Improved muconic acid production from genetically engineered microorganisms

a technology of genetic engineering and genetic engineering, applied in the direction of lysine, transferase, ligase, etc., can solve the problems of environmental damage to current synthesis of adipic acid, poor synthesis effect of adipic acid, and high cost of medium components (aromatic amino acids and vitamins), so as to enhance the activity of one or more enzymes involved in the central aromatic biosynthetic pathway within the microbial cell, and enhance the activity of one or

Inactive Publication Date: 2020-06-11
PTT GLOBAL CHEMICAL PUBLIC COMPANY LIMITED
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Benefits of technology

[0010]In another embodiment of the present invention, the activity of one or more of the enzymes involved in the central aromatic biosynthetic pathway within the microbial cell is enhanced. In one aspect of the present invention, the enhancement of the activity of one or more enzymes involved in the operation of an aromatic pathway and/or a muconic acid pathway is accomplished through genetic manipulation. In a preferred aspect of the present invention, the expression of one or more of the genes coding for the proteins AroF, AroG, AroH, AroB, TktA, TalB, AroZ, QutC, Qa-4, AsbF, QuiC, AroY, Rpe, Rpi, Pps, CatA and CatX or their homologs or analogs are enhanced leading to the increased activity of said proteins. Rpe is a ribulose-5-phosphate epimerase, Rpi is a ribulose-5-phosphate isomerase, and Pps is a phosphoenol pyruvate synthetase (Neidhardt and Curtiss, 1996). If the host strain is yeast, for example Saccharomyces cerevisiae, or a filamentous fungus, for example, Neurospora crassa, several of the enzymes that catalyze reactions in the shikimate pathway can be combined into one large protein or polypeptide, called Aro1p, encoded by the ARO1 gene in the case of S. cerevisiae. Aro1p combines the functions of AroB, AroD, AroE, AroK (or AroL), and AroA). As such, for the purposes of this invention, Aro1p, and ARO1, or a portion thereof, can be used as a substitute, or in addition to, AroB, AroD, AroE, AroK, and/or AroA.
[0011]In yet another embodiment of the present invention, flux through erythrose-4-phosphate within the bacterial cell is enhanced by means of overexpressing enzymes in the operation of the pentose phosphate pathway. In one aspect of the present invention, the expression of transaldolase enzyme, for example one coded by the talB or talA gene is enhanced by genetic modification. In another aspect of the present invention, the expression of a gene encoding transketolase enzyme, for example, the tktA gene is enhanced by genetic manipulations. In yet another aspect of the present invention, the expression of the genes encoding either or both ribulose-5-phosphate epi

Problems solved by technology

Current synthesis of adipic acid is environmentally harmful releasing nitrous acid (Xie et al., 2014).
However, the prior art process for muconic acid production suffered from sig

Method used

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  • Improved muconic acid production from genetically engineered microorganisms
  • Improved muconic acid production from genetically engineered microorganisms
  • Improved muconic acid production from genetically engineered microorganisms

Examples

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example 1

Increasing Expression of aroG and aroF Genes

[0100]The tyrR gene of E. coli can be mutated by any one of a number of well-known methods, such as chemical or radiation mutagenesis and screening (for example by PCR and DNA sequencing) or selection for analog resistance (for example, resistance to 4-fluorotyrosine), transposon mutagenesis, bacteriophage Mu mutagenesis, or transformation. In a preferred embodiment, the mutation in tyrR gene is a null mutation (a mutation that leaves no detectable activity), and in a more preferable embodiment, at least a portion of the tyrR gene is deleted. This can be accomplished, for example, by using a two-step transformation method using linear DNA molecules (Jantama et al, 2008a; Jantama et al, 2008b). In the first step, a camR, sacB cassette is integrated at the tyrR locus to replace most or all of tyrR open reading frame by double recombination and selecting for chloramphenicol resistance. In the second step, a linear DNA comprising a deleted ver...

example 2

Feedback Resistant AroG and AroF

[0102]Mutations in the aroG gene that lead to a feedback resistant AroG enzyme (3-deoxy-D-arabinoheptulosonate-7-phosphate synthase or DAHPS) are well known in the art (Shumilin et al, 1999; Kikuchi et al, 1997; Shumilin et al, 2002). Also well known are methods for creating, identifying, and characterizing such mutations (Ger et at, 1994, Hu et al., 2003). A preferable mutation is one that leads to complete resistance to inhibition by phenylalanine. Any of the known published feedback resistant mutations can be introduced into an aroG gene contained in the chromosome or on a plasmid by any of a number of well known methods, one example of which is mutagenic PCR in which the desired mutation is synthesized as part of a PCR priming oligonucleotide (Hu et al., 2003). Correct installation of the mutation is confirmed by DNA sequencing. The sequence of the wild type aroG gene from E. coil C is given in SEQ ID No. 18. A preferred mutation is a point mutati...

example 3

Deletion of aroE from Chromosomal DNA and Muconic Acid Production

[0109]In this example the effect of overexpression of aroB and aroG on multicopy plasmids as well as the expression of genes coding for proteins functional in the muconic acid pathway was investigated. Strain MYR34 containing a deletion in the aroE gene coding for shikimate dehydrogenase was used as parent strain in these studies. The deletion of chromosomal copy of aroE was accomplished in a fashion similar to that described above in Example 1. When MYR34 was transformed with the plasmid pCP32AMP overexpressing the aroG gene coding for DAHP synthase protein functional in the shikimic acid pathway, there was a significant increase in the accumulation of DHS. When MYR34 was transformed with the plasmid expressing aroB from a constitutive promoter, no significant increase in the accumulation of DHS was noticed. However, when the E. coli strain MYR34 was transformed with the plasmid expressing both aroB and aroG genes, th...

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Abstract

The subject of this invention is improvements in the yield and titer of biological production of muconic acid by fermentation. Increased activity of one or more enzymes involved in the muconic acid pathway leads to increased production of muconic acid.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is the U.S. national stage application of International Application No. PCT / US2017 / 020263 filed Mar. 1, 2017, which claims priority to U.S. Provisional Patent Application No. 62 / 302,558 filed Mar. 2, 2016.REFERENCE TO SEQUENCE LISTING[0002]This application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 20, 2020, is named 524823US_ST25.txt and is 344 kb in size.FIELD OF THE INVENTION[0003]The present invention is in the field of producing renewable chemical feedstocks using biocatalysts that have been genetically engineered in the central aromatic biosynthetic pathway. More specifically, the present invention provides processes for improving the production of muconic acid :from renewable carbon resources using genetically modified biocatalysts.BACKGROUND OF THE INVENTION[0004]Adipic acid is a major...

Claims

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Application Information

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IPC IPC(8): C12P7/46C12N9/88C12N15/70
CPCC12N9/88C12P7/46C12N15/70C12Y402/01118C12N15/52C12P7/44C12Y401/01063C12Y101/01025C12Y207/01002C12Y205/01054C12Y401/01031C12Y604/01001
Inventor SILLERS, RYANHERMANN, THERONSPENCER, MICHELLEUDANI, RUSSELLYOCUM, R. ROGERS
Owner PTT GLOBAL CHEMICAL PUBLIC COMPANY LIMITED
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