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At least 397 records · Page 22

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DOE OSTI2025

Taxogenomic analysis of Pichia senei sp. nov. and new insights into hybridization events in the Pichia cactophila species complex

Three strains of a novel yeast species were isolated from necrotic cactus tissues of Cereus saddianus and Micranthocereus dolichospermaticus and from phytotelmata of Bromelia karatas. DNA sequence analysis of the Internal Transcribed Spacer (ITS) region and D1/D2 domains of the large subunit ribosomal RNA, along with whole genome phylogenomic analysis, showed that this yeast is most closely related to Pichia insulana, Pichia cactophila, and Pichia inconspicua. The new species differs by 10–13 nucleotide substitutions from these species in D1/D2 sequences and exhibits <90% genome-wide average nucleotide identity to them. The name Pichia senei sp. nov. is proposed for the novel species, which is homothallic and produces asci with one to four hat-shaped ascospores. The holotype is CBS 16311 (MycoBank MB 858723). Taxogenomic analyses of the P. cactophila species complex, including P. senei, provide new insights about the hybridizations events that shaped this group. Pichia insulana and P. inconspicua are identified as the parental lineages that originated P. cactophila, and P. senei also appears closely related to one of the progenitors of P. inconspicua. We assess phylogeny, heterozygosity, and ploidy to explore the processes shaping diversity, showing how genomic data support yeast species delimitation and reveal complex hybridization.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Attraction and Longevity of 2- and 3-Component Food Cone Lures for the Caribbean Fruit Fly, Anastrepha suspensa (Diptera: Tephritidae)

Abstract The Caribbean fruit fly, Anastrepha suspensa (Loew) (Diptera: Tephritidae), is a quarantine pest of Citrus spp. and a production pest of guava and other specialty fruits in Florida. Effective monitoring lures and traps are needed for early pest detection and timely initiation of control measures. As part of a continued effort to identify attractive synthetic lures for the Caribbean fruit fly, we conducted field tests in Homestead, Florida to compare the efficacy and longevity of commercial 2- and 3-component cone lures (2C [ammonium acetate and putrescine], 3C [ammonium acetate, putrescine, and trimethylamine]), the current standards used by regulatory agencies, versus the traditional liquid protein bait consisting of hydrolyzed torula yeast and borax as a positive control. Additional lures were also field-aged and periodically brought into the laboratory to quantify residual chemical contents. Traps baited with the torula yeast-borax mixture captured the highest mean number of A. suspensa, and traps baited with the commercial 2C lures captured more flies than the 3C lures. Traps baited with torula yeast-borax also captured the highest number of nontarget Diptera. Captures with all three treatments were significantly biased toward females. Attractiveness of the 2C lure began to drop after 6–8 wk, and the 3C lure after 5–6 wk. Overall, these data suggest that the 2C cone lure is more attractive to A. suspensa than the 3C cone lure under field conditions in south Florida, and that the 2C lures are attractive for up to 8 wk.

Entomology↗

DOE OSTI2022

Functional Divergence in a Multi-gene Family Is a Key Evolutionary Innovation for Anaerobic Growth in Saccharomyces cerevisiae

The amplification and diversification of genes into large multi-gene families often mark key evolutionary innovations, but this process often creates genetic redundancy that hinders functional investigations. When the model budding yeast Saccharomyces cerevisiae transitions to anaerobic growth conditions, the cell massively induces the expression of seven serine/threonine-rich anaerobically-induced cell wall mannoproteins (anCWMPs): TIP1, TIR1, TIR2, TIR3, TIR4, DAN1, and DAN4. Here, we show that these genes likely derive evolutionarily from a single ancestral anCWMP locus, which was duplicated and translocated to new genomic contexts several times both prior to and following the budding yeast whole genome duplication (WGD) event. Based on synteny and their phylogeny, we separate the anCWMPs into four gene subfamilies. To resolve prior inconclusive genetic investigations of these genes, we constructed a set of combinatorial deletion mutants to determine their contributions toward anaerobic growth in S. cerevisiae. We found that two genes, TIR1 and TIR3, were together necessary and sufficient for the anCWMP contribution to anaerobic growth. Overexpressing either gene alone was insufficient for anaerobic growth, implying that they encode non-overlapping functional roles in the cell during anaerobic growth. We infer from the phylogeny of the anCWMP genes that these two important genes derive from an ancient duplication that predates the WGD event, whereas the TIR1 subfamily experienced gene family amplification after the WGD event. Taken together, the genetic and molecular evidence suggests that one key anCWMP gene duplication event, several auxiliary gene duplication events, and functional divergence underpin the evolution of anaerobic growth in budding yeasts.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

