CN116162559A - 一种利用重组解脂耶氏酵母合成对羟基苯甲醇的方法 - Google Patents
一种利用重组解脂耶氏酵母合成对羟基苯甲醇的方法 Download PDFInfo
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- hydroxybenzyl alcohol
- yarrowia lipolytica
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Abstract
本发明属于微生物发酵技术领域,提供了一种利用重组解脂耶氏酵母合成对羟基苯甲醇的方法,通过引入外源的羧酸还原酶基因,促进对羟基苯甲醇在微生物细胞的胞外合成与积累,有效的提高了对羟基苯甲醇的产量,为代谢工程改造高效生产对羟基苯甲醇奠定了基础,为对羟基苯甲醇微生物发酵生产提供了更优的潜在选择。
Description
技术领域
本发明涉及微生物发酵技术领域,具体地,涉及一种合成对羟基苯甲醇的重组解脂耶氏酵母菌株构建方法及应用。
背景技术
对羟基苯甲醇,又称:4-羟基苯甲醇,是一种重要的医药中间体,主要通过结构修饰用于药物和香料的合成。对羟基苯甲醇也是我国著名中草药天麻的有效成分之一,是天麻在人体内的主要代谢产物。现代药理学实验表明,对羟基苯甲醇具有调节中枢神经系统的能力,包括镇静、抗惊厥、抗抑郁、抗炎症、促进记忆和神经保护等方面,具有潜在的营养和医药市场价值。目前对羟基苯甲醇的制备工艺主要包括直接提取法和化学合成法,直接提取法是利用有机溶剂直接从中药材天麻中提取,该方法提取效率低,且成本较高;化学合成法,主要涉及还原性试剂硼氢化钠、硼氢化钾等合成对羟基苯甲醇,生产过程中产生大量无机废料,污染环境,不利于大规模生产,因此开发绿色高效的生物合成方法具有重要的研究意义。
解脂耶氏酵母(Yarrowialipolytica)是一种重要的非常规酵母,因其属于食品安全级菌株,并且对多种有机溶剂、高渗透压和酸碱环境的耐受性强以及具有多底物利用能力和高产油脂的能力而受到广泛关注,逐渐成为合成生物学研究与代谢工程改造理想的底盘菌株。近年来,分子生物学技术的进步促进了各种改造解脂耶氏酵母的基因工程工具的蓬勃发展,人们已成功构建解脂耶氏酵母细胞工厂用于生产各种脂类、萜类、黄酮类、聚酮类化合物等,例如二十二碳六烯酸、赤藓糖醇、芳樟醇、法尼烯、角鲨烯、番茄红素和虾青素等,解脂耶氏酵母表现出巨大的工业化应用潜力。
但是,目前解脂耶氏酵母应用于合成对羟基苯甲醇的研究报道很少,之前的研究表明羧酸还原酶是对羟基苯甲醇生物合成中的关键酶,但是目前已鉴定用于对羟基苯甲醇生物合成的诺卡氏菌来源的羧酸还原酶基因(CAR)需要与枯草芽孢杆菌来源的磷酸泛乙烯基转移酶(Sfp)共表达,将羧酸还原酶从apo转化为holo形式才能发挥作用,随后醛通过内源性醇脱氢酶(ADH)转化为相应的醇。该过程较为复杂,同时目前产量难以满足实际生产的需求。
发明内容
本发明的目的是为了克服现有技术存在的微生物用于生产对羟基苯甲醇时,需要多种基因共同作用并且对羟基苯甲醇产量仍较低的问题,提供一种合成对羟基苯甲醇的重组解脂耶氏酵母菌株及其构建方法和发酵合成对羟基苯甲醇的方法,该重组菌株发酵合成对羟基苯甲醇的产量较高,为对羟基苯甲醇的微生物发酵生产提供了更优的潜在选择。
为了实现上述目的,本发明提供了一种利用重组解脂耶氏酵母合成对羟基苯甲醇的方法,包括:
(1)构建重组解脂耶氏酵母;
(2)将重组解脂耶氏酵母接种至发酵培养基中进行发酵培养;
(3)加入对羟基苯甲酸继续发酵培养得发酵液。
本发明中所述构建重组解脂耶氏酵母的方法包括:对出发菌株引入含有羧酸还原酶基因的重组载体,得到重组解脂耶氏酵母。
