According to the official statistics from the World Health Organization (WHO), cardiovascular disease (CVD) is the leading cause of death and disability worldwide, causing 17.9 million deaths per year. Statistics in Taiwan also indicate the seriousness of CVD locally.

  One important cause of CVD is the accumulation of oxidized low-density lipoprotein (LDL) in blood vessels. Taking Omega-3 polyunsaturated fatty acid (PUFA) supplements, including DHA and EPA, could effectively slow CVD progression.

  The human body can synthesize DHA from α-linolenic acid (ALA), which is abundant in some plant oils. However, the conversion rate is extremely low, reported to be from 2-10% (Chiu, Su et al. 2008) or even as low as 0.01% (Hussein, Ah-Sing et al. 2005). Therefore, DHA-rich foods, such as fish, or DHA supplements are the two main sources for humans to get enough DHA. Notably, most DHA supplements are also purified from fish, suggesting that supply of supplemental DHA will become a problem as the marine resources are exhausted.

  Since fish is almost the only source of DHA, we attempted to figure out if we could generate an alternative source of DHA by synthetic biology. Accordingly, we first found that oily fish, such as salmon or sardines, do not generate DHA by themself. Instead, these fish uptake DHA-rich algae and accumulate DHA in the body. Exploration of DHA-generating algae and other organisms showed that the deep-sea bacteria Moritella marina MP-1 may be the excellent target for the following reasons (Yazawa 1996):

a. The genome sequence of MP-1 is known, suggesting that we have a chance to build the biobricks for DHA production.

b. The pfa genes responsible for DHA production are known (Okuyama, Orikasa et al. 2007).

c. It had been shown that the ectopic expression of pfa genes in E. coli induced DHA generation (Orikasa, Nishida et al. 2006).

  Following the research reported by Orisaka et. al., we extracted the pfa gene sequences from MP-1 genome, and found that the five pfa genes, pfa A, pfa B, pfa C, pfa D and pfa E are 7959 bp, 2652bp, 6036bp, 1617bp and 864bp in length, respectively (Orikasa, Nishida et al. 2006). Because of the length limitation of each plasmid, we decided to clone these pfa genes into different vectors. To balance the final length of each vector, we divided the pfa genes into two groups. The pfa A and pfa D belong to group 1 (9576bp in total) and others belong to group 2 (9552bp in total). Because gene expression in prokaryotes is poly-cistronic, we inserted a ribosome binding site before each gene to regulate the protein translation.

  While deciding a promoter for protein expression, it is common to select the lac-induced promoter to induce protein expression through lactose analog supplement. However, due to the extraordinary size of the pfa A gene, it is highly possible that expression would form inclusion bodies and thus loss of activity.

  Since MP-1 is a deep-sea bacteria grown at low temperature, and low temperature promotes the proper folding of large proteins (i.e. pfa A), we decided to apply the cold-inducible promoter cspA for pfa A expression (Vasina and Baneyx 1997).

  Accordingly, we designed the pfa A and pfa D genes under the control of the CspA promoter, while pfa B', pfa C and pfa E genes are under the control of the lac inducible promoter.

  After we constructed the plasmid design, we consulted different experts for suggestions. The secondary PI, Dr. Lee, admired the cspA promoter designs and gave some suggestions for dealing with protein aggregation. However, unexpectedly, another adviser, Dr. G. Rau, sent some references showing that DHA and EPA had different effects in CVD prevention (Mason, Sherratt et al. 2022, Sherratt, Libby et al. 2022). Further consultation with a cardiologist (name not shown, by request) from National Taiwan University Hospital supported the opinion that EPA, but not DHA, is functional in CVD prevention .

  EPA can prevent CVDs in different ways. In arteries, EPA preserves the membrane structure and regulates cholesterol distribution. In blood cells, EPA can split heme into several reducers to remove the free radicals, and reduce oxidative LDL accumulation. Consequently, EPA prevents the inflammation caused by oxidative LDL. In the vascular environment, EPA can also relax the surrounding smooth muscle by enhancing nitric oxide production to avoid severe CVDs (Peter, John et al. 2022).

  To switch from DHA to EPA production without dramatically changing the whole design, we turned back to survey references indicating that the DHA to EPA switch is feasible. Fortunately, we found that pfa B is responsible for the final product formation. Importantly, the swapping of pfa B from Moritella marina with pfa B' from Shewanella pneumatophori could shift the product from DHA to EPA (Orikasa, Tanaka et al. 2009).

  Finally, we exchanged the Moritella marina pfa B with Shewanella pneumatophori pfa B' and established a switchable PUFA generation system.

Reference:

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