Engineering a thermoregulated intein-modified xylanase into maize for consolidated lignocellulosic biomass processing.

Plant cellulosic biomass is an abundant, low-cost feedstock for producing biofuels and chemicals. Expressing cell wall-degrading (CWD) enzymes (e.g. xylanases) in plant feedstocks could reduce the amount of enzymes required for feedstock pretreatment and hydrolysis during bioprocessing to release soluble sugars. However, in planta expression of xylanases can reduce biomass yield and plant fertility. To overcome this problem, we engineered a thermostable xylanase (XynB) with a thermostable self-splicing bacterial intein to control the xylanase activity. Intein-modified XynB (iXynB) variants were selected that have <10% wild-type enzymatic activity but recover >60% enzymatic activity upon intein self-splicing at temperatures >59 °C. Greenhouse-grown xynB maize expressing XynB has shriveled seeds and low fertility, but ixynB maize had normal seeds and fertility. Processing dried ixynB maize stover by temperature-regulated xylanase activation and hydrolysis in a cocktail of commercial CWD enzymes produced >90% theoretical glucose and >63% theoretical xylose yields.

Global and grain-specific accumulation of glycoside hydrolase family 10 xylanases in transgenic maize (Zea mays).

In planta expression of cell wall degrading enzymes is a promising approach for developing optimized biomass feedstocks that enable low-cost cellulosic biofuels production. Transgenic plants could serve as either an enzyme source for the hydrolysis of pretreated biomass or as the primary biomass feedstock in an autohydrolysis process. In this study, two xylanase genes, Bacillus sp. NG-27 bsx and Clostridium stercorarium xynB, were expressed in maize (Zea mays) under the control of two different promoters. Severe phenotypic effects were associated with xylanase accumulation in maize, including stunted plants and sterile grains. Global expression of these xylanases from the rice ubiquitin 3 promoter (rubi3) resulted in enzyme accumulation of approximately 0.01 mg enzyme per gram dry weight, or approximately 0.1% of total soluble protein (TSP). Grain-specific expression of these enzymes from the rice glutelin 4 promoter (GluB-4) resulted in higher-level accumulation of active enzyme, with BSX and XynB accumulating up to 4.0% TSP and 16.4% TSP, respectively, in shriveled grains from selected T0 plants. These results demonstrate the potential utility of the GluB-4 promoter for biotechnological applications. The phenotypic effects of xylanase expression in maize presented here demonstrate the difficulties of hemicellulase expression in an important crop for cellulosic biofuels production. Potential alternate approaches to achieve xylanase accumulation in planta without the accompanying negative phenotypes are discussed.

The complete genome of Teredinibacter turnerae T7901: an intracellular endosymbiont of marine wood-boring bivalves (shipworms).

Here we report the complete genome sequence of Teredinibacter turnerae T7901. T. turnerae is a marine gamma proteobacterium that occurs as an intracellular endosymbiont in the gills of wood-boring marine bivalves of the family Teredinidae (shipworms). This species is the sole cultivated member of an endosymbiotic consortium thought to provide the host with enzymes, including cellulases and nitrogenase, critical for digestion of wood and supplementation of the host's nitrogen-deficient diet. T. turnerae is closely related to the free-living marine polysaccharide degrading bacterium Saccharophagus degradans str. 2-40 and to as yet uncultivated endosymbionts with which it coexists in shipworm cells. Like S. degradans, the T. turnerae genome encodes a large number of enzymes predicted to be involved in complex polysaccharide degradation (>100). However, unlike S. degradans, which degrades a broad spectrum (>10 classes) of complex plant, fungal and algal polysaccharides, T. turnerae primarily encodes enzymes associated with deconstruction of terrestrial woody plant material. Also unlike S. degradans and many other eubacteria, T. turnerae dedicates a large proportion of its genome to genes predicted to function in secondary metabolism. Despite its intracellular niche, the T. turnerae genome lacks many features associated with obligate intracellular existence (e.g. reduced genome size, reduced %G+C, loss of genes of core metabolism) and displays evidence of adaptations common to free-living bacteria (e.g. defense against bacteriophage infection). These results suggest that T. turnerae is likely a facultative intracellular ensosymbiont whose niche presently includes, or recently included, free-living existence. As such, the T. turnerae genome provides insights into the range of genomic adaptations associated with intracellular endosymbiosis as well as enzymatic mechanisms relevant to the recycling of plant materials in marine environments and the production of cellulose-derived biofuels.