进一步地,所述出发菌株为解脂耶氏酵母,优选地,所述出发菌株为亮氨酸营养缺陷型解脂耶氏酵母Po1g。
进一步地,所述羧酸还原酶基因的氨基酸序列如Seq_1所示,所述羧酸还原酶基因的核苷酸序列如Seq_2所示。
进一步地,所述重组载体为将羧酸还原酶基因整合至PYLXP’表达载体构建而成,所述PYLXP’的核苷酸序列如Seq_3所示。
本发明中所述羧酸还原酶基因片段可以通过如下方式获得:根据本领域公知的数据库(例如GenBank数据库,
https://www.ncbi.nlm.nih.gov/genbank/)中公开的来自Neurosporacrassa羧酸还原酶基因(GeneID:3871967)序列进行人工合成。
本发明所述的构建重组解脂耶氏酵母,可以先构建重组载体,再将重组载体导入出发菌株(如解脂耶氏酵母)中。所述重组载体能够将出发菌株(如解脂耶氏酵母)中外源羧酸还原酶基因过表达。本领域已知构建重组载体的各种方法,以用来将目的基因片段连接至载体来制备基因过表达重组载体,例如但不限于,经典的“酶切-连接”方法、Invitrogen公司开发的Gateway克隆系统、Clontech公司开发的Creator克隆系统、StephenElledge实验室开发的Univector克隆系统以及基于IIs型限制性内切酶的GoldenGate克隆法。所述将重组载体导入出发菌株(如解脂耶氏酵母)中,例如但不限于显微注射、基因枪、转化(例如电转化)、感染或转染。上述显微注射、基因枪、转化、感染或转染均为本领域常规操作。例如,转化是指通过采用分子生物学和基因工程中的一些已知方法处理细胞,使经处理后的细胞处于感受态,并由此与外源DNA接触,从而使外源DNA进入处于感受态的细胞中。常用的转化方法包括原生质体转化法、化学转化法和电穿孔转化法。感染是指用经人工改造的噬菌体活病毒作载体,将该载体与目的DNA序列重组后,在体外用噬菌体或病毒的外壳蛋白将重组DNA包装成有活力的噬菌体或病毒,从而以感染的方式使重组DNA进入宿主细胞中。转染是指通过CaCl2、电穿孔等方法将细胞处理成感受态细胞,然后使该感受态细胞接受重组的噬菌体DNA。
在将重组载体导入出发菌株(如解脂耶氏酵母)中之后,可通过筛选标记(例如抗性基因)筛选出阳性克隆,并通过菌落PCR进行验证、或者通过DNA测序进行验证,从而得到所述的用于合成对羟基苯甲醇的重组菌株。
本发明中将重组解脂耶氏酵母接种至发酵培养基中进行发酵培养,具体是挑选重组菌株的单菌落于种子培养基中培养,得到种子液;再将种子液接种至发酵培养基中进行发酵,得到发酵液。所述发酵培养基的组分包括碳源、氮源、硫酸盐或腺嘌呤中的至少一种氨基酸。
进一步地,所述发酵培养基的组分包括碳源、氮源、硫酸盐、腺嘌呤、L-精氨酸、L-天门冬氨酸、L-组氨酸、L-异亮氨酸、L-赖氨酸、L-甲硫氨酸、L-苯丙氨酸、L-苏氨酸、L-色氨酸、L-缬氨酸和尿嘧啶。
进一步地,所述发酵培养基的组分包括:葡萄糖30-50g/L,硫酸铵0.8-1.3g/L,YNB(无氨基酵母氮源)1.5-2g/L,腺嘌呤0.01-0.02g/L,L-精氨酸0.02-0.08g/L,L-天门冬氨酸0.05-1g/L,L-组氨酸0.01-0.03g/L,L-异亮氨酸0.02-0.08g/L,L-赖氨酸0.02-0.08g/L,L-甲硫氨酸0.01-0.03g/L,L-苯丙氨酸0.02-0.08g/L,L-苏氨酸0.05-0.15g/L,L-色氨酸0.02-0.08g/L,L-缬氨酸0.05-0.25g/L,尿嘧啶0.01-0.03g/L。
本发明所述将重组解脂耶氏酵母接种至发酵培养基中进行发酵培养,其重组解脂耶氏酵母的接种量为发酵培养基体积的4-6%,培养温度为25-35℃,转速为180-250rpm,培养时间为72-96h。
