Team RoSynth has designed a 3D bioprinter capable of printing microbe-laden hydrogels, utilizing an engineered bacteria and yeast hydrogel parallel culture system for the efficient synthesis of plant-derived molecules. As a proof of concept, we attempted to synthesize rosmarinic acid, a small molecule with vast therapeutic and culinary applications, with the objective of expanding our project to create additional crucial botanical compounds using a cutting-edge synthetic biology approach.
At the heart of our project, we aspire to unite solutions for the impacts of climate change and growing human demand on agricultural production with emerging techniques in microbial printing. Botanical compounds can function as essential drugs, flavoring agents, dyes, and pesticide alternatives, and are incorporated into all manner of products both mundane and vital. Humans rely on plants and the molecules they produce for so many aspects of our lives, and our need is only growing.
Due to a surge of public interest in nontoxic and eco-friendly alternatives to synthetic chemicals, the global market of natural colorants is growing faster than the entire color market [1]. Similarly, the market for natural flavorings was worth $8.63B in 2021 and has been growing rapidly since, with a majority of modern consumers avoiding artificial food colorings and flavors [2]. A prime example of our intense demand for botanical compounds and the shortcomings of current production techniques is that of drug shortages experienced in the United States in the wake of the SARS-CoV-2 pandemic. According to a Senate report, new drug shortages in the US increased nearly 30% between 2021 and 2022, reaching a record five-year high of 295 active drug shortages [3]. A large proportion of these shortages involve plant-derived drugs whose supply is limited by a shortage of raw materials in the global supply chain. Roughly 80% of the raw materials in the pharmaceuticals sold in the U.S. are imported from abroad. In the case of medicinal plants, this is often because the plants themselves require particular climate conditions for cultivation. Issues experienced by foreign suppliers in locations such as India, Europe, and China can lead to disruptions in the American drug supply. Problems of availability can arise as a result of economical or armed conflict, political upheaval, degradation or contamination during transport, environmental conditions, or decreased crop yields [4]. Similar supply chain issues plague nonmedical botanicals, which are also predominantly imported for commercial use.
Not only do current markets and supply chains place medicine and industry in a perilous position, but many of these indispensable plant species themselves are coming under threat due to changing climates, invasive pests and diseases, and unsustainable farming practices as a result of high demand. There are between 50,000 and 80,000 flowering plant species used for medicinal purposes worldwide, while the current loss of plant species is between 100 and 1000 times higher than the expected natural extinction rate. This data implies that Earth is losing at least one potential major drug every 2 years [5].
Our iGEM team is based in Rochester, NY, and adjacent to the Finger Lakes region, a major agricultural area in New York State. While all manner of crops are grown around us in both small-scale and industrial settings, the increasingly severe impacts of climate change in New York State are anticipated to decrease crop yields over the coming years and impact local supply of plants and plant-based compounds. Existing local cultivators of herbs that contain valuable organic compounds are likely to suffer material and economic losses in the coming years [6], and there are not many to begin with. This is because competition with commercial growers overseas is fierce, and the world herb market itself is volatile [7].
New York State alone is home to hundreds of biotechnology and pharmaceutical companies [8] and thousands of food and beverage manufacturing companies [9], many of which have been negatively impacted by a lack of local supply for herbs and plant-derived molecules in their development processes. Healthcare facilities across New York, including the 7 hospitals and 29 other patient care facilities operated by University of Rochester Medicine [10], have felt the strain of pharmaceutical shortages resulting in part from a lack of local source of botanicals. Our project will fill this niche by offering a stable, agriculture-independent, and affordable production source of plant-derived compounds not just for our Rochester community, but for anyone across the world who replicates our technique. Our project was created to protect ecosystems, the economy, and the lives of patients both in our immediate community and across the globe by providing accessible biosynthesis solutions for essential botanical molecules. In the long term, we plan to extend our project’s applications to the synthesis of botanical compounds which cannot be agriculturally produced in our area, bringing previously international, expensive, and potentially unstable supplies of life-saving, versatile molecules right to our doorstep, and soon the doorsteps of all those who need them most.
