Proposed Implementation

Introduction

We started our project MonChassis with the goal of manufacturing monoterpenoids in a sustainable and economical way. The current method for producing monoterpenoids is energy and cost intensive, unsustainable, has low efficiencies and relays on physical and chemical extraction from different plants (Wu and Maravelias, 2018). However, industrial extraction of monoterpenoids is hampered by the influence of external factors which affect growth conditions of plants and therefore batch composition of the extract. Although monoterpenoids are used in many areas of our daily life, such as the pharmaceutical and cosmetic, the food and beverage industry, agriculture and many more, there are no alternative production methods to solve the current and upcoming problems like increasing demands (Verified Market Research, 2022), high production costs (Wu and Maravelias, 2018) and cultivation challenges through climate change (Cho, 2022). To tackle these challenges, we designed MonChassis, a manufacturing platform to produce monoterpenoids. In the following text, we would like to present our considerations for the successful implementation of MonChassis.

Steps towards real world implementation

In our business plan, we considered several aspects that need to be addresses when thinking about the successful implementation of MonChassis. First, to accelerate manufacturing of MonChassis and be economically competitive, we thought about ways to increase the production of the precursor GPP and NPP in Saccharomyces cerevisiae and Yarrowia lipolytica. So, we aimed to introduce the Saccharomyces cerevisiae genes SctHMGR and ScERG13 to eradicate the bottleneck of the mevalonate pathway. We tried to reduce the amount of produced farnesyl pyrophosphate (FPP) and increase the amount of GPP through insertion of the AgtGPPS2 and GgMFPS144 genes into the YlERG20 locus of Y. lipolytica. Moreover, we wanted to introduce the SltNPPS1 with the aim of NPP production as well as the insertion of the promoter YlpCTR1 upstream of the YlERG20 gene. Thereby a reduction of the amount of FPP and the decreasing competition for isopentenyl pyrophosphate and dimethylallyl pyrophosphate (IPP and DMAPP) would be the desired effects. In the end, NPP and GPP should be converted into α-pinene via the PptAPS. This would enable us to achieve higher α-pinene concentrations that pave the way towards scale-up production in fermenters. Our expression in yeast is already well established and manufacturing in large bioreactors is feasible. Nevertheless, it is necessary to optimize and screen the precursor extractions. Further research and trials of the different strains for example on successful integration of homology flanks or gene knock-out of ROX1 are needed. Second, since our approach to synthesize valuable monoterpenoids is based on electrically driven enzymes, it can easily be scaled up by the simple usage of electricity instead of NADPH (nicotinamide adenine dinucleotide phosphate). The current use of our BioReactor for Enzymatic ElectroSynthesis (BREES) so far only allows a small output but is scalable when considering up-scaling requirements like a bigger technical realization of our BREES, the cultivation of yeasts in a fermenter, or an optimized electron transport through the linker enzyme for precursor oxidization. Modelling not only helped us performing troubleshooting but also improving the yield by methods like media optimization.

Third, we got in touch with our end customers who currently fight against the bark beetle. In a conversion with Dr. Dr. Gabriela Lobinger, we learned that verbenone – which is already tested as a repellent against bark beetle, is very volatile and therefore poses challenges in widespread application. For this problem, a solution is still being sought through various studies. Another problem users of verbenone currently face is inconsistency of verbenone isomerism from chemical extractions as the efficacy of verbenone is greatly affected when the isomerism is altered even slightly. In contrast, a yeast based monoterpenoid production can selectively control isomerism, resulting in uniform production and standardized large-scale quality. Moreover, we discussed ways of deployment and spreading of verbenone to the designated forests. Here, we learned from Dr. Dr. Gabriela Lobinger that the method of choice would be a micro encapsuled liquid product, embedded in uric acid. This is an approved and biologically safe method and therefore would not have a negative impact when applied.

Safety aspects

One yeast of our choice, feasible for monoterpenoid production is S. cerevisiae. Discussing aspects of security with Dr. Christian Schulze Gronover, we learned that this yeast has the GRAS status (Generally recognized as safe), is well-known and very well studied. Critical danger should therefore not exist when working with this strain in enclosed environments like a fermenter. That also concerned our Y. lipolytica, the other yeast strain of choice.

In context of large-scale application, tolerance and toxicity towards the environment need to be investigated. Until today, there is no data highlighting verbenone´s potential to be harmful to the environment, as verbenone is also a natural messenger used by the bark beetle itself. Nevertheless, effects on the behavior of bark beetles and a few subgroups are to be expected but necessary for effective control. Further studies should be performed as to the potential impact of verbenone on the interaction between organisms and how affected insects respond to permanent presence of verbenone. This could reveal potential challenges with the long-term use of verbenone in ecosystems.

Other challenges to consider

Other important factors towards the successful implementation of MonChassis are aspects of commercialization and distribution of yeast produced monoterpenoids. To this end we developed a business plan with guiding milestones paving the way towards a possible market implementation. Moreover, we noticed through our work in human practices and education that it is critical to inform and discuss with the broad society. This helps to enlighten people to use biotechnologically manufactured products, both because this approach is more sustainable, inexpensive and prejudices and concerns can be addressed. Another challenge to consider is receiving long-term financial support, to be able to continue the project. Networking, as in human practice work, exposure to businesses, influencers and other scientists can ensure such support. Logistics is another challenge to consider when distributing produced monoterpenes world wide. It is necessary to implement safety guidelines and processes for packaging and transport because of safety regulations by freight forwarders and national or international laws. Therefore, trained staff and certificates for working with these substances are needed.

All in all, MonChassis is the first step towards bioeconomy of monoterpenoids and many more challenges lie ahead on the road. Being economically competitive is of high importance in context of industrial manufacturing and considerations in our business plan helped us to address these challenges. Through our engineering we set the basis to develop an economically competitive manufacturing process based on yeast, opening the vast applications of monoterpenoids.

Business plan

Monoterpenoids and their associated class of terpenes hold great potential from a scientific as well as an economic point of view. The total market volume of terpenes is estimated to grow with approximately 8% annually until 2027. Consequently, even a small transformation of the production technique towards a more sustainable and efficient method in the future might also lead to a growing advantage concerning the economic value, reduction of greenhouse gas emission, and avoidance of pollution. As part of the solution, we, the iGEM Team Münster, present our project MonChassis, a biotechnological approach for monoterpenoid production. We are 23 young and highly motivated students who want to contribute to solving the problem of monoterpenoid biosynthesis.

Current production methods are based on physical and chemical extraction from plants or on chemical synthesis from fossil fuels. The composition of monoterpenoids in essential oils in plant extracts heavily relies on the plants’ environment, therefore leading to inconsistent quality and quantity in isomer composition with changing growth conditions. Yet, these chemical substances are used in many general manufacturing processes as well as in specific fields of application. The main users are the fragrance, flavour, and food industry as well as the chemical and health care sector.

We aim to use genetically modified yeasts and a cell-free conversion system to produce our desired monoterpenoid. With our approach, we want to replace the inefficient and unsustainable production of monoterpenoids and take a step towards a biobased industry. Further information regarding our business plan for our project MonChassis can be found in the attached file.