Enzyme production is still a huge concern for Indonesian domestic producers as the lack of knowledge and technology plays a huge part in the development of an efficient system.
Why is it important?
Based on an article published by Agency for the Assessment and Application of Technology (BPPT), Indonesia in December 2019, many efforts have been made by Indonesian enzyme industries to meet the domestic enzyme needs . Yet Indonesia still has to import 90% of its enzyme demand. We then decided to look into this further by holding a discussion with a BPPT researcher and a researcher who worked for one of the major enzyme producers, Amano Enzyme, to pinpoint the exact obstacles that are causing this directly in the field. Our conversations with the stakeholders, more on Integrated Human Practices, lead us to find out that there are still plenty of barriers to produce high quality enzymes with a lower cost. Mainly because of the lack of knowledge and technical or technological difficulties faced by the stakeholders and local industries, including the low yield of enzymes produced because the yield would most likely be diluted by the cultivation media. In order to make it more concentrated, the product has to go through complicated, multiple downstream processes such as ultrafiltration and microfiltration, which are expensive.
What are we doing?
Motivated to tackle the industrial problems currently experienced by Indonesian national enzyme producers, we think synthetic biology could be the solution to make a system able to produce enzymes with a higher yield. This is done by exploiting the natural mechanism of vesicle-producing Gram-negative bacteria coupled with the utilization of an enhanced outer membrane protein, eCPX, as the site of attachment for the protein of interest (PoI). eCPX or enhanced circularly permuted outer membrane protein OmpX is a modified outer membrane protein or OmpX and has both of its termini exposed on the exterior to the cell . The eCPX in our project will be linked with a specific protease linker between them and the PoI, providing a specific cleaving site.
eCPX will be expressed on the cell membrane and will act as the site for fusion with the PoI. eCPX that will be expressed abundantly, due to the use of a strong signal promoter on the circuit design, will be linked with the PoI through a TEV protease-specific linker. Therefore when the eCPX is produced on the cell membrane, it will automatically attach the PoI on its terminals which are exposed on the external of the cells. With the strong expression of eCPX, it is hoped that the PoI will be concentrated on the cell membrane. This will allow for easier observation to analyze the yield for recombinant protein expression.
Also due to the addition of a specific linker, the harvesting process would be way easier and simpler because only a specific type of protease would be able to cleave the PoI off of the vesicles or cell membrane. On the industrial scale, simplified protein harvesting method means a simpler downstream process that would therefore reduce the cost for processing and also save time because less steps are required to get a purified and cleaner yield.
The goal of downstream processing is to recover enzymes at a high yield, purity, and concentration while still cost-effective, resulting in a form that is stable, safe, and easy to use. Enzymes might require gentle processing for purification to avoid protein unfolding or denaturation. To achieve the most efficient way of downstream processing and address the limitations currently present in enzyme production, we propose to express our protein of interest on the outer membrane proteins of the host cell. We believe that our project is a useful application of synthetic biology as the simplification of the downstream process on enzyme production would lead to better efficiency in enzyme productions in the future.
Stated by Ojima et al. in 2020 , insertion of protease site-specific cleavage sequences between OmpW and target protein will enable a smart purification process for the target protein. We found that this motivation is suitable for the use of eCPX in our project. While allowing for a better and easier purification process, the utilization of eCPX would also allow for both of its termini to be used for recombinant protein expression and attachment. From Rice et al., in 2008 , it was stated that enhanced CPX (eCPX) is able to be localized on the outer membrane as efficiently as the non-permuted parent.
COVID-19 impacted all of the projects of the team and it is no exception to us. Because of the pandemic, we couldn't get lab access as soon as we had planned, causing a three-month delay for the lab work. While waiting for the lockdown to be over, we were occupying ourselves on designing our parts and researching journals to make our system functional with the time limit that we have.