Biosynthesis - the genetic engineering of living organisms to produce complex molecules for human use - offers an attractive alternative to the extraction of plant-based compounds from commercial crops and to traditional chemical synthesis in terms of chemical yields, production time, energy and water consumption, cost, and overall dependability. Not just any standard biosynthesis will do, however.
Existing, solely bacteria or yeast-based systems for the production of plant-derived chemicals such as rosmarinic acid possess major limitations. First, such small molecule products involve a long biosynthetic pathway, which requires traditional biosynthesis techniques to place a high metabolic load on engineered organisms. This curbs the organisms’ growth, and ultimately production efficiency. Using a co-culture distributes the metabolic load between bacteria and yeast for improved synthesis efficacy. Additionally, co-culturing the organisms exploits their individual strengths: Bacteria tend to synthesize smaller molecules more efficiently while yeast is more effective in assembling smaller molecules into a larger product. One of the drawbacks, however, of using a bacteria and yeast co-culture is that the bacteria outcompete the yeast over time. Thus, our solution is to employ 3D-printed cultures of E. coli and S. cerevisiae in adjacent but separate hydrogels that are submerged in media ("parallel cultures"), allowing pathway intermediates to pass between the bacteria and yeast hydrogels for synthesis of rosmarinic acid while preventing the bacterial culture’s invasion of yeast and preserving biological containment. Our first home-built, two-channel 3D bioprinter will provide a model for utilizing microbe-laden hydrogel systems for inexpensive and customizable synthesis.
The idea to apply a co-culture of yeast and bacteria to biosynthesis was inspired by research conducted on hydrogel co-cultures of microbes. Previous work developed a microbe-laden hydrogel platform to spatially organize individual microbial populations into hydrogel constructs for the production of both small molecules and active peptides [11]. This strategy enables a portable, reusable, and on-demand capacity for small molecule and pharmaceutical production from a variety of microorganisms. Our project uses the microbe-laden hydrogel technique to synthesize endangered plant compounds, and improves upon this system by designing an accompanying two-channel printer to facilitate the compartmentalization of yeast and bacteria. 3D printing of microbes has been studied extensively in recent years, tackling the coupling of living organisms in bioink designs [12]. However, previous research has focused on printing a single strain of bacteria or organism. Our approach to 3D printing addresses bioink designs for two organisms. We aim to maximize the production of our compound by constructing our hydrogels to complement both organisms and diffusion of our compounds. To quantify cell viability and migration within the gels, we tested the microbe-laden hydrogels with fluorescent-expressing bacteria and yeast.
Developing production of plant-derived compounds, specifically rosmarinic acid, has been tested in bacteria and yeast individually. A biosynthetic pathway of rosmarinic acid using microbial co-cultures composed of multiple metabolically engineered E. coli strains inspired our engineered production pathway for rosmarinic acid [13]. Taking considerations from their work, we mapped out our chemical pathways in a similar fashion. However, we engineered the E. coli and S. cerevisiae to carry out different jobs by expressing separate enzyme genes along the production pathway. We first transformed our E. coli strain, BL21, with plasmids, pSB3T5 and pSB1C3, containing the genes that express L-DHL, HpaB, and HpaC. The intermediate produced by the E. coli then allowed our engineered S. cerevisiae strain, S288c, to continue the synthesis. The engineered S. cerevisiae strain was transformed with plasmids pRS426, pRS403, and pRS405 to express TAL, TyrB, HpaB, HpaC, 4Cl, and RAS. Utilizing S. cerevisiae to complete the pathway was derived from previous research concluding that it more effectively synthesizes large molecules compared to E. coli [14]. We created our own custom plasmids to incorporate enzyme genes into bacteria and yeast, and will integrate genes into yeast chromosomes to improve production.
Overall, our project has aspired to improve upon many techniques of facilitating cooperation between microbes and aiding the extraction process to provide an efficient, accessible synthesis solution to any chemical or therapeutic in short supply.