Cloning And Expression Of Erythrose Reductase Genes Of Trichos Poronoides Oedocephalis

Erythritol is four carbon polyol with highly natural added values, good thermal stability, low sweetness, zero calorific value, no moisture absorption and not produce dental caries so that it can be used as filling type sweetener. It widely exists in the nature, such us wine, soy sauce, honey, pollen grain and animal milk, etc. Its appearance is white crystalline, the sweetness is only about 70% of the sucrose. It can be used for low calorific value food, diabetic food and many kinds of health food, candy, chocolate and lactobacillus beverages products. Due to its high security for food, its demand in market will increase in future. So how to develop a strain which has higher yield is the key to the content of recent research task.Erythritol mainly obtained by osmophilic yeast fermentation through the pentose phosphate pathway. First, glycolysis makes the most carbon into enough reducing power to generate Erythritol, which secreted to the extracellular. As the key enzyme, erythrose reductase is the final enzyme catalyzed the reaction, has direct effects on erythritol generating. So far the research for erythrose reductase is quite little, for the majority of erythritol producing strains, the gene encoding an erythrose reductase is still unknown. Today, in producing of erythritol reports, only the genes of er 1,er 2,er 3, which respectively codes erythrose reductase ER- I, ERII, ER- III for Trichosporonoides megachiliensis SNG- 42 and genes of erythrose reductase in Candida Magnoliae JH110 were identified.This study selected strain osmophilic yeast Trichosporonoides oedocephalis(ATCC 16958). Although the production of erythritol by this strain increased after optimized cultivation, but its production was still difficult to reach large scale of producing standard. Therefore, this research hopes to improve the yield of erythritol by the means of genetic engineering, making the genes of erythrose reducase successfully expressed in prokaryotic bacteria. By cloning the gene of erythrose reductase in this strain, then transformed the cloned gene into E.coli BL-21(DE3), and made successful induced expression. Then provided an eminent strain for the large-scale production of erythritol and also provided excellent gene templates for subsequent work of researching on erythrose reductase activity and three-dimensional modeling. Thus improved the sugar alcohol yield fundamentally. The main research content is as follows:(1)Construction of cloning vectors: Used Primer 5.0 software to analysis the gene sequence of erythrose reductase published in GeneBank, obtaining erythrose reductase gene from existing strains, sub cloning the key enzyme genes, and obtaining the key functional gene sequence(ER). Connected it to the cloning vector pMD18-t to get the cloned strain pM-ER.(2)Construction of prokaryotic expressing vector: Further connected the obtained enzyme gene to the prokaryotic expressing vector PET-28a(+), then constructed prokaryotic gene recombined plasmid(pE-ER) and transformed it into competent E.coli BL21(DE3) strains. Established apparent screening method, completed sequencing, and obtained the expressed strain pE-ER.(3)Identification of expression: Through inducing by inducer IPTG and identifying by analysis of SDS-PAGE and Western Blotting to prove that the constructed recombined expressing strains of pE- ER successfully completed the expression in expressing host bacterium BL21(DE3).