Abstract:Efficient saccharification of lignocellulosic biomass requires concerted development of a pretreatment method, an enzyme cocktail and an enzymatic process, all of which are adapted to the feedstock. Recent years have shown great progress in most aspects of the overall process. In particular, increased insights into the contributions of a wide variety of cellulolytic and hemicellulolytic enzymes have improved the enzymatic processing step and brought down costs. Here, we review major pretreatment technologies a… Show more
“…On land, the most abundant carbohydrate is plant biomass‐derived cellulose, a rich source of glucose in soil and herbivorous habitats. Due to the recalcitrant crystalline structure of cellulose, only specialized species possess the consortium of enzymes required to fully break it down, including oxygen‐dependent lytic polysaccharide monooxygenases (LPMOs), cellobiohydrolases (CBHs), and other GH types (Vaaje‐Kolstad et al ., 2010; Horn et al ., 2012; Østby et al ., 2020). Although Bacteroidetes are abundant in cellulose‐rich environments, and cellulolytic species are known, no PUL from an isolated species has yet been conclusively shown to target cellulose.…”
Section: Puls Are Specialized Saccharolytic Systems With Functional Homology To the Paradigmatic Starch Utilization Systemmentioning
The Bacteroidetes phylum is renowned for degradation of a wide range of complex carbohydrates, a trait that has enabled its dominance in many diverse environments. The best studied species inhabit the human gut microbiome and use Polysaccharide Utilisation Loci (PULs), discrete genetic structures that encode proteins involved in the sensing, binding, deconstruction, and import of target glycans. In many environmental species, polysaccharide degradation is tightly coupled to the phylum-exclusive Type IX Secretion System (T9SS), which is used for the secretion of certain enzymes and is linked to gliding motility. In addition, within specific species these two adaptive systems (PULs and T9SS) are intertwined, with PUL-encoded enzymes being secreted by the T9SS. Here, we discuss the most noteworthy PUL and non-PUL mechanisms that confer specific and rapid polysaccharide degradation capabilities to the Bacteroidetes in a range of environments. We also acknowledge that literature showcasing examples of PULs is rapidly expanding and developing a set of assumptions can be hard to track back to original findings. Therefore, we present a simple universal description of conserved PUL functions and how they are determined, while proposing a common nomenclature describing PULs and their components, to simplify discussion and understanding of PUL systems. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as
“…On land, the most abundant carbohydrate is plant biomass‐derived cellulose, a rich source of glucose in soil and herbivorous habitats. Due to the recalcitrant crystalline structure of cellulose, only specialized species possess the consortium of enzymes required to fully break it down, including oxygen‐dependent lytic polysaccharide monooxygenases (LPMOs), cellobiohydrolases (CBHs), and other GH types (Vaaje‐Kolstad et al ., 2010; Horn et al ., 2012; Østby et al ., 2020). Although Bacteroidetes are abundant in cellulose‐rich environments, and cellulolytic species are known, no PUL from an isolated species has yet been conclusively shown to target cellulose.…”
Section: Puls Are Specialized Saccharolytic Systems With Functional Homology To the Paradigmatic Starch Utilization Systemmentioning
The Bacteroidetes phylum is renowned for degradation of a wide range of complex carbohydrates, a trait that has enabled its dominance in many diverse environments. The best studied species inhabit the human gut microbiome and use Polysaccharide Utilisation Loci (PULs), discrete genetic structures that encode proteins involved in the sensing, binding, deconstruction, and import of target glycans. In many environmental species, polysaccharide degradation is tightly coupled to the phylum-exclusive Type IX Secretion System (T9SS), which is used for the secretion of certain enzymes and is linked to gliding motility. In addition, within specific species these two adaptive systems (PULs and T9SS) are intertwined, with PUL-encoded enzymes being secreted by the T9SS. Here, we discuss the most noteworthy PUL and non-PUL mechanisms that confer specific and rapid polysaccharide degradation capabilities to the Bacteroidetes in a range of environments. We also acknowledge that literature showcasing examples of PULs is rapidly expanding and developing a set of assumptions can be hard to track back to original findings. Therefore, we present a simple universal description of conserved PUL functions and how they are determined, while proposing a common nomenclature describing PULs and their components, to simplify discussion and understanding of PUL systems. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as
“…Through their oxidative mechanism of attacking the cellulose [ 4 – 8 ], these enzymes provide a different mode of bond breakage than the hydrolases, i.e., the exo-acting cellobiohydrolases (CBH) and the endo-acting endoglucanases (EG). The mode of interaction between LPMOs and hydrolytic enzymes, including CBHs and EGs, has been studied by several groups (e.g., [ 9 – 12 ]), as reviewed recently by Østby et al [ 13 ]. On the one hand, LPMOs, similarly to EGs, generate free chain ends upon endolytic cleavage of cellulose where CBHs can start their action and cleave off cellobiose units in a processive manner.…”
Background
Biochemical conversion of lignocellulosic biomass to simple sugars at commercial scale is hampered by the high cost of saccharifying enzymes. Lytic polysaccharide monooxygenases (LPMOs) may hold the key to overcome economic barriers. Recent studies have shown that controlled activation of LPMOs by a continuous H2O2 supply can boost saccharification yields, while overdosing H2O2 may lead to enzyme inactivation and reduce overall sugar yields. While following LPMO action by ex situ analysis of LPMO products confirms enzyme inactivation, currently no preventive measures are available to intervene before complete inactivation.
