CN103890186A

CN103890186A - Microorganisms and methods for producing acrylate and other products from homoserine

Info

Publication number: CN103890186A
Application number: CN201280048633.9A
Authority: CN
Inventors: 徐隽; C·W·桑德斯; P·R·格林; J·E·维拉斯奎茨
Original assignee: Procter and Gamble Ltd
Current assignee: Procter and Gamble Ltd; Procter and Gamble Co
Priority date: 2011-10-05
Filing date: 2012-10-05
Publication date: 2014-06-25
Also published as: CA2849823A1; EP2764111A2; WO2013052717A3; WO2013052717A2; AU2012318597A2; AU2012318597A1

Abstract

This invention relates to microorganisms that convert a carbon source to acrylate or other desirable products using homoserine and 2-keto-4-hydroxybutyrate as intermediates. The invention provides genetically engineered microorganisms that carry out the conversion, as well as methods for producing acrylate by culturing the microorganisms. Also provided are microorganisms and methods for converting homoserine to 3-hydroxypropionyl-CoA, 3-hydroxypropionate (3HP), poly-3-hydroxypropionate and 1,3-propanediol.

Description

Produce microorganism and the method for acrylate and other products by homoserine

Technical field

The present invention relates to microorganism, it is converted into acrylate or other desired product as intermediate by carbon source with homoserine and 2-ketone group-4 hydroxybutyric acid ester.The invention provides carry out described conversion through genetically engineered microorganism, and the method for producing acrylate by cultivating described microorganism.The microorganism and the method that homoserine are converted into 3-hydroxyl propionyl-CoA, 3-hydroxy propionate (3HP), poly--3-hydroxy propionate and 1,3-PD are also provided.

Background technology

A kind of organic chemicals for the preparation of superabsorbent polymers (for diaper), plastics, coating, paint vehicle, caking agent and tackiness agent (for leather, paper wood and textile product) is vinylformic acid.Vinylformic acid (IUPAC:prop-2-enoic acid) is the simplest unsaturated carboxylic acid.

Traditionally, vinylformic acid is made up of propylene.Propylene is from the by product of the olein refining as from oil (, crude oil) and Sweet natural gas production.Produce relevant disadvantage to conventional acrylic and be that oil is non-renewable raw material and olein refining processing pollution environment.Due to expense and environmental problem, utilize the vinylformic acid synthetic method of other raw material to be not yet used in purposes widely.These raw materials for example comprise, acetylene, ketene and ethene cyanalcohol.

For avoiding the production based on oil, investigator has proposed for the production of acrylic acid other method, and it relates to the sugar-fermenting being undertaken by the microorganism of through engineering approaches.The people such as Straathof, Appl Microbiol Biotechnol, 67:727-734 (2005) discusses the conceptual fermentation process for carrying out vinylformic acid production by sugar.The method proposing in described article is carried out via Beta-alanine, methyl citrate, malonyl--CoA or propanedioic acid methyl esters-CoA intermediate in microorganism.The another kind of method of describing in the U.S. Patent Publication 2011/0125118 of Lynch relates in microflora produces 3-hydroxy-propionic acid by synthesis gas component as carbon source, subsequently described 3-hydroxy-propionic acid is converted into vinylformic acid.

Propose in the method for manufacturing other organic chemicals in genetically engineered microorganism.Referring to for example, the U.S. Patent Publication 2011/0014669 that on January 20th, 2011 announces, it relates to Pidolidone is converted into BDO.

Due to the annual vinylformic acid of producing at least four hundred ten thousand tonnes, be there is to demand in the high performance-price ratio environment protection method of its production for being undertaken by renewable carbon source in the art.

Summary of the invention

Homoserine is intermediate in amino acid Threonine and methionine(Met) biosynthesizing.Homoserine is made by glucose in natural situation in bacteria Escherichia coli (E.coli) and numerous other biology.The step explanation that it is homoserine that Fig. 1 illustrates conversion of glucose.

The present invention utilizes homoserine and 2-ketone group-4 hydroxybutyric acid ester to prepare acrylate (chemical species of vinylformic acid under neutral pH) and other product of paying close attention to as intermediate.Fig. 2 and 3 illustrates an example of the imagination approach for prepare acrylate, 3-hydroxy propionate, poly--3-hydroxy propionate, 1,3-PD and 3-hydroxyl propionyl-CoA by homoserine.Microorganism is not manufactured acrylate and described other products in natural situation, but microorganism (such as bacterium, yeast, fungi and algae) carries out genetic modification according to the present invention and carries out the conversion in described approach.Microorganism includes but not limited to intestinal bacteria bacterium.

produce acrylate

In first aspect, the invention provides first kind microorganism, homoserine is converted into acrylate by it, wherein said microbial expression recombination, described recombination coding desaminase or transaminase; Desaturase or decarboxylase; Dehydratase; And thioesterase, the combination of phosphotransferase/kinases or acyl group-CoA transferring enzyme.

Described desaminase or transaminase catalysis are converted into homoserine the reaction of 2-ketone group-4 hydroxybutyric acid ester.In certain embodiments, described desaminase or transaminase are transaminase, L-amino acid oxidase or L-amino acid dehydrogenase.Transaminase includes but not limited to L-glutamic acid-oxaloacetic acid transaminase, L-glutamic acid-pyruvate aminotransferase, L-Aspartic acid salt: 2-oxoglutaric acid transaminase and ALANINE: 2-oxoglutaric acid transaminase.The aminoacid sequence of transaminases more as known in the art is shown in SEQ ID NO:2,4,6,8,10 and 12.The exemplary DNA sequence dna of encoding those transaminase is respectively shown in SEQ ID NO:1,3,5,7,9 and 11.The aminoacid sequence of L-amino acid oxidases more as known in the art is shown in SEQ ID NO:14 and 16.The exemplary DNA sequence dna of encoding those L-amino acid oxidase is respectively shown in SEQ ID NO:13 and 15.The aminoacid sequence of L-amino acid dehydrogenases more as known in the art is shown in SEQ ID NO:18 and 20.The exemplary DNA sequence dna of encoding those L-amino acid dehydrogenase is respectively shown in SEQ ID NO:17 and 19.

Described desaturase catalysis is converted into 2-ketone group-4 hydroxybutyric acid ester the reaction of 3-hydroxyl propionyl-CoA.In certain embodiments, described desaturase is 2-ketoacid dehydrogenase (or α ketoacid dehydrogenase).Desaturase includes but not limited to pyruvic oxidase, 2-ketone group-glutatate dehydrogenase or branched-chain keto acids desaturase.Pyruvic oxidase as known in the art is pyruvic oxidase PDH, and the aminoacid sequence of its subunit is shown in SEQ ID NO:30,32 and 34.The exemplary DNA sequence dna of encoding those subunit is respectively shown in SEQ ID NO:29,31 and 33.2-ketone group-glutatate dehydrogenase as known in the art comprises three subunits similarly, and its aminoacid sequence is shown in SEQ ID NO:36,38 and 40.The exemplary DNA sequence dna of encoding those subunit is respectively shown in SEQ ID NO:35,37 and 39.Branched-chain keto acids desaturase as known in the art is branched-chain keto acids desaturase BKD, and the aminoacid sequence of its subunit is shown in SEQ ID NO:22,26,24 and 28.The exemplary DNA sequence dna of encoding those subunit is respectively shown in SEQ ID NO:21,25,23 and 27.

Described dehydratase catalysis is converted into 3-hydroxyl propionyl-CoA the reaction of acryloyl-CoA.In certain embodiments, described dehydratase is 3-hydroxyl propionyl-CoA-dehydratase.The aminoacid sequence of 3-hydroxyl propionyl-CoA-dehydratase as known in the art is shown in SEQ ID NO:48.Encode the exemplary DNA sequence dna of described 3-hydroxyl propionyl-CoA-dehydratase shown in SEQ ID NO:47.

Described thioesterase, the combination of described phosphotransferase/kinases or the catalysis of described acyl group-CoA transferring enzyme are converted into acryloyl-CoA the reaction of acrylate.In certain embodiments, described thioesterase is acryloyl-CoA thioesterase.The aminoacid sequence of phosphoric acid acryloyl transferring enzyme as known in the art is shown in SEQ ID NO:50.Encode the exemplary DNA sequence dna of described phosphoric acid acryloyl transferring enzyme shown in SEQ ID NO:49.The kinase whose aminoacid sequence of acrylate as known in the art is shown in SEQ ID NO:52.The kinase whose exemplary DNA sequence dna of the described acrylate of encoding is shown in SEQ ID NO:51.The aminoacid sequence of acyl group-CoA transferring enzyme as known in the art is shown in SEQ ID NO:46.Encode the exemplary DNA sequence dna of described acyl group-CoA transferring enzyme shown in SEQ ID NO:45.

In second aspect, the invention provides first kind method, it cultivates described first kind microorganism to produce acrylate for the production of acrylate in described method.First kind method for the production of acrylate is converted into homoserine 2-ketone group-4 hydroxybutyric acid ester, 2-ketone group-4 hydroxybutyric acid ester is converted into 3-hydroxyl propionyl-CoA, 3-hydroxyl propionyl-CoA is converted into acryloyl-CoA, and then acryloyl-CoA is converted into acrylate.

In the third aspect, the invention provides Equations of The Second Kind microorganism, homoserine is converted into acrylate by it, wherein said microbial expression recombination, described recombination coding: desaminase or transaminase, decarboxylase, desaturase, dehydratase and thioesterase.

Described desaminase or transaminase catalysis are converted into homoserine the reaction of 2-ketone group-4 hydroxybutyric acid ester.In certain embodiments, described desaminase or transaminase are transaminase, L-amino acid oxidase or L-amino acid dehydrogenase.Transaminase includes but not limited to L-glutamic acid-oxaloacetic acid transaminase, L-glutamic acid-pyruvate aminotransferase, L-Aspartic acid salt: 2-oxoglutaric acid transaminase, ALANINE: 2-oxoglutaric acid transaminase.The aminoacid sequence of transaminases more as known in the art is shown in SEQ ID NO:2,4,6,8,10 and 12.The exemplary DNA sequence dna of encoding those transaminase is respectively shown in SEQ ID NO:1,3,5,7,9 and 11.The aminoacid sequence of L-amino acid oxidases more as known in the art is shown in SEQ ID NO:14 and 16.The exemplary DNA sequence dna of encoding those L-amino acid oxidase is shown in SEQ ID NO:13 and 15.The aminoacid sequence of L-amino acid dehydrogenases more as known in the art is shown in SEQ ID NO:18 and 20.The exemplary DNA sequence dna of encoding those L-amino acid dehydrogenase is shown in SEQ ID NO:17 and 19.

Described decarboxylase catalysis is converted into 2-ketone group-4 hydroxybutyric acid ester the reaction of 3-hydroxyl-propionic aldehyde.In certain embodiments, described decarboxylase is 2-keto acid decarboxylase.Described 2-keto acid decarboxylase includes but not limited to that 2-keto acid decarboxylase KdcA(is shown in SEQ ID NO:54) and derivative.The exemplary DNA sequence dna of encoded K dcA is shown in SEQ ID NO:53.

Described desaturase catalysis is converted into 3-hydroxyl-propionic aldehyde the reaction of 3-hydroxyl propionyl-CoA.In certain embodiments, described desaturase is propionic aldehyde desaturase.Propionic aldehyde desaturase includes but not limited to PduP.The aminoacid sequence of PduP propionic aldehyde desaturases more as known in the art is shown in SEQ ID NO:60 and 62.The exemplary DNA sequence dna of coding PduP propionic aldehyde desaturase is respectively shown in SEQ ID NO:59 and 61.

Described dehydratase catalysis is converted into 3-hydroxyl propionyl-CoA the reaction of acryloyl-CoA.In certain embodiments, described dehydratase is 3-hydroxyl propionyl-CoA dehydratase.The aminoacid sequence of 3-hydroxyl propionyl-CoA dehydratase as known in the art is shown in SEQ ID NO:48.Encode the exemplary DNA sequence dna of described 3-hydroxyl propionyl-CoA dehydratase shown in SEQ ID NO:47.

Described thioesterase catalysis is converted into acryloyl-CoA the reaction of acrylate.In certain embodiments, described thioesterase is acryloyl-CoA thioesterase.Acryloyl-CoA thioesterase includes but not limited to that intestinal bacteria TesB(is shown in SEQ ID NO:90), comprise E324D surrogate (shown in SEQ ID NO:92) derived from the thioesterase of clostridium propionicum (Clostridium propionicum), and comprise E325D surrogate (shown in SEQ ID NO:94) derived from the thioesterase of Erichsen Megasphaera (Megasphaera elsdenii).The exemplary DNA sequence dna of these acryloyl-CoA thioesterases of encoding carries out codon optimized at SEQ ID NO:89,91(for intestinal bacteria respectively) and 93(carry out codon optimized for intestinal bacteria) shown in.