yihuishen/metabolic_flux_regulation

Metabolic efficiency profoundly influences organismal fitness. Heterotrophs, from yeast to mammals, derive usable energy primarily through glycolysis and respiration. While respiration is more energy-efficient, some cells favor glycolysis even when oxygen is available (aerobic glycolysis, Warburg effect). A leading explanation is that glycolysis is more efficient in terms of ATP production per unit mass of protein (i.e. faster). Through quantitative flux analysis and proteomics, we find however that mitochondrial respiration is actually more proteome-efficient than aerobic glycolysis. This is shown across yeasts, T cells, cancer cells, and tissues and tumors in vivo. Instead of aerobic glycolysis being valuable for fast ATP production, it correlates with high glycolytic protein expression, which is valuable for hypoxic growth. Aerobic glycolytic yeasts do not excel at aerobic growth, but outgrow respiratory cells in oxygen limitation. Thus, aerobic glycolysis emerges from cells maintaining a proteome conducive to both aerobic and hypoxic growth.

Shen, Yihui↗

DOE OSTI2024

yihuishen/T_cell_MFA

Shen, Yihui↗

DOE OSTI2022

raogroupuiuc/lipo11557_growth

Project: Systems analysis of Lipomyces starkeyi during growth on various plant-based sugars Authors: Anshu Deewan*, Jing-Jing Liu*, Sujit Sadashiv Jagtap, Eun Ju Yun, Hanna E Walukiewicz, Yong-Su Jin, Christopher V Rao (* - co-authors) Abstract : Oleaginous yeasts have received significant attention due to their substantial lipids storage capability. The accumulated lipids can be utilized directly or processed into various bioproducts and biofuels. Lipomyces starkeyi is an oleaginous yeast capable of using multiple plant-based sugars, such as glucose, xylose, and cellobiose. It is, however, a relatively unexplored yeast due to limited knowledge about its physiology. In this study, we have evaluated the growth of L. starkeyi on different sugars and performed transcriptomic and metabolomic analyses to understand the underlying mechanisms of sugar metabolism. Principal component analysis showed clear differences resulting from growth on different sugars. We have further reported various metabolic pathways activated during growth on these sugars. We also observed non-specific regulation in L. starkeyi and have updated the gene annotations for the NRRL Y-11557 strain. This analysis provides a foundation for understanding the metabolism of these plant-based sugars and potentially valuable information to guide the metabolic engineering of L. starkeyi to produce bioproducts and biofuels.

Deewan, Anshu↗

DOE OSTI2022

Metabolic engineering of Rhodotorula toruloides IFO0880 improves C16 and C18 fatty alcohol production from synthetic media

Abstract Background The oleaginous, carotenogenic yeast Rhodotorula toruloides has been increasingly explored as a platform organism for the production of terpenoids and fatty acid derivatives. Fatty alcohols, a fatty acid derivative widely used in the production of detergents and surfactants, can be produced microbially with the expression of a heterologous fatty acyl-CoA reductase. Due to its high lipid production, R. toruloides has high potential for fatty alcohol production, and in this study several metabolic engineering approaches were investigated to improve the titer of this product. Results Fatty acyl-CoA reductase from Marinobacter aqueolei was co-expressed with SpCas9 in R. toruloides IFO0880 and a panel of gene overexpressions and Cas9-mediated gene deletions were explored to increase the fatty alcohol production. Two overexpression targets ( ACL1 and ACC1 , improving cytosolic acetyl-CoA and malonyl-CoA production, respectively) and two deletion targets (the acyltransferases DGA1 and LRO1 ) resulted in significant (1.8 to 4.4-fold) increases to the fatty alcohol titer in culture tubes. Combinatorial exploration of these modifications in bioreactor fermentation culminated in a 3.7 g/L fatty alcohol titer in the LRO1 Δ mutant. As LRO1 deletion was not found to be beneficial for fatty alcohol production in other yeasts, a lipidomic comparison of the DGA1 and LRO1 knockout mutants was performed, finding that DGA1 is the primary acyltransferase responsible for triacylglyceride production in R. toruloides , while LRO1 disruption simultaneously improved fatty alcohol production, increased diacylglyceride and triacylglyceride production, and increased glucose consumption. Conclusions The fatty alcohol titer of fatty acyl-CoA reductase-expressing R. toruloides was significantly improved through the deletion of LRO1 , or the deletion of DGA1 combined with overexpression of ACC1 and ACL1 . Disruption of LRO1 surprisingly increased both lipid and fatty alcohol production, creating a possible avenue for future study of the lipid metabolism of this yeast.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2019

