The impact of hydrogen peroxide supply on LPMO activity and overall saccharification efficiency of a commercial cellulase cocktail.

BACKGROUND: The discovery of enzymes named lytic polysaccharide monooxygenases (LPMOs) has had a major impact on the efficiency of current commercial cellulase cocktails for saccharification of lignocellulosic biomass. However, the notion that LPMOs use molecular oxygen as a co-substrate and require two externally delivered electrons per catalytic cycle poses a challenge in the development of efficient large-scale industrial processes. Building on the recent discovery that H2O2, rather than O2, is the co-substrate of LPMOs, we show here how cellulose degradation by the LPMO-containing commercial cellulase cocktail Cellic® CTec2 can be controlled and boosted by supplying the reaction with H2O2. RESULTS: The controlled supply of anaerobic hydrolysis reactions with H2O2 and sub-stoichiometric amounts of reductant increased apparent LPMO activity by almost two orders of magnitude compared to standard aerobic reactions utilizing O2 and stoichiometric amounts of reductant. Improved LPMO activity was correlated with enhanced saccharification rates and yields for a model cellulosic substrate (Avicel) as well as industrial lignocellulosic substrates (sulfite-pulped Norway spruce and steam-exploded birch), although the magnitude of the effects was substrate dependent. Improvements in lignocellulose conversions were achieved at low H2O2 feeding rates (in the range of 90-600 µM h-1). Tight control of LPMO reactions by controlled supply of H2O2 under anaerobic conditions was possible. CONCLUSION: We report saccharification rates and yields for a model substrate (Avicel) and industrial lignocellulosic substrates that, at low H2O2 feeding rates, are higher than those seen under standard aerobic conditions. In an industrial setting, controlling and supplying molecular oxygen and stoichiometric amounts of reductant are challenging. The present report shows that the use of small amounts of a liquid bulk chemical, H2O2, provides an alternative to the currently available processes, which likely is cheaper and more easy to control, while giving higher product yields.

Oxidative cleavage of polysaccharides by monocopper enzymes depends on H2O2.

Enzymes currently known as lytic polysaccharide monooxygenases (LPMOs) play an important role in the conversion of recalcitrant polysaccharides, but their mode of action has remained largely enigmatic. It is generally believed that catalysis by LPMOs requires molecular oxygen and a reductant that delivers two electrons per catalytic cycle. Using enzyme assays, mass spectrometry and experiments with labeled oxygen atoms, we show here that H2O2, rather than O2, is the preferred co-substrate of LPMOs. By controlling H2O2 supply, stable reaction kinetics are achieved, the LPMOs work in the absence of O2, and the reductant is consumed in priming rather than in stoichiometric amounts. The use of H2O2 by a monocopper enzyme that is otherwise cofactor-free offers new perspectives regarding the mode of action of copper enzymes. Furthermore, these findings have implications for the enzymatic conversion of biomass in Nature and in industrial biorefining.

Enzymatic degradation of sulfite-pulped softwoods and the role of LPMOs.

BACKGROUND: Recent advances in the development of enzyme cocktails for degradation of lignocellulosic biomass, especially the discovery of lytic polysaccharide monooxygenases (LPMOs), have opened new perspectives for process design and optimization. Softwood biomass is an abundant resource in many parts of the world, including Scandinavia, but efficient pretreatment and subsequent enzymatic hydrolysis of softwoods are challenging. Sulfite pulping-based pretreatments, such as in the BALI™ process, yield substrates that are relatively easy to degrade. We have assessed how process conditions affect the efficiency of modern cellulase preparations in processing of such substrates. RESULTS: We show that efficient degradation of sulfite-pulped softwoods with modern, LPMO-containing cellulase preparations requires the use of conditions that promote LPMO activity, notably the presence of molecular oxygen and sufficient reducing power. Under LPMO activity-promoting conditions, glucan conversion after 48-h incubation with Cellic® CTec3 reached 73.7 and 84.3% for Norway spruce and loblolly pine, respectively, at an enzyme loading of 8 mg/g of glucan. The presence of free sulfite ions had a negative effect on hydrolysis efficiency. Lignosulfonates, produced from lignin during sulfite pretreatment, showed a potential to activate LPMOs. Spiking of Celluclast®, a cellulase cocktail with low LPMO activity, with monocomponent cellulases or an LPMO showed that the addition of the LPMO was clearly more beneficial than the addition of any classical cellulase. Addition of the LPMO in reactions with spruce increased the saccharification yield from approximately 60% to the levels obtained with Cellic® CTec3. CONCLUSIONS: In this study, we have demonstrated the importance of LPMOs for efficient enzymatic degradation of sulfite-pulped softwood. We have also shown that to exploit the full potential of LPMO-rich cellulase preparations, conditions promoting LPMO activity, in particular the presence of oxygen and reducing equivalents are necessary, as is removal of residual sulfite from the pretreatment step. The use of lignosulfonates as reductants may reduce the costs related to the addition of small molecule reductants in sulfite pretreatment-based biorefineries.

