CN110628758A - 一种提高寇氏隐甲藻dha产率和得率的方法 - Google Patents
一种提高寇氏隐甲藻dha产率和得率的方法 Download PDFInfo
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Abstract
本发明公布了一种通过ARTP诱变获得高DHA产率和得率的寇氏隐甲藻ATCC30556突变藻种的方法。其特征是包含如下步骤:(1)对寇氏隐甲藻ATCC30556进行ARTP诱变,获得诱变藻种后,利用淀粉能够和碘相结合呈现紫色的原理,对转化子进行通量筛选,挑选平板上与出发株相比,菌落直径较大并且菌体呈现淡紫色的菌落;(2)利用摇瓶发酵,复筛上述(1)中获得的候选诱变藻种,获得生长速率、油脂和DHA产率都显著性提高,同时淀粉含量显著降低的藻种;(3)对(2)中获得的诱变藻种利用5‑L发酵罐进行表型进一步确认,确定其DHA产率及得率。利用本发明的方法获得的寇氏隐甲藻突变藻种与出发藻种相比,DHA产率和得率分别提高到原来的1.69倍和1.28倍,达到57.7mg/L/h和51.96mg/g,相比于其他的育种方法,本发明获得的DHA产率和得率是目前报道的最高值,在DHA产业化领域具有重要的应用前景。
Description
技术领域
本发明公开一种提高寇氏隐甲藻DHA产率和得率的方法,具体涉及一种高产DHA诱变藻株的获得方法及应用。属于工业微生物领域。
背景技术
DHA(二十二碳六烯酸),是正常脑功能所必需的主要脂质,因而被称为脑黄金。DHA主要存在于人脑中的灰质部分,其所占比例约为人脑组织细胞总脂含量的10%。DHA作为一种重要的不饱和脂肪酸存在于人脑神经细胞膜中,并且是最先被大脑细胞吸收利用的脂肪酸之一。DHA主要分布在脑细胞的突触中,对于神经递质传递途径、突触传递途径和信号传导均有影响作用。DHA也是视网膜组织中的重要组成成分,约占视网膜柱细胞中总脂肪酸含量的60%。DHA通过强化视网膜细胞和脑组织之间的联系网络,传递光刺激,从而使眼睛更好地发挥功能。因此视网膜细胞中的DHA含量至关重要。DHA对血小板凝集有抑制作用,因此摄入足量的DHA可以防止血栓形成,也可以防治心肌梗塞和脑梗塞等疾病。
利用海洋微藻异养发酵生产DHA是目前重要的DHA生产方法。裂殖壶藻(Schizochytrium)是一种富含DHA的微藻种属,也是目前在DHA发酵生产中使用最为广泛的藻株。隐甲藻中DHA占长链不饱和脂肪酸的比例大于99%,并且在过去30年里未见有关隐甲藻产生毒素的报道,也是第一个被FDA批准用于生产DHA的微藻。
目前,实际隐甲藻发酵过程中生产应用的一个重要瓶颈问题是生产成本偏高,这是由于DHA生产率和产量相对较低造成。提高隐甲藻DHA产率和得率的一直是研究的热点,例如,
在补料分批培养中通过控制乙醇进料提高DHA产率和得率。通过开发连续供氮策略以减轻补料分批培养期间的高氮抑制,提高葡萄糖培养条件下DHA的产率和得率。然而,仍然需要进一步努力提高DHA的生产率和产量,降低发酵成本。
由于相对较高的阳性突变率和更稳定的突变体遗传表型,ARTP诱变育种技术被认为是一种强大的微生物育种技术。ARTP诱变技术已成功应用于细菌,真菌和微藻。ARTP诱变育种技术也在寇氏隐甲藻中有所应用。例如,ARTP诱变结合基于稀禾定的筛选获得名为M-1-2的寇氏隐甲藻突变体。M-1-2的生物量和油脂含量分别增加了24.32%和7.05%。需要将ARTP诱变育种技术与高通量的筛选方法相结合,高效诱变同时实现高通量的筛选,才能获得性状优良的寇氏隐甲藻诱变藻种。
