Construction Of High Biotin-producing Strains And Optimization Of Fermentation Processes

Biotin,also known as vitamin H or coenzyme R,is essential to all organisms for maintaining a normal physiological function.It is involved in multiple biochemical reactions,such as carboxylation,decarboxylation and transcarboxylation.Biotin has been widely used in medicine,food and feed industries because of its biological versatility.In this study,biotin operon genes from different sources were cloned and expressed in Escherichia coli BL21(DE3).Three biotin-producing strains were constructed and the fermentation processes for them were optimized.The biotin operon from Bacillus subtilis 168,bioWAFDB,was cloned and ligated into vector pET28a.The recombinant plasmid was transformed into E.coli BL21(DE3)to construct the recombinant strain BM01(E.coli BL21(DE3)/pET28a-bioWAFDB).The biotin operon from Pseudomonas putida KT2440,bioBFHCD,together with the gene coding 7,8-Diammoniononanoate transferase,bioA,were also cloned and ligated into the double promoter vector pACYCDuet-1.The recombinant plasmid was transformed into E.coli BL21(DE3)to obtain the recombinant strain PM01(E.coli BL21(DE3)/pACYCDuet-bio BFHCD-bioA).Preliminary study between these two biotin-producing strains showed that the P.putida KT2440 biotin operon exhibited a stronger biotin-synthesizing ability in E.coli than that from Bacillus subtilis 168.However,neither E.coli BL21(DE3)nor P.putida KT2440 contain the pimeloyl-CoA synthase coding gene,bioW.To synthesize biotin from pimelic acid as biosynthetic precursor,it is necessary to introduce heterogenous pimeloyl-CoA synthase gene into host bacteria.The supply of coenzyme S-adenosyl-L-methionine(SAM),which is involved in several steps of the biotin synthesis pathway in the host bacteria,may also be an important factor in biotin synthetical metabolism.Therefore,an expression plasmid pETDuet-bioW-sam2 was constructed using the expression vector pETDuet-1.The gene coding pimeloyl-CoA synthase,bioW,was cloned from B.subtilis 168.The gene coding S-adenosine-L-methionine(SAM)synthetase,sam2,was cloned from S.cerevisiae ZJU001.Because the vector pACYCDuet can be used in combination with pETDuet-1,a recombinant strain PM02(E.coli BL21(DE3)/pACYCDuet-bioBFHCD-bioA,pETDuet-biow-sam2)was constructed.The fermentation processes were optimized for the three recombinant strains.Studies on the influence of carbon and nitrogen sources showed that glucose and glycerin are suitable carbon sources,and NH4C1,peptone and yeast extract are suitable nitrogen sources.Pimelic acid could significantly increase the biotin production in BM01 and PM02 which contains the pimeloyl-CoA synthase coding gene,bioW Studies on the metal ions showed that Fe2+ and Fe3+ promoted biotin synthesis.With 5mM of FeSO4 in the broth,the biotin production in PM02 increased to 60 mg/L.Both SAM and L-methionine(L-Met)could increase the production of biotin and L-Met worked better in PM02.With 200 mg/L L-methionine(L-Met)in the broth,the biotin production in PM02 reached 102 mg/L,which was 50 times as that of original E.coli.During fed-batch fermentations in a 15 L tank,the production of biotin in BM01,PM01 and PM02 reached 211 mg/L,260 mg/L and 417 mg/L,respectively.The biotin production in PM02 was 200 times as that of original E.coli.