Source: SAN DIEGO STATE UNIVERSITY submitted to NRP
USING ARTIFICIAL INTELLIGENCE AND ROBUST PROTEIN DESIGN METHODS FOR HIGHLY EFFICIENT PRODUCTION OF MILK PROTEINS VIA BACTERIAL FERMENTATION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1032168
Grant No.
2024-67017-42678
Cumulative Award Amt.
$300,000.00
Proposal No.
2023-10521
Multistate No.
(N/A)
Project Start Date
Jul 15, 2024
Project End Date
Jul 14, 2026
Grant Year
2024
Program Code
[A1364]- Novel Foods and Innovative Manufacturing Technologies
Recipient Organization
SAN DIEGO STATE UNIVERSITY
(N/A)
SAN DIEGO,CA 92182
Performing Department
(N/A)
Non Technical Summary
Global weather patterns have been changing unpredictably as the average planet surface temperature has been steadily increasing over the last 50 to 60 years. Global warming is increasing the intensity of dangerous weather events, such as tornadoes and hurricanes and inducing climate change, which has resulted in more floods and droughts. Our research functions to reduce the greenhouse gas (GHG) emissions associated with dairy farming. Sources of GHG emissions on dairy farms include diesel-powered machinery, methane emissions from cow digestive systems, nitrous oxide emissions from fertilized soils, as well as the significant energy used for mass production of fertilizers. We believe it is crucial that everything possible should be done in order to reduce our collective carbon footprint.We are not proposing the elimination of dairy farming, which has historically provided high-quality nutrition to generations of Americans. What we are proposing is to engineer an alternative method of producing certain milk proteins, which can be utilized to produce different types of cheeses and other nutritious food products. Our method has a lower carbon footprint as it entails the use of precision bacterial fermentation to produce milk casein proteins. For much of human history, we have been perfecting the use of microorganism fermentation, which utilizes beneficial bacteria and yeast to produce many foods such as cheeses, yogurts, beer, wine, and many types of breads. Our research will result in enhanced bacterial fermentation by combining artificial intelligence with robust biotechnology to produce nutritious milk proteins more sustainably and with a lower carbon footprint.
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
50250101000100%
Knowledge Area
502 - New and Improved Food Products;

Subject Of Investigation
5010 - Food;

Field Of Science
1000 - Biochemistry and biophysics;
Goals / Objectives
Overarching Research Goal: The overarching goal of this research project is to engineer a rapid, effective, and sustainable protein expression/purification system used to produce nutritious milk proteins (caseins) via precision bacterial fermentation. The system will enable casein protein production with a lower carbon footprint relative to producing milk proteins via traditional dairy farming.Immediate Research Goal: The immediate research goal is to engineer increased, yet transient solubility/stability into the αS1-casein milk protein. To achieve this, small stable proteins, referred to as transient tertiary structure (TTS) tags, will be expressed as N-terminal and C-terminal fusions with the αS1-casein milk protein. The goal of this form of protein engineering is to impart greater solubility on the αS1-casein and thus render this protein more straightforward to produce, isolate, and purify. Following high-level expression, the TTS tags will be catalytically removed from the casein proteins. We will continue to use the AI program AlphaFold, in combination with rational design, to generate fusion variants of αS1-casein using designed Gβ1 TTS tags. Relying on this approach, we have already generated preliminary data indicating that this research approach is highly feasible.Objective 1:Produce αS1-casein fusions with increased solubility and higher levels of bacterial expression.Thus far we have engineered one variant with a wild-type Gβ1 TTS tag expressed as a fusion to the N-terminus of αS1-casein. This variant exhibited favorable solubility properties relative to the wild-type protein and therefore we intend to express WT Gβ1 TTS tags as fusions to both the N- and C-terminus of αS1-casein. Increases in solubility of the associated inclusion bodies for these variants will be analyzed using standard solubility tests that include but are not limited to variable centrifugation/gel electrophoresis, circular dichroism, and NMR.Milestone: Generate 2 to 3 αS1-casein-TTS tag fusion variants that are