Propionate CoA transferase for P. putida
This is the DNA coding sequence for the enzyme Propionate CoA. The enzyme normally catalyzes the reaction: acetyl-CoA + propanoate <--> acetate + propanoyl-CoA. Transferase. This mutated sequence has one amino acid substitution: A243T (compared to the wild type enzyme) and is codon optimized for Pseudomonas putida in order to produce (D)-Lactyl-CoA. It extend the BBa_K1211001 biobrick from Yale 2013 and have the same purpose.
We use this enzyme as the first enzyme in the pathway to create PLA or Polylactic acid. Lactate can also act as acceptors for the enzyme. In our project, the enzyme will catalyze the reaction: acetyl-CoA + lactate <--> acetate + Lactyl-CoA.
The wild type sequence is from Clostridium propionicum genome.
This is the naturally-occurring RBS found in the regulatory region of the DntR gene. gataaaaacaagaggaaaacaa
RBS + PhaC (sp. MBEL 6-19) + RBS + PCT (CP)
PCR using primers with biobrick prefix and surfix, and then cloned into pSB1AC3.
Engineered D-Lactate dehydrogenase (D-LDH*)
This is the coding sequence of lactate deshydrogenase that can super efficiently use NADPH and NADH as cofactor
Pseudomonas sp. MBEL 6-19 PHA synthase 1
This is the coding sequence for a Polyhydroxyalkanoate (PHA) synthase 1 from Pseudomonas resinovorans. The enzyme takes the monomer (D)-lactyl-CoA and creates the PLA homopolymer. This sequence is codon optimized for pseudomonas putida it includes four mutations which were found to increase yields of PLA (Yang et al.) site-directed mutagenesis, the mutation leads to production of high molecular weights of P(3HB-co-LA); site-directed mutagenesis, the type II PHA synthase 1 is engineered to accept short-chain-length hydroxyacyl-CoAs including lactyl-CoA and 3-hydroxybutyryl-CoA as substrates and support the synthesis of P(3HB-co-LA) by site-directed mutagenesis of four sites, i.e. E130, S325, S477, and Q481 We note that unlike BioBrick BBa K1211002 Yale 2013, one of the mutation is not on the Serine 477 on Serine 475. which is able to do the reaction: lactylCoA + PLA(n) = PLA(n+1).
We used this sequence as second enzyme in order to produce PLA. In fact it will take lactyl coA as substract and polymerise it into PLA homopolymere
It is the coding of pseudomonas putida constitutive promotor
This promotor will constitutively express LDH