In fourth aspect, the invention provides Equations of The Second Kind method, it cultivates described Equations of The Second Kind microorganism to produce acrylate for the production of acrylate in described method.Equations of The Second Kind method for the production of acrylate is converted into homoserine 2-ketone group-4 hydroxybutyric acid ester, 2-ketone group-4 hydroxybutyric acid ester is converted into 3-hydroxyl-propionic aldehyde; 3-hydroxyl-propionic aldehyde is converted into 3-hydroxyl-propionyl-CoA; 3-hydroxyl-propionyl-CoA is converted into acryloyl-CoA, and then acryloyl-CoA is converted into acrylate.

produce 3-hydroxy propionate

Aspect the 5th; the invention provides the 3rd quasi-microorganism; homoserine is converted into 3-hydroxy propionate by it, wherein said microbial expression recombination, described recombination coding: desaminase or transaminase, desaturase or decarboxylase and acyl group-CoA transferring enzyme or thioesterase.

Described desaminase or transaminase catalysis are converted into homoserine the reaction of 2-ketone group-4 hydroxybutyric acid ester.In certain embodiments, described desaminase or transaminase are transaminase, L-amino acid oxidase or L-amino acid dehydrogenase.Transaminase includes but not limited to L-glutamic acid-oxaloacetic acid transaminase, L-glutamic acid-pyruvate aminotransferase, L-Aspartic acid salt: 2-oxoglutaric acid transaminase, ALANINE: 2-oxoglutaric acid transaminase.The aminoacid sequence of transaminases more as known in the art is shown in SEQ ID NO:2,4,6,8,10 and 12.The exemplary DNA sequence dna of encoding those transaminase is respectively shown in SEQ ID NO:1,3,5,7,9 and 11.The aminoacid sequence of L-amino acid oxidases more as known in the art is shown in SEQ ID NO:14 and 16.The exemplary DNA sequence dna of encoding those L-amino acid oxidase is respectively shown in SEQ ID NO:13 and 15.The aminoacid sequence of L-amino acid dehydrogenases more as known in the art is shown in SEQ ID NO:18 and 20.The exemplary DNA sequence dna of encoding those L-amino acid dehydrogenase is shown in SEQ ID NO:17 and 19.

Described desaturase or decarboxylase catalysis are converted into 2-ketone group-4 hydroxybutyric acid ester the reaction of 3-hydroxyl propionyl-CoA.In certain embodiments, described desaturase is 2-ketoacid dehydrogenase (or α ketoacid dehydrogenase).Desaturase includes but not limited to pyruvic oxidase, 2-ketone group-glutatate dehydrogenase or branched-chain keto acids desaturase.Pyruvic oxidase as known in the art is pyruvic oxidase PDH, and the aminoacid sequence of its subunit is shown in SEQ ID NO:30,32 and 34.The exemplary DNA sequence dna of encoding those subunit is respectively shown in SEQ ID NO:29,31 and 33.2-ketone group-glutatate dehydrogenase as known in the art comprises three subunits similarly, and its aminoacid sequence is shown in SEQ ID NO:36,38 and 40.The exemplary DNA sequence dna of encoding those subunit is respectively shown in SEQ IDNO:35,37 and 39.Branched-chain keto acids desaturase as known in the art is branched-chain keto acids desaturase BKD, and the aminoacid sequence of its subunit is shown in SEQ ID NO:22,26,24 and 28.The exemplary DNA sequence dna of encoding those subunit is respectively shown in SEQ ID NO:21,25,23 and 27.

Described acyl group-CoA transferring enzyme or the catalysis of described acyl group-CoA thioesterase are converted into 3-hydroxyl propionyl-CoA the reaction of 3-hydroxy propionate.The thioesterase of imagination includes but not limited to that intestinal bacteria TesB(is shown in SEQ ID NO:90), comprise E324D surrogate (shown in SEQ ID NO:92) derived from the thioesterase of clostridium propionicum (C.propionicum), and comprise E325D surrogate (shown in SEQ ID NO:94) derived from the thioesterase of Erichsen Megasphaera (M.elsdenii).Exemplary (carrying out codon optimized for the intestinal bacteria) DNA sequence dna of these thioesterases of encoding carries out codon optimized at SEQ ID NO:89,91(for intestinal bacteria respectively) and 93(carry out codon optimized for intestinal bacteria) shown in.

Aspect the 6th, the invention provides the 3rd class methods, it cultivates described the 3rd quasi-microorganism to produce 3-hydroxy propionate for the production of 3-hydroxy propionate in described method.Homoserine is converted into 2-ketone group-4 hydroxybutyric acid ester by described the 3rd class methods, 2-ketone group-4 hydroxybutyric acid ester is converted into 3-hydroxyl-propionic aldehyde, 3-hydroxyl-propionic aldehyde is converted into 3-hydroxyl propionyl-CoA, and then 3-hydroxyl propionyl-CoA is converted into 3-hydroxy propionate.

Aspect the 7th, the invention provides the 4th quasi-microorganism, homoserine is converted into 3-hydroxy propionate by it, wherein said microbial expression recombination, described recombination coding: desaminase or transaminase, decarboxylase and desaturase.

Described desaturase catalysis is converted into 3-hydroxyl-propionic aldehyde the reaction of 3-hydroxyl propionyl.In certain embodiments, described desaturase is aldehyde dehydrogenase.The aminoacid sequence of aldehyde dehydrogenase as known in the art is shown in SEQ ID NO:56 and 58.The exemplary DNA sequence dna of encoding said aldehyde dehydrogenase is respectively shown in SEQ ID NO:55 and 57.

In eight aspect, the invention provides the 4th class methods, it cultivates described the 3rd quasi-microorganism to produce 3-hydroxy propionate for the production of 3-hydroxy propionate in described method.Homoserine is converted into 2-ketone group-4 hydroxybutyric acid ester by described the 4th class methods, and 2-ketone group-4 hydroxybutyric acid ester is converted into 3-hydroxyl-propionic aldehyde, and 3-hydroxyl-propionic aldehyde is converted into 3-hydroxy propionate.

Aspect the 9th; the invention provides the 5th quasi-microorganism; homoserine is converted into 3-hydroxy propionate by it, wherein said microbial expression recombination, described recombination coding: desaminase or transaminase, decarboxylase, desaturase and acyl group-CoA transferring enzyme or thioesterase.

Described 3-hydroxyl propionyl-CoA transferring enzyme or thioesterase catalysis are converted into 3-hydroxyl propionyl-CoA the reaction of 3-hydroxy propionate.The thioesterase of imagination includes but not limited to that intestinal bacteria TesB(is shown in SEQ ID NO:90), comprise E324D surrogate (shown in SEQ ID NO:92) derived from the thioesterase of clostridium propionicum, and comprise E325D surrogate (shown in SEQ ID NO:94) derived from the thioesterase of Erichsen Megasphaera.The exemplary DNA sequence dna of these acryloyl-CoA thioesterases of encoding carries out codon optimized at SEQ ID NO:89,91(for intestinal bacteria respectively) and 93(carry out codon optimized for intestinal bacteria) shown in.

Aspect the tenth, the invention provides the 5th class methods, it cultivates described the 5th quasi-microorganism to produce 3-hydroxy propionate for the production of 3-hydroxy propionate in described method.The 5th class methods for the production of acrylate are converted into homoserine 2-ketone group-4 hydroxybutyric acid ester, 2-ketone group-4 hydroxybutyric acid ester are converted into 3-hydroxyl-propionic aldehyde, 3-hydroxyl-propionic aldehyde is converted into 3-hydroxyl propionyl-CoA, 3-hydroxyl propionyl-CoA is converted into 3-hydroxy propionate.

produce poly--3-hydroxy propionate

In the tenth one side, the invention provides the 6th quasi-microorganism, it is converted into homoserine poly--3-hydroxy propionate, wherein said microbial expression recombination, described recombination coding: desaminase or transaminase, desaturase or decarboxylase and PHA synthase.

Described desaturase or decarboxylase catalysis are converted into 2-ketone group-4 hydroxybutyric acid ester the reaction of 3-hydroxyl propionyl-CoA.In certain embodiments, described desaturase is 2-ketoacid dehydrogenase (or α ketoacid dehydrogenase).Desaturase includes but not limited to pyruvic oxidase, 2-ketone group-glutatate dehydrogenase or branched-chain keto acids desaturase.Pyruvic oxidase as known in the art is pyruvic oxidase PDH, and the aminoacid sequence of its subunit is shown in SEQ ID NO:30,32 and 34.The exemplary DNA sequence dna of encoding those subunit is respectively shown in SEQ ID NO:29,31 and 33.2-ketone group-glutatate dehydrogenase as known in the art comprises three subunits similarly, and its aminoacid sequence is shown in SEQ ID NO:36,38 and 40.The exemplary DNA sequence dna of encoding those subunit is respectively shown in SEQ ID NO:35,37 and 39.Branched-chain keto acids desaturase as known in the art is branched-chain keto acids desaturase BKD, and the aminoacid sequence of its subunit is shown in SEQ ID NO:22,26,24 and 28.The exemplary DNA sequence dna of encoding those subunit is respectively shown in SEQ ID NO:21,25,23 and 27.

The catalysis of described PHA synthase is converted into 3-hydroxyl propionyl-CoA the reaction of the gather-3-hydroxy alkane acid ester that comprises 3-hydroxy propionate monomer.Described polymkeric substance can have in carboxyl end coenzyme A (CoA) molecule.The aminoacid sequence of PHA synthase as known in the art is shown in SEQ ID NO:42.Encode the exemplary DNA sequence dna of described PHA synthase shown in SEQ ID NO:41.

Aspect the 12, the invention provides the 6th class methods, it is for the production of poly--3-hydroxy propionate, in described method, cultivate described the 6th quasi-microorganism poly-to produce-3-hydroxy propionate.Homoserine is converted into 2-ketone group-4 hydroxybutyric acid ester by described the 6th class methods, 2-ketone group-4 hydroxybutyric acid ester is converted into 3-hydroxyl propionyl-CoA, and 3-hydroxyl propionyl-CoA is converted into poly--3-hydroxy propionate.

At the tenth three aspects:, the invention provides the 7th quasi-microorganism, it is converted into homoserine poly--3-hydroxy propionate, wherein said microbial expression recombination, described recombination coding: desaminase or transaminase, desaturase, decarboxylase and PHA synthase.

Described desaturase catalysis is converted into 3-hydroxyl-propionic aldehyde the reaction of 3-hydroxyl propionyl-CoA.In certain embodiments, described desaturase is propionic aldehyde desaturase.Propionic aldehyde desaturase includes but not limited to PduP.The aminoacid sequence of coding PduP propionic aldehyde desaturase as known in the art is shown in SEQ ID NO:60 and 62.The exemplary DNA sequence dna of coding PduP propionic aldehyde desaturase is respectively shown in SEQ ID NO:59 and 61.

Aspect the 14, the invention provides the 7th class methods, it cultivates described the 7th quasi-microorganism to produce poly-3-hydroxy propionate for the production of poly--3-hydroxy propionate in described method.Homoserine is converted into 2-ketone group-4 hydroxybutyric acid ester by described the 7th class methods, 2-ketone group-4 hydroxybutyric acid ester is converted into 3-hydroxyl-propionic aldehyde, 3-hydroxyl-propionic aldehyde is converted into 3-hydroxyl propionyl-CoA, and then 3-hydroxyl propionyl-CoA is converted into poly--3-hydroxy propionate.

produce 3-hydroxyl propionyl-CoA

Aspect the 15, the invention provides the 8th quasi-microorganism, homoserine is converted into 3-hydroxyl propionyl-CoA by it, wherein said microbial expression recombination, described recombination coding: desaminase or transaminase, decarboxylase or desaturase.

Aspect the 16, the invention provides the 8th class methods, it cultivates described the 8th quasi-microorganism to produce 3-hydroxyl propionyl-CoA for the production of 3-hydroxyl propionyl-CoA in described method.Described the 8th class methods are converted into homoserine 2-ketone group-4 hydroxybutyric acid ester and then 2-ketone group-4 hydroxybutyric acid ester are converted into 3-hydroxyl propionyl-CoA.

Aspect the 17, the invention provides the 9th quasi-microorganism, homoserine is converted into 3-hydroxyl propionyl-CoA by it, wherein said microbial expression recombination, described recombination coding: desaminase or transaminase, decarboxylase and desaturase.

In the tenth eight aspect, the invention provides the 9th class methods, it cultivates described the 9th quasi-microorganism to produce 3-hydroxyl propionyl-CoA for the production of 3-hydroxyl propionyl-CoA in described method.Homoserine is converted into 2-ketone group-4 hydroxybutyric acid ester by described the 9th class methods, 2-ketone group-4 hydroxybutyric acid ester is converted into 3-hydroxyl-propionic aldehyde, and 3-hydroxyl-propionic aldehyde is converted into 3-hydroxyl propionyl-CoA.

produce 1,3-PD

Aspect the 19, the invention provides the tenth quasi-microorganism, it is converted into 1 by homoserine, ammediol, wherein said microbial expression recombination, described recombination coding: desaminase or transaminase, decarboxylase and 1,3-PD desaturase or aldehyde reductase.