Complete genome sequence of the complex carbohydrate-degrading marine bacterium, Saccharophagus degradans strain 2-40 T.

The marine bacterium Saccharophagus degradans strain 2-40 (Sde 2-40) is emerging as a vanguard of a recently discovered group of marine and estuarine bacteria that recycles complex polysaccharides. We report its complete genome sequence, analysis of which identifies an unusually large number of enzymes that degrade >10 complex polysaccharides. Not only is this an extraordinary range of catabolic capability, many of the enzymes exhibit unusual architecture including novel combinations of catalytic and substrate-binding modules. We hypothesize that many of these features are adaptations that facilitate depolymerization of complex polysaccharides in the marine environment. This is the first sequenced genome of a marine bacterium that can degrade plant cell walls, an important component of the carbon cycle that is not well-characterized in the marine environment.

CelAB, a multifunctional cellulase encoded by Teredinibacter turnerae T7902T, a culturable symbiont isolated from the wood-boring marine bivalve Lyrodus pedicellatus.

We characterized a multifunctional cellulase (CelAB) encoded by the endosymbiont Teredinibacter turnerae T7902(T). CelAB contains two catalytic and two carbohydrate-binding domains, each separated by polyserine linker regions. CelAB binds cellulose and chitin, degrades multiple complex polysaccharides, and displays two catalytic activities, cellobiohydrolase (EC 3.2.1.91) and beta-1,4(3) endoglucanase (EC 3.2.1.4).

Genomic and proteomic analyses of the agarolytic system expressed by Saccharophagus degradans 2-40.

Saccharophagus degradans 2-40 (formerly Microbulbifer degradans 2-40) is a marine gamma-subgroup proteobacterium capable of degrading many complex polysaccharides, such as agar. While several agarolytic systems have been characterized biochemically, the genetics of agarolytic systems have been only partially determined. By use of genomic, proteomic, and genetic approaches, the components of the S. degradans 2-40 agarolytic system were identified. Five agarases were identified in the S. degradans 2-40 genome. Aga50A and Aga50D include GH50 domains. Aga86C and Aga86E contain GH86 domains, whereas Aga16B carries a GH16 domain. Novel family 6 carbohydrate binding modules (CBM6) were identified in Aga16B and Aga86E. Aga86C has an amino-terminal acylation site, suggesting that it is surface associated. Aga16B, Aga86C, and Aga86E were detected by mass spectrometry in agarolytic fractions obtained from culture filtrates of agar-grown cells. Deletion analysis revealed that aga50A and aga86E were essential for the metabolism of agarose. Aga16B was shown to endolytically degrade agarose to release neoagarotetraose, similarly to a beta-agarase I, whereas Aga86E was demonstrated to exolytically degrade agarose to form neoagarobiose. The agarolytic system of S. degradans 2-40 is thus predicted to be composed of a secreted endo-acting GH16-dependent depolymerase, a surface-associated GH50-dependent depolymerase, an exo-acting GH86-dependent agarase, and an alpha-neoagarobiose hydrolase to release galactose from agarose.

Complete cellulase system in the marine bacterium Saccharophagus degradans strain 2-40T.

Saccharophagus degradans strain 2-40 is a representative of an emerging group of marine complex polysaccharide (CP)-degrading bacteria. It is unique in its metabolic versatility, being able to degrade at least 10 distinct CPs from diverse algal, plant and invertebrate sources. The S. degradans genome has been sequenced to completion, and more than 180 open reading frames have been identified that encode carbohydrases. Over half of these are likely to act on plant cell wall polymers. In fact, there appears to be a full array of enzymes that degrade and metabolize plant cell walls. Genomic and proteomic analyses reveal 13 cellulose depolymerases complemented by seven accessory enzymes, including two cellodextrinases, three cellobiases, a cellodextrin phosphorylase, and a cellobiose phosphorylase. Most of these enzymes exhibit modular architecture, and some contain novel combinations of catalytic and/or substrate binding modules. This is exemplified by endoglucanase Cel5A, which has three internal family 6 carbohydrate binding modules (CBM6) and two catalytic modules from family five of glycosyl hydrolases (GH5) and by Cel6A, a nonreducing-end cellobiohydrolase from family GH6 with tandem CBM2s. This is the first report of a complete and functional cellulase system in a marine bacterium with a sequenced genome.

