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

Outputs
Target Audience:Xylome research and administrative staff. This is research designed to increase our production of omega-3 fatty acids. We regularly review all data and their implications for production. Changes/Problems:None What opportunities for training and professional development has the project provided?Economic Benefits: Successful development of a yeast-based platform to produce tropical oil substitutes could significantly reduce U.S. reliance on imported palm and coconut oils. Domestic production of these oils using renewable feedstocks will reduce transportation costs while creating new revenue streams for ethanol producers through value-added byproducts. This can potentially increase profitability across the grain industry in the Midwest - particularly as transportation moves from petroleum to electric vehicles. Social and Environmental Benefits: This project offers a compelling solution to deforestation's environmental and social impacts, including biodiversity loss and displacement of indigenous communities caused by tropical oil production. The domestic production of a palm oil substitute on cellulosic residues will also contribute to national food security by reducing pressure on global supply chains. Benefits to Commercial Sector, Federal Government, and Researchers: Commercial Sector: Food manufacturers, cosmetic companies, and biofuel producers will benefit from a stable, sustainable, and cost-effective domestic source of palm oil. This will enhance consumer demand for environmentally responsible products. Federal Government: This research aligns with the USDA's goals of promoting sustainable agricultural practices and advancing bioenergy technologies. It also supports federal initiatives aimed at reducing deforestation and mitigating climate change. Researchers: The research community will benefit from the insights gained into optimizing enzyme production in oleaginous yeast, which could have applications beyond tropical oil substitutes. The findings could inform future research into biofuels, bioplastics, and other high value bioproducts derived from renewable feedstocks. (b) Estimated Total Cost of the Approach Relative to Benefits The total estimated cost of the Phase I effort is justified by significant technical and economic benefits. The investment required to optimizeL. starkeyistrains and scale the production process in Phase II will be offset by reduced reliance on imported oils, increased profitability for ethanol producers, and expanded rural job creation. Furthermore, the cost-effectiveness of using cellulosic residues as feedstocks makes this approach highly competitive compared to conventional tropical oil production. By repurposing agricultural byproducts, we can create a low-cost, high-yield system that maximizes the value of domestic resources while minimizing waste. (c) Policy Issues and Decisions The successful development of a yeast-based platform for tropical oil production could influence several important policy areas: Sustainable Agriculture and Bioenergy: The proposed process will use ethanol stillage and corn stover - which do not compete with other agricultural products. The USDA's bioenergy and sustainability goals will benefit from the advancement of a process that improves the economics of grain ethanol production. This project could lead to policy support for further research and commercialization of yeast-based biofuel and bioproduct technologies. Climate Change and Deforestation Mitigation: Policymakers focused on climate change mitigation will see this project as a valuable tool for reducing the environmental impact of the global food supply chain because the technology will provide an alternative to palm oil and other tropical oils linked to deforestation. Trade and Food Security: With the potential to reduce U.S. dependence on imported tropical oils, this research could affect trade policies and enhance national food security by providing a stable, domestic source of these vital products. In summary, the Phase I effort provides a strong foundation for Phase II by addressing critical technical challenges and demonstrating the feasibility of producing tropical oils using cellulosic feedstocks. This project's economic, environmental, and social benefits make it a high-value opportunity that aligns with federal sustainability and bioenergy goals while addressing key market needs. Successful outcomes from this research can influence policy decisions related to sustainable agriculture, climate change, and trade. Technical Background for Proposed Work The initial step of protein secretion involves the transfer of a nascent or fully synthesized protein across the endoplasmic reticulum (ER) membrane. This process can occur either co-translationally or post-translationally; both use the Sec61 complex. However, the pathways differ based on the signal peptide's characteristics and the involvement of other molecular components. In co-translational translocation, the ribosome and the nascent polypeptide chain are targeted to the ER membrane while protein synthesis is still ongoing. This pathway predominantly involves the signal recognition particle (SRP) and its receptor (SR). With more hydrophobic signal peptides, SRP has a higher affinity, making co-translational translocation the preferred pathway.