CN108486160A - 高密度发酵合成莱鲍迪苷a的方法 - Google Patents
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Abstract
本发明公开了一种高密度发酵合成莱鲍迪苷A的方法。首先,敲除编码细胞壁合成酶系的基因,增加细胞壁通透性的同时实现莱鲍迪苷A的发酵合成。其次,发酵获得的菌体应用于全细胞催化反应,实现莱鲍迪苷A的高收率。高密度发酵过程中,菌体量长到(10~25)g/L(DCW)时,以恒溶氧指数补料的方式流加培养基,细胞生长到稳定期菌体密度OD600达到100~150;菌体生长到稳定期,以恒速补料的方式流加培养基,发酵结束发酵液中莱鲍迪苷A的含量为13.7±1.6 g/L。发酵收集的菌体使用磷酸钾缓冲清洗处理后,用于50 L全细胞催化反应,体系中莱鲍迪苷A的浓度为28.2±4.9 g/L。该方法很好解决细胞发酵甜菊糖转化率低的问题,操作简单易于扩大生产,具有良好的制备莱鲍迪苷A的前景。
Description
技术领域
本发明涉及一种高密度发酵合成莱鲍迪苷A的方法,属于生物工程技术领域。
背景技术
莱鲍迪苷A(Rebaudioside A,RA苷)是从甜叶菊(Stevia rebaudiana Bert)中提取的天然甜味剂,是甜菊糖的主要成份之一。RA苷具有良好的耐高温、耐酸碱性,在常温、相对湿度60%条件下,其可以稳定保存两年。RA苷是理想的天然甜味剂,零热量、甜度高、口感佳,且不含不良余味,其甜度约为蔗糖的350~450倍。然而,市售甜菊糖主要以甜菊糖苷(Stevioside,St苷),甜菊糖苷味质较差,略带甘苦味和甘草异味,影响其在食品工业中的应用。因此,增加RA苷在甜菊糖总苷中的比例,将会有效改善甜菊糖味质。
近年来,已有许多成功合成RA苷的策略。譬如,直接提取法,酶促修饰法,全细胞催化法,发酵法等。直接从甜叶菊中提取莱鲍迪苷A是工业化生产的方式,然而,这种方式致使糖苷中含有植物残留物,比如色素、脂类、蛋白质、酚类等等,降低了其味质。酶促修饰法利用酶的转糖基作用,向甜菊糖苷上引入葡萄糖基合成莱鲍迪苷A。(Park S H,Kim J E,YoonR Y,et al.METHOD FOR PREPARING REBAUDIOSIDE A FROM STEVIOSIDE:,EP2963122[P].2016.)利用蔗糖合酶水解蔗糖生成的尿苷二磷酸葡萄糖(UDPG)作为糖基供体,偶联甜叶菊糖基转移酶UGT76G1合成莱鲍迪苷A。酶促修饰法的中间产物UDP对糖基化反应有抑制作用;而且,游离酶稳定性差,难以循环利用。发酵法正积极研发,该方法利用细胞内代谢途径,微生物利用基本营养物质从头合成莱鲍迪苷A及其甜菊糖系列物质。目前,发酵合成甜菊糖系列产品已有报到,(Mikkelsen M D,Hansen J,Simon E,et al.METHODS FORIMPROVED PRODUCTION OF REBAUDIOSIDE D AND REBAUDIOSIDE M:WO,WO/2014/122227[P].2014.),从头合成路线长,产率低,甜菊糖产量低仅有~2.5g/L。因为从头合成莱鲍迪苷A途径中关键酶未解析及能量供给不足等问题。
目前发酵法代谢合成的莱鲍迪苷A,主要存在于细胞内,胞内累计量有限且胞内物质复杂不利于提取纯化,(Lehmann M,Trueheart J,Zwartjens P,et al.DITERPENEPRODUCTION:WO,EP2806754[P].2014.)。
发明内容
本发明的目的之一在于提供一株细胞壁通透性良好的能够高密度发酵生产莱鲍迪苷A的基因工程菌;
本发明的另一个目的在于:提供上述基因工程菌的构建方法;
