Breeding Of High-yielding β-carotene Strain Blakeslea Trispora And Optimization Of Its Fementation Technique

β-Carotene is a kind of important carotenoid widely used in the fields of medicine,functional food,and cosmetics.β-Carotene with cis and trans all two isomers,studies have shown that cis isomer ofβ-carotene with higher nutritional value and anti-vasclular disease.However,the cis isomer is mainly derived from naturalβ-carotene rather than synthetic.Production of naturalβ-carotene by the microorganisms of Blakeslea trispora（B.trispora）is one of the main pathways,but the microorganisms have some problems because of its disadvantages such as the poor stability and low content ofβ-carotene.In this study,the preservation of B.trispora in our laboratory for starting strain,to establish a separate single colony method of filamentous fungi and high-throughput screening strategy screening a stable high-yielding strain of the filamentous fungi B.trispora.The high-yield strains is hope to been obtained combined with mutagenesis breeding technology of B.trispora.The concrete results are as following:（1）A new method of separation of single colony of filamentous fungi was established,which effectively solved the problem of filamentous fungi of pure screening.This study screening for a screening agent clethodim to separate filamentous fungi.The 0.01%-0.04%（v/v）clethodim in PDA solid plate can make the B.trispora（-）form obvious single colony prevented degradation strains and guaranteed the stability of strain performance.The fermentation performance ofβ-carotene in B.trispora（-）single colony also increased significantly,whereas the high concentration of clethodim can inhibit the growth of hypha.This technology can also be applied in the cultivation of Mortierella and Aspergillus nidulans.（2）A high-throughput screening strategies of B.trispora（-）was established,which successfully bred for high yield mutant strains.Firstly,the rapid detection of the number of spores method was established by OD value and the mutant of B.trispora（-）was selected by the use of lovastatin in PDA plate.The high yield spore ability of B.trispora（-）was screened by physical and chemical mutagenesis,which is 4.87-fold higher than the parent strain of the spore production quantity.Secondly,the best conditions of high-throughput method for screening B.trispora strains that produce high yields ofβ-carotene in 96-well deep-hole culture plate was identified based on 2,3,5-triphenyltetrazolium chloride dyeing.That is,the best fermentation time and the dyeing time was 2 d and 8 hours.The best concentrations of TTC and oscillation dissolution time were 1.2%and 1.5 hours,respectively.The absorption was measured at485 nm on an enzyme-standardized instrument.According to theβ-carotene production and TTC staining degree is proportional to screen mutant strains.Using this screening method,the mutant XL2（-）,obtained rapidly via repeated gradient promotion mutagenesis by N-methyl-N′-nitro-N-nitrosoguanidine and ultraviole,attainedβ-carotene yields of1.34±0.06 g/L,which is 121.9-fold higher than the parent strain.The result showed the hereditary stability was fine when continuous transfer of culture of strain Blakeslea trispora XL2（-）reached 6 generations from one tube to anther.The expressions of hmgr,ipi,carG,carRA and carB involving theβ-carotene biosynthetic pathway at 64 hours was detected by real-time quantitative PCR,the expression level of carG,carRA,and carB in mutant XL2（-）was significantly higher than parent strain.The mutation strain appears to improve the production ofβ-carotene by strengtheningβ-carotene metabolic flow.（3）Optimum the fermentation conditions of B.trispora strains that produce high yields ofβ-carotene.Firstly,the single factor optimization of fermentation conditions were50 mL broth’s volume in 250 mL shake flasks and B.trispora XL2（-）and B.trispora（+）was inoculated seed medium firstly,then mixed fermentation.Then via response surface method,the optimal medium was obtained,which was contained（g/L）:soybean oil 20,corn steep powder 13.96,corn starch 40,KH₂PO4 3.17,MgSO₄?7H₂O 1,glucose 10,soybean meal 30,Span 20 2,butylated hydroxytoluene（BHT）4,VB₁ 0.0001.Under the optimum conditions,the yield ofβ-carotene was improved significantly which the maximum yield ofβ-carotene was 1.41±0.09 g/L,which is 128.2-fold higher than the parent strain B.trispora（-）.The above research improved the B.trispora fermentation to produceβ-carotene significantly,laid a good foundation for the industrialization of B.trispora fermentation.