Progress 09/01/23 to 08/31/24

Outputs
Target Audience:The target audiences we focused on during the reporting period included companies that use taurine to make products: scientists and manufacturers who formulate and make the products; business leaders who need to meet consumer demand for healthier and sustainable ingredients; and supply chain buyers who require reliability and fast turnaround. Our target audience also included scientists who are interested in developing new technologies to improve the production yield of sulfur-based compounds. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We have provided one-on-one mentorship to a recent PhD graduate and have provided opportunities to build his skill set with novel approaches within metabolic engineering and cloning. How have the results been disseminated to communities of interest?Our major outreach has been to potential customers. We have submitted a PCT patent application and a provisional application. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we plan to complete the second half of our stated goals, namely: Objective 3: To increase taurine 5-fold by using peptide scaffolding in the taurine biosynthetic and precursor pathways We plan to proceed with the scaffolding which is the physical combination of enzymes to increase flux through targeted molecules. We plan to use peptide linkers as scaffolds as described in the proposal. Objective 4: To develop an effective DSP protocol for bio-based taurine that achieves 80% yield and 97% purity We will develop an efficient and cost-effective downstream-processing(DSP) protocol. To do so, we will first run a 10-L batch-fed fermentation using our best-performing line to obtain material to develop the DSP protocol. We plan to follow the protocol that we have outlined in the proposal.

Impacts
What was accomplished under these goals? Problemyour project addresses:Taurine is an essential nutrient for humans and animals found in hundreds of everyday products including nutraceuticals, food, beverages, infant formula, personal care products, and pet food. Taurine is needed for cardiovascular, skeletal muscle, vision, and nervous system function. Itis naturally occurring in meat and other animal products, but it is not found in plants, fungi, or most single-cell organisms. Today, nearly all the 160,000 tons of taurine aremanufactured in China from petrochemicals, whichare harmful to people, animals, and the environment. The process uses a carcinogenic and hazardous air pollutant as an intermediate and has toxic byproducts. In addition, the process has high carbon emissions. Bio-based processes can reduce carbon emissions by up to 80-90%. The overall project goal is to produce a cost-competitive, non-petroleum source of taurine by applying a biology-based solution that uses clean and renewable resources.Bio-based taurine meets consumers' demand for healthy and sustainable ingredients and has societal benefits on the environment and human health. Who or what will be most immediately helped by your work, and how. (1) Manufacturers of products that use taurine in their products will benefit from having non-petroleum-based taurine. This will address the demands of their customers, the consumers, who want sustainably produced ingredients. Manufacturers will also benefit from lower carbon emissions from a non-petroleum-based taurine. Impending regulations require companies to report on the greenhouse gas emissions of their production process and supply chain. (2) Humans and animals will benefit from cleaner air and water from a bio-based process of taurine production. (3) The environment will benefit from a bio-based process of taurine production, which has lower carbon emissions and no toxic by-products or carcinogenic intermediates to pollute the air and water. Following are the results from the Phase II experiments to date: Objective 1: To increase taurine levels 5-fold by overexpressing genes in the serine precursor biosynthetic pathway Major activities: The taurine biosynthetic pathway requires nitrogen, carbon, and sulfur. For Nataur's taurine pathway, carbon is supplied from components in the serine pathway. To increase taurine levels, we modified the serine pathway to funnel more serine to the taurine pathway and thereby increase the carbon flow. Standard molecular biology protocols were used, and the cells were grown in a basic media under standard growing conditions. Data collected: Samples were collected at 48 hours, and the taurine content was determined by HPLC analysis. Cells were pelleted out of the solution, and the taurine content of the liquid (supernatant) was assessed. Summary statistic and discussion of results: The median level of taurine produced by the microbes with the serine-modified pathway was 4.8 times higher than the level of taurine produced by the microbes without the modified serine pathway. Providing more carbon to the taurine pathway significantly increased taurine production. Objective 2: To increase taurine levels 3-fold by deregulating genes in the sulfur pathway Major activities:Sulfur-based precursors for taurine biosynthesis come from the sulfur uptake and reduction pathways. To increase activity in the sulfur uptake and reduction pathways, we deregulated the expression of key genes in the pathways by performing a series of knock-ins. Standard molecular biology protocols were used, and the cells were grown in a basic media under standard growing conditions. Data collected: Samples were collected at 48 hours, and the taurine content was determined by HPLC analysis. Summary statistic and discussion of results: The median level of taurine produced by microbes within the deregulated sulfur pathways was 20% higher than the level of taurine produced by microbes without a deregulated sulfur pathway. Higher taurine levels were achieved when the sulfur pathway was more active. Describe the key outcomes realized.Agoalof this Phase II project is to further increase taurine production by optimizing carbon and sulfur utilization in the taurine biosynthetic pathway. In the first year of the grant period, we focused on increasing the availability of carbon skeletons and sulfur and as such increased taurine production nearly 6-fold.

Publications