With a diverse and widely distributed global resource base, woody biomass is a compelling organic feedstock for conversion to renewable liquid fuels. In California, woody biomass comprises the largest fraction of underutilized biomass available for biofuel production, but conversion to fuels is challenged both by recalcitrance to deconstruction and by toxicity towards downstream saccharification and fermentation due to organic acids and phenolic compounds generated during pre-treatment. In this study, we optimize pretreatment and scale-up of an integrated one-pot process for deconstruction of California woody biomass using the ionic liquid cholinium lysinate [Ch][Lys] as a pretreatment solvent. By evaluating the impact of solids loading, solids removal, yeast acclimatization, fermentation temperature, fermentation pH, and nutrient supplementation on final ethanol yields and titers, we achieve nearly full conversion of both glucose and xylose to ethanol with commercial C5-utilizing Saccharomyces cerevisiae. We then demonstrate process scalability in a 680 L pilot-scale fermentation, achieving >80 % deconstruction efficiency, >90 % fermentation efficiency, 27.7 g/L ethanol titer, and >80 % ethanol distillation efficiency from the ILcontaining hydrolysate post-fermentation. This fully integrated process requires no intermediate separations and no intermediate detoxification of 1 the hydrolysate. Using an integrated biorefinery model, current performance results in a minimum ethanol selling price of $8.8/gge. Reducing enzyme loading along with other minor process improvements can reduce the ethanol selling price to $3/gge. This study is the largest-scale demonstration of ionic liquid pretreatment and biofuel conversion known to date, and the overall biomass-to-ethanol efficiencies are the highest reported to date for any ionic liquid-based biomass to biofuel conversion.

Corynebacterium glutamicum is an ideal microbial chassis for production of valuable bioproducts including amino acids and next generation biofuels. Here we resequence engineered isopentenol (IP) producing C. glutamicum BRC-JBEI 1.1.2 strain and assess differential transcriptional profiles using RNA sequencing under industrially relevant conditions including scale transition and compare the presence vs absence of an ionic liquid, cholinium lysinate ([Ch][Lys]). Analysis of the scale transition from shake flask to bioreactor with transcriptomics identified a distinct pattern of metabolic and regulatory responses needed for growth in this industrial format. These differential changes in gene expression corroborate altered accumulation of organic acids and bioproducts, including succinate, acetate, and acetoin that occur when cells are grown in the presence of 50 mM [Ch][Lys] in the stirred-tank reactor. This new genome assembly and differential expression analysis of cells grown in a stirred tank bioreactor clarify the cell response of an C. glutamicum strain engineered to produce IP.

Background Rhodosporidium toruloides is capable of co-utilization of complex carbon sources and robust growth from lignocellulosic hydrolysates. This oleaginous yeast is therefore an attractive host for heterologous production of valuable bioproducts at high titers from low-cost, deconstructed biomass in an economically and environmentally sustainable manner. Here we demonstrate this by engineering R. toruloides to produce the polyketide triacetic acid lactone (TAL) directly from unfiltered hydrolysate deconstructed from biomass with minimal unit process operations. Results Introduction of the 2-pyrone synthase gene into R. toruloides enabled the organism to produce 2.4 g/L TAL from simple media or 2.0 g/L from hydrolysate produced from sorghum biomass. Both of these titers are on par with titers from other better-studied microbial hosts after they had been heavily engineered. We next demonstrate that filtered hydrolysates produced from ensiled sorghum are superior to those derived from dried sorghum for TAL production, likely due to the substantial organic acids produced during ensiling. We also demonstrate that the organic acids found in ensiled biomass can be used for direct synthesis of ionic liquids within the biomass pretreatment process, enabling consolidation of unit operations of in-situ ionic liquid synthesis, pretreatment, saccharification, and fermentation into a one-pot, separations-free process. Finally, we demonstrate this consolidation in a 2 L bioreactor using unfiltered hydrolysate, producing 3.9 g/L TAL. Conclusion Many steps involved in deconstructing biomass into fermentable substrate can be combined into a distinct operation, and directly fed to cultures of engineered R. toruloides cultures for subsequent valorization into gram per liter titers of TAL in a cost-effective manner.

Corynebacterium glutamicum is an ideal microbial chassis for the production of valuable bioproducts including amino acids and next-generation biofuels. Here we resequence engineered isopentenol (IP) producing C. glutamicum BRC-JBEI 1.1.2 strain and assess differential transcriptional profiles using RNA sequencing under industrially relevant conditions including scale transition and compare the presence vs. absence of an ionic liquid, cholinium lysinate ([Ch][Lys]). Analysis of the scale transition from shake flask to bioreactor with transcriptomics identified a distinct pattern of metabolic and regulatory responses needed for growth in this industrial format. These differential changes in gene expression corroborate altered accumulation of organic acids and bioproducts, including succinate, acetate, and acetoin that occur when cells are grown in the presence of 50mM [Ch][Lys] in the stirred-tank reactor. This new genome assembly and differential expression analysis of cells grown in a stirred tank bioreactor clarify the cell response of a C. glutamicum strain engineered to produce IP.