E.i. Dupont De Nemours and Company

E. I. Du Pont Nemours and Co.

Xiao-Song Tang

Der-Ing Liao

Katharine Janet Gibson

Wei Wei Qi

Fateme Sima Sariaslani

Qiong Cheng

Luan Tao

Todd M Vannelli

Joanne Y Shyr

Arie Ben-Bassat

Bruce Aaron Diner

Douglas Brian Jordan

Anthony Arthur Gatenby

Sima Sariaslani

Joseph Milano

Sequences of B-dependent dehydratases with improved reaction kinetics are presented. Use of these B-dependent dehydratases reduce the rate of the enzyme's suicide inactivation in the presence of glycerol and 1,3-propanediol. The enzymes were created using error-prone PCR and oligonucleotide-directed mutagenesis to target the DhaB1 gene, which encodes the &#x3b1;-subunit of glycerol dehydratase. Mutants with improved reaction kinetics were rapidly identified using high throughput assays.

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B12 dependent dehydratases with improved reaction kinetics

A method for the recombination of a gene is disclosed. The method involves the design of unpaired forward and reverse primers having homology to the 5' end of one template and to the 3&#x2032; end of another template. Short primer extension periods results in a recombined template having paired 5&#x2032; and 3&#x2032; ends that can then be amplified. The amplified sample is devoid of any parental template.

Method for the recombination of genetic elements

Protein engineered CrtO ketolases isolated fromAN12 are provided having increased carotenoid ketolase activity. Methods using the present CrtO ketolases are also provided for increasing ketocarotenoid production in suitable production hosts.

Carotenoid ketolase genes with improved ketocarotenoid yield

Genes encoding phenylalanine ammonia-lyase (PAL), tyrosine ammonia lyase (TAL) and phenylalanine hydroxylase (PAH) have been introduced into a host organism for the production of Para-hydroxycinnamic acid (PHCA). The introduction of these genes results in the redirection of carbon flow in the host, optimizing the flow of carbon from glucose to PHCA. The intermediates, tyrosine and cinnamic acid are also produced.

Methods for the production of tyrosine, cinnamic acid and para-hydroxycinnamic acid using recombinant microorganisms

An in vivo method for the production of pHS via a recombinant host cell is disclosed. The host cell expresses at least one gene encoding a polypeptide having para-hydroxycinnamic acid decarboxylase activity in combination with either at least one gene encoding a polypeptide having tyrosine ammonia lyase activity or at least one gene encoding a polypeptide having phenylalanine ammonia lyase activity.

Microbial conversion of glucose to para-hydroxystyrene

Methods for the production of tyrosine, cinnamic acid and para-hydroxycinnamic acid

Protein engineered nucleic acid fragments encoding a CrtO ketolase and a CrtZ hydroxylase are provided with increased astaxanthin synthesis activity. Methods using the present nucleic acid fragments are also provided for increasing or altering astaxanthin production in suitable production hosts.

Bioproduction of astaxanthin using mutant carotenoid ketolase and carotenoid hydroxylase genes

The present invention provides several methods for biological production of para-hydroxycinnamic acid (PHCA). The invention is also directed to the discovery of new fungi and bacteria that possess the ability to convert cinnamate to PHCA. The invention relates to developing of a new biocatalyst for conversion of glucose to PHCA by incorporation of the wild type PAL from the yeast into underlining the ability of the wildtype PAL to convert tyrosine to PHCA. The invention is also directed to developing a new biocatalyst for conversion of glucose to PHCA by incorporation of the wildtype PAL from the yeast plus the plant cytochrome P-450 and the cytochrome P-450 reductase into In yet another embodiment, the present invention provides for the developing of a new biocatalyst through mutagenesis of the wild type yeast PAL which possesses enhanced tyrosine ammonia-lyase (TAL) activity.

Polynucleotide encoding a mutant Rhodotorula glutinis tyrosine ammonia lyase polypeptide

The present invention provides several methods for biological production of para-hydroxycinnamic acid (PHCA). The invention is also directed to the discovery of new fungi and bacteria that possess the ability to convert cinnamate to PHCA. The invention relates to developing of a new biocatalyst for conversion of glucose to PHCA by incorporation of the wild type PAL from the yeast into underlining the ability of the wildtype PAL to convert tyrosine to PHCA. The invention is also directed to developing a new biocatalyst for conversion of glucose to PHCA by incorporation of the wildtype PAL from the yeast plus the plant cytochrome P-450 and the cytochrome P-450 reductase into . In yet another embodiment, the present invention provides for the developing of a new biocatalyst through mutagenesis of the wild type yeast PAL which possesses enhanced tyrosine ammonia-lyase (TAL) activity.

Bioproduction of para-hydroxycinnamic acid

D1 protease has been isolated from the alga ( ), wheat, and Synechocystis PCC 6803 and the genes encoding these enzymes have been cloned and sequenced. Native or recombinantly produced enzyme has been used to develop assays to detect herbicidal compositions capable of inhibiting the D1 protease enzyme.

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Hockessin, DE, United States of America

Xiao-Song Tang