Survey of the genome of opitutus terrae and other organisms for novel carbohydrate-active enzyme specificities

Lignocellulose is the major component of the plant cell wall and is a sustainable source of inexpensive abundant biomass. Efficient degradation of the lignocellulosic polysaccharides, cellulose and hemicellulose, is required if the plant cell wall is to be used as a resource for renewable biofuels. Microorganisms have the ability to catalyse the degradation of such bio-material through a cascade of enzyme activities into fermentable sugars and therefore are considered to be a major resource of biocatalysts for the emerging biofuel industry. The stability of the component polysaccharides and the complexity of the plant cell wall are reflected in the diverse range of functions and substrate specificities of lignocellulosic degrading enzymes. Part of this work describes the identification of a novel GH8 endo-xylanase, OtXyn8A, from the soil bacterium, Opitutus terrae. GH8 is a family in which there is only a limited amount of data available on the xylanase substrate specificity in comparison to families GH10 and GH11 in which xylanases are well established. With this in mind, OtXyn8A is the only endo-xylanase characterised from GH8 that primarily releases xylobiose from its substrates. Synergy between O. terrae enzymes was partially investigated within this study with the identification of a gene cluster within the bacterial genome. Genes organised within this cluster encoded products required for the degradation of xylan substrates and so the associated enzymes were cloned, expressed and subsequently determined for activity. Combined activities of gene products from the cluster exhibited synergy in the hydrolysis of 4-O-methyl glucuronoxylan. While surveying the genome of O. terrae, the multiplication of genes encoding GH43 enzymes was also investigated. Genes encoding GH43 enzymes were cloned, expressed and investigated for catalytic activity. Three arabinofuranosidases from O. terrae and one from Lactobacillus brevis were characterised, including the characterisation of an exo-1,5-L-arabinofuranosidase. Furthermore, a β-xylosidase from O. terrae was characterised which exhibited dual functionality as it catalysed the release of arabinose in addition to xylose from arabino-xylooligosaccharides

Mechanoenzymatic reactions for the hydrolysis of PET

Recent advances in the enzymatic degradation of poly(ethylene terphthalate) (PET) have led to a number of PET hydrolytic enzymes and mutants being developed. With the amount of PET building up in the natural world, there is a pressing need to develop scalable methods of breaking down the polymer into its monomers for recycling or other uses. Mechanoenzymatic reactions have gained traction recently as a green and efficient alternative to traditional biocatalytic reactions. For the first time we report increased yields of PET degradation by whole cell PETase enzymes by up to 27-fold by utilising ball milling cycles of reactive aging, when compared with typical solution-based reactions. This methodology leads to up to a 2600-fold decrease in the solvent required when compared with other leading degradation reactions in the field and a 30-fold decrease in comparison to reported industrial scale PET hydrolysis reactions

Design and use of de novo cascades for the biosynthesis of new benzylisoquinoline alkaloids

The benzylisoquinoline alkaloids (BIAs) are an important group of secondary metabolites from higher plants and have been reported to show significant biological activities. The production of BIAs through synthetic biology approaches provides a higher‐yielding strategy than traditional synthetic methods or isolation from plant material. However, the reconstruction of BIA pathways in microorganisms by combining heterologous enzymes can also give access to BIAs through cascade reactions. Most importantly, non‐natural BIAs can be generated through such artificial pathways. In the current study, we describe the use of tyrosinases and decarboxylases and combine these with a transaminase enzyme and norcoclaurine synthase for the efficient synthesis of several BIAs, including six non‐natural alkaloids, in cascades from l‐tyrosine and analogues

An Integrated Biorefinery Concept for Conversion of Sugar Beet Pulp into Value-added Chemicals and Pharmaceutical Intermediates

Over 8 million tonnes of sugar beet are grown annually in the UK. Sugar beet pulp (SBP) is the main by-product of sugar beet processing which is currently dried and sold as a low value animal feed. SBP is a rich source of carbohydrates, mainly in the form of cellulose and pectin, including D-glucose (Glu), L-arabinose (Ara) and D-galacturonic acid (GalAc). This work describes the technical feasibility of an integrated biorefinery concept for fractionation of SBP and conversion of these monosaccharides into value-added products. SBP fractionation is initially carried out by steam explosion under mild conditions to yield soluble pectin and insoluble cellulose fractions. The cellulose is readily hydrolysed by cellulases to release Glu that can then be fermented by a commercial Yeast strain to produce bioethanol with a high yield. The pectin fraction can be either fully hydrolysed, using physico-chemical methods, or selectively hydrolysed, using cloned arabinases and galacturonases, to yield Ara-rich and GalAc-rich streams. These monomers can be separated using either Centrifugal Partition Chromatography (CPC) or ultrafiltration into streams suitable for subsequent enzymatic upgrading. Building on our previous experience with transketolase (TK) and transaminase (TAm) enzymes, the conversion of Ara and GalAc into higher value products was explored. In particular the conversion of Ara into L-gluco-heptulose (GluHep), that has potential therapeutic applications in hypoglycaemia and cancer, using a mutant TK is described. Preliminary studies with TAm also suggest GluHep can be selectively aminated to the corresponding chiral aminopolyol. Current work is addressing upgrading of the remaining SBP monomer, GalAc, and modelling of the biorefinery concept to enable economic and Life Cycle Analysis (LCA)