本发明还有一个目的在于:利用上述基因工程菌高密度发酵,并合成莱鲍迪苷A。
为了实现上述目的,本发明采用的技术方案为:
首先构建一株敲除编码细胞壁功能基因的酿酒酵母,弱化细胞壁的通透性。酵母细胞壁主要有葡聚糖、甘露聚糖、几丁质、蛋白质等交联而成。鉴于细胞壁成份多样性,合成相关组成的酶多样,其编码相关酶的基因不局限所述部分。
比如,编码葡聚糖酶的基因有DSE2、GSC2、FKS1、KRE1、KRE5、KRE6、KRE9、KNH1、ROT1、SKN1、RHO1、FKS3、BIG1等等;编码甘露聚糖酶的基因有MNN9、OCH1、MNN1、KTR2、KTR3、YUR1、KTR4、KTR6、KRE2、ANP1等等;编码几丁质酶的基因有SKT5、SHC1、CHS3、CHS2、CHS1等等;以及,编码葡聚糖与几丁质交联酶的基因有CRH1、UTR2、CRR1等等。以及,编码各成份不同链接方式酶的基因。敲除相关功能的基因都可以实现细胞的通透性。本发明实施例分别构建敲除DSE2、GSC2、FKS1、MNN9、OCH1、CHS3、CRH1基因的五株菌,得到了细胞壁通透性良好的酿酒酵母,敲除其他相关基因也具备类似效果,本发明不一一列举说明。
设计带有上述基因DSE2、GSC2、FKS1、MNN9、OCH1、CHS3、CRH1同源臂的上游、下游引物,PCR扩增质粒pPIC9K(Invitrogen)中G418完整表达框基因。琼脂糖凝胶纯化回收上述基因片段。应用化学转化法,将纯化后的片段转化入酿酒酵母感受态中。将酵母转化液涂布于YPDA(含有一定浓度的G418抗性)培养基平板,30℃生长直至长出单菌落,菌落PCR验证阳性克隆,并保存菌种。
使用重叠PCR将启动子分别跟UGT76G1、UGPase连接,组成完整表达框。上述基因可以仅表达UGT76G1酶,亦可共表达UGT76G1酶和UGPase酶。将上述单一表达框或者组合表达框连接到酵母穿梭型质粒pESC上,构建多个载体,导入大肠杆菌DH5α感受态中,复制扩增质粒。
从上述DH5α中提取的质粒为模板,设计带有σ或△Leu2同源臂的引物PCR扩增UGT76G1,UGPase基因,利用同源重组的方式,将单一表达框UGT76G1或者组合表达框UGT76G1和UGPase,分别或者共同整合到酵母基因组上。
所述启动子类型不局限于TEF1、TEF2、PGK1、HXT7、GPM1、CDC5、FBA1、ENO2、GAL1、GAL10、ALA1;终止子类型不局限于CYC1、TDH1、FBA1、PGI1、ENO2、TDH2、ADH2。
培养构建得到的重组酿酒酵母,利用细胞代谢途径产莱鲍迪苷A,将重组酿酒酵母接种至发酵初始培养基中,温度为20~40℃,溶氧维持在10~50%范围内,pH维持在4~5,以恒溶氧、恒pH、比速率μ为指标流加补料培养基,流加方程式为:
其中:μ(t)-发酵过程中细胞数的比生长速率h-1;
YX/N-细胞生长的得率,可以是底物消耗、氧消耗、碳等为基准的细胞得率;
m-为保持系数[g/(g·h)-0.025];
X0、V0-补料开始时细胞干重(g/L)和发酵液体积(L);
发酵结束,收集发酵菌体和发酵液,所得发酵液分离得到莱鲍迪苷A,所得菌体进一步用于全细胞催化合成莱鲍迪苷A。
所述发酵初始培养基(g/L)为:碳源10~100,酵母膏3~50,蛋白胨3~50,氯化钠0.1~10,甜菊糖苷1~50,硫酸铵0.5~10,硫酸镁0.5~8,硫酸锌0.06~0.1,硫酸亚铁0.05~0.1,磷酸(2~10)mL/L。所述碳源为葡萄糖、糖蜜、麦芽糖、半乳糖或者蔗糖中的一种或几种。
