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Summary Conversion of nongrain biomass into liquid fuel is a sustainable approach to energy demands as global population increases. Previously, we showed that iron can act as a catalyst to enhance the degradation of lignocellulosic biomass for biofuel production. However, direct addition of iron catalysts to biomass pretreatment is diffusion‐limited, would increase the cost and complexity of biorefinery unit operations and may have deleterious environmental impacts. Here, we show a new strategy for in planta accumulation of iron throughout the volume of the cell wall where iron acts as a catalyst in the deconstruction of lignocellulosic biomass. We engineered CBM‐IBP fusion polypeptides composed of a carbohydrate‐binding module family 11 (CBM11) and an iron‐binding peptide (IBP) for secretion into Arabidopsis and rice cell walls. CBM‐IBP transformed Arabidopsis and rice plants show significant increases in iron accumulation and biomass conversion compared to respective controls. Further, CBM‐IBP rice shows a 35% increase in seed iron concentration and a 40% increase in seed yield in greenhouse experiments. CBM‐IBP rice potentially could be used to address iron deficiency, the most common and widespread nutritional disorder according to the World Health Organization.

Doc number: 495 Abstract Background: New lignocellulolytic enzymes are needed that maintain optimal activity under the harsh conditions present during industrial enzymatic deconstruction of biomass, including high temperatures, the absence of free water, and the presence of inhibitors from the biomass. Enriching lignocellulolytic microbial communities under these conditions provides a source of microorganisms that may yield robust lignocellulolytic enzymes tolerant to the extreme conditions needed to improve the throughput and efficiency of biomass enzymatic deconstruction. Identification of promising enzymes from these systems is challenging due to complex substrate-enzyme interactions and requirements to assay for activity. In this study, metatranscriptomes from compost-derived microbial communities enriched on rice straw under thermophilic and mesophilic conditions were sequenced and analyzed to identify lignocellulolytic enzymes overexpressed under thermophilic conditions. To determine differential gene expression across mesophilic and thermophilic treatments, a method was developed which pooled gene expression by functional category, as indicated by Pfam annotations, since microbial communities performing similar tasks are likely to have overlapping functions even if they share no specific genes. Results: Differential expression analysis identified enzymes from glycoside hydrolase family 48, carbohydrate binding module family 2, and carbohydrate binding module family 33 domains as significantly overexpressed in the thermophilic community. Overexpression of these protein families in the thermophilic community resulted from expression of a small number of genes not currently represented in any protein database. Genes in overexpressed protein families were predominantly expressed by a single Actinobacteria genus, Micromonospora . Conclusions: Coupling measurements of deconstructive activity with comparative analyses to identify overexpressed enzymes in lignocellulolytic communities provides a targeted approach for discovery of candidate enzymes for more efficient biomass deconstruction. Glycoside hydrolase family 48 cellulases and carbohydrate binding module family 33 polysaccharide monooxygenases with carbohydrate binding module family 2 domains may improve saccharification of lignocellulosic biomass under high-temperature and low moisture conditions relevant to industrial biofuel production.

Abstract Genome analysis of the Gram-positive cellulolytic bacterium Clostridium thermocellum revealed the presence of multiple negative regulators of alternative σ factors. Nine of the deduced proteins share a strong similarity in their N-terminal sequences to the Bacillus subtilis membrane-associated anti-σI factor RsgI and have an unusual domain organization. In six RsgI-like proteins, the C-terminal sequences contain predicted carbohydrate-binding modules. Three of these modules were overexpressed and shown to bind specifically to cellulose and/or pectin. Bioinformatic analysis of >1200 bacterial genomes revealed that the C. thermocellum RsgI-like proteins are unique to this species and are not present in other cellulolytic clostridial species (e.g. Clostridium cellulolyticum and Clostridium papyrosolvens). Eight of the nine genes encoding putative C. thermocellum RsgI-like anti-σ factors form predicted bicistronic operons, in which the first gene encodes a putative alternative σ factor, similar to B. subtilisσI, but lacking in one of its domains. These observations suggest a novel carbohydrate-sensing mechanism in C. thermocellum, whereby the presence of polysaccharide biomass components is detected extracellularly and the signal is transmitted intracellularly, resulting in the disruption of the interaction between RsgI-like proteins and σI-like factors, the latter of which serve to activate appropriate genes encoding proteins involved in cellulose utilization.

