$1B).Producers in the food-ingredient sector have expressed interest in using sustainable production processes to build and maintain customer loyalty. Consumers are becoming more health-conscious and making buying decisions based on products that contain healthy ingredients. Tau has been linked with overall human wellness and longevity. The inclusion of sustainable Tau in alternative protein products would increase the nutritional value of food, promote wellness, and as such reduce societal health-related costs. Marketing of the sustainable ingredient would increase consumer awareness of the health benefits of Tau, which we expect to translate into increased consumption of products with Tau as an ingredient and improved wellness.' />
Source: NATAUR LLC submitted to
MICROBIAL FERMENTATION OF TAURINE FOR THE SUSTAINABLE PRODUCTION OF A NUTRITIONAL INGREDIENT
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
SMALL BUSINESS GRANT
Reporting Frequency
Annual
Accession No.
1028400
Grant No.
2022-33530-36945
Cumulative Award Amt.
$174,999.00
Proposal No.
2022-00771
Multistate No.
(N/A)
Project Start Date
Jul 1, 2022
Project End Date
Feb 28, 2023
Grant Year
2022
Program Code
[8.5]- Food Science & Nutrition
Recipient Organization
NATAUR LLC
6204 BLACKBURN LN
BALTIMORE,MD 212122515
Performing Department
(N/A)
Non Technical Summary
Taurine (Tau) is an essential nutrient for humans and animals; it is needed for cardiovascular, skeletal muscle, vision, and nervous system function. Tau is naturally occurring in meat and other animal products, but it is not found in plants, fungi, or most single-cell organisms. As we shift to more plant-based food and feed diets, Tau must be added as an ingredient or taken as a supplement to maintain healthy Tau levels. The vast majority of Tau is manufactured in China from petrochemical-based processes that use carcinogenic and hazardous compounds. Without an alternative, consumers will be dependent on the use of a petroleum-based ingredient, international trade agreements, supply chain disruptions, and the environment will be increasingly polluted by hazardous waste by-products and the negative effects of global transport. A real need exists for a non-petroleum-based source of Tau that could be used as an ingredient in products made with alternative proteins.The overall project goal is to apply a biology-based solution that uses clean and renewable resources to produce a cost-competitive source of Tau. Our approach is to use recombinant microbes, grown in a fermentor with a simple sugar and natural compounds, to serve as factories. This approach is similar to that which has been used to make hundreds of safe and environmentally friendly products used in everyday life. A cost-effective, sustainable production of Tau by microbial fermentation would make it a superior choice for Tau as an ingredient in our customers' (and partners' customers') products.Successful completion of the project and commercialization would result in a non-petroleum source of Tau that could have societal benefits on the environment and human health. Currently, Tau is not produced in the US. The vast majority of the Tau production currently occurs overseas using a petroleum-based chemical process. This scenario presents several global problems, specifically, environmental degradation and supply chain disruptions. Businesses that utilize Tau in their products understand these problems. Petroleum-based Tau is currently being purchased from overseas and used as an ingredient in many products including infant formula, pet food, animal feed, energy & sports drinks, and personal-care products. The successful completion of this project would pave the way for the establishment of a sustainable Tau production industry in the US. The growth of the industry will provide jobs in and around the communities of the production facilities and throughout the supply chain. An indirect economic benefit of a US-based Tau industry is the infusion of funds back into the US economy. An even greater benefit to the US economy could be realized if the entire Tau production business were relocated from Asia to the US (expected annual sales >$1B).Producers in the food-ingredient sector have expressed interest in using sustainable production processes to build and maintain customer loyalty. Consumers are becoming more health-conscious and making buying decisions based on products that contain healthy ingredients. Tau has been linked with overall human wellness and longevity. The inclusion of sustainable Tau in alternative protein products would increase the nutritional value of food, promote wellness, and as such reduce societal health-related costs. Marketing of the sustainable ingredient would increase consumer awareness of the health benefits of Tau, which we expect to translate into increased consumption of products with Tau as an ingredient and improved wellness.