straightforward to extract from inclusion bodies and have a good level of solubility in the context of the fused protein construct.Timeline: Protein expression and testing, Months 1 - 4.Objective 2: Continue to use the AI program AlphaFold to engineer variants of the Gβ1 TTS tags with the goal of increasing the solubility and expression levels of αS1-casein. Rational design, based on human intuition and molecular visualization (Pymol), results in a series of candidate mutant proteins that are rapidly 'filtered' and critically assessed using the program AlphaFold. The resulting mutant Gβ1 TTS tags, which provide greater transient tertiary interactions, will be expressed as fusions with αS1-casein.Milestone: Generate 2 to 3 engineered αS1-casein-TTS tag fusions using the described AlphaFold-based methods.Timeline: AlphaFold design: Months 1-2. Gene design, protein expression, and assessment: Months 2 - 6.Objective 3: Design additional TTS tags based on the Gβ1 variants that have been previously engineered to dimerize upon binding metals. These Gβ1 variants dimerize upon binding divalent transition metals and are referred to as Metal-controlled dimer (MCD) proteins. The MCDs afford unprecedented chemical and temporal control over dimerization. The MCDs will be expressed as N- and C-terminal fusions of αS1-casein for enhanced solubilization from inclusion bodies during purification. Upon expression, the divalent transition metal Zn2+ will be added to bacterial cultures to drive MCD dimerization and reduce strong intermolecular interactions that cause insoluble inclusion bodies. During inclusion body workup, the metal chelating chemical EDTA will be added to the inclusion body solution to dissociate the dimers and enhance αS1-casein fusion folding and solubility.Milestone: Express MCD TTS tags as N- and C-terminus fusions with αS1-casein and test the addition of different metal concentrations to enhance inclusion body purification.Timeline: Gene design, protein expression, and assessment: Months 5 - 8.Objective 4: Cleave the fusion proteins produced from Objectives 1 thru 3with the TEV protease and induce precipitationin a manner similar to cheese curds. The precipitated curds will be formulated into different types of cheese products by varying ingredient components such as water, fat, salt, lactic acid, stabilizers, and flavors. The rheological properties (elastic modulus, viscous modulus, complex modulus, etc.), textural properties (firmness, stickiness, meltability, stretchability, etc.), color and browning, microscopic structures, and volatile profiles of the cheese products will be characterized and compared to dairy cow produced αS1-casein.Milestone: Cleave successful variants from Objectives 1 thru 3 and analyze the textural and structural properties of the produced material.Timeline: Cleavage and cheese curd quality assessment, Months 7 - 12.Objective 5: The fusion proteins produced from Objectives 1 thru 3 will be tested for phosphorylation by the casein Kinase I and 2 enzymes displayed on the surface of E. coli using the Bacterial Surface Display (BSD) system. Catalysis will be tested in the context of the Gβ1-αS1-casein fusions both before and after cleavage with the TEV protease. Functional and nutritional properties of cheese made with the phosphorylated casein will be evaluated as described in Objective 4.Milestone: Subclone the gene for casein Kinase I and 2 enzymes into the BSD system. Phosphorylate αS1-casein and test for degree and pattern of phosphorylation. Compare degree of phosphorylation to verify similarity to the same protein from standard milk. Test functional properties of the phosphorylated casein.Timeline: Subcloning of the gene for casein Kinase I enzyme into the BSD system, test degree and pattern of phosphorylation, test casein functional properties. Months 12 - 20.Objective 6:This objectiveis a proof-of-concept study that aims to develop a novel expression system for robust surface expression (and possibly secretion) of target proteins in a methane-consuming host (the 20ZR methanotrophic bacterial strain) and demonstrate its applicability for production of Gβ1-αS1-casein variants. We will focus on surface (S)-layer proteins as a target fusion protein for surface level expression of Gβ1-αS1-casein variants.Milestone: Subclone the gene(s) for select Gβ1-αS1-casein variants into 20ZR engineered plasmids so they are expressed as S-layer protein fusions, or possibly as secretion proteins.Timeline: Subcloning of the gene for Gβ1-αS1-casein variants, workup and assessment, Months 16 - 24.