Described 1,3-PD desaturase or aldehyde reductase catalysis are converted into 3-HPA the reaction of 1,3-PD.The aminoacid sequence of 1,3-PD desaturases more as known in the art is shown in SEQ ID NO:64,66,68,70 and 72.Encode the exemplary DNA sequence dna of described 1,3-PD desaturase respectively shown in SEQ ID NO:63,65,67,69 and 71.

Aspect the 20, the invention provides the tenth class methods, it cultivates described the tenth quasi-microorganism to produce 1,3-PD for the production of 1,3-PD in described method.Homoserine is converted into 2-ketone group-4 hydroxybutyric acid ester by described the tenth class methods, 2-ketone group-4 hydroxybutyric acid ester is converted into 3-hydroxyl-propionic aldehyde, and then 3-hydroxyl-propionic aldehyde is converted into 1,3-PD.

increase the carbon stream to homoserine

In the 20 one side, the invention provides microorganism, it comprises other genetic modification to increase the carbon stream to homoserine, and it increases the production of acrylate or other products of the present invention then.Described microorganism shows one or more in following characteristics.

In certain embodiments, compare with corresponding wild-type microorganisms, described microorganism shows the carbon stream to the increase of oxaloacetic acid.Described microbial expression recombination, described recombination is for example encoded, phosphoric acid enol pyruvic acid carboxylase or pyruvate carboxylase (or both).Described phosphoric acid enol pyruvic acid carboxylase includes but not limited in the phosphoenolpyruvic acid shown in SEQ ID NO:84.Encode the exemplary DNA sequence dna of described phosphoric acid enol pyruvic acid carboxylase shown in SEQ ID NO:83.Described pyruvate carboxylase includes but not limited at the pyruvate carboxylase shown in SEQ ID NO:86 and 88.Encode the exemplary DNA sequence dna of described pyruvate carboxylase shown in SEQ ID NO:85 and 87.

In certain embodiments, compare with corresponding wild-type microorganisms, described microorganism shows the E.C. 2.7.2.4. feedback inhibition of reduction.One or more in the gene of described microbial expression coded polypeptide, described gene includes but not limited to S345F ThrA (SEQ ID NO:76), T352I LysC (SEQ ID NO:78) and MetL (SEQ ID NO:74).The exemplary encoding sequence of coding said polypeptide is respectively shown in SEQ ID NO:75, SEQ ID NO:77 and SEQ ID NO:73.

In certain embodiments, compare with corresponding wild-type microorganisms, described microorganism shows lysA genetic expression or the diaminapimelate decarboxylase activity of reduction.In certain embodiments, compare with corresponding wild-type biology, the dapA that described microorganism shows reduction expresses or dihydropyridine acid synthase activity.The exemplary DNA sequence dna of lysA encoding sequence as known in the art is shown in SEQ ID NO:113.It is coded in the aminoacid sequence shown in SEQ ID NO:114.The exemplary DNA sequence dna of dapA encoding sequence as known in the art is shown in SEQ ID NO:115.It is coded in the aminoacid sequence shown in SEQ ID NO:116.

In certain embodiments, compare with corresponding wild-type microorganisms, described microorganism shows metA genetic expression or the homoserine succinyl transferase active of reduction.The exemplary DNA sequence dna of metA encoding sequence as known in the art is shown in SEQ ID NO:79.It is coded in the aminoacid sequence shown in SEQ ID NO:80.

In certain embodiments, compare with corresponding wild-type microorganisms, described microorganism shows thrB genetic expression or the homoserine kinase activity of reduction.The exemplary DNA sequence dna of thrB encoding sequence as known in the art is shown in SEQ ID NO:81.It is coded in the aminoacid sequence shown in SEQ ID NO:82.

In certain embodiments, described microorganism is not expressed eda gene.The exemplary DNA sequence dna of eda encoding sequence as known in the art is shown in SEQ ID NO:43.It is coded in the aminoacid sequence shown in SEQ IDNO:44.

Aspect the 22, the invention provides the microorganism of cultivating through further modifying to produce the method for product of the present invention.

thioesterase

At the 20 three aspects:, the invention provides thioesterase, its intermediate that is hydrolyzed metabolic pathway as herein described is to produce desired final product.In this regard, microbial expression recombination of the present invention, the nucleotide sequence that described recombination comprises the thioesterase of encoding, described thioesterase has for the activity of coenzyme A (CoA) that attaches to two carbon, three carbon or four carbochains, for example, such as three carbon or four carbochains that comprise two keys (wrapping double bond containing three carbon or four carbochains between C2 and C3).In certain embodiments, described thioesterase hydrolysis acryloyl-CoA is to form vinylformic acid.(or in addition) alternatively, in certain embodiments, described thioesterase hydrolysis crotonyl-CoA is to form β-crotonic acid.

This aspect of the present invention is at least in part according to the use judgement of thioesterase, and described thioesterase has for example, activity for the double bond containing short carbon chain of bag (, being less than four carbon on main chain).Although identified the thioesterase of the saturated short carbon chain of hydrolysis, script is unexpected can act on unsaturated carbon chains to identified thioesterase.Can expect that thioesterase shows substrate specificity highly to avoid being hydrolyzed acetyl-CoA for short carbon chain, described acetyl-CoA is crucial to lipogenesis.Unexpectedly, identifies that hydrolysis attaches to thioesterase and the successfully generation (or excessive generation) in host cell of the CoA intermediate of short unsaturated carbon chains.

Exemplary thioesterase includes but not limited to from colibacillary TesB and from its homologues of different organisms.In this regard, described host cell optionally comprises polynucleotide, described polynucleotide comprise nucleotide sequence, described nucleic acid sequence encoding is identical (for example with the aminoacid sequence at least 80% shown in SEQ ID NO:90 (TesB), 85%, 90%, 95%, 99% or 100% is identical), and coding has the polypeptide (, described polypeptide hydrolyze thioester key) of thioesterase activity.Encode the exemplary DNA sequence dna of described TesB aminoacid sequence shown in SEQ ID NO:89.The aminoacid sequence of other known thioesterase is shown in SEQ ID NO:96,98,100,102,104,106 and 108.Encode the DNA sequence dna of exemplary codon optimized (carrying out for intestinal bacteria) of described thioesterase shown in SEQ ID NO:95,97,99,101,103,105 and 107.

The thioesterase of through engineering approaches is also applicable in the present invention.For example, this enzyme thioesterase activity is given in the sudden change in the avtive spot of CoA transferring enzyme, significantly reduces (and if non-elimination) transferase active simultaneously.In many aspects, the use of thioesterase is better than the use of CoA transferring enzyme by discharging the energy being associated with CoA key.Described energy discharges and drives completing of vinylformic acid or β-crotonic acid path.Modifying CoA transferring enzyme comprises the alternative aminoacid replacement of the amino acid playing a role as catalytic carboxylate radical using the illustrative methods that obtains thioesterase activity.Be applicable to modifying and include but not limited to acetyl-CoA transferring enzyme, propionyl-CoA transferring enzyme and butyryl-CoA transferring enzyme for the CoA transferring enzyme in the scope of the invention.In one aspect, the aminoacid sequence that thioesterase of the present invention comprises propionyl-CoA transferring enzyme, wherein catalytic glutaminic acid residue is replaced such as aspartic acid with alternative amino acid.Propionyl-CoA the transferring enzyme that is applicable to sudden change comprises the propionyl-CoA transferring enzyme from clostridium propionicum (C.propionicum) and Erichsen Megasphaera (M.elsdenii).Glutaminic acid residue 324 and glutaminic acid residue 325 are respectively the catalytic carboxylate radical in propionyl-CoA transferring enzyme and Erichsen Megasphaera propionyl-CoA transferring enzyme of clostridium propionicum.Because catalytic carboxylate radical is guarded in CoA transferring enzyme, by with for example, the aminoacid sequence of clostridium propionicum propionyl-CoA transferring enzyme carries out sequence alignment and identifies from the catalytic amino-acid residue in the propionic acid CoA transferring enzyme in other source.Similarly, by with for example, the aminoacid sequence of clostridium propionicum propionyl-CoA transferring enzyme carries out sequence alignment and identifies for example, catalytic amino-acid residue in other CoA transferring enzyme (, acetyl-CoA transferring enzyme or butyryl radicals-CoA transferring enzyme).Propionyl-CoA transferring enzyme of clostridium propionicum is the example that is applicable to the sequence comparing with other CoA transferring enzyme.Should be appreciated that the sequence that can compare other CoA transferase sequences is to identify the conservative L-glutamic acid catalytic residue for suddenling change.Should also be understood that the sudden change CoA transferring enzyme with thioesterase activity can produce by the encode nucleotide sequence of polynucleotide of existing CoA transferring enzyme of change, or generate new polynucleotide by the encoding sequence based on CoA transferring enzyme and produce.Therefore, in these embodiments, host cell of the present invention optionally comprises polynucleotide, described polynucleotide comprise nucleotide sequence, described nucleic acid sequence encoding and SEQ ID NO:92(are derived from the thioesterase of clostridium propionicum, comprise E324D surrogate) or SEQ ID NO:94(derived from the thioesterase of Erichsen Megasphaera, comprise E325D surrogate) shown in aminoacid sequence at least 80% identical (for example, 85%, 90%, 95%, 99% or 100% is identical) and coding there are the polynucleotide of thioesterase activity.Encode exemplary (carrying out codon optimized for intestinal bacteria) DNA sequence dnas of this two kinds of thioesterases respectively shown in SEQ ID NO:91 and 93.The aminoacid sequence of other through engineering approaches thioesterase is shown in SEQ ID NO:109,110,111 and 112.

the enzyme separating

In certain embodiments, the enzyme of separation can be used for one or more steps of describing in catalysis aspect of the present invention.Advantage can comprise higher products collection efficiency, the easier product recovery from the more strong solution of acellular related impurities, wider possible reaction conditions, the use of more cheap reactor.

Accompanying drawing explanation

Fig. 1 illustrates the step that glucose transforms to homoserine.

Fig. 2 illustrates the step of being produced acrylate, 3-hydroxyl propionyl-CoA, 3-hydroxy propionate and poly--3-hydroxy propionate in the inventive method by homoserine.

Fig. 3 illustrates the step of being produced acrylate, 3-hydroxy propionate, 1,3-PD and 3-hydroxyl propionyl-CoA in the inventive method by homoserine.

Fig. 4 illustrate 2-ketone group-4 hydroxybutyric acid ester and L-glutamic acid with the L-homoserine that there is (reaction) and do not there is (contrast) Pf_AT transaminase with single ion monitoring (SIM) the LC-MS chromatogram of α – ALPHA-KG after hatching.

Fig. 5 illustrates the original speed of deaminizing, and it is the function of L-homoserine concentration to Pf_AT transaminase.

Fig. 6 illustrates that the 3-hydroxyl propionyl-CoA being undertaken by L-homoserine by D-AAO and 2-Ketoglutarate desaturase or D-AAO, KdcA decarboxylase and the catalysis of PduP desaturase produces.

Fig. 7 is illustrated in the HPLC chromatogram of the acryloyl-CoA sample with (top) or after not hatching with (bottom) dehydratase, thereby confirms that only the 3-hydroxyl propionyl-CoA in the time that this enzyme exists forms.

Fig. 8 illustrates by dehydratase catalysis and produces 3-hydroxyl propionyl-CoA by acryloyl-CoA.

Fig. 9 is illustrated in the 3-hydroxyl propionyl-CoA consumption after hatching with PHA synthase, thus the formation that shows to gather (3-hydroxy propionate).

Figure 10 illustrates the thioesterase activity for acryloyl-CoA substrate.Activity is monitored by the optical density(OD) under 412nm (OD).

Figure 11 illustrates the thioesterase activity for capryloyl-CoA substrate.Activity is monitored by the optical density(OD) under 412nm (OD).

Figure 12 illustrates the thioesterase activity for acryloyl-CoA substrate.Activity is monitored by the optical density(OD) under 412nm (OD).

Figure 13 illustrates the thioesterase activity for acryloyl-CoA substrate.Activity is monitored by the optical density(OD) under 412nm (OD).

Figure 14 illustrates the thioesterase activity for capryloyl-CoA substrate.Activity is monitored by the optical density(OD) under 412nm (OD).

Figure 15 illustrates the thioesterase activity for capryloyl-CoA substrate.Activity is monitored by the optical density(OD) under 412nm (OD).

Embodiment

definition

The invention provides product vinylformic acid and acrylate.As understood in the art, acrylate is acrylic acid carboxylate anion (, conjugate base).The pH of product solution determines in prepared product that acrylate is to acrylic acid relative quantity, according to Martin Henderson-Ha Sai Bach (Henderson-Hasselbalch) equation { pH=pKa+log ([A ^-]/[HA], wherein pKa Wei – log (Ka).Ka is acrylic acid acid dissociation constant.The pKa of vinylformic acid in water is approximately 4.35.Therefore, neutral pH place or near, vinylformic acid will exist mainly as carboxylate anion.As used herein, both are all intended to " vinylformic acid " and " acrylate " contain each other.