Xylose utilization stimulates mitochondrial production of isobutanol and 2-methyl-1-butanol in Saccharomyces cerevisiae

Abstract Background Branched-chain higher alcohols (BCHAs), including isobutanol and 2-methyl-1-butanol, are promising advanced biofuels, superior to ethanol due to their higher energy density and better compatibility with existing gasoline infrastructure. Compartmentalizing the isobutanol biosynthetic pathway in yeast mitochondria is an effective way to produce BCHAs from glucose. However, to improve the sustainability of biofuel production, there is great interest in developing strains and processes to utilize lignocellulosic biomass, including its hemicellulose component, which is mostly composed of the pentose xylose. Results In this work, we rewired the xylose isomerase assimilation and mitochondrial isobutanol production pathways in the budding yeast Saccharomyces cerevisiae . We then increased the flux through these pathways by making gene deletions of BAT1 , ALD6 , and PHO13 , to develop a strain (YZy197) that produces as much as 4 g/L of BCHAs (3.10 ± 0.18 g isobutanol/L and 0.91 ± 0.02 g 2-methyl-1-butanol/L) from xylose. This represents approximately a 28-fold improvement on the highest isobutanol titers obtained from xylose previously reported in yeast and the first report of 2-methyl-1-butanol produced from xylose. The yield of total BCHAs is 57.2 ± 5.2 mg/g xylose, corresponding to ~ 14% of the maximum theoretical yield. Respirometry experiments show that xylose increases mitochondrial activity by as much as 7.3-fold compared to glucose. Conclusions The enhanced levels of mitochondrial BCHA production achieved, even without disrupting ethanol byproduct formation, arise mostly from xylose activation of mitochondrial activity and are correlated with slow rates of sugar consumption.

09 BIOMASS FUELS↗

DOE OSTI2023

Rapid and robust squashed spore/colony PCR of industrially important fungi

Fungi have been utilized for centuries in medical, agricultural, and industrial applications. Development of systems biology techniques has enabled the design and metabolic engineering of these fungi to produce novel fuels, chemicals, and enzymes from renewable feedstocks. Many genetic tools have been developed for manipulating the genome and creating mutants rapidly. However, screening and confirmation of transformants remain an inefficient step within the design, build, test, and learn cycle in many industrial fungi because extracting fungal genomic DNA is laborious, time-consuming, and involves toxic chemicals. In this study we developed a rapid and robust technique called “Squash-PCR” to break open the spores and release fungal genomic DNA as a template for PCR. The efficacy of Squash-PCR was investigated in eleven different filamentous fungal strains. Clean PCR products with high yields were achieved in all tested fungi. Spore age and type of DNA polymerase did not affect the efficiency of Squash-PCR. However, spore concentration was found to be the crucial factor for Squash-PCR in Aspergillus niger, with the dilution of starting material often resulting in higher PCR product yield. We then further evaluated the applicability of the squashing procedure for nine different yeast strains. We found that Squash-PCR can be used to improve the quality and yield of colony PCR in comparison to direct colony PCR in the tested yeast strains. The developed technique will enhance the efficiency of screening transformants and accelerate genetic engineering in filamentous fungi and yeast.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Data for System Analysis of Lipomyces starkeyi During Growth on Various Plant‑Based Sugars

Oleaginous yeasts have received significant attention due to their substantial lipid storage capability. The accumulated lipids can be utilized directly or processed into various bioproducts and biofuels. Lipomyces starkeyi is an oleaginous yeast capable of using multiple plant-based sugars, such as glucose, xylose, and cellobiose. It is, however, a relatively unexplored yeast due to limited knowledge about its physiology. In this study, we have evaluated the growth of L. starkeyi on different sugars and performed transcriptomic and metabolomic analyses to understand the underlying mechanisms of sugar metabolism. Principal component analysis showed clear differences resulting from growth on different sugars. We have further reported various metabolic pathways activated during growth on these sugars. We also observed non-specific regulation in L. starkeyi and have updated the gene annotations for the NRRL Y-11557 strain. This analysis provides a foundation for understanding the metabolism of these plant-based sugars and potentially valuable information to guide the metabolic engineering of L. starkeyi to produce bioproducts and biofuels.