[1, 2] In post-translational translocation, the protein is fully synthesized in the cytosol before it is translocated across the ER membrane. This pathway involves the Sec61 channel but utilizes different auxiliary components, including the cytosolic chaperones Hsp70 and Hsp40, which keep the newly synthesized protein in an unfolded state suitable for translocation. In yeast, Kar2p and the Sec63 complex are required for post-translational translocation. The SNARE protein Snc1 is particularly important in vesicle trafficking, membrane fusion, and protein secretion, [3] and overexpression of Hac1 can enhance secretion.[4-6] Overexpression of the endoplasmic reticulum chaperone Bip and the disulfide isomerase Pdi1p can enhance protein folding, [7] particularly when expression of the ATPase Pmr1p is altered.[8] Disruption or partial deletions of key components in the Trichoderma reesei secretion pathway have been shown to enhance secretion.[9] Technical Objectives of the Research and Development Effort This research aims to establish the technical feasibility of producing pail oil substitutes using Lipomyces starkeyi by improving enzyme production, secretion, and lipid accumulation on cellulosic feedstocks, such as pretreated corn stover. To achieve this, the following technical objectives and associated research questions must be addressed: Objective 1: Optimize Enzyme Production for Efficient Cellulose Hydrolysis Technical Question: Can we genetically engineerL. starkeyito secrete higher enzyme levels by modifying the secretory pathway? Approach: Leverage genetic engineering techniques to delete or overexpress secretory pathway components and determine the effect on the secretion of cellulases, hemicellulases, and cellobiohydrolases. Enzyme production will be assessed using an enzymatic assay of the fermentation broth. Expected Outcome: Increased production of cellulases, hemicellulases, and cellobiohydrolases. Objective 2: Enhance Digestion of Cellulosic Material by Increased Enzyme Production Technical Question: Does increased cellulosic enzymes using objective one result in higher degradation of Avicel and pre-treated deacetylated corn stover? Approach: The yeast produced in objective one will be grown in defined media, and the supernatant will be isolated and used to digest Avicel or pre-treated deacetylated corn stover. The percent digestion will be calculated by the amount of insoluble fiber left. Expected Outcome: We expect increased digestion of the Avicel or deacetylated corn stover from yeast made in objective 1. How have the results been disseminated to communities of interest?Objective 3: Enhance the Production of Oil from Cellulosic Material Technical Question: Can we increase oil production from pre-treated deacetylated corn stover by fermentation with enhanced secreted Lipomyces strains from Objective 1? Approach: Fermentation of pre-treated deacetylated corn stover by fermentation with enhanced secreted Lipomyces strains from Objective 1 in bioreactors, calculating the percentage of cellulosic sugar converted into oil. Expected Outcome: Enhanced secretion of functional enzymes, leading to more efficient cellulose hydrolysis, better utilization of cellulosic feedstocks, and higher production of oil. Objective 4: Assess the Commercial Potential and Scale-Up Feasibility Technical Question: Can the process developed in Objective 3 use corn stover as a feedstock to compete with palm oil production? Approach: Perform an initial techno-economic analysis (TEA) to evaluate the costs, energy inputs, and environmental impacts of scaling the process from lab to pilot scale. Expected Outcome: A detailed assessment of the process's commercial viability, identifying key parameters and indicators that need improvement for viable commercial production. Work Plan This section outlines the research and development tasks required to achieve the technical objectives and determine the feasibility of producing palm oil substitutes using genetically engineered Lipomyces starkeyi on cellulosic feedstocks. This plan is structured around four technical objectives and focuses on optimizing enzyme production, improving cellulose digestion, enhancing oil production, and assessing commercial potential. The work will be conducted at Xylome's laboratories, leveraging internal resources and external expertise when necessary. Objective 1: Optimize Enzyme Production for Efficient Cellulose Hydrolysis Goal: Modify the secretory pathway ofL. starkeyito increase enzyme secretion (cellulases, hemicellulases, and cellobiohydrolases). Task 1.1: Deletion of the Lipomyces starkeyi PEP1, DSC2, and TUL1 genes A ?lig4 strain of CEL5001, which has been engineered with cellulases and xylanases (PPDA1-TrEGII + PCYC1-TrCBHII + PACT1-TrCBH1 + PACO1-ThXYN2 + PPDE3-TrLPMO) will be created using drug resistance markers and a standard lithium acetate