CN116179377A - 一种利用解脂亚罗酵母将葡萄糖酸(盐)转化生成赤藓糖醇的方法 - Google Patents
一种利用解脂亚罗酵母将葡萄糖酸(盐)转化生成赤藓糖醇的方法 Download PDFInfo
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- CN116179377A CN116179377A CN202211386829.0A CN202211386829A CN116179377A CN 116179377 A CN116179377 A CN 116179377A CN 202211386829 A CN202211386829 A CN 202211386829A CN 116179377 A CN116179377 A CN 116179377A
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- Prior art keywords
- gluconic acid
- yarrowia lipolytica
- erythritol
- gluconate
- salt
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- 239000000174 gluconic acid Substances 0.000 title claims abstract description 57
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Abstract
本发明涉及一株解脂亚罗酵母将葡萄糖酸(盐)转化生成赤藓糖醇的方法,该酵母经人工突变获得变异株,将葡萄糖酸(盐)经微生物发酵转化为赤藓糖醇。通过该法在30℃的温度下摇瓶发酵72小时可以将葡萄糖酸(盐)转化生成7.6%(w/v)的赤藓糖醇,基于消耗葡萄糖酸的转化率可达到54%以上。可利用的底物包括葡萄糖酸、葡萄糖酸盐及酯类衍生物,也能利用葡萄糖酸钠结晶母液等原料进行转化。
Description
技术领域
本发明涉及工业微生物的改造和应用,属于生物工程技术领域。
背景技术
葡萄糖酸是一种无腐蚀性、无毒、可生物降解的弱有机酸(pKa =3.86)。在自然界中主要存在于植物体、水果、酒和蜂蜜中。葡萄糖酸是D-葡萄糖的醛基(C1)氧化成羧基而产生的。在中性pH的水溶液中,葡萄糖酸形成与其环酯D-葡糖酸δ-内酯(1,5-葡糖酸内酯)平衡的葡糖酸离子。葡糖酸作为调味剂被使用于各种食品中,包括肉、酒和乳制品。由于其与二价和三价金属离子形成水溶性复合物(螯合物)的能力,在治疗性钙和/或铁给药期间经常用作抗衡离子。出于同样的原因,它也用于清除金属或其他表面的钙质和铁锈沉积物,葡萄糖酸钠作为混凝土减水剂和家用洗涤剂中的用量也非常可观。当今的葡萄糖酸及其衍生物的年市场容量约为800,000吨,是一种常见的生物基原料。但对于一些特殊的场合,如食用果汁的降酸、金属表面处理及化学镀废液和工业生产葡萄糖酸盐过程中副产的大量葡萄糖酸(盐)的回收和减排,无论从环境角度还是资源节约角度,对葡萄糖酸的再资源化利用都具有相当的价值。
