Construction Of Recombinant Bacillus Subtilis For Synthesis Of Guanidinoacetate

Guanidinoacetate（GAA）is a naturally occurring amino acid derivative in vertebrates.It has many physiological functions,such as promoting energy metabolism and affecting nervous regulation,which has been widely used as nutraceutical and pharmaceutical intermediates.At present,the industrial production of GAA is mainly based on the chemical synthesis method.However,there are some problems such as product pollution,purification complexity,environmental pollution,and so on.Therefore,the development of microbial chassis cells for the synthesis of GAA is of great significance to provide an environmentally safe and more efficient GAA production method.Although biosynthesis of GAA using Escherichia coli as a host has been achieved,the potential risk of endotoxin contamination may affect its application in the food and pharmaceutical industries.In this study,Bacillus subtilis,generally regarded as food safe strain,was used as the production host.Through the construction and optimization of the GAA heterologous synthesis pathway,blocking of substrate degradation pathway and optimization of ornithine cycle,B.subtilis was constructed to synthesize GAA using glycine and arginine as substrates,which provides an effective food safety production method for GAA.The main research results and conclusions are as follows:（1）The heterologous synthesis pathway of GAA using arginine and glycine as substrates was constructed.The gene aga T encoding L-arginine:glycine amidotransferase（Aga T）from Amycolatopsis kentuckyensis has been reported to be the best to catalyze the synthesis of GAA in E.coli.Firstly,the aga T was cloned into the expression vector p HT01 and transformed into B.subtilis 168.Four constitutive promoters with different strengths P_{lyt R},P₄₃,P_333,and P₅₆₆regulated the expression of Aga T.While with GFP as a reporter protein,the relative fluorescence intensity under the control of promoter P₅₆₆ was 1.7-fold than P_{lyt R},1.6-fold than P_43,and 1.9-fold than P₃₃₃.It was confirmed that the regulation level of Aga T controlled by P₅₆₆was the highest.（2）The Ribosome binding site（RBS）and N-terminal coding sequence（NCS）of Aga T were optimized.Firstly,a mutant library of RBS and NCS of Aga T was constructed by designing degenerate primers.Using GFP as a reporter protein,the mutant with high fluorescence intensity was obtained by flow sorting technique.After whole-cell catalysis,the biosynthesis of GAA was achieved in B.subtilis for the first time in this study.Next,we replaced B.subtilis 168 host with BS168-NM6 host.At the same time,we optimized the conditions of whole-cell catalysis from the substrate concentration and OD₆₀₀ of strain.The titer of GAA was 0.24 g·L^-1,which was further increased by 3.9-fold.（3）The effect of blocking the degradation pathways of the substrates on the titer of GAA was investigated.Firstly,the first gene arg I of arginine degradation pathways and gcv P of glycine degradation pathways were separately knocked out.It was found that knocking out arg I gene alone was more effective than knocking out gcv P alone and the cell growth was not affected.The titer of GAA was increased to 0.46 g·L^-1.Next,the related genes of substrate degradation pathway were knocked out in combination.It was found that knocking out arginine catabolic pathway genes arg I and nos A was more effective than knocking out both glycine and arginine catabolic pathway.The titer of GAA of the obtained strain B6-2 was increased to 0.91g·L^-1.（4）The natural ornithine cycle in B.subtilis was optimized to promote the conversion of ornithine to arginine,which alleviated the inhibition of Aga T by ornithine on the one hand,and increased the arginine required for GAA biosynthesis on the other hand.The genes related to the ornithine cycle,as well as the car AB,gln A,and asp A gene were overexpressed on the B6-2 genome under the regulation of the P_veg promoter.The maximum titer of GAA biosynthesis by whole-cell catalysis of strain B10 was 4.26 g·L^-1 with a maximum productivity of 1.58 g·L^-1·h^-1.The conversion rate of glycine and arginine was 13.7%and 31.7%,respectively.