Gene Transfer Enhances Transglutaminase Production Of Streptomyces Mobaraensis

Transglutaminase?TGase,EC2.3.2.13?is a wildly used food additive.It can make protein crosslink and change the food texture.Nowadays,MTG is produced by microbial fermentation,and the most commonly used strain is Streptomyces mobaraensis.This MTG production method takes shorter time with lower cost.Also,MTG produced by microorganism still has activity without Ca²⁺ in the environment.However,the MTG production and heat stability needs improving.Obtaining better strains by microbial breeding is the key to solve these problems.Most studies used random mutagenesis to screen for better strains,but we henced characteristics by genetic recombination in this study.Strain HS9 could produce MTG in shorter time,and the MTG produced by HS9 had higher heat stability,while PoE was a strain with high yield.We uesd protoplasts fusion and vector-free DNA electrotransformation to make genes from HS9 and PoE recombine.After screening,we got a strain with advantages of two parent strains.It had higher production,shorter fermentation time and the MTG had higher heat stability.The results of this research are as follows:?1?We analyzed advantages and disadvantages of different mutant strains to choose parent strains.PoE was a mutant strain and its MTG activity was 8.65U/mL,while HS9 was 4.43U/mL.However,HS9 needed shorter fermentation time.The enzyme activity of fermentation broth reached its highest after fermentation for 36h,which was 18h earlier than using PoE as producing strains.Also,MTG produced by HS9 had higher heat stability in 60?.These two strains could be differentiated by morphology and TG gene sequence.In addition,PoE contained glucose in cell wall and had higher WPG ratio than HS9.?2?We had to optimize conditions for preparing protoplasts before protoplasts fusion and vector-free DNA electrotransformation.High purity and formation ratio made the higher recombination rate.The optimum condition for preparing HS9 protoplasts were:adding 34%sucrose,1%Gly and glass beads in YEME medium,treating 106/mL protoplasts with 6mg/mL lysozyme for 120min,using buffer solution containing Zn²⁺?Fe3+?Cu²⁺?Mn²⁺?Mo6+,separating mycelium from protoplasts by centrifugation.The optimum condition for preparing PoE protoplasts were mostly same as HS9,but the lysozyme treating time was 60min and need?-glucanase assisting.The HS9 protoplasts preparing by optimum condition purity was 98.87%and formation ratio was 88.15%,while PoE protoplasts purity was 95.73%and formation ratio was 91.27%.?3?Protoplasts fusion was a method of gene transfer.We inactivated HS9 protoplasts by treating with ultraviolet light for 2min and inactivated PoE protoplasts by heat treatment in 60? for 40min.The Inactivation death ratio reached to 99%.Then we optimized conditions for protoplasts fusion.The optimum condition for fusion was treating 106/mL protoplasts with 50%PEG-2000 and the fusion ratio reached to 96.54%.The variable coefficient of 96-well plate fermentation system was 20%,so we considered strains improved 20%enzyme activity as high-producing strains.Also we found strains with higher MTG enzyme activity after fermentation for 3d in 96-well plate fermentation system to shorten fermentation time and mesure enzyme activity in 60? to improve heat stability.The proportion of strains got from protoplasts fusion with higher MTG production,shorter fermentation time and higher heat stability was 6.79%.?4?Vector-free DNA electrotransformation was another method of gene transfer.We cut genome into little pieces of different sizes by Xho I and EcoRI.Then we optimized conditions for vector-free DNA electrotransformation.The optimum condition for electrotransformation was giving HS9 protoplasts and PoE DNA an electric shock on 1.5kV voltage and recovering for 120min after electric shock.The transfer efficiency was 33.8×104CFU/?g.The proportion of strains got from vector-free DNA electrotransformation with higher MTG production,shorter fermentation time and higher heat stability was 11.92%,which was higher than the proportion of strains got from protoplasts fusion.?5?The proportion of strains got from vector-free DNA electrotransformation had higher MTG production than HS9 was 100%,had higher MTG production than PoE was 56%,had shorter fermentation time was 89%and had higher heat stability was 70%.All these are higher than the proportion of strains got from protoplasts fusion.We obtained a high production strain DZ10 and its enzyme activity reached to 8.06U/mL after fermentation for 32h.The MTG produced by DZ10 had higher heat stability and the optimal reaction temperature was 55?.In conclusion,this study established protoplasts fusion breeding and vector-free DNA electrotransformation breeding methods and screened a strain had higher production,shorter fermentation time and the MTG had higher heat stability by gene transfer and recombination.