淀粉和油脂是寇氏隐甲藻体内的主要存储物质,同时他们使用共同的前体物质,因此阻断淀粉合成被认为是提高寇氏隐甲藻藻体内部油脂含量的一种有效策略。但是这通常会导致生长的减弱,影响DHA产率和得率。研究发现含有淀粉的寇氏隐甲藻菌落很容易被碘蒸气染色,可以实现平板上高效的筛选。因此,可以利用碘蒸法对含有寇氏隐甲藻的平板进行染色筛选,获得菌落直径较大同时颜色较淡的藻种。
本发明提供了一种通过阻断淀粉合成,增加流向生长和DHA合成的碳代谢流,快速获得性状优良的寇氏隐甲藻藻种的方法。
发明内容
本发明的目的是提供一种提高寇氏隐甲藻DHA产率和得率的方法。
本发明公布了一种通过ARTP诱变和碘蒸法相结合的筛选方法获得高生长和高DHA含量的寇氏隐甲藻藻种的方法。具体步骤包括:
1.将寇氏隐甲藻细胞接种于C9N2液体培养,25℃,180rpm培养36小时,取10μL菌液(细胞浓度约为106-107mL-1),均匀涂布于ARTP仪器专用小铁片上,利用ARTP诱变仪(无锡源清天木生物科技有限公司SERIAL NO:M114852322403N)进行诱变,诱变时间为40s,功率为100W。诱变结束后,将载有诱变菌液的小铁片放入500μL的C9N2液体培养基中,吹打混匀。取50μL稀释后的菌液,均匀涂布到C9N2固体平板培养基上。将平板培养基放在25℃培养箱中培养,培养10-15天后,利用牙签将新菌落转移到新的C9N2固体平板培养基上,培养5-7天。
2.将固体碘倒在平板培养皿中,用5L的烧杯倒扣平板,形成一个密闭的环境。在5L烧杯底部放置一个含有隐甲藻诱变藻种的平板,固体碘升华形成的碘蒸气能均匀地将平板中各个单菌落进行染色(熏蒸后示意图,见图1)。整个实验在通风橱中进行,熏蒸时间为2-3min。熏蒸完毕后将目标菌株用灭过菌的移液枪枪头转移到C9N2液体培养基中培养,进行下一步的摇瓶验证实验。
3.将步骤2中筛选出的隐甲藻诱变藻种及出发株,同时接入C27N6液体培养基中,每隔24小时取样1mL,用紫外可见分光光度计在波长490nm的条件下测定OD值,绘制生长曲线,筛选生长未受到损伤的藻种,如图2所示,突变株1的生长与出发株基本一致.
4.将步骤3中突变株1与出发藻种等OD同时接入C27N6液体培养基中,每隔24小时取样1mL,用紫外可见分光光度计在波长490nm的条件下测定OD值,绘制生长曲线(图3A),并在培养72、84、96和108小时时,采用称重法测定细胞干重(图3中的B图),测定96小时时出发藻种和突变藻种1的油脂含量和DHA占总油脂比例(图3中的C、D图),测定72、84和96小时时出发藻种和突变藻种1的淀粉含量(图3中的E图)。
5.将在摇瓶中筛选出的优良菌株进行发酵罐放大培养。出发藻种和诱变藻种1在C9N2液体培养基转接两代,然后转接到200mL的C27N6的培养基中进行种子培养,培养时间为72小时。72小时后将种子液接到装有2LC27N6的培养基的5L发酵罐中进行发酵培养。接种量为10%,发酵温度控制在25℃,转速在200rpm-800rpm,保持溶氧在30%以上,葡萄糖浓度保持在15g/L以上。补料葡萄糖浓度为479g/L,补料酵母粉浓度为375g/L。每隔12小时测定一次葡萄糖消耗量并及时补糖,补氮策略为12h-24h的补氮速率为1.2mL/h,24h-96h为2.1mL/h。取合适的时间点进行油脂和淀粉含量的验证,如图4所示。
所述步骤1,2,所述C9N2液体培养基是由葡萄糖9g,酵母浸粉2g,海盐25g,加水至1L而成,pH为6.5;
所述步骤1所述C9N2固体平板培养基是由葡萄糖9g,酵母浸粉2g,海盐25g,琼脂1.5g,加水至1L而成,pH为6.5;
所述步骤3,4,5所述C27N6培养基是由葡萄糖27g,酵母浸粉6g,海盐25g,加水至1L而成,pH为6.5。
本发明所述的利用特定的诱变技术对隐甲藻进行改造获得高生长和高DHA含量的藻种,特定的诱变技术是ARTP诱变技术,筛选方法是碘蒸平板筛选法。
