucleotide sequence having at least 80% sequence identity with SEQ ID No. 35; or (f) a nucleotide sequence having at least 65% sequence identity with SEQ ID No. 35 wherein said sequence preferably encodes or is complementary to a sequence encoding a nystatin PKS enzyme or a part thereof.

Abstract: The invention provides a nucleic acid molecule comprising: (a) a nucleotide sequence as shown in SEQ ID No. 35; or (b) a nucleotide sequence which is the complement of SEQ ID No. 35; or (c) a nucleotide sequence which is degenerate with SEQ ID No. 35; or (d) a nucleotide sequence hybridising under conditions of high stringency to SEQ ID No. 35, to the complement of SEQ ID No. 35, or to a hybridisation probe derived from SEQ ID No. 35 or the complement thereof; or (e) a nucleotide sequence having at least 80% sequence identity with SEQ ID No. 35; or (f) a nucleotide sequence having at least 65% sequence identity with SEQ ID No. 35 wherein said sequence preferably encodes or is complementary to a sequence encoding a nystatin PKS enzyme or a part thereof.

Abstract: It is described biologically pure bacterial cultures of mutant strains of Pseudomonas fluorescens, which producee large amounts of alginate. The alginate may contain a certain determined content of mannuronate and guluronate residues, possible residues, possible presence and determined level of acetyl groups in the alginate, and a desired molecular weight of the alginate. Also high yielding of the alginate. Also high yielding mutants with regulation of alginate production, is described. The invention further provides methods for producing new mutant provides methods for producing new mutant strains of Pseudomonas fluorescens and variants thereof, and use of the resulting strains in alginate production.

Abstract: The invention provides a nucleic acid molecule comprising: (a) a nucleotide sequence as shown in SEQ ID No. 35; or (b) a nucleotide sequence which is the complement of SEQ ID No. 35; or (c) a nucleotide sequence which is degenerate with SEQ ID No. 35; or (d) a nucleotide sequence hybridising under conditions of high stringency to SEQ ID No. 35, to the complement of SEQ ID No. 35, or to a hybridisation probe derived from SEQ ID No. 35 or the complement thereof; or (e) a nucleotide sequence having at least 80% sequence identity with SEQ ID No. 35; or (f) a nucleotide sequence having at least 65% sequence identity with SEQ ID No. 35 wherein said sequence preferably encodes or is complementary to a sequence encoding a nystatin PKS enzyme or a part thereof.

Abstract: The invention provides an isolated DNA molecule comprising a promoter sequence, said promoter sequence being a Pm or a Pu promoter of a TOL plasmid, said promoter sequence having sequence modifications in its 10 region together with DNA constructs, expression vectors and transformed cells containing such molecules. Also provided by the invention is a method for assaying promoter activity, comprising expressing in an antibiotic-susceptible (i.e. sensitive) host cell, an antibiotic resistance gene under the control of the promoter to be assayed, and assessing the growth of said cell in the presence of said antibiotic.

Abstract: The present invention provides an expression vector comprising an RK2 minimum replicon together with an expression cassette comprising the regulatory functions of a TOL plasmid, and, in particular, an expression vector comprising a RK2 minimum replicon together with a promoter Pm and/or Pu and a corresponding regulatory gene xylS and/or xylR as derived from a TOL plasmid. Such expression vectors may be used to express desired genes in a wide range of gram negative and gram positive bacterial hosts.

Abstract: DNA compounds encompassing sequences coding for enzymes having mannuronan C-5-epimerase activity are disclosed and a process for the preparation of such enzymes. The genetic sequences and enzymes prepared may be used in the production of alginates having a definite G/M ratio and block structure. Alginates having a definite G/M ratio may also be produced by selective inactivation of the genetic sequences.