protocol and selected for growth in the presence of an antibiotic. Deletion of lig4 enables targeted knockouts. A PCR-based protocol will confirm the ?lig4 genotype. In the ?lig4 strain, a DNA fragment composed of a 1 kb 5' flanking region of the gene to be disrupted, a selective marker, and 1 kb of the 3' flanking region of the gene to be disrupted will be transformed, and transformants selected by the antibiotic. A PCR-based protocol will do confirmation of the ?pep1 genotype. Task 1.2: Overexpression of LsSNC1, LsHAC1, LsBIP1, LsPDI1, and LsSEC61 A plasmid will be constructed that places either the LsSNC1, LsHAC1, LsBIP1, LsPDI1, or LsSEC61codon-optimized open reading frame under the control of a strong promoter, TDH3, and the ACT1 terminator. This cassette will then be placed in a disruption cassette for POX1 that contains a selective marker. The cassette will be transformed into the ?lig4 deletion strain, CEL5001, to integrate fatty-acyl coenzyme A oxidase, POX1. PCR will confirm its deletion and correct integration of the overexpression cassettes. Task 1.3: Testing strains to increase enzyme production Strains developed in Task 1.1 and Task 1.2 will be grown in minimal media in a 250 ml flask to mid-log phase (OD600 from 10 to 20). Cells will be spun down, and the supernatant will be passed through a 0.2 µm filter. Enzymatic assays, cellulase, hemicellulase, and xylanase levels. The enzyme level will be compared between the parent strain (?lig4 Cel5001 strain) and the deletions or overexpressing genes. The top four strains with enhanced enzyme production will be identified. Expected Output: At least four strains will have an improved enzyme production. Timeline: Months 1-5. Objective 2: Enhance Digestion of Cellulosic Material by Increased Enzyme Production Task 2.1: Cellulose Digestion Assays Using Avicel and Corn Stover: Goal: Evaluate the efficacy of the engineered strains from Objective 1 to break down cellulose. Grow engineered L. starkeyi strains in optimized media identified in Task 1.2. Harvest the culture supernatant and apply it to Avicel and pre-treated deacetylated corn stover as substrates. Measure cellulose degradation gravimetrically (weighing residual insoluble fibers) and by DNS (assaying release of reducing sugars). Expected Output: Quantitative data showing the improved degradation of cellulosic substrates by enhanced enzyme production and identification of the top two strains. Timeline: Months 6. Objective 3: Enhance the Production of Oil from Cellulosic Material Task 3.1: Fermentation of Corn Stover with Engineered Lipomyces starkeyi: Goal: Ferment pre-treated deacetylated corn stover with engineeredL. starkeyistrains to produce oil. Scale-up fermentation in seven-liter bioreactors under optimized conditions for enzyme production and oil accumulation. Monitor the fermentation process by sampling culture broth at regular intervals, measuring cell density (OD600), sugar consumption (HPLC), and oil production (lipid extraction and gravimetric analysis). Expected Output: Data on lipid accumulation and sugar-to-oil conversion rates. Timeline: Months 7-8. Task 3.2: Lipid Extraction and Characterization Goal: Extract and characterize the lipids produced from corn stover. Extract lipids using hexane or chloroform extraction. Analyze the lipid composition viaGC-MSfor fatty acid profiles, focusing on the production of triglycerides like palm oil. Expected Output: Characterization of lipid composition and yields from fermentation. Timeline: Months 7-8. Objective 4: Assess the Commercial Potential and Scale-Up Feasibility Task 4.1: Perform a Techno-Economic Analysis (TEA) Goal: Evaluate the costs and economic feasibility of scaling the process from lab to pilot scale. Collect data on raw material costs (corn stover, enzymes), equipment costs, and operational expenses (labor, energy) based on lab-scale experiments. Use industry-standard tools (e.g., SuperPro Designer) to model the scale-up and perform a cost analysis. Expected Output: A detailed cost estimate for commercial production, including sensitivity analysis to identify key cost drivers. Timeline: Month 8 Project Management and Milestones The project will be managed by the Principal Investigator (PI) and executed by the R&D team at Xylome. Regular meetings will be held to monitor progress, troubleshoot issues, and ensure timely completion of tasks. Milestones are the following: Milestone Timeline Deliverables Complete genetic engineering ofL. starkeyi Months 1-5 Engineered strains with enhanced enzyme secretion Cellulose digestion assays completed Month 6 Data on cellulose degradation Fermentation and lipid production completed Months 7-8 Oil yield data and lipid characterization Techno-economic and life-cycle assessments Month 8 Reports on cost analysis and environmental impact Related Research or Research and Development Significant research and development activities are directly related to the proposed effort of engineering L. starkeyi to produce tropical oil substitutes on cellulosic feedstocks. The Principal Investigator (PI) and the proposing small business concern (SBC), Xylome