Zhiyang Dong

Metatranscriptomic analyses of plant cell wall polysaccharide degradation by microorganisms in the cow rumen.

ABSTRACT The bovine rumen represents a highly specialized bioreactor where plant cell wall polysaccharides (PCWPs) are efficiently deconstructed via numerous enzymes produced by resident microorganisms. Although a large number of fibrolytic genes from rumen microorganisms have been identified, it remains unclear how they are expressed in a coordinated manner to efficiently degrade PCWPs. In this study, we performed a metatranscriptomic analysis of the rumen microbiomes of adult Holstein cows fed a fiber diet and obtained a total of 1,107,083 high-quality non-rRNA reads with an average length of 483 nucleotides. Transcripts encoding glycoside hydrolases (GHs) and carbohydrate binding modules (CBMs) accounted for ∼1% and ∼0.1% of the total non-rRNAs, respectively. The majority (∼98%) of the putative cellulases belonged to four GH families (i.e., GH5, GH9, GH45, and GH48) and were primarily synthesized by Ruminococcus and Fibrobacter. Notably, transcripts for GH48 cellobiohydrolases were relatively abundant compared to the abundance of transcripts for other cellulases. Two-thirds of the putative hemicellulases were of the GH10, GH11, and GH26 types and were produced by members of the genera Ruminococcus, Prevotella, and Fibrobacter. Most (∼82%) predicted oligosaccharide-degrading enzymes were GH1, GH2, GH3, and GH43 proteins and were from a diverse group of microorganisms. Transcripts for CBM10 and dockerin, key components of the cellulosome, were also relatively abundant. Our results provide metatranscriptomic evidence in support of the notion that members of the genera Ruminococcus, Fibrobacter, and Prevotella are predominant PCWP degraders and point to the significant contribution of GH48 cellobiohydrolases and cellulosome-like structures to efficient PCWP degradation in the cow rumen.

Five mutations in N-terminus confer thermostability on mesophilic xylanase

The termini of a pair of xylanases, one of mesophilic and one of thermophilic origin, was studied by molecular dissection and systematic mutagenesis. The thermostability of the mesophilic xylanase SoxB from Streptomyces olivaceovirdis was significantly improved by substituting its 33 N-terminal amino acid residues with the corresponding residues of the thermophilic xylanase TfxA from Thermomonospora fusca. Five amino acid substitutions, which clustered in one of the regions of the N-terminus, were discovered, for the first time, to account for the majority of the improvement in thermostability of SoxB. Further systematic mutagenesis and analysis of the five mutations demonstrated that comprehensive synergism of the five mutations was involved in conferring the thermostability on the SoxB. Moreover, when the five thermostabilizing mutations were introduced into two other G/11 xylanases, SlxB from Streptomyces lividans and AnxB from Aspergillus niger, their thermostabilities were also dramatically enhanced.

Crystal Structure of Group II Chaperonin in the Open State

Thermosomes are group II chaperonins responsible for protein refolding in an ATP-dependent manner. Little is known regarding the conformational changes of thermosomes during their functional cycle due to a lack of high-resolution structure in the open state. Here, we report the first complete crystal structure of thermosome (rATcpnβ) in the open state from Acidianus tengchongensis. There is a ∼30° rotation of the apical and lid domains compared with the previous closed structure. Besides, the structure reveals a conspicuous hydrophobic patch in the lid domain, and residues locating in this patch are conserved across species. Both the closed and open forms of rATcpnβ were also reconstructed by electron microscopy (EM). Structural fitting revealed the detailed conformational change from the open to the closed state. Structural comparison as well as protease K digestion indicated only ATP binding without hydrolysis does not induce chamber closure of thermosome.

Metabolomics analysis reveals large effect of roughage types on rumen microbial metabolic profile in dairy cows

The aim of our study was to determine the effect of diets with different types of roughage on the ruminal microbial metabolite profile in dairy cows. Holstein dairy cows were fed a diet containing either corn stover (CS group) or a mixture of alfalfa hay, Leymus chinensis hay and corn silage (MF group) at 0700 and 1900 h daily. Rumen fluid was sampled from each cow through a ruminal cannula at 0630 and 1030 h, and the mixed ruminal fluid from 3 day in each cow was analysed using nuclear magnetic resonance (NMR) spectroscopy. A multivariate analysis revealed a significant difference between the ruminal metabolome of the CS and MF groups at both time points. The MF group had higher levels of acetate, valerate, hydrocinnamate and methylamine and lower levels of glucose, glycine, propionate and isovalerate than those in the CS group. Our results showed that different types of roughages can significantly influence the ruminal microbial metabolome, especially with regard to organic acids, amines and amino acids.

Design of an ectoine-responsive AraC mutant and its application in metabolic engineering of ectoine biosynthesis

Advanced high-throughput screening methods for small molecules may have important applications in the metabolic engineering of the biosynthetic pathways of these molecules. Ectoine is an excellent osmoprotectant that has been widely used in cosmetics. In this study, the Escherichia coli regulatory protein AraC was engineered to recognize ectoine as its non-natural effector and to activate transcription upon ectoine binding. As an endogenous reporter of ectoine, the mutated AraC protein was successfully incorporated into high-throughput screening of ectoine hyper-producing strains. The ectoine biosynthetic cluster from Halomonas elongata was cloned into E. coli. By engineering the rate-limiting enzyme L-2,4-diaminobutyric acid (DABA) aminotransferase (EctB), ectoine production and the specific activity of the EctB mutant were increased. Thus, these results demonstrated the effectiveness of engineering regulatory proteins into sensitive and rapid screening tools for small molecules and highlighted the importance and efficacy of directed evolution strategies applied to the engineering of genetic components for yield improvement in the biosynthesis of small molecules.