进一步地优选为,初始培养中(g/L),糖蜜30,酵母膏3,蛋白胨3,甜菊苷30,硫酸铵3,硫酸镁0.5,氯化钠0.1,硫酸锌0.06,硫酸亚铁0.05,磷酸2mL/L,pH维持在4~5,温度为30℃,溶氧维持在10~50%范围内。
所述补料培养基为:碳源3~50,酵母膏3~50,蛋白胨3~50,甜菊糖苷10~200,氯化钠0.1~10,硫酸镁0.5~8,C/N为1,培养过程中用100%氨水、100%磷酸控制发酵液pH为4~5。
补料流加的方式,以恒溶氧、恒pH、比速率μ为参考标准。其中,恒溶氧调节的方式:溶氧减少增大转速增加发酵液中的含氧量,反之降低转速。溶氧降低说明发酵液中细胞快速增长,此时营养成份难以维持其生长,向其中增加碳源,比如葡萄糖、糖蜜等。
恒pH补料的方式:以pH升高为标志,向发酵液中补料,维持C/N比为1。
恒比速率μ补料的方式:生长过程中加入碳源与氮源维持恒定的比生长速率μ为0.1-0.5。
所述全细胞催化合成莱鲍迪苷A的催化反应体系为:基因工程菌的用量按湿菌体计为1~100g/L;葡萄糖的用量为20~120g/L;甜菊甙的用量为1~100g/L;镁离子用量为1~l0g/L;反应pH为6.8~8.0,反应温度为20~42℃,反应时间为8~96h。
本发明弱化酵母细胞壁降低胞内酶与底物的反应阻力,增加催化反应的效率。与传统的物理、化学方法相比,分子生物学方法对细胞破坏小、能充分保证细胞活性,尤其对涉及辅酶再生以及复杂生化反应的体系更具优势。此外,将表达UGT76G1和UGPase的基因整合到酵母染色体上,实现这两个酶的高效表达。相比较于酶促修饰法,全细胞催化的方法可以胞内辅酶体系实现高效转化率。
一方面,细胞培养过程中加入甜菊苷,在细胞生长表达目的蛋白的同时,可以实现莱鲍迪苷A的合成。在细胞培养周期,高密度发酵累积细胞质,增加目的蛋白比例,从而提高细胞催化甜菊糖苷合成莱鲍迪苷A的转化能力。此外,培养结束,直接分离发酵液即得到所需要的产品。这比(Jens H L,Hicks P M,Michael N,et al.RECOMBINANT PRODUCTION OFSTEVIOL GLYCOSIDES:EP2742142[P].2014.)所述过程更加简单易于扩大。另一方面,发酵培养的细胞也可以应用于全细胞催化反应。离心发酵液收集菌泥,并建立催化体系,结果表明细胞的二次使用,进一步提高莱鲍迪苷A的收率。
有益效果:
本发明通过敲除酵母细胞壁功能基因,增加细胞通透性,提高底物与胞内酶的接触,提高细胞转化合成莱鲍迪苷A的能力。
发酵过程中酶的稳定表达影响莱鲍迪苷A的产量,本发明将目的基因利用同源重组的方式整合到细胞的基因组上,实现酶稳定高效的表达。
本发明很好解决细胞转化甜菊糖苷合成莱鲍迪苷A的能力,通过高密度发酵一步糖基化合成莱鲍迪苷A,莱鲍迪苷A的产量达到13.7±1.6g/L。
高密度发酵获得的菌株,简单处理后可以继续应用于全细胞催化,并且不需要添加细胞通透剂,工程菌合成RA苷的浓度达到28.2±4.9g/L。
附图说明
图1酿酒酵母催化合成莱鲍迪苷A途径;
图2高密度发酵过程中的菌体量;
图3发酵与全细胞催化合成莱鲍迪苷A的量;
图4启动子对UGT76G1和UGPase酶活的影响。
具体实施方式
根据下述实施例,可以更好地理解本发明。然而,本领域的技术人员容易理解,实施例所描述的内容仅用于说明本发明,而不应当也不会限制权利要求书中所详细描述的本发明。
以下实施例中所采用的酵母为酿酒酵母YPH499(Invitrogen)。
本发明包括如下步骤实现:
1.设计带有FKS1、GSC2、OCH1、MNN9、DSE2、CHS3、CRH1基因同源臂的上游、下游引物,PCR扩增带有G418完整表达框抗性的基因。琼脂糖凝胶纯化回收上述基因片段。
