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Building a stronger bioeconomy requires production capabilities that are largely generated through microbial genetic engineering. Plant feedstocks can additionally be genetically engineered to generate desirable feedstock traits and provide precursors for direct microbial conversion into desired products. The oleaginous yeast Rhodosporidium toruloides is a promising organism for this type of conversion as it can grow on a wide range of deconstructed biomass and consume a variety of carbon sources. Here, we leveraged R. toruloides native p-coumaric acid consumption pathway to accumulate protocatechuate (PCA) from 4-hydroxybenzoate (4HBA) released from a sorghum feedstock line genetically engineered to overproduce 4HBA. We did so by generating and evaluating an R. toruloides strain that accumulates PCA, RSΔ12623. We then show that at two scales a cholinium lysinate pretreatment with enzymatic saccharification successfully extracts 95% of the 4HBA from the engineered sorghum biomass while producing deconstructed lignin that can be more efficiently depolymerized in a subsequent thermochemical reaction. We also demonstrate that strain RSΔ12623 can convert more than 95% of 4HBA to PCA while consuming >95% of the glucose and >80% of the xylose present in sorghum hydrolysates. Finally, to evaluate the scalability of such fermentations, we conducted the conversion of 4HBA to PCA in a 2 L bioreactor under controlled conditions. This work demonstrates the potential of purposefully producing aromatic precursors in planta that can be liberated during biomass deconstruction for direct microbial conversion to desirable bioproducts. [ABSTRACT FROM AUTHOR]

Generating value from lignin through depolymerization and biological conversion to valuable fuels, chemicals, or intermediates has great promise but is limited by several factors including lack of cost-effective depolymerization methods, toxicity within the breakdown products, and low bioconversion of the breakdown products. High yield depolymerization of natural lignins requires cleaving carbon–carbon bonds in addition to ether bonds. To address that need, we report that a chelator-mediated Fenton reaction can efficiently cleave C–C bonds in sulfonated polymers at or near room temperature, and that unwanted repolymerization can be minimized through optimizing reaction conditions. This method was used to depolymerize lignosulfonate from Mw = 28 000 g mol−1 to Mw = 800 g mol−1. The breakdown products were characterized by SEC, FTIR and NMR and evaluated for bioavailability. The breakdown products are rich in acid, aldehyde, and alcohol functionalities but are largely devoid of aromatics and aliphatic dienes. A panel of nine organisms were tested for the ability to grow on the breakdown products. Growth at a low level was observed for several monocultures on the depolymerized lignosulfonate in the absence of glucose. Much stronger growth was observed in the presence of 0.2% glucose and for one organism we demonstrate doubling of melanin production in the presence of depolymerized lignosulfonate. The results suggest that this chelator-mediated Fenton method is a promising new approach for biological conversion of lignin into higher value chemicals or intermediates. [ABSTRACT FROM AUTHOR]

Pretreatment is an essential step to enable the efficient conversion of lignocellulosic biomass to biofuels and bioproducts. The most effective pretreatment methods currently in use are based on severe thermochemical approaches, which are costly and energy-intensive. Here we explored whether the common practice of ensiling grassy biomass, such as sorghum, could be used as a pre-processing step to increase the conversion efficiency under milder pretreatment conditions. We determined the impact of replacing dry sorghum biomass with ensiled sorghum biomass on the deconstruction efficiency, process economics, and carbon footprint of a lignocellulosic biorefinery that employs a separation-free ionic liquid pretreatment coupled to enzymatic saccharification and microbial conversion. Our results indicate that the use of ensiled biomass allowed for a 50% reduction in both the amount of ionic liquid (from 5 to 2.5% (w/w) as measured by initial pretreatment loading) and the time required for enzymatic saccharification (from 72 h to 24 h) without sacrificing efficiency. We show that the resulting hydrolysate can be used to cultivate an engineered strain of Rhodosporidium toruloides to convert >90% of the monomeric sugars into bisabolene, a promising intermediate to biofuels and bioproducts. Overall, we estimate that the replacement of field-dried biomass sorghum with ensiled sorghum in combination with an ionic liquid-based deconstruction process could reduce the minimum selling price and carbon footprint of biofuel production in a biorefinery by at least 13% and 8.2%, respectively. [ABSTRACT FROM AUTHOR]

To date, information on biomass deconstruction and advanced biofuels production using pilot-scale pretreatments is scarce and typically derived from single feedstocks, making comparisons complicated. This work evaluates the effect of subjecting four different feedstocks (agave bagasse [AG], corn stover [CS], sugarcane bagasse [SC] and wheat straw [WS]) to pretreatment at 180 °C for 20 min in a 200 kg day−1 pilot-scale continuous tubular reactor constituted by three sequential stages (compression, autohydrolysis and steam explosion). Correlations between the structural changes and differences in digestibility on pretreated biomass were determined. It was determined that xylan removal by pretreatment generated more crystalline and energy dense materials, with higher lignin content and S/G ratio. Optimal reaction conditions for enzymatic saccharification of pretreated feedstocks at lower enzyme doses generated glucose conversions up to 80%. Finally, it was demonstrated that the yeast Rhodosporidium toruloides was able to grow in all hydrolysates, exhibiting simultaneous C5 and C6 sugar consumption and bisabolene production. [ABSTRACT FROM AUTHOR]

Achieving low cost and high efficiency lignocellulose deconstruction is a critical step towards widespread adoption of lignocellulosic biofuels. Certain ionic liquid (IL)-based pretreatment processes effectively reduce recalcitrance of lignocellulose to enzymatic degradation but require either costly separations following pretreatment or novel IL compatible processes to mitigate downstream toxicity. Here we demonstrate at benchtop and pilot bioreactor scales a separation-free, intensified process for IL pretreatment, saccharification, and fermentation of sorghum biomass to produce the sesquiterpene bisabolene, a precursor to the renewable diesel and jet fuel bisabolane. The deconstruction process employs the IL cholinium lysinate ([Ch][Lys]), followed by enzymatic saccharification with the commercial enzyme cocktails Cellic CTec2 and HTec2. Glucose yields above 80% and xylose yields above 60% are observed at all scales tested. Unfiltered hydrolysate is fermented directly by Rhodosporidium toruloides– with glucose, xylose, acetate and lactate fully consumed during fermentation at all scales tested. Bisabolene titers improved with scale from 1.3 g L−1 in 30 mL shake flasks to 2.2 g L−1 in 20 L fermentation. The combined process enables conversion of saccharified IL-pretreated biomass directly to advanced biofuels with no separations or washing, minimal additions to facilitate fermentation, no loss of performance due to IL toxicity, and simplified fuel recovery via phase separation. This study is the first to demonstrate a separation-free IL based process for conversion of biomass to an advanced biofuel and is the first to demonstrate full consumption of glucose, xylose, acetate, and lactic acid in the presence of [Ch][Lys]. [ABSTRACT FROM AUTHOR]