As used herein, " amplification ", " amplification " or " amplification " refer to any method or the rules that polynucleotide sequence are copied into greater amount polynucleotide molecule, for example, by reverse transcription, polymerase chain reaction and ligase chain reaction.

As used herein, " antisense sequences " refers to specifically the sequence with the second polynucleotide sequence hybridization.For example, antisense sequences is to transcribe the reverse DNA sequence dna of normal orientation with respect to it.Antisense sequences can be expressed the rna transcription thing (for example, it can hybridize on said target mrna molecule by Wo Sen-Ke Like (Watson-Crick) base pairing) with the said target mrna complementary element at host cell inner expression.

As used herein, " cDNA " refers to strand or the double chain form DNA complementary or identical with mRNA.

As used herein, " complementation " refers to the polynucleotide paired with the second polynucleotide base.In other words, " complementation " relation between two single-chain nucleic acid sequences of annealing by base pairing is described.For example there are the polynucleotide and the polynucleotide complementation with sequence 5'-TCGGAC-3' of sequence 5'-GTCCGA-3'.

As used herein, " conservative replacement " refers to and uses similar aminoacid replacement in function for the amino acid in polypeptide.In other words, conservative replacement relates to the amino-acid residue replacement amino-acid residue with similar side chain.This area has defined the amino-acid residue family with similar side chain, they comprise amino acid (for example Methionin with basic side chain, arginine and Histidine), there is the amino acid (for example aspartic acid and L-glutamic acid) of acid side-chain, there is amino acid (for example glycine of uncharged polar side chain, l-asparagine, glutamine, Serine, Threonine, tyrosine and halfcystine), there is amino acid (for example L-Ala of non-polar sidechain, α-amino-isovaleric acid, leucine, Isoleucine, proline(Pro), phenylalanine, methionine(Met) and tryptophane), there is amino acid (for example Threonine of β-side chain side chain, α-amino-isovaleric acid and Isoleucine) and there is amino acid (for example tyrosine of aromatic side chains, phenylalanine, tryptophane and Histidine).

As used herein, " corresponding wild-type microorganisms " be naturally occurring microorganism, its should with microbial of the present invention together, difference be described naturally occurring microorganism without genetically engineered to express any recombination.

As used herein, " coding " refers to the natural characteristics of Nucleotide as other polymkeric substance and the synthetic template action of macromole.Except as otherwise noted, " nucleotide sequence of encoding amino acid sequence " comprises all nucleotide sequences for degeneracy version the same acid sequence of encoding each other.

As used herein, " endogenous " refers to polynucleotide natural expression or primary, polypeptide or other compound in organism or cell., endogenous polynucleotide, polypeptide or other compound are not external sources.For example, when described cell initially-separate is during from natural origin, " endogenous " polynucleotide or peptide are present in cell.

As used herein, " expression vector " refers to the carrier that comprises recombination of polynucleotide, and described polynucleotide comprise to be operably connected to intends the expression control sequenc that lists of nucleotides sequence of expressing.That for example suitable expression vector can be self-replicating or be integrated into the plasmid in karyomit(e).

As used herein, " exogenous " refers to any polynucleotide or the polypeptide that non-natural exists in the specific cells of expecting to express or organism.Exogenous polynucleotide, polypeptide or other compound are not endogenic.

As used herein, " homoserine " comprises enantiomorph, such as L-homoserine and D-homoserine.

As used herein, " hybridization " comprises any process that nucleic acid chains engages by base pairing with complementary nucleic acid chain.Therefore, this term refers to that target sequence is attached to the complementary ability in test (, target) sequence, or vice versa.

As used herein, " hybridization conditions " is conventionally by " severity " degree classification of measuring under the condition of hybridizing.Severity degree can be based on nucleic acid for example melt temperature (Tm) in conjunction with complex compound or probe.For example, " maximum severity " is conventionally in about Tm-5 ℃ (lower 5 ° than the Tm of probe) lower generation; " high severity " is than the low about 5-10 ° of Tm; " middle severity " is than the low about 10-20 ° of the Tm of this probe; And " low severity " is than the low about 20-25 ° of Tm.Alternatively or in addition, hybridization conditions can be based on hybridization salt or ionic strength conditions and/or the washing of one or many severity.For example, the extremely low severity of 6 × SSC=; 3 × SSC=is low to moderate middle severity; Severity in l × SSC=; And the high severity of 0.5 × SSC=.In function, maximum stringency can be used for identifying the nucleotide sequence that has strictly (, approximately 100%) identity with hybridization probe or approach strict identity; And high stringency is for the identification of having approximately 80% or the nucleotide sequence of higher sequence identity with described probe.

As used herein, " identical " or the per-cent " identity " that relate to two or more polynucleotide or peptide sequence refer to that two or more sequences are in the time being used sequence comparison algorithm or compared and compared maximum match by visual detection, and they are identical or have identical Nucleotide or the amino-acid residue of prescribed percentage.

In " microorganism " of the present invention natural situation of expression recombination, do not exist.In other words, described microorganism be artificial and through genetically engineered with express recombination.Microorganism of the present invention has carried out genetic modification to express recombination, and described recombination coding carries out homoserine and transforms necessary enzyme to desired product.Microorganism of the present invention is bacterium, yeast, fungi and algae.Bacterium includes but not limited to coli strain K, B or C.The microorganism that the toxicity of product of the present invention is had more to resistance is preferred.The present invention for example imagines vegetable cell, using the alternative cell as microorganism (in the production of poly--3-hydroxy propionate), its not naturally occurring (artificial) and the genetically engineered recombination that carries out conversion detailed in this article with expression.

As used herein, " naturally occurring " refers to natural visible material.For example be present in polypeptide or polynucleotide sequence in organism (comprising virus), it can separate and not yet in laboratory, be subject to artificial having a mind to and modify from natural origin, is naturally occurring.As used herein, " naturally occurring " and " wild-type " is synonym.

As used herein, in the time describing being related between two DNA regions or two polypeptide regions, " being operably connected " refers to that described region is relevant in function each other.For example, promotor may be operably coupled to encoding sequence in the situation that its control sequence is transcribed; Ribosome bind site is placed in the position that allows translation and may be operably coupled to encoding sequence in the case of it; And sequence is brought into play function (such as the secretion of the protein by participating in mature form) and be may be operably coupled to peptide as signal sequence in the case of it.

As used herein, the recombination of " cross and express " produces than the corresponding more RNA of naturally occurring gene and/or protein in microorganism.Measuring RNA and method of protein is well known in the art.Cross and express and can determine by measuring protein active, such as enzymic activity.According to embodiments of the invention, " cross and express " more measures at least 3%, at least 5%, at least 10%, at least 20%, at least 25% or at least 50%.Cross the natural polynucleotide that the polynucleotide of expressing are generally host cell, the product amount of its generation is greater than the amount of normal presence in host cell.Cross to express for example and without restriction by polynucleotide being may be operably coupled to the promotor different from this polynucleotide natural promoter or described polynucleotide that will additional copies and import in host cell and realize.

As used herein, " polynucleotide " refer to the polymkeric substance being made up of Nucleotide.Polynucleotide can be isolated fragment form or can be the component of larger nucleotide sequence construct, it is derived from the nucleotide sequence separating at least one times, and the quantity of this nucleotide sequence or concentration can be by standard molecular biology method as used cloning vector to identify, handle and reclaim this sequence and component nucleotide sequence thereof.In the time that DNA sequence dna for nucleotide sequence (being A, T, G, C) represents, this also comprises RNA sequence (being A, U, G, C), wherein " U " replacement " T ".In other words, " polynucleotide " refer to the nucleotide polymer (fragment or entity separately) removing from other Nucleotide or assembly or element, for example expression vector or the polycistron sequence that can be larger constructs.Polynucleotide comprise DNA, RNA and cDNA sequence.

As used herein, " polypeptide " refers to the polymkeric substance being made up of amino-acid residue, and it can comprise or can not comprise modified forms as phosphate and formyl radical.

As used herein, " primer " refers to polynucleotide primers is placed under the synthetic condition of induction, can hybridize to specifically the polynucleotide of specifying on polynucleotide template and the synthetic start-up point of complementary polynucleotide being provided.

As used herein, " recombination of polynucleotide " refers to the polynucleotide with the sequence not linking together in natural situation.Recombination of polynucleotide can be included in suitable carrier, and described carrier can be used for transforming suitable host cell.The host cell that comprises recombination of polynucleotide is called " recombinant host cell ".Then described polynucleotide are expressed in recombinant host cell to generate, for example, " recombinant polypeptide ".

As used herein, " recombinant expression vector " refers to for example, DNA construct for expressing polynucleotide (coding is expected the polynucleotide of polypeptide).Recombinant expression vector can comprise for example transcribes subunit, the described subunit of transcribing comprises that the genetic elements (i) with genetic expression regulatory function is as the subassembly of promotor and enhanser, (ii) be transcribed into mRNA and translate into structure or the encoding sequence of protein, and (iii) suitable transcribe and translate start and terminator sequence.Build in any suitable manner recombinant expression vector.The character of carrier is not crucial, and can use any carrier to comprise plasmid, virus, phage and transposon.May include but not limited to karyomit(e), non-chromosome and synthetic DNA sequence by carrier for of the present invention, for example, bacterial plasmid; Phage DNA; Yeast plasmid; And derived from the carrier of the combination of plasmid and phage DNA, from viral DNA, such as cowpox, adenovirus, fowl pox, baculovirus, SV40 and pseudorabies virus.

As used herein, " recombination " is not naturally occurring gene.Recombination is artificial.Recombination comprises protein coding sequence, and it is operatively connected to expression control sequenc.Embodiment includes but not limited to introduce the foreign gene of microorganism, operatively be connected in allogeneic promoter (, in natural situation, be not attached to the promotor of described protein coding sequence) endogenous protein encoding sequence and (for example there is modified protein coding sequence, the protein coding sequence that coded amino acid changes, or the protein coding sequence being optimized for the expression in described microorganism).Described recombination is maintained in the genome of described microorganism, is maintained on the plasmid in described microorganism or is maintained on the phage in described microorganism.

As used herein, " reduction " is expressed as the RNA lower than the expression of corresponding natural horizontal or protein expression.Measuring RNA and method of protein is well known in the art.The expression reducing can be measured by measuring protein active, such as enzymic activity.Depend on embodiments of the invention, " reduction " is at least 3%, at least 5%, at least 10%, at least 20%, at least 25% or at least 50% amount still less.

As used herein, " specific hybrid " refers to that polynucleotide are preferential in conjunction with, duplex or hybridize on specific nucleotide sequence under critical conditions.

As used herein, " critical conditions " refers to that probe will preferentially hybridize on its target subsequence under this condition, and lower or do not hybridize completely with the degree of other sequence hybridization.

As used herein, " homology substantially " or " substantially the same " that relates to two nucleic acid or polypeptide refers generally to two or more sequences or subsequence in the time using sequence comparison algorithm or range estimation relatively and compare maximum match, has at least 40%, 60%, 80%, 90%, 95%, 96%, 97%, 98% or 99% Nucleotide or amino-acid residue identity.Substantially the samely can be present in any suitable region of sequence, for example length is at least about the region of 50 residues, and length is at least about the region of 100 residues, or length is at least about the region of 150 residues.In certain embodiments, in office one or two of sequence compares in biological polymer total length substantially the same.

polynucleotide

The polynucleotide of one or more enzymic activitys of the step in path of the present invention of encoding can be derived from any source.Depend on embodiments of the invention, described polynucleotide separate from natural source, such as bacterium, algae, fungi, plant or animal; Produce (for example, the nucleotide sequence of polynucleotide carries out codon optimized for specific host cell such as intestinal bacteria) by semi-synthetic route; Or de novo synthesis.In certain embodiments, the substrate specificity based on for example enzyme or the enzyme activity level in given host cell are favourable from particular source selection enzyme.In the present invention aspect some, described enzyme and corresponding polynucleotide are natural to be present in host cell and to expect expression polynucleotide.In this regard, in certain embodiments, the polynucleotide of additional copies are introduced in host cell to increase enzyme concentration.In certain embodiments, crossing of endogenous polynucleotide expressed and can be realized by raising endogenesis promoter activity or described polynucleotide being operatively connected in to more powerful allogeneic promoter.

The polynucleotide of exogenous enzymes and their correspondences are also applicable to the present invention, and can change according to certain enzyme used feature or the final product of biosynthetic pathway.

The present invention's imagination, polynucleotide of the present invention can be through through engineering approaches to comprise that alternative degenerate code is to optimize the expression of described polynucleotide in specified microorganisms.For example, at described DNA sequence dna, by expression in escherichia coli in the situation that, polynucleotide can be through through engineering approaches to comprise preferred codon in intestinal bacteria.Codon optimized method is well known in the art.

enzyme variants

In certain embodiments, analogue or the variant of the polypeptide of described microorganisms codase activity.The aminoacid sequence variant of described polypeptide comprise replacement, insert or disappearance variant, and variant can with the polypeptide of unmodified homology or substantially the same substantially.In certain embodiments, described variant retains at least some biological activitys of described polypeptide, for example catalytic activity.Other variant comprises the variant of described polypeptide, and it retains at least about 50%, preferably at least about 75%, more preferably at least about 90% biological activity.