Project Methods
The research methods employed to sustainably produce casein milk proteins through precise bacterial fermentation will follow a systematic approach based on standard scientific methodology, with unique aspects tailored to the specific objectives of this project:Experimental Design and Protein Engineering: The project will begin with the design of casein fusion proteins engineered for increased solubility/stability. This will involve the engineering of transient tertiary structure (TTS) tags designed to increase the solubility and bacterial expression of milk casein proteins. Unique to this project is the precision required for effective or protein engineering to ensure high level, consistent, and sustainable production of the targeted casein milk proteins.Protein Production Optimization: Once the casein fusion proteins have been successfully engineered, protein production will be optimized to maximize yield and purity. This will involve controlling factors such as solubility, temperature, pH, nutrient availability, and oxygen levels.Protein Purification and Characterization: Following protein expression, the produced proteins will be subjected to purification to remove impurities and isolate the target casein milk proteins. Various chromatographic techniques will be tested and employed for purification. The purified proteins will be characterized using analytical methods such as SDS-PAGE, circular dichroism, solubility tests, NMR, and other spectroscopic techniques used to confirm their increased solubility and assess their characteristics relative to milk proteins obtained from dairy cows. Assessment will entail characterizing rheological properties (elastic modulus, viscous modulus, complex modulus, etc.), textural properties (firmness, stickiness, meltability, stretchability, etc.), color and browning, microscopic structures, and volatile profiles of the cheese products.Data Analysis and Interpretation: The results obtained following protein production and characterization will be analyzed statistically to identify optimal conditions for increased production and high purity. Deviations from expected outcomes will be carefully examined and interpreted to refine experimental procedures and enhance protein yield and quality.Knowledge Dissemination and Outreach: Efforts will be made to disseminate the findings of the research to both scientific and non-scientific audiences. This will include publishing research articles in relevant journals, presenting findings at conferences and seminars, and potentially engaging with industry stakeholders.Impact Evaluation: The scientific impact of this project will be assessed through metrics such as publications, presentations, collaborations, and potential adoption of the developed methods by the scientific community. Additionally, the project's potential to contribute to sustainable food production practices and reduce reliance on traditional dairy farming will be evaluated.By following this systematic approach, the project aims to advance scientific knowledge in the field of protein design, while also contributing to the development of sustainable and innovative solutions for casein milk protein production.The scientific methods employed to achieve the research goals of this project include standard biochemical and molecular biology methods and technologies. These include, but are not limited to the following:Recombinant DNA technologiesBiochemical techniques and methodologies including buffer preparation, pH titration, bacterial media preparation, etc.Autoclaving of bacterial media and agar plates for antibiotic selection.Buffer preparations for optimal bacterial growth and protein production.PCR based sub-cloning, DNA amplification, and mutagenesis methods.Bacterial transformation with recombinant DNA plasmids.IPTG induced bacterial protein expression.T7-based bacterial protein expression and purification.Bacterial cell lysis via sonication or freeze/thaw methodsPAGE and agar gel electrophoresisHPLC and FPLC column chromatographyStructure-based Rational Protein DesignComputational chemistry methods and programs including the molecular visualization program Pymol, the AI-based program AlphaFold, and Molecular Dynamics calculations run using the programs Amber and GROMACS.

