Comparative Analysis Of Gene Diversity And Transcript Profiles Of Xylanases And Phytases In The Sheep Rumen And Characterization Of Several Novel Xylanases And Phytases

Carbon and phosphorus are essential elements to living organisms. Herbivorous animals obtainthese elements from plant-based feed, of which lingocellulose and phytase represent the main storedforms of carbon and phosphorus, although these can not be digested by monogastric animals. Ruminantanimals harbor varieties of microorganisms in the rumen that secrete different enzymes and have theability to easily utilize these substrates. Currently, metagenomic sequencing method has been used tostudy ruminal micro-organisms leading to the identification of a great variety of glycoside hydrolasesand other enzymes. To date, there is scant information on ruminal enzymes at the transcriptional level.This study analyzes the genetic composition and expression characteristics of ruminal xylanase andphytases during the feeding cycles of ruminants in order to identify the functional genes and to providemore information on the ruminal environment.The rumen of Small Tail Han sheep was selected as the excellent microorganism source to studythe ruminal xylanases of glycoside hydrolase （GH） family10and cysteine phytases. DNA and cDNAlibraries of xylanase and phytase during a feeding cycle were constructed, and their diversity at thetranscriptional level was studied. Analysis of the rumen pH, bacterial population, and enzymatic activityduring a feeding cycle indicated that rumen pH and xylanase activity showed similar trends. The valuesdecreased with feeding for4h, then increased up to the initial level. The trend of bacterial populationwas different, showing the highest at12h and reduced then to the initial level. The results reveal thatthe rumen environment is dynamic with regular changes during feeding cycles.According to the changing rule of rumen environment, four cDNA libraries at0h,4h,9h and16hand one DNA library at16h of GH10xylanases were constructed, and44distinct gene fragments withidentities of <95%were identified. Genes in the DNA and cDNA libraries at16h showed significantdivergence with only nine genes identical. The result indicated that evaluating functional genes at thetranscription level is a more reliable indicator for understanding fluctuations than that of genomic level.Comparison of the relative abundance of xylanase genes indicated that six xylanase genes were detectedat all time points of the feeding cycle and showed a complex trend of gene expression over24h. Furtherquantitative analysis with qPCR indicated that these six representative genes showed similar trends atthe genomic level but varied at transcriptional level. These results reveal that xylanases have complexdynamics of expression in the rumen. Correlation analysis indicated that the rumen is a dynamicecosystem where the transcript profiles of xylanase genes are closely related to ruminal conditions,especially rumen pH and bacterial population.Using the same strategy, four cDNA libraries at0h,4h,9h and16h and one DNA library at9hof cysteine phytases were constructed, and a total of46unique fragments （<95%identities） of cysteinephytase genes were retrieved at both genomic and transcriptional levels. Most of these fragments hadlow identities （30–70%） with known sequences. Comparison of the cDNA and DNA libraries at9hindicated that the constitution and abundance of cysteine phytase genes were divergent, and morefunctional genes were identified at the transcriptional level. Furthermore, comparative analysis of thecysteine phytase during the feeding cycle shows the complex dynamic expression of phytase in therumen.Of the large number of novel gene fragments retrieved from the sheep rumen, ten full-length xylanase genes and four phytase genes were cloned, and three of them were chosen for further studies.Recombinant XynB and XynC were produced in Escherichia coli and had similar enzymatic properties.With beechwood xylan as the substrate, XynB and XynC showed the specific activities of73.9U/mgand142.3U/mg, respectively. Both enzymes showed the maximal activity at pH6.0. The enzymes werestable between pH5.0and8.0, retaining more than80%of their initial activity after incubation at39°Cfor1h. The optimal temperature for enzymatic activity was40°C, and the enzymes exhibitedapproximately60%relative activity at20°C. Recombinant XynB and XynC were thermostable at40°C,retaining90%and86%of their relative activities after incubation for1h, respectively. These enzymecharacteristics are similar to the physiological conditions of rumen （39°C and pH5.0–7.0）, revealingthat ruminal xylanases and rumen may adapt to each other in a mutual selection style.XynA was the most predominant gene at the transcriptional level and encoded a407-residuepolypeptide. It consisted of a signal peptide of24amino acids, a catalytic motif of GH10, and aproline-rich C-terminal sequence of60amino acids without homolog. To determine its function, matureXynA and its C terminus-truncated mutant derivative XynA-Tr were both expressed in E. coli. TheC-terminal oligopeptide had significant effects on the function and structure of XynA. Its presenceincreased the temperature optimum （from45C to50C） and broadened the ranges of temperature andpH optima （from40–50C to40–60C and from pH5.5–6.5to pH5.0–7.5）. It also improved thespecific activity (1135U mg^–1v.s.97U mg^–1) and catalytic efficiency (133.5ml s^–1mg^–1v.s.7.3mls^–1mg^–1). Analysis of the secondary structures with circular dichroism spectroscopy indicated that XynAhad more α-helical components. Binding analysis with isothermal titration calorimetry showed thatXynA had greater binding capacity to xylooligosaccharides, which was essential to the improvement ofcatalytic efficiency. Similar results were obtained when fused the C-terminal sequence to anotherxylanase （XynB） of the same source and same family. This result suggested the C-terminal oligopeptideis versatile to other xylanases and reveals an engineering strategy to improve the enzyme catalyticperformance.In summary, this study has identified a large amount of functional genes in the rumen of Small TailHan sheep based on the transcriptional analysis of ruminal GH10xylanases and cysteine phytasesduring a feeding cycle. The changes in xylanase and phytase genes are revealed by comparing theconstitution and abundance of genes between the genomic and transcriptional levels. By analyzing thecomplexity and dynamics of ruminal enzymes at the molecular level, this study provides valuableinformation for the exploitation of the microbial source in the rumen. Characterization of therepresentative xylanase （XynA, XynB and XynC） not only verifies the functions of these genes, but alsoprovides a strategy for xylanase improvement based on the study of the relation between structure andfunction.