2.使用酿酒酵母高效转化方法,将上述纯化的DNA片段转入酿酒酵母YPH499(MATaura3-52 lys2-801amber ade2-101ochre trp1-Δ63 his3-Δ200 leu2-Δ1)中,双挑鉴定阳性克隆,构建成功的菌株命名为YLY01(△gene)。
3.将构建成功的单菌落置于YPDA(G418终浓度500μg/mL)液体培养基中,30℃摇床培养8-48h,并保存菌种YLY01(△gene)。
4.表达目的蛋白菌株的构建。
5.将密码子优化后的基因UGT76G1(S.rebaudiana)以及UGPase(Saccharomycescerevisiae)构建到质粒pESC上,其中HXT7启动子控制UGT76G1表达,终止子为ADH2;TEF1启动子控制UGPase表达,终止子为ADH2,成功构建质粒pSECL
6.将构建的质粒pSECL导入大肠杆菌DH5α中,双挑验证并测序。将测序成功的菌落置于LB液体培养基中过夜生长,用于保存菌种。
7.利用同源重组的方法将目的基因插入到酿酒酵母σ位点。设计带有σ位点同源臂引物,PCR扩增构建质粒上的UGT76G1、UGPase基因,导入酿酒酵母YLY01(△gene)中,成功构建一株工程菌命名为YLY02。
8.工程菌YLY02高密度发酵合成莱鲍迪苷A。
9.初始培养基,碳源为糖蜜,氮源为蛋白胨和酵母膏,以及微量元素,甜菊糖苷。初始培养中(g/L),糖蜜30,酵母膏3,蛋白胨3,甜菊苷的浓度为50,硫酸铵3,硫酸镁0.5,氯化钠0.1,硫酸锌0.06,硫酸亚铁0.05,磷酸2mL/L,pH维持在4~5,温度为30℃,溶氧维持在10~50%范围内。
10.补料培养基碳源与氮源为主,并保证C/N为1。培养过程中用100%氨水、100%磷酸控制发酵液pH为4~5。
11.补料流加的方式,以恒溶氧、恒pH、比速率μ为参考标准。
12.其中,恒溶氧调节的方式。溶氧减少增大转速增加发酵液中的含氧量,反之降低转速。溶氧降低说明发酵液中细胞快速增长,此时营养成份难以维持其生长,向其中增加碳源,比如葡萄糖、糖蜜等。
13.恒pH补料的方式。以pH升高为标志,向发酵液中补料,维持C/N比为1。
14.恒比速率μ补料的方式。生长过程中加入碳源与氮源维持恒定的比生长速率μ为0.1-0.5。
15.高密度发酵的细胞应用全细胞催化甜菊苷合成莱鲍迪苷A。
16.催化体系。基因工程菌的用量按湿菌体计为1~100g/L;葡萄糖的用量为20~120g/L;甜菊甙的用量为1~100g/L;镁离子用量为1~l0g/L;反应pH为6.8~8.0,反应温度为20~42℃,反应时间为8~96h。
实施例1:编码细胞壁合成酶基因的敲除
在NCBI数据库中查找基因FKS1、GSC2、OCH1、MNN9、DSE2、CHS3、CRH1序列,并设计带有其基因同源臂序列引物PCR扩增上述基因,反应体系为:
ddH2O:19μL
P3:2μL
P4:2μL
模板:2μL
2×Phanta Master Mix:25μL
总体积:50μL
其中变性、退后温度及其相应时间根据引物计算得到。
表1 引物序列
实施例2:重组菌YLY01(△fks1)、YLY01(△gsc2)、YLY01(△och1)、YLY01(△mnn9)、YLY01(△dse2)、YLY01(△chs2)、YLY01(△crh1)构建
将实施例1中的NEO基因用琼脂糖凝胶分离回收,应用化学转化法,将纯化后的片段转化入酿酒酵母感受态YPH499(Invitrogen)中。将酵母转化液涂布于YPDA(含有一定浓度的G418抗性)培养基平板(YPDA:酵母膏10g/L,蛋白胨20g/L,葡萄糖20g/L,腺嘌呤0.75g/L),30℃生长直至长出单菌落,菌落PCR验证阳性克隆。将阳性克隆菌命名为YLY01(△fks1)、YLY01(△gsc2)、YLY01(△och1)、YLY01(△mnn9)、YLY01(△dse2),YLY01(△chs2)、YLY01(△crh1)并保存菌种。
