Adele Kim Williamson

Biochemical Characterization of a Family 15 Carbohydrate Esterase from a Bacterial Marine Arctic Metagenome.

Background The glucuronoyl esterase enzymes of wood-degrading fungi (Carbohydrate Esterase family 15; CE15) form part of the hemicellulolytic and cellulolytic enzyme systems that break down plant biomass, and have possible applications in biotechnology. Homologous enzymes are predicted in the genomes of several bacteria, however these have been much less studied than their fungal counterparts. Here we describe the recombinant production and biochemical characterization of a bacterial CE15 enzyme denoted MZ0003, which was identified by in silico screening of a prokaryotic metagenome library derived from marine Arctic sediment. MZ0003 has high similarity to several uncharacterized gene products of polysaccharide-degrading bacterial species, and phylogenetic analysis indicates a deep evolutionary split between these CE15s and fungal homologs. Results MZ0003 appears to differ from previously-studied CE15s in some aspects. Some glucuronoyl esterase activity could be measured by qualitative thin-layer chromatography which confirms its assignment as a CE15, however MZ0003 can also hydrolyze a range of other esters, including p-nitrophenyl acetate, which is not acted upon by some fungal homologs. The structure of MZ0003 also appears to differ as it is predicted to have several large loop regions that are absent in previously studied CE15s, and a combination of homology-based modelling and site-directed mutagenesis indicate its catalytic residues deviate from the conserved Ser-His-Glu triad of many fungal CE15s. Taken together, these results indicate that potentially unexplored diversity exists among bacterial CE15s, and this may be accessed by investigation of the microbial metagenome. The combination of low activity on typical glucuronoyl esterase substrates, and the lack of glucuronic acid esters in the marine environment suggest that the physiological substrate of MZ0003 and its homologs is likely to be different from that of related fungal enzymes.

Recombinant expression and purification of an ATP-dependent DNA ligase from Aliivibrio salmonicida

The genome of the psychrophilic fish-pathogen Aliivibrio salmonicida encodes a putative ATP-dependent DNA ligase in addition to a housekeeping NAD-dependent enzyme. In order to study the structure and activity of the ATP dependent ligase in vitro we have undertaken its recombinant production and purification from an Escherichia coli based expression system. Expression and purification of this protein presented two significant challenges. First, the gene product was moderately toxic to E. coli cells, second it was necessary to remove the large amounts of E. coli DNA present in bacterial lysates without contamination of the protein preparation by nucleases which might interfere with future assaying. The toxicity problem was overcome by fusion of the putative ligase to large solubility tags such as maltose-binding protein (MBP) or Glutathione-S-transferase (GST), and DNA was removed by treatment with a nuclease which could be inhibited by reducing agents. As the A. salmonicida ATP-dependent DNA ligase gene encodes a predicted leader peptide, both the full-length and mature forms of the protein were produced. Both possessed ATP-dependent DNA ligase activity, but the truncated form was significantly more active. Here we detail the first reported production, purification and preliminary characterization of active A. salmonicida ATP-dependent DNA ligase.

Cold shock induction of recombinant Arctic environmental genes

BackgroundHeterologous expression of psychrophilic enzymes in E. coli is particularly challenging due to their intrinsic instability. The low stability is regarded as a consequence of adaptation that allow them to function at low temperatures. Recombinant production presents a significant barrier to their exploitation for commercial applications in industry.MethodsAs part of an enzyme discovery project we have investigated the utility of a cold-shock inducible promoter for low-temperature expression of five diverse genes derived from the metagenomes of marine Arctic sediments. After evaluation of their production, we further optimized for soluble production by building a vector suite from which the environmental genes could be expressed as fusions with solubility tags.ResultsWe found that the low-temperature optimized system produced high expression levels for all putatively cold-active proteins, as well as reducing host toxicity for several candidates. As a proof of concept, activity assays with one of the candidates, a putative chitinase, showed that functional protein was obtained using the low-temperature optimized vector suite.ConclusionsWe conclude that a cold-shock inducible system is advantageous for the heterologous expression of psychrophilic proteins, and may also be useful for expression of toxic mesophilic and thermophilic proteins where properties of the proteins are deleterious to the host cell growth.

Enzyme-adenylate structure of a bacterial ATP-dependent DNA ligase with a minimized DNA-binding surface

The enzyme–adenylate structure of a bacterial ATP-dependent DNA ligase (ADL), which does not have any additional DNA-binding domains, is similar to minimal viral ADLs that comprise only the core catalytic domains. The bacterial ADL also lacks the unstructured loops which are involved in DNA binding in the viral ADLs, implying that it must instead use short well structured motifs of the core domains to engage its substrate.

Draft Genome Sequence of the Actinomycete Rhodococcus sp. Strain AW25M09, Isolated from the Hadsel Fjord, Northern Norway.

ABSTRACT The cold-adapted Rhodococcus sp. strain AW25M09 was isolated from an Atlantic hagfish caught off the shore of northern Norway as part of an ongoing bioprospecting project that aims to identify novel bacteria with biotechnological potential. Here, we present the 5.8-Mb draft genome sequence, together with details regarding the origin of the strain and its sequence assembly.

