Metabolic Engineering Of Bacillus Subtilis To Synthesize 3’-sialyllactose

Human milk oligosaccharides（HMOs）are the third most nutritious component of breast milk,after fat and lactose.As an important component of HMOs,3′-sialyllactose（3′-SL）plays a key role in the development and maturation of the infant immune system.It has shown broad market prospects in the pharmaceutical and food industries,especially in the infant formula industry.Since the content of 3′-SL in the animal emulsion is very low,which is not conducive to extraction,and the enzyme catalysis method is expensive and difficult to scale.By contrast,microbial synthesis of 3′-SL is safe,low-cost,and easy to scale.In this study,the food-safety grade microorganism Bacillus subtilis was used to construct a recombinant strain for the de novo synthesis of 3′-SL.Firstly,the heterologous synthetic pathway of 3′-SL was introduced into B.subtilis.Then,the expressions of key genes,utilization of substrates and metabolic flux were regulated.The details are as follows:（1）Constructing 3′-SL de novo biosynthesis pathway.Firstly,the de novo synthetic pathway of 3′-SL was constructed by using B.subtilis 3D6.2,which could produce N-acetylneuraminic acid（Neu5Ac）and stored in the laboratory,as the initial strain.The neu A（encoding CMP-Neu5Ac synthetase）and nst（encodingα-2,3-sialyltransferase）from Nesseria meningitides were expressed by a plasmid p HT01 to obtain p P₁-neu A-P₁-nst.the recombinant plasmid was transformed into 3D6.2 and the initial titer of 3′-SL was 3.9 mg/L by shaking-flask fermentation and LC/MS analysis.（2）Reconstruction and optimization of 3′-SL synthesis pathway.Five types of promoters and three types of RBS with different strengths were selected to optimize the expression of two key enzymes Neu A and Nst in the 3′-SL biosynthetic pathway.The 3′-SL production reached119.6 mg/L.Lactose is an important substrate for the synthesis of 3′-SL.In order to improve the conversion rate of lactose,the genes related to the decomposition of lactose were further knocked out,and the 3′-SL titer of recombinant strain BLa K1 was 344.7 mg/L,which was 188%higher than that of strain BZ27.（3）Enhancing metabolic flux of 3′-SL synthetic pathway by self-assembly of key enzymes.Five types of protein scaffolds（PDZ,GBD,SH3,CC-Di-A,and CC-Di-B）were selected for fusion expression with key enzymes Neu A and Nst to improve the metabolic flux of the 3′-SL synthetic pathway.Seven recombinant plasmids were constructed and transferred into strain Z1.The fermentation results showed that CC-Di-A and CC-Di-B significantly increased the 3′-SL production.The 3′-SL titer of strain BLa S7 reached 678.9 mg/L,which increased by 97%compared with BLa K1.（4）Optimizing the stoichiometry of enzyme assemblies.First,to ensure sufficient expression of nst in optimizing the stoichiometry,the low copy plasmid p HT01 and high copy plasmid p P43NMK were used to express neu A and nst,respectively.The fermentation verified that the using of two plasmids had no adverse effects on cell growth and 3′-SL synthesis.Then,it was proven that the introduction of the above protein scaffolds had no adverse effect on the activity of pathway enzymes by investigating the cell growth and 3′-SL titer before and after fusing CC-Di-A and CC-Di-B.Finally,when the ratio of CC-Di-A and CC-Di-B was adjusted to 2:1,the 3′-SL titer reached 1252.1 mg/L.