The High-yield Mechanism Of Microbial Transglutaminase From Streptomyces Mobaraensis

Transglutaminase(EC 2.3.2.13,TG,protein-glutamine y-glutamytransferase)is a kind of transferase which can catalyze acyl transfer between protein moleculer or intramolecular.Transglutaminase widely originates from animals,plants and microorganism.Among them,anzyme from microorganism(microbial transglutaminase,MTG)has many advantages such as small moleculer weight,Ca2+-independent,substrate-specificity and shorter growth period,that it is widely used in food,medicine and textile industry and so on.At present,streptomyces mobaraensis is regarded as the main fermentation strain to produce transglutaminase in the industry.The Production of MTG from wild streptomyces mobaraensis is relatively low.This results in the high costs of production and it cannot applied in large scale industrial production.To solve the problems,rearchers used traditional mutagenesis technique and genetic engineering to get highly-yield strain and largely improve the production of MTG.However,traditional mutagenesis technique needs heavy workload and negative mutant is much more than positive mutant.The yield of genetic engineering was still low.Besides that,enzyme remoulded by genomics engineering cannot be applied in food and the operation is too complex to realize industrial production.This leads to low efficiency and more screening.All in all,the high yield mechanism of MTG from Streptomyces mobaraensis is of vital importance.This article constrastive analyze the highily-yield strain and parent strain from the aspect of protein level,genome level and transcriptional level.Secreting of MTG in or out of cell,genomic sequences and translation level were compared between these two strains to make clear the high yield mechanism.Genomic level and transcriptional level difference between highly-yield strain and parent strain.At first,whole genome sequenceing of these two strains was done and then using bioinformatics to analyze,forecast and annotation the whole genome.According to the results,sequences of pre-pro-MTG are totally the same between these two strains.Besides that,sequences of regulation factors related to maturity of MTG,such as TAMEP,SM-TAP,are all the same.So the improvement of enzyme yield has nothing to do with the sequences of MTG and regulation factors.However,there are dozens of point mutation no matter in the coding region or non-coding region through SNP analysis.The most important thing is that there is only one point mutation in the non-coding area of MTG and it exactly right locate in the promoter area of MTG by promoter prediction tool.Besides,other mutation locus locate in the gene related to metabolism pathway such as carbon and nitrogen metabolism.They may also affect the synthesis and secretion of MTG.Comparison of transglutaminase yield inside the cell.Guo Weiting had proved that pro-MTG yield of highly-yield strain had remarkablely higher than which of parent strain when pro-MTG was secreted to extracelluar.As it is well known that MTG exists in the cell in the form of pre-pro-MTG.Chosing the fetmentation liquid of 24h,44h and 50h of the two strains at the same time to extract intracellular proteins and detect the protein content through western blot.The transport rate of highly-yield strain is so fast that pre-pro-MTG is immediately transferred to the extracellular while pre-pro-MTG in parent strain is partly turned to mature enzyme in the cell that it cannot totally transferred to extracelluar.In addition,taking 16sRNA as reference,relative expression quantity of highly-yield is about 6.9 when it is 0.1 of parent strain through the analysis of MTG transcriptional level.P-value is less than 0.0001.All in all,the relative expression is of significant difference between highly-yield strain and parent strain.Combining the difference of genome and transcriptional level,studies were done towards the relationship of non-coding area mutation of MTG and transcriptional discrepancy.pSET152 was used as the plasmid vector and sfGFP was used as the report gene.It had been proved that the non-coding area mutation led to the transcriptional discrepancy of sfGFP which was about 138 in highly-yield strain and 51 in parent strain.The p-value was about 0.0021.Therefore,The mutation in the non-coding area was the reason which led to the increasing of transcriptional level of pre-pro-MTG in highly-yield strain.The increasing of the MTG yield is by the reason of the mutation of non-coding area of MTG gene sequence which leads to the significant improvement of transcriptional relative expression quantity.As a result,the production of MTG from Streptomyces mobaraensis is increased.