Quantifying microbial robustness in dynamic environments using microfluidic single-cell cultivation.

BACKGROUND: Microorganisms must respond to changes in their environment. Analysing the robustness of functions (i.e. performance stability) to such dynamic perturbations is of great interest in both laboratory and industrial settings. Recently, a quantification method capable of assessing the robustness of various functions, such as specific growth rate or product yield, across different conditions, time frames, and populations has been developed for microorganisms grown in a 96-well plate. In micro-titer-plates, environmental change is slow and undefined. Dynamic microfluidic single-cell cultivation (dMSCC) enables the precise maintenance and manipulation of microenvironments, while tracking single cells over time using live-cell imaging. Here, we combined dMSCC and a robustness quantification method to a pipeline for assessing performance stability to changes occurring within seconds or minutes. RESULTS: Saccharomyces cerevisiae CEN.PK113-7D, harbouring a biosensor for intracellular ATP levels, was exposed to glucose feast-starvation cycles, with each condition lasting from 1.5 to 48 min over a 20 h period. A semi-automated image and data analysis pipeline was developed and applied to assess the performance and robustness of various functions at population, subpopulation, and single-cell resolution. We observed a decrease in specific growth rate but an increase in intracellular ATP levels with longer oscillation intervals. Cells subjected to 48 min oscillations exhibited the highest average ATP content, but the lowest stability over time and the highest heterogeneity within the population. CONCLUSION: The proposed pipeline enabled the investigation of function stability in dynamic environments, both over time and within populations. The strategy allows for parallelisation and automation, and is easily adaptable to new organisms, biosensors, cultivation conditions, and oscillation frequencies. Insights on the microbial response to changing environments will guide strain development and bioprocess optimisation.

Integration of subcritical water extraction and treatment with xylanases and feruloyl esterases maximises release of feruloylated arabinoxylans from wheat bran.

Wheat bran is an abundant and low valued agricultural feedstock rich in valuable biomolecules as arabinoxylans (AX) and ferulic acid with important functional and biological properties. An integrated bioprocess combining subcritical water extraction (SWE) and enzymatic treatments has been developed for maximised recovery of feruloylated arabinoxylans and oligosaccharides from wheat bran. A minimal enzymatic cocktail was developed combining one xylanase from different glycosyl hydrolase families and a feruloyl esterase. The incorporation of xylanolytic enzymes in the integrated SWE bioprocess increased the AX yields up to 75%, higher than traditional alkaline extraction, and SWE or enzymatic treatment alone. The process isolated AX with tailored molecular structures in terms of substitution, molar mass, and ferulic acid, which can be used for structural biomedical applications, food ingredients and prebiotics. This study demonstrates the use of hydrothermal and enzyme technologies for upcycling agricultural side streams into functional bioproducts, contributing to a circular food system.

Performance and robustness analysis reveals phenotypic trade-offs in yeast.

To design strains that can function efficiently in complex industrial settings, it is crucial to consider their robustness, that is, the stability of their performance when faced with perturbations. In the present study, we cultivated 24 Saccharomyces cerevisiae strains under conditions that simulated perturbations encountered during lignocellulosic bioethanol production, and assessed the performance and robustness of multiple phenotypes simultaneously. The observed negative correlations confirmed a trade-off between performance and robustness of ethanol yield, biomass yield, and cell dry weight. Conversely, the specific growth rate performance positively correlated with the robustness, presumably because of evolutionary selection for robust, fast-growing cells. The Ethanol Red strain exhibited both high performance and robustness, making it a good candidate for bioproduction in the tested perturbation space. Our results experimentally map the robustness-performance trade-offs, previously demonstrated mainly by single-phenotype and computational studies.

The lignocellulosic biorefinery concept is sound: a commentary on Zhao et al.

In the opinion paper by Zhao et al. 'Making the biochemical conversion of lignocellulose more robust', the authors claim that '…lignocellulose biorefinery is conceptually wrong'. In response, we argue that this claim itself has already been proved wrong by several companies.

Four ways of implementing robustness quantification in strain characterisation.

