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.

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.

BioBricks to be made:

Parts:

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