Production of ent-kaurene from lignocellulosic hydrolysate in Rhodosporidium toruloides.

BACKGROUND: Rhodosporidium toruloides has emerged as a promising host for the production of bioproducts from lignocellulose, in part due to its ability to grow on lignocellulosic feedstocks, tolerate growth inhibitors, and co-utilize sugars and lignin-derived monomers. Ent-kaurene derivatives have a diverse range of potential applications from therapeutics to novel resin-based materials. RESULTS: The Design, Build, Test, and Learn (DBTL) approach was employed to engineer production of the non-native diterpene ent-kaurene in R. toruloides. Following expression of kaurene synthase (KS) in R. toruloides in the first DBTL cycle, a key limitation appeared to be the availability of the diterpene precursor, geranylgeranyl diphosphate (GGPP). Further DBTL cycles were carried out to select an optimal GGPP synthase and to balance its expression with KS, requiring two of the strongest promoters in R. toruloides, ANT (adenine nucleotide translocase) and TEF1 (translational elongation factor 1) to drive expression of the KS from Gibberella fujikuroi and a mutant version of an FPP synthase from Gallus gallus that produces GGPP. Scale-up of cultivation in a 2 L bioreactor using a corn stover hydrolysate resulted in an ent-kaurene titer of 1.4 g/L. CONCLUSION: This study builds upon previous work demonstrating the potential of R. toruloides as a robust and versatile host for the production of both mono- and sesquiterpenes, and is the first demonstration of the production of a non-native diterpene in this organism.

Conversion of poplar biomass into high-energy density tricyclic sesquiterpene jet fuel blendstocks.

BACKGROUND: In an effort to ensure future energy security, reduce greenhouse gas emissions and create domestic jobs, the US has invested in technologies to develop sustainable biofuels and bioproducts from renewable carbon sources such as lignocellulosic biomass. Bio-derived jet fuel is of particular interest as aviation is less amenable to electrification compared to other modes of transportation and synthetic biology provides the ability to tailor fuel properties to enhance performance. Specific energy and energy density are important properties in determining the attractiveness of potential bio-derived jet fuels. For example, increased energy content can give the industry options such as longer range, higher load or reduced takeoff weight. Energy-dense sesquiterpenes have been identified as potential next-generation jet fuels that can be renewably produced from lignocellulosic biomass. RESULTS: We developed a biomass deconstruction and conversion process that enabled the production of two tricyclic sesquiterpenes, epi-isozizaene and prespatane, from the woody biomass poplar using the versatile basidiomycete Rhodosporidium toruloides. We demonstrated terpene production at both bench and bioreactor scales, with prespatane titers reaching 1173.6 mg/L when grown in poplar hydrolysate in a 2 L bioreactor. Additionally, we examined the theoretical fuel properties of prespatane and epi-isozizaene in their hydrogenated states as blending options for jet fuel, and compared them to aviation fuel, Jet A. CONCLUSION: Our findings indicate that prespatane and epi-isozizaene in their hydrogenated states would be attractive blending options in Jet A or other lower density renewable jet fuels as they would improve viscosity and increase their energy density. Saturated epi-isozizaene and saturated prespatane have energy densities that are 16.6 and 18.8% higher than Jet A, respectively. These results highlight the potential of R. toruloides as a production host for the sustainable and scalable production of bio-derived jet fuel blends, and this is the first report of prespatane as an alternative jet fuel.

Brachytic2 mutation is able to counteract the main pleiotropic effects of brown midrib3 mutant in maize.

Maize is the basis of nutrition of domesticated herbivores and one of the most promising energy crops. The presence of lignin in the cell wall, tightly associated to carbohydrates, prevents the physical access of enzymes such as cellulase, limiting the carbohydrate degradability and consequently the energy value. To increase the utilization of the biomass cellulose content, the challenge of breeding programs is to lower or modify the lignin components. In maize several mutations are able to modify the lignin content and in particular the mutation in brown midrib3 (bm3) gene appeared as one of the most promising in breeding programs. Unfortunately this mutation has several negative pleiotropic effects on various important agronomic traits such as stay green, lodging and susceptibility to several infections.The maize Brachyitic 2 (br2) gene encodes for a putative protein involved in polar movement of auxins. br2 mutant plants are characterized by shortening of lower stalk internodes, unusual stalk strength and tolerance to wind lodging, darker leaves persisting longer in the active green state in comparison to wild type plants, suggesting a possible utilization of br2 plants to counteract the negative effects of the bm3 mutation. In this work, we report the generation and a preliminary characterization of the double mutant bm3 br2, suggesting the potential use of this new genetic material to increase biomass cellulose utilization.

Nanoscale structure of the cell wall protecting cellulose from enzyme attack.