进一步地,在发酵40-55h时向发酵培养基中加入对羟基苯甲酸,所述对羟基苯甲酸的加入量为每1L发酵培养基加入0.5-1g。
本发明所述重组解脂耶氏酵母在制备对羟基苯甲醇中的应用。
本发明中,可以通过已知的方法分离所得发酵液中的对羟基苯甲醇,例如,先将所述发酵液中的菌体去除,将出去菌体后的发酵液进行浓缩使产物结晶等诸如此类的方法分离对羟基苯甲醇。
本发明中,还可以通过已知方法对所述发酵液中的对羟基苯甲醇或从所述发酵液中分离得到的对羟基苯甲醇进行检测。例如,可以通过高效液相色谱法和诸如此类的方法来对对羟基苯甲醇的含量进行检测。
本发明与现有技术相比,具有以下有益效果或者优点:
(1)本发明提供的重组解脂耶氏酵母通过引入外源的羧酸还原酶基因,促进对羟基苯甲醇在微生物细胞的胞外合成与积累,有效的提高了对羟基苯甲醇的产量,为代谢工程改造高效生产对羟基苯甲醇奠定了基础,为对羟基苯甲醇微生物发酵生产提供了更优的潜在选择。
(2)在本发明最优选实施方式中,重组解脂耶氏酵母菌株发酵72h得到的发酵液中对羟基苯甲醇含量为275mg/L,相比于未重组解脂耶氏酵母菌株显著提高了对羟基苯甲醇的产量。
(3)本发明提供的重组解脂耶氏酵母构建方法简单,便于使用,具有良好的应用前景。
附图说明
图1为对羟基苯甲醇生物合成过程示意图。
图2为重组质粒pYLXP’-CAR-PPTcg-1的结构图,其中pTEF表示启动子,XPR2表示终止子。
图3为重组质粒pYLXP’-NcCAR的结构图,其中pTEF表示启动子,XPR2表示终止子。
图4为对羟基苯甲醇的标准品和实施例3获得的重组解脂耶氏酵母菌株发酵上清液的液相检测图谱。
图5为实施例3和对比例1获得的发酵液的上清液中对羟基苯甲醇的含量图。
具体实施方式
以下对本发明的具体实施方式进行详细说明。应当理解的是,此处所描述的具体实施方式仅用于说明和解释本发明,并不用于限制本发明。
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值,这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说,各个范围的端点值之间、各个范围的端点值和单独的点值之间,以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围,这些数值范围应被视为在本文中具体公开。
本发明使用的术语“增加”或“提高”通常都意味着统计学显著量的增加。然而,为避免疑义,术语“增加”或“提高”表示相比参比水平(例如出发菌株中的水平)增加至少10%,例如增加至少约20%、或是更大量的增加。
本发明提供了一种利用重组解脂耶氏酵母合成对羟基苯甲醇的方法,其最优选的实施方式为:
如图1所示,选择出发菌株为亮氨酸营养缺陷型解脂耶氏酵母Po1g。通过已知的构建重组载体方法将羧酸还原酶基因整合至PYLXP’表达载体构建重组载体,再将重组载体导入出发菌株中得到重组解脂耶氏酵母。
所述羧酸还原酶基因的氨基酸序列如Seq_1所示。
所述羧酸还原酶基因的核苷酸序列如Seq_2所示。
所述PYLXP’的核苷酸序列如Seq_3所示。
然后,挑选重组解脂耶氏酵母的单菌落于种子培养基中,在温度为25-35℃,转速为180-250rpm的条件下进行种子培养40-60h,得到所述种子液;再将所述种子液以体积比4-6体积%的接种量接种至所述发酵培养基中,在温度为28-35℃,转速为180-250rpm的条件下进行发酵培养40-55h,向发酵液中加入对羟基苯甲酸使得其添加量为0.5-1g/L,再进行发酵培养20-48h,得到发酵液。
