Abstract: In order to study the feasibility of using konjac powder for preparation of konjac kombucha,the gene cel9 C coding β- glucanase of Clostridium josui was cloned into the expression vector p ESP- 2- 2 and the recombinant plasmid was transformed into Schizosaccharomyces pombe,to obtain the engineered yeast strain y AS56,and the engineered yeast was used in konjac kombucha fermentation. Using total acid as the response value,the fractional factorial design and central composite design were applied to optimize the fermentation conditions. Two significant factors,loading volume and time,were selected among seven factors. The optimal conditions of fermentation were as following:green tea 0.4%,sucrose 7.5%,konjac powder 0.25%,inoculum size 10%,ratio of cells concentration of Saccharomyces cerevisiae / engineered yeast 0.5,loading volume 98.7 m L,time 124 h. The results of confirmation test with optimal parameters showed that the production of total acid was(27.88±0.26) g/kg,close to the predictive value,being increased by 41.45% compared with the un-optimized conditions. This indicated that using konjac powder and engineered yeast strain for preparation of konjac kombucha was feasible.