Members of the Bacteroidetes phylum in the human colon deploy an extensive number of proteins to capture and degrade polysaccharides. Operons devoted to glycan breakdown and uptake are termed polysaccharide utilization loci or PUL. The starch utilization system (Sus) is one such PUL and was initially described in Bacteroides thetaiotaomicron (Bt). BtSus is highly conserved across many species, except for its extracellular & alpha;-amylase, SusG. In this work, we show that the Bacteroides ovatus (Bo) extracellular & alpha;-amylase, BoGH13A(Sus), is distinguished from SusG in its evolutionary origin and its domain architecture and by being the most prevalent form in Bacteroidetes Sus. BoGH13A(Sus) is the founding member of both a novel subfamily in the glycoside hydrolase family 13, GH13_47, and a novel carbohydrate-binding module, CBM98. The BoGH13A(Sus) CBM98-CBM48-GH13_47 architecture differs from the CBM58 embedded within the GH13_36 of SusG. These domains adopt a distinct spatial orientation and invoke a different association with the outer membrane. The BoCBM98 binding site is required for Bo growth on polysaccharides and optimal enzymatic degradation thereof. Finally, the BoGH13A(Sus) structure features bound Ca2+ and Mn2+ ions, the latter of which is novel for an & alpha;-amylase. Little is known about the impact of Mn2+ on gut bacterial function, much less on polysaccharide consumption, but Mn2+ addition to Bt expressing BoGH13A(Sus) specifically enhances growth on starch. Further understanding of bacterial starch degradation signatures will enable more tailored prebiotic and pharmaceutical approaches that increase starch flux to the gut.

Noncatalytic carbohydrate-binding modules (CBMs), which are found in a variety of carbohydrate-degrading enzymes, have been grouped into sequence-based families. CBMs, by recruiting their appended enzymes onto the surface of the target substrate, potentiate catalysis particularly against insoluble substrates. Family 6 CBMs (CBM6s) display unusual properties in that they present two potential ligand-binding sites termed clefts A and B, respectively. Cleft B is located on the concave surface of the β-sandwich fold while cleft A, the more common binding site, is formed by the loops that connect the inner and the outer β-sheets. Here, we report the biochemical properties of CBM6-1 from Cellvibrio mixtus CmCel5A. The data reveal that CBM6-1 specifically recognizes β1,3-glucans through residues located both in cleft A and in cleft B. In contrast, a previous report showed that a CBM6 derived from a Bacillus halodurans laminarinase binds to β1,3-glucans only in cleft A. These studies reveal a different mechanism by which a highly conserved protein platform can recognize β1,3-glucans.

Anaerobic digestion of highly polymerized biomass by microbial communities present in diverse microbial ecosystems is an indispensable metabolic process for biogeochemical cycling in nature and for industrial activities required to maintain a sustainable society. Therefore, the evaluation of the complicated microbial metabolomics presents a significant challenge. We here describe a comprehensive strategy for characterizing the degradation of highly crystallized bacterial cellulose (BC) that is accompanied by metabolite production for identifying the responsible biocatalysts, including microorganisms and their metabolic functions. To this end, we employed two-dimensional solid- and one-dimensional solution-state nuclear magnetic resonance (NMR) profiling combined with a metagenomic approach using stable isotope labeling. The key components of biocatalytic reactions determined using a metagenomic approach were correlated with cellulose degradation and metabolic products. The results indicate that BC degradation was mediated by cellulases that contain carbohydrate-binding modules and that belong to structural type A. The degradation reactions induced the metabolic dynamics of the microbial community and produced organic compounds, such as acetic acid and propionic acid, mainly metabolized by clostridial species. This combinatorial, functional and structural metagenomic approach is useful for the comprehensive characterization of biomass degradation, metabolic dynamics and their key components in diverse ecosystems.

In the starch processing industry including the food and pharmaceutical industries, α-amylase is an important enzyme that hydrolyses the α-1,4 glycosidic bonds in starch, producing shorter maltooligosaccharides. In plants, starch molecules are organised in granules that are very compact and rigid. The level of starch granule rigidity affects resistance towards enzymatic hydrolysis, resulting in inefficient starch degradation by industrially available α-amylases. In an approach to enhance starch hydrolysis, the domain architecture of a Glycoside Hydrolase (GH) family 13 α-amylase from Aspergillus niger was engineered. In all fungal GH13 α-amylases that carry a carbohydrate binding domain (CBM), these modules are of the CBM20 family and are located at the C-terminus of the α-amylase domain. To explore the role of the domain order, a new GH13 gene encoding an N-terminal CBM20 domain was designed and found to be fully functional. The starch binding capacity and enzymatic activity of N-terminal CBM20 α-amylase was found to be superior to that of native GH13 without CBM20. Based on the kinetic parameters, the engineered N-terminal CBM20 variant displayed surpassing activity rates compared to the C-terminal CBM20 version for the degradation on a wide range of starches, including the more resistant raw potato starch for which it exhibits a two-fold higher Vmax underscoring the potential of domain engineering for these carbohydrate active enzymes.

