Method for producing vectors containing a gene coding for an enzyme having reduced or deactivated feedback inhibition and the use thereof for producing amino acids and nucleotides

Patent metadata

Field	Value
Publication number	US-9644226-B2
Application number	US-201314422394-A
Country	US
Kind code	B2
Filing date	Jul 23, 2013
Priority date	Aug 22, 2012
Publication date	May 9, 2017
Grant date	May 9, 2017

Abstract

A method for producing vectors containing a gene coding for an enzyme having reduced or deactivated feedback inhibition and to the use thereof for producing amino acids and nucleotide. Genes coding for feedback-inhibited enzymes are mutagenized in vitro, —ligated into vectors in a further step—the vectors are each transformed in a microorganism containing a metabolite sensor in a further step, which brings about the synthesis of a fluorescent protein at an increased metabolite concentration, —whereupon microorganisms exhibiting increased or maximum fluorescence are selected; and —the vectors that contain a gene coding for an enzyme having reduced or deactivated feedback inhibition are isolated from microorganisms having increased or maximum fluorescence.

First claim

The invention claimed is:

1. A method for producing vectors encoding a feedback inhibited enzyme, the enzyme comprising a mutation resulting in the enzyme having reduced or deactivated feedback inhibition by a metabolite, wherein the method comprises
mutating genes encoding the feedback-inhibited enzyme in vitro,
ligating the mutated genes into vectors wherein the ligated mutated genes are each operably linking to an expression control sequence,
transforming the ligated vectors into a population of microorganisms that each contain a fluorescent metabolite sensor which synthesizes a fluorescent protein in the presence of the metabolite wherein microorganisms comprising the ligated vectors with the mutations resulting in the enzyme having reduced or deactivated feedback inhibition synthesize the fluorescent protein at increased concentration compared to the concentration of fluorescent protein in a non-transformed cell presence of the vector comprising and isolating said plurality of microorganisms exhibiting the increased fluorescent protein concentration, and
wherein the vectors encoding the enzyme having reduced or deactivated feedback inhibition are isolated from the plurality of microorganisms having the increased fluorescence, and wherein the metabolite sensor comprises at least one metabolite sensor selected from the group of pSenLys, pSenArg, pSenSer, pSenOAS and pJC1-lrp-brnF-eyfp.

2. The method according to claim 1 , wherein said mutating comprises random mutagenesis.

3. The method according to claim 1 wherein the vectors comprise at least one of a plasmid, an insertion element, a transposon and a phage.

4. The method according to claim 1 , wherein the gene encoding the feedback-inhibited enzyme is from a yeast or a bacterium.

5. The method according to claim 1 , wherein the feedback inhibited enzyme is 2-isopropylmalate synthase, acetohydroxy acid synthase, acetylglutamate synthase, anthranilate synthase, asparagine synthetase, aspartate transcarbamylase, aspartate kinase, ATP-phosphoribosyl transferase, carbamoyl phosphate synthetase, chorismatemutase I, chorismatemutase II, cysteine synthase, D-3-phosphoglycerate dehydrogenase, 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, dihydrodipicolinate synthase, glutamate dehydrogenase, glutamate synthase, glutamine synthetase, glutamyl kinase, homocysteinetransmethylase, homoserine O-acetyltransferase, homoserine O-succinyltransferase, homoserine dehydrogenase, methionine synthase, phosphoribosylphrophosphate synthase, prephenate dehydrogenase I, prephenate dehydrogenase II, pyrroline-5-carboxylate reductase, ribose 1,5-bisphosphokinase, serinace-tyl(succinyl) transferase, threonine ammoniumlyase or tyrosine aminotransferase.

6. The method according to claim 1 , wherein isolating said plurality of microorganisms exhibiting the increase fluorescent protein concentration comprises selecting for isolation microorganisms from among said plurality of microorganisms that have relatively highest fluorescence.

7. The method according to claim 1 , wherein production of the isolated vectors is achieved by transforming the isolated vectors into a population of second microorganisms.

8. The method according to claim 1 , wherein the genes encoding the enzyme with reduced or deactivated feedback inhibition are isolated from the vectors and sub-cloned into a second set of vectors for improved expression of the enzymes wherein the second set of ligated vectors are transformed into a production strain.

9. The method according to claim 1 , wherein the genes encoding the enzyme having reduced or deactivated feedback inhibition are introduced into the chromosome of a production strain.

10. The method according to claim 1 , wherein the population of microorganism containing the gene encoding the enzyme having reduced or deactivated feedback inhibition is a bacterium or a yeast.

11. The method for producing vectors according to claim 1 , wherein the isolated plurality of microorganisms further comprises a biosynthesis pathway for an amino acid or a nucleotide wherein the mutation results in enhanced production of the amino acid or the nucleotide.

12. The method according to claim 11 , wherein the produced amino acid comprises at least one component from the group comprising L-valine, L-leucine, L-asparagine, L-lysine, L-methionine, L-threonine, L-isoleucine, L-histidine, L-glutamate, L-glutamine, proline, L-arginine, L-tryptophan, L-tyrosine, L-phenyalanine, L-serine and L-cysteine.

13. The method according to claim 11 , wherein the produced nucleotide comprises at least one component from the group comprising orotidine-5′-phosphate, 5-phosphoribosyl diphosphate or inosine-5′-phosphate.

Assignees

• Forschungszentrum Juelich Gmbh

Inventors

• Schendzielorz Georg
• Binder Stephan
• Eggeling Lothar
• Bott Michael

Classifications

• C12P13/08

  Lysine; Diaminopimelic acid; Threonine; Valine · CPC title

• C12N9/1217 Primary

  Phosphotransferases with a carboxyl group as acceptor (2.7.2) · CPC title

• C12Y207/02004

  Aspartate kinase (2.7.2.4) · CPC title

• C12P19/34 Primary

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• Y02P20/52

  using catalysts, e.g. selective catalysts · CPC title