Introduction

Pyrethroids are commonly used to combat aphid pests, making up 84% of all insecticides used for agriculture within the UK. However, they are highly toxic to bees, fish and aquatic invertebrates. In the interest of protecting local ecosystems and developing low-cost pesticide in-field testing, we took an integrated and dynamic approach utilising synthetic biology. Here, we report the successful development of a cheap rapid biosensor for λ-cyhalothrin based on aptamer technology which produces an easy-to-read colour change. We demonstrated our biosensor’s target could be extended to fenitrothion, a second pesticide, with only minor modifications. To enhance pyrethroid breakdown, we engineered a whole-cell catalyst utilising a carboxylesterase from Bacillus velezensis sd. To allow membrane insertion of the expressed carboxylesterase, we linked it to a modified anchor protein isolated from Pseudomonas syringae. To explore growth-production trade-offs, we developed an ordinary differential equation model of microbial gene expression and growth. Working under guidance from farmers and other stakeholders, we developed a spatial model for optimising potential deployment strategies in the field.

The Problem

Pyrethroids are a class of pesticides which currently make up 84% of all insecticides and nematicides used in the UK and are highly toxic to fish, bees, and aquatic invertebrates^[1][2]. It has become a replacement for neonicotinoid pesticides which have seen bans in recent years, contributing to its skyrocketing use (Figure 1). In the climate of COVID-19 pandemic and Russia-Ukraine conflict exacerbating growing food security concerns, the UK government is calling for greater local food production, which drives up the use of pesticides within the country^[3]. Moreover, the development of pyrethroid resistance in pests such as aphids has led to farmers resorting to higher doses of pesticides within shorter intervals out of desperation^[4]. This overuse of pyrethroid resulted in build-ups of the toxic compound in soil and water systems. The contamination reduces the ability of fish and aquatic invertebrates to maintain ionic balances through affecting control of ATPases associated with active transport^[5]. These problems are not limited to the UK and can be observed in other parts of the world. As the older generation pesticides are being banned and newer pyrethroid pesticides become more prevalent, pyrethroid overuse would inevitably become a global concern. Although pesticides can adversely affect biodiversity, their use is vital in meeting the growing food demand around the world. Bioremediation of pesticides provides a promising, economically viable solution to minimise the impact agriculture has on the environment at a time where food security is a vital concern around the world. Thus, our project ensures these pyrethroid pesticides can be used in a sustainable way. Find out more about the current state of the field on our background page.

PyRe - Our Solution

We chose λ-cyhalothrin as our target, in accordance with the guidance we gathered from our stakeholders and characterisation from existing literature. Its toxicity, half-life and potential carboxylesterases for degradation are well described. As a class II pyrethroid, it exhibits toxicity by reducing ATPase channel control significantly^[5]. It is also accompanied by a half-life well above 90 days dependent on soil type and other environmental factors^[3][6]. Find out more about λ-cyhalothrin here.

Part One: Aptamer-based Biosensor
Our sole initial goal was to prevent pyrethroid build-ups in the environment and minimise its impact on agricultural industries and the livelihoods of farmers. After discussions with various stakeholders such as Syngenta and Severn Trent, our project evolved to incorporate an in-field, cell-free pesticide detection system to guide the deployment of our whole-cell degradation system.
Our biosensing system utilises aptamers which are highly specific to our target λ-cyhalothrin for pesticide, and gold nanoparticles which have capacity for colour change when aggregated for visualisation of detected pesticide^[7]. In the presence of λ-cyhalothrin, the system exhibits an indicative colour change from red to blue. Find out more on our design page.

Part Two: Degradation
With a clear target compound and a chassis E. coli (Our Parts) for which we wanted to carry out the task, we conducted extensive literature reviews to find enzymes for degrading λ-cyhalothrin. Based on previous research by Jing et al.^[8], it was found that carboxylesterase CarCB2 from Bacillus velezensis sd. linked to an ice nucleation protein (INP) from Pseudomonas syringae could catalyse the first step of λ-cyhalothrin biodegradation in an extended pathway (Figure 2). The enzyme could maintain a higher turnover when expressed on the cell surface than when expressed in the cytosol. Additionally, this eliminated the need for a membrane transport mechanism to uptake the pesticide, although it was later made known to us by Syngenta that λ-cyhalothrin could readily diffuse across the cell membrane.

While it would be attractive to aim for maximum expression and hence maximum degradation, this would impose a metabolic burden on the host cell. Therefore, it was important that we take into consideration the trade-offs within a system with limited resources. This is explored by our modelling team.

Part Three: Biocontainment
As our project requires environmental deployment of a GMO, we had clear concerns around biocontainment and GMO legislations. To focus on the safe deployment of our engineered bacteria, we reached out for a partnership with CyanoClean (iGEM Concordia University 2022), who were also working within pesticide remediation, specifically targeting the organophosphate fenitrothion. CyanoClean agreed to develop a toxin/antitoxin kill switch for our degradation pathway intermediate 3-PBH. In return, we at Pyre adapted our aptamer-based biosensing system for their target pesticide, fenitrothion. This partnership completed the trifecta of sensing, degradation and biocontainment for the biodegradation of λ-cyhalothrin.
Recognising that public opinions around GMO’s though our survey is vital to potential legislation changes in the future, we laid out outreach plans to target a wide range of age groups and specialities through collaborations with Warwick Institute of Synthetic Biology (WISB) and Warwick Institute of Engagement (WIE) which aimed raise awareness among the general public through the use of interactive resources.

Future Vision

At Pyre we have developed an extensive plan for future development of the project into a viable product. Initially, we aimed to develop a pyrethroid testing kit, encompassing a selection of biosensors to facilitate environmental cleanup and the mapping of water quality. Although we focused on the deployment of our chassis in fields, our conversations with Severn Trent show potential deployment in water treatment to be viable in the long term and computational modelling of downstream enzymes has laid the groundwork to allow for complete degradation of λ-cyhalothrin.