实施例3 启动子对基因UGT76G1和UGPase表达水平的影响
筛选启动子提高目的基因的转录水平,本例子选用如下启动子,TEF1、PGK1、HXT7、GPM1、CDC5、FBA1、ENO2、GAL10、ALA1。使用同源重组的方式将选择的启动子分别与目的基因连接,比较不同启动子调节下UGT76G1与UGPase酶活。
将构建成功的工程菌转接100mL YPDA液体培养基中,30℃摇床生长24h。离心收集上述菌泥,用100mM、pH8.0磷酸缓冲清洗菌泥三遍。用超声破碎仪破碎细胞20min,破碎液12000rpm离心30min,收集上清酶液。
UGT76G1酶活力单位定义为:在上述反应条件下,30℃,1min催化形成1μmol莱鲍迪甙A所需要的酶量为1个活力单位(U)。
UGPase酶活力单位定义为:每毫克细胞蛋白每分钟消耗1nmol的NADP为一个酶活力单位。
利用UGPase可逆反应,其催化反应生成1-磷酸葡萄糖,在磷酸葡萄糖变位酶和6-磷酸葡萄糖脱氢酶作用下将NADP转化为NADPH,在340nm的吸光值增加速率反映了UGPase活性。
实验结果表明,UGT76G1酶活力在HXT7p作用下,最高达2.281±0.032mU/mg(总蛋白);UGPase酶活力在TEF1p作用下,最高达16.874±0.972mU/mg(总蛋白)。
实施例4目的基因UGT76G1和UGPase的构建。
使用重叠PCR将启动子HXT7与UGT76G1、TEF1与UGPase、PGK1与Leu2相连,组成一个完整表达框。这三个表达框可以单一,也可以组合连接到酵母穿梭型质粒(Invitrogen)上,构建多个载体。导入大肠杆菌DH5α感受态中,复制扩增质粒。
实施例5:重组菌YLY02(△gene,ugt76g1)
以实施例4中的质粒为模板扩增UGT76G1,将PCR片段琼脂糖凝胶纯化回收后,导入工程菌YLY01(△fks1)、YLY01(△gsc2)、YLY01(△och1)、YLY01(△mnn9)、YLY01(△dse2),YLY01(△chs2)、YLY01(△crh1)以同源重组的方式整合到酵母基因组上,构建一株重组酿酒酵母命名为YLY02(△fks1,ugt76g1)、YLY02(△gsc2,ugt76g1)、YLY02(△och1,ugt76g1)、YLY02(△mnn9,ugt76g1)、YLY02(△dse2,ugt76g1)、YLY02(△chs2,ugt76g1)、YLY02(△crh1,ugt76g1)。
实施例6:重组菌YLY02(△gene,ugt76g1,ugpase)构建
以实施例4中的质粒为模板扩增UGT76G1和UGPase完整表达框,将PCR片段琼脂糖凝胶纯化回收后,应用化学转化法将其导入工程菌YLY01(△fks1)、YLY01(△gsc2)、YLY01(△och1)、YLY01(△mnn9)、YLY01(△dse2)、YLY01(△chs2)、YLY01(△crh1)(实施例2中构建),以同源重组的方式整合到酵母基因组上,构建一株重组酿酒酵母命名为YLY02(△fks1,ugt76g1,ugpase),YLY02(△gsc2,ugt76g1,ugpase),YLY02(och1,ugt76g1,△ugpase),YLY02(mnn9,ugt76g1,ugpase),YLY02(Δdse2,ugt76g1,ugpase),YLY02(△chs2,ugt76g1,ugpase),YLY02(△crh1,ugt76g1,ugpase)。
