Preparation Of Trehalose By Catalytic Cellobiose Based On OtsAB Pathway

This paper focuses on the feasibility of synthesizing trehalose with cellobi ose as a substrate.Escherichia coli was used as host cell,and the synthesis pathway of cellobiose to trehalose was constructed by using multiple plasmid expression system.The key factors affecting trehalose synthesis were discussed.Finally,a TCA cycle was established.A joint energy regeneration system that increases trehalose production and conversion.The major results are as follows:(1)In this paper,the 6-phosphate trehalose synthase(OtsA)and 6-phosphate trehalose phosphatase(OtsB)derived from Pseudomonas stutzeri A1501 were cloned to construct the OtsAB pathway for the synthesis of trehalose.This pathway was introduced exogenously into E.coli BL21(DE3)to obtain a recombinant strain E.coli BL01.Using glucose as a substrate,E.coli BL01 synthesized trehalose by whole cell catalysis,and the trehalose yield was 0.25 g/L after 9 h.(2)Glucose is the product of the hydrolysis of cellobiose,which requires expensive commercial beta-glucosidase.Therefore,the development of trehalose by the use of cellobiose-producing microorganisms can alleviate these problems.Based on E.coli BL01,E.coli BL05 was constructed by cloning the UDP-glucose pyrophosphorylase gene galU from E.coli BL21 and the cellobiose phosphorylase gene cepA from the natural cellulose-decomposing bacteria Saccharophagus degradans.E.coli BL05 can realize the biotransformation of cellobiose to trehalose.When trehalose is synthesized by 20 g/L cellobiose as a substrate by whole cell catalysis,10 mM validamycin A is added,36 h later.The highest yield of trehalose is about 1.3 g/L.The effects of Jinggangmycin,temperature and iodoacetic acid on trehalose production were also investigated.The results showed that the addition of 10 mM validamycin A significantly inhibited the d egradation of trehalose and increased the production of trehalose;increasing the temperature and adding iodoacetic acid did not help to increase the yield of trehalose.(3)The supply of substrate and energy,the degradation of trehalose,and the diversion of other metabolic branches all affect the production of trehalose.In order to overcome the above limitation factors and increase the cellobiose to trehalose conversion rate,this study knocked out the zwf and pgi genes on the basis of E.coli YW-3 to construct E.coli ZL-1A,which was heterologously expressed.Glucose-1-phosphate is used as a substrate to synthesize GDP-glucose GDP mannose pyrophosphorylase gene gmppb and glucose-6-phosphate and GDP-glucose as substrate to synthesize trehalose-6-phosphate trehalose-6-phosphate The enzyme gene otsA realizes the conversion of cellobiose to trehalose.At the same time,the effect of trisodium citrate on the synthesis of trehalose was investigated.When adding 10 g/L trisodium citrate,the yield of trehalose obtained from E.coli YW-3A was 0.41 g/L.The increase without adding was about 10 times;the yield of trehalose when adding 10 g/L trisodium citrate to E.coli ZL-1A was 0.12 g/L,which was about 6 times higher than that without addition.