Animal Health Component
67%
Research Effort Categories
Basic
(N/A)
Applied
67%
Developmental
33%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7014010104080%
7014010100020%
Knowledge Area
701 - Nutrient Composition of Food;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1040 - Molecular biology; 1000 - Biochemistry and biophysics;
Goals / Objectives
The overall project goal is to produce a sustainable, non-petroleum-based, cost-competitive source of taurine (Tau) that could be added as a nutritional ingredient in alternative protein products. This Phase I project aims to establish a minimum viable process for the commercial production of Tau using microbial fermentation. The technical objectives of this projectare (1) to achieve Tau levels of 0.4 g per liter of media (g/l) in a laboratory shaker by genetically modifying the Tau biosynthetic pathway and the microbial strain; (2) to achieve Tau levels of 1.5g/l in a laboratory shaker by optimizing the formulation of the growth media containing the optimized Tau-producing microbe; and (3) to achieve Tau levels of 25 g/l with the optimized growth media and Tau-producing microbe in a 10-L fermentor with optimized fermentation conditions.
Project Methods
The plan is to use genetic engineering to introduce a constitutive promoter into the Tau biosynthetic pathway, knock out the Tau degradation pathways, and modulate the global regulator of sulfur metabolism. Media formulation and fermentation parameters will be optimized to further increase Tau production and demonstrate scalability using fermentation.We plan to start with the microbial strain that we engineered to increase levels of Tau precursors and to secrete Tau when transformed with the appropriate Tau biosynthetic pathway. These cells will be used as the basis of further genetic engineering in the proposed project. We plan to replace the inducible promoter with a proprietary constitutive promoter on the plasmid of the Tau biosynthetic pathway and test its effects on Tau production. The effectiveness of the constitutive promoter to increase Tau will be tested against that of the inducible promoter. The presence of the promoter/Tau gene construct will be confirmed by PCR using standard molecular biology protocols. Three (3) independent colonies will be grown under standard growth conditions. Tau levels will be determined by HPLC. Descriptive statistics of the Tau values will be calculated, and the promoter associated with the higher median Tau level will be used going forward.We plan to knockout (KO) the Tau degradation enzymes and combinations thereof (total of 7 KO lines) and determine the effect on Tau production. For each cell line, 3 independent colonies will be picked, PCR confirmed, grown under standard conditions, and Tau determined. Descriptive statistics will be calculated on the Tau levels and the cell line with the highest median Tau level will be moved forward to the next step.We plan to modulate the expression of the global sulfur transcription regulator. We will use a Knock-In approach to replace the native promoter of the regulator with an inducible promoter. Three (3) independent colonies will be selected in each of the 2 cell lines. The independent colonies will be PCR confirmed, grown under standard conditions, and induced by the inducer in late-log phase. Tau levels will be determined by HPLC. Descriptive statistics will be calculated on the Tau values, and the cell line that has the highest median Tau level will be used as the Production Strain going forward.The effects of medium composition on biomass and Tau production will be assessed in a high throughput culture format. Glucose, ammonium, sulfate, and phosphate concentrations are expected to have a significant impact on Tau production. A 4-factor, 2-level factorial design will be used to identify the critical factors and define the concentration ranges. An overnight culture of the Production Strain will serve as the inoculum for each of the wells. Samples will be scanned, and OD will be recorded every 30 min over 24 hours. At 24 hours, each of the 48 samples will be analyzed for Tau by HPLC. The Tau level, growth rate, and final biomass yield will be used to perform the statistical analysis. From the resulting model, the predicted optimum concentrations of each component will be verified. Four of the predicted optimum formulations will be prepared and tested for Tau production in duplicate 25 ml shake flasks. A surface-response methodology at 3 or 5 component levels will also be used to design a set of experiments aimed at identifying the optimum medium composition for batch mode production. The formulation will be subject to two rounds of formulation review: the first to define the boundaries of the composition and the second to refine the optimum levels of each component. The results of the second set of experiments are expected to produce a model that will predict the optimum formulation. Four of the prediction formulations will be tested in 25 ml shake-flask cultures.The top two media formulations will be used to cultivate the Production Strain in batch mode fermentations. We will grow the cells in a small scalable fermentor (10L), and production will be monitored. Each fermentation will be performed in duplicate. The growth rate and yield, glucose consumption, and Tau levels will be determined at selected time points during the fermentation. At the end of each fermentation, the cells will be removed and the clarified culture medium reserved for Tau determinations.

