Synthetic Biology Applications in Food Safety and Security

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This entry is adapted from the peer-reviewed paper 10.3390/synbio1010006

With an ever-growing global population, issues of food safety and security, as well as addressing pollution and striving for sustainability are of the utmost importance. Synthetic biology offers a unique, exciting range of solutions to address these urgent and growing challenges.

food safety food security biosensors biomanufacturing sustainability synthetic biology environmental biotechnology

1. Introduction

Synthetic biology is a broad, interdisciplinary field leveraging engineering, life sciences, and data science to engineer biological systems with new and useful properties ^[1]. The growing interest in synthetic biology research is reflected in the substantial increase in the number and diversity of technologies and companies attempting to address challenges in a variety of fields including medicine, agriculture, energy, and consumer products. The synthetic biology market is growing at nearly 20% compound annual growth rate and anticipated to exceed USD 10 billion in the next 5 years ^[1]. This rapid growth is largely driven by the global challenges resulting from climate change and chemical overaccumulation. Biologically derived technologies and materials often do not contribute to the negative environmental impacts that usually characterize petrochemically derived materials, and therefore can be considered a more sustainable route.

2. Food Safety and Security

According to the USDA, 13.5 million people experienced food insecurity in 2021 ^[2]. A major contributor to this widespread issue is the overabundance of resource competition from undesirable biomass that ultimately leads to decreased crop yields and increased food prices ^[3]. While herbicides are an attractive solution, their broad specificity becomes quickly problematic as desirable crops succumb to treatment as well ^[4]. Additionally, according to the Food and Agriculture Organization of the United Nations, approximately 20–40% of crops worldwide are lost due to insect pests each year, also contributing to food insecurity ^[5].

2.1. Herbicide Tolerance

Generating crops with herbicide resistance or tolerance through protein engineering has become an attractive area in agricultural research to address food security. Commonly, the target sites for most herbicides are enzymes involved in plant growth and development, with different herbicides targeting different enzymes. To eliminate weeds, herbicides are attracted to and bind these various enzymes which inhibit their activity leading to altered growth and ultimately plant death ^[6]. However, the problem then becomes that there is no distinction between these target sites in weeds versus those in food crops, resulting in both weed and crop death and reducing crop yields as well. Murphy and Tranel (2019) published an exhaustive review of herbicide target sites, along with known mutations to confer resistance ^[6].

Enzymes involved in amino acid synthesis or other biosynthesis pathways to support plant growth are major sites of action for herbicides. The target site for common globally used herbicides such as those in the sulfonylurea (SU) and pyrimidinyl-benzoate (PYB) families is acetohydroxyacid synthase (AHAS) ^[7]. The function of AHAS in plants is to promote growth and development through its role in branched amino acid (BCAA) production ^[8]. By engineering target sites such as AHAS, decreased affinity for herbicides can be acquired which will allow the properly functioning enzyme to promote healthy plant growth. For example, weed varieties have developed tolerance to herbicides due to mutations in AHAS. This natural phenomenon inspired the engineering of the enzyme based on these mutations for herbicide-tolerant (HT) crops. Rather than experimentally investigating a pool of mutants, Fang et al. (2020) used a molecular docking approach to explore the W548 residue, a common resistance mutation site ^[8]. Previous studies have only looked at amino acid substitutions, however, through molecular docking validated by in vitro studies, this study found that the deletion of W548 led to multi-family herbicide tolerance and performed better than those of previous substitution mutations ^[8]. Another enzyme crucial to the production of aromatic amino acids to support plant growth is 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), that is targeted by glyphosate herbicides. Various weed species including Conyza sumatrensis and Eleusine indica are known to have specific mutations in EPSPS, which are P106T and double-mutant T102I/P106S (TIPS), respectively, making them resistant to glyphosate ^[9]. The double mutant has proven higher glyphosate resistance than P106T, showing that evolutionary mutations in EPSPS can inform the future design of herbicide-resistant crops ^[9]. Recently, a study incorporated the TIPS mutation into the EPSPS gene in rice, resulting in glyphosate tolerance ^[10]. Additionally, Ortega et al. (2018) mutated the EPSPS gene from Capsicum annum (chili pepper) to introduce the TIPS mutation and then looked at the use of different promoters to determine which led to the expression of the most highly resistant EPSPS to confer glyphosate resistance in chili peppers ^[11]. Identifying evolutionary changes in weed species that confer resistance to herbicides and then harnessing synthetic biology techniques to create and express these mutations transgenically is an effective way for food crops to combat herbicide treatment and to maintain global food security.

