【作者】 张磊；

【导师】 余龙江；

【作者基本信息】 华中科技大学 ， 生物化工， 2017， 硕士

【摘要】 β-胡萝卜素是一类重要的类胡萝卜素,在医药领域、食品领域和化妆品领域等方面具有广泛应用。β-胡萝卜具有顺式和全反式两种异构体,研究表明,顺式异构体的β-胡萝卜素具有更高营养价值和抗心血管疾病等功能,该异构体主要来源于天然β胡萝卜素,而非人工合成。利用三孢布拉霉菌株发酵是生产天然β-胡萝卜素的主要途径之一,但仍存在产量不高、菌种稳定性较差等关键问题。因此,选育三孢布拉霉稳定高产β-胡萝卜素菌株成为研究重点。为此,本研究以实验室保藏的三孢布拉霉（Blakeslea trispora）为出发菌株,通过建立丝状真菌分离单菌落方法以及高产β-胡萝卜素的三孢布拉霉菌株高通量筛选方法,并结合三孢布拉霉诱变选育技术,以期选育获得高产菌株,并通过发酵工艺优化,提高β-胡萝卜素产量,取得的主要研究结果如下:（1）建立了一种丝状真菌分离单菌落方法,可有效解决丝状真菌平板分纯筛选难题。本研究获得一种丝状真菌筛选剂烯草酮,在烯草酮与PDA固体培养基体积比为0.01%-0.04%时,可使三孢布拉霉形成明显单菌落,更好的防止菌株退化,保证菌株性能的稳定性且该三孢布拉霉单菌落发酵β-胡萝卜素性能也得到明显提高,而烯草酮浓度进一步提高则会抑制菌丝生长。本技术在产油脂丝状真菌被孢霉（Mortierella）、模式真菌构巢曲霉（Aspergillus nidulans）也可以很好的推广应用。（2）建立了三孢布拉霉高通量筛选方法,并成功选育获得β-胡萝卜素高产突变株。首先,建立了利用OD值快速检测孢子数量的方法,以及利用洛伐他汀定向筛选三孢布拉霉的方法,通过理化诱变筛选获得高产孢能力的负菌,使其产孢量较出发菌株提高了4.87倍;其次,结合TTC染色,确定了96深孔板高通量筛选高产β-胡萝卜素突变株的最佳条件:即,在三孢布拉霉发酵时间2天后,用1.2%TTC染色8 h,二甲基亚砜振荡萃取1.5 h,萃取液在酶标仪上485 nm下检测菌体TTC染色程度,根据β-胡萝卜素产量与TTC染色程度成正比筛选,利用上述筛选技术,获得β-胡萝卜素高产突变菌株Blakeslea trispora XL2（-）,β-胡萝卜素产量最高为1.34±0.06g/L,相对于原始出发菌株Blakeslea trispora（-）提高了121.9倍。对菌株Blakeslea trispora XL2（-）连续6次传代进行遗传稳定性评价,结果表明,Blakeslea trispora XL2（-）可稳定遗传。实时荧光定量分析了β-胡萝卜素代谢途径中的5个关键酶基因表达,发现突变株XL2中carG,carB和carRA显著提高,强化了β-胡萝卜素的代谢流,提高了β-胡萝卜素的产量。（3）优化了β-胡萝卜素发酵条件。首先,确定了单因素优化的发酵条件:装液量50 mL/250 mL,三孢布拉霉突变株XL2（-）和正菌先接入种子培养基培养再混合发酵;其次,通过响应面分析,优化了发酵培养基（g/L）:大豆油20,玉米浆干粉13.96,磷酸二氢钾3.17,玉米粉40,葡萄糖10,黄豆粉30,硫酸镁1,Span 20 2,BHT 4,VB₁ 0.0001。最后,采用上述优化条件,使β-胡萝卜素产量显著提高,最高达1.41±0.09 g/L,较出发菌株Blakeslea trispora（-）提高了128.2倍。以上研究大幅提高了三孢布拉霉发酵生产β-胡萝卜素的水平,为其产业化应用奠定了较好的基础。更多还原

【Abstract】 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.更多还原