Optimization Of Eiythritol Production By Torula Sp.B84512and Its Gene GPD1

Erythritol, a four-carbon sugar alcohol or polyol, is well known as a food additive. Dueto its low-calorie, high tolerance and anti-caries properties, more and more people committedto develop the erythritol application value. The study used fed-batch method to improve theproductivity of erythritol by strain Torula sp.B84512. We found that byproduct glycerol wasaccumulated while sythnsied the erythritol. Therefore, we decided to gene knockout the keyenzyme3-phosphate glycerol dehydrogenase (GPD1). By screened the Haploid strains ofTorula sp. B4512and constructed the GPD1deletion strain Torula sp. B4512-7△GPD1andTorula sp. B4512-9△GPD1. Then intergrated two haploid strains into the diploid Torula sp.B4512-79△GPD1strain. Comparaed the fermentation ability between recombinantion strainTorula sp. B84512-79△GPD1and original strain Torula sp. B4512. The results were asfollows:(1) Used fed-batch method to improve the productivity of erythritol by strain Torulasp.B84512. When the total glucose concentration was reached50%, the maximum erythritolproduct was253g/L, the yield was1.03g/(L h). The cytoplasmic glyceraldehyde3-phosphate dehydrogenation (isozyme GPD1) was the key enzyme in glycerol metabolic.Sowe decided to knockout the GPD1gene in Torula sp.B84512to induce the productivity oferythritol.(2) Filtered the Torula sp.B84512haploid,3haploid strains could produce high yieldof erythritol relatively and named Torula sp.B84512-7, Torula sp.B84512-8and Torulasp.B84512-9. PCR method was used to verify the haplotypes of Haploid strains. Theexperimental results showed that Torula sp.B84512-7and Torula sp.B84512-8were alphatype, and Torula sp.B84512-9were a type. Cultured three strains in fermentation mediums,Torula sp.B84512-7could produce more erythritol than Torula sp.B84512-8.So Torulasp.B84512-7and Torula sp.B84512-9strain was used for gene knockout.(3) Based on the theory of cre-loxp to knockout the GPD1gene in Torula sp. B84512-7and Torula sp. B84512-9, we constructed the Torula sp. B84512-7△GPD1strain and Torulasp. B84512-9△GPD1strain. Two haploid recombinant bacterias were hybrided to form theTorula sp.B84512-79△GPD1strain. Compared the GPD1enzyme activity between therecombinant strains Torula sp.B84512-△GPD1and the original strains Torula sp.B84512, theresult showed that the GPD1enzyme activity of Torula sp.B84512-△GPD1was lower thanthe original strain Torula sp.B84512. But the Torula sp.B84512-79△GPD1strain may beunable to resist the osmotic pressure and growth in fermentation medium slowly.