Results
Here, we carried out enzymatic saccharification of the model cellulose Avicel with an LPMO-containing enzyme preparation (Cellic CTec3) and H2O2 feed at 1 L bioreactor scale and followed the oxidation–reduction potential and H2O2 concentration in situ with corresponding electrode probes. The rate of oxidation of the reductant as well as the estimation of the amount of H2O2 consumed by LPMOs indicate that, in addition to oxidative depolymerization of cellulose, LPMOs consume H2O2 in a futile non-catalytic cycle, and that inactivation of LPMOs happens gradually and starts long before the accumulation of LPMO-generated oxidative products comes to a halt.
Conclusion
Our results indicate that, in this model system, the collapse of the LPMO-catalyzed reaction may be predicted by the rate of oxidation of the reductant, the accumulation of H2O2 in the reactor or, indirectly, by a clear increase in the oxidation–reduction potential. Being able to monitor the state of the LPMO activity in situ may help maximizing the benefit of LPMO action during saccharification. Overcoming enzyme inactivation could allow improving overall saccharification yields beyond the state of the art while lowering LPMO and, potentially, cellulase loads, both of which would have beneficial consequences on process economics.
“…Moreover, Ara f can be further substituted with a 5- O linked feruloyl residue (Fa-Ara f ) or with a complex oligomeric side chain [ 11 ]. For most applications, such as fermentation or chemical conversion, xylan requires degradation into smaller oligosaccharides or monosaccharides [ 1 – 4 , 12 , 13 ]. Enzymatic routes to xylan deconstruction employ Carbohydrate Active enZymes (CAZymes), which are classified into protein domain families by the CAZy database; www.cazy.org [ 14 ].…”
Background
Nowadays there is a strong trend towards a circular economy using lignocellulosic biowaste for the production of biofuels and other bio-based products. The use of enzymes at several stages of the production process (e.g., saccharification) can offer a sustainable route due to avoidance of harsh chemicals and high temperatures. For novel enzyme discovery, physically linked gene clusters targeting carbohydrate degradation in bacteria, polysaccharide utilization loci (PULs), are recognized ‘treasure troves’ in the era of exponentially growing numbers of sequenced genomes.
Results
We determined the biochemical properties and structure of a protein of unknown function (PUF) encoded within PULs of metagenomes from beaver droppings and moose rumen enriched on poplar hydrolysate. The corresponding novel bifunctional carbohydrate esterase (CE), now named BD-FAE, displayed feruloyl esterase (FAE) and acetyl esterase activity on simple, synthetic substrates. Whereas acetyl xylan esterase (AcXE) activity was detected on acetylated glucuronoxylan from birchwood, only FAE activity was observed on acetylated and feruloylated xylooligosaccharides from corn fiber. The genomic contexts of 200 homologs of BD-FAE revealed that the 33 closest homologs appear in PULs likely involved in xylan breakdown, while the more distant homologs were found either in alginate-targeting PULs or else outside PUL contexts. Although the BD-FAE structure adopts a typical α/β-hydrolase fold with a catalytic triad (Ser-Asp-His), it is distinct from other biochemically characterized CEs.
Conclusions
The bifunctional CE, BD-FAE, represents a new candidate for biomass processing given its capacity to remove ferulic acid and acetic acid from natural corn and birchwood xylan substrates, respectively. Its detailed biochemical characterization and solved crystal structure add to the toolbox of enzymes for biomass valorization as well as structural information to inform the classification of new CEs.
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