Conversion↗

DOE OSTI2023

Data for Glucose Assimilation Rate Determines the Partition of Flux at Pyruvate Between Lactic Acid and Ethanol in Saccharomyces cerevisiae

Engineered Saccharomyces cerevisiae expressing a lactic acid dehydrogenase can metabolize pyruvate into lactic acid. However, three pyruvate decarboxylase (PDC) isozymes drive most carbon flux toward ethanol rather than lactic acid. Deletion of endogenous PDCs will eliminate ethanol production, but the resulting strain suffers from C2 auxotrophy and struggles to complete a fermentation. Engineered yeast assimilating xylose or cellobiose produce lactic acid rather than ethanol as a major product without the deletion of any PDC genes. We report here that sugar flux, but not sensing, contributes to the partition of flux at the pyruvate branch point in S. cerevisiae expressing the Rhizopus oryzae lactic acid dehydrogenase (LdhA). While the membrane glucose sensors Snf3 and Rgt2 did not play any direct role in the option of predominant product, the sugar assimilation rate was strongly correlated to the partition of flux at pyruvate: fast sugar assimilation favors ethanol production while slow sugar assimilation favors lactic acid. Applying this knowledge, we created an engineered yeast capable of simultaneously converting glucose and xylose into lactic acid, increasing lactic acid production to approximately 17 g L−1 from the 12 g L−1 observed during sequential consumption of sugars. This work elucidates the carbon source-dependent effects on product selection in engineered yeast.

Conversion↗

DOE OSTI2025

Data for "RT-EZ: A Golden Gate Assembly Toolkit for Streamlined Genetic Engineering of Rhodotorula toruloides"

For economic and sustainable biomanufacturing, the oleaginous yeast Rhodotorula toruloides has emerged as a promising platform for producing biofuels, pharmaceuticals, and other valuable chemicals. However, genetic manipulation of R. toruloides has been limited by its high GC content and the lack of a replicating plasmid, necessitating gene integration into the genome of the yeast. To address these challenges, we developed the RT-EZ ( R. toruloides Efficient Zipper) toolkit, a versatile tool based on Golden Gate assembly, designed to streamline R. toruloides engineering with improved efficiency and flexibility. The RT-EZ toolkit simplifies vector construction by incorporating new features such as bidirectional promoters and 2A peptides, color-based screening using RFP, and sequences optimized for both Agrobacterium tumefaciens-mediated transformation (ATMT) and easy linearization, enabling straightforward selection and transformation. Notably, the RT-EZ kit can be used to construct an expression cassette with four different genes in one assembly reaction, significantly improving vector construction speed and efficiency. The utility of the RT-EZ toolkit was demonstrated through the successful synthesis of arachidonic acid in R. toruloides by coexpressing fatty acid elongases and desaturases. This result underscores the potential of the RT-EZ toolkit to advance synthetic biology in R. toruloides , providing a streamlined method for addressing genetic engineering challenges in the yeast.

gene editing↗

DOE OSTI2021

Data for Cloning and Characterization of a Panel of Mitochondrial Targeting Sequences for Compartmentalization Engineering in Saccharomyces cerevisiae

Mitochondrion is generally considered as the most promising subcellular organelle for compartmentalization engineering. Much progress has been made in reconstituting whole metabolic pathways in the mitochondria of yeast to harness the precursor pools (i.e., pyruvate and acetyl-CoA), bypass competing pathways, and minimize transportation limitations. However, only a few mitochondrial targeting sequences (MTSs) have been characterized (i.e., MTS of COX4), limiting the application of compartmentalization engineering for multigene biosynthetic pathways in the mitochondria of yeast. In the present study, based on the mitochondrial proteome, a total of 20 MTSs were cloned and the efficiency of these MTSs in targeting heterologous proteins, including the Escherichia coli FabI and enhanced green fluorescence protein (EGFP) into the mitochondria was evaluated by growth complementation and confocal microscopy. After systematic characterization, six of the well-performed MTSs were chosen for the colocalization of complete biosynthetic pathways into the mitochondria. As proof of concept, the full α-santalene biosynthetic pathway consisting of 10 expression cassettes capable of converting acetyl-coA to α-santalene was compartmentalized into the mitochondria, leading to a 3.7-fold improvement in the production of α-santalene. The newly characterized MTSs should contribute to the expanded metabolic engineering and synthetic biology toolbox for yeast mitochondrial compartmentalization engineering.