木质纤维素是自然界最为丰富的可再生生物质,将这类资源转化为大宗燃料和化学品极具吸引力,而在其生物利用过程中,用于纤维素降解的纤维素酶用量居高不下仍然是降低加工成本的技术瓶颈之一。近期发现的裂解性多糖单加氧酶(LPMO)可加速纤维素降解作用,可极大地提高纤维素水解的效率和糖产率,为高效利用木质纤维素资源开辟了一条新的途径。然而,LPMOs所催化的纤维素的氧化水解,得到的氧化纤维素降解产物(包括纤维二糖酸和葡萄糖酸)在总水解产物中的比例很高,为了减少这些降解产物的糖损失,需要解决包括葡萄糖酸在内的糖酸的利用问题才能使得该途径更具技术优势。
目前利用生化方法处理葡萄糖酸的方法之一就是通过所谓的鲁夫递降反应将分子中的羧基进行脱去反应得到少一个碳原子的戊糖,即将葡萄糖酸钙和葡萄糖酸铁一起用双氧水氧化。过程中使用了强氧化剂,而且得到的D-阿拉伯糖没有显著的现实利用价值,与葡萄糖酸一样也是微生物难利用的碳源物质,加之还会产生较多的副产物,并损失一个碳原子,所以实用价值不大。
解脂亚罗酵母(Yarrowialipolytica)是一种非发酵性的好氧酵母菌种。由于其安全、易培养的特点,目前被广泛的用于脂肪酶、有机酸等产物的生产并被广泛用作基因工程宿主菌用于外源基因的表达。然而解脂亚罗酵母的可利用碳源谱却非常狭窄,一般仅能利用葡萄糖等简单糖和n-烃类等碳氢化合物,因而对该菌种的应用会带来一定的限制。但相关研究表明,解脂亚罗酵母的磷酸戊糖途径非常活跃,这一利用葡萄糖经由6-磷酸葡萄糖酸为中间体的代谢支路会产生大量的还原性辅酶NADPH,供生物体用于脂质和糖醇等物质的合成。因此有望改造和开发此菌种将葡萄糖酸(盐)作为有效碳源进行有用目标化合物的生物合成的能力。目前,解脂亚罗酵母是用于市场非常广阔的天然代糖赤藓糖醇生产的实用菌种中是唯一被国际公认安全(GRAS)的菌种,摆脱现有原料单一,开发新的生产原料对改善和提高赤藓糖醇生产过程的经济性无疑具有重要的意义。
在理论上,为了获得可利用葡萄糖酸盐的解脂亚罗酵母菌种,要求菌种具有葡萄糖酸(盐)转运蛋白(gntT)、葡萄糖酸激酶(gntK)等基因的功能,以使葡萄糖酸可从基质进入细胞并转化为可以被菌种利用的磷酸化的葡萄糖酸,从而在进入磷酸戊糖途径后被利用。而实际上,野生型的解脂亚罗酵母菌种虽然具有葡萄糖酸激酶的活性,但尚未认证出葡萄糖酸(盐)转运蛋白的存在,故一般认为解脂亚罗酵母不具备同化该物质的能力。
目前,为了扩大解脂亚罗酵母的底物利用能力,一般采取构建含外源特殊底物利用关键基因的菌种的方法加以解决。例如,可以通过在解脂亚罗酵母中表达来自酿酒酵母的蔗糖酶基因(Suc)等来提高菌种利用这种双糖的能力。但是迄今实际使用这种方法的成功率较低,对底盘菌种的生产性状的影响可能较大,还需要进行全局性的细胞生物学优化才能获得可供工业应用的良好菌株。而且目前在很多国家和地区,利用基因工程方法获得的工程菌在食品及其配料中的应用还面临法规和伦理学方面的限制,所以采用经典方法进行菌种选育是比较通行的技术路线。
由于葡萄糖酸(盐)在适宜酵母生长的pH中性条件下为离子状态,而多数生物的葡萄糖酸(盐)转运蛋白都是与氢离子偶联的同向转运体(symporter),故可考虑通过建立从外向内的氢离子梯度辅助实现葡萄糖酸(盐)的跨膜转运目的。为此以能耐受高浓度的盐类底物的高渗透压,并能适应较低pH生长环境性状的变异株为筛选目标的解脂亚罗酵母菌种改造路线,可能成为解决该菌种对葡萄糖酸(盐)的利用瓶颈的技术方案。
本发明通过对耐高渗的解脂亚罗酵母进行理化和自然诱变改造,然后定向筛选具有高葡萄糖酸(盐)同化能力的变异株,并将其定向应用于赤藓糖醇的生物转化。
发明内容
本发明的目的是提供一种可利用葡萄糖酸(盐)的解脂亚罗酵母菌株的选育方法,并利用该菌株进行以赤藓糖醇为目标产物的发酵转化。