Engineering low-temperature expression systems for heterologous production of cold-adapted enzymes

ABSTRACT Production of psychrophilic enzymes in the commonly used mesophilic expression systems is hampered by low intrinsic stability of the recombinant enzymes at the optimal host growth temperatures. Unless strategies for low-temperature expression are advanced, research on psychrophilic enzymes may end up being biased toward those that can be stably produced in commonly used mesophilic host systems. Two main strategies are currently being explored for the development of low-temperature expression in bacterial hosts: (i) low-temperature adaption of existing mesophilic expression systems, and (ii) development of new psychrophilic hosts. These developments include genetic engineering of the expression cassettes to optimize the promoter/operator systems that regulate heterologous expression. In this addendum we present our efforts in the development of such low-temperature expression systems, and speculate about future advancements in the field and potential applications.

High quality draft genome sequence of Streptomyces sp. strain AW19M42 isolated from a sea squirt in Northern Norway

Here we report the 8 Mb high quality draft genome of Streptomyces sp. strain AW19M42, together with specific properties of the organism and the generation, annotation and analysis of its genome sequence. The genome encodes 7,727 putative open reading frames, of which 6,400 could be assigned with COG categories. Also, 62 tRNA genes and 8 rRNA operons were identified. The genome harbors several gene clusters involved in the production of secondary metabolites. Functional screening of the isolate was positive for several enzymatic activities, and some candidate genes coding for those activities are listed in this report. We find that this isolate shows biotechnological potential and is an interesting target for bioprospecting.

Structural insight into a CE15 esterase from the marine bacterial metagenome

The family 15 carbohydrate esterase (CE15) MZ0003, which derives from a marine Arctic metagenome, has a broader substrate scope than other members of this family. Here we report the crystal structure of MZ0003, which reveals that residues comprising the catalytic triad differ from previously-characterized fungal homologs, and resolves three large loop regions that are unique to this bacterial sub-clade. The catalytic triad of the bacterial CE15, which includes Asp 332 as its third member, closely resembles that of family 1 carbohydrate esterases (CE1), despite the overall lower structural similarity with members of this family. Two of the three loop regions form a subdomain that deepens the active site pocket and includes several basic residues that contribute to the high positive charge surrounding the active site. Docking simulations predict specific interactions with the sugar moiety of glucuronic-acid substrates, and with aromatically-substituted derivatives that serve as model compounds for the lignin-carbohydrate complex of plant cell walls. Molecular dynamics simulations indicate considerable flexibility of the sub-domain in the substrate-bound form, suggesting plasticity to accommodate different substrates is possible. The findings from this first reported structure of a bacterial member of the CE15 family provide insight into the basis of its broader substrate specificity.

Complete genome sequence of Halomonas sp. R5-57

The marine Arctic isolate Halomonas sp. R5-57 was sequenced as part of a bioprospecting project which aims to discover novel enzymes and organisms from low-temperature environments, with potential uses in biotechnological applications. Phenotypically, Halomonas sp. R5-57 exhibits high salt tolerance over a wide range of temperatures and has extra-cellular hydrolytic activities with several substrates, indicating it secretes enzymes which may function in high salinity conditions. Genome sequencing identified the genes involved in the biosynthesis of the osmoprotectant ectoine, which has applications in food processing and pharmacy, as well as those involved in production of polyhydroxyalkanoates, which can serve as precursors to bioplastics. The percentage identity of these biosynthetic genes from Halomonas sp. R5-57 and current production strains varies between 99 % for some to 69 % for others, thus it is plausible that R5-57 may have a different production capacity to currently used strains, or that in the case of PHAs, the properties of the final product may vary. Here we present the finished genome sequence (LN813019) of Halomonas sp. R5-57 which will facilitate exploitation of this bacterium; either as a whole-cell production host, or by recombinant expression of its individual enzymes.

Analysis of the distribution and evolution of the ATP-dependent DNA ligases of bacteria delineates a distinct phylogenetic group ‘Lig E’

Prior to the discovery of a minimal ATP‐dependent DNA ligase in Haemophilus influenzae, bacteria were thought to only possess a NAD‐dependent ligase, which was involved in sealing of Okazaki fragments. We now know that a diverse range of bacterial species possess up to six of these accessory bacterial ATP‐dependent DNA ligases (b‐ADLs), which vary in size and enzymatic domain associations. Here we compare the domain structure of different types of b‐ADLs and investigate their distribution among the bacterial domain to describe possible evolutionary trajectories that gave rise to the sequence and structural diversity of these enzymes. Previous biochemical and genetic analyses have delineated three main classes of these enzymes: Lig B, Lig C and Lig D, which appear to have descended from a common ancestor within the bacterial domain. In the present study, we delineate a fourth group of b‐ADLs, Lig E, which possesses a number of unique features at the primary and tertiary structural levels. The biochemical characteristics, domain structure and inferred extracellular location sets this group apart from the other b‐ADLs. The results presented here indicate that the Lig E type ligases were horizontally transferred into bacteria in a separate event from other b‐ADLs possibly from a bacteriophage.