其中,种子培养基含有葡萄糖15-25g/L,硫酸铵4-6g/L,无氨基酵母氮源(YNB)1.5-2g/L,腺嘌呤0.01-0.02g/L,L-精氨酸0.02-0.08g/L,L-天门冬氨酸0.05-1g/L,L-组氨酸0.01-0.03g/L,L-异亮氨酸0.02-0.08g/L,L-赖氨酸0.02-0.08g/L,L-甲硫氨酸0.01-0.03g/L,L-苯丙氨酸0.02-0.08g/L,L-苏氨酸0.05-0.15g/L,L-色氨酸0.02-0.08g/L,L-缬氨酸0.05-0.25g/L,尿嘧啶0.01-0.03g/L;
发酵培养基含有葡萄糖30-50g/L,硫酸铵0.8-1.3g/L,YNB(无氨基酵母氮源)1.5-2g/L,腺嘌呤0.01-0.02g/L,L-精氨酸0.02-0.08g/L,L-天门冬氨酸0.05-1g/L,L-组氨酸0.01-0.03g/L,L-异亮氨酸0.02-0.08g/L,L-赖氨酸0.02-0.08g/L,L-甲硫氨酸0.01-0.03g/L,L-苯丙氨酸0.02-0.08g/L,L-苏氨酸0.05-0.15g/L,L-色氨酸0.02-0.08g/L,L-缬氨酸0.05-0.25g/L,尿嘧啶0.01-0.03g/L。
以下将通过实施例对本发明进行详细描述。但这些实施例仅用于说明本发明而不用来限制本发明的范围。以下实施例中,除非另有说明,所使用的实验方法均为本领域技术人员所熟知的常规方法。
以下实施例中,解脂耶氏酵母Po1g为解脂耶氏酵母ATCC20460的衍生菌株,由马里兰大学PengXu教授直接提供,其购自中国台湾Yeastern Biotech(益生生技)公司;pYLXP’等质粒为自行构建;除非特别说明,所用试剂、培养基均为市售商品,所用方法均为常规方法。
1、培养基与试剂
亮氨酸缺陷型平板:葡萄糖20g/L,硫酸铵5g/L,无氨基酵母氮源YNB1.7g/L,半硫酸腺嘌呤10mg/L,L-精氨酸0.05g/L,L-天门冬氨酸0.08g/L,L-组氨酸0.02g/L,L-异亮氨酸0.05g/L,L-赖氨酸0.05g/L,L-甲硫氨酸0.02g/L,L-苯丙氨酸0.05g/L,L-苏氨酸0.1g/L,L-色氨酸0.05g/L,L-酪氨酸0.05g/L,L-缬氨酸0.14g/L,尿嘧啶0.02g/L,琼脂20g/L;
YPD培养基:酵母提取物10g/L,蛋白胨20g/L,葡萄糖20g/L;
种子培养基:葡萄糖20g/L,YNB(无氨基酵母氮源)1.7g/L,硫酸铵5g/L,腺嘌呤0.01g/L,L-精氨酸0.05g/L,L-天门冬氨酸0.08g/L,L-组氨酸0.02g/L,L-异亮氨酸0.05g/L,L-赖氨酸0.05g/L,L-甲硫氨酸0.02g/L,L-苯丙氨酸0.05g/L,L-苏氨酸0.1g/L,L-色氨酸0.05g/L,L-缬氨酸0.14g/L,尿嘧啶0.02g/L;
发酵培养基:葡萄糖40g/L,YNB(无氨基酵母氮源)1.7g/L,硫酸铵1.1g/L,腺嘌呤0.01g/L,L-精氨酸0.05g/L,L-天门冬氨酸0.08g/L,L-组氨酸0.02g/L,L-异亮氨酸0.05g/L,L-赖氨酸0.05g/L,L-甲硫氨酸0.02g/L,L-苯丙氨酸0.05g/L,L-苏氨酸0.1g/L,L-色氨酸0.05g/L,L-缬氨酸0.14g/L,尿嘧啶0.02g/L。
2、对羟基苯甲醇含量采用高效液相色谱(HPLC)检测方法:Agilent1200,VWD检测器,ZORBAXEclipsePlusC18色谱柱(4.6×100mm,3.5μm,Agilent),流动相:以40%(v/v)甲醇水溶液为流动相,流速为0.6mL/min,检测光谱为225nm,柱温为35℃,进样体积为10μL,对羟基苯甲醇的保留时间为4.08min左右。
制备例1
