Optimization Of Fermentation Conditions Of Rhodosporidium Toruloides And Enhancement Of Synthesis Of Limonene

Limonene is a valuable monoterpenoid with diverse applications in food,medicine,agriculture,etc.Currently,the commercial production of limonene from citrus pomace cannot meet the evergrowing market demand.With the development of synthetic biology,microbial biosynthesis is offering a sustainable alternative to plant-based extraction.However,the performance of engineered strains can be affected by many factors including the microbial host,the cultivation conditions,etc.Therefore,the potential fermentation conditions influencing the production of limonene by engineered strains were studied by single factor and orthogonal experiments.The conclusions of this paper are as follows:（1）The engineering strains in the laboratory were tested for stability screening and basic performance of producing limonene.After three rounds of transformation screening,the engineering bacteria 3#showed relatively stable and high yield in the three screening,and the titer of limonene was 52.5 mg/L.This strain was determined as the research object.（2）The optimal combination of experiments is:The optimized medium was MM,with a working volume of 50 m L,inoculum OD₆₀₀=0.6,22℃,p H 6,and a dodecane coverage（v/v）of 20%.The final limonene titer was improved from 52.5 to 358.1 mg/L,a 586%net increase in titer and 34.8-fold improvement in production efficiency(mg/OD₆₀₀)on250-m L flasks.（3）The preliminary amplification experiment was carried out in a 3 L fermenter,421.5 mg/L limonene was obtained.（4）Finally,the solution of grape shoots hydrolysate was used as the medium for de novo synthesis of limonene.Under the condition of no additional nutrient elements,9.8mg/L limonene was obtained.This was the first time that the solution of grape shoots hydrolysate was used as the medium to produce limonene,and the conversion of low-value compound to high-value compound was realized.23.8 mg/L limonene was obtained by adding 2 g/L ammonium sulfate and 1 g/L potassium dihydrogen phosphate to the hydrolysate of grape branches.