Agrivida, Inc.

Binzhang Shen

R Michael Raab

Gabor Lazar

Humberto De La Vega

James Reasoner Apgar

Philip A Lessard

Jason Eric Donald

Oleg Bougri

Michael R Raab

Methods for enhancing phytase thermal stability by fusing binding elements to target phytases are provided. Engineered phytases that include binding elements fused to target phytases to cause cyclization of the engineered phytases and enhance thermal stability of the target phytases are described. Engineered nucleic acids encoding engineered phytases and hosts engineered to express engineered nucleic acids are also provided. Methods for incorporating engineered phytases in animal feed and animal feed including the same are described.

Animal feed with engineered phytases

Engineered phytases in animal feed

Methods for producing a trans-splicing intein-modified protease with enhanced solubility and regulating its activity are described. Intein-modified proteases having enhanced solubility and polynucleotides encoding the same are provided. Methods of storing trans-splicing proteases are also described.

Methods and compositions for stabilizing trans-splicing intein modified proteases

Methods for producing intein-modified proteases are provided. Expression cassettes and vectors for using to genetically engineer hosts are described. Hosts genetically engineered to express one or more intein-modified proteases using expression cassettes and vectors of the invention are also provided. Methods to produce a protease and regulate its activity are described.

Intein-modified proteases, their production and industrial applications

Engineered phytases and methods of using the same

Intein modified proteins, isolated nucleic acids encoding intein modified proteins, fragments of intein modified proteins, isolated nucleic acids encoding fragments of intein modified proteins, transgenic plants containing any of the foregoing, and antibodies recognizing epitopes on intein modified proteins are provided.

Intein-modified enzymes, their production and industrial applications

A method of predicting an intein insertion site in a protein that will lead to a switching phenotype is provided. The method includes identifying a plurality of C/T/S sites within the protein; selecting from the plurality of C/T/S/sites those that are ranked 0.75 or higher by a support vector machine, within ten angstroms of the active site of the protein, and at or near a loop-&#x3b2;-sheet junction or a loop-&#x3b1;-helix junction. A method of controlling protein activity and hosts including proteins with controlled activity are also provided. Also, intein modified proteins and plants containing intein modified proteins are provided.

Intein-modified enzymes, their production and industrial application

Methods for expressing multiple proteins by constructing transformation vectors that include multiprotein expression cassettes and transforming hosts with vectors and by engineering hosts expressing multiprotein units are provided. Multiprotein units that include multiple proteins fused to modified inteins capable of effecting splicing of the multiprotein units are described. Expression cassettes that include nucleic acids encoding multiprotein units and hosts including the expression cassettes are also provided.

Multiprotein expression cassettes

A method of predicting an intein insertion site in a protein that will lead to a switching phenotype is provided. The method includes identifying a plurality of C/T/S sites within the protein; selecting from the plurality of C/T/S/ sites those that are ranked 0.75 or higher by a support vector machine, within ten angstroms of the active site of the protein, and at or near a loop-&#x3b2;-sheet junction or a loop-&#x3b1;-helix junction. A method of controlling protein activity and hosts including proteins with controlled activity are also provided. Also, intein modified proteins and plants containing intein modified proteins are provided.

In planta consolidated bioprocessing has the advantages of decreasing biomass pretreatment costs, utilizing excess plant protein production capacity for enzyme production, and decreasing mass transfer resistance of enzyme diffusion to its substrate. However, in planta expression of cell wall degrading (CWD) enzymes often leads to detrimental plant phenotypes that impact crop yield. To provide in planta CWD enzyme activity without any adverse phenotype, a thermostable xylanase, XynB (EC 3.2.1.8), was engineered with a thermoregulated intein, Tth-HB27 DnaE-1 (Tth intein), that controls its hydrolytic activity through conditional intein splicing. Maize plants expressing the heat inducible Tth intein-modified XynB developed normally, yet possessed enhanced post harvest glucose production from dried corn stover. Expression of CWD enzymes as dormant, intein-modified proteins that can be activated by heat treatment after harvest provides the basis for developing a novel cellulosic processing trait in plants.

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Boston, MA, United States of America

Binzhang Shen