In this work we will enhance the in vivo residence time of probiotic yeast.

This fundamental research is motivated by three major global challenges that directly involve the transformation of gas molecules: carbon dioxide (CO2) capture for greenhouse gas mitigation, CO2 conversion to fuels and chemicals, and nitrogen (N2) gas conversion to biologically available ammonia to meet growing fertilizer demand. The research focuses on creating and investigating multi-functional interfaces that durably immobilize enzymes near their gaseous substrates while simultaneously delivering essential chemical and electrical reducing equivalents and removing reaction products to achieve maximum catalytic rates. Biocatalytic systems to be explored are: conversion of CO2 to bicarbonate catalyzed by carbonic anhydrase, reduction of CO2 to formate catalyzed by formate dehydrogenase, and reduction of N2 to ammonia catalyzed by nitrogenase. We envision that minimization of reaction barriers near immobilized biocatalyst interfaces involving gas molecule conversions will lead to transformative innovations that help overcome global sustainability challenges.

Carotenoids are naturally occurring red, orange, and yellow pigments found mainly in plants. They may function as vitamin A precursors, have important antioxidant, immunological or mediate metabolic functions. Absorption of carotenoids takes place in the small intestine, but the overall absorption efficiency from whole foods is low (~5-20%) at the level of small intestine and the remaining unabsorbed carotenoids move into the colon. Here, carotenoids can be further released and metabolized within the colon or within colon epithelial cells. The overall objective of this proposal is to determine the impact of carotenoids on obesity-associated gut dysfunction and gut microbiota. Our central hypothesis is that carotenoids escaping the absorption in the small intestine can be transformed by the gut microbiota in the colon, and these undigested carotenoids are available to the gut epithelium and protect against obesity-associated gut dysfunction. In aim 1, we will elucidate the molecular mechanisms of action of major dietary carotenoids on inflammatory transcription factors and gut barrier integrity in colon epithelial cells. In aim 2, we will determine the impact of carotenoids on obesity-associated gut dysfunction and metabolic endotoxemia, and intestinal barrier function in a diet-induced obesity animal model. In aim 3, we will characterize diet-induced changes in the gut microbiota resulting from a carotenoid-rich diet, utilizing metagenomics and metabolomics. We believe that the results of this study will increase demand of carotenoid rich plants. Finally, the findings of this proposal will be translated to commercial use of plants rich in carotenoids.

We propose to engineer yeast to deliver intracellular molecules to the mammalian gut

PreMiEr������������������s microbiome engineering framework will enable the development of a wide range of transformative technologies that solve societal challenges at the interface of health and the environment. However, the dissemination of these same technologies is not without risk as it relies on the responsible development and societal acceptance of microbiome engineering approaches. Thus, in this research core, we will consider the ethical, societal, and policy implications of PreMiEr������������������s evolving microbiome engineering discoveries. There have been national calls for cross-disciplinary and integrated work to better understand the social implications of microbiome science and engineering [1]. In parallel, there is increasing awareness that challenges at the nexus of human- and natural-world coupled systems cannot be solved by technology alone. Through the research and deliberative engagement approaches described below, PreMiEr research will embrace the concept of responsible research and innovation and its elements of anticipation, deliberation, reflexivity, and responsiveness [6]. Particular areas of inquiry will be on social equity of microbiome engineering, ownership and privacy of microbiome data and information, and ethical implications including informed consent, consumer and patient autonomy, beneficence, non-malfeasance, and procedural justice. Core B will also work with the natural scientists and engineers in other thrusts and cores to help identify and address policy and societal questions associated with risk governance and analysis, oversight of microbiome engineering, and equitable distribution of risks and benefits.

