Manipulation Of Geobacillus Thermoglucosidasius For Overproduction Of Riboflavin

Compared with the normal temperature fermentation process of normal temperature microorganisms,the use of thermophilic microorganisms for high temperature fermentation in industrial production has the potential advantages of improving the biochemical reaction rate,reducing the risk of bacterial contamination and reducing the energy consumption of fermentation cooling.Thermophile Geobacillus thermoglucosidasius can make full use of lignocellulosic hydrolysates at an optimal growth temperature of 60℃ with generation time of 16 minutes,so it was an excellent candidate strain for microbial fermentation at high-temperature.G.thermoglucosidasius,as a production strain,can be resistant to high temperature,grow rapidly and make use of cheap carbon source.It has great development value because of the combination of industrial production needs such as reducing the use of cooling water,reducing the risk of bacterial contamination,shortening the fermentation period and reducing the cost of raw or auxiliary materials.A fast and convenient genetic manipulation method of Geobacillus thermoglucosidasius was developed by using heat-resistant green fluorescent marker in this study.Based on this method,the chassis of G.thermoglucosidasius was modified by metabolic engineering generation by generation,and the yield of riboflavin at high fermentative temperature from scratch and from low to high was realized.The first generation riboflavin strain Rib-Gtd was obtained including the introduction of heterogenic riboflavin biosynthesis gene cluster,with riboflavin yield up to 28.7 mg/L.The second generation riboflavin strain Rib-Gtd1 was obtained by point mutation of ribCGtg to weaken riboflavin cell consumption,with riboflavin yield up to 84.6 mg/L.PurRGtg,the regulatory protein of purine pathway,was eliminated from inhibiting the purine pathway to obtain the third generation riboflavin strain Rib-Gtd2,with riboflavin yield up to 110.7 mg/L.In order to avoid the metabolic flow of purine synthesis pathway to adenine,purAGtg was further knocked out to increase the supply of precursor to obtain the fourth-generation riboflavin strain Rib-Gtd3,and the riboflavin production reached 171.6 mg/L.The fifth generation riboflavin strain Rib-Gtd4 was obtained by knockout of ccpNGtg regulating center carbon metabolism to riboflavin production,and the riboflavin yield reached 260±15 mg/L.Lactate dehydrogenase gene ldhGtg was knocked out to block the production of by-product lactic acid.The 6th generation riboflavin strain Rib-Gtd5 was obtained with riboflavin yield up to 445.8 mg/L.Finally,the engineered strain produced 1034.5 mg/L riboflavin yield by fermenting 12-h in a mineral salt medium with a glucose-xylose mixture as a carbon source.In this study,a series of metabolic engineering modifications were carried out on G.thermoglucosidasius to achieve high temperature fermentation of riboflavin in thermophilic bacteria.This study shows that G.thermoglucosidasius is expected to overcome the natural defects of the normal temperature process,break through many limiting factors of riboflavin production in normal temperature fermentation,become a high yield host of riboflavin fermentation at high temperature in industrial production,have the potential of further transformation and the prospect of improving the market competitiveness of riboflavin industry in China.