隐甲藻突变藻种生长、淀粉、油脂和DHA含量分析。利用富集培养基对所得到的隐甲藻藻种突变体进行生长、淀粉、油脂和DHA含量分析。生长测定采用两种方法(1)利用紫外分光光度计,选择OD490nm波长,每组做三个平行;(2)称重法,每组做三个平行。淀粉含量测定:收集5.0OD细胞培养液,离心,倒掉上清,用1mL的PBS溶液洗一遍,然后加入1mL 6mol/L的HCl和0.5mL的ddH2O,在100℃下反应30min,将细胞提取物恢复到室温,加入0.5mL 6mol/L的NaOH,用0.2μm的过滤膜过滤,测出滤液中还原糖的含量,计算得到淀粉的含量。油脂分析方法:3500×g离心10分钟收集细胞,并用PBS缓冲液(NaCl 137mmol/L,KCl 2.7mmol/L,Na2HPO4 10mmol/L,KH2PO4 2mmol/L)洗涤细胞一次,并冻成干粉。利用含有0.01%丁羟甲苯的氯仿/甲醇溶液(2:1,v/v)抽提25mg藻粉,总共抽提3到4次,然后利用1mL的1.0M KCl和蒸馏水洗涤,最后用真空浓缩仪抽干,获得总油脂含量。DHA含量测定:称量大约25mg藻粉,加入4mL溶液(2mL甲醇+2mL氯仿+0.6mL硫酸)98℃反应2小时,冷却后,加入2mL水,7400×g离心10分钟,收集下层有机相。利用GC/MS仪器测定DHA含量,GC/MS仪器型号:GC7890-MSD5975。柱子型号:HP-5MS毛细管柱(30m×250mm id),采用NIST/EPA/NIH质谱库识别DHA峰图。
最终,通过本发明的方法,获得的隐甲藻突变藻种,与出发藻种相比,DHA产率和得率分别提高到原来的1.69倍和1.28倍,达到57.7mg/L/h和51.96mg/g。
附图说明
图1为本发明筛选淀粉缺陷型突变体的示意图。
图2为本发明采用摇瓶培养方法复筛突变藻种。其中●红色代表出发藻种;★紫色代表突变藻种1;◆绿色代表突变藻种2;■黄色代表突变藻种3;▲蓝色代表突变藻种4。
图3为本发明摇瓶培养条件下藻种生长、油脂、淀粉及DHA含量测定。其中■红色代表诱变藻种1,▲黑色代表出发藻种。A)生96长曲线及耗糖量测定,方块代表生长曲线,圆圈代表耗糖量;B)藻体干重;C)油脂含量;D)DHA占总脂肪酸比例;E)淀粉含量测定。*代表p<0.05;**代表p<0.01。
图4为本发明5-L发酵罐培养条件下藻种生长、油脂、淀粉及DHA含量测定。其中■红色代表诱变藻种1,▲黑色代表出发藻种。A)生长曲线;B)油脂含量;C)DHA占总脂肪酸比例;D)淀粉含量测定;E)DHA生产效率;F)DHA产率*代表p<0.05;**代表p<0.01。
具体实施方式
下面的实施例是为了使本领域的技术人员更好地理解本发明但并不用于限制本发明。
下面通过实施例并结合附图对本发明作进一步详细说明。
实施例1
1.ARTP诱变寇氏隐甲藻细胞。接种寇氏隐甲藻细胞于C9N2液体培养,到指数生长期,取10μL菌液,均匀涂布于ARTP仪器专用小铁片上,利用ARTP诱变仪(无锡源清天木生物科技有限公司SERIAL NO:M114852322403N)进行诱变,诱变时间为40s,功率为100W。诱变结束后,将载有诱变菌液的小铁片放入500μL的C9N2液体培养基中,吹打混匀。取50μL稀释后的菌液,均匀涂布到C9N2固体平板培养基上。将平板培养基放在25℃培养箱中培养,培养10-15天后,利用牙签将新菌落转移到新的C9N2固体平板培养基上,培养5-7天。
2.碘蒸发筛选淀粉缺陷突变藻种。在5L烧杯底部放置一个含有隐甲藻诱变藻种的平板,固体碘升华形成的碘蒸气能均匀地将平板中各个单菌落进行染色,熏蒸时间为2-3min。熏蒸完毕后将目标菌株用灭过菌的移液枪枪头转移到C9N2液体培养基中培养,进行下一步的摇瓶验证实验。
