Pumping out module

Through the production increase and control module, we have been able to greatly increase the amounts of specific FFAs produced by our engineered E.coli. For further application in industrial respect, however, we are still facing the challenge in intercellular fatty acids extraction. Generally speaking, most of the fatty acids synthesized in microbial would be stored in cells as the form of triglyceride. And the separation of fatty acids from cells requires complex and energy-intensive processing, high production cost and might cause serious environmental pollution. Therefore, in this module, we are trying to enhance the pumping out function of fatty acids in E.coli by engineering tools.

The gene we choose

Among the related researches conducted before, we found the overexpression of endogenous or exogenous thioesterases would facilitate extracellular fatty acids production to some extent. Also, many researchers have paid attention to two genes in cross-membrane uptake process of long-chain fatty acids, fadD and fadL. Such experiments have proved the knockout or inactivation of them contributed to enhance the exportation of fatty acids in E.coli. While in our project, we mostly concentrate on an ATP-binding cassette transporter protein, MsbA.

Specifically, MabA found in E.coli is an integral membrane protein. It is essential for the transport of a wide variety of substrate molecules from the cytoplasmic leaflet to the periplasmic leaflet of the inner membrane, especially lipid A and lipopolysaccharide (LPS). Related structural studies have proved it might function as a lipid flippase, which has provoked some thoughts about its possibility in fatty acids transporta. Through the over-expression of MsbA in E.coli, Xin et al. have observed an obvious increase in the ratio of extracellular fatty acids, even though the corresponding mechanism still remains unknown. Apart from this, however, few researchers have reported its similar function in facilitating fatty acids secretion in E. coli. Given that, we would like to further testify this in our module, hoping it could really enhance our fatty acids transport to the culture medium.

How to construct this module and how it works

At the beginning, we thought the higher the expression level of MsbA, the more the exported FFAs. Therefore, we employed the most common strong promoter T7 to initiate the transcription of msbA gene. And then we introduce the assembled plasmid into E.coli BL21 in order to detect its transcribed level. However, we were frustrated to find this batch of E.coli grew extremely slow in the culture. In fact, it never met the basic standard for QPCR (OD600 reached about 0.6). Why did it happen? Was there something wrong with the plasmid we constructed?

After consulting our instructor and the advisors, it turned out that we overlooked the toxic effects of member protein on E.coli itself. It is widely accepted that the overexpression of membrane proteins tends to breed noxious influence on cell viability. To release this effect as much as possible, therefore, we tried to enhance the MsbA expression only when high amounts of fatty acids were present and needed to be exported. And this reminded us of the operon we used to regulate the production of FFAs. It would be perfect if we could find a promoter regulated by the amount of fatty acids in E.coli. Luckily, we found it in E.coli’s fatty acids pathway.

The fadD gene encodes a crucial enzyme for fatty acids degradation in E.coli. When enough fatty acids have been produced or the cell needs energy for living activity, fatty acids will work as a signal, relieving the regulatory protein from fadD promoter, enabling its transcription and starting the degradation of fatty acids. Therefore, we isolated the fadD promoter from E.coli and assembled it with msbA gene in the same plasmid (see picture below). Theoretically, when fatty acids have been highly produced, MsbA would be automatically expressed, thereby enhancing the pumping out function for fatty acids.

Reference