实施例7:重组菌YLY02(△gene,ugt76g1,ugpase)和YLY02(△gene,ugt76g1)活化
将-80℃保存的甘油管菌体于冰上融化,转接100μL菌液涂布于YPDA固体平板上,30℃培养箱生长。挑选长出的单菌落涂布于SC-Leu营养缺陷型固体平板上,30℃培养箱生长。YPDA(g/L):酵母膏10,蛋白胨20,葡萄糖20,腺嘌呤0.75,琼脂糖20,SC-Leu(g/L):YNB6.7,葡萄糖20,腺嘌呤0.1,半胱氨酸0.1,精氨酸0.1,尿嘧啶0.1,苏氨酸0.1,赖氨酸0.1,色氨酸0.1,组氨酸0.05,天冬氨酸0.05,甲硫氨酸0.05,异亮氨酸0.05,苯丙氨酸0.05,丝氨酸0.05,脯氨酸0.05,酪氨酸0.05,缬氨酸0.05,琼脂糖20。
实施例8:高密度发酵YLY02(△gen2,ugt76g1,ugpase)合成莱鲍迪苷A
将营养缺陷型筛选的种子液生长至OD600=15,按3%接种量转接于发酵罐中。发酵罐初始培养基浓度(g/L):蛋白胨3,葡萄糖30,甜菊糖苷30,硫酸铵3,硫酸镁0.5,氯化钠0.1,硫酸锌0.06,硫酸铁0.05,酵母膏3。生长条件:温度30℃,磷酸和氨水控制pH4~5,溶氧与转速串级并控制溶氧为30%。补料培养基1浓度(g/L):葡萄糖200,酵母膏50,硫酸镁12,甜菊糖苷100。补料培养基2浓度(g/L):葡萄糖200,甜菊糖苷200;
酵母生长到对数生长期按补料培养基1补料。细胞干重为15~20g/L,流加培养基用量,按公式计算-发酵过程中细胞数的比生长速率h-1
YX/N-细胞生长的得率,可以是底物消耗、氧消耗、碳等为基准的细胞得率。
m-为保持系数[g/(g·h)-0.025]
X0,V0-补料开始时细胞干重(g/L)和发酵液体积(L)
酵母生长到稳定期后按补料培养基2补料。此时,以恒定比生长速率μ=0.24h-1,流加培养基,稳定菌株的正常代谢从而合成莱鲍迪苷A,该过程持续24-72h,测得细胞密度OD600为168.5,高效液相色谱仪测定发酵液中RA苷为13.7±1.6g/L。
表2 高密度发酵YLY02(△gene,ugt76g1,ugpase)合成RA
实施例9:高密度发酵YLY02(△gene,ugt76g1)合成莱鲍迪苷A
培养过程如同实施例8中。最终检测发酵液中的RA苷浓度为5.4±0.8g/L。
表3 高密度发酵YLY02(△gene,ugt76g1)合成RA
实施例10:重组菌YLY02(△gene,ugt76g1,ugpase)催化甜菊苷合成莱鲍迪苷A
取实施例8中的湿菌体,并用50mM、pH8.0的磷酸钾缓冲清洗三遍。离心的沉淀转移至50L反应罐中,建立全细胞催化反应体系。此反应体系为40L,其中菌体2.0g/L,底物St甙50g/L;葡萄糖60g/L;MgCl2 6g/L;用pH为8.0的磷酸钾缓冲液定容至7L,30℃,200rpm,反应一段时间后,取样品灭活离心,经高效液相色谱仪测得反应体系中RA苷28.2±4.9g/L。
表4 全细胞YLY02(△gene,ugt76g1,ugpase)催化合成RA
实施例11:重组菌YLY02(△gene,ugt76g1)催化甜菊苷合成莱鲍迪苷A
取实施例9中的湿菌体,并用50mM、pH8.0的磷酸钾缓冲清洗三遍。催化体系如实施例10中。最终测得反应液中RA苷的浓度为15.6±3.1g/L。
表5 全细胞YLY01(△gene,ugt76g1,ugpase)催化合成RA
序列表
<110> 兴化格林生物制品有限公司
南京工业大学
<120> 高密度发酵合成莱鲍迪苷A的方法
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Claims (10)
