Genome Sequencing And Analysis Of Alicyclobacillus Tengchongensis CGMCC1504and Characterization Of Its Carboxylesterase

Alicyclobacillus tengchongensis CGMCC1504is a thermalacidophilus which isone of extremophiles isolated from a hot spring in Tengchong, Yunnan, China.Extremophiles usually evolve unique adaption mechianism when they in extremeenvironments. These organisms have diversity of species, genetic composition andecological function. At the same time, they may have different metabolic pathwaysand genetic background, that have great value for genetic research and biotechnologyapplications. They are a potential source of microbial for novel enzyme resourcedevelopment. To better understand the basic genetic characterisitics, environmentaladaption mechianism, metabolic pathways and novel enzymes found of thisextremophile from genomic perspective, we carry out the whole genome sequencingof A. tengchongensis CGMCC1504. Furthermore, two carboxylesterases (CarE3andCarE5) from the functional annotation of A. tengchongensis CGMCC1504wassuccessfully cloned and expressed in E. coli BL21(DE3), studied the characterizationof enzymes, the degradation of pesticides, and construction of the cell surface displaysystem. This thesis mainly as following:1. Genome sequencing and bioinformatics analysis of A. tengchongensisCGMCC1504The nucleotide sequence was determined using an Illumina Solexa sequencer. A.tengchongensis CGMCC1504genome consists of a chromosome (2.81Mb), GCcontent of53.98%, coded2867of CDs, gene average length is2,506,377, gene lengthaccount for89.19%of the proportion of the genome,98of tandem repeat regions,5of5s_rRNAs,60of tRNAs,121of transposons,1of CRISPR-Cas system,1ofgenomic islands, none prophage. Synteny analysis of six Alicyclobacillus showedclose genetic relationship between A. tengchongensis CGMCC1504and A.hesperidum URH17-3-68that has better co-linearity than others. Core/Pan analysis ofsix Alicyclobacillus showed they have1704of core genes and6651of pan genes thathave a larger proportion of the total coding genes. It also indicated the existence of genetic diversity among different Alicyclobacillus. Furthermore, A. tengchongensisCGMCC1504has an intact Embden-Meyerhof-Parnas (EMP) pathway, pentosephosphate pathway (PPP) and tricarboxylicacid cycle (TCA). The complete primarymetabolite pathway can not only meet the needs of physiological activities but alsoprovide the primary metabolites for the secondary metabolite pathway, and producemany kinds of bioactive secondary metabolites. It is also found that having a varietyof glycan biosynthesis and metabolism, a variety of xenobiotics biodegradation andmetabolism, nitrogen metabolism, sulfur metabolism, lipid metabolism, secretoryprotein secretionwith typeⅡsecretion system and type Ⅲ secretion system. Thesemay be the why that they adapt to the extreme environment.Two carboxylesterases (CarE3and CarE5) were screened from the functionalgenes annotation of A. tengchongensis CGMCC1504that were deposited in GenBankunder accession numbers JQ034612and JX101458, respectively. A1542bp-longORF that encoded for a513amino acid protein was found, and with the predictedmolecular weight of56kDa and57.8kDa, respectively, the theoretical isoelectricpoint (pI) of5.52and4.75, respectively. CarE3and CarE5have the highest aminoacid similarity with57%and81%, respectively. According to the reported thedegradation pesticide of carboxylesterase in amino acids with highest homology lessthan40%, they are two novel esterases for hydrolysis pesticides. Secondary structureprediction, both of CarE3and CarE5have cysteine which may be formation ofdisulfide bonds. Multiple sequence alignment analysis CarE3and CarE5with thespecific type B carboxylesterase conserved region (FGGDPDRVTIAGQSAG)182-197and (FGGDPENITIGGQSAG)191-206, respectively, with esterase family conservativefive-peptide structure Gly193Gln194Ser195Ala196Gly197and Gly202Gln203Ser204Ala205Gly206, respectively. Ser195and Ser204are the active center of CarE3and CarE5,respectively, Ser195/Glu333/His424and Ser204/Glu325/His415are catalytic triad domain ofCarE3and CarE5, respectively,(HGGG)115-118and (FGGG)117-120are the oxygen ionresidue of CarE3and CarE5, respectively. CarE3and CarE5are the/β-hydrolase(CL0028) PF00135member that have typical esterase/lipase superfamily domainsand belong to the esterase family. In addition, the amino acid sequence phylogeneticanalysis that both CarE3and CarE5belong to the lipase family members VII. SWISS-MODEL homology modeling showed CarE3and CarE5with template2ogsAonly have the highest sequence similarities30.44%and28.02%, respectively.2. Prokaryotic expression system of CarE3and CarE5and enzyme characteriza-tionCarE3and CarE5were heterologous expression in E. coli.BL21(DE3), respectiv-ely. The optimum temperature of recombinant CarE3and CarE5is65°C and60°C,respectively. They are active under a broad temperature range (6-85°C) and a broadpH range (pH4.8-8.0). The two enzymes are stable at room temperature and pH7.0.CarE3will lost activity after incubation at60°C for50min, while CarE5has87%activity remainedat60°C for10min, and after50min, only36%residual activityremainder. The recombinant CarE3and CarE5have optimum activity at pH7.0. Theyare unstable in acidic or alkaline conditions. Zn2+has a strong inhibitory effect forenzymes, and the stability of the enzymes are independent on the Ca2+. Other organicsolvents have a different inhibition for the enzymes except for dichloromethane. BothDTT and beta-mercaptoethanol have inhibition for the carboxylesterases. Reducingagent can destroy the disulfide bonds and generate cysteine residues (-SH). Enzymekinetics studies have shown that both CarE3and CarE5have higher alpha-naphthylacetate catalytic activity than that of beta-naphthyl acetate. It indicates that theconfiguration of the substrate has a great influence on the catalytic activity of theenzyme.3. Degradation of pesticides by the recombinant CarE3and CarE5Pyrethroids (tetramethrin and cyfluthrin), carbamates (carbaryl, Fenobucarb andanti-pirimicarb) and the organophosphorus pesticides (malathion, parathion anddichlorvos) are organic pesticides. The recombinant CarE3and CarE5for degradationof pesticides showed that CarE3degradate cyfluthrin, carbaryl, and malathion, whileCarE5degradate cyfluthrin and malathion. The recombinant CarE3(1U) candegradat-e55.7%of cyfluthrin (5mg/L),41.18%of carbaryl (5mg/L), and47.1%ofmalathion (5mg/L) within60min at pH7.0and37°C. At the same conditions, therecombinant of CarE5(1U) can hydrolyze58.14%of cyfluthrin (5mg/L) and70.5%of malathion (5mg/L) within100min.4. Cell surface display of CarE3and CarE5and whole-cell activity on E. coli. BL21(DE3)Fusion CarE3or CarE5respectively formed recombination plasmids pET28ND3,pET28NCD3, pET28ND5and pET28NCD5using synthetic N-or NC-terminaldomain of ice nucleation protein (inaK) from Pseudomonas syringae. Expression ofcarboxylesterase in E. coli BL21(DE3) under T7promoter and0.05mM of IPTGinduced, the activity of whole cell dry reached595.6U/g,539.5U/g,153.96U/g and78.7U/g, respectively. The inaK-N terminal fusion of carboxylesterase activity wassignificantly higher than that of inaK-NC terminal fusion. The activity of whole celldry can keep stability up to67d when storage at room temperature.