CN108048494B - 一种利用生物酶合成1,3-丙二醇的方法 - Google Patents
一种利用生物酶合成1,3-丙二醇的方法 Download PDFInfo
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- CN108048494B CN108048494B CN201711470812.2A CN201711470812A CN108048494B CN 108048494 B CN108048494 B CN 108048494B CN 201711470812 A CN201711470812 A CN 201711470812A CN 108048494 B CN108048494 B CN 108048494B
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Abstract
本发明公开了一种利用生物酶合成1,3‑丙二醇的方法。本发明提供了DERA蛋白或所述DERA蛋白相关生物材料在合成1,3‑丙二醇中的应用,以及利用DERA蛋白和yqhD蛋白生物合成1,3‑丙二醇的具体方法。本发明构建并实现了1,3‑丙二醇的新途径,此途径不需要辅酶B12的参与,有潜力成为1,3‑丙二醇工业生产的新方法。
Description
技术领域
本发明涉及生物酶合成领域,具体涉及一种利用生物酶合成1,3-丙二醇的方法。
背景技术
1,3-丙二醇是一种重要的化学中间体,可以参与聚合反应生产聚醚、聚氨酯和聚酯。与1,2-丙二醇、丁醇和乙二醇为前体合成的聚合物相比,以1,3-丙二醇为前体合成的聚合物具有更好的性能和稳定性,因此被广泛应用于纺织业、塑剂、冷却剂等。如1,3-丙二醇和对苯二甲酸发生聚合反应形成的聚对苯二甲酸丙二醇酯(PTT),主要用于制造地毯、纺织纤维,用这种聚合物制成的纺织品具有良好的拉伸弹性、复苏性、防沾污性;在发动机冷却配方中加1,3-丙二醇,可以增强热稳定性并且减少腐蚀性,而且比用乙二醇作冷却剂毒性小。近来还发现1,3-丙二醇还可以用于热塑性塑料、涂料和胶片的制造,如用1,3-丙二醇合成的热塑性聚氨酯(TPU),改善了热水解性和热稳定性。
1,3-丙二醇合成方法主要分为化学合成法和微生物合成法,化学法主要有环氧乙烷羰基化法和丙烯醛水合氢化法。目前通用的微生物法生产1,3-丙二醇主要是利用微生物将甘油转化为1,3-丙二醇。生物柴油生产过程中会生成一部分副产物甘油,以此为发酵底物生产1,3-丙二醇,可大大降低生产成本,但是该方法需要使用甘油脱水酶和辅酶B12。而甘油脱水酶(glycerol dehydratase)是甘油代谢还原途径中的限速酶,它是由dhaB1、dhaB2、dhaB3三个基因编码合成的三个亚基组成,并且在辅酶B12的参与下,催化甘油脱去一分子水形成3-羟基丙醛。辅酶B12易与甘油相互作用,使得C-Co键发生断裂,形成了5'-脱氧腺苷和烷基氰钴胺素类似物,烷基氰钴胺素类似物可以与甘油脱水酶紧密结合使之失去催化活性,甘油脱水反应停止。甘油脱水酶的这种现象严重影响甘油的转化。除了上述情况外,甘油脱水酶在氧存在的情况下也会失活,研究表明氧的存在对甘油脱水酶的酶活有强烈的影响,与无氧情况下的甘油脱水酶相比,暴露在氧中的甘油脱水酶在5min内酶活下降80%,并且在60min后酶活彻底丧失。同样甘油浓度对甘油脱水酶也有抑制作用,浓度越高抑制作用越大。
发明内容
为了解决上述技术问题,本发明提供了一种利用生物酶DERA催化合成1,3-丙二醇的方法,该方法不需要辅酶B12的参与。
第一,本发明要求保护DERA蛋白或所述DERA蛋白相关生物材料在合成1,3-丙二醇中的应用。
其中,所述DERA蛋白具体可为如下任一所示蛋白质:
(A1)氨基酸序列为SEQ ID No.2的蛋白质;
(A2)将SEQ ID No.2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质;
(A3)与(A1)或(A2)所限定的氨基酸序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同源性且具有相同功能的蛋白质;
(A4)在(A1)-(A3)中任一所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白。
所述DERA蛋白相关生物材料为能够表达所述DERA蛋白的核酸分子,或含有所述核酸分子的表达盒、重组载体、重组菌或转基因细胞系。
进一步地,所述应用为所述“DERA蛋白或所述DERA蛋白相关生物材料”和“yqhD蛋白或所述yqhD蛋白相关生物材料”在合成1,3-丙二醇中的应用。