Family 6 carbohydrate binding modules in beta-agarases display exquisite selectivity for the non-reducing termini of agarose chains.

Carbohydrate recognition is central to the biological and industrial exploitation of plant structural polysaccharides. These insoluble polymers are recalcitrant to microbial degradation, and enzymes that catalyze this process generally contain non-catalytic carbohydrate binding modules (CBMs) that potentiate activity by increasing substrate binding. Agarose, a repeat of the disaccharide 3,6-anhydro-alpha-L-galactose-(1,3)-beta-D-galactopyranose-(1,4), is the dominant matrix polysaccharide in marine algae, yet the role of CBMs in the hydrolysis of this important polymer has not previously been explored. Here we show that family 6 CBMs, present in two different beta-agarases, bind specifically to the non-reducing end of agarose chains, recognizing only the first repeat of the disaccharide. The crystal structure of one of these modules Aga16B-CBM6-2, in complex with neoagarohexaose, reveals the mechanism by which the protein displays exquisite specificity, targeting the equatorial O4 and the axial O3 of the anhydro-L-galactose. Targeting of the CBM6 to the non-reducing end of agarose chains may direct the appended catalytic modules to areas of the plant cell wall attacked by beta-agarases where the matrix polysaccharide is likely to be more amenable to further enzymic hydrolysis.

Saccharophagus degradans gen. nov., sp. nov., a versatile marine degrader of complex polysaccharides.

Gammaproteobacteria belonging and related to the genus Microbulbifer are an emerging group of complex carbohydrate-degrading marine bacteria. Previously, all of the representatives were placed within Microbulbifer or were unclassified. Recently, a new genus, Teredinibacter, represented by a single species, Teredinibacter turnerae, was formed to include an endosymbiotic branch of these organisms. In this study, based on 16S rRNA gene sequence similarity and phenotypic analyses, a new genus, Saccharophagus, is proposed to accommodate the most versatile marine carbohydrate degrader yet identified, Saccharophagus degradans gen. nov., sp. nov. 2-40(T) (=ATCC 43961(T)=DSM 17024(T)). S. degradans strain 2-40(T) can degrade 10 tested complex polysaccharides: agar, alginate, chitin, cellulose, fucoidan, laminarin, pectin, pullulan, starch and xylan. S. degradans 2-40(T) shares 90.5% 16S rRNA gene sequence similarity with the type strain of the Microbulbifer type species, Microbulbifer hydrolyticus IRE-31(T), and 91.5% with T. turnerae T7902(T), and can be further distinguished from members of these two genera by 16S rRNA gene cluster analysis, the ability to utilize 10 different complex polysaccharides as sole carbon sources, a significantly lower G+C content and differences in fatty acid content. The three genera of complex polysaccharide-degrading, marine bacteria now encompass 20 strains from diverse marine niches.

Identification and analysis of polyserine linker domains in prokaryotic proteins with emphasis on the marine bacterium Microbulbifer degradans.

Polyserine linkers (PSLs) are interdomain, serine-rich sequences found in modular proteins. Though common among eukaryotes, their presence in prokaryotic enzymes is limited. We identified 46 extracellular proteins involved in complex carbohydrate degradation from Microbulbifer degradans that contain PSLs that separate carbohydrate-binding domains or catalytic domains from other binding domains. In nine M. degradans proteins, PSLs also separated amino-terminal lipoprotein acylation sites from the remainder of the polypeptide. Furthermore, among the 76 PSL proteins identified in sequence repositories, 65 are annotated as proteins involved in complex carbohydrate degradation. We discuss the notion that PSLs are flexible, disordered spacer regions that enhance substrate accessibility.

Chitinase B of "Microbulbifer degradans" 2-40 contains two catalytic domains with different chitinolytic activities.