本发明的解脂亚罗酵母变异株77,分类学名称为解脂耶氏酵母Yarrowia lipolytica,寄存于中国微生物菌种保藏管理委员会普通微生物中心,地址:北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,保藏编号:CGMCC No.25431,保藏日期:2022年7月29日。
为实现上述目的,本发明采用如下技术方案。
一、本发明的解脂亚罗酵母菌种改造技术,是以野生型的解脂亚罗酵母为出发株,利用理化、分子生物学方法使其发生遗传变异。从中筛选出葡萄糖酸盐转化能力强的变异株。
进一步的,该操作按如下步骤进行:
(1) 利用EMS、NTG及等电离射线和离子体等化学及物理诱变方法和其他分子诱变方法处理野生型解脂亚罗酵母细胞使之发生随机突变。
(2) 将通过(1)处理后的酵母细胞在酸性的以葡萄糖酸盐为唯一碳源的合成培养基中进行多轮转接培养,以富集能在酸性条件下同化葡萄糖酸盐的酵母突变体。
(3) 从(2)步骤富集后的酵母培养物在添加制霉菌素的以赤藓糖醇为唯一碳源的合成培养基上进行第二轮富集,进一步富集具赤藓糖醇利用缺陷性状的解脂亚罗酵母突变体。
(4) 对(3)步骤富集后的酵母培养物进行稀释并在YPDA培养基平板上进行单菌落分离,挑选出可利用葡萄糖酸(盐)合成赤藓糖醇的酵母变异株。
二、利用获得的解脂亚罗酵母变异株对各类葡萄糖酸(盐)底物进行赤藓糖醇的发酵转化。
进一步,具体步骤如下:
(1)菌种活化和培养
将变异株在含葡萄糖酸盐的YDPA固体培养基平板上划线,30±1℃培养72小时,挑取平板上的单菌落,接种发酵培养基,30±1℃摇床培养24小时,获得种子液。
(2) 发酵培养
将种子液以0.1 – 20%接种量接种到发酵培养基中,30±1℃摇床培养48-96小时,获得发酵液。
其中,上述步骤(1)中所述YPDA培养基配方为:1L纯水中加入葡萄糖酸钠20g,葡萄糖20g,酵母粉10g,蛋白胨 20g,忍天青 10mg,琼脂20g,115℃灭菌30分钟。
上述步骤(1)中所述种子培养基配方为:1 L水中加入葡萄糖酸钠20g,酵母浸粉3g,蛋白胨5g,葡萄糖20g,酵母粉10g,蛋白胨 20g,115℃灭菌30分钟。
上述步骤(2)中所述发酵培养基配方为:1 L水中加入酵母浸粉3g,蛋白胨5g,葡萄糖钠300g,115℃灭菌30分钟。
(4) 发酵过程葡萄糖(酸)和葡萄糖的检测方法
将发酵液稀释并经0.22μm滤膜过滤后,采用HPLC的方式检测过滤液中葡萄糖(酸)和赤藓糖醇的含量。色谱条件如下: 检测器,示差检测器;色谱柱,H型强阳离子交换柱;柱温,65℃;流动相,7mMH2SO4水溶液;流速,0.7 mL/min;进样体积,10μL。
本发明成功实现了从解脂亚罗酵母野生株出发,通过变异株文库的酸性环境下的葡萄糖酸同化功能富集和赤藓糖醇同化功能弱化筛选,获得能高效转化葡萄糖酸盐为赤藓糖醇的解脂亚罗酵母的变异株。使用该菌株能够高效转化葡萄糖酸盐(酯)以及葡萄糖酸钠母液进行赤藓糖醇的发酵,具有重要的经济和环保价值。
本发明具有的突出特点是:
(1)本发明提供了对野生型解脂亚罗酵母采用复合诱变,结合性状富集过程中的抗生素和酸性诱导等处理,获得具葡萄糖酸盐同化能力的酵母变异株的方法,该方法具有很高的定向筛选效率。
(2)利用上述方法获得的解脂亚罗酵母变异株可以在96小时内转化20%的葡萄糖酸钠为10%以上的赤藓糖醇,转化率可达到50%以上,使得该菌种能够利用更加低质的碳源生产有用化合物。
附图说明
