In Moritella marina and Shewanella pneumatophori, the pfa gene cluster encodes five open reading frames, namely pfa A, pfa B, pfa C, pfa D and pfa E. Each gene contains up to several functional domains to perform carboxylation, dehydration, and reduction of the ketoacyl group and double bonds of the carbon chain in the PKS pathway. In Moritella marina, the pfa genes work together to generate docosahexaenoic acid (DHA), while Shewanella pneumatophori generates eicosapentaenoic acid (EPA). The exact functions of pfa A-D genes are unclear. To the best of our knowledge, the pfa B gene is the key molecule to determine the final product (e.g. EPA and DHA).
The acetyl-CoA carboxylase of C. glutamicum is composed of a biotin carboxylase (ACCBC), a biotin carboxyl carrier protein (ACCE), and a transcarboxylase (ACCD1). They carboxylated acetyl-CoA into malonyl-CoA, which is necessary for the elongation of nascent fatty acid. The carboxylation occurs in two steps. In the first step, the ATP-dependent biotin carboxylase (ACCBC) catalyzes the transfer of a carboxyl group to biotin, which is linked to the biotin carboxyl carrier protein (ACCE). In the second step, the transcarboxylase (ACCD1) transferred the carboxyl group from carboxybiotin to acetyl-CoA to form malonyl-CoA.
In our project, the pfa A gene is co-expressed with pfa D gene under the control of CspA cold-inducible promoter, while the pfa B, pfa C and pfa E genes are co-expressed under the control of lac-inducible promoter. The AccBC gene is co-expressed with AccD1 and AccE genes under the control of lac-inducible promoter.
| Part Number | Description | Length (bp) |
|---|---|---|
| BBa_K4131000 | AccD1 gene (C. glutamicum), codon optimized for E. coli expression | 1632 |
| BBa_K4131001 | AccBC gene (C. glutamicum), codon optimized for E. coli expression | 1776 |
| BBa_K4131002 | AccE gene (C. glutamicum), codon optimized for E. coli expression | 249 |
| BBa_K4131003 | Original pfa A gene (Moritella marina) | 7959 |
| BBa_K4131004 | pfa B gene (Moritella marina), codon optimized for E. coli expression. | 2652 |
| BBa_K4131005 | Original pfa C gene (Moritella marina) | 6036 |
| BBa_K4131006 | Original pfa D gene (Moritella marina) | 1617 |
| BBa_K4131007 | pfa E gene (Moritella marina), codon optimized for E. coli expression. | 864 |
| BBa_K4131008 | pfa B gene (Shewanella pneumatophori), codon optimized for E. coli expression. | 2343 |
| BBa_R0010 | Lac promoter and operator | 200 |
| BBa_K3778013 | RBS (Ribosome-binding site) | 23 |
| BBa_K3778018 | T7 terminator | 39 |
| BBa_K328001 | cspA Promoter + Cold Box + UTR + SD + DB | 321 |
| Part Number | Description | Length (bp) |
|---|---|---|
| BBa_K4131010 | The lac-induced expressing cassette of pfa B, pfa C and pfa E (DHA) | 9919 |
| BBa_K4131011 | The lac-induced expressing cassette of pfa B, pfa C and pfa E (EPA) | 9610 |
| BBa_K4131012 | The lac-induced expressing cassette of AccBC, AccD1 and AccE | 4024 |
| BBa_K4131013 | The Cold-induced expressing cassette of pfa A and pfa D | 10027 |