Conversion↗

DOE OSTI2022

Data for Metabolic Engineering of Rhodotorula toruloides IFO0880 Improves C16 and C18 Fatty Alcohol Production from Synthetic Media

The oleaginous, carotenogenic yeast Rhodotorula toruloides has been increasingly explored as a platform organism for the production of terpenoids and fatty acid derivatives. Fatty alcohols, a fatty acid derivative widely used in the production of detergents and surfactants, can be produced microbially with the expression of a heterologous fatty acyl-CoA reductase. Due to its high lipid production, R. toruloides has high potential for fatty alcohol production, and in this study several metabolic engineering approaches were investigated to improve the titer of this product. Fatty acyl-CoA reductase from Marinobacter aqueolei was co-expressed with SpCas9 in R. toruloides IFO0880 and a panel of gene overexpressions and Cas9-mediated gene deletions were explored to increase the fatty alcohol production. Two overexpression targets (ACL1 and ACC1, improving cytosolic acetyl-CoA and malonyl-CoA production, respectively) and two deletion targets (the acyltransferases DGA1 and LRO1) resulted in significant (1.8 to 4.4-fold) increases to the fatty alcohol titer in culture tubes. Combinatorial exploration of these modifications in bioreactor fermentation culminated in a 3.7 g/L fatty alcohol titer in the LRO1Δ mutant. As LRO1 deletion was not found to be beneficial for fatty alcohol production in other yeasts, a lipidomic comparison of the DGA1 and LRO1 knockout mutants was performed, finding that DGA1 is the primary acyltransferase responsible for triacylglyceride production in R. toruloides , while LRO1 disruption simultaneously improved fatty alcohol production, increased diacylglyceride and triacylglyceride production, and increased glucose consumption. The fatty alcohol titer of fatty acyl-CoA reductase-expressing R. toruloides was significantly improved through the deletion of LRO1, or the deletion of DGA1 combined with overexpression of ACC1 and ACL1. Disruption of LRO1 surprisingly increased both lipid and fatty alcohol production, creating a possible avenue for future study of the lipid metabolism of this yeast.

Conversion↗

DOE OSTI2026

Data for Mitochondrial ATP Generation is More Proteome Efficient than Glycolysis

Metabolomics↗

DOE OSTI2023

Dissecting the Division of Labor in Microbial Consortia for the Production of Biofuels and Chemicals (Final Technical Report)

The overarching objective of this project is to elucidate the fundamental design rules for microbial division of labor (DOL), the core of ecosystem organization, in the context of artificial yeast—lactic acid bacteria consortia that produce advanced biofuel and chemical from cellulosic biomass. Over the course of four years, we have successfully achieved the objective through three lines of research that combines experiment with mathematical modeling. Specifically, we have shown that compositional and temporal DOL is effective in modulating mixed sugar fermentation by an engineered consortium and uncovered general quantitative criteria for conditions under which microbial DOL outperforms single superbugs for simultaneous utilization of mixed substrates. We also demonstrated, in synthetic consortia of yeast and lactic acid bacteria, that partitioning the labor for substrate breakdown and end-product synthesis can allow an efficient production of 2-butanol that is difficult otherwise for a single yeast strain. Additionally, by systematically probing the stability of an engineered cooperative consortium, we found that the stability of microbial symbiosis is governed by the topological structure of the underlying cellular interactions rather than specific microbial species and that rational modulation of the interactions may facilitate the restoration of collapsed consortia as well as the intervention of target communities. Together, this project has advanced the fundamental knowledge of microbial DOL in terms of its strength in mixed substrate fermentation, production of complex chemicals, and ecological system stability in the context of microbial consortia. It also provides valuable insights into the design, construction and optimization of artificial consortia for the utilization of cellulosic biomass and economic production of biofuel and chemicals.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Enhancing organic matter removal, product recovery, and hydrogen generation from fermentation wastewater