Corporation, have laid the groundwork for the current project. Key efforts that support this proposal expand upon previous work and provide insight into related commercial innovations follow: Previous and Ongoing R&D by Xylome Corporation Xylome has been at the forefront of developing lipid-producing yeast strains and has conducted several research initiatives that underpin this project. Notably, Xylome has worked on advancingL. starkeyifor commercial use in various applications, including aquaculture, biofuels, food, cosmetics, and pharma. What do you plan to do during the next reporting period to accomplish the goals? USDA-NIFA-SBIR Aquaculture Feed Program (2018 USDA-NIFA-SBIR-GRANT 006428): Xylome previously received USDA SBIR funding for a project titled "Aquaculture Feeds from Grain Ethanol Stillage," which focused on utilizing stillage as a cost-effective feedstock for producing omega-3-rich lipids inL. starkeyi. This research demonstrated thatL. starkeyican be engineered to grow on ethanol byproducts and accumulate lipids, which are then used to produce high-value omega-3 oils. The proposed project builds upon this foundation by shifting the focus from omega-3 production to synthesizing palm oil substitutes. DOE Grant (DE-EE0008497) - Biodiesel and Higher Value Products from Stillage Fiber: In a DOE-funded project, Xylome successfully engineered L. starkeyi to express cellulases (CBHI, CBHII, EGII) and xylanases (XYN2) to convert cellulosic fibers into fermentable sugars which L. starkeyi transformed into lipids. This effort set the stage for further optimization. The current proposal expands endogenous enzyme production by supplementation with commercial cellulases to create a viable process for making tropical oil substitutes. USDA-NIFA-SBIR Polyunsaturated Fatty Acids (2023 USDA-NIFA-SBIR GRANT 13841528): Building on the success of prior efforts, Xylome is currently engaged in research focused on producing polyunsaturated fatty acids (PUFAs) inL. starkeyifor use in aquaculture feeds. This work leverages genetic engineering to introduce elongases and desaturases intoL. starkeyi, boosting its ability to produce long-chain PUFAs. While the focus of the proposed project is on saturated and monounsaturated fats found in tropical oils, this research demonstrates the technical capacity for tailoring lipid biosynthesis pathways inL. starkeyi. How the Proposed Effort Expands on Related Work The proposed project builds directly on these prior R&D activities by taking the following steps: Enhancing Enzyme Production: Previous work focused on demonstrating the ability ofL. starkeyito break down cellulosic materials using heterologous cellulases. The current project seeks to improve the secretion and activity of these enzymes, ensuring more efficient hydrolysis of feedstocks, leading to higher sugar yields for lipid production. Optimizing Lipid Accumulation: Building on lipid accumulation research from past projects, the current effort aims to fine-tune lipid biosynthetic pathways to maximize the production of oils that closely resemble those from tropical sources. Utilizing Cellulosic Feedstocks: While previous projects demonstratedL. starkeyi's growth on stillage, this project will expand the application to a broader range of cellulosic feedstocks, including agricultural residues, thus increasing the technology's feedstock flexibility and commercial potential of the technology. Planned Coordination with Outside Sources To ensure the successful completion of this project, Xylome will coordinate with government and industry partners specializing in cellulosic feedstock, fermentation, and techno-economic analysis. Collaborations with enzyme manufacturers and feedstock suppliers will optimize biological and logistical aspects of production. University and Government Collaborations: Xylome will engage academic and government researchers with enzyme engineering and secretion expertise. These collaborations will provide access to cutting-edge tools and methodologies for enhancing enzyme production and secretion. Industry Partnerships: Xylome has established relationships with ethanol producers and enzyme companies, providing access to industrial-scale stillage and feedstock supply chains. These partnerships will test and scale the technology developed in this project. Commercial Innovations in the Market Tropical oils, such as palm and coconut oil, are produced using environmentally destructive agricultural practices in tropical regions. The global market for these oils is vast, driven by their use in food, cosmetics, and industrial products. However, there is increasing demand for sustainable alternatives due to deforestation and habitat destruction associated with conventional palm oil production. Several companies are developing microbial platforms to produce oils, but most focus on algae-based systems or microbial fermentation of simple sugars. Xylome's approach is differentiated by its emphasis on using low-cost, cellulosic feedstocks--such as ethanol stillage and corn stover- to reduce production costs and environmental impact significantly. Microbial Oil Production: Companies like C16 Biosciences and Yali Bio are developing microbial oil production platforms using yeast or other microbes. However, these companies typically rely on glucose or other simple sugars as feedstocks, which increases production costs. Xylome's use of cellulosic feedstocks provides a competitive advantage by lowering input costs and leveraging byproducts from existing ethanol production facilities. Sustainable Palm Oil Alternatives: The push for sustainable palm oil alternatives has gained momentum, with consumers and industries seeking more eco-friendly options. While synthetic biology approaches to produce palm oil analogs exist, Xylome's use of L. starkeyi and cellulosic feedstocks positions it to meet this demand at lower costs and with fewer environmental consequences. Contribution to Innovation and Commercial Impact The proposed research will lead to a significant innovation by developing a commercially viable process for producing tropical oils from cellulosic materials. If successful, this approach will provide an alternative to environmentally harmful palm oil production, reduce reliance on tropical agriculture, and create new market opportunities for domestic ethanol producers. The ability to utilize low-cost feedstocks, such as ethanol stillage and corn stover, for oil production could also reduce costs, making the technology competitive with traditional palm oil and related products. Conclusion This project builds upon previous research and expands the technical capabilities ofL. starkeyias a platform for producing valuable oils. It addresses a significant market need for sustainable tropical oil alternatives. It has the potential to substantially improve the economics of ethanol production by providing a high-value product from stillage. The proposed effort will deliver environmental and economic benefits by reducing deforestation, expanding domestic bio-based manufacturing, and offering a sustainable, cost-effective alternative to tropical oils. The Market Opportunity The global market for tropical edible oils such as palm oil and coconut oil is vast, with palm oil alone valued at approximately $60 billion in 2023. Palm oil is essential in numerous industries, including food, cosmetics, pharmaceuticals, and biofuels. Xylome's palm oil replacement has a fatty acid composition highly similar to palm oil. It is a colorless and odorless solid at room temperature and a liquid at body temperature. These features make it an essential constituent of many food products, such as margarine, ice cream, and sauces. However, current production methods are linked to severe environmental issues, such as deforestation, habitat destruction, and carbon emissions, prompting a global search for sustainable alternatives. This project aims to address these challenges by producing sustainable tropical oil substitutes through the fermentation ofLipomyces starkeyion cellulosic feedstocks, such as ethanol stillage.

Impacts
What was accomplished under these goals? Responsiveness to USDA SBIR/STTR Program Priorities This work strongly connects to agriculturally related manufacturing technology and feedstock availability that avoid environmentally destructive practices. The project will make tropical palm oil and coconut oil from corn stover by cultivating an engineered lipogenic yeast on cellulosic feedstocks rather than by harvesting drupes as is currently practiced. We have engineered Lipomyces starkeyi XYL403 to synthesize cellulases and xylanases to convert agricultural residues into tropical oils. The work will stimulate technological innovation and profitability of domestic grain ethanol fermentations, expand employment opportunities in the rural Midwest, and increase domestic food security. This application supports NIFA Challenge Areas: in Bioenergy by improving the economics of grain ethanol production and in Water conservation and recovery by disposing of dissolved organics from ethanol manufacture. Identification and Significance of the Problem or Opportunity Tropical palm and coconut oils are in high demand. Almost 50% of all products found in the grocery store contain palm oil or its products. Global demand for these oils by the food, cosmetic, and biofuel industries continues to grow. These oils are essential components of food ingredients, bio-based fuels, and cosmetics. Their increasing use is destroying tropical forests and wildlife. According to the World Wildlife Fund (WWF), the palm oil industry is a leading cause of tropical deforestation, contributing to significant greenhouse gas emissions, habitat loss, and long-term ecosystem disruption. We propose cultivating Lipomyces starkeyi on low-cost, abundant, and renewable cellulosic feedstocks. Xylome's technology will make tropical oils from deacetylated, fiberized corn stover and ethanol stillage. This will provide consumers with an abundant, sustainable, inexpensive source of tropical oils. Background and Rationale This proposal builds on two USDA SBIR Phase I and Phase II grants from the aquaculture program entitled "Aquaculture feeds from grain ethanol stillage" and "Heterologous