Replace variant and conventionally change an amino acid on one or more sites in protein into another.This replacement can be conservative replacement, i.e. the amino acid substitution of an analogous shape and electric charge for amino acid.Conservative replacement for example comprises, below changes: L-Ala is to Serine; Arginine is to Methionin; L-asparagine is to glutamine; Aspartate is to L-glutamic acid; Halfcystine is to Serine; Glutamine is to l-asparagine; L-glutamic acid is to aspartate; Isoleucine is to leucine or α-amino-isovaleric acid; Leucine is to α-amino-isovaleric acid or Isoleucine; Methionin is to arginine; Methionine(Met) is to leucine or Isoleucine; Phenylalanine is to tyrosine, leucine or methionine(Met); Serine is to Threonine; Threonine is to Serine; Tryptophane is to tyrosine; Tyrosine is to tryptophane or phenylalanine; And α-amino-isovaleric acid is to Isoleucine or leucine.Herein for an example of nomenclature of indicating amino acid substitution for " S345F ThrA ", the naturally occurring tryptophane wherein existing on the 345th in naturally occurring ThrA enzyme is replaced by phenylalanine.

In some cases, described microorganism comprises one or more of polynucleotide exogenous or that excessively express as herein described analogue or variant.Nucleotide sequence variant comprises one or more replacements, insertion or disappearance, and variant can with unaltered polynucleotide homology or substantially the same substantially.Polynucleotide variant or analogue encoding mutant enzyme, described enzyme has at least part of activity of unaltered enzyme.Alternatively, polynucleotide variant or the analogue coding aminoacid sequence identical with unaltered polynucleotide.The general coding of for example codon optimized sequence aminoacid sequence identical with parent/native sequences, but the codon of preferentially expressing comprised in specific host biology.

" derived from " polypeptide of organism or one or more modifications that polynucleotide comprise naturally occurring aminoacid sequence or nucleotide sequence, and show and (if not better) active (for example at least 70%, at least 80%, at least 90%, at least 95%, at least 100% or at least 110% natural enzyme activity level) like natural Enzymes.For example, in some degree, improve enzymic activity by orthogenesis parent/naturally occurring sequence.Alternatively or in addition, enzyme encoding sequence undergos mutation to realize feedback resistance.

expression vector/transfer enters microorganism

Expression vector for recombination can produce the expression to set up described gene in microorganism in any suitable manner.Expression vector includes but not limited to plasmid and phage.This expression vector can comprise the exogenous polynucleotide that may be operably coupled to Expression element, and described Expression element is for example promotor, enhanser, ribosome bind site, operon and activation sequence.This type of Expression element can be regulatable, for example derivable (by adding inductor).Alternatively or in addition, expression vector can comprise the polynucleotide of additional copies, its coding may be operably coupled to the natural gene product of Expression element.The representative example of available allogeneic promoter includes but not limited to: 35 tumor-necrosis factor glycoproteinss of the retroviral long end of LTR() or SV40 promotor, intestinal bacteria lac, tet or trp promotor, phageλ promotor and known control protokaryon or eukaryotic cell or their virus in other promotor of genetic expression.In one aspect, expression vector also comprises the proper sequence that amplification is expressed.Expression vector can comprise and promotes polynucleotide to sneak into the element of cellular genome.

To in expression vector or other polynucleotide introducing cell, can use any suitable method to complete, for example conversion, electroporation, microinjection, microinjection bombardment, calcium phosphate precipitation, modified phosphate calcium deposit, cation lipid processing, light perforation, fusion method, receptor-mediated transfer or polybrene precipitation.Alternatively, can by with viral vector infection, put together, transduce or other usability methods imports expression vector or other polynucleotide.

culture

The microorganism that the present invention comprises recombination is cultivated under the condition that is suitable for described Growth of Cells and described genetic expression.Expressing the microorganism of one or more polypeptide can identify by any applicable method, for example, screen, screened or screened by Southern engram analysis the protein of expression by PCR.In certain embodiments, can be by comprising selected marker in DNA construct, then under the condition of cell survival that is only applicable to expressing selected marker, cultivate the microorganism that comprises selected marker and select the microorganism that comprises described polynucleotide.Can (for example under the existence of finite concentration medicine, cultivate the microorganism through genetic modification that comprises amplifiable marker gene by the microorganism of cultivating through genetic modification under conditions suitable, under described concentration, the microorganism that only comprises multiple copied amplifiable marker gene can survive) the further DNA construct that increases and import.

In certain embodiments, the described microorganism bacterial cell of genetic modification (for example through) has best growth temperature, the lower temperature of temperature for example facing than growing under normal circumstances and/or fermenting.In addition, in certain embodiments, cell of the present invention is showing growth at the temperature that the normal growth of the type cell and/or leavening temperature are higher and is declining than being conventionally present in.

The microorganism that is applicable to grow is all applicable to method of the present invention with any cell culture condition of the synthetic product of paying close attention to.

reclaim

Method of the present invention optionally comprises product removal process.The recovery of acrylate, 3-hydroxyl propionyl-CoA, 3-hydroxy propionate, poly--3-hydroxy propionate or 1,3-PD can be implemented by method as known in the art.For example, acrylate can reclaim by distillating method, extracting process, crystallization method or their combination; 3-hydroxy propionate can reclaim as the description in U.S.'s publication application 2011/038364 or international publication WO2011/0125118; Polyhydroxyalkanoatefrom can be according to Yu and Chen, Biotechnol Prog, 22 (2): the description in 547-553 (2006) is reclaimed; And 1,3 propylene glycol can be according to the people such as United States Patent (USP) 6428992 or Cho, Process Biotechnology, 41 (3): the description in 739-744 (2006) is reclaimed.

example

Following instance further describes and shows the embodiment in the scope of the invention.Given example is only for illustrating, and unintelligible for being limitation of the present invention.Example 1 to 6 description is the structure at the different plasmids of the heterogenous expression of intestinal bacteria for protein; Example 7 and 8 is described conversion and the cultivation of coli strain; Example 9 and 10 is described the purifying of multiple proteins; Example 12 is described the quantivative approach to acyl group-CoA molecule; Example 11 and 13 to 16 is described the reconstruction in vitro of the multiple proteins enzymic activity of describing in the present invention; The 3-hydroxy-propionic acid that example 17 is described in the intestinal bacteria of through engineering approaches is produced.

example 1

the expression vector of aminotransferase gene

Build the coli expression carrier for the production of restructuring transaminase.Utilize the pET30a carrier (Novagen, EMD Chemicals, Gibbstown, NJ, catalogue #69909-30) for express the protein that is connected in N-terminal hexahistidine tag under T7 promotor to set up conventional Strategies For The Cloning.Implement the modification to pET30a carrier by the DNA sequence dna between SphI and XhoI site being replaced to synthetic DNA sequence dna (SEQ ID NO:117) (GenScript, Piscataway, NJ).In the carrier of this gained (called after pET30a-BB), the XbaI site in lac operon is removed and the region in coding zymoplasm, S-label and enteropeptidase site is replaced to the sequence of coding factor Xa recognition site.In addition, modify described multiple clone site to comprise EcoRV, EcoRI, BamHI, SacI and PstI site.

Carry out codon optimized for the expression in intestinal bacteria to multiple aminotransferase gene.For being conducive to clone, also from described gene order, remove common restriction site: AvrII; BamHI; BglII; BstBI; EagI; EcoRI; EcoRV; HindIII; KpnI; NcoI; NheI; NotI; NspV; PstI; PvuII; SacI; SalI; SapI; SfuI; SpeI; XbaI; XhoI.In addition, the 5' sequence (SEQ ID NO:118) that moves ahead is added to the next-door neighbour upstream of initiator codon, and will after SpeI, NotI and PstI restriction site 3', connect sequence (SEQ ID NO:119) and add to the downstream of terminator codon.The synthetic sequence (GenScript, Piscataway, NJ) of optimizing also enters pET30a-BB carrier at KpnI and PstI site clone.The plasmid of gained and coded protein are described in table 1.

table 1: the plasmid list of the different transaminases of encoding

example 2

for the expression vector of side chain 2-keto acid decarboxylase (KdcA)

Build the coli expression carrier for generation of restructuring 2-keto acid decarboxylase (KdcA).Lactococcus lactis (Lactococcus lactis) side chain 2-keto acid decarboxylase gene carries out codon optimized for the expression in intestinal bacteria, and removes common restriction site: AvrII; BamHI; BglII; BstBI; EagI; EcoRI; EcoRV; HindIII; KpnI; NcoI; NheI; NotI; NspV; PstI; PvuII; SacI; SalI; SapI; SfuI; SpeI; XbaI; XhoI is to be conducive to clone.In addition, described sequence comprises additional EcoRI, NotI, XbaI restriction site and the ribosome bind site (RBS) at the 5' place of ATG initiator codon and SpeI, NotI and the Pst restriction site at terminator codon 3' place.Synthesize the sequence (SEQ ID NO:124) (GenScript, Piscataway, NJ) of optimizing and enter pET30a-BB carrier at EcoRI and PstI site clone.The expression vector called after pET30a-BB Ll KDCA of the KdcA (SEQ ID NO:54) of the coding N-terminal histidine mark of gained.

example 3

for the expression vector of coenzyme-A acidylate propionic aldehyde desaturase (PduP)

Build the coli expression carrier for generation of restructuring coenzyme-A acidylate propionic aldehyde desaturase (PduP).Salmonella enteritidis (Salmonella enterica) coenzyme-A acidylate propionic aldehyde dehydrogenase gene carries out codon optimized for the expression in intestinal bacteria, and removes common restriction site: AvrII; BamHI; BglII; BstBI; EagI; EcoRI; EcoRV; HindIII; KpnI; NcoI; NheI; NotI; NspV; PstI; PvuII; SacI; SalI; SapI; SfuI; SpeI; XbaI; XhoI is to be conducive to clone.In addition, described sequence comprises additional EcoRI, NotI, XbaI restriction site and the ribosome bind site (RBS) at the 5' place of ATG initiator codon and SpeI, NotI and the PstI restriction site at terminator codon 3' place.Synthesize the sequence (SEQ ID NO:125) (GenScript, Piscataway, NJ) of optimizing and enter pET30a-BB carrier at EcoRI and PstI site clone.The PduP (SEQ ID NO:60) of the gained expression vector codes N-terminal histidine mark version of called after pET30a-BB Se PDUP.

example 4

for the expression vector of poly-(3-hydroxybutyrate ester) polysaccharase (PhaC or PHA synthase)

Build the coli expression carrier for generation of poly-(3-hydroxybutyrate ester) polysaccharase of restructuring.Hookworm is coveted poly-(3-hydroxybutyrate ester) polysaccharase (phaC) of copper bacterium (Cupriavidus necator) and carries out codon optimizedly for the expression in intestinal bacteria, and removes common restriction site: AvrII; BamHI; BglII; BstBI; EagI; EcoRI; EcoRV; HindIII; KpnI; NcoI; NheI; NotI; NspV; PstI; PvuII; SacI; SalI; SapI; SfuI; SpeI; XbaI; XhoI is to be conducive to clone.In addition, described sequence comprises additional EcoRI, NotI, XbaI restriction site and the ribosome bind site (RBS) at the 5' place of ATG initiator codon and SpeI, NotI and the PstI restriction site at terminator codon 3' place.Synthesize the sequence (SEQ ID NO:126) (GenScript, Piscataway, NJ) of optimizing and enter pET30a-BB carrier at EcoRI and PstI site clone.The PHA synthase (SEQ ID NO:42) of the gained expression vector codes N-terminal histidine mark version of called after pET30a-BB Cn PHAS.

example 5

for the expression vector of 3-hydroxyl propionyl-CoA-dehydratase

Build the coli expression carrier for generation of restructuring 3-hydroxyl propionyl-CoA-dehydratase.3-hydroxyl propionyl-CoA-dehydrase gene of diligent metal coccus (Metallosphaera sedula) carries out codon optimized for the expression in intestinal bacteria, and removes common restriction site: AvrII; BamHI; BglII; BstBI; EagI; EcoRI; EcoRV; HindIII; KpnI; NcoI; NheI; NotI; NspV; PstI; PvuII; SacI; SalI; SapI; SfuI; SpeI; XbaI; XhoI is to be conducive to clone.In addition, described sequence comprises additional EcoRI, NotI, XbaI restriction site and the ribosome bind site (RBS) at the 5' place of ATG initiator codon and SpeI, NotI and the PstI restriction site at terminator codon 3' place.Synthesize the sequence (SEQ ID NO:127) (GenScript, Piscataway, NJ) of optimizing and enter pET30a-BB carrier at EcoRI and PstI site clone.The dehydratase (SEQ ID NO:48) of the gained expression vector codes N-terminal histidine mark version of called after pET30a-BB Ms3HP-CD.

