Cloning And Expression Of Lipoxygenase Gene From Anabaena Sp. PCC 7120

Lipoxygenase（LOX） can catalyze the oxidation of polyunsaturated fatty acids containing the cis,cis-1,4-pentadiene moiety such as Linoleic acid and a-linolenic acid. In flour processing, this oxidizing reaction will result in the formation of unstable hydroperoxides which can induce polymerization of gluten proteins to strengthen the role of gluten and damage double bond of carotenoids, and so white the flour. Thus, these functions of lipoxygenase can reduce or replace usage of potassium bromate and benzoyl peroxide. But it is rare that lipoxygenase serve as a food additive directly in food processing applications. The following may be the main reasons:（1） there is high level of lipoxygenase in soybean so we can add soybean powder directly to flour to improve flour procession quality, but the soybean powder has complex composition and condition is difficult to control; （2） extract pure enzyme from plant materials costs too high （3） high-level expression of recombinant lipoxygenase through genetic engineering has not been reported.In this study, the lipoxygenase gene from Anabaena sp. PCC 7120 was cloned, site-directed mutagenesis was designed for determination the conserved active sites, vectors used for expression lipoxygenase gene in B.subtilis was constructed, and a new method for direct genetic manipulation of B. subtilis 168 that removes the antibiotic resistance gene used as a selectable marker was developed. The detailed works were described as following:1. Searched in the NCBI database, a fragment in Anabaena sp. PCC 7120 genome was found belong to Lipoxygenase gene （LOX） family, with an allene monooxygenase gene （AOS） in the same open reading frame which can encoding a bifunctional enzyme AOS-LOX. The LOX’s conservative active sites were found at C-side of the dual-function enzyme though bioinformatics analysis software. Primers were designed according to the reported Anabaena sp. PCC 7120 AOS-LOX gene sequence （NC₀03267）, and an open reading frame of 1368bp which contains conservative active sites of LOX that encode 455 amino acids was amplified by PCR. The PCR products was designed as ana-LOX and the conservative active sites were predicted as His197, His202, His369, Asn373, and Ile455.2. E.coli was used as a host for recombinant ana-LOX expression. And pET-23a-ana-LOX and two molecular chaperone-fused recombinant expression vectors pET-32a-ana-LOX and pGS-21a-ana-LOX were constructed. The three recombinant expression were transformed into E.coli BL21 （DE3） supplemented with 100μg/ml IPTG for induction, and grown at 16℃and 30℃. All of the three recombinant recombinant vectors expressed active ana-LOX. The level of ana-LOX activity at 16℃was higher than that of at 30℃and the pET-32a-ana-Lox expressed the highest activity at 16℃. Though single-factor experiments of the recombinant E.coli BL21 （DE3）/pET-32a-ana-Lox we found that the best fermentation medium was TY; optimal concentration of lactose as inducer was 1g/L; the best pH value of initial fermentation medium was 7.0; OD600=0.4 was the best time to induce; optimal induction time was 12h.3. The enzyme was purified by Ni-NTA chelating affinity chromatography, with 61.92% yield and specific activity of 1.372×104 u/mg. The optimum temperature and pH of the purified recombinant enzyme were 45℃and 6.0, respectively. The recombinant enzyme was relative stable at 20℃with half-lives of 25,10,8min at 30℃,40℃,50℃, respectively. The enzyme was activated by Fe2+ up to 181.91% of activity, and was strongly inhibited by Cu2+, Fe3+ up to 100%. Mg2+ and Ca2+ have a weak activation while Na+, Zn2+ and Mn2+ have an opposite effect.4.25 mutagenesis of ana-LOX was constructed by site-directed, they were 3 at His197,3 at His202,3 at His369,3 at Asn373,4 at Ile455,3 at Ile326,3 at Asn419, and 3 at His364. All of these mutagenesis were transformed into E.coli BL21 （DE3） for expression and the results confirmed that the active site residues of ana-LOX were His197, His202, His369, Asn373, and Ile455. Then we designed a gradual reduction from the 5’end of the ana-LOX gene to determine the mini-ana-LOX was 1254bp. We also found that with the length of ana-LOX gradually shorten from 1254bp the enzyme activity became lower. When the gene shortened to 1230bp no enzyme activity can be detected.5. Eleven expression vectors of B. subtilis were constructed. pHPQR, pHPY and pHPYR were inducible expression vectors that secreted by Sec-pathway. The pHPQR was constructed based on the laboratory pre-constructed vector pHPQ. PamyE-PrsA-TrpA, expression fragments of co-expression molecular chaperone PrsA, was cloned into pHPQ to generate pHPQR. The SacB promoter, the SsacB signal peptide and the P43-SacY （expression fragments of co-expression enhancer SacY） were cloned into pHB-hc, yielding pHPY. PamyE-PrsA-TrpA and co-expression molecular chaperone PrsA were cloned into pHPY resulting pHPYR. pHPSQ, pHPSB, pHPSQR and pHPSBR were constitutive expression vectors that secreted by Sec-pathway. The P43 promoter and MCS were cloned into pHB-hc to generate pHP43. And Sec-type signal peptide SamyQ and SnprB were cloned into MCS of pHP43 respectively, yielding pHPSQ and pHPSB. Based on these two vectors, PamyE-PrsA-TrpA was cloned into pHPSQ and pHPSB, resulting pHPSQR and pHPSBR. pHPSC, pHPSD, pHPSCA and pHPSDA were constitutive that secreted by Tat-pathway. And Tat-type signal peptide SymZC and SphoD were cloned into MCS of pHP43, we got pHPSC and pHPSD. And PamyE-PspA-TrpA was cloned into pHPSC and pHPSD respectively to generate pHPSCA and pHPSDA.6. Twelve ana-LOX B. subtilis recombinant expression vectors were constructed. Ana-LOX gene was cloned into above-mentioned expression vectors to generate four induced recombinant expression vectors named pHPQ-ana-LOX, pHPQR-ana-LOX, pHPY-ana-LOX and pHPYR-ana-LOX; four constitutive, Sec-pathway recombinant expression vectors named pHPSQ-ana-LOX, pHPSQR-ana-LOX, pHPSB-ana-LOX and pHPSBR-ana-LOX; four constitutive, Tat-pathway recombinant expression vectors named pHPSC-ana-LOX, pHPSCA-ana-LOX, pHPSD-ana-LOX and pHPSDA-ana-LOX. PrsA promotes protein secretion by the Sec pathway. PrsA, molecular chaperone in the B. subtilis Sec pathway, was used to facilitate ana-LOX secretion. When ana-LOX was expressed and secred from the protease-deficient strain B.subtilis WB800, LOX activity was 46U/mL when secretion was directed by SnprB, and 32U/mL when directed by SamyQ at 16℃,84h.7. A new method for direct genetic manipulation of B. subtilis 168 that removes the antibiotic resistance gene used as a selectable marker was developed. This method relies on the Pspac promoter, which was regulated by the LacI repressor, to confer conditional lysine auxotrophy BS-PS, replacing the native PlysA promoter with the Pspac promoter. Homologous plasmid integration by a single cross-over yields a strain in which the target gene in BS-PS-PI was duplicated with a modified copy. Because of this unstable genome structure, homologous recombination between the duplicated sequences leads to another single cross-over. Recombination strains can be selected and distinguished easily, and the method allows the use of lacI for repeated modifications of B. subtilis 168. We used this method to inactivate aprE and nprE in B. subtilis 168.