Team:Alberta-North-RBI E/genetics

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Genetics

We plan to use a single metabolic pathway for the conversion of glucose to aromatic chemicals. We are currently engineering a Pseudomonas putida strain containing a “switch” system enabling the manufacture of either shikimic acid (SA), 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.



Our metabolic engineering strategy is based on the invention of Dr. John Frost (US Patent No. 5168056, 6613552). 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. To enhance the yield of our desired products, two additional categories of changes will be made to our host strain: (1) Enzymatic activities which direct metabolites away from the desired pathway will be abolished by removing the underlying endogenous genes and (2) transporter proteins responsible for re-uptake of SA or CA from the medium will be removed in the same manner.


Pathway.jpg


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.


Our genetic switch will be designed to tightly regulated gene expression. This will be accomplished by using both an inducer module and a repressor module so that strong expression of the regulated gene is only possible when a dual signal is given. One signal will cause an “inducer protein” to bind a cis regulatory element upstream of the promoter, where it can recruit cell machinery to initiate transcription. This signal alone, however, would be insufficient to allow expression. A repressor cis element located between the inducer cis element and the promoter is normally bound by a “repressor protein” which prevents movement of the transcription machinery down the gene. A second signal will cause the “repressor protein” to dissociate from it's cis element – thus clearing the transcription machinery's path and allowing expression. The exact details of this regulatory system are still under investigation – the design of this system may become a focus of next year's Alberta North iGEM team.


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

PPS_3480

P. putida

Constitutively upregulated

DAHP synthase

PPS_1503

P. putida

Constitutively upregulated, Thr326Pro = feedback insensitive

DHQ synthase

PPS_4923

P. putida

Constitutively upregulated

DHQ dehydratase

PPS_0557

P. putida

Constitutively upregulated

Shikimate dehydrogenase

PPS_0037

P. putida

Constitutively upregulated

Shikimate kinase

PPS_4924

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 knocked out:

Shikimate kinase

PPS_4924

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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