example 6

for the expression vector of acyl-CoA thioesterase

Build the coli expression carrier that is as short as the acyl-CoA thioesterase of medium chain for generation of restructuring.Carry out codon optimizedly from different biological thioesterase genes for the expression in intestinal bacteria, and remove common restriction site: BamHI, BglII, BstBI, EcoRI, HindIII, KpnI, PstI, NcoI, NotI, SacI, SalI, XbaI and XhoI to be conducive to clone.In addition, described sequence comprises the additional BamHI at 5' place and SacI and the HindIII restriction site at XbaI restriction site and ribosome bind site and terminator codon 3' place of ATG initiator codon.Synthesize the sequence (GenScript, Piscataway, NJ or GeneArt, Invitrogen, Carlsbad, CA) of optimizing and enter pET30a carrier at BamHI and SacI site clone.The plasmid of gained and coded protein are described in table 2.

table 2: the plasmid list of the different thioesterases of encoding

example 7

colibacillary conversion

Then use described recombinant plasmid transformed chemoreception state One Shot BL21 (DE3) pLysS Bacillus coli cells (Invitrogen, Carlsbad, CA).Melt the cell of single bottle on ice and gently mix with 10ng plasmid DNA.Described bottle is hatched on ice 30 minutes.Hatch 30 seconds and put back to rapidly additional 2 minutes on ice of short duration at 42 ℃ described bottle.Add 37 ℃ SOC substratum 250 μ L aliquots containigs and described vial level is fixed on shaking culture case and at 37 ℃, under 225rpm, hatch 1 hour.The aliquots containig of 20 μ L and 200 μ L cells is coated on LB agar plate, and it is supplemented with suitable microbiotic (50 μ g/mL kantlex; 34 μ g/mL paraxin) to select respectively to carry the cell of recombinant plasmid and pLysS plasmid, overnight incubation at 37 ℃ subsequently.Separate single bacterium colony isolate, in 5mL selectivity LB liquid nutrient medium, cultivate and use

the miniature test kit of preparing of the centrifugal plasmid of Spin Miniprep test kit (Qiagen, Valencia, CA) separates recombinant plasmid.Plasmid DNA is carried out gel electrophoresis by the restriction digest to AflIII and is characterized.

example 8

the cultivation of coli strain and the expression of recombinant protein

Suitable microbiotic (34 μ g/mL paraxin will be supplemented with; 50 μ g/mL kantlex) the aliquots containig (15mL) of LB liquid nutrient medium be vaccinated the different coli strains from freezing glycerine stock.Culture is vibrated to hatch with 250rpm at 25 ℃.(150mL, comprises 34 μ g/mL paraxin to LB liquid nutrient medium, 50 μ g/mL kantlex; Balance at 25 ℃) in 1 to 2.8L recessed edge Erlenmeyer flask by overnight culture with～0.1 600nm under optical density(OD) (OD) inoculate.At 25 ℃, continue cultivation and monitor optical densities with 250rpm vibration until～0.4 A ₆₀₀.By adding 1M IPTG(Teknova, Hollister, CA; 1mM ultimate density) induce the production of recombinant protein.Before by centrifugal collection, at 25 ℃, further hatch culture 24h with 250rpm vibration.Cell mass stores until use at-80 ℃.

example 9

recombinant protein separates

The recombinant protein of His mark uses the Sepharose CL-6B resin (Ni-NTA, Qiagen, Valencia, CA) of nickel-nitrilotriacetic acid(NTA) coupling to separate as follows by fixing metal affinity chromatography (IMAC).Melt cell mass on ice and be suspended in (20mM sodium phosphate in the binding buffer liquid of 20mL, 500mM NaCl, 20mM imidazoles, pH7.4), it is supplemented with the N,O-Diacetylmuramidase of 1mg/mL and 1 completely without EDTA protease inhibitor (Roche Applied Science, Indianapolis, IN).At 4 ℃, hatch sample 30 minutes with the rotation of 30rpm, carry out subsequently French and crush processing (1000psi).By carry out sedimentation cell fragment with the centrifugal 1h of 15,000 × g at 4 ℃.Supernatant liquor is transferred on the 5mL base for post of Ni-NTA resin, it carries out balance with binding buffer liquid.Described Ni-NTA resin is resuspended in described supernatant liquor and at 4 ℃ and uses the slow mixing of rolling to hatch 60 minutes.By run by gravity remove unconjugated material and by run by gravity with 20 times of column volume (CV, binding buffer liquid 100mL) is subsequently with dcq buffer liquid (the 20mM sodium phosphate of 10 times of CV (50mL), 500mM NaCl, 100mM imidazoles, pH7.4) wash this resin.The protein of the run by gravity wash-out institute combination of the elution buffer (20mM sodium phosphate, 500mM NaCl, 500mM imidazoles, pH7.4) by 10 times of CV (50mL) also assigns to its classification to collect.Analyze for protein content by SDS-PAGE and analyze the aliquots containig of wash-out, converged and concentrate to there is the Amicon Ultra-15 centrifugal filter (EMD Millipore, Billerica, MA) that 30kD nominal molecular weight holds back.Use PD-10 desalination strain (GE Healthcare Biosciences, Pittsburgh, PA) concentrated protein isolate is carried out desalination wash-out and is entered store buffer liquid (50mM HEPES, the 300mM NaCl of 3.5mL, 20% glycerine, pH7.3) in.

example 10

the separation of restructuring thioesterase

The restructuring thioesterase of His mark uses the sepharose base magnetic beads (His Mag Sepharose Ni) with nickel ion to separate as follows by IMAC.1 × the BugBuster(that at room temperature melts cell mass and be suspended in 1.7mL is without primary amine; There is 1 μ L/mL Benzonase nuclease; Be respectively Novagen#70923-3 and 70750-3).At room temperature hatch sample 30 minutes with 60rpm rotation.By within centrifugal 10 minutes, carrying out sedimentation cell fragment with 14,000rpm.Described supernatant liquor is transferred to His Mag Sepharose Ni (GE Healthcare Biosciences, Piscataway, NJ#28-9799-17) bead, it is according to test kit explanation (20mM sodium phosphate in binding buffer liquid, 500mMNaCl, 20mM imidazoles, pH7.4) carry out balance.Described bead is suspended in described supernatant liquor and with slow tumble mixed and is hatched 60 minutes.Then in the binding buffer liquid of 800 μ L, washing described bead amounts to 5 times and each washing is carried out to hatch～3-5 minute of slow tumble mixed.In the elution buffer (20mM sodium phosphate, 500mM NaCl, 500mM imidazoles, pH7.4) of 300 μ L by slow tumble mixed 5 minutes by restructuring thioesterase wash-out from described bead.

example 11

the reconstruction in vitro of transaminase and liquid chromatography associating mass spectrum (LC-MS) are analyzed

By the activity (example 9) that the LC-MS of expection product is analyzed to test purified restructuring transaminase.In independently reacting, with the ultimate density of 0.27mg/mL, various enzymes are added to reaction buffer (20mM potassiumphosphate, 500mM sodium-chlor, pH8).L-homoserine (Sigma, St.Louis, MO; Catalogue #H6515) or different amino acid substrate (Sigma, St.Louis, MO) add with the ultimate density of 1mM.Secondary substrate is α-ketoglutaric acid (disodium salt, dehydration; Sigma, St.Louis, MO; Catalogue #75892) or pyruvic acid (Sigma, St.Louis, MO; Catalogue #P2256), be separately 1mM ultimate density.Pyridoxal 5'-phosphoric acid salt hydrate (Sigma, St.Louis, MO; Catalogue #P9255) add with the ultimate density of 50 μ M.At room temperature hatching described reaction spends the night.After hatching, each solution uses to be had the Amicon Ultra centrifugal filter device (EMD Millipore, Billerica, MA) that 3kDa nominal molecular weight holds back and filters, and it uses ultrapure water to carry out pre-wash.Collect permeate and at-20 ℃, store until LC-MS analyzes.

By the aliquots containig dilution 50-100 of reaction mixture × and by the high performance liquid chromatography associating mass spectrum (LC-MS) under negative mode, use EFI ionization (ESI) Fourier transform track trap MS (Exactive Model; Thermo Fisher, San Jose, CA) under 50,000 resolving power, analyze.Use ZIC-pHILIC post (2.1 × 100mm, 5 μ m polymkeric substance, Sequant, EMD Millipore, catalogue #1504620001; Darmstadt, Germany) and 2mM ammonium formiate in 85% acetonitrile/15% water separate with the flow of 200 μ L/ minutes as mobile.LC-MS analyzes demonstration, and every kind of tested enzyme (table 1) produces the product of expection in the time that the substrate desirable with it combines, and all enzymes all produce 2-ketone group-4 hydroxybutyric acid ester (Fig. 4) in the time combining with L-homoserine.

the spectrophotometric spectra assay method that uses transaminase to carry out

For further confirming the enzymic activity of described transaminase, purified recombinant protein carries out spectrophotometric spectra analysis in a series of conjugate enzyme reaction.In independently reacting, with the ultimate density of 0.27mg/ml, Pf AT transaminase is added to (pH8.0 in 100mM potassium phosphate buffer; Sigma, St.Louis, MO).L-homoserine (Sigma, St.Louis, MO; Catalogue #H6515) or Valine (Sigma, St.Louis, MO; Catalogue #V0500) add as substrate using the ultimate density of 10-25mM.Described transamination enzyme reaction coupling generates reduced form β-Reduced nicotinamide-adenine dinucleotide (NADH) with dehydrogenase reaction, and it can detect by spectrophotometric spectra.β-Reduced nicotinamide-adenine dinucleotide (NAD ⁺; Sigma, St.Louis, MO; Catalogue #N8410) add with the ultimate density of 3mM.Pyridoxal 5'-phosphoric acid salt hydrate (Sigma, St.Louis, MO; Catalogue #P9255) add with the ultimate density of 50 μ M.The dehydration of α-ketoglutaric acid disodium salt (Sigma, St.Louis, MO; Catalogue #75892) add with the ultimate density of 1mM as secondary substrate.From Pidolidone desaturase (Sigma, St.Louis, the MO of beef liver; Catalogue #G2626) add with 10U/mL.Each reaction added in the quartz cuvette of 1mL and in spectrophotometer, under 340nm, follow the tracks of in time the formation of NADH.As expection, the initial velocity that L-homoserine transforms depends on its concentration (Fig. 5).Even use in high density also unrealized with L-homoserine saturated to enzyme.

example 12

acyl group-CoA level is as the tolerance of enzymic activity: liquid chromatography associating mass spectrum (LC-MS).

for prepared by the culture of Escherichia coli that acyl-CoA horizontal analysis is carried out

Prepare (deuterium) stable, mark and comprise the main mixture of interior target, described main mixture comprises d ₃the 200 μ L50 μ g/mL stostes of-3-hydroxymethyl glutaryl-CoA(in 10ml15% trichoroacetic acid(TCA)).The aliquots containig (500 μ L) of main mixed thing is added in 2mL pipe.By silicone oil (AR200; Sigma, St.Louis, MO; Catalogue #85419; 800 μ L) be laid on the mixed thing of this master.The aliquots containig of culture of Escherichia coli (800 μ L) is gently laid on silicone oil.In Eppendorf5417C whizzer at 4 ℃ with 20,000 × g to described sample centrifugal 5 minutes.Forward in blank pipe and on dry ice the aliquots containig (300 μ L) of the layer that comprises main mixture to freezing 30 minutes.

to the measurement of acyl group-CoA level

Use the acyl-CoA content of LC-MS/MS working sample.Individual acyl group-CoA standard substance is purchased from Sigma (St.Louis, MO) and in methyl alcohol, make the stoste of 500 μ g/ml.Synthesizing propylene acyl-CoA similarly preparation.Collection analysis thing, and prepare the standard substance with all analytes by the trichoroacetic acid(TCA) dilution with 15%.Comprise target automatic sampler bottle in 10 μ L50 μ g/mL and be prepared by 500 μ L working standard things are transferred to for the standard substance of regression analysis.Interior mark (the d of sample peak area (or height) to stable labelling ₃-3-hydroxymethyl glutaryl-CoA) be normalized.By LCMS/MS on Sciex API5000 mass spectrograph, working sample in positively charged ion Turbo Ion Spray.Use Phenomenex Onyx Monolithic C18 post (2 × 100mm) and mobile phase A(5mM ammonium acetate by RPLC (HPLC), 5mM dimethyl butylamine and 6.5mM acetic acid) and B(0.1% formic acid, in acetonitrile) separate under the flow of 0.6mL/ minute with the gradient of describing in table 3.

mobile phase composite during table 3:LC-MS/MS analyzes

Time	Mobile phase A(%)	Mobile phase B(%)
			0 minute	97.5	2.5
1.0 minute	97.5	2.5
			2.5 minute	91.0	9.0
5.5 minute	45	55
			6.0 minute	45	55
6.1 minute	97.5	2.5
			7.5 minute	-	-
9.5 minute	Finish operation	?