图1 解脂亚罗酵母变异株的葡萄糖酸钠发酵产物的HPLC图。
图2 解脂亚罗酵母变异株发酵葡萄糖酸钠葡萄糖酸钠结晶母液产物的HPLC图。
具体实施方式
下面通过实施例进一步阐明本发明,实施例将有助于理解本发明,但不仅限于此。
实施例1. 解脂亚罗酵母变异株的获得
(1)接种活化的解脂亚罗酵母CGMCC No.1431到YPD培养基中,摇瓶培养24小时后,在无菌离心管加入菌液1ml,离心除去上清中的培养基,将沉淀的酵母细胞用pH6.0的磷酸缓冲溶液洗涤3次,再用缓冲液调整OD600达到约为1.0后,加入200ppm的NTG,在30℃下振荡处理30分钟。在1000g下离心10min除去上清液中的诱变剂以终止反应,沉淀酵母细胞用pH6.0的50mM磷酸缓冲溶液洗涤3次后,将其接种到100毫升的GnYNB培养基中,28℃下振荡培养至OD600达到约为0.5后,吸取1ml菌液到另一瓶100毫升的GnYNB培养基中,如此重复转接5次后,取1ml菌液将其接种到100毫升的MT+1%赤藓糖醇的无氮源培养基中摇瓶振荡培养8小时,然后加入终浓度为5g/l的硫酸铵无菌溶液,28℃下振荡培养8小时,再加入制霉菌素至最终浓度20μg/ml继续振荡培养3小时。将处理后的菌液适当稀释涂布在GnYNBA平板上,于30℃下培养3天后。挑选较大的表面光滑的单菌落进行发酵性能验证。
所述的YPD培养基配方为:酵母浸粉3.0g,蛋白胨5.0g,无水葡萄糖50g,加水到1.0升,115℃灭菌30分。
所述的GnYNB培养基配方为:YNB 6.7g(过滤灭菌),葡萄糖酸钠 15.0g,琼脂 20g,加水到1.0升,用2M的盐酸调整pH3.5,115℃灭菌30分。
所述的GnYNBA培养基配方为:YNB 6.7g(过滤灭菌),葡萄糖酸钠 15.0g,琼脂20g,加水到1.0升,用2M的盐酸调整pH3.5,115℃灭菌30分。
所述的MT培养基的配方为:
NH4H2PO4 5 g/l
KH2PO4 2.5g/l
MgSO4•7H2O 1 g/l
盐酸硫胺素 0.3mg/l
微量元素溶液 1ml/l
微量元素溶液组成:(mg/100 ml):
Ca(NO3)2 •4H2O 2000
FeCl3•6H2O 200
H3BO3 50
CuSO4•5H2O 10
MnSO4•4H20 40
ZnSO4•7H2O 40
Na2MoO4 20
CoCl2 10
KI 10
所述的NTG是指N-甲基-N’-硝基-N-亚硝基胍。
所述的磷酸缓冲溶液是指用浓NaOH溶液调整50mM的磷酸溶液至pH6.0获得的溶液。
(2)将步骤(1)在GnYNBA培养基上培养3天的平板上的单菌落挑出,接种于2.5mlYPD培养基中,在30℃下振荡培养过夜,然后接种于装入500mL三角瓶中,该瓶中装有25mlGnYS培养基。将接种后的三角瓶在在30℃下,以250rpm转速回转振荡培养72小时,然后用HPLC分析发酵产物,色谱图见附图1。根据分析结果挑选出转化赤藓糖醇效率高的突变株。
所述的GnYS培养基配方为:酵母粉 5g(过滤灭菌),柠檬酸铵5g,磷酸二氢钾 1g,硫酸镁 0.5g,葡萄糖酸钠 15.0g,琼脂 20g,加水到1.0升,115℃灭菌30分。
实施例2. 解脂亚罗酵母CGMCC 25431利用葡萄糖酸钠发酵赤藓糖醇的发酵
在装有25ml的灭菌葡萄糖酸钠发酵培养基的500ml的三角瓶中,接入在30℃下用YPD培养基培养24h的解脂亚罗酵母变异株CGMCC 25431种子液2.5ml(接种比1:10),在30±1℃,以280rpm转速回转振荡发酵72h,分析结果见下表:
初始葡萄糖酸内酯g/l(以葡萄糖酸计) | 72h时葡萄糖酸内酯g/l(以葡萄糖酸计) | 消耗葡萄糖酸内酯g/l(以葡萄糖酸计) | 72h时赤藓糖醇g/l | 赤藓糖醇转化率% |
180 | 36.5 | 143.5 | 73.2 | 51.0% |
在72小时的发酵过程中,基于消耗葡萄糖酸的赤藓糖醇的转化率达到51.0%。
所述的葡萄糖酸钠发酵培养基成分为:葡萄糖酸钠20%(w/v),柠檬酸铵 0.5(w/v),酵母浸出物 0.5(w/v),磷酸二氢钾 0.05%,硫酸镁 0.025%。
实施例3. 解脂亚罗酵母CGMCC 25431利用葡萄糖酸铵发酵赤藓糖醇
在装有25ml的灭菌葡萄糖酸铵培养基的500ml的三角瓶中,接入2.5ml在30℃下用YPD培养基培养24h的解脂亚罗酵母变异株CGMCC 25431种子液(OD600=15),在30±1℃,280rpm摇床上进行72h摇瓶发酵,发酵液中消耗的葡萄糖酸铵量与产生的赤藓糖醇如下表:
初始葡萄糖酸内酯g/l(以葡萄糖酸计) | 72h时葡萄糖酸内酯g/l(以葡萄糖酸计) | 消耗葡萄糖酸内酯g/l(以葡萄糖酸计) | 72h时赤藓糖醇g/l | 赤藓糖醇转化率% |
185 | 50.3 | 134.7 | 61.0 | 45.3% |
在72小时的发酵过程中,基于消耗葡萄糖酸铵的赤藓糖醇的转化率达到45.3%。
所述的葡萄糖酸铵培养基成分为:葡萄糖酸铵20%(w/v),酵母粉 0.5(w/v),硫酸镁 0.025%,磷酸二氢钾0.05%(v/v)。
实施例4. 利用葡萄糖酸内酯的赤藓糖醇发酵
在装有25ml的灭菌葡萄糖酸内酯培养基的500ml的三角瓶中,接入2.5ml在30℃下用YPD培养基培养24h的解脂亚罗酵母变异株CGMCC 25431种子液(OD600=15),在30±1℃,280rpm摇床上进行72h摇瓶发酵,发酵液中消耗的葡萄糖酸内酯量与产生的赤藓糖醇如下表:
初始葡萄糖酸内酯g/l(以葡萄糖酸计) | 72h时葡萄糖酸内酯g/l(以葡萄糖酸计) | 消耗葡萄糖酸内酯g/l(以葡萄糖酸计) | 72h时赤藓糖醇g/l | 赤藓糖醇转化率% |
198 | 57.1 | 140.9 | 76.1 | 54.0% |
葡萄糖酸内酯是葡萄糖酸分子内脱水形成的内酯类化合物,溶于水后呈较强的酸性,用适量的氢氧化钠中和pH到4.5以下后,经过72小时的发酵,基于消耗葡萄糖酸内酯的赤藓糖醇的转化率可达到54.0%。
所述的葡萄糖酸内酯培养基成分为:葡萄糖酸内酯18%(w/v),酵母粉 0.5(w/v),磷酸氢二铵0.45%,硫酸镁 0.025%,磷酸二氢钾0.05%(v/v)。
实施例5. 利用葡萄糖钠母液发酵赤藓糖醇
葡萄糖酸钠母液是葡萄糖酸钠生产过程中的副产物,由于杂质多粘度大,难以从中分离出更多的葡萄糖酸钠结晶物。其主要成分包括:固形物含量 50%,葡萄糖酸钠 30%,还原糖15%。
在装有25ml的灭菌母液培养基的500ml的三角瓶中,接入在30℃下用YPD培养基培养24h的解脂亚罗酵母CGMCC 25431种子液2.5ml(接种比1:10),在30±1℃,以280rpm转速回转振荡发酵72h,分析结果见下表:
初始葡萄糖酸钠母液g/l(以葡萄糖酸计) | 72h时葡萄糖酸钠g/l(以葡萄糖酸计) | 消耗葡萄糖酸钠g/l(以葡萄糖酸计) | 72h时赤藓糖醇g/l | 赤藓糖醇转化率% |