BACKGROUND: In industrial bioprocesses, microorganisms are generally selected based on performance, whereas robustness, i.e., the ability of a system to maintain a stable performance, has been overlooked due to the challenges in its quantification and implementation into routine experimental procedures. This work presents four ways of implementing robustness quantification during strain characterisation. One Saccharomyces cerevisiae laboratory strain (CEN.PK113-7D) and two industrial strains (Ethanol Red and PE2) grown in seven different lignocellulosic hydrolysates were assessed for growth-related functions (specific growth rate, product yields, etc.) and eight intracellular parameters (using fluorescent biosensors). RESULTS: Using flasks and high-throughput experimental setups, robustness was quantified in relation to: (i) stability of growth functions in response to the seven hydrolysates; (ii) stability of growth functions across different strains to establish the impact of perturbations on yeast metabolism; (iii) stability of intracellular parameters over time; (iv) stability of intracellular parameters within a cell population to indirectly quantify population heterogeneity. Ethanol Red was the best-performing strain under all tested conditions, achieving the highest growth function robustness. PE2 displayed the highest population heterogeneity. Moreover, the intracellular environment varied in response to non-woody or woody lignocellulosic hydrolysates, manifesting increased oxidative stress and unfolded protein response, respectively. CONCLUSIONS: Robustness quantification is a powerful tool for strain characterisation as it offers novel information on physiological and biochemical parameters. Owing to the flexibility of the robustness quantification method, its implementation was successfully validated at single-cell as well as high-throughput levels, showcasing its versatility and potential for several applications.

Arabinoxylan source and xylanase specificity influence the production of oligosaccharides with prebiotic potential.

Cereal arabinoxylans (AXs) are complex polysaccharides in terms of their pattern of arabinose and ferulic acid substitutions, which influence their properties in structural and nutritional applications. We have evaluated the influence of the molecular structure of three AXs from wheat and rye with distinct substitutions on the activity of ß-xylanases from different glycosyl hydrolase families (GH 5_34, 8, 10 and 11). The arabinose and ferulic acid substitutions influence the accessibility of the xylanases, resulting in specific profiles of arabinoxylan-oligosaccharides (AXOS). The GH10 xylanase from Aspergillus aculeatus (AcXyn10A) and GH11 from Thermomyces lanuginosus (TlXyn11) showed the highest activity, producing larger amounts of small oligosaccharides in shorter time. The GH8 xylanase from Bacillus sp. (BXyn8) produced linear xylooligosaccharides and was most restricted by arabinose substitution, whereas GH5_34 from Gonapodya prolifera (GpXyn5_34) required arabinose substitution and produced longer (A)XOS substituted on the reducing end. The complementary substrate specificity of BXyn8 and GpXyn5_34 revealed how arabinoses were distributed along the xylan backbones. This study demonstrates that AX source and xylanase specificity influence the production of oligosaccharides with specific structures, which in turn impacts the growth of specific bacteria (Bacteroides ovatus and Bifidobacterium adolescentis) and the production of beneficial metabolites (short-chain fatty acids).

ScEnSor Kit for Saccharomyces cerevisiae Engineering and Biosensor-Driven Investigation of the Intracellular Environment.

In this study, the three-step build-transform-assess toolbox for real-time monitoring of the yeast intracellular environment has been expanded and upgraded to the two-module ScEnSor (S. cerevisiae Engineering + Biosensor) Kit. The Biosensor Module includes eight fluorescent reporters for the intracellular environment; three of them (unfolded protein response, pyruvate metabolism, and ethanol consumption) were newly implemented to complement the original five. The Genome-Integration Module comprises a set of backbone plasmids for the assembly of 1-6 transcriptional units (each consisting of promoter, coding sequence, and terminator) for efficient marker-free single-locus genome integration (in HO and/or X2 loci). Altogether, the ScEnSor Kit enables rapid and easy construction of strains with new transcriptional units as well as high-throughput investigation of the yeast intracellular environment.

Structural and functional investigation of a fungal member of carbohydrate esterase family 15 with potential specificity for rare xylans.

