CCU_TAIWAN contributed to polyunsaturated fatty acids (PUFAs) production by providing new biobricks and expression methods.
To engineer the PUFA-producing system in E. coli, we focused on the pfa genes from the deep sea bacterium Moritella marina and Shewanella pneumatophori. In the deep sea bacterium, the pfa genes are translated into protein and cooperate together as a megasynthase to synthesize PUFA by the polyketide synthase (PKS) pathway. The pfa A-D genes encode proteins containing many functional domains for PKS pathway, such as acyl carrier protein (ACP), 3-Ketoacyl-ACP reductase (KR), 3-Ketoacyl synthase (KS), 3-Hydroxydecanoyl-ACP dehydratase (DH), and acyltransferase (AT). The pfa E encodes phosphopantetheinyl transferase (PPTase). Each functional domain plays a role in modification of final products, carbon chain elongation and carrying intermediate products.
In brief, the PKS pathway starts from the activation of ACP by PPTase. After that, the AT domain will transfer the malonyl-CoA, which is the raw material of PUFA, to the ACPs. The backbone of malonyl-ACP will be further elongated by KS and AT domains, while KR, DH, ER domains are responsible for modification of the intermediate products. Finally, the PUFA will be produced.
Figure 1: The ACP proteins are activated by the PPTase, giving ACPs
the ability to transfer the intermediate product to interact with other functional domains.
Figure 2: The AT domain transfers the substrate to the ACP protein.
Figure 3: the elongation and modification of the backbone of PUFA.
The functional domains:
KS: Catalyzes a decarboxylative Claisen-like condensation reaction to extend the carbon chain of
polyketide.
DH: Reduces hydroxyl groups to enoyl groups.
KR: Reduces β-ketone groups to hydroxyl groups.
ER: Reduces enoyl groups to alkyl groups.
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 each pfa gene are unclear. To the best of our knowledge, the pfa B is the key molecule to determine the final product (e.g. EPA and DHA).
Figure 4: The DHA-producing gene clusters
Figure 5: The EPA-producing gene clusters
On the other hand, we also enhanced PUFA production through expressing acetyl-CoA carboxylase (ACC) from Corynebacterium glutamicum with pfa genes. The ACC protein is a multisubunit complex containing three subunits encoded by AccBC, AccD1, and AccE. The function of ACC is biosynthesis of malonyl-CoA, the raw material of PUFA. With greater raw material, more PUFA can be produced by E. coli through our PUFA-producing system.
Figure 6: The function and components of ACC.
1. We provided biobricks containing the coding sequences of multisubunit enzyme pfa from the docosahexaenoic acid (DHA)-producing deep sea bacteria Moritella marina.
In Moritella marina, the DHA production is mediated by multisubunit enzyme pfa through the Polyketide synthase (PKS) pathway (Okuyama, Orikasa et al. 2007). The multisubunit enzyme pfa includes five pfa genes, including pfa A, pfa B, pfa C, pfa D and pfa E. It has been reported that the ectopic co-expression of pfa A, pfa B, pfa C, pfa D and pfa E genes in E.coli. successfully induced DHA production (Orikasa, Nishida et al. 2006).
2. We provided a biobrick containing pfa B coding sequence from the eicosapentaenoic acid (EPA)-producing deep sea bacteria Shewanella pneumatophori.
In Shewanella pneumatophori, the multisubunit enzyme pfa responsible for EPA biosynthesis is encoded by pfa A, pfa B', pfa C, pfa D and pfa E (Orikasa, Yamada et al. 2004). Orikasa et al. indicated that the replacement of pfa B from Moritella marina with pfa B' from Shewanella pneumatophori could enhance the EPA production of multisubunit pfa from Moritella marina (Orikasa, Tanaka et al. 2009).
3. We provided biobricks containing the coding sequences of multisubunit acetyl-CoA carboxylase (ACC) from Corynebacterium glutamicum.
The multisubunit enzyme ACC is responsible for the biosynthesis of malonyl-CoA, which is the raw material necessary for PUFA production. The ACC enzyme contains three Acc genes, including AccBC, AccD1 and AccE from Corynebacterium glutamicum. The ectopic expression of ACC enzyme can increase the rate of fatty acid synthesis (Davis, Solbiati et al. 2000).
4. We improved the biobricks of AccBC, AccD1 and AccE.
In the biobrick, we optimized the codons of AccBC, AccD1 and AccE for E. coli expression. We also eliminate restriction enzyme sites of NdeI, EcoRI, SpeI, Pst1, XbaI, and NotI.
5. We designed the expression method for expressing large protein and swapping genes
Among the five pfa genes (pfa A, pfa B, pfa C, pfa D and pfa E), pfa A and pfa C genes are the largest (~8 kb and ~6 kb, respectively). Therefore, we decided to clone these two genes into different vectors to avoid the potential expression problem (Rosano and Ceccarelli 2014). The largest subunit, pfa A was cloned into the pColdI vector with pfa D (1.6 kb) for expression, since this cold shock protein activated vector is suited for expressing large proteins. The pfa C gene is cloned into pSTV28 vector with pfa B' (2.3 kb) and pfa E (0.6 kb) genes. Accordingly, these two pfa-expressing vectors of similar size may avoid the problem of transformation efficiency caused by plasmid size (Rosano and Ceccarelli 2014).
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