LPMOs in cellulase mixtures affect fermentation strategies for lactic acid production from lignocellulosic biomass.

Enzymatic catalysis plays a key role in the conversion of lignocellulosic biomass to fuels and chemicals such as lactic acid. In the last decade, the efficiency of commercial cellulase cocktails has increased significantly, in part due to the inclusion of lytic polysaccharide monooxygenases (LPMOs). However, the LPMOs' need for molecular oxygen to break down cellulose demands reinvestigations of process conditions. In this study, we evaluate the efficiency of lactic acid production from steam-exploded birch using an LPMO-containing cellulase cocktail in combination with lactic acid bacteria, investigating both separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF). While the SSF set up generally has been considered to be more efficient because it avoids sugar accumulation which may inhibit the cellulases, the SHF set up in our study yielded 26-32% more lactic acid than the SSF. This was mainly due to competition for oxygen between LPMOs and the fermenting organisms in the SSF process, which resulted in reduced LPMO activity and thus less efficient saccharification of the lignocellulosic substrate. By means of aeration it was possible to activate the LPMOs in the SSF, but less lactic acid was produced due to a shift in metabolic pathways toward production of acetic acid. Overall, this study shows that lactic acid can be produced efficiently from lignocellulosic biomass, but that the use of LPMO-containing cellulase cocktails in fermentation processes demands re-thinking of traditional process set ups due to the requirement of oxygen in the saccharification step. Biotechnol. Bioeng. 2017;114: 552-559. © 2016 Wiley Periodicals, Inc.

Toward "homolactic" fermentation of glucose and xylose by engineered Saccharomyces cerevisiae harboring a kinetically efficient l-lactate dehydrogenase within pdc1-pdc5 deletion background.

l-Lactic acid is an important platform chemical and its production from the lignocellulosic sugars glucose and xylose is, therefore, of high interest. Tolerance to low pH and a generally high robustness make Saccharomyces cerevisiae a promising host for l-lactic acid fermentation but strain development for effective utilization of both sugars is an unsolved problem. The herein used S. cerevisiae strain IBB10B05 incorporates a NADH-dependent pathway for oxidoreductive xylose assimilation within CEN.PK113-7D background and was additionally evolved for accelerated xylose-to-ethanol fermentation. Selecting the Plasmodium falciparum l-lactate dehydrogenase (pfLDH) for its high kinetic efficiency, strain IBB14LA1 was derived from IBB10B05 by placing the pfldh gene at the pdc1 locus under control of the pdc1 promotor. Strain IBB14LA1_5 additionally had the pdc5 gene disrupted. With both strains, continued l-lactic acid formation from glucose or xylose, each at 50 g/L, necessitated stabilization of pH. Using calcium carbonate (11 g/L), anaerobic shaken bottle fermentations at pH ≥ 5 resulted in l-lactic acid yields (YLA ) of 0.67 g/g glucose and 0.80 g/g xylose for strain IBB14LA1_5. Only little xylitol was formed (≤0.08 g/g) and no ethanol. In pH stabilized aerobic conversions of glucose, strain IBB14LA1_5 further showed excellent l-lactic acid productivities (1.8 g/L/h) without losses in YLA (0.69 g/g glucose). In strain IBB14LA1, the YLA was lower (≤0.18 g/g glucose; ≤0.27 g/g xylose) due to ethanol as well as xylitol formation. Therefore, this study shows that a S. cerevisiae strain originally optimized for xylose-to-ethanol fermentation was useful to implement l-lactic acid production from glucose and xylose; and with the metabolic engineering strategy applied, advance toward homolactic fermentation of both sugars was made. Biotechnol. Bioeng. 2017;114: 163-171. © 2016 Wiley Periodicals, Inc.