167.5 | 48.6 | 118.9 | 56.6 | 47.6% |
葡萄糖酸钠结晶母液中含有较多的可溶性杂质,发酵效率比单纯的原料稍低,但经过72小时的发酵,基于消耗葡萄糖酸盐的赤藓糖醇的转化率也可达到47.6%,而原料中该酵母难同化的低聚糖类物质仍然得以保留(发酵过程中糖酸成分的HPLC图见附图2)。
所述的母液培养基成分为:酵母粉 0.5(w/v),无水葡萄糖10%(w/v),磷酸氢二铵0.2(w/v),硫酸镁 0.025%,葡萄糖酸钠母液30%(v/v)。
实施例6. 野生型解脂亚罗酵母CGMCC 1431的葡萄糖酸钠发酵(对照)
在装有25ml的灭菌葡萄糖酸钠发酵培养基的500ml的三角瓶中,接入在30℃下用YPD培养基培养24h的解脂亚罗酵母野生株CGMCC 1431种子液2.5ml(OD600=15),在30±1℃,以280rpm转速回转振荡发酵96h,分析结果见下表:
初始葡萄糖酸钠g/l(以葡萄糖酸计) | 96h时葡萄糖酸钠g/l(以葡萄糖酸计) | 消耗葡萄糖酸钠g/l(以葡萄糖酸计) | 96h时赤藓糖醇g/l | 赤藓糖醇转化率% |
180 | 175 | 5.2 | 0.2 | 3.8% |
经过96小时的发酵,野生型的解脂亚罗酵母除了由种子带入的少量赤藓糖醇外,基本不利用葡萄糖酸(盐)生产赤藓糖醇。
所述的葡萄糖酸钠发酵培养基成分为:葡萄糖酸钠20%(w/v),柠檬酸铵 0.5(w/v),酵母浸出物 0.5(w/v),磷酸二氢钾 0.05%,硫酸镁 0.025%。
Claims (5)
1.一种利用解脂亚罗酵母将葡萄糖酸(盐)转化生成赤藓糖醇的方法。
2.如权利要求1所述解脂亚罗酵母,其特征在于系自野生菌种经人工突变,利用酸性葡萄糖酸碳源和抗生素富集处理后筛选得到解脂亚罗酵母变异株。
3.如权利要求2所述解脂亚罗酵母变异株,其特征还在于,变异株之一寄存于中国微生物菌种保藏管理委员会普通微生物中心,分类学名称为解脂亚罗酵母(Yarrowia lipolytica),地址:北京市朝阳区北辰西路1号院3号,保藏编号:CGMCC 25431,保藏日期:2022年7月29日。
4.权利要求1所述葡萄糖酸(盐),包括但不限于葡萄糖酸、葡萄糖酸甲(乙)酯、葡萄糖酸内酯、葡萄糖酸钠、葡萄糖酸钾、葡萄糖酸钙、葡萄糖酸镁、葡萄糖酸锌盐及酯,或以上物质的混合物,其在培养基中的质量浓度范围(以葡萄糖酸计)为5%~40%。
5.权利要求1所述利用解脂亚罗酵母将葡萄糖酸(盐)转化生成赤藓糖醇的方法,包括以下步骤:
(1)将权利要求3任一项所述的菌种采用如下培养基制备种子液:葡萄糖0~20%(w/v),葡萄糖酸钠0~20%(w/v),酵母浸出物0.03~2.5%(w/v),蛋白胨0.03~2.5%(w/v),磷酸二氢钾0.02~0.5%(w/v),柠檬酸铵0.1~0.75%(w/v);磷酸氢二铵0~0.75%(w/v);
(2)将上述种子液按照0.5-50%的比例接入发酵培养基,该培养基采用如下组成:葡萄糖酸(盐)0~20%(w/v),酵母浸出物0.03~2.5%(w/v),蛋白胨0.03~2.5%(w/v),磷酸二氢钾0.02~0.5%(w/v),柠檬酸铵0.1~0.75%(w/v);磷酸氢二铵0~0.75%(w/v),在25~33℃下摇瓶发酵60~120h,摇瓶转速200~400rpm,得到以赤藓糖醇为主要产物的发酵液。
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