Progress 07/15/24 to 07/14/25

Outputs
Target Audience:Target Audience The target audience for this reporting period primarily consisted of undergraduate and graduate students, educators, academic researchers, and industry professionals engaged in sustainable protein production and synthetic biology. These individuals and groups are considered target audiences due to their direct involvement in and associated benefits from the research, education, and outreach activities associated with this project. 1. Graduate Students (Primary Research Trainees): The core research project is the PhD dissertation project for a second-year PhD student working in the Love Laboratory. Two MS students (one in their first year, one in their second year) are also directly working on this project. These graduate students were the principal trainees and contributors to experimental design, data collection, and dissemination efforts. They received extensive training in protein biochemistry, molecular biology, protein design, microbial fermentation, and computational modeling (including the use of the AI program AlphaFold 3). This directly aligns with the project's scientific goals and the NIFA mission of workforce development. 2. Undergraduate Research Students: During the Fall 2024 and Spring 2025 semesters, approximately 5-7 undergraduate students participated in hands-on laboratory research as part of the project team. These students engaged in experimental work under the supervision of the graduate students and Prof. John Love, the PI. This experience contributed to the students STEM education through mentored research and experiential learning, which are key educational goals of NIFA-funded projects. 3. University-Level Course Integration - "Sustainable Biochemistry": The research content from this project was directly integrated into the Spring 2024 and 2025 offering of an upper-division undergraduate course titled Sustainable Biochemistry, which enrolled approximately 45 students. Course modules included case studies on microbial production of milk proteins, protein design using AlphaFold 3, and the environmental advantages of precision fermentation. Students were introduced to current challenges and innovations in sustainable food protein production, making this course an important dissemination pathway for both educational and societal impact. 4. Scientific Community (Presentations and Conferences): Project outcomes were shared with the broader scientific community through formal presentations: A seminar was presented at the 2025 Spring American Chemical Society (ACS) National Meeting in San Diego titled "Utilizing the AI protein design tool AlphaFold to enhance precision bacterial fermentation of bovine αs1-casein". The audience included academic researchers, students, and professionals in biotechnology and food science. A similar seminar was presented at the 2025 SDSU EDUFAN Symposium, an interdisciplinary event attended by undergraduate and graduate students, postdoctoral researchers, faculty, and sustainability-focused stakeholders. This event particularly emphasized outreach to university-level audiences with an interest in sustainable food systems. 5. Graduate Student Dissemination Activities: Sierra Murrell, PhD student, presented a poster titled "Using the AI program AlphaFold to engineer transient tertiary structure (TTS) tags to enhance αs1-casein solubility" at the 2025 ACS Spring Meeting. Mia Bartolovich, MS student, presented her poster "Bacterial Surface Expression of the Kinases FAM20C and CK2 for Milk Protein Phosphorylation" at both the 2025 SDSU Student Research Symposium and the 2025 SDSU EDUFAN Symposium. These venues reached diverse student and faculty audiences across multiple disciplines including biology, biochemistry, chemical engineering, and sustainability science. 6. Industry Stakeholders - Fermented Protein Producers: Another important audience includes biotechnology companies actively developing nutritious food proteins through microbial fermentation. These include companies like Perfect Day, Formo, Better Dairy, Bon Vivant, ImaginDairy (focusing on milk proteins), and Onego Bio and the EVERY Company (focused on egg proteins). These organizations represent a rapidly growing segment of the food biotechnology industry and may benefit from the project's findings, particularly with respect to protein solubility, phosphorylation, and expression system optimization. Dissemination to this group has occurred informally through networking at professional meetings and will continue via planned publications and possible partnerships. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This USDA NIFA-funded research project has provided extensive training and professional development opportunities to different groups of students, which include undergraduate, MS, and PhD students. Graduate Students: A second-year PhD student, Sierra Murrell, has been fully engaged in this project as her dissertation research. She has led key efforts in gene cloning, protein expression optimization, and resolubilization of alpha-S1-casein fusion proteins. She also led the design and execution of solubility assays and worked closely with undergraduates in multiple mentoring roles. A first-year MS student and a second-year MS student were directly involved in constructing expression vectors, optimizing inclusion body processing, and testing solubilization conditions. The second-year MS student, Mia Bartolovich, independently prepared and presented her research at two institutional conferences, gaining vital experience in scientific communication and project ownership. Undergraduate Students: Between five and seven undergraduates participated in this research during the Fall 2024 and Spring 2025 semesters. These students were actively involved in wet-lab experimentation, including bacterial culture, protein extraction, SDS-PAGE analysis, and CD spectropolarimetry. Their training was delivered through guided mentorship from the PI and graduate students. The project introduced them to AI-assisted protein design, molecular biology, and sustainability-focused biotechnology. Course Integration: The PI integrated project material into a Spring 2025 undergraduate course, "Sustainable Biochemistry," reaching approximately 50 students. The class explored the role of recombinant protein