In plant cell walls, covalent bonds between polysaccharides and lignin increase recalcitrance to degradation. Ester bonds are known to exist between glucuronic acid moieties on glucuronoxylan and lignin, and these can be cleaved by glucuronoyl esterases (GEs) from carbohydrate esterase family 15 (CE15). GEs are found in both bacteria and fungi, and some microorganisms also encode multiple GEs, although the reason for this is still not fully clear. The fungus Lentithecium fluviatile encodes three CE15 enzymes, of which two have previously been heterologously produced, although neither was active on the tested model substrate. Here, one of these, LfCE15C, has been investigated in detail using a range of model and natural substrates and its structure has been solved using X-ray crystallography. No activity could be verified on any tested substrate, but biophysical assays indicate an ability to bind to complex carbohydrate ligands. The structure further suggests that this enzyme, which possesses an intact catalytic triad, might be able to bind and act on more extensively decorated xylan chains than has been reported for other CE15 members. It is speculated that rare glucuronoxylans decorated at the glucuronic acid moiety may be the true targets of LfCE15C and other CE15 family members with similar sequence characteristics.

Investigating the role of AA9 LPMOs in enzymatic hydrolysis of differentially steam-pretreated spruce.

BACKGROUND: To realize the full potential of softwood-based forest biorefineries, the bottlenecks of enzymatic saccharification of softwood need to be better understood. Here, we investigated the potential of lytic polysaccharide monooxygenases (LPMO9s) in softwood saccharification. Norway spruce was steam-pretreated at three different severities, leading to varying hemicellulose retention, lignin condensation, and cellulose ultrastructure. Hydrolyzability of the three substrates was assessed after pretreatment and after an additional knife-milling step, comparing the efficiency of cellulolytic Celluclast + Novozym 188 and LPMO-containing Cellic CTec2 cocktails. The role of Thermoascus aurantiacus TaLPMO9 in saccharification was assessed through time-course analysis of sugar release and accumulation of oxidized sugars, as well as wide-angle X-ray scattering analysis of cellulose ultrastructural changes. RESULTS: Glucose yield was 6% (w/w) with the mildest pretreatment (steam pretreatment at 210 °C without catalyst) and 66% (w/w) with the harshest (steam pretreatment at 210 °C with 3%(w/w) SO2) when using Celluclast + Novozym 188. Surprisingly, the yield was lower with all substrates when Cellic CTec2 was used. Therefore, the conditions for optimal LPMO activity were tested and it was found that enough O2 was present over the headspace and that the reducing power of the lignin of all three substrates was sufficient for the LPMOs in Cellic CTec2 to be active. Supplementation of Celluclast + Novozym 188 with TaLPMO9 increased the conversion of glucan by 1.6-fold and xylan by 1.5-fold, which was evident primarily in the later stages of saccharification (24-72 h). Improved glucan conversion could be explained by drastically reduced cellulose crystallinity of spruce substrates upon TaLPMO9 supplementation. CONCLUSION: Our study demonstrated that LPMO addition to hydrolytic enzymes improves the release of glucose and xylose from steam-pretreated softwood substrates. Furthermore, softwood lignin provides enough reducing power for LPMOs, irrespective of pretreatment severity. These results provided new insights into the potential role of LPMOs in saccharification of industrially relevant softwood substrates.

Enzymatic debranching is a key determinant of the xylan-degrading activity of family AA9 lytic polysaccharide monooxygenases.

BACKGROUND: Previous studies have revealed that some Auxiliary Activity family 9 (AA9) lytic polysaccharide monooxygenases (LPMOs) oxidize and degrade certain types of xylans when incubated with mixtures of xylan and cellulose. Here, we demonstrate that the xylanolytic activities of two xylan-active LPMOs, TtLPMO9E and TtLPMO9G from Thermothielavioides terrestris, strongly depend on the presence of xylan substitutions. RESULTS: Using mixtures of phosphoric acid-swollen cellulose (PASC) and wheat arabinoxylan (WAX), we show that removal of arabinosyl substitutions with a GH62 arabinofuranosidase resulted in better adsorption of xylan to cellulose, and enabled LPMO-catalyzed cleavage of this xylan. Furthermore, experiments with mixtures of PASC and arabinoglucuronoxylan from spruce showed that debranching of xylan with the GH62 arabinofuranosidase and a GH115 glucuronidase promoted LPMO activity. Analyses of mixtures with PASC and (non-arabinosylated) beechwood glucuronoxylan showed that GH115 action promoted LPMO activity also on this xylan. Remarkably, when WAX was incubated with Avicel instead of PASC in the presence of the GH62, both xylan and cellulose degradation by the LPMO9 were impaired, showing that the formation of cellulose-xylan complexes and their susceptibility to LPMO action also depend on the properties of the cellulose. These debranching effects not only relate to modulation of the cellulose-xylan interaction, which influences the conformation and rigidity of the xylan, but likely also affect the LPMO-xylan interaction, because debranching changes the architecture of the xylan surface. CONCLUSIONS: Our results shed new light on xylanolytic LPMO9 activity and on the functional interplay and possible synergies between the members of complex lignocellulolytic enzyme cocktails. These findings will be relevant for the development of future lignocellulolytic cocktails and biomaterials.