1.高密度发酵合成莱鲍迪苷A的方法,其特征在于,包括如下步骤:
1)构建一株细胞壁通透性良好的酵母,将目的基因UGT76G1和UGPase整合到所构建的酵母基因组上,得到基因工程菌;
2)培养步骤1)构建得到的基因工程菌,利用细胞代谢途径产莱鲍迪苷A,将基因工程菌接种至发酵初始培养基中,温度为20~40℃,溶氧维持在10~50%范围内,pH维持在4~5,以恒溶氧、恒pH、比速率μ为指标流加补料培养基,流加方程式为:
;
3)发酵结束,收集发酵菌体和发酵液,所得发酵液分离得到莱鲍迪苷A,所得菌体进一步用于全细胞催化合成莱鲍迪苷A。
2.根据权利要求1所述的高密度发酵合成莱鲍迪苷A的方法,其特征在于,步骤1)中所述细胞壁通透性良好的酵母是通过敲除酵母的细胞壁功能基因所得,所述细胞壁功能基因包括但不限于编码葡聚糖酶的基因、编码甘露聚糖酶的基因、编码几丁质酶的基因、编码葡聚糖与几丁质交联酶的基因、编码葡聚糖、甘露聚糖、几丁质、蛋白质交联成份不同链接方式酶的基因。
3.根据权利要求1所述的高密度发酵合成莱鲍迪苷A的方法,其特征在于,步骤1)中使用重叠PCR将启动子与终止子连接到UGT76G1和UGTase两个DNA片段上,构成完整表达框,将表达框分别或者共同整合到酵母细胞σ、△Leu2位点。
4.根据权利要求1所述的高密度发酵合成莱鲍迪苷A的方法,其特征在于,步骤2)中所述发酵初始培养基(g/L)为:碳源10~100,酵母膏3~50,蛋白胨3~50,氯化钠0.1~10,甜菊糖苷1~50,硫酸铵0.5~10,硫酸镁0.5~8,硫酸锌0.06~0.1,硫酸亚铁0.05~0.1,磷酸 (2~10)mL/L。
5.根据权利要求1所述的高密度发酵合成莱鲍迪苷A的方法,其特征在于,步骤2)中所述补料培养基为:碳源:3~50,酵母膏3~50,蛋白胨3~50,氯化钠0.1~10,甜菊糖苷10~200,硫酸镁0.5~8。
6.根据权利要求2所述的高密度发酵合成莱鲍迪苷A的方法,其特征在于,编码葡聚糖酶的基因包括DSE2、GSC2、FKS1、KRE1、KRE5、KRE6 、KRE9、KNH1、ROT1、SKN1;编码甘露聚糖酶的基因包括MNN9、OCH1、MNN1、KTR2、KTR3、YUR1、KTR4、KTR6、KRE2、ANP1;编码几丁质酶的基因包括SKT5、SHC1、CHS3、CHS2、CHS1;编码葡聚糖与几丁质交联酶的基因包括CRH1、UTR2、CRR1。
7.根据权利要求3所述的高密度发酵合成莱鲍迪苷A的方法,其特征在于,启动子类型b包括但不局限于TEF1、TEF2、PGK1、HXT7、GPM1、CDC5、FBA1、ENO2、GAL1、GAL10、ALA1;终止子类型包括但不局限于CYC1、TDH1、FBA1、PGI1、ENO2、TDH2、ADH2。
8.根据权利要求4所述的高密度发酵合成莱鲍迪苷A的方法,其特征在于,所述碳源为葡萄糖、糖蜜、麦芽糖、半乳糖或者蔗糖中的一种或几种,保证C/N为1。
9.根据权利要求1所述的高密度发酵合成莱鲍迪苷A的方法,其特征在于,所述培养基中甜菊糖苷的纯度为10~98%;或者直接使用甜叶菊提取液。
10.根据权利要求4所述的高密度发酵合成莱鲍迪苷A的方法,其特征在于,所述全细胞催化合成莱鲍迪苷A的催化反应体系为:基因工程菌的用量按湿菌体计为1~100 g/L;葡萄糖的用量为 20~120 g/L;甜菊甙的用量为 1~100 g/L;镁离子用量为 1~l0 g/L;反应 pH为 6. 8~8.0,反应温度为 20~42 ℃,反应时间为 8~96 h。
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