High Expression Of Bovine Lactoferrin In Bacillus Subtilis And Rational Design Of Its Iron Saturation

Lactoferrin（LF）is a multifunctional iron-binding protein,with the activities of antibacterial,antioxidant,antitumor,antiviral etc.It is widely used in food,cosmetics,and pharmaceutical industries.The functionalities and characteristics of lactoferrin including structural elucidation,heterologous expression,iron saturation and the heat stability improvement,are always the research hotspot domestically and overseas.Although it is reported of the heterologous expression of lactoferrin in the systems of animals,plants,and microbial cells,little can take into account safety,efficiency and rational design.In this study,according to the reported amino acid sequences of lactoferrin antimicrobial peptides,and the mechanism of iron binding and iron releasing,we constructed recombinant Bacillus subtilis168 containing bovine lactoferrin N-lobe（BLF-N）and C-lobe（BLF-C）and realized their soluble expression and studied their antibacterial activities.Taking BLF-N with stronger antibacterial activity as the research object,the high-efficiency expression of BLF-N was achieved through promoter engineering and codon optimization.Through the rational design on the Fe³⁺binding sites of BLF-N,its iron saturation and the protein heat stability were improved.Finally,at the level of both shake flask and 10 L fermentor,through a two-stage fermentation process by regulating temperature and p H value,the cell-growth and protein expression level of mutant bacteria were improved by optimizing iron saturation and stability improvement.The main research contents are as follows:（1）The soluble expression of BLF-N and BLF-C were achieved and their antibacterial properties were studied.The target genes of BLF-N and BLF-C were fished,and the recombinant strains E.coli BL21/p ET-BLF-N and E.coli BL21/p ET-BLF-C were constructed.SDS-PAGE results showed both recombinant BLF-N and BLF-C were expressed as inclusion bodies.Five different plasmids were selected to construct recombinant B.subtilis168.The expression levels of BLF-N in B.subtilis168/p AX-BLF-N,B.subtilis168/p HT-BLF-N,B.subtilis168/p WB-BLF-N,B.subtilis168/p HY-BLF-N and B.subtilis168/p MA-BLF-N were6.3,9.8,12.1,10.5 and 12.0 mg·L^-1by RT-PCR.The expression levels of BLF-C in B.subtilis168/p AX-BLF-C,B.subtilis168/p HT-BLF-C,B.subtilis168/p WB-BLF-C,B.subtilis168/p HY-BLF-C and B.subtilis168/p MA-BLF-C were 2.9,5.6,7.1,6.2 and 8.3mg·L^-1,respectively.The expression levels of BLF-N and BLF-C were the highest with genes on vector p MA0911.The inhibition rates of BLF-C purified at 10 mg·m L^-1on E.coli JM109,P.aeruginosas and S.aureus were 100%,100%and 48.4%,while the inhibition rates of purified BLF-N at 200 ng·m L^-1on the three tested bacteria were 100%,70.3%and 41.5%,respectively.The minimum inhibitory concentrations of BLF-C to E.coli JM109,P.aeruginosas and S.aureus were 4,8 and 16 mg·m L^-1respectively,while the minimum inhibitory concentrations of BLF-N to the three bacteria were 128,256 and 512μg·m L^-1respectively.Those results indicate that the antibacterial activity of BLF-N is much higher than that of BLF-C.（2）The expression level of BLF-N was improved through promoter engineering and codon optimization and its antibacterial activity after optimization was studied.Recombinant B.subtilis168/p MA0911-P_cat-BLF-N,B.subtilis168/p MA0911-P_{sac B}-BLF-N,B.subtilis168/p MA0911-P₄₃-BLF-N,B.subtilis168/p MA0911-P_veg-BLF-N and B.subtilis168/p MA0911-P_{ser A}-BLF-N with different promoters were constructed.RT-PCR results showed that the promoters P_veg,P₄₃and P_{ser A}increased the expression of m RNA by240.2%,10.4%and 161.5%respectively,and the protein expression by 80.3%,3.5%and55.4%respectively.Therefore,B.subtilis168/p MA0911-P_veg-BLF-N was selected for the subsequent codon optimization,and its 16 rare codons were replaced with the most frequently used synonymous codons to construct the recombinant strain B.subtilis168/p MA0911-P_veg-m BLF-N.The m RNA expression was increased by 164.3%and the protein expression was increased by 52.7%after codon optimization.The antibacterial activity was tested by he filter plate method and the Flores-Villase?or method.The results showed that the optimization of promoter and codon did not change the antibacterial activity of BLF-N against E.coli JM109,P.aeruginosa and S.aureus,which was probably caused by the destruction of the cell membrane of gram-negative or positive bacteria by the alkaline residues lined outside the B molecule in LF.