Directed Evolution of (R)-2-Hydroxyglutarate Dehydrogenase Improves 2-Oxoadipate Reduction by 2 Orders of Magnitude.

Pathway engineering is commonly employed to improve the production of various metabolites but may incur in bottlenecks due to the low catalytic activity of a particular reaction step. The reduction of 2-oxoadipate to (R)-2-hydroxyadipate is a key reaction in metabolic pathways that exploit 2-oxoadipate conversion via α-reduction to produce adipic acid, an industrially important platform chemical. Here, we engineered (R)-2-hydroxyglutarate dehydrogenase from Acidaminococcus fermentans (Hgdh) with the aim of improving 2-oxoadipate reduction. Using a combination of computational analysis, saturation mutagenesis, and random mutagenesis, three mutant variants with a 100-fold higher catalytic efficiency were obtained. As revealed by rational analysis of the mutations found in the variants, this improvement could be ascribed to a general synergistic effect where mutation A206V played a key role since it boosted the enzyme's activity by 4.8-fold. The Hgdh variants with increased activity toward 2-oxoadipate generated within this study pave the way for the bio-based production of adipic acid.

Data mining of Saccharomyces cerevisiae mutants engineered for increased tolerance towards inhibitors in lignocellulosic hydrolysates.

The use of renewable plant biomass, lignocellulose, to produce biofuels and biochemicals using microbial cell factories plays a fundamental role in the future bioeconomy. The development of cell factories capable of efficiently fermenting complex biomass streams will improve the cost-effectiveness of microbial conversion processes. At present, inhibitory compounds found in hydrolysates of lignocellulosic biomass substantially influence the performance of a cell factory and the economic feasibility of lignocellulosic biofuels and chemicals. Here, we present and statistically analyze data on Saccharomyces cerevisiae mutants engineered for altered tolerance towards the most common inhibitors found in lignocellulosic hydrolysates: acetic acid, formic acid, furans, and phenolic compounds. We collected data from 7971 experiments including single overexpression or deletion of 3955 unique genes. The mutants included in the analysis had been shown to display increased or decreased tolerance to individual inhibitors or combinations of inhibitors found in lignocellulosic hydrolysates. Moreover, the data included mutants grown on synthetic hydrolysates, in which inhibitors were added at concentrations that mimicked those of lignocellulosic hydrolysates. Genetic engineering aimed at improving inhibitor or hydrolysate tolerance was shown to alter the specific growth rate or length of the lag phase, cell viability, and vitality, block fermentation, and decrease product yield. Different aspects of strain engineering aimed at improving hydrolysate tolerance, such as choice of strain and experimental set-up are discussed and put in relation to their biological relevance. While successful genetic engineering is often strain and condition dependent, we highlight the conserved role of regulators, transporters, and detoxifying enzymes in inhibitor tolerance. The compiled meta-analysis can guide future engineering attempts and aid the development of more efficient cell factories for the conversion of lignocellulosic biomass.

Quantification of Microbial Robustness in Yeast.