Novel Glycoside Hydrolases Identified by Screening a Chinese Holstein Dairy Cow Rumen-Derived Metagenome Library

ABSTRACT One clone encoding glycoside hydrolases was identified through functional screening of a rumen bacterial artificial chromosome (BAC) library. Of the 68 open reading frames (ORFs) predicted, one ORF encodes a novel endo-β-1,4-xylanase with two catalytic domains of family GH43 and two cellulose-binding modules (CBMs) of family IV. Partial characterization showed that this endo-xylanase has a greater specific activity than a number of other xylanases over a wide temperature range at neutral pH and could be useful in some industrial applications.

Multistate folding of a hyperthermostable Fe-superoxide dismutase (TcSOD) in guanidinium hydrochloride: The importance of the quaternary structure.

Superoxide dismutases (SODs), which are the first line of cellular defense against the toxic effects of reactive oxygen species, are metalloenzymes that catalyze the disproportionation of superoxide radicals to produce oxygen and hydrogen peroxide. Although much effort has been devoted to the folding mechanisms of Cu/Zn-SODs, little is known about the folding of Fe-SODs. In this research, the equilibrium unfolding and refolding of TcSOD, a tetrameric hyperthermostable Fe-SOD, were investigated by circular dichroism, intrinsic fluorescence, ANS fluorescence, size-exclusion chromatography and cross-linking experiments. The results herein suggested that the guanidine hydrochloride-induced unfolding of TcSOD involved a stable monomeric intermediate and a possible tetrameric intermediate. The Gibbs free energy of TcSOD dissociation was about 3-fold larger than that of the monomeric intermediate unfolding, which suggested that the quaternary structure plays a crucial role in TcSOD stability. A comparison of the thermodynamic parameters between TcSOD and other SODs also suggested that the stability of quaternary structure might be responsible for the hyperthemostability of TcSOD.

Seven N-terminal Residues of a Thermophilic Xylanase Are Sufficient to Confer Hyperthermostability on Its Mesophilic Counterpart

Xylanases, and especially thermostable xylanases, are increasingly of interest for the deconstruction of lignocellulosic biomass. In this paper, the termini of a pair of xylanases, mesophilic SoxB and thermophilic TfxA, were studied. Two regions in the N-terminus of TfxA were discovered to be potentially important for the thermostability. By focusing on Region 4, it was demonstrated that only two mutations, N32G and S33P cooperated to improve the thermostability of mesophilic SoxB. By introducing two potential regions into SoxB in combination, the most thermostable mutant, M2-N32G-S33P, was obtained. The M2-N32G-S33P had a melting temperature (Tm) that was 25.6°C higher than the Tm of SoxB. Moreover, M2-N32G-S33P was even three-fold more stable than TfxA and had a Tm value that was 9°C higher than the Tm of TfxA. Thus, for the first time, the mesophilic SoxB “pupil” outperformed its thermophilic TfxA “master” and acquired hyperthermostability simply by introducing seven thermostabilizing residues from the extreme N-terminus of TfxA. This work suggested that mutations in the extreme N-terminus were sufficient for the mesophilic xylanase SoxB to acquire hyperthermostability.

Flexible interwoven termini determine the thermal stability of thermosomes.

Group II chaperonins, which assemble as double-ring complexes, assist in the refolding of nascent peptides or denatured proteins in an ATP-dependent manner. The molecular mechanism of group II chaperonin assembly and thermal stability is yet to be elucidated. Here, we selected the group II chaperonins (cpn-α and cpn-β), also called thermosomes, from Acidianus tengchongensis and investigated their assembly and thermal stability. We found that the binding of ATP or its analogs contributed to the successful assembly of thermosomes and enhanced their thermal stabilities. Cpn-β is more thermally stable than cpn-α, while the thermal stability of the hetero thermosome cpn-αβ is intermediate. Cryo-electron microscopy reconstructions of cpn-α and cpn-β revealed the interwoven densities of their non-conserved flexible N/C-termini around the equatorial planes. The deletion or swapping of their termini and pH-dependent thermal stability assays revealed the key role of the termini electrostatic interactions in the assembly and thermal stability of the thermosomes.

Formation of High-Order Oligomers by a Hyperthemostable Fe-Superoxide Dismutase (tcSOD)

Hyperthermostable proteins are highly resistant to various extreme conditions. Many factors have been proposed to contribute to their ultrahigh structural stability. Some thermostable proteins have larger oligomeric size when compared to their mesophilic homologues. The formation of compact oligomers can minimize the solvent accessible surface area and increase the changes of Gibbs free energy for unfolding. Similar to mesophilic proteins, hyperthermostable proteins also face the problem of unproductive aggregation. In this research, we investigated the role of high-order oligomerization in the fight against aggregation by a hyperthermostable superoxide dismutase identified from Tengchong, China (tcSOD). Besides the predominant tetramers, tcSOD could also form active high-order oligomers containing at least eight subunits. The dynamic equilibrium between tetramers and high-order oligomers was not significantly affected by pH, salt concentration or moderate temperature. The secondary and tertiary structures of tcSOD remained unchanged during heating, while cross-linking experiments showed that there were conformational changes or structural fluctuations at high temperatures. Mutational analysis indicated that the last helix at the C-terminus was involved in the formation of high-order oligomers, probably via domain swapping. Based on these results, we proposed that the reversible conversion between the active tetramers and high-order oligomers might provide a buffering system for tcSOD to fight against the irreversible protein aggregation pathway. The formation of active high-order oligomers not only increases the energy barrier between the native state and unfolded/aggregated state, but also provides the enzyme the ability to reproduce the predominant oligomers from the active high-order oligomers.