Alginate lyases degrade alginate into oligosaccharides, of which the biological activities have vital roles in various fields. Some alginate lyases contain one or more carbohydrate-binding modules (CBMs), which assist the function of the catalytic modules. However, the precise function of CBMs in alginate lyases has yet to be fully elucidated. We have identified a new multi-domain alginate lyase, TsAly7B, in the marine bacterium Thalassomonas sp. LD5. This novel lyase contains an N-terminal CBM9, an internal CBM32, and a C-terminal polysaccharide lyase family 7 (PL7) catalytic module. To investigate the specific function of each of these CBMs, we expressed and characterized the full-length TsAly7B and three truncated mutants: TM1 (CBM32-PL7), TM2 (CBM9-PL7), and TM3 (PL7 catalytic module). CBM9 and CBM32 could enhance the degradation of alginate. Notably, the specific activity of TM2 was 7.6-fold higher than that of TM3. CBM32 enhanced the resistance of the catalytic module to high temperatures. In addition, a combination of CBM9 and CBM32 showed enhanced thermostability when incubated at 80 °C for 1 h. This is the first report that finds CBM9 can significantly improve the ability of enzyme degradation. Our findings provide new insight into the interrelationships of tandem CBMs and alginate lyases and other polysaccharide-degrading enzymes, which may inspire CBM fusion strategies.

One of the critical steps in lignocellulosic deconstruction is the hydrolysis of crystalline cellulose by cellulases. Endoglucanases initially facilitate the breakdown of cellulose in lignocellulosic biomass and are further aided by other cellulases to produce fermentable sugars. Furthermore, if the endoglucanase is processive, it can adsorb to the smooth surface of crystalline cellulose and release soluble sugars during repeated cycles of catalysis before dissociating. Most glycoside hydrolase family 9 (GH9) endoglucanases have catalytic domains linked to a CBM (carbohydrate-binding module) (mostly CBM3) and present the second-largest cellulase family after GH5. GH9 endoglucanases are relatively less characterized. Bacillus licheniformis is a mesophilic soil bacterium containing many glycoside hydrolase (GH) enzymes. We identified an endoglucanase gene, gh9A , encoding the GH9 family enzyme H1AD14 in B. licheniformis and cloned and overexpressed H1AD14 in Escherichia coli . The purified H1AD14 exhibited very high enzymatic activity on endoglucanase substrates, such as β-glucan, lichenan, Avicel, CMC-Na (sodium carboxymethyl cellulose) and PASC (phosphoric acid swollen cellulose), across a wide pH range. The enzyme is tolerant to 2 M sodium chloride and retains 74% specific activity on CMC after 10 days, the highest amongst the reported GH9 endoglucanases. The full-length H1AD14 is a processive endoglucanase and efficiently saccharified sugarcane bagasse. The deletion of the CBM reduces the catalytic activity and processivity. The results add to the sparse knowledge of GH9 endoglucanases and offer the possibility of characterizing and engineering additional enzymes from B. licheniformis toward developing a cellulase cocktail for improved biomass deconstruction. Key points • H1AD14 is a highly active and processive GH9 endoglucanase from B. licheniformis. • H1AD14 is thermostable and has a very long half-life. • H1AD14 showed higher saccharification efficiency than commercial endoglucanase

Enzymatic degradation of abundant renewable polysaccharides such as cellulose and starch is a field that has the attention of both the industrial and scientific community. Most of the polysaccharide degrading enzymes are classified into several glycoside hydrolase families. They are often organized in a modular manner which includes a catalytic domain connected to one or more carbohydrate-binding modules. The carbohydrate-binding modules (CBM) have been shown to increase the proximity of the enzyme to its substrate, especially for insoluble substrates. Therefore, these modules are considered to enhance enzymatic hydrolysis. These properties have played an important role in many biotechnological applications with the aim to improve the efficiency of polysaccharide degradation. The domain organization of glycoside hydrolases (GHs) equipped with one or more CBM does vary within organisms. This review comprehensively highlights the presence of CBM as ancillary modules and explores the diversity of GHs carrying one or more of these modules that actively act either on cellulose or starch. Special emphasis is given to the cellulase and amylase distribution within the filamentous microorganisms from the genera of Streptomyces and Aspergillus that are well known to have a great capacity for secreting a wide range of these polysaccharide degrading enzyme. The potential of the CBM and other ancillary domains for the design of improved polysaccharide decomposing enzymes is discussed.