(1)使用SnaBI和KpnI将质粒pYLXP’(核苷酸序列如Seq_3所示)酶切线性化,同时使用SnaBI和KpnI酶切基因合成的获得来源于Nocardiaiowensis的羧酸还原酶基因CAR(核苷酸序列如Seq_4所示)和来源于Corynebacteriumglutamicum的磷酸泛酰巯基乙胺基转移酶基因PPTcg-1(核苷酸序列如Seq_5所示)通过酶切连接的方式获得重组质粒pYLXP’-CAR、pYLXP’-PPTcg-1;然后使用使用NheI和NotI将质粒pYLXP’-CAR酶切线性化,同时使用NotI和AvrII将质粒pYLXP’-PPTcg-1酶切线性化,通过酶切连接的方式获得重组质粒pYLXP’-CAR-PPTcg-1(结构图如图2所示);
(2)采用醋酸锂法的转化方法将重组质粒pYLXP’-CAR-PPTg-1转化至出发菌株Po1g菌株中,具体过程如下:将Po1g菌株在2mL的YPD培养基中培养至指数生长期(16-24h),的收集1mL发酵液中的Po1g菌体,离心弃上清后,加入含90μL的50体积%PEG4000溶液,5μL醋酸锂(2M),5μL的单链DNA(鲑鱼精子,购买于Sigma-Aldrich,货号D7656)和5μL的重组质粒Po1gpYLXP’-CAR-PPTg-1,混匀后在37℃孵浴1h后,涂布亮氨酸缺陷型平板,筛选得到解脂耶氏酵母Po1gpYLXP’-CAR-PPTcg-1。
实施例1
重组质粒pYLXP’-NcCAR的构建。
使用SnaBI和KpnI将质粒pYLXP’(核苷酸序列如Seq_3所示)进行酶切线性化,再采用引物NcCAR_F/NcCAR_R(核苷酸序列如Seq_6和Seq_7所示),人工合成获得的来源于Neurosporacrassa的羧酸还原酶NcCAR基因片段为模板扩增得到,之后采用Gibson组装的方式将线性化的pYLXP’和扩增得到的NcCAR基因片段进行组装,获得重组质粒pYLXP’-NcCAR(核苷酸序列如Seq_8所示,结构图如图3所示),将重组质粒pYLXP’-NcCAR由上海生工测序验证正确后用于实施例2中进行基因过表达。
实施例2
将外源基因引入解脂耶氏酵母。
以解脂耶氏酵母Yarrowia lipolytica Po1g为出发菌株,将Yarrowialipolytica Po1g菌株在2mL的YPD培养基中培养至指数生长期(16-24h),收集1mL的发酵液中的Yarrowia lipolytica Po1g菌体,离心弃上清后,加入含90μL的50体积%的PEG4000溶液,5μL醋酸锂(2M),5μL的单链DNA(鲑鱼精子)和5μL实施例1获得的pYLXP’-NcCAR重组质粒,混匀后在37℃下孵育1h后,涂布亮氨酸缺陷型平板,筛选得到解脂耶氏酵母Po1gpYLXP’-NcCAR。
实施例3
重组菌株发酵合成对羟基苯甲醇。
(1)挑取实施例2中解脂耶氏酵母Po1g pYLXP’-NcCAR,接种于种子培养基,在温度为30℃、转速为220rpm的条件下培养48h,得到重组解脂耶氏酵母种子液;
(2)将步骤(1)得到的重组解脂耶氏酵母种子液以发酵培养基体积5%的接种量接种至发酵培养基中,在温度为30℃,转速为220rpm的条件下进行发酵培养48h,向发酵液中加入对羟基苯甲酸使得添加量为1g/L,再进行发酵培养24h,得到发酵液;发酵结束后通过高效液相色谱测定方法测定发酵上清液中对羟基苯甲醇的含量为275mg/L。
实施例4
重组菌株发酵合成对羟基苯甲醇。
(1)挑取实施例2中解脂耶氏酵母Po1g pYLXP’-NcCAR,接种于种子培养基,在温度为30℃、转速为220rpm的条件下培养48h,得到重组解脂耶氏酵母种子液;
(2)将步骤(1)得到的重组解脂耶氏酵母种子液以发酵培养基体积5%的接种量接种至发酵培养基中,在温度为30℃,转速为220rpm的条件下进行发酵培养72h,得到发酵液;发酵结束后通过高效液相色谱测定方法测定发酵上清液中对羟基苯甲醇的含量(如图4所示)。