production in agriculture and introduced AlphaFold as a tool in synthetic biology. This provided students with contextual understanding and technical literacy in the emerging field of bacterial fermentation of important food proteins. Mentorship and Cross-Level Training: The project has fostered layered mentorship: the PI mentors all graduate students, who in turn mentor undergraduates. This tiered structure cultivates leadership skills and technical proficiency across all levels. Conference Participation: Students and the PI presented research at the 2025 American Chemical Society national meeting, the SDSU EDUFAN Symposium, and the SDSU Student Research Symposium. These experiences offered opportunities to interact with industry representatives, faculty from other institutions, and peers, strengthening professional networks and science communication skills. Together, these experiences have provided an immersive training environment. Students have gained hands-on laboratory skills, bioinformatics expertise, presentation experience, and exposure to the broader scientific and food science communities, which are key competencies for careers in food tech, biotechnology, and academia. How have the results been disseminated to communities of interest?The outcomes of this project have been shared with academic, industry, and student audiences through multiple channels of dissemination during the reporting period. Scientific Conferences: A seminar presentation titled "Utilizing the AI protein design tool AlphaFold to enhance precision bacterial fermentation of bovine alpha-S1-casein" was delivered by the PI at the 2025 Spring American Chemical Society (ACS) National Meeting in San Diego, California. This national forum reached a broad audience of scientists, graduate students, industry representatives, and educators in biochemistry and synthetic biology. The same seminar was delivered at the 2025 SDSU EDUFAN Symposium, an interdisciplinary event that brought together stakeholders in sustainable food, agriculture, and energy systems. Attendees included undergraduate and graduate students, postdoctoral scholars, and faculty with an interest in sustainable biotechnology. Poster Presentations: PhD student Sierra Murrell presented a poster titled "Using the AI program AlphaFold to engineer transient tertiary structure (TTS) tags to enhance alpha-S1-casein solubility" at the 2025 ACS National Meeting. This presentation fostered one-on-one dialogue with researchers from across the country. MS student Mia Bartolovich presented her poster "Bacterial Surface Expression of the Kinases FAM20C and CK2 for Milk Protein Phosphorylation" at both the 2025 SDSU Student Research Symposium and the SDSU EDUFAN Symposium. Academic Integration: The research was embedded into a Spring 2025 undergraduate course, "Sustainable Biochemistry," attended by 40 students. The project served as a real-world case study on protein production via bacterial fermentation, with discussions on protein structure, microbial expression systems, and the application of AI in protein design in biochemistry. This instructional strategy broadened awareness and enhanced learning outcomes. Near-Term Plans for Broader Dissemination: We plan to prepare manuscripts for publication in high-quality, peer-reviewed research journals. These will provide the means of communicating our findings to global scientific communities and industry professionals. Industry Awareness: Given the growing relevance of precision fermentation in the food-tech sector, this work also indirectly targets companies pursuing similar goals--such as Perfect Day, Formo, Better Dairy, Bon Vivant, and ImaginDairy. While direct industry outreach has not occurred yet, our planned publications and future presentations aim to reach these stakeholders. What do you plan to do during the next reporting period to accomplish the goals?The next reporting period will focus on advancing the remaining objectives of this research project, building on the successful developments of Year 1. Objective 4 (TEV Cleavage and Curd Formation): We will cleave the solubilized alpha-S1-casein fusion proteins using the TEV protease. Cleavage will be confirmed by SDS-PAGE, size exclusion chromatography coupled to multi-on the light scattering (SEC MALS), and mass spectrometry. Following successful cleavage, we will initiate cheese curd formation by adjusting pH and ionic strength, mimicking cheese-production processes. Functional testing of curds will entail profiling the textural, rheological, and flavor aspects of the produced milk protein. As described, this will be conducted in collaboration with colleagues in food science and engineering. Objective 5 (Kinase-Assisted Phosphorylation): We will initiate phosphorylation assays using the surface-expressed FAM20C and CK2 kinases. Reactions will be optimized for buffer conditions, temperature, and ATP concentration. Circular dichroism (CD) spectroscopy will be used to monitor structural changes in alpha-S1-casein as a proxy for phosphorylation. Phosphorylation efficiency will be compared to native milk-derived casein, providing validation of our recombinant approach. Objective 6 (Methanotroph-Based Expression System): We will begin cloning select alpha-S1-casein fusion variants into plasmids suitable for expression in the 20ZR methanotrophic bacterial strain. Initial transformation and protein expression experiments will commence late in the next reporting period. Manuscript Preparation and Dissemination: We plan to prepare and submit one or more peer-reviewed journal articles detailing the advancements in TTS tag engineering, AlphaFold 3 modeling, our unique kinase expression platform, and the physical chemical properties of the produce proteins. We will continue conference dissemination and anticipate submitting abstracts to the next ACS national meeting and relevant biotechnology symposia. Student Training and Mentorship: Graduate and undergraduate students will continue refining laboratory techniques, data analysis, and presentation skills. New undergraduates will be recruited to join the project, ensuring continuity of training and building team capacity. These next steps are designed to complete unfinished objectives, deepen impact, and prepare for broader applications of this research in economically viable protein biomanufacturing.