Stable cell performance in a fluctuating environment is essential for sustainable bioproduction and synthetic cell functionality; however, microbial robustness is rarely quantified. Here, we describe a high-throughput strategy for quantifying robustness of multiple cellular functions and strains in a perturbation space. We evaluated quantification theory on experimental data and concluded that the mean-normalized Fano factor allowed accurate, reliable, and standardized quantification. Our methodology applied to perturbations related to lignocellulosic bioethanol production showed that the industrial bioethanol producing strain Saccharomyces cerevisiae Ethanol Red exhibited both higher and more robust growth rates than the laboratory strain CEN.PK and industrial strain PE-2, while a more robust product yield traded off for lower mean levels. The methodology validated that robustness is function-specific and characterized by positive and negative function-specific trade-offs. Systematic quantification of robustness to end-use perturbations will be important to analyze and construct robust strains with more predictable functions.

Robustness: linking strain design to viable bioprocesses.

Microbial cell factories are becoming increasingly popular for the sustainable production of various chemicals. Metabolic engineering has led to the design of advanced cell factories; however, their long-term yield, titer, and productivity falter when scaled up and subjected to industrial conditions. This limitation arises from a lack of robustness - the ability to maintain a constant phenotype despite the perturbations of such processes. This review describes predictable and stochastic industrial perturbations as well as state-of-the-art technologies to counter process variability. Moreover, we distinguish robustness from tolerance and discuss the potential of single-cell studies for improving system robustness. Finally, we highlight ways of achieving consistent and comparable quantification of robustness that can guide the selection of strains for industrial bioprocesses.

Comparison of Six Lytic Polysaccharide Monooxygenases from Thermothielavioides terrestris Shows That Functional Variation Underlies the Multiplicity of LPMO Genes in Filamentous Fungi.

Lytic polysaccharide monooxygenases (LPMOs) are mono-copper enzymes that oxidatively degrade various polysaccharides. Genes encoding LPMOs in the AA9 family are abundant in filamentous fungi while their multiplicity remains elusive. We describe a detailed functional characterization of six AA9 LPMOs from the ascomycetous fungus Thermothielavioides terrestris LPH172 (syn. Thielavia terrestris). These six LPMOs were shown to be upregulated during growth on different lignocellulosic substrates in our previous study. Here, we produced them heterologously in Pichia pastoris and tested their activity on various model and native plant cell wall substrates. All six T. terrestris AA9 (TtAA9) LPMOs produced hydrogen peroxide in the absence of polysaccharide substrate and displayed peroxidase-like activity on a model substrate, yet only five of them were active on selected cellulosic substrates. TtLPMO9A and TtLPMO9E were also active on birch acetylated glucuronoxylan, but only when the xylan was combined with phosphoric acid-swollen cellulose (PASC). Another of the six AA9s, TtLPMO9G, was active on spruce arabinoglucuronoxylan mixed with PASC. TtLPMO9A, TtLPMO9E, TtLPMO9G, and TtLPMO9T could degrade tamarind xyloglucan and, with the exception of TtLPMO9T, beechwood xylan when combined with PASC. Interestingly, none of the tested enzymes were active on wheat arabinoxylan, konjac glucomannan, acetylated spruce galactoglucomannan, or cellopentaose. Overall, these functional analyses support the hypothesis that the multiplicity of the fungal LPMO genes assessed in this study relates to the complex and recalcitrant structure of lignocellulosic biomass. Our study also highlights the importance of using native substrates in functional characterization of LPMOs, as we were able to demonstrate distinct, previously unreported xylan-degrading activities of AA9 LPMOs using such substrates. IMPORTANCE The discovery of LPMOs in 2010 has revolutionized the industrial biotechnology field, mainly by increasing the efficiency of cellulolytic enzyme cocktails. Nonetheless, the biological purpose of the multiplicity of LPMO-encoding genes in filamentous fungi has remained an open question. Here, we address this point by showing that six AA9 LPMOs from a single fungal strain have various substrate preferences and activities on tested cellulosic and hemicellulosic substrates, including several native xylan substrates. Importantly, several of these activities could only be detected when using copolymeric substrates that likely resemble plant cell walls more than single fractionated polysaccharides do. Our results suggest that LPMOs have evolved to contribute to the degradation of different complex structures in plant cell walls where different biomass polymers are closely associated. This knowledge together with the elucidated novel xylanolytic activities could aid in further optimization of enzymatic cocktails for efficient degradation of lignocellulosic substrates and more.