Progress 07/01/22 to 02/28/23

Outputs
Target Audience:The target audienceconsists of companies whose customers are interested in wellness orsustainable products and ingredients. The target market sectors include producers of plant-based proteins, human and animal nutrition, and sports drinks. Market reports indicate that42%of surveyed consumersworldwide consider wellness a top priority, and 67%of surveyed consumers make purchasing decisions based onenvironmental friendliness. To maintain competitiveness in the marketplace, business leaders, product developers, scientists, marketing managers, and supply chain managers across various industries need to consider the health benefits and sustainability of their ingredients and processes. Our target audience also encompasses scientists and academics interested in understanding the interactions of carbon, nitrogen, and sulfur within the highly regulated sulfur metabolic pathway in microbes. Additionally, it includesthose who are interested in developing new technologies that integrate the disciplines of molecular biology, synthetic biology, and metabolic engineering to enhance the production yield of other sulfur-based compounds. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project offered training opportunities through one-on-one mentorship of the principal investigator (PI) with thetechnician. The technician received training and developed proficiency in various molecular biology techniques, which included the following:restriction enzyme and PCR gene mapping, PCR overlap gene assembly, DNA fragment purification, ligations, and transformations. How have the results been disseminated to communities of interest?The results have been disseminated through social media (LinkedIn), social interactions at Baltimore regional business networking events, and discussions with the team of our manufacturing and distributing partner. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The Phase I project aimed to increase taurine production to a level of minimum commercial viability. Three objectives were proposed for increasing taurine levels:(1) optimizing the taurine-producing microbial strain through genetic improvements, (2) optimizingthe formulation of the growth media, and (3) growingthe microbes in afermentor. Completion of these three objectives resulted in a 189-fold increase in taurine production, reaching thelevel of minimum commercial viability. Objective 1: To increase taurine levels by optimizing the microbial strainthrough genetic improvements Several experiments were conductedto achieve Objective 1. These experiments helped identify key components that needed modificationin the microbial strain to increasetaurine production,specifically: (1) the promoter (genetic on/off switch), (2) the specific taurine-producing genes, and (3) specific genes in the sulfur pathway. Taurine production increased by 22.1 times as a result ofgenetic improvements in the microbial strain. Objective 2: To increase taurine levels by optimizing the formulation of the growth media Since the composition of the media is crucial for optimal taurine production during fermentation, we aimed to determine the media components that enhance cell growth and taurine production efficiency. We tested the following components at various concentrations: NaH2PO4, K2HPO4, (NH4)2SO4, and glucose in vessels and scales of different sizes. Samples were collected at 28 hours, and the taurine content was determined by HPLC analysis. The optimal media formulation increased taurine production by 20% compared to the basic media. We identified several componentsin the media that maximizetaurine production. The media formulation that yielded the highest level of taurine wasused in the subsequent experiment conducted in a fermentor. Objective 3: To increase taurine levels by switching from a shake flask to a fermentor To increase taurine production, we transitionedfrom a shake flask system to a fermentor. A simple batch-fed experiment was conducted at the 10 L scale. Samples were collected every hour for up to 41 hours. At each time point, cell density was determined using a spectrophotometer,and taurine content was determined through HPLC analysis. Taurine production was7 times higher when the microbes were grown in a fermentor compared to taurine levelsin theshake flask system. IMPACT STATEMENT The overall increase in taurine produced and transported outside the microbes was 189 times higher than the initial amount produced within the microbes at the beginning of the project. The taurine level achieved at the end of the project was sufficiently high to establish acommercially viable product. The success of the Phase I project enabled Nataur to finalize a Manufacturing Agreement with a global bio-manufacturingand distributing company. Additionally, the announcement of the Manufacturing Agreement attracted over a dozen potential customers interested in obtaining samples of our bio-based taurine. The Phase 1 project'sgreatest impact lies in the application ofknowledge regarding taurine production in microbes. Specifically, we gained insights into the biological constraints that limit taurineproduction within the microbial system, as well as the potential modifications to enhance taurine production.Additionally, we identified the compounds in the media that either inhibit or facilitate taurine production.This knowledge will be implemented in our Phase II project, with the ultimate goal of achievingcommercial production of sustainable,bio-basedtaurine. Our primary stakeholders willbenefit from having a sustainable source of taurinefor their products, meeting the demands of their customers.Furthermore, society as a whole cangreatly benefit from the nutrificationof plant-based food with taurine.

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