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Team:Alberta-North-RBI E/genetics
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Enzymatic activities which lead from SA to phenylalanine (Phe) will be abolished by removing the underlying endogenous genes. These endogenous genes, in addition to a codon-optimized Rhodosporidium toruloides gene encoding for a phenylalanine ammonia-lysase (PAL) enzyme, will be re-introduced at an extra-chromosomal locus as modified genes containing inducible promoter/repressor “switches” allowing us to direct metabolism toward CA at will. The “switch” mechanism will require “cis” DNA elements immediately upstream of each enzyme’s coding sequence (CDS). In addition, each regulatory system (ie. induction, repression) will require a constitutively-regulated “trans” element encoded at a different locus which produces the protein component. | Enzymatic activities which lead from SA to phenylalanine (Phe) will be abolished by removing the underlying endogenous genes. These endogenous genes, in addition to a codon-optimized Rhodosporidium toruloides gene encoding for a phenylalanine ammonia-lysase (PAL) enzyme, will be re-introduced at an extra-chromosomal locus as modified genes containing inducible promoter/repressor “switches” allowing us to direct metabolism toward CA at will. The “switch” mechanism will require “cis” DNA elements immediately upstream of each enzyme’s coding sequence (CDS). In addition, each regulatory system (ie. induction, repression) will require a constitutively-regulated “trans” element encoded at a different locus which produces the protein component. | ||
To enhance the yield of our desired products, two additional categories of changes will be made to our host strain. Enzymatic activities which direct metabolites away from the desired pathway will be abolished by removing the underlying endogenous genes and transporter proteins responsible for re-uptake of SA or CA from the medium will be removed in the same manner. | To enhance the yield of our desired products, two additional categories of changes will be made to our host strain. Enzymatic activities which direct metabolites away from the desired pathway will be abolished by removing the underlying endogenous genes and transporter proteins responsible for re-uptake of SA or CA from the medium will be removed in the same manner. | ||
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Revision as of 22:56, 14 October 2012
Genetics
We plan to use a single metabolic pathway for the conversion of glucose to aromatics. We are currently engineering a Pseudomonas putida strain containing a “switch” system enabling the manufacture of either shikimic acid (SA) or cinnamic acid (CA) as a major product. Future embodiments of this strain will contain additional directing mechanisms whereby a multitude of derivative chemicals can be favored.
The enzymatic activities leading to the production of SA will be enhanced by the addition of extra-chromosomal copies of endogenous genes modified such that expression is constitutive. Where necessary, these genes will be further modified through site-directed mutagenesis to abolish feedback regulation. Optimization of metabolism to SA is based on the invention(s) described in US patents 5168056 and 6613552.
Enzymatic activities which lead from SA to phenylalanine (Phe) will be abolished by removing the underlying endogenous genes. These endogenous genes, in addition to a codon-optimized Rhodosporidium toruloides gene encoding for a phenylalanine ammonia-lysase (PAL) enzyme, will be re-introduced at an extra-chromosomal locus as modified genes containing inducible promoter/repressor “switches” allowing us to direct metabolism toward CA at will. The “switch” mechanism will require “cis” DNA elements immediately upstream of each enzyme’s coding sequence (CDS). In addition, each regulatory system (ie. induction, repression) will require a constitutively-regulated “trans” element encoded at a different locus which produces the protein component.
To enhance the yield of our desired products, two additional categories of changes will be made to our host strain. Enzymatic activities which direct metabolites away from the desired pathway will be abolished by removing the underlying endogenous genes and transporter proteins responsible for re-uptake of SA or CA from the medium will be removed in the same manner.
BioBricks to be made:
|
Part name |
Gene/Locus |
Source |
Note |
|
Constitutive promoter |
n/a |
Synthetic |
General part |
|
Inducible promoter (cis) |
n/a |
Synthetic |
General part |
|
Inducible promoter (trans) |
n/a |
Synthetic |
General part |
|
Inducible repressor (cis) |
n/a |
Synthetic |
General part |
|
Inducible repressor (trans) |
n/a |
Synthetic |
General part |
|
Ribosome binding site |
n/a |
Synthetic |
General part |
|
Transcription terminator |
n/a |
Synthetic |
General part |
|
Transketolase |
tktA |
P. putida |
Constitutively upregulated |
|
DAHP synthase |
aroF^FBR |
P. putida |
Constitutively upregulated, Thr326Pro = feedback insensitive |
|
DHQ synthase |
aroB |
P. putida |
Constitutively upregulated |
|
DHQ dehydratase |
aroD |
P. putida |
Constitutively upregulated |
|
Shikimate dehydrogenase |
aroE |
P. putida |
Constitutively upregulated |
|
Shikimate kinase |
aroK |
P. putida |
Removed and reintroduced as inducible |
|
3-phosphoshikimate 1-carboxyvinyltransferase |
PPS_1411 |
P. putida |
Removed and reintroduced as inducible |
|
Chorismate synthase |
PPS_1471 |
P. putida |
Removed and reintroduced as inducible |
|
Chorismatemutase |
PPS_1410 |
P. putida |
Removed and reintroduced as inducible |
|
Aromatic amino acid aminotransferase |
PPS_1561 |
P. putida |
Removed and reintroduced as inducible |
|
Aromatic amino acid aminotransferase |
PPS_3083 |
P. putida |
Removed and reintroduced as inducible |
|
Histidinol-phosphate aminotransferase |
PPS_0995 |
P. putida |
Removed and reintroduced as inducible |
|
Phenylalanine ammonia-lysase |
|
R. toruloides |
Introduced as inducible |
Genes to be removed:
|
Shikimate kinase |
aroK |
P. putida |
Removed and reintroduced as inducible |
|
3-phosphoshikimate 1-carboxyvinyltransferase |
PPS_1411 |
P. putida |
Removed and reintroduced as inducible |
|
Chorismate synthase |
PPS_1471 |
P. putida |
Removed and reintroduced as inducible |
|
Chorismatemutase |
PPS_1410 |
P. putida |
Removed and reintroduced as inducible |
|
Aromatic amino acid aminotransferase |
PPS_1561 |
P. putida |
Removed and reintroduced as inducible |
|
Aromatic amino acid aminotransferase |
PPS_3083 |
P. putida |
Removed and reintroduced as inducible |
|
Histidinol-phosphate aminotransferase |
PPS_0995 |
P. putida |
Removed and reintroduced as inducible |
|
Pyrroloquinolinequinone |
PPS_3064 |
P. putida |
Removed |
|
Anthranilate synthase component I |
PPS_0413 |
P. putida |
Removed |
|
Anthranilate synthase component II |
PPS_0416 |
P. putida |
Removed |
|
Para-aminobenzoate synthase subunit I |
PPS_1911 |
P. putida |
Removed |
|
Shikimate transporter |
shiA |
P. putida |
Removed |
|
Cinnamic acid transporter |
|
|
Removed |
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