Structure-function analysis of two closely related cutinases from Thermobifida cellulosilytica.

Cutinases can play a significant role in a biotechnology-based circular economy. However, relatively little is known about the structure-function relationship of these enzymes, knowledge that is vital to advance optimized, engineered enzyme candidates. Here, two almost identical cutinases from Thermobifida cellulosilytica DSM44535 (Thc_Cut1 and Thc_Cut2) with only 18 amino acids difference were used for a rigorous biochemical characterization of their ability to hydrolyze poly(ethylene terephthalate) (PET), PET-model substrates, and cutin-model substrates. Kinetic parameters were compared with detailed in silico docking studies of enzyme-ligand interactions. The two enzymes interacted with, and hydrolyzed PET differently, with Thc_Cut1 generating smaller PET-degradation products. Thc_Cut1 also showed higher catalytic efficiency on long-chain aliphatic substrates, an effect likely caused by small changes in the binding architecture. Thc_Cut2, in contrast, showed improved binding and catalytic efficiency when approaching the glass transition temperature of PET, an effect likely caused by longer amino acid residues in one area at the enzyme's surface. Finally, the position of the single residue Q93 close to the active site, rotated out in Thc_Cut2, influenced the ligand position of a trimeric PET-model substrate. In conclusion, we illustrate that even minor sequence differences in cutinases can affect their substrate binding, substrate specificity, and catalytic efficiency drastically.

Inhibition of LPMOs by Fermented Persimmon Juice.

Fermented persimmon juice, Kakishibu, has traditionally been used for wood and paper protection. This protective effect stems at least partially from inhibition of microbial cellulose degrading enzymes. The inhibitory effect of Kakishibu on lytic polysaccharide monooxygenases (LPMOs) and on a cocktail of cellulose hydrolases was studied, using three different cellulosic substrates. Dose dependent inhibition of LPMO activity by a commercial Kakishibu product was assessed for the well-characterized LPMO from Thermoascus aurantiacus TaAA9A, and the inhibitory effect was confirmed on five additional microbial LPMOs. The model tannin compound, tannic acid exhibited a similar inhibitory effect on TaAA9A as Kakishibu. It was further shown that both polyethylene glycol and tannase can alleviate the inhibitory effect of Kakishibu and tannic acid, indicating a likely mechanism of inhibition caused by unspecific tannin-protein interactions.

Quantifying Oxidation of Cellulose-Associated Glucuronoxylan by Two Lytic Polysaccharide Monooxygenases from Neurospora crassa.

Family AA9 lytic polysaccharide monooxygenases (LPMOs) are abundant in fungi, where they catalyze oxidative depolymerization of recalcitrant plant biomass. These AA9 LPMOs cleave cellulose and some also act on hemicelluloses, primarily other (substituted) ß-(1→4)-glucans. Oxidative cleavage of xylan has been shown for only a few AA9 LPMOs, and it remains unclear whether this activity is a minor side reaction or primary function. Here, we show that Neurospora crassa LPMO9F (NcLPMO9F) and the phylogenetically related, hitherto uncharacterized NcLPMO9L from N. crassa are active on both cellulose and cellulose-associated glucuronoxylan but not on glucuronoxylan alone. A newly developed method for simultaneous quantification of xylan-derived and cellulose-derived oxidized products showed that NcLPMO9F preferentially cleaves xylan when acting on a cellulose-beechwood glucuronoxylan mixture, yielding about three times more xylan-derived than cellulose-derived oxidized products. Interestingly, under similar conditions, NcLPMO9L and the previously characterized McLPMO9H, from Malbranchea cinnamomea, showed different xylan-to-cellulose preferences, giving oxidized product ratios of about 0.5:1 and 1:1, respectively, indicative of functional variation among xylan-active LPMOs. Phylogenetic and structural analysis of xylan-active AA9 LPMOs led to the identification of characteristic structural features, including unique features that do not occur in phylogenetically remote AA9 LPMOs, such as four AA9 LPMOs whose lack of activity toward glucuronoxylan was demonstrated in the present study. Taken together, the results provide a path toward discovery of additional xylan-active LPMOs and show that the huge family of AA9 LPMOs has members that preferentially act on xylan. These findings shed new light on the biological role and industrial potential of these fascinating enzymes. IMPORTANCE Plant cell wall polysaccharides are highly resilient to depolymerization by hydrolytic enzymes, partly due to cellulose chains being tightly packed in microfibrils that are covered by hemicelluloses. Lytic polysaccharide monooxygenases (LPMOs) seem well suited to attack these resilient copolymeric structures, but the occurrence and importance of hemicellulolytic activity among LPMOs remain unclear. Here, we show that certain AA9 LPMOs preferentially cleave xylan when acting on a cellulose-glucuronoxylan mixture, and that this ability is the result of protein evolution that has resulted in a clade of AA9 LPMOs with specific structural features. Our findings strengthen the notion that the vast arsenal of AA9 LPMOs in certain fungal species provides functional versatility and that AA9 LPMOs may have evolved to promote oxidative depolymerization of a wide variety of recalcitrant, copolymeric plant polysaccharide structures. These findings have implications for understanding the biological roles and industrial potential of LPMOs.

