Progress 11/15/23 to 11/14/24
Outputs
Target Audience:We have engaged with dairy organizations and creameries, which are interested in our work on turning acid whey and dairy industry byproducts into natural food and feed ingredients. Our next step is to keep expanding our network within the dairy industry and the industries using carotenoids in the formulation of their own products. The purpose of this is to share our research outcomes with a broader audience and ultimately explore commercialization opportunities and transfer our technology to the marketplace, aiming to maximize its environmental and societal impact. Through our engagement with diverse stakeholders, we can ensure that our bioprocess addresses real-world challenges, promoting a sustainable and circular bioeconomy. Such collaborations may be pursued with any animal feed-producing company seeking to use cost-competitive natural ingredients in the formulation of their feed products. For instance, our products, belonging to the carotenoid family, have several applications as food pigments and animal feed additives. On an animal farm level, a carotenoid-rich feed additive has the potential to reduce the overall cost of the feed and increase farmers' profitability. Moreover, our biomanufacturing method is more sustainable than synthetic alternatives and aligns with the consumer demand for natural and environmentally friendly products, which is likely to provide a competitive edge in the market. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?We continue fostering a learning environment by working with, educating, and mentoring an extremely diverse group of people allowing them to actively participate in the research process. Undergraduate students, visiting PhD students, and postdoctoral associates have been introduced to the project and they areengaging in research and activities designed to promote intellectual growth and improve their ability to design and conduct innovative research. This is achieved through a comprehensive mentoring program that includes regular meetings, participation in weekly seminars, networking opportunities, teamwork, and close collaboration with other research group members. How have the results been disseminated to communities of interest? We have disseminated the results of our research to various stakeholders through discussions and presentations given to executives of Dairy Management Inc., other ag-tech companies, and suppliers of food colorants for the food and beverage industries. Engaging with them offered us networking opportunities and increased exposure that could eventually lead to collaborations and commercialization opportunities. Moreover, we gained valuable insights into current market preferences and trends in the food and beverage industry that will help us modify our bioprocess to better align with the industry's demands. In addition, an ag-tech company with operations and interests in animal feed production offered to provide us with access to fermentation facilities where we can run larger-scale bioreactor experiments to validate our strains and optimize the parameters of our bioprocess. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period we plan to: [1] Continue improving the engineered strains through metabolic engineering to improve productivity and enhance robustness. Specifically, building upon our previous results, we aim to identify the optimal combination of gene copy numbers in carotenoid biosynthetic pathways and target genes related to oxidative stress defense pathways and cell morphology.[2] Validate the newly constructed strains in larger-scale bioreactor runs at industrially relevant conditions using raw AW or preferably blends with other concentrated streams of whey. Before running the larger-scale bioreactor, we first conduct a fermentation process parameter optimization using high-throughput microbioreactors and 3-L lab-scale glass vessel reactors. [3] Finally, conduct a comprehensive techno-economic analysis based on the data generated during the larger bioreactor runs employing the process simulator SuperPro Designer. Different scenarios including the use of various whey blends as feedstock will be evaluated. We will also perform a sensitivity analysis, which is critical to identify the variables with the highest impact on the feasibility of our bioprocess and better prepare for potential risks. A Life Cycle Assessment assessing the environmental footprint of our process will also be conducted.
Impacts
What was accomplished under these goals?
