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476 records · Page 27

Microbial Pathways for Cost-Effective Low-Carbon Renewable Indigoidine

Indigoidine is a bioadvantaged platform molecule with diverse applications, including use as a textile dye, biotransistor, biosolar cell, biosensor, and food coloring. There are multiple microbial hosts and carbon sources that can be used and optimized for its production, yet there is limited guidance for which options have the greatest commercial potential. Here, we consider five different host microbes and combine genome-scale metabolic models with techno-economic and lifecycle assessment models. Pseudomonas putida currently outperforms synthetic indigo production and other indigoidine-producing hosts, using glucose, xylose, and lignin-derived aromatics to produce indigoidine at a minimum selling price of $2.9/kg and a greenhouse gas (GHG) footprint of 3.5 kgCO 2e /kg. Optimizing pathways-achieving 90% of the theoretical indigoidine yield from sugars and aromatics-can reduce costs 6-7-fold and GHG emissions 3-10-fold. From a cost perspective, microbes that co-utilize aromatics are advantageous, while selecting hosts that coproduce other value-added molecules can reduce GHG emissions. System-wide improvements and the use of a low-cost, low-carbon nitrogen source are crucial for commercial viability in all cases.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Phylogenetic diversity of light-dependent phosphorylation of Thr78 in Rubisco activase

Rubisco activase is an ATP-dependent chaperone that facilitates dissociation of inhibitory sugar phosphates from the catalytic sites of Rubisco during photosynthesis. In Arabidopsis, Rubisco activase is negatively regulated by dark-dependent phosphorylation of Thr78. The prevalence of Thr78 in Rubisco activase was investigated across sequences from 91 plant species, finding that 29 (∼32%) species shared a threonine in the same position. Analysis of seven C3 species with an antibody raised against a Thr78 phospho-peptide demonstrated that this position is phosphorylated in multiple genera. However, light-dependent dephosphorylation of Thr78 was observed only in Arabidopsis. Further, phosphorylation of Thr78 could not be detected in any of the four C4 grass species examined. The results suggest that despite conservation of Thr78 in Rubisco activase from a wide range of species, a regulatory role for phosphorylation at this site is more limited. Furthermore, this provides a case study for how variation in post-translational regulation can amplify functional divergence across the phylogeny of plants beyond what is explained by sequence variation in a metabolically important protein.

Arabidopsis↗

Data for Phylogenetic diversity of light-dependent phosphorylation of Thr78 in Rubisco activase

Rubisco activase is an ATP-dependent chaperone that facilitates dissociation of inhibitory sugar phosphates from the catalytic sites of Rubisco during photosynthesis. In Arabidopsis, Rubisco activase is negatively regulated by dark-dependent phosphorylation of Thr78. The prevalence of Thr78 in Rubisco activase was investigated across sequences from 91 plant species, finding that 29 (∼32%) species shared a threonine in the same position. Analysis of seven C3 species with an antibody raised against a Thr78 phospho-peptide demonstrated that this position is phosphorylated in multiple genera. However, light-dependent dephosphorylation of Thr78 was observed only in Arabidopsis. Further, phosphorylation of Thr78 could not be detected in any of the four C4 grass species examined. The results suggest that despite conservation of Thr78 in Rubisco activase from a wide range of species, a regulatory role for phosphorylation at this site is more limited. This provides a case study for how variation in post-translational regulation can amplify functional divergence across the phylogeny of plants beyond what is explained by sequence variation in a metabolically important protein.

photosynthesis↗

Managing flowering time in Miscanthus and sugarcane to facilitate intra- and intergeneric crosses