Phylogenetic analysis and in-depth characterization of functionally and structurally diverse CE5 cutinases.

Cutinases are esterases that release fatty acids from the apoplastic layer in plants. As they accept bulky and hydrophobic substrates, cutinases could be used in many applications, ranging from valorization of bark-rich side streams to plastic recycling. Advancement of these applications, however, requires deeper knowledge of cutinases' biodiversity and structure-function relationships. Here, we mined over 3000 members from carbohydrate esterase family 5 for putative cutinases and condensed it to 151 genes from known or putative lignocellulose-targeting organisms. The 151 genes were subjected to a phylogenetic analysis, which showed that cutinases with available crystal structures were phylogenetically closely related. We then selected nine phylogenic diverse cutinases for recombinant production and characterized their kinetic activity against para-nitrophenol substrates esterified with consecutively longer alkyl chains (pNP-C2 to C16). Each investigated cutinase had a unique activity fingerprint against the tested pNP substrates. The five enzymes with the highest activity on pNP-C12 and C16, indicative of activity on bulky hydrophobic compounds, were selected for in-depth kinetic and structure-function analysis. All five enzymes showed a decrease in kcat values with increasing substrate chain length, whereas KM values and binding energies (calculated from in silico docking analysis) improved. Two cutinases from Fusarium solani and Cryptococcus sp. exhibited outstandingly low KM values, resulting in high catalytic efficiencies toward pNP-C16. Docking analysis suggested that different clades of the phylogenetic tree may harbor enzymes with different modes of substrate interaction, involving a solvent-exposed catalytic triad, a lipase-like lid, or a clamshell-like active site possibly formed by flexible loops.

RNA sequencing reveals metabolic and regulatory changes leading to more robust fermentation performance during short-term adaptation of Saccharomyces cerevisiae to lignocellulosic inhibitors.

BACKGROUND: The limited tolerance of Saccharomyces cerevisiae to inhibitors is a major challenge in second-generation bioethanol production, and our understanding of the molecular mechanisms providing tolerance to inhibitor-rich lignocellulosic hydrolysates is incomplete. Short-term adaptation of the yeast in the presence of dilute hydrolysate can improve its robustness and productivity during subsequent fermentation. RESULTS: We utilized RNA sequencing to investigate differential gene expression in the industrial yeast strain CR01 during short-term adaptation, mimicking industrial conditions for cell propagation. In this first transcriptomic study of short-term adaption of S. cerevisiae to lignocellulosic hydrolysate, we found that cultures respond by fine-tuned up- and down-regulation of a subset of general stress response genes. Furthermore, time-resolved RNA sequencing allowed for identification of genes that were differentially expressed at 2 or more sampling points, revealing the importance of oxidative stress response, thiamin and biotin biosynthesis. furan-aldehyde reductases and specific drug:H+ antiporters, as well as the down-regulation of certain transporter genes. CONCLUSIONS: These findings provide a better understanding of the molecular mechanisms governing short-term adaptation of S. cerevisiae to lignocellulosic hydrolysate, and suggest new genetic targets for improving fermentation robustness.