Impacts
What was accomplished under these goals? The overarching aim of this research is to engineer a precision bacterial fermentation platform to produce the milk protein alpha-S1-casein. This will potentially provide significantly increased economic benefits by enabling high-yield, scalable microbial production of milk proteins. During this reporting period, major progress was made on five of the six core objectives outlined in the proposal. Objective 1: We successfully cloned and expressed wild-type alpha-S1-casein in E. coli, and, not unexpectedly, the expression levels were low. A significant advance was achieved by fusing the wild-type Gβ1 tag to the N-terminus of alpha-S1-casein, which markedly improved expression. This validates our hypothesis that TTS (Transient Tertiary Solubility) tags can enhance solubility and expression of alpha-S1-casein. Gel electrophoresis confirmed an increased yield, which demonstrates progress toward scalable production. Objective 2: We advanced our use of AlphaFold 3, the most recent version of the AI-driven protein structure prediction tool. While we have not yet used it to redesign TTS tags (due to success in Objective 3), improved features of AlphaFold 3 enabled first time modeling of alpha-S1-casein in monomeric, dimeric, and trimeric forms, both phosphorylated and unphosphorylated. This has deepened our understanding of how phosphorylation alters casein secondary structure, particularly alpha-helical content. This exciting finding will enable us to monitor alpha-S1-casein phosphorylation using circular dichroism. Objective 3: We created two novel chimeric alpha-S1-casein constructs by separately fusing the constitutive dimer (M2) and a metal-controlled dimer (MCD-C1) to the N- and C-termini of alpha-S1-casein. These variants, termed M2-alphaS1-M2 and MC1-alphaS1-MC1, exhibited significantly increased expression. Following sonication and centrifugation, the proteins were confirmed to pellet into the insoluble fraction. Extensive optimization of resolubilization conditions (buried salt concentrations, pH, chaotropic chemicals, detergents) raised soluble yield from ~1% to ~50%. This is a major milestone. Objective 4: While we have not yet initiated TEV protease cleavage or cheese curd formation, this work will soon commence upon confirming phosphorylation (Objective 5). Cleavage efficiency and functional cheese production assays are planned for the next phase. Objective 5: We have successfully cloned the two kinases, FAM20C and CK2, into the bacterial surface display system previously developed in the Love Laboratory. This marks a critical advancement that will enable us to begin phosphorylation assays on the expressed alpha-S1-casein variants. Phosphorylation is central to the physical-chemical properties and nutritional function of milk proteins, and these assays will be critical for downstream objectives. Objective 6: Not yet initiated, as planned. This methane-oxidizing bacterial platform will be explored in Year 2 once expression and phosphorylation systems are optimized. Impact and Outcomes: Our approach has demonstrated the feasibility of precision microbial casein production, with validated significant increases in expression, increases in solubility, and the ability to model phosphorylation affects. These results provide a tangible step toward sustainable production of not only alpha-S1-casein but also other milk proteins and milk and other nutritionally important proteins from other species. This platform could help meet global protein demands as the human population continues to expand.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2025 Citation: Presentation given by Prof. John Love at the Annual American Chemicals Society (ACS) meeting held in San Diego, CA on March 25, 2025 PAPER ID: 4192991 PAPER TITLE: Utilizing the AI protein design tool AlphaFold to enhance precision bacterial fermentation of bovine alpha-s1-casein SESSION: Extraction, Recombinant Production, and Function of Proteins of Food Safety and Food Manufacturing Importance PRESENTATION FORMAT: Oral - In-person DAY & TIME OF PRESENTATION: Tuesday, March 25, 2025 from 3:40 PM ORGANIZING DIVISIONS/COMMITTEES: AGFD: Division of Agricultural and Food Chemistry Authors: John Love, Sierra Murrell, Mia Bartolovich, Changqi Liu, Jing Zhao 1. Department of Chemistry and Biochemistry, San Diego State University, San Diego, CA 92182 2. School of Exercise and Nutritional Sciences, San Diego State University, San Diego, CA 92182 Abstract: The primary focus of this research is to develop an effective milk protein (casein) production system through precision bacterial fermentation. The