对比例1
按照实例3的方法发酵合成对羟基苯甲醇,不同的是,将重组解脂耶氏酵母菌株替换为制备例1得到的解脂耶氏酵母菌株Po1gpYLXP’-CAR-PPTcg-1,得到的上清液中对羟基苯甲醇的含量为127mg/L。
参见图5,比较实施例3与对比例1可以看出,实施例3中得到的重组解脂耶氏酵母经发酵获得的发酵液中胞外对羟基苯甲醇的产量明显提高。采用本发明的构建方法对出发菌株解脂耶氏酵母菌株Po1g通过过表达外源羧酸还原酶NcCAR改造得到的重组菌株发酵合成对羟基苯甲醇能够更显著提升的发酵效果,得到的发酵上清液中对羟基苯甲醇的含量可达到275mg/L,与解脂耶氏酵母Po1gpYLXP’-CAR-PPTcg-1相比提高了1.2倍。
以上详细描述了本发明的优选实施方式,但是,本发明并不限于此。在本发明的技术构思范围内,可以对本发明的技术方案进行多种简单变型,包括各个技术特征以任何其它的合适方式进行组合,这些简单变型和组合同样应当视为本发明所公开的内容,均属于本发明的保护范围。
Claims (9)
1.一种利用重组解脂耶氏酵母合成对羟基苯甲醇的方法,其特征在于,包括:
(1)构建重组解脂耶氏酵母;
(2)将重组解脂耶氏酵母接种至发酵培养基中进行发酵培养;
(3)加入对羟基苯甲酸继续发酵培养得发酵液。
2.根据权利要求1所述的合成对羟基苯甲醇的方法,其特征在于,所述构建重组解脂耶氏酵母的方法为:对出发菌株引入含有羧酸还原酶基因的重组载体,得到重组解脂耶氏酵母。
3.根据权利要求2所述的合成对羟基苯甲醇的方法,其特征在于,所述出发菌株为亮氨酸营养缺陷型解脂耶氏酵母Po1g。
4.根据权利要求1所述的合成对羟基苯甲醇的方法,其特征在于,所述将重组解脂耶氏酵母接种至发酵培养基中进行发酵培养包括,挑选重组菌株的单菌落于种子培养基中培养,得到种子液;再将种子液接种至发酵培养基中进行发酵,得到发酵液。
5.根据权利要求4所述的合成对羟基苯甲醇的方法,其特征在于,所述发酵培养基的组分包括碳源、氮源、硫酸盐或腺嘌呤中的至少一种氨基酸。
6.根据权利要求4所述的合成对羟基苯甲醇的方法,其特征在于,所述发酵培养基的组分包括碳源、氮源、硫酸盐、腺嘌呤、L-精氨酸、L-天门冬氨酸、L-组氨酸、L-异亮氨酸、L-赖氨酸、L-甲硫氨酸、L-苯丙氨酸、L-苏氨酸、L-色氨酸、L-缬氨酸和尿嘧啶。
7.根据权利要求4所述的合成对羟基苯甲醇的方法,其特征在于,所述发酵培养基的组分包括:葡萄糖30-50g/L,硫酸铵0.8-1.3g/L,YNB(无氨基酵母氮源)1.5-2g/L,腺嘌呤0.01-0.02g/L,L-精氨酸0.02-0.08g/L,L-天门冬氨酸0.05-1g/L,L-组氨酸0.01-0.03g/L,L-异亮氨酸0.02-0.08g/L,L-赖氨酸0.02-0.08g/L,L-甲硫氨酸0.01-0.03g/L,L-苯丙氨酸0.02-0.08g/L,L-苏氨酸0.05-0.15g/L,L-色氨酸0.02-0.08g/L,L-缬氨酸0.05-0.25g/L,尿嘧啶0.01-0.03g/L。
8.根据权利要求1或4所述的合成对羟基苯甲醇的方法,其特征在于,重组解脂耶氏酵母的接种量为发酵培养基体积的4-6%,培养温度为25-35℃,转速为180-250rpm,培养时间为72-96h。
9.根据权利要求1或4所述的合成对羟基苯甲醇的方法,其特征在于,在发酵培养40-55h时向发酵培养基中加入对羟基苯甲酸,所述对羟基苯甲酸的加入量为每1L发酵培养基加入0.5-1g。
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