Miscanthus is a close relative of Saccharum and a potentially valuable genetic resource for improving sugarcane. Differences in flowering time within and between Miscanthus and Saccharum hinders intra- and interspecific hybridizations. A series of greenhouse experiments were conducted over three years to determine how to synchronize flowering time of Saccharum and Miscanthus genotypes. We found that day length was an important factor influencing when Miscanthus and Saccharum flowered. Sugarcane could be induced to flower in a central Illinois greenhouse using supplemental lighting to reduce the rate at which days shortened during the autumn and winter to 1 min d -1 , which allowed us to synchronize the flowering of some sugarcane genotypes with Miscanthus genotypes primarily from low latitudes. In a complementary growth chamber experiment, we evaluated 33 Miscanthus genotypes, including 28 M . sinensis , 2 M . floridulus , and 3 M . ×giganteus collected from 20.9° S to 44.9° N for response to three day lengths (10 h, 12.5 h, and 15 h). High latitude-adapted M . sinensis flowered mainly under 15 h days, but unexpectedly, short days resulted in short, stocky plants that did not flower; in some cases, flag leaves developed under short days but heading did not occur. In contrast, for M . sinensis and M . floridulus from low latitudes, shorter day lengths typically resulted in earlier flowering, and for some low latitude genotypes, 15 h days resulted in no flowering. However, the highest ratio of reproductive shoots to total number of culms was typically observed for 12.5 h or 15 h days. Latitude of origin was significantly associated with culm length, and the shorter the days, the stronger the relationship. Nearly all entries achieved maximal culm length under the 15 h treatment, but the nearer to the equator an accession originated, the less of a difference in culm length between the short-day treatments and the 15 h day treatment. Under short days, short culms for high-latitude accessions was achieved by different physiological mechanisms for M . sinensis genetic groups from the mainland in comparison to those from Japan; for mainland accessions, the mechanism was reduced internode length, whereas for Japanese accessions the phyllochron under short days was greater than under long days. Thus, for M . sinensis , short days typically hastened floral induction, consistent with the expectations for a facultative short-day plant. However, for high latitude accessions of M . sinensis , days less than 12.5 h also signaled that plants should prepare for winter by producing many short culms with limited elongation and development; moreover, this response was also epistatic to flowering. Thus, to flower M . sinensis that originates from high latitudes synchronously with sugarcane, the former needs day lengths >12.5 h (perhaps as high as 15 h), whereas that the latter needs day lengths <12.5 h.

54 ENVIRONMENTAL SCIENCES↗

Integration of genome-scale metabolic model with biorefinery process model reveals market-competitive carbon-negative sustainable aviation fuel utilizing microbial cell mass lipids and biogenic CO 2

Producing scalable, economically viable, low-carbon biofuels or biochemicals hinges on more efficient bioconversion processes. While microbial conversion can offer robust solutions, the native microbial growth process often redirects a large fraction of carbon to CO 2 and cell mass. By integrating genome-scale metabolic models with techno-economic and life cycle assessment models, this study analyzes the effects of converting cell mass lipids to hydrocarbon fuels, and CO 2 to methanol on the facility’s costs and life-cycle carbon footprint. Results show that upgrading microbial lipids or both microbial lipids and CO 2 using renewable hydrogen produces carbon-negative bisabolene. Additionally, on-site electrolytic hydrogen production offers a supply of pure oxygen to use in place of air for bioconversion and fuel combustion in the boiler. To reach cost parity with conventional jet fuel, renewable hydrogen needs to be produced at less than $\$2.2$ to $\$3.1$/kg, with a bisabolene yield of 80% of the theoretical yield, along with cell mass and CO 2 yields of 22 wt% and 54 wt%, respectively. The economic combination of cell mass, CO 2 , and bisabolene yields demonstrated in this study provides practical insights for prioritizing research, selecting suitable hosts, and determining necessary engineered production levels.