Condition on mass spectrograph is: DP160, CUR30, GS165, GS265, IS4500, CAD7, temperature 650C.Transition for multiple-reaction monitoring (MRM) is described at table 4.

table 4: different acyl-CoA molecule is carried out to the description of quantitative parameter

Compound	Precursor ion ¹	Product ion ¹	Collision energy	CXP
					3-hydroxyl propionyl-CoA ²	840.3	333.2	45	13
N-propionyl-CoA	824.3	317.2	41	32
					Succinyl--CoA	868.2	361.1	49	38
Isobutyryl-CoA	838.3	331.2	43	21
					Lactoyl-CoA	840.3	333.2	45	38
Acryloyl-CoA	822.4	315.4	45	36
					Coenzyme A	768.3	261.2	45	34
Isovaleryl-CoA	852.2	345.2	45	34
					Malonyl--CoA	854.2	347.2	41	36
Acetyl-CoA	810.3	303.2	43	30
					d ₃-3-hydroxymethyl glutaryl-CoA	915.2	408.2	49	13

1 energy, with voltmeter, analyzes for MS/MS

2 based on n-propionyl-CoA response quantitatively

example 13

the 3-hydroxyl propionyl-CoA produced in vitro of carrying out with 2-keto acid decarboxylase or desaturase

In the first assay method, D-homoserine (2mM; Acros, Geel, Belgium; Catalogue #348362500) and D-AAO (1U/mL; Sigma, St.Louis, MO; Catalogue #A5222) and beef liver catalase (600U/mL; Sigma, St.Louis, MO; Catalogue #C40) under the existence of HEPES damping fluid (50mM, pH7.3), hatch together.At room temperature hatch after 2-4h, in this solution, add coenzyme A (2mM), β-NAD ⁺(2mM), diphosphothiamine (0.2mM) and MgCl ₂(2mM), and by described component further with business Pigs Hearts α-ketoglutaric acid ester desaturase (1.0mg/mL; Sigma, St.Louis, MO; Catalogue #K1502) hatch together or do not hatch therewith.

In the second assay method, D-homoserine (2mM; Acros, Geel, Belgium; Catalogue #348362500) and D-AAO (1U/mL; Sigma, St.Louis, MO; Catalogue #A5222) and beef liver catalase (600U/mL; Sigma, St.Louis, MO; Catalogue #C40) under the existence of HEPES damping fluid (50mM, pH7.3), hatch together.At room temperature hatch after 2-4h, in this solution, add coenzyme A (2mM), β-NAD ⁺(2mM), diphosphothiamine (0.2mM) and MgCl ₂(2mM) and by described component further hatch or do not hatch therewith together with the 2-of purifying keto acid decarboxylase KdcA (1.8 μ M) and propionic aldehyde desaturase PduP (1.8 μ M).

By at room temperature overnight incubation of described sample, carry out subsequently LC-MS analysis to measure the concentration of 3-hydroxyl propionyl-CoA, described in example 12.The described product (Fig. 6) of significant quantity only in the time that existing, desaturase (and decarboxylase) detected.

example 14

the 3-hydroxyl propionyl-CoA being undertaken by acryloyl-CoA with 3-hydroxyl propionyl-CoA-dehydratase is raw produce

Acryloyl-CoA (1mM) is hatched or is not hatched therewith under the existence of HEPES damping fluid (50mM, pH7.3) together with 3-hydroxyl propionyl-CoA-dehydratase (20 μ M).At room temperature hatch after 2-4h, use absorption and the Waters Atlantis T3 post (Waters under Agilent1100 system (Agilent, Santa Clara, CA) monitoring 254nm by high performance liquid chromatography (HPLC), Milford, MA; Catalogue #186003748) analyze aliquots containig.Movement is 0.1% the phosphoric acid of 0.1% phosphoric acid (A) in water and (B) in 80% acetonitrile/20% water mutually.Analyte carried out isocratic elution in 12 minutes with the 2%B in A, carry out subsequently the linear gradient elution from 2% to 35%B in A in 18 minutes.HPLC analyzes and shows the consumption of acryloyl-CoA and the formation (Fig. 7) of different absorptivity molecules.The essence identity 3-hydroxyl propionyl-CoA of described reaction product is analyzed and is confirmed by LC-MS, as (Fig. 8) described in example 12.

example 15

the reconstruction in vitro of PHA synthase

3-hydroxy-propionic acid (5mM; Aldrich, St.Louis, MO; Catalogue #AMS000335), coenzyme A (2mM), ATP (6mM), MgCl ₂(2mM) and solution and the Acetyl-CoA synthetase (5U/mL of HEPES damping fluid (50mM, pH7.3); Sigma, St.Louis, MO; Catalogue #A1765) to hatch together, it carries out or does not carry out therewith with together with purified PHA synthase (1 μ M).At room temperature hatch after 2-4h, as analyzed to measure the concentration of 3-hydroxyl propionyl-CoA described in example 12 by LC-MS.In the time that PHA synthase exists, the concentration of 3-hydroxyl propionyl-CoA is remarkable minimizing (Fig. 9) compared with the sample without enzyme.

example 16

thioesterase activation measurement: Ellman reagent

For measuring relative thioesterase enzymic activity, use Ellman reagent.Also be called DTNB(5,5'-bis-sulphur two (2-nitrobenzoic acid)).Measuring damping fluid is 50mM KCl, 10mM HEPES (pH7.4).In ethanol, prepare the Ellman reagent stoste of 10mM.In mensuration damping fluid, acryloyl-CoA substrate stoste is prepared into 10mM.

For every kind of enzyme and the substrate of test, described reaction is as follows: the Ellman reagent stoste of 10mM is diluted to 50 μ M ultimate densities measuring in damping fluid.Add acryloyl-CoA stoste so that the ultimate density of 90 μ M to be provided.In the untreated droplet degree of 96 hole polystyrene flat board, add described Ellman reagent/acryloyl-CoA mixture (95 μ L/ hole).Preparation without the matching response of substrate in contrast.In flat board independently, the enzyme of purifying is carried out to 1:3 serial dilution measuring in damping fluid, and 5 μ L are added in reacting hole.Come 60 minutes estimation thioesterase activity by the optical density(OD) (OD) of measuring in microplate reader under 412nm.Calculate relative activity by the OD of the sample that comprises substrate (412nm) being deducted without the OD (412nm) of substrate contrast.

Two kinds of thioesterases (EcTesB and CpTT) all demonstrate the hydrolytic activity for acryloyl-CoA substrate, and described activity increases (Figure 10) along with the increase of thioesterase amount.EcTesB also has activity (Figure 11) to other substrate.EcTesB is hydrolyzed capryloyl-CoA, even under the EcTesB of relatively low amount.By contrast, CpTT only demonstrates the increase (Figure 11) of capryloyl-CoA hydrolysis under the thioesterase of maximum.Other thioesterase demonstrates less for acryloyl-CoA or the thioesterase activity (Figure 10 and 11) of nothing, and they point out described recombinase to have activity (Figure 12 and 13) to the obvious hydrolysis of capryloyl-CoA.For confirming that described thioesterase has activity on tested coenzyme A substrate, use liquid chromatography associating mass spectrum (LC-MS) analytic sample, described in example 12.

monitoring by LC-MS to substrate and product

In the assay method of the generation of the free sulfhydryl groups based on from acryloyl-CoA, EcTesB and CpTT demonstrate acryloyl-CoA thioesterase activity.As the further test to this thioesterase activity, it can be used for observing the disappearance of substrate and the appearance of product.Therefore, LC-MS is used for the amount of substrate and the product of monitoring the analysis of carrying out with these enzymes, described in example 12.The increase of the amount of EcTesB and acryloyl-CoA hydrolysis is relevant, if the disappearance of passing through Ellman reagent and acryloyl-CoA is to (table 1) as shown in the detection of coenzyme A.Show according to each analysis, along with described enzyme is diluted, thioesterase activity level reduces.These results support EcTesB as the function to the activated thioesterase of acryloyl-CoA tool.

relative activity and the acryloyl-CoA of table 5:TesB thioesterase sample are quantitative.Described activity (OD under 412nm) refers to the survey of the colour-change based in the situation that Ellman reagent exists determine method.The measurement of acryloyl-CoA is based on LC/MS.

By different assay methods, EcTesB and CpTT all show acryloyl-CoA hydrolytic activity (table 6) separately.According to the detection of carrying out with Ellman reagent, every kind of enzyme causes the increase of coenzyme A.Every kind of enzyme also causes the type figure of the variation in LC-MS analysis, and described thioesterase causes the minimizing of acryloyl-CoA and the increase (table 6) of coenzyme A.

table 6: coenzyme A and acryloyl-CoA to thioesterase sample are quantitative.Described activity (412nm under OD) refer to the assay method of the colour-change based in the situation that Ellman reagent exists.Third alkene acyl-CoA and coenzyme A are measured and are analyzed based on LC-MS.

example 17

the production of 3-hydroxy-propionic acid in the intestinal bacteria (E.coli) of through engineering approaches

the increase of this examples show 3-hydroxy-propionic acid in intestinal bacteria (E.coli) host cell produced, so rear its can be converted into poly--3-hydroxy-propionic acid or vinylformic acid.Create coli strain to cross the vacation of expression fluorescence the branched-chain-amino-acid aminotransferase (Pf AT) of Zymomonas mobilis (P.fluorescens) is (at SEQ ID NO:8 shown in), the side chain 2-keto acid decarboxylase (KdcA) of lactobacillus lactis (L.lactis) is (at SEQ ID? shown in NO:54), Salmonella enteritidis (S.enterica) coenzyme-A acidylate propionic aldehyde desaturase (PduP) (shown in SEQ ID NO:60) and hookworm are in some cases coveted copper bacterium (C.necator) poly-(3-hydroxybutyrate ester) polysaccharase (PhaC) (shown in SEQ ID NO:42).

In this example, Pseudomonas fluorescens ( p.fluorescens) branched-chain-amino-acid aminotransferase (SEQ ID NO:8) promote the conversion to 2-ketone group-4 hydroxybutyric acid ester of L-homoserine.Side chain 2-keto acid decarboxylase (KdcA, shown in SEQ ID NO:540) catalysis 2-ketone group-4 hydroxybutyric acid ester of lactobacillus lactis (L.lactis) is to the conversion of 3-hydroxyl-propionic aldehyde.Salmonella enteritidis (S.enterica) coenzyme-A acidylate propionic aldehyde desaturase (PduP, shown in SEQ ID NO:60) catalysis 3-hydroxyl-propionic aldehyde is to the conversion of 3-hydroxyl propionyl-CoA.Thioesterase catalysis 3-hydroxyl propionyl-CoA is to the conversion of 3-hydroxy propionate.Alternatively, poly-(3-hydroxybutyrate ester) polysaccharase (PhaC, shown in SEQ ID NO:42) of the greedy copper bacterium (C.necator) of hookworm can catalysis 3-hydroxyl propionyl-CoA to the conversion of poly--3-hydroxy propionate.

plasmid construction

build intestinal bacteria (E.coli) expression vector, it expresses restructuring for crossing

Pseudomonas fluorescens ( p.fluorescens) branched-chain-amino-acid aminotransferase (Pf AT) and poly-(3-hydroxybutyrate ester) polysaccharase (PhaC) of the greedy copper bacterium (C.necator) of hookworm.By use restriction enzyme XbaI and PstI to the double digestion of pET30a-BB Cn PHAS by from pET30a-BB Cn PHAS(example 4) enter pET30a-BB Pf AT(example 1 through poly-(3-hydroxybutyrate ester) polysaccharase (phaC) clone of the greedy copper bacterium (C.necator) of codon optimized hookworm).Cn PHAS is carried out to band separation, use QIAquick Gel Extraction Kit (Qiagen, Carlsbad, CA) purifying and be connected (Fast-Link Epicentre Biotechnologies with the pET30a-BB Pf AT carrier through SpeI/PstI-digestion, Madison, WI).Use and connect mixture to transform OneShot Top10 ^tMbacillus coli cells (Invitrogen, Carlsbad, CA).

Melt the cell of single bottle on ice and be gently connected mixture with 2 μ L and mix.Described bottle is hatched on ice 30 minutes.Hatch 30 seconds and put back to rapidly additional 2 minutes on ice of short duration at 42 ℃ described bottle.Add the SOC substratum of 250 μ L37 ℃ and described vial level be fixed on shaking culture case and hatch 1 hour under 37 ℃ and 225rpm.The aliquots containig of 20 μ L and 200 μ L cells is coated on LB agar, and it is supplemented with kantlex (50 μ g/mL).Separate single bacterium colony isolate and cultivate in 5mL has the LB liquid nutrient medium of kantlex (50 μ g/mL).Separate described recombinant plasmid and by the gel electrophoresis of AflIII restriction digestion product is characterized with Qiagen Plasmid Mini Kit.The plasmid called after pET30a-BB Pf AT_Cn PHAS of gained.