The cell wall structure protects cellulose from enzymatic attack and its successive fermentation. The nature of this protection consists in the very complex macroscopic and microscopic structure of cell wall that limits transport. Explaining this kind of protection is critical in future research to improve cell polymer availability for enzymatic attack. This research shows that the complete description of the cell wall topography at a nanoscale level allows a mechanistic understanding of cellulose protection. For this purpose, we used gas adsorption methods (CO(2) at 273 K and N(2) at 77 K) to detect mesoporosity (pore size of 1.5-30 nm diameter; MeS) and microporosity (pore size of 0.3-1.5 nm diameter; MiS) of the cell wall of five energy crops, i.e., giant cane, rivet wheat straw, miscanthus, proso millet, and sorghum. The presence of both hemicelluloses in the spaces between cellulose fibrils and the unhydrolyzable and highly cross-linked lignocarbohydrate complex (LCC) determines a microporous (80% pores having diameters below 0.8 nm) structure of the cell wall that prevents the cellulase enzymes from coming into direct contact with the cellulose, as their sizes exceed the cell wall pore size. On the other hand, the removal of the hemicelluloses and of the LCC complex determines a reduction of the MiS and an increase of the available surface for enzymatic attack, i.e., pores >5 nm diameter. This was confirmed by the good negative (r = -0.87, P < 0.001, n = 11) and positive (r = 0.78, P < 0.005, n = 11) correlations found for microporosity and mesoporosity (pores of diameters >5 nm), respectively, vs the glucose production, by cellulase enzyme attack in specific enzymatic hydrolysis tests performed on biomass samples.

Idiopathic thrombocytopenic purpura in a patient with situs inversus totalis: case report and literature review.

Situs inversus totalis is a rare recessive autosomal congenital abnormality in which the mediastinal and abdominal organs are in a mirrored position when compared to the usual topography. The literature reports some cases of situs inversus totalis and concomitant conditions: spinal abnormalities, cardiac malformations and hematological diseases, such as idiopathic thrombocytopenic purpura, which is an autoimmune disease that causes thrombocytopenia due to platelet destruction or suppression of its production. This article aimed to report the coexistence of situs inversus totalis and idiopathic thrombocytopenic purpura.

One-pot bio-derived ionic liquid conversion followed by hydrogenolysis reaction for biomass valorization: A promising approach affecting the morphology and quality of lignin of switchgrass and poplar.

The use of bio-derived ionic liquids (e.g., cholinium lysinate) in a one-pot process was evaluated on overall sugar and lignin yields as a function of two model woody and herbaceous feedstocks, switchgrass and poplar, with emphasis on the study of physical and chemical alterations in lignin structure, by performing a detailed mass balance analysis and chemical characterization. Multiple chromatographic and spectroscopic analytical techniques were applied tracking lignin reactivity and partitioning during the ionic liquid one-pot conversion. Depolymerization efficiency of the lignin-rich residue derived from the whole process was investigated as a function of different temperatures and pressures during catalytic hydrogenolysis by Ni(SO)4. This study validates the potential of ionic liquid one pot process as an integrated approach for full exploitation of lignocellulosic feedstocks. The insights gained will contribute to the design of future conversion routes for efficient biomass deconstruction and lignin valorization.

Parametric study for the optimization of ionic liquid pretreatment of corn stover.

A parametric study of the efficacy of the ionic liquid (IL) pretreatment (PT) of corn stover (CS) using 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]) and cholinium lysinate ([Ch][Lys]) was conducted. The impact of 50% and 15% biomass loading for milled and non-milled CS on IL-PT was evaluated, as well the impact of 20 and 5mg enzyme/g glucan on saccharification efficiency. The glucose and xylose released were generated from 32 conditions - 2 ionic liquids (ILs), 2 temperatures, 2 particle sizes (S), 2 solid loadings, and 2 enzyme loadings. Statistical analysis indicates that sugar yields were correlated with lignin and xylan removal and depends on the factors, where S did not explain variation in sugar yields. Both ILs were effective in pretreating large particle sized CS, without compromising sugar yields. The knowledge from material and energy balances is an essential step in directing optimization of sugar recovery at desirable process conditions.

Idiopathic thrombocytopenic purpura in a patient with situs inversus totalis: case report and literature review / Púrpura trombocitopênica idiopática em paciente com situs inversus totalis: relato de caso e revisão da literatura

ABSTRACT Situs inversus totalis is a rare recessive autosomal congenital abnormality in which the mediastinal and abdominal organs are in a mirrored position when compared to the usual topography. The literature reports some cases of situs inversus totalis and concomitant conditions: spinal abnormalities, cardiac malformations and hematological diseases, such as idiopathic thrombocytopenic purpura, which is an autoimmune disease that causes thrombocytopenia due to platelet destruction or suppression of its production. This article aimed to report the coexistence of situs inversus totalis and idiopathic thrombocytopenic purpura.

RESUMO Situs inversus totalis é uma anormalidade congênita autossômica recessiva rara em que os órgãos mediastinais e abdominais encontram-se em posição espelhada em relação à topografia habitual. A literatura relata alguns casos de concomitância do situs inversus totalis com outras condições: anomalias espinhais, malformações cardíacas e doenças hematológicas, como púrpura trombocitopênica idiopática, que é uma doença autoimune com plaquetopenia, devido à destruição dos trombócitos ou supressão da sua produção. Esse artigo teve o objetivo de relatar coexistência de situs inversus totalis e púrpura trombocitopênica idiopática.