The daunting challenges associated with capturing and recycling microplastic particles are that common processes for particle capture such as filtration are cost-prohibitive and that energy-efficient approaches to depolymerization do not yet exist. We propose innovative solutions to both challenges. Guided by multi-scale computational and machine-learning methodologies (Hall and You), our team aims to develop innovative strategies that use active colloidal systems that recognize and remove microplastic particles from water (Velev and Abbott), and then subsequently transform the captured plastics into valuable chemical feed stocks via microbial systems optimized by directed evolution (Crook). Through this approach, our team will advance E3P goals of enabling processes that eliminate plastic waste (Thrust 3) and permit depolymerization of polymers (Thrust 2). Our approach fuses a series of ambitious efforts, including (i) the computational design of peptides that will be optimized to recognize specific polymeric surfaces, (ii) the design of next-generation ����������������active��������������� particle microcleaners that have fibrillar coronas and move autonomously in aqueous environments, thus enabling efficient capture of microplastics, and (iii) data-driven optimization of efficient microbial biocatalysts for depolymerization, achieved by bioprospecting of natural plastic degraders, metabolic engineering of rapidly-growing marine bacteria, and high-throughput directed evolution. To intensify this integrated capture and depolymerization process, we will also develop a new class of liquid crystal-based sensors (integrating the designer peptides mentioned above) that will monitor process conditions and increase its throughput using modern artificial intelligence and deep learning algorithms. This comprehensive approach will build the basis of a circular plastics economy.

These studies will enable us to determine specific formulations of nCAP-PGMs that will promote growth in lowland and upland switchgrass varieties.

More than a third of crop yields are currently lost due to abiotic and biotic stressors such as drought, pests, and disease. These stressors are expected to worsen in a warmer, drier future, resulting in crop yields further declining ~25%; however, breeding is only expected to rescue 7-15% of that loss [1]. The plant microbiome is a new avenue of plant management that may help fill this gap. All plants have fungi living inside their leaves (����������������foliar fungal endophytes���������������). This is an ancient and intimate relationship in which the fungi affect plant physiology, biotic and abiotic stress tolerance, and productivity. For example, some foliar fungi prevent or delay onset of major yield-limiting diseases caused by pathogens such as Fusarium head blight [2]. Foliar endophytes also reduce plant water loss by up to half and delay wilting by several weeks [3, 4]. Endophyte effects on plants occur via diverse genes and metabolites, including genes involved in stress responses and plant defense [5]. Genes and metabolites also predict how interactions in fungal consortia affect host stress responses, which is important for developing field inoculations [6]. Because newly emergent leaves lack fungi, endophytes are also an attractive target for manipulation (particularly compared to soils, where competition with the existing microbial community inhibits microbial additives). We propose to address the role of endophytic ����������������mycobiomes��������������� in stress tolerance of five North Carolina food, fiber, and fuel crops (corn, hemp, soybean, switchgrass, wheat), and to develop tools that can push this field beyond its current limits. Our major objectives (Fig. 1) are to: 1. Identify key microbiome scales to optimally manage endophytes 2. Determine fungal mechanisms via greenhouse tests, modeling, and genetic engineering 3. Build tools for field detection of endophytes 4. Understand the regulatory environment and engage diverse stakeholders Results of these objectives will allow us to make significant progress in both understanding the basic biology of plant-fungal interactions and managing those interactions in real-world settings. Our extension efforts will also bring these ideas to the broader community. Finally, we will also be well positioned to pursue several future research endeavors supported by federal granting agencies.

We will engineer probiotic microbes to produce vitamins.

Beneficial microorganisms can greatly improve crop plant performance, motivating their use as seed inoculants. However, exogenous microbes are often outcompeted in the field, which limits their utility and reveals fundamental gaps in our understanding of root colonization. The rationale for this proposal is that the genes that are most important for colonizing the root are largely unknown. Recent work using comparative genomics and knockout mutants has provided the first insights into the genes involved in root colonization. However, a complete picture of colonization must include how to enhance it, an understanding that is not currently available. Since root colonization is multifaceted (encompassing interactions with other microbes, the host, and abiotic soil conditions) it is expected that a diversity of microbial and plant genes will impact root colonization. To parse this complexity, we will use a functional metagenomics approach to screen for genes conferring improved colonization maize under three different nutrient conditions. We will then probe the functions encoded by colonization-enhancing genes through carbon utilization plates, lectin arrays, and co-culture experiments. This work will therefore advance our understanding of root colonization and our ability to identify microbes that exert beneficial effects for prolonged periods. In prior work by our group, functional metagenomic screens have proven successful to identify genes that enhance the colonization of probiotics in the mammalian gut. We hypothesize that the rhizosphere microbiota contains genes that, when expressed in root-associated microbes, enhance their persistence on roots.