Existing ethanol biorefineries produce billions of gallons of wastewater (stillage) that must be treated extensively prior to discharge or concentrated in an energy intense evaporation step to produce animal feed as a value-added bioproduct. Yeasts and filamentous fungi were screened for growth and reduction of soluble organic compounds, carbohydrates, and protein from stillage to identify species capable of rapidly concentrating organic compounds into readily separable biomass in an attempt to concentrate the product using a much more efficient centrifugation step. Yeasts amenable to production of biomass derived products including bio-oils (Yarrowia lipolytica and Rhodotorula pallida), chitosan (Aspergillus niger) and animal feed (Cyberlindnera jadinii) were identified as well as microbes capable of efficient production of small organic acids with applications as bioderived polymers including 3-hydroxypropionic acid (engineered A. niger) and lactic acid using a co-culture of Debaryomyces udenii and Lactobacillus pentosus. The filamentous fungus A. niger eliminated all specifically measured soluble organics (glycerol, acetate, lactate, ethanol, and citrate) in under 48 hours and reduced total soluble protein content by 46% and total carbohydrate content by 83% within 96 hours, the greatest reduction in organic content observed. During this process an A. niger strain engineered to produce 3-hydroxypropionic acid achieved a yield of 0.35 C-mol 3HP / C-mol non-protein organics consumed at an overall rate of 0.09 g/Lh in unmodified stillage without pH control. Stillage treated by the fodder yeast C. jadinii as well as stillage containing acetic or lactic acid was found to be most suitable for growth by electrogenic bacteria with applications in production of electricity or hydrogen from wastewater. This work establishes fungal and bacterial strains appropriate for biological treatment of stillage to produce biomass derived products, soluble commodity chemicals, energy, and treated water to reduce the energy intensity and improve the economics of ethanol biorefineries.

09 BIOMASS FUELS↗

DOE OSTI2020

Creating multifunctional synthetic lichen platforms for sustainable biosynthesis of biofuel precursors

In this project, we were creating a sustainable platform for biofuel production, utilizing carbon-fixing autotrophs to supply oxygen and organic substrates to heterotrophic partners, which in turn produce carbon dioxide to feed the autotrophs. This symbiotic lichen community could lower the input cost, optimize metabolic exchanges and improve the generation of biofuel precursors through multi-omics driven genetic engineering. The cyanobacteria Synechococcus elongatus (S. elongatus) was used as the primary autotroph to provide oxygen and organic substrates, especially sucrose, to a co-culture system. The strain with overexpression of sucrose transporter cscB demonstrated a significant increase in sucrose production under salt stress as what we expected. We also implemented 13C metabolic flux analysis on the sucrose secreting strain S. elongatus cscB-NaCl. Next, transporters proteins like glutamate exporter mscCG from Corynebacterium glutamicum was overexpressed in S. elongatus to improve metabolite exchange. We provided sucrose and glutamate to filamentous fungi to enable their growth and production of biochemicals using substrates from the cyanobacterium. Two fungi ( Aspergillus nidulans and Aspergillus niger ) and two yeast strains ( Rhodotorula toruloides and Lipomyces starkeyi ) served as heterotrophs in this system. They were co-cultured with S.elongatus under different pH condition, and growth on different carbon source to understand the symbiotic system and optimize the parameters for production. The two fungi strained grew much better at pH 7, 8 and 9 with Yeast Nitrogen Base (YNB ) supplementation. Two yeast strains grew well on pH 7 and 8 with YNB supplementation. All four species achieved the highest biomass level when utilizing sucrose as the main carbon sources, which demonstrates a great pairing with S.elongatus . In addition to investigate the metabolite exchange and growth condition of the co-culture system, we incorporated an Aspergillus strain expressing three genes (PAND, BAPAT and HPDH). Expression of these three genes enabled production of 3-hydroxypropionic acid (3HP) with a titer of 3-6 g/L regardless of pH and other nutrient conditions. Moreover, we developed a computational model with different heterotrophic fungi symbiosing with S.elongatus to evaluate the exchange of hundreds of metabolites. The accuracy and sensitivity had been optimized by high-throughput phenotyping assays. This project demonstrated ways to enhance a synthetic lichen platform’s efficiency and scalability to create a robust system for sustainable bioproduct synthesis. The project has enabled us to explore the technological and commercial potential of sustainable lichen co-culture. The project has also trained the next generation of scientists to address sustainability, carbon fixation, and design solutions to contemporary global challenges. The lichen platform represented a novel approach to sustainably producing bioproducts with a reduced carbon footprint and lower costs.

09 BIOMASS FUELS↗