highly nutritious casein proteins, produced in mammalian mammary glands, are relatively unstructured and exist as suspended colloids in milk. This biophysical feature presents challenges for bacterial production of casein proteins, yet also opportunities. We are exploiting the relatively insoluble nature of the unphosphorylated milk protein, alpha-s1-casein, to simplify purification. Our strategy combines chemical intuition with molecular visualization and the AI-based program AlphaFold. Our design approach entails fusing small, soluble proteins to the N- and C-termini of alpha-s1-casein, which function as transient tertiary-structure tags (TTS). AlphaFold is being used to design the TTS tags such that they form weak associations with alpha-s1-casein, thus imparting increased structural stability on the entire chimeric protein construct. The TTS tags function to impart transient solubility on the casein proteins, which renders them slightly more soluble, and reduces the complexity of the purification steps needed to produce pure milk protein. Following preliminary purification, the semi-soluble fused construct will be tested for native-like phosphorylation using the kinases 20FamC and CK2 (casein kinase 2). These kinases will be produced on the surface of E. coli using a Bacterial Surface Display (BSD) system. Following phosphorylation, the TTS tags will be cleaved from alpha-s1-casein using the site-specific TEV protease. After cleavage, the solubility tags will remain in solution and alpha-s1-casein will be induced to precipitate, mimicking casein curd formation in cheese production. Purified alpha-s1-casein will then be tested for structural, functional, and nutritional properties and for its potential use in cheeses and other food products.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2025 Citation: Presentation given by Dr. John Love at the Annual SDSU EDUFAN Symposium; meeting held at SDSU on April 18, 2025 PRESENTATION TITLE: Utilizing the AI protein design tool AlphaFold to enhance precision bacterial fermentation of bovine alpha-s1-casein PRESENTATION FORMAT: Oral - In-person DAY & TIME OF PRESENTATION: Tuesday, April 18, 2025 from 3:00 PM Authors: John Love, Sierra Murrell, Mia Bartolovich, Changqi Liu, Jing Zhao Abstract: The primary focus of this research is to develop an effective milk protein (casein) production system through precision bacterial fermentation. The highly nutritious casein proteins, produced in mammalian mammary glands, are relatively unstructured and exist as suspended colloids in milk. This biophysical feature presents challenges for bacterial production of casein proteins, yet also opportunities. We are exploiting the relatively insoluble nature of the unphosphorylated milk protein, alpha-s1-casein, to simplify purification. Our strategy combines chemical intuition with molecular visualization and the AI-based program AlphaFold. Our design approach entails fusing small, soluble proteins to the N- and C-termini of alpha-s1-casein, which function as transient tertiary-structure tags (TTS). AlphaFold is being used to design the TTS tags such that they form weak associations with alpha-s1-casein, thus imparting increased structural stability on the entire chimeric protein construct. The TTS tags function to impart transient solubility on the casein proteins, which renders them slightly more soluble, and reduces the complexity of the purification steps needed to produce pure milk protein. Following preliminary purification steps, the semi-soluble fused construct will be tested for native-like phosphorylation using the kinases 20FamC and CK2 (casein kinase 2). These kinases will be produced on the surface of E. coli using a Bacterial Surface Display (BSD) system. Following phosphorylation, the TTS tags will be cleaved from alpha-s1-casein using the site-specific TEV protease. After cleavage, the solubility tags will remain in solution and alpha-s1-casein will be induced to precipitate, mimicking casein curd formation in cheese production. Purified alpha-s1-casein will then be tested for structural, functional, and nutritional properties and for its potential use in cheeses and other food products.