3.摇瓶发酵验证突变藻种。将步骤2中筛选出的隐甲藻诱变藻种及出发株,同时接入C27N6液体培养基中,每隔24小时取样1mL,用紫外可见分光光度计在波长490nm的条件下测定OD值,绘制生长曲线,筛选生长未受到损伤的藻种。
4.摇瓶发酵测定突变藻种油脂含量和淀粉含量。将步骤3筛选出的诱变藻种与出发藻种等OD同时接入C27N6液体培养基中,每隔24小时取样1mL,用紫外可见分光光度计在波长490nm的条件下测定OD值,绘制生长曲线,并在培养72、84、96和108小时时,采用称重法测定细胞干重,测定96小时时出发藻种和突变藻种1的油脂含量和DHA占总油脂比例,测定72、84和96小时时出发藻种和突变藻种1的淀粉含量。
5.5-L发酵罐测定突变藻种的表型。将在摇瓶中筛选出的优良菌株进行发酵罐放大培养。出发藻种和诱变藻种在C9N2液体培养基转接两代,然后转接到200mL的C27N6的培养基中进行种子培养,培养时间为72小时。72小时后将种子液接到装有2LC27N6的培养基的5L发酵罐中进行发酵培养。接种量为10%,发酵温度控制在25℃,转速在200rpm-800rpm,保持溶氧在30%以上,葡萄糖浓度保持在15g/L以上。补料葡萄糖浓度为479g/L,补料酵母粉浓度为375g/L。每隔12小时测定一次葡萄糖消耗量并及时补糖,补氮策略为12h-24h的补氮速率为1.2mL/h,24h-96h为2.1mL/h。取合适的时间点进行油脂和淀粉含量的验证。
经过上述实施例,DHA产率和得率分别提高到原来的1.69倍和1.28倍,达到57.7mg/L/h和51.96mg/g。
Claims (8)
1.一种提高寇氏隐甲藻DHA产率和得率的方法,其特征在于:通过减少藻体内部淀粉含量,提高流向生长和DHA的碳代谢流,获得高生长和高DHA含量的藻种,具体操作包括:
(1)利用ARTP技术诱变及碘蒸平板法初步筛选直径较大、菌落颜色较淡的寇氏隐甲藻诱变藻种;
(2)采用摇瓶发酵,复筛获得的诱变藻种,确定最终诱变藻种;
(3)采用5-L发酵罐对最终诱变藻种的表型进一步确认,确定其DHA产率及得率。
2.根据权利要求1的方法,其特征在于,所述藻种为隐甲藻属(包括隐甲藻但不限定于隐甲藻);所用的诱变和筛选方法是ARTP诱变和碘蒸平板筛选方法。
3.根据权利要求1的方法,其特征在于,步骤(1)所用的ARTP诱变时间是0-1分钟。
4.根据权利要求1的方法,其特征在于,步骤(1)所用的碘是固体碘;熏蒸时间是2-3分钟。
5.根据权利要求1的方法,步骤(1)所用的平板是C9N2固体平板,是由葡萄糖9g,酵母浸粉2g,海盐25g,琼脂糖20g加水至1L而成,pH为6.5。
6.根据权利要求1的方法,其特征在于,步骤(2)所述摇瓶筛选所用的培养基是由葡萄糖2~80g,酵母浸粉0.5~20g,蛋白胨0.25~10g,海盐1~40g,加水至1L而成,pH为7.0。
7.根据权利要求1的方法,其特征在于,步骤(3)所述5-L发酵罐进行筛选所用的培养基是由葡萄糖2~80g,酵母浸粉0.5~20g,蛋白胨0.25~10g,海盐1~40g,加水至1L而成,pH为7.0,补料葡萄糖浓度479g/L,补料酵母粉浓度375g/L,每隔12小时测定一次葡萄糖消耗量并及时补糖,保持残糖量在15g/L,补氮策略为12h-24h的补氮速率为1.2mL/h,24h-96h为2.1mL/h。
8.一种寇氏隐甲藻诱变的方法,其特征在于基于减少藻体内部淀粉含量,提高流向生长和DHA的碳代谢流,获得高生长和高DHA含量的藻种。
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