Our proposed technology aims to engineer yeast cells to render them capable of (a) using the fermentable compounds present in whey, namely, lactose, galactose, and lactic acid, as carbon sources and (b) synthesizing value-added compounds that can serve as food or feed ingredients, such as carotenoids. During the previous reporting period, we demonstrated the microbial production of carotenoids in yeast cells using acid whey as feedstock. First, we identified the optimal biosynthetic genes for synthesizing the carotenoids lycopene, beta-carotene, and astaxanthin and developed a method for their extraction from the cell biomass. During this reporting period, we have improved intracellular carotenoid accumulation by partitioning the carbon flux between isoprenoid and lipid synthesis. Lipid bodies inYarrowia lipolyticacreate hydrophobic pockets that facilitate carotenoid accumulation and storage. By balancing the flux distribution between carotenoid and lipid synthesis, through carbon to nitrogen ratio adjustments we achieved higher productivity and content (amount of product per amount of dry cell weight) levels. Besides raw AW, we also used concentrated AW as feedstock to produce lycopene. The engineered cells were able to utilize the concentrated feedstock; however, the higher initial amount of lactic acid inhibited cell growth leading to longer fermentation times. Overexpression of lactate importers, such as the lactate permease, in combination with the enzymes that are responsible for lactate conversion to pyruvate, led to a lag phase decrease. In addition, we also focused on the production of alpha-carotene from AW. This compound serves not only as provitamin A but also as a precursor to lutein, another industrially significant natural product. Targeting the biosynthetic enzymes to lipid bodies resulted in a 10-fold increase in alpha-carotene concentration. In the case of astaxanthin synthesis, we initially fused the two key enzymes responsible for the conversion of beta-carotene to astaxanthin, namely beta-carotene ketolase and hydroxylase. Our findings demonstrated that this fused enzyme outperformed the separate expression of individual enzymes. Subsequently, we proceeded to assess the efficiency of pathway expression within various compartments. This localization of the biosynthetic pathway within subcellular organelles led to a significant enhancement in the conversion rates. Ultimately, our goal is to manufacture lower-cost microbial animal feed and ingredients. Drawing from our initial Techno-Economic Analyses (TEAs) conducted using industrial glucose as the primary feedstock, it becomes evident that approximately 50-60% of the overall operating expenses are attributed to raw material costs. Based on that, the availability of a feedstock, like acid whey, that carries either negligible or minimal cost for producing ingredients and animal feed would allow us produce carotenoids at more competitive prices, thus capturing a significant share of the target market.
Publications
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Progress 11/15/22 to 11/14/23
Outputs
Target Audience:During the reporting period, we engaged with Dairy Management Inc. (DMI), which is an umbrella organization representing more than 29,000 dairy farmers. DMI is a strategic consultant and resource to businesses and organizations in the food and beverage sector, spanning the full range of uses and applications of dairy products. DMI expressed their keen interest in our work focusing on the bioconversion of acid whey to natural food and feed ingredients and their eagerness to facilitate the development and deployment of such technologies that valorize byproducts of dairy manufacturing operations that may be harmful to the environment and raise sustainability concerns. Specifically, DMI agreed to provide us with contacts for the supply of the quantities of acid whey and other dairy byproducts required to run lab-scale experiments and pilot operations. In addition, as a next step, we aim to identify appropriate and relevant industry meetings through DMI's network to facilitate the information transfer of the research outcomes to the broader dairy industry. Furthermore, our outreach efforts included a customer discovery phase where we interviewed decision-makers from creameries that produce acid whey. The consensus among them was the need to employ an alternative to the prevailing industry practice of transporting acid whey to wastewater treatment facilities. Through this phase, we also learned about other dairy byproducts that pose sustainability issues that we could target through our research. Beyond the dairy industry, we reached out to end users of the products we aim to synthesize using our technology. We gained insights into the industry's needs and potential applications through these interactions. Our products belong to the carotenoid family and have many applications as food colorants, fortificants, and animal feed additives. Notably, we observed a growing interest in natural products, especially in the food and agriculture sectors, mainly due to the growing consumer awareness of the impact of synthetic ingredients on health and the environment. Currently, most carotenoids in the market are synthetic, as their natural counterparts extracted from natural sources or through fermentation processes tend to be expensive. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During the project, we aimed to foster a learning environment by offering training opportunities and mentoring to undergraduate students through MIT's Undergraduate Research Opportunities Program (UROP), allowing them to actively participate in the research process. The purpose of this program was to provide them with practical skills in the field of microbial fermentation and metabolic engineering through their hands-on involvement. Additionally, we have introduced visiting PhD students to the project and offered them an opportunity to contribute to the research project and learn new biotechnological methods. These efforts were complemented by active mentoring by the PI, including the participation of researchers in group seminars and one-on-one sessions with the PI and senior postdocs. How have the results been disseminated to communities of interest?We have disseminated the results of our research to various stakeholders through discussions and presentations given to executives of Dairy Management Inc., other ag-tech companies, and suppliers of food colorants for the food and beverage industries. Engaging with them offered us networking opportunities and increased exposure that could eventually lead to collaborations and commercialization opportunities. Moreover, we gained valuable insights into current market preferences and trends in the food and beverage industry that will help us modify our bioprocess to better align with the industry's demands. In addition, an ag-tech company with operations and interests in animal feed production offered to provide us with access to fermentation facilities where we can run larger-scale bioreactor experiments to validate our strains and optimize the parameters of our bioprocess. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period we plan to: [1] Continue improving the engineered strains through metabolic engineering. We will attempt to localize the enzymes in intracellular regions that are close to their substrates for enhanced catalytic efficiency. We will also investigate fusing specific enzymes to alleviate intermediate metabolite accumulation. For instance, increasing the physical proximity between enzymes could minimize the substrate-enzyme distance and increase reaction rates. [2] Investigate utilization of concentrated acid whey or a combination of acid whey and other types of whey generated during the manufacturing of cheese. We will also explore the addition of supplemental nitrogen sources in AW to regulate intracellular hydrophobicity. [3] Conduct larger-scale bioreactor runs at industrially relevant conditions. We will assess our engineered strains using raw AW or blends with other types of whey and optimize fermentation process parameters. Initially, we will employ 3-10 L lab-scale bioreactors, and upon satisfactory results, we will use larger volume fermenters (600 L). [4] Perform techno-economic analysis based on large-scale data. Process modeling will be carried out using a process simulator (SuperPro Designer), and the economic assessment will include an estimation of the equipment and operating costs, followed by profitability assessment
Impacts
What was accomplished under these goals?
The acidic and salty taste, high ash content, and low protein levels of acid whey (AW) have limited its utility in the food industry. Disposing of AW in water streams is problematic due to its high organic content (mainly lactose) while using it as fertilizer raises odor and runoff concerns. Enriching animal feed with AW has faced challenges as animals reduce food intake. Finding cost-effective and eco-friendly AW reuse solutions has been elusive to date. Our project has the double goal of bioremediating dairy industry waste and, at the same time, creating value by synthesizing natural food and feed ingredients. To that end, during the reporting period, we demonstrated the microbial production of carotenoids in yeast cells using acid whey as feedstock. First, we identified the optimal biosynthetic genes for synthesizing the carotenoids lycopene, beta-carotene, and astaxanthin and developed a method for their extraction from the cell biomass. These naturally occurring molecules are used in various markets, from food supplements to cosmetics, but currently must be harvested directly from a plant source, which is expensive. By introducing the carotenoid biosynthetic genes to our host microorganism, together with the genes responsible for acid whey consumption, we achieved the construction of yeast strains that can completely consume all the objectionable compounds found in acid whey and produce these valuable carotenoids. In addition, we further improved the biosynthesis of lycopene and beta-carotene through metabolic engineering. As increased amounts of lycopene inhibited beta-carotene synthesis, we employed a method that diverted metabolic flux away from the inhibitory metabolite. As a result, we constructed a strain capable of selectively producing high levels of beta-carotene from acid whey without any substrate inhibition effect. For lycopene production, we used a highly efficient combination of enzymes that resulted in high titers of lycopene with undetectable levels of β-carotene. All the carbon resources present in acid whey were fully consumed in both cases, significantly minimizing its organic load. Based on the market needs, we can quickly shift the production from one carotenoid to another by using the appropriate engineered strain. This technology can impact the dairy industry and companies that employ carotenoids in formulating their products. The dairy industry can utilize an otherwise wasted byproduct and create additional revenue. At the same time, food or animal feed companies can benefit from the supply of low-cost natural ingredients to use as colorants or additives. For instance, carotenoid-containing animal feed can improve animal health, regulate the color of egg yolks or fish meat, or adjust broiler skin to market demands. Overall, our technology is designed to be readily adopted by the industry to create new revenue streams for economic growth and contribute to a robust domestic supply of food and feed ingredients, strengthening the US position in the global bioeconomy landscape.
Publications
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