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2025 Citation: Poster presentation given by PhD student Sierra Murrell at the Annual American Chemicals Society (ACS) meeting held in San Diego, CA on March 25, 2025 POSTER TITLE: Using the AI program AlphaFold to engineer transient tertiary structure (TTS) tags to enhance alpha-S1-casein solubility SESSION: Extraction, Recombinant Production, and Function of Proteins of Food Safety and Food Manufacturing Importance PRESENTATION FORMAT: Poster - In-person DAY & TIME OF PRESENTATION: Monday March 24, 2025 ORGANIZING DIVISIONS/COMMITTEES: AGFD: Division of Agricultural and Food Chemistry Authors: Sierra Murrell, Mia Bartolovich, Changqi Liu, Jing Zhao, John Love Abstract: The primary aim of this research is to produce the nutritious proteins found in milk (alpha-S1-casein) using precision bacterial fermentation. The production of feedstock agricultural products (e.g., soy, corn, and grains) for milk production requires significant amounts of water. For example, approximately 120 gallons of water are required to produce four ounces of cheese using traditional methods. We believe that the production of the alpha-S1-casein milk protein via bacterial fermentation is potentially more economical with a reduced requirement for water. When casein proteins are expressed in E. coli, they tend to aggregate into inclusion bodies due to the lack of structure and high expression rates driven by strong transcriptional promoters. A negative aspect of this is exemplified by the irreversible aggregation of egg white proteins in boiled eggs. If the aggregation of an expressed protein is not extensive, then it is possible to re-solubilize the proteins from looser aggregates. There is a spectrum of inclusion body properties that range from completely insoluble aggregates to loosely packed gels that are straightforward to re-solubilize and purify. The more folded an expressed protein is, the less prone it is to form insoluble inclusion bodies. This is precisely why we are using the AI program, AlphaFold, to design transient tertiary structure (TTS) tags that are expressed as fusions to the N- and C- termini of alpha-S1-casein. The function of the TTS tags is to impart transient structure on alpha-S1-casein, enabling a looser protein association within inclusion bodies, which will greatly enhance our ability to quickly isolate and purify nutritious milk proteins. Purified alpha-S1-casein will be tested for structural and functional properties and its potential use in cheeses and other food products. Once the process is developed and optimized for cost-effective alpha-S1-casein production, we will apply the same methods for large-scale production of the other caseins: alpha-S1-casein, beta-casein, and kappa-casein.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2025 Citation: Poster presentation given by MS student Mia Bartolovich at the Annual Student Research Symposium held at SDSU on February 28, 2025 and the Annual SDSU EDUFAN Symposium on April 18, 2025 POSTER TITLE: Bacterial Surface Expression of the Kinases FAM20C and CK2 for Milk Protein Phosphorylation PRESENTATION FORMAT: Poster - In-person Authors: Mia Bartolovich, Sierra Murrell, John Love Abstract: To potentially provide a secondary source of nutritious milk proteins, this research focuses on the biosynthesis of phosphorylated milk proteins, specifically alpha-S1-casein, using bacterial fermentation. Caseins are the primary proteins within milk, constituting approximately 80% of total milk proteins, and are inherently insoluble, forming micellar structures essential for milk stability. Phosphorylation is a critical post-translational modification to caseins, and it facilitates the stabilization of calcium phosphate nanoclusters within casein micelles, influencing their structural and functional properties. To replicate native phosphorylation processes that occur in the bovine mammary gland, we are developing a Bacterial Surface Display (BSD) system in E. coli. The BSD system enables the surface expression of kinases FAM20C and CK2, which efficiently phosphorylate serine and threonine residues in the milk protein alpha-S1-casein. A fluorescent mCherry label is incorporated into the BSD construct for visual confirmation of kinase expression on the bacterial surface. To overcome the inherent disorder and solubility challenges of caseins, we utilize the AI program AlphaFold3 to design Transient Tertiary Structure (TTS) tags, which provide temporary structural stabilization during protein production and purification. Once phosphorylated, the TTS tags are cleaved using the TEV protease, yielding functional alpha-S1-casein with properties comparable to its natural milk proteins. Additionally, AlphaFold3 structural predictions suggest that phosphorylation induces alpha-helical formation, indicating a shift from a disordered to a more ordered tertiary structure. This work lays the foundation for a sustainable, recombinant approach to producing phosphorylated milk proteins, offering a lower carbon footprint for the production of highly nutritious milk proteins.