Chitinase B of "Microbulbifer degradans" 2-40 is a modular protein that is predicted to contain two glycoside hydrolase family 18 (GH18) catalytic domains, two polyserine domains, and an acidic repeat domain. Each of the GH18 domains was shown to be catalytically active against chitin. Activity assays reveal that the amino-terminal catalytic domain (GH18(N)) releases methylumbelliferone from 4'-methylumbelliferyl-N,N'-diacetylchitobiose 13.6-fold faster than the carboxy-terminal catalytic domain (GH18(C)) and releases chitobiose from the nonreducing end of chitooligosaccharides, therefore functioning as an exochitinase. GH18(C) releases methylumbelliferone from 4'-methylumbelliferyl-N,N',N"-triacetylchitotriose 2.7-fold faster than GH18(N) and cleaves chitooligosaccharides at multiple bonds, consistent with endochitinolytic activity. Each domain was maximally active from 30 to 37 degrees C and from pH 7.2 to 8.0 and was not affected by Mg(2+), Mn(2+), Ca(2+), K(+), EDTA, EGTA, or 1.0 M NaCl. The activity of each domain was moderately inhibited by Ni(2+), Sr(2+), and Cu(2+), while Hg(2+) completely abolished activity. When the specific activities of various recombinant portions of ChiB were calculated by using native chitin as a substrate, the polypeptide containing the endo-acting domain was twofold more active on native chitin than the other containing the exo-acting domain. The presence of both domains in a single reaction increased the amount of reducing sugars released from native chitin to 140% above the theoretical combined rate, indicating that the domains function cooperatively to degrade chitin. These data demonstrate that the GH18 domains of ChiB have different activities on the same substrate and function cooperatively to enhance chitin depolymerization.

Detection and characterization of chitinases and other chitin-modifying enzymes.

Multiple industrial and medical uses of chitin and its derivatives have been developed in recent years. The demand for enzymes with new or desirable properties continues to grow as additional uses of chitin, chitooligosaccharides, and chitosan become apparent. Microorganisms, the primary degraders of chitin in the environment, are a rich source of valuable chitin-modifying enzymes. This review summarizes many methods that can be used to isolate and characterize chitin-modifying enzymes including chitin depolymerases, chitodextrinases, chitin deacetylases, N-acetylglucosaminidases, chitin-binding proteins, and chitosanases. Chitin analogs, zymography, detection of reducing sugars, genomic library screening, chitooligosaccharide electrophoresis, degenerate PCR primer design, thin layer chromatography, and chitin-binding assays are discussed.

Genomic analysis and initial characterization of the chitinolytic system of Microbulbifer degradans strain 2-40.

The marine bacterium Microbulbifer degradans strain 2-40 produces at least 10 enzyme systems for degrading insoluble complex polysaccharides (ICP). The draft sequence of the 2-40 genome allowed a genome-wide analysis of the chitinolytic system of strain 2-40. The chitinolytic system includes three secreted chitin depolymerases (ChiA, ChiB, and ChiC), a secreted chitin-binding protein (CbpA), periplasmic chitooligosaccharide-modifying enzymes, putative sugar transporters, and a cluster of genes encoding cytoplasmic proteins involved in N-acetyl-D-glucosamine (GlcNAc) metabolism. Each chitin depolymerase was detected in culture supernatants of chitin-grown strain 2-40 and was active against chitin and glycol chitin. The chitin depolymerases also had a specific pattern of activity toward the chitin analogs 4-methylumbelliferyl-beta-D-N,N'-diacetylchitobioside (MUF-diNAG) and 4-methylumbelliferyl-beta-D-N,N',N"-triacetylchitotrioside (MUF-triNAG). The depolymerases were modular in nature and contained glycosyl hydrolase family 18 domains, chitin-binding domains, and polycystic kidney disease domains. ChiA and ChiB each possessed polyserine linkers of up to 32 consecutive serine residues. In addition, ChiB and CbpA contained glutamic acid-rich domains. At 1,271 amino acids, ChiB is the largest bacterial chitinase reported to date. A chitodextrinase (CdxA) with activity against chitooligosaccharides (degree of polymerization of 5 to 7) was identified. The activities of two apparent periplasmic (HexA and HexB) N-acetyl-beta-D-glucosaminidases and one cytoplasmic (HexC) N-acetyl-beta-D-glucosaminidase were demonstrated. Genes involved in GlcNAc metabolism, similar to those of the Escherichia coli K-12 NAG utilization operon, were identified. NagA from strain 2-40, a GlcNAc deacetylase, was shown to complement a nagA mutation in E. coli K-12. Except for the GlcNAc utilization cluster, genes for all other components of the chitinolytic system were dispersed throughout the genome. Further examination of this system may provide additional insight into the mechanisms by which marine bacteria degrade chitin and provide a basis for future research on the ICP-degrading systems of strain 2-40.