Build intestinal bacteria (E.coli) expression vector, it is for crossing coenzyme A acidylate propionic aldehyde desaturase (PduP) and lactobacillus lactis (L.lactis) the side chain 2-keto acid decarboxylase (KdcA) of expressing restructuring Salmonella enteritidis (S.enterica).By use restriction enzyme XbaI and PstI to the double digestion of pET30a-BB Ll KDCA by from pET30a-BB Ll(example 2) enter pET30a-BB Se PDUP(example 3 through codon optimized lactobacillus lactis (L.lactis) side chain 2-keto acid decarboxylase (kdcA) clone).LlKDCA fragment is carried out to band separation, use QIAquick Gel Extraction Kit (Qiagen, Carlsbad, CA) purifying and be connected (Fast-Link Epicentre Biotechnologies with the pET30a-BB Se PDUP carrier through SpeI/PstI-digestion, Madison, WI).Use and connect mixture conversion OneShot Top10 ^tMintestinal bacteria (E.coli) cells (Invitrogen, Carlsbad, CA).Melt the cell of single bottle on ice and be gently connected mixture with 2 μ L and mix.Described bottle is hatched on ice 30 minutes.Hatch 30 seconds and put back to rapidly additional 2 minutes on ice of short duration at 42 ℃ described bottle.Add the SOC substratum of 250 μ L37 ℃ and described vial level be fixed on shaking culture case and hatch 1 hour under 37 ℃ and 225rpm.The aliquots containig of 20 μ L and 200 μ L cells is coated on LB agar, and it is supplemented with kantlex (50 μ g/mL).Separate single bacterium colony isolate and it is cultivated in the 5mL LB liquid nutrient medium with kantlex (50 μ g/mL), and described recombinant plasmid separates and carries out gel electrophoresis by the restriction digestion product to AflIII with Qiagen Plasmid Mini Kit and characterizes.The plasmid called after pET30a-BB Se PDUP_Ll KDCA of gained.

For being conducive to and the cotransformation of pET30a-BB Pf AT or pET30a-BB Pf AT_Cn PHAS, by using limiting enzyme EcoRI and PstI, to the double digestion of pET30a-BB Se PDUP_Ll KDCA, by codon optimized Salmonella enteritidis (S.enterica) coenzyme-A acidylate propionic aldehyde desaturase (pduP) and lactobacillus lactis (L.lactis) side chain 2-keto acid decarboxylase (kdcA) gene pairs, subclone from pET30a-BB Se PDUP_Ll KDCA enters pCDFDuet-1 carrier (Novagen, EMD Chemicals, Gibbstown, NJ; Catalogue #71340-3).Described Se PDUP_Ll KDCA fragment separates through band, use QIAquick Gel Extraction Kit (Qiagen, Carlsbad, CA) purifying be connected (Fast-Link Epicentre Biotechnologies with the pCDFDuet-1 with EcoRI/PstI-digestion, Madison, WI).Use and connect mixture conversion OneShot Top10 ^tMintestinal bacteria (E.coli) cells (Invitrogen, Carlsbad, CA).Melt the cell of single bottle on ice and be gently connected mixture with 2 μ L and mix.Described bottle is hatched on ice 30 minutes.Hatch 30 seconds and put back to rapidly additional 2 minutes on ice of short duration at 42 ℃ described bottle.Add the SOC substratum of 250 μ L37 ℃ and described vial level be fixed on shaking culture case and hatch 1 hour under 37 ℃ and 225rpm.The aliquots containig of 20 μ L and 200 μ L cells is coated on LB agar, and it is supplemented with Trobicin (50 μ g/mL).Separate single bacterium colony isolate, it is cultivated in the 5mL LB liquid nutrient medium with Trobicin (50 μ g/mL), and described recombinant plasmid separates and carries out gel electrophoresis by the restriction digestion product to AflIII with Qiagen Plasmid Mini kit and characterizes.The plasmid called after pCDFDuet-1Se PDUP_Ll KDCA of gained.

the cotransformation of intestinal bacteria (E.coli)

Described recombinant plasmid and empty parent vector are for cotransformation chemoreception state BL21 (DE3) pLysS Bacillus coli cells (Invitrogen, Carlsbad, CA), and it is undertaken by following combination:

PET30a-BB Pf AT_Cn PHAS and pCDFDuet-1Se PDUP_Ll KDCA

PET30a-BB Pf AT and pCDFDuet-1Se PDUP_Ll KDCA

PET30a-BB and pCDFDuet-1

Melt the cell of single bottle on ice and gently mix with 50ng plasmid DNA.Described bottle is hatched on ice 30 minutes.Hatch 30 seconds and put back to rapidly additional 2 minutes on ice of short duration at 42 ℃ described bottle.Add the SOC substratum of 250 μ L37 ℃ and described vial level be fixed on shaking culture case and hatch 1 hour under 37 ℃ and 225rpm.The aliquots containig of 20 μ L and 200 μ L cells is coated on LB agar, and it is supplemented with suitable microbiotic (50 μ g/mL kantlex; 50 μ g/mL Trobicins; 34 μ g/mL paraxin) to select respectively to carry the cell of restructuring pET30a-BB, pCDFDuet-1 plasmid and pLysS plasmid, and at 37 ℃ overnight incubation.Separate single bacterium colony isolate, it is cultivated in 5mL selectivity LB liquid nutrient medium, and described recombinant plasmid uses

spin Miniprep (Qiagen, Valencia, CA) separates and carries out gel electrophoresis by the restriction digestion product to AflIII and characterizes.

strain culturing

With the single colony-forming unit of intestinal bacteria (E.coli) BL21 (DE3) the pLysS cell of described plasmid cotransformation, for inoculating the aliquots containig (25mL) of basic M9 liquid nutrient medium, it is supplemented with suitable microbiotic (34 μ g/mL paraxin, 50 μ g/mL kantlex and 50 μ g/mL Trobicins).At 37 ℃ with the vibration of 250rpm by described culture night incubation and use it for the fresh basic M9 substratum (50mL) of inoculation, described culture medium supplemented has identical microbiotic.Under conditions of similarity after night incubation, by the aliquots containig of culture, for inoculating one group of new M9 liquid nutrient medium (50mL), described substratum has microbiotic (34 μ g/mL paraxin, 50 μ g/mL kantlex and 50 μ g/mL Trobicins) and is supplemented with or is not supplemented with L-homoserine (1g/L; Sigma, St.Louis, MO), subsequently at 25 ℃ with 250rpm oscillation incubation.When reaching approximately 0.2 OD ₆₀₀time, by adding the ultimate density of 1M IPTG(1mM of 50 μ L; Teknova, Hollister, CA) carry out induced protein and express, subsequently at 25 ℃ with 250rpm oscillation incubation 17h.By centrifugal collecting cell and by Acrodisc Syringe Filters(0.2 μ m HT Tuffryn film; Lower protein combination; Pall Corporation, Ann Arbor, MI) filtering supernatant, and freezing on dry ice before being stored at-80 ℃, until analyze.

intestinal bacteria (E.coli) by through engineering approaches detect 3-hydroxy-propionic acid

at 1:1MeOH:H ₂ in O, prepare 100 μ g/mL's ¹³ c ₃ mark lactic acid inner mark solution.At 1:1 meOH:H ₂ in O with the 3-of 1 μ g/mL, 2.5 μ g/mL, 5 μ g/mL, 10 μ g/mL and 25 μ g/mL hydroxy-propionic acid concentration is prepared external standard solution.The filtering supernatant of 900 μ L or external standard are added to 100 μ L's in inner mark solution.Make these solution stand ion and get rid of liquid chromatography (LC) separation and mass spectrum (MS) inspection survey.

Described LC separation condition is as follows: sample/standard substance of 10 μ L is expelled on Thermo Fisher Dionex ICE-AS1 (4 × 250mm) post (having protector), and what described post moved 1mM hyptafluorobutyric acid under the flow of 0.15mL/ minute moves phase without gradient.With 0.15mL/ minute by the 20mM NH in MeCN ₄oH introduces the effluent of described post.

MS testing conditions is as follows: Sciex API-4000MS moves and monitor m/z89 to 59(unit resolution rate under negative ion mode) the transition of 3-hydroxy-propionic acid and m/z92 to 45(unit resolution rate) ¹³c ₃the lactic acid transition of-mark.The residence time using is 300ms, go a bunch electromotive force to be set as-38, entrance electromotive force is set as-10, collision gas is set as 12, and curtain gas setting is 15, and ion source gas 1 is set as 55, ion source gas 2 is set as 55, ionspray voltage is set as-3500, and Temperature Setting is 650, interface heater starting.For 3-hydroxy-propionic acid, collision energy be set as-16 and collision set outlet electromotive force be set as-9.For ¹³c ₃the lactic acid of-mark, collision energy be set as-18 and collision set outlet electromotive force be set as-16.

The result of analyzing is shown in table 7.Described data acknowledgement, expresses and when supplementing L-homoserine in substratum, produces the 3-hydroxy-propionic acid level increasing when Pf AT, KdcA and PduP cross.When not to adding exogenous homoserine in described substratum and/or in the time there is empty pET30a-BB and pCDF Duet-1 carrier, endogenous L-homoserine and Escherichia coli protein may be supported the generation of a small amount of 3-hydroxy-propionic acid.

table 7: the intestinal bacteria by through engineering approaches produce 3-hydroxy-propionic acid

Although the present invention describes by specific embodiment, should be appreciated that those skilled in the art should be susceptible to modification and correction form.Therefore, should only apply as the restriction as shown in claims for the present invention.

The all files of mentioning in the application is incorporated to hereby by reference in full.

Claims

1. a method that homoserine is converted into 3-hydroxyl propionyl-CoA, comprises the following steps:

A) homoserine is converted into 2-ketone group-4 hydroxybutyric acid ester, wherein this conversion is by least one enzyme catalysis that is selected from transaminase, L-amino acid oxidase and L-amino acid dehydrogenase; And

B) 2-ketone group-4 hydroxybutyric acid ester is converted into 3-hydroxyl propionyl-CoA, wherein this conversion is by least one enzyme catalysis of combination that is selected from 2-ketoacid dehydrogenase and 2-keto acid decarboxylase and desaturase.

2. method according to claim 1, wherein recombinant microorganism is crossed and is expressed one or more genes homoserine is converted into 3-hydroxyl propionyl-CoA.

3. method according to claim 2, gather-3-hydroxyl alkane acid ester synthase of wherein said microbial expression is further 3-hydroxyl propionyl-CoA is converted into poly--3-hydroxy alkane acid ester, and described gather-3-hydroxy alkane acid ester comprises 3-hydroxy propionate monomer.

4. method according to claim 1, further comprising the steps of:

C) 3-hydroxyl propionyl-CoA is converted into acryloyl-CoA, wherein this conversion is by dehydratase catalysis; And

D) acryloyl-CoA is converted into vinylformic acid, wherein this conversion is by least one enzyme catalysis that is selected from thioesterase, CoA-transferring enzyme and phosphotransferase and kinase whose combination.

5. method according to claim 4, wherein homoserine is converted into vinylformic acid by recombinant microorganism.

6. method according to claim 1, wherein 3-hydroxyl propionyl-CoA is further converted to 3-hydroxy-propionic acid by microorganism, and described microbial expression is selected from the enzyme of transferring enzyme and thioesterase.

7. a method that homoserine is converted into 3-HPA, comprises the following steps:

B) 2-ketone group-4 hydroxybutyric acid ester is converted into 3-HPA, wherein this conversion is by the catalysis of 2-keto acid decarboxylase.

8. method according to claim 7, wherein recombinant microorganism is crossed and is expressed at least one gene and homoserine is converted into 3-HPA.

9. method according to claim 7, wherein 3-HPA is further converted to 1,3-PD.

10. method according to claim 7, wherein 3-HPA is further converted to 1,3-PD, and wherein said conversion is by the enzyme catalysis that is selected from 1,3-PD desaturase and aldehyde reductase.

11. methods according to claim 7, are wherein used desaturase that 3-HPA is further converted to 3-hydroxyl propionyl-CoA.

12. methods according to claim 11, wherein microorganism crosses and expresses at least one gene and homoserine is converted into 3-hydroxyl propionyl-CoA.

13. methods according to claim 12, gather-3-hydroxyl alkane acid ester synthase of wherein said microbial expression is further 3-hydroxyl propionyl-CoA is converted into poly--3-hydroxy alkane acid ester, and described gather-3-hydroxy alkane acid ester comprises 3-hydroxy propionate monomer.

14. methods according to claim 12, wherein 3-hydroxyl propionyl-CoA is further converted to vinylformic acid, said method comprising the steps of:

A) 3-hydroxyl propionyl-CoA is converted into acryloyl-CoA, wherein this conversion is by the catalysis of hydroxyl acyl group-CoA dehydratase; And

B) acryloyl-CoA is converted into vinylformic acid, wherein this conversion is by least one enzyme catalysis that is selected from thioesterase, CoA-transferring enzyme and phosphotransferase and kinase whose combination.

15. methods according to claim 12, wherein the reorganized microorganism of 3-hydroxyl propionyl-CoA is further converted to 3-hydroxy-propionic acid, and described recombinant microorganisms express is selected from the enzyme of CoA transferring enzyme or CoA thioesterase.