Team:Hamburg/Implementation

Implementation

How would we implement our project idea, the “Electron Transferrestrial”, in the real world? And why are we so convinced of our project’s relevance?

Our goal was to develop a platform for microbial sesquiterpenoid synthesis. Sesquiterpenoids are of great significance due to their role as pharmaceuticals, like artemisinin, zerumbone or parthenolide. But also, certain food additives, odorants, flavourings and sweeteners are based on the sesquiterpenoid backbone and part of our everyday life. Unfortunately, extracting sesquiterpenoids out of plant compounds or synthesizing them through chemical catalysis is associated with high costs and high complexity. Therefore, our team focused on biological production as a sustainable and efficient alternative.

To reach our goal and to improve current processes, we decided to optimize E. coli for the synthesis of certain sesquiterpenoids. We did not just want to achieve the expression of the CYP enzymes, catalyzing the final reaction step of the terpenoids synthesis. But we also wanted to improve the existing system by fusing the CYP enzymes with their complementary reductases. The reductases are responsible for the electron transfer between reduction equivalents and the CYP enzymes catalyzing the reaction. By bringing CYP and reductase closer together, we expect to improve the reaction regarding time and efficiency. However, for implementing the whole terpenoids synthetic pathway, our modified E. coli also needed to produce precursors like DMAPP and IPP, leading to the synthesis of FPP. For this reason, by means of the Keasling plasmids, we implemented the mevalonate pathway in E. coli . But that’s not all: to further optimize the synthetic pathway, we also required a high NADH production rate in E. coli . Therefore, we increased the regeneration of reduction equivalents by integrating the glucose 1-dehydrogenase 2 in our process. Besides E. coli as a host organism, we also tried to establish this process in cyanobacteria. Fortunately, they already contain the MEP pathway to synthesize the required precursors DMAPP and IPP for terpenoid production. Furthermore, the fusion of certain CYP enzymes to ferredoxin leads to a direct use of the photosystems electrons. Through photosynthesis coupled sesquiterpenoid production we aimed to pursue a different approach regarding use of energy in the host organisms.

Optimizing E. coli and cyanobacteria to produce certain terpenoids by integrating the mentioned mechanisms was our main goal. However, for industrial implementation, an upscaling of microbial cultivation in bioreactors would be required, which poses further challenges.

Opportunities

Let us show you the future opportunities for the “Electron Transferrestrial”. First of all, one would be able to use genetically optimized host organisms like E. coli or cyanobacteria for terpenoid production. This can be achieved by integrating genes for the precursor synthesis or for dehydrogenases delivering high amounts of NAD(P)H into the genome. Moreover, the synthetic pathway in the host organisms could be improved by integrating a secretion system for the produced terpenoids. This would be achieved by developing a specific carrier targeting the sesquiterpenoid. The extraction process would be simplified and more sustainable due to the longer use of bacterial cells. Also, a regulatory system, possibly based on small regulatory RNAs or promoter specificity, would allow further metabolic control in biological synthetic pathways. We could pave the way for biological terpenoid production by genomic modification with more efficient host organisms.

Furthermore, inventing a tool for fusing CYP enzymes with matching reductases for many different applications by bioinformatic analysis would constitute an additional opportunity. Running a high number of experimental series regarding the suitability and efficiency of different CYP enzymes fused with various reductases, could lay the foundation for an algorithm. This algorithm might calculate the optimal combination of the applicant's CYP enzyme with a reductase and even predict a suitable linker regarding length and composition for the fusion process. By analyzing natural fusion complexes, our bioinformatics team used similarities of these linkers to design our artificial fusion constructs. An improved tool incorporating structural data of the fusion enzymes would lay the foundation for systematic improvement of terpenoid production.

Challenges

For the implementation of our system, the scale-up process might pose a challenge. Synthesis in industrial scale requires different growth and expression conditions among other things. Parameters like the power input per volume related to gassing strategies, mixing times, nutrient supply, pH value, waste removal, temperature and cell density have to be considered, tested and permanently measured. For the cultivation of cyanobacteria further parameters like wavelength and illumination time play an important role as well. Upscaling can be implemented by increasing the cell cultures’ volumes step by step, slightly changing the growth conditions and selecting the optimal cell growth parameters to continue with the next step. Varying the different parameters, analyzing their combined effects and optimizing them in an ongoing process takes a lot of time. Therefore, by only optimizing the conditions for cultivation and protein expression in the lab, we are far away from industrial implementation.

An essential challenge requiring further work is the cytotoxicity of high amounts of phosphates, possibly fortified during terpenoid synthesis. The precursors for sesquiterpenoid production of the MEP and MVA pathway (DMAPP, IPP and FPP) can damage the host organism and even lead to its death. It would be necessary to accelerate every step of the chemical conversion to prevent the accumulation of intermediate products. Another possibility would be the regulation of FPP synthesis by including biosensors that are activated or deactivated depending on the FPP concentration. A common example are FPP dependent promoters or repressors regulating gene expression. With further optimization, cytotoxicity will no longer be the limiting factor of terpenoid biosynthesis. Moreover, industrial isolation and purification of the terpenoid products, like artemisinin, humulene or parthenolide, could involve effort. One possible method would be disrupting the bacterial cell walls and separating the product by Reversed Phase Chromatography, which would require the adjustment of retention times. Although, aiming for high efficiency, extracting the products without killing the microbes would be more sustainable. In this context, one could develop a secretion system, specifically transporting the terpenoids in the bacterial environment. Developing this system would certainly take time, which is why one could also focus on different approaches. In our project we, therefore, worked with a two-phase system, overlaying the cell cultures with dodecane and extracting the lipophilic sesquiterpenes out of the medium. In the following process, terpenoids in the dodecane layer could be directly detected via GC-MS. The two-phased extraction system could also be used for industrial scale production of sesquiterpenoids. Due to their volatility, lighter terpenoids, like limonene, could even be separated by gassing out from the growth medium and subsequent condensation.

To establish the mentioned tool for matching CYP enzymes and reductases as well as choosing a linker with the right length and composition, further research is required. A database with test results and efficiency for a variety of fusion enzymes for different applications would be needed.

Safety considerations

The risk of the terpenoid synthesis system for the environment and society is very low, since the production could take place in E. coli and cyanobacteria. E. coli as well as cyanobacteria are classified as risk group 1 organisms and are not associated with disease in humans or negative environmental impacts. Furthermore, bioreactors are closed systems, subject to strict physical biocontainment procedures. Most certainly, there will not be a release of genetically modified organisms into the environment, as long as waste is filtered and disposed correctly.

Even if the organisms would be accidently released, there would be a possibility to stop their spread easily. If necessary, the organisms could also be modified with a kill-switch or missing a vital metabolic pathway, making them dependent on specific growth-media.

Moreover, the synthesized products should be tested for their quality, toxicity and purity regularly before distribution.

https://www.frontiersin.org/articles/10.3389/fmicb.2018.02460/full