Breeding Of Rhodotorula Toruloides With High Stress Tolerance By Heavy Ion Mutation Combined With Laboratory Adaptive Evolution

Lignocellulose is one of the most abundant renewable resources in the world,but at present,it is facing many challenges to produce microbial lipids by biorefinery.Fermentable sugar is produced in the process of lignocellulose hydrolysis,but at the same time,various toxic by-products are also produced,which are unfavorable to the growth and fermentation of microorganisms and also affect the industrialization process of microbial lipids.Therefore,it is very importent to improve the tolerance of strains to hydrolysate environment.In this thesis,Rhodotorula toruloides Y4,an oleaginous yeast,was mutated by heavy ion by using ¹²C⁶⁺heavy ion beam,and the mutant strain was adaptively evolved in the laboratory,finally the strain could tolerate high-solid loading straw hydrolysate and was applied to the production of microbial lipids.The research results are as follows:1.The heave ion beam of ¹²C⁶+was obtained by using the heavy ion accelerator,and R.toruloides Y4 was mutated by heavy ions.By calculating the survival rate of the strain,fitting the biological parameters such as the maximum specific growth rate and lag phase with the mathematical model,the changes of the strain’s ability to tolerate hydrolysate before and after mutagenesis were compared,and the effect of heavy ion mutagenesis and the solid loading limit of R.toruloides Y4 to tolerate hydrolysate were evaluated.The cell survival rate of the strain was about 50%after 200 Gy irradiation.R.toruloides Y4 can tolerate 12%（w/w）of the maximum solid load of the hydrolysate.Compared with the control,the maximum specific growth rate of the mutant strain in the hydrolysate is obviously increased,the lag phase is obviously shortened,and the maximunm solid load of the hydrolysate that the mutant strain can tolerate is also slightly increased.2.The strain obtained by heavy ion mutation was adaptively evolved in the laboratory.The laboratory evolution was carried out in the hydrolysate of straw with gradually increased solid loading.After 9 generations,the strain could grow in the hydrolysate with 20%solid loading.After single colony isolation,two strains L11 and L12 were obtained,which can tolerate 17%of the hydrolysate,and the capacity of R.toruloides was increased by 42%.The fermentation conditions of L11 were optimized by means of fermentation engineering,and L11 could finally tolerate 20%solid loading of hydrolysate to produce microbial lipids,the lipid titer reached to 6.91 g/L,the lipid content was up to 52.2%,and the fermentation lag phase is shortened from 5 days to 1day.3.Tracking the preparation process of high-solid soliding straw hydrolysate,using modern analytical instruments to determine the composition of the hydrolysate,comparing the tolerance of R.toruloides Y4 and mutant strain L11 to different types of lignocellulose hydrolysate by-products.The main by-products affecting the growth of the strain were phenolic compounds and the tolerance of L11 to p-hydroxybenzoic acid,syringic acid and levulinic acid was improved.