（3）The iron binding sites were rationally modified for protein stability improvement.Mutants D60Y,Y92D,Y192D and H253D were implemented in the distorted octahedron formed by the coordination of BLF-N lobe with Fe³⁺.Compared with the wild type,mutant D60Y showed the maximum iron binding level of 91.3%at p H 7.0.The combined mutations D60Y/Y92D and D60Y/Y192D enhanced the binding force of the protein to iron.Among them,D60Y/Y92D had the strongest binding force to iron,and the saturated iron content was93.4%.The results of circular dichroism showed that the T_mof wild type was 70.0?C at p H7.0,the mutations of D60Y/Y92D and D60Y/Y192D increased T_mby 2.0-3.4?C.The results of intrinsic tryptophan fluorescence spectrometry indicated the maximum fluorescence emission of BLF-N and its mutants were increased gradually with the decrease of p H,and showed a red shift.The maximum fluorescence emission of wild-type BLF-N,mutants D60Y/Y92D,D60Y/Y192D,Y92D/H253D and Y192D/H253D were 332-335 nm at p H 7.0,and their tryptophan fluorescence intensity value was 13.2-20.4 a.u.The mutant D60Y/Y92D has the maximum fluorescence emission value and the lowest fluorescence intensity value,which reflects the lowest red shift degree,the low protein unfolding level,and the most stable structure.The results of 8-anilino-1-naphthalene sulfonate（ANS）binding fluorescence intensity showed that the maximum emission wavelength of BLF-N and its mutant decreased with p H and showed a blue shift after combined with ANS.Compared with the wild-type,the mutants D60Y/Y92D and D60Y/Y192D showed the lower ANS fluorescence blue shift,indicating a lower degree of of proteolysis and better structural stability.（4）The fermentation conditions were optimized at the level of shake flask and 10 L tank to improve the yield of target protein.The recombinant B.subtilis168/p MA0911-P_veg-D60Y/Y92D with improved iron saturation and stability was used as the research object.Through the optimization of shake flask fermentation,it was determined that the seed age was 11 h,the inoculation amount was 4%,glucose and peptone were used as the optimal carbon and nitrogen sources.In the initial 0-7 h,p H 7.5 was conducive to the growth of the recombinant bacteria,and during 7-16 h,p H 7.0 was more conducive to protein accumulation.The optimal induction temperature of 28?C and the optimal fermentation duration of 25.5 h were used to induce the protein expression.The Integrated Optical Density（IOD）value of the target protein was as high as 68.03%.According to the single factor test results,using three factors and three levels to optimize the response surface,it was obtained the multiple regression equation of response value IOD value（Y）,fermentation p H（A）,fermentation temperature（B）and fermentation time（C）as below,Y=-2089.69+441.33A+18.22B+25.34C+0.005AB-0.711AC+0.039BC-30.11A²-0.31B²-0.42C².The extent of influence on IOD was fermentation time（29）fermentation p H（29）fermentation temperature.In 10 L tank fermentation,the bacteria was cultured at 300 r·min^-1.In the initial0-7 h,p H was set at 7.5 under 30?C;during 7-25 h,p H was adjusted at 7.0 under 28?C to induce the protein expression.Two-stage regulating process of temperature and p H was adopted.The recombinant protein was purified and its purity reached 94.3%.And 23.5 mg BLF-N/D60Y/Y92D protein per liter fermentation broth was obtained with an increase by 2.4times compared with before optimization.