09 BIOMASS FUELS↗

Genetics and Genomics of Pathogen Resistance in Switchgrass (Final Report)

This project was funded by DOE under Grant no. DE-SC0016108. Originally approved for the 2016-2019 period, two no-cost extensions were solicited and approved, which prolonged the lifespan through July 2021. This final report informs on the results obtained so far from the research implemented. The research hinged on integrating genomics (genomic selection, RNAseq, virus-plant interactions) with classical genetics (conventional breeding) to incorporate durable resistance to fungal (rust) and viral (mosaic) diseases in switchgrass (Panicum virgatum) populations being bred for bioenergy. Higher biomass yield, higher quality (low lignin content), and durable disease resistance are key features to make lignocellulosic switchgrass feedstocks economically competitive and sustainable. Genomic selection is being applied on three generations of a switchgrass population derived from crossing two ecotypes (Kanlow as lowland female and Summer as upland male) with differential performance in terms of biomass yield and quality, disease resistance, and winter survivability. Target populations were screened for rust and mosaic in field and/or lab and phenotyped for biomass yield and quality traits. Genetic analyses were applied across generations to capture the joint inheritance of the targeted traits and predict breeding values for parents and progeny with greater accuracy. Parental and a panel of different switchgrass populations were genotyped with the DArTseq technology to develop SNP (0, 1, 2) and in-silico (presence/absence) DArT markers. Rust inoculations techniques were developed and applied successfully on switchgrass. The original populations (Kanlow and Summer) were sequenced with RNAseq to capture the gene expression profiles across sequential time-points and appraise the basis of greater resistance in the Kanlow vs the Summer ecotype. Constructs of PMV and sPMV mosaic virus were assembled and tested first on proso millet to find the best protocol to use later on switchgrass. Results from the preliminary analyses indicate that 1) ample additive genetic variation is available for selection and improving this inter-ecotypic population for yield, quality, and disease traits, 2) significant gains are to be expected with the genetic correlations being favorable between yield and lignin content and between yield and disease ratings, 3) substantial differences exist in the genetic regions controlling rust resistance in the two ecotypes, 4) co-infection with PMV isolates from Nebraska and its satellite from Kansas elicit severe mosaic symptoms, and 5) two different genetic systems are responsible for imparting resistance to rust and virus in switchgrass.

59 BASIC BIOLOGICAL SCIENCES↗

Structural and Interactional Analysis of the Flavonoid Pathway Proteins: Chalcone Synthase, Chalcone Isomerase and Chalcone Isomerase-like Protein

Chalcone synthase (CHS) and chalcone isomerase (CHI) catalyze the first two committed steps of the flavonoid pathway that plays a pivotal role in the growth and reproduction of land plants, including UV protection, pigmentation, symbiotic nitrogen fixation, and pathogen resistance. Based on the obtained X-ray crystal structures of CHS, CHI, and chalcone isomerase-like protein (CHIL) from the same monocotyledon, Panicum virgatum, along with the results of the steady-state kinetics, spectroscopic/thermodynamic analyses, intermolecular interactions, and their effect on each catalytic step are proposed. In addition, PvCHI’s unique activity for both naringenin chalcone and isoliquiritigenin was analyzed, and the observed hierarchical activity for those type-I and -II substrates was explained with the intrinsic characteristics of the enzyme and two substrates. The structure of PvCHS complexed with naringenin supports uncompetitive inhibition. PvCHS displays intrinsic catalytic promiscuity, evident from the formation of p-coumaroyltriacetic acid lactone (CTAL) in addition to naringenin chalcone. In the presence of PvCHIL, conversion of p-coumaroyl-CoA to naringenin through PvCHS and PvCHI displayed ~400-fold increased Vmax with reduced formation of CTAL by 70%. Supporting this model, molecular docking, ITC (Isothermal Titration Calorimetry), and FRET (Fluorescence Resonance Energy Transfer) indicated that both PvCHI and PvCHIL interact with PvCHS in a non-competitive manner, indicating the plausible allosteric effect of naringenin on CHS. Significantly, the presence of naringenin increased the affinity between PvCHS and PvCHIL, whereas naringenin chalcone decreased the affinity, indicating a plausible feedback mechanism to minimize spontaneous incorrect stereoisomers. These are the first findings from a three-body system from the same species, indicating the importance of the macromolecular assembly of CHS-CHI-CHIL in determining the amount and type of flavonoids produced in plant cells.

59 BASIC BIOLOGICAL SCIENCES↗