First observation of Chapman rearrangement of a pseudosaccharyl ether in the solid state: the thermal isomerization of 3-(methoxy)-1,2-benzisothiazole 1,1-dioxide revisited

3-(Methoxy)-1,2-benzisothiazole 1,1-dioxide, a pseudosaccharyl ether, was long ago known to undergo a thermal Chapman-like [1,3′]-isomerization to the corresponding N-methyl pseudosaccharin at temperatures above its melting point (ca. 184 °C) [Hettler H., Tetrahedron Lett.1968, 15, 1793]. In the present study, it is shown that this rearrangement can also take place in the solid state, at temperatures as low as 150 °C. This was the first observation of a Chapman-like [1,3′]-isomerization in pseudosaccharyl ethers in the solid state. The study has been carried out by a multidisciplinary approach using temperature dependent infrared spectroscopy, differential scanning calorimetry (DSC), and polarized light thermomicroscopy, complemented by theoretical methods. Graphical abstrac

Y Chromosome Sequences Reveal a Short Beringian Standstill, Rapid Expansion, and early Population structure of Native American Founders

The Americas were the last inhabitable continents to be occupied by humans, with a growing multidisciplinary consensus for entry 15-25 thousand years ago (kya) from northeast Asia via the former Beringia land bridge [1-4]. Autosomal DNA analyses have dated the separation of Native American ancestors from the Asian gene pool to 23 kya or later [5, 6] and mtDNA analyses to ∼25 kya [7], followed by isolation ("Beringian Standstill" [8, 9]) for 2.4-9 ky and then a rapid expansion throughout the Americas. Here, we present a calibrated sequence-based analysis of 222 Native American and relevant Eurasian Y chromosomes (24 new) from haplogroups Q and C [10], with four major conclusions. First, we identify three to four independent lineages as autochthonous and likely founders: the major Q-M3 and rarer Q-CTS1780 present throughout the Americas, the very rare C3-MPB373 in South America, and possibly the C3-P39/Z30536 in North America. Second, from the divergence times and Eurasian/American distribution of lineages, we estimate a Beringian Standstill duration of 2.7 ky or 4.6 ky, according to alternative models, and entry south of the ice sheet after 19.5 kya. Third, we describe the star-like expansion of Q-M848 (within Q-M3) starting at 15 kya [11] in the Americas, followed by establishment of substantial spatial structure in South America by 12 kya. Fourth, the deep branches of the Q-CTS1780 lineage present at low frequencies throughout the Americas today [12] may reflect a separate out-of-Beringia dispersal after the melting of the glaciers at the end of the Pleistocene.status: publishe

Y chromosome sequences reveal a short beringian standstill, rapid expansion, and early population structure of native american founders

The Americas were the last inhabitable continents to be occupied by humans, with a growing multidisciplinary consensus for entry 15–25 thousand years ago (kya) from northeast Asia via the former Beringia land bridge [1, 2, 3, 4]. Autosomal DNA analyses have dated the separation of Native American ancestors from the Asian gene pool to 23 kya or later [5, 6] and mtDNA analyses to ∼25 kya [7], followed by isolation (“Beringian Standstill” [8, 9]) for 2.4–9 ky and then a rapid expansion throughout the Americas. Here, we present a calibrated sequence-based analysis of 222 Native American and relevant Eurasian Y chromosomes (24 new) from haplogroups Q and C [10], with four major conclusions. First, we identify three to four independent lineages as autochthonous and likely founders: the major Q-M3 and rarer Q-CTS1780 present throughout the Americas, the very rare C3-MPB373 in South America, and possibly the C3-P39/Z30536 in North America. Second, from the divergence times and Eurasian/American distribution of lineages, we estimate a Beringian Standstill duration of 2.7 ky or 4.6 ky, according to alternative models, and entry south of the ice sheet after 19.5 kya. Third, we describe the star-like expansion of Q-M848 (within Q-M3) starting at 15 kya [11] in the Americas, followed by establishment of substantial spatial structure in South America by 12 kya. Fourth, the deep branches of the Q-CTS1780 lineage present at low frequencies throughout the Americas today [12] may reflect a separate out-of-Beringia dispersal after the melting of the glaciers at the end of the Pleistocene