2.2. Insecticidal Activity

Lepidopteran pests such as Spodoptera frugiperda and Coleoptera pests such as Diabrotica virgifera virgifera (western corn root worm, WCRW) pose a major threat to crop yields, so providing food crops with the machinery to combat these insects is crucial to addressing food security. Bacillus thuringiensis (Bt), a spore-forming bacterium, is known for releasing proteins with insecticidal activity and researchers have found applications for these proteins in transgenic plants to improve insect resistance. One class of these proteins is the insecticidal crystalline (Cry) proteins which were commercially used to protect food crops from insect pests. However, as the commercialization of these proteins becomes more popular, like antibiotic usage in humans, there is a risk of resistance mechanisms developing. Therefore, continually engineering new insecticidal proteins could help the agricultural world combat potential resistance. Cry proteins have three domains that each provide different functional characteristics to the protein. In recent work, Liu et al. (2022) engineered new Cry proteins through domain swapping. In Liu’s work, domain III in the N-terminal, which is responsible for interaction with insect receptors, was swapped with domain III regions from other Cry proteins to create new, novel proteins providing insect resistance ^[12]. Understanding the functional purpose of the N-terminal domains in combination with domain swapping led to the generation of new, unique Cry proteins such as Cry1Ab-Gc, which provides resistance to two lepidopteran insects in rice and maize ^[12]. Another Cry protein from Bt, Cry8Hb, was found to be effective against WCRWs ^[13]. Hou et al. (2019) discovered that fusing maltose binding protein (MBP) to Cry8Hb results in the increased solubility of the protein in the insect midgut, leading to the enhancement of its insecticidal effect. Following these results, DNA shuffling was used on a newly synthesized gene, IP3-1, which closely resembled Cry3 to discover proteins that had increased solubility and therefore higher anti-WCRW activity, but this time without the MBP fusion partner. The mutations generated from DNA shuffling increased the solubility and anti-WCRW activity in the same region where MBP was likely bound to fused proteins. These results suggest that the increased solubility of Cry proteins in the insect gut leads to their enhanced ability to protect food crops from insects and this can be accomplished via MBP fusion or mutations in specific residues ^[13].

Additionally, Bt produces another category of insecticidal proteins during its vegetative growth phase called vegetative insecticidal proteins (Vip). Like the aforementioned work, Gomis-Cebolla et al. (2020) used domain swapping between Vip proteins to give this category of insecticidal protein greater solubility and stability in the insect gut as well. Vip3A proteins are known to have insecticidal activity, while Vip3B and Vip3C are lacking. By domain swapping, a new Vip protein, Vip3_ch2, was identified to have increased activity against S. frugiperda ^[14]. Furthermore, these studies determined which domains were most important for toxicity and stability to inform the future of engineering novel Vip proteins ^[14].

2.3. Environmental Change Tolerance

The increasing occurrence of late spring and early fall flash freezes can have damaging effects on crop yields ^[15]^[16]. Providing plants with better resistance to cold temperatures will help prepare them for changing climate conditions ^[17]. Improving the efficiency of photosynthesis is another way in which crops can be enhanced to be better suited for their environment ^[18].

Crops with better cold resistance offer a wider range of growing locations and seasons while also helping to reduce the amount of crops lost to frost damage ^[15]. Xu et al. (2020) used a point mutation on the low-temperature tolerance 1 (LTT1) gene of rice for increased seed setting during low-temperature stresses while not hindering crop yields during normal growing conditions. Since previous work has faced difficulties identifying reliable cold tolerance genes, the discovery of LTT1 allows the future hybridization of rice plants with increased cold tolerance using internal mechanisms that do not affect other important plant functions such as the development of pollen and tapetum ^[17]. Wani et al. (2021) were able to express a cold response protein1 (BOCRP1), which is constitutively expressed in the leaves, roots, and stems of the cabbage species, in a tomato plant susceptible to cold temperatures. The modified tomato plants exhibited increased seed germination and root length in addition to the improved tolerance of cold stresses ^[19]. These results provide insight for future work in improving the cold tolerance crops while not hampering the normal growth and development of the plant ^[19].