Harnessing the potential of LPMO-containing cellulase cocktails poses new demands on processing conditions.

BACKGROUND: The emerging bioeconomy depends on improved methods for processing of lignocellulosic biomass to fuels and chemicals. Saccharification of lignocellulose to fermentable sugars is a key step in this regard where enzymatic catalysis plays an important role and is a major cost driver. Traditionally, enzyme cocktails for the conversion of cellulose to fermentable sugars mainly consisted of hydrolytic cellulases. However, the recent discovery of lytic polysaccharide monooxygenases (LPMOs), which cleave cellulose using molecular oxygen and an electron donor, has provided new tools for biomass saccharification. RESULTS: Current commercial enzyme cocktails contain LPMOs, which, considering the unique properties of these enzymes, may change optimal processing conditions. Here, we show that such modern cellulase cocktails release up to 60 % more glucose from a pretreated lignocellulosic substrate under aerobic conditions compared to anaerobic conditions. This higher yield correlates with the accumulation of oxidized products, which is a signature of LPMO activity. Spiking traditional cellulase cocktails with LPMOs led to increased saccharification yields, but only under aerobic conditions. LPMO activity on pure cellulose depended on the addition of an external electron donor, whereas this was not required for LPMO activity on lignocellulose. CONCLUSIONS: In this study, we demonstrate a direct correlation between saccharification yield and LPMO activity of commercial enzyme cocktails. Importantly, we show that the LPMO contribution to overall efficiency may be large if process conditions are adapted to the key determinants of LPMO activity, namely the presence of electron donors and molecular oxygen. Thus, the advent of LPMOs has a great potential, but requires rethinking of industrial bioprocessing procedures.

Process intensification through microbial strain evolution: mixed glucose-xylose fermentation in wheat straw hydrolyzates by three generations of recombinant Saccharomyces cerevisiae.

BACKGROUND: Lignocellulose hydrolyzates present difficult substrates for ethanol production by the most commonly applied microorganism in the fermentation industries, Saccharomyces cerevisiae. High resistance towards inhibitors released during pretreatment and hydrolysis of the feedstock as well as efficient utilization of hexose and pentose sugars constitute major challenges in the development of S. cerevisiae strains for biomass-to-ethanol processes. Metabolic engineering and laboratory evolution are applied, alone and in combination, to adduce desired strain properties. However, physiological requirements for robust performance of S. cerevisiae in the conversion of lignocellulose hydrolyzates are not well understood. The herein presented S. cerevisiae strains IBB10A02 and IBB10B05 are descendants of strain BP10001, which was previously derived from the widely used strain CEN.PK 113-5D through introduction of a largely redox-neutral oxidoreductive xylose assimilation pathway. The IBB strains were obtained by a two-step laboratory evolution that selected for fast xylose fermentation in combination with anaerobic growth before (IBB10A02) and after adaption in repeated xylose fermentations (IBB10B05). Enzymatic hydrolyzates were prepared from up to 15% dry mass pretreated (steam explosion) wheat straw and contained glucose and xylose in a mass ratio of approximately 2. RESULTS: With all strains, yield coefficients based on total sugar consumed were high for ethanol (0.39 to 0.40 g/g) and notably low for fermentation by-products (glycerol: ≤0.10 g/g; xylitol: ≤0.08 g/g; acetate: 0.04 g/g). In contrast to the specific glucose utilization rate that was similar for all strains (qGlucose ≈ 2.9 g/gcell dry weight (CDW)/h), the xylose consumption rate was enhanced by a factor of 11.5 (IBB10A02; qXylose = 0.23 g/gCDW/h) and 17.5 (IBB10B05; qXylose = 0.35 g/gCDW/h) as compared to the qXylose of the non-evolved strain BP10001. In xylose-supplemented (50 g/L) hydrolyzates prepared from 5% dry mass, strain IBB10B05 displayed a qXylose of 0.71 g/gCDW/h and depleted xylose in 2 days with an ethanol yield of 0.30 g/g. Under the conditions used, IBB10B05 was also capable of slow anaerobic growth. CONCLUSIONS: Laboratory evolution of strain BP10001 resulted in effectively enhanced qXylose at almost complete retention of the fermentation capabilities previously acquired by metabolic engineering. Strain IBB10B05 is a sturdy candidate for intensification of lignocellulose-to-bioethanol processes.