Synthesis of Polyunsaturated Fatty Acids for Aquaculture." This application also builds on a DOE grant entitled Biodiesel and Higher Value Products from Stillage Fiber, which enabled Xylome to engineer the native L. starkeyi NRRL Y-11557 with cellulases and xylanases. The genetically engineered L. starkeyi has some capacity to convert cellulosic biomass into lipids, but its enzyme production is limited - likely by its secretion pathway. L. starkeyi natively produces glucoamylase, but we have engineered it to overproduce five cellulases, xylanases, and lignin peroxide monooxygenase (LPMO). These additional enzymes probably overwhelm the existing secretion pathway. Excess protein for secretion is known to confound the process. The proposed research focuses on the exogenous application and enhances in-situ secretion of cellulases and hemicellulases. Domestic production of tropical oils will reduce reliance on imports and support the growth of rural bio-economies. Market Needs and End-User Considerations The global market for palm oil is valued at over $60 billion annually, with demand expected to increase due to wide application in the food, cosmetics, and biofuel sectors. However, the environmental and social impacts of the oil harvest drive demand for sustainable alternatives. End-users seek sustainable, ethically sourced oils to meet production needs. Recent EU legislation requiring companies to source oil from 100% deforestation-free land has restricted supply. Companies face heightened scrutiny to meet these stringent environmental standards. Producing oils using cellulolytic L. starkeyi on cellulosic feedstocks offers an environmentally sustainable alternative that aligns with evolving consumer preferences and regulatory pressures. Using stillage and corn stover as feedstocks supports the biofuel industry by creating a sustainable revenue stream for ethanol producers. The production of oils from cellulosic residues reduces the environmental footprint of oil production. These factors align with USDA's goals of promoting sustainable agricultural practices and advancing bioenergy technologies. Creation of Cellulolytic Lipomyces In previous research sponsored by the DOE, Xylome engineered wild-type (Y11557) and hyper-lipogenic (Xyl403) strains of L. starkeyi with five different cellulases and xylanases (Table 1). Table 1. strain information Strain Background Foreign Genes Expressed Y11557 Wild type None XYL403 Hyper-lipogenic None (overexpressed endogenous genes) 5001 Y11557 ChimericCBHI, TrCBHII, TrEGII, ThXYN2, TrCEL61b 5401 XYL403 ChimericCBHI, TrCBHII, TrEGII, ThXYN2, AnAxhA The two cellulolytic strains differed slightly in that Y11557 was transformed with Trichoderma reesei CEL61b (TrCEL61b), which is a lignin peroxide monooxygenase (LPMO), and the Xyl403 strain was transformed with Aspergillus niger arabinoxylanase (AnAxhA). While the wild-type Y11557 and Xyl403 strains do not exhibit cellulolytic or xylanolytic activities, those transformed with cellulases and xylanases do. Role of the Proposed Research The following activities play crucial roles in advancing sustainable production of tropical oils: Optimization of Enzyme Production in L. starkeyi: By increasing the expression and secretion of cellulases and hemicellulases, we will enable more efficient degradation of deacetylated fiberized corn stover. This will result in higher lipid yields and a more economically viable process for producing tropical oil substitutes. Development of a Scalable, Sustainable Platform:This research will create a scalable production platform for generating oils domestically, reducing reliance on imported palm and coconut oils. Meeting End-User Demand for Sustainable Oils:Our research directly responds to market needs for sustainable, ethically sourced oils in the food, cosmetic, and biofuel industries and supports consumer preferences and regulatory requirements. In summary, this research will provide the technological advancements necessary to meet the growing demand for tropical oils in a sustainable and environmentally responsible way that benefits the environment and rural economies while addressing critical market needs. Relationship with Research or Research and Development The proposed Phase I effort will lay the groundwork for larger-scale commercial applications that follow in Phase II. By optimizing enzyme production in L. starkeyi in Phase I, we will address the key technical challenges to achieve efficient conversion of cellulosic feedstocks into lipids. The improvements in enzyme production and biomass conversion rates will enable a more robust, scalable process for tropical oil production in Phase II. (a) Technical, Economic, Social, and Other Benefits Technical Benefits: Phase I will demonstrate that increased cellulase and hemicellulase production in L. starkeyi can produce higher lipid yields when grown on cellulosic feedstocks. Success in Phase I will lead to a refined technical approach that we will scale in Phase II, optimizing enzyme expression and feedstock utilization for maximum lipid production.

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