The photosynthesis process in crops can also benefit from the introduction of new proteins to the crop’s chloroplasts. Gomez et al. (2018) showed that two flavodiiron proteins (Flvs) from cyanobacteria, Flv1 and Flv3, were able to increase the photosynthesis performance of tobacco plants during light transition periods, such as sunset or shading caused by passing clouds, while maintaining performance under constant lighting conditions ^[18]. These Flvs are responsible for relieving the excess energy of the photosynthetic electron transport chain of cyanobacteria and perform a similar function in the Nicotiana tabacum chloroplasts ^[18]. The introduction of Flvs to act as electron sinks during light transition periods could allow other field crops to exhibit photosynthesis protection and efficiency that will lead to improved plant health and yields ^[18].

References

Synthetic Biology Market Size, Share, Trends, by Technology, by Tools, by Application, by End-Use, and by Region Forecast to 2030; Synthetic Biology Market; Research and Markets. 2022. Available online: https://www.researchandmarkets.com/reports/5648701/synthetic-biology-market-size-share-trends-by (accessed on 1 October 2022).
USDA ERS-Key Statistics & Graphics. Available online: https://www.ers.usda.gov/topics/food-nutrition-assistance/food-security-in-the-u-s/key-statistics-graphics/ (accessed on 23 September 2022).
Daramola, O.S.; Adigun, J.A.; Olorunmaiye, P.M. Challenges of weed management in rice for food security in Africa: A review. Agric. Trop. Subtrop. 2020, 53, 107–115.
Chen, Z.; Wang, Z.; Heng, Y.; Li, J.; Pei, J.; Cao, Y.; Deng, X.W.; Ma, L. Generation of a series of mutant lines resistant to imidazolinone by screening an EMS-based mutant library in common wheat. Crop J. 2020, 9, 1030–1038.
Sarkozi, A. New Standards to Curb the Global Spread of Plant Pests and Diseases; Food and Agriculture Organization of the United Nations: Rome, Italy, 2019.
Murphy, B.P.; Tranel, P.J. Target-Site Mutations Conferring Herbicide Resistance. Plants 2019, 8, 382.
Acetohydroxyacid Synthase Inhibitors (AHAS/ALS). Modern Crop Protection Compounds; Jeschke, P., Witschel, M., Krämer, W., Schirmer, U., Eds.; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, Germany, 2019; pp. 33–171. ISBN 978-3-527-69926-1.
Fang, J.; Wan, C.; Wang, W.; Ma, L.; Wang, X.; Cheng, C.; Zhou, J.; Qiao, Y.; Wang, X. Engineering Herbicide-Tolerance Rice Expressing an Acetohydroxyacid Synthase with a Single Amino Acid Deletion. Int. J. Mol. Sci. 2020, 21, 1265.
Fonseca, E.C.M.; da Costa, K.S.; Lameira, J.; Alves, C.N.; Lima, A.H. Investigation of the target-site resistance of EPSP synthase mutants P106T and T102I/P106S against glyphosate. RSC Adv. 2020, 10, 44352–44360.
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Ortega, J.L.; Rajapakse, W.; Bagga, S.; Apodaca, K.; Lucero, Y.; Sengupta-Gopalan, C. An intragenic approach to confer glyphosate resistance in chile (Capsicum annuum) by introducing an in vitro mutagenized chile EPSPS gene encoding for a glyphosate resistant EPSPS protein. PLoS ONE 2018, 13, e0194666.
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Wani, U.M.; Majeed, S.T.; Raja, V.; Wani, Z.A.; Jan, N.; Andrabi, K.I.; John, R. Ectopic expression of a novel cold-resistance protein 1 from Brassica oleracea promotes tolerance to chilling stress in transgenic tomato. Sci. Rep. 2021, 11, 16574.

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Update Date: 29 Nov 2022

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