其中,所述yqhD蛋白为如下任一所示蛋白质:
(B1)氨基酸序列为SEQ ID No.5的蛋白质;
(B2)将SEQ ID No.5所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质;
(B3)与(B1)或(B2)所限定的氨基酸序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同源性且具有相同功能的蛋白质;
(B4)在(B1)-(B3)中任一所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白。
所述yqhD蛋白相关生物材料为能够表达所述yqhD蛋白的核酸分子,或含有所述核酸分子的表达盒、重组载体、重组菌或转基因细胞系。
进一步地,所述核酸分子可以是DNA,如cDNA、基因组DNA或重组DNA;所述核酸分子也可以是RNA,如mRNA等。所述重组载体可为重组表达载体,也可为重组克隆载体。所述表达盒可由能够启动所述核酸分子转录的启动子,所述核酸分子,以及转录终止序列组成。
第二,本发明要求保护一种合成的1,3-丙二醇方法。
本发明所提供的合成的1,3-丙二醇方法,可为如下方法I或方法II。
方法I:一种合成的1,3-丙二醇方法,包括如下步骤:
(a1)以甲醛和乙醛作为底物,经DERA蛋白催化反应生成3-羟基丙醛;
(a2)以3-羟基丙醛作为底物,经yqhD蛋白催化反应生成1,3-丙二醇。
所述DERA蛋白为前文(A1)-(A4)任一所示蛋白;所述yqhD蛋白为前文(B1)-(B4)任一所示蛋白。
方法II:一种合成的1,3-丙二醇方法,包括如下步骤:
(b1)以乙醇为底物经yqhD蛋白催化反应生成乙醛;
(b2)以乙醛和甲醛作为底物,经DERA蛋白催化反应生成3-羟基丙醛;
(b3)以3-羟基丙醛作为底物,经yqhD蛋白催化反应生成1,3-丙二醇;
所述DERA蛋白为前文(A1)-(A4)任一所示蛋白;所述yqhD蛋白为前文(B1)-(B4)任一所示蛋白。
在所述方法中,所述DERA蛋白和所述yqhD蛋白均可以粗酶液、粗酶液冻干粉、纯酶或全细胞的形式发生催化作用。
进一步,所述粗酶液、粗酶液冻干粉和纯酶均可按照包括如下步骤的方法制备得到:在宿主细胞中表达所述DERA蛋白和/或所述yqhD蛋白,得到重组细胞;裂解所述重组细胞获得所述粗酶液、粗酶液冻干粉或纯酶。所述全细胞均按照包括如下步骤的方法制备得到:在宿主细胞中表达所述DERA蛋白和/或所述yqhD蛋白,得到的重组细胞即为所述全细胞。
再进一步,所述重组细胞具体可按照包括如下步骤的方法制备获得:向所述宿主细胞导入能够表达所述DERA蛋白和/或所述yqhD蛋白的核酸分子,经诱导培养后获得表达所述DERA蛋白和/或所述yqhD蛋白的所述重组细胞。
更进一步,所述“能够表达所述DERA蛋白和/或所述yqhD蛋白的核酸分子”是通过重组载体的形式导入到所述宿主细胞中的。其中,所述重组载体可为携带有所述DERA蛋白和/或所述yqhD蛋白的编码基因的细菌质粒(如在细菌中表达的基于T7启动子的表达载体,具体如pET-28a等)、噬菌体、酵母质粒(如YEp系列载体等)或逆转录病毒包装质粒。
进一步地,所述宿主细胞可为原核细胞或低等真核细胞。
更进一步地,所述原核细胞具体可为细菌;所述低等真核细胞具体可为酵母细胞。
在本发明的一个实施例中,所述宿主细胞具体为大肠杆菌,更加具体的为E.coliBL21(DE3)。相应的,所述诱导培养为向培养体系中加IPTG至终浓度0.1-2mM(具体如0.5mmol/L),10℃-37℃(具体如16℃)诱导培养2-24小时(具体如16小时)。
在本发明中,所述方法I具体包括如下步骤:配制含有甲醛、乙醛、所述DERA蛋白和所述yqhD蛋白的反应体系,将所述反应体系于20℃-37℃(具体如37℃)反应0.5小时以上(具体如4h),然后从反应产物中获得1,3-丙二醇。
其中,将所述反应体系于20℃-37℃(具体如37℃)反应0.5小时以上(具体如4h),还包括60-100℃(具体如70℃)加热2min以上(具体如3min)使蛋白沉淀,后离心(如12000rpm离心25min)除去蛋白沉淀的步骤。
另外,所述方法I的反应体系中还可含有能够为反应外加还原力的物质以及NADPH。
在本发明中,所述能够为反应外加还原力的物质具体为葡萄糖-6-磷酸和葡萄糖-6-磷酸脱氢酶。反应过程中,葡萄糖-6-磷酸脱氢酶将葡萄糖-6-磷酸脱氢的过程会为所述yqhD蛋白催化3-羟基丙醛生成1,3-丙二醇的步骤提供还原力。
在所述方法中,所述反应可在pH为7-9的缓冲液中进行。
在本发明中,所述反应具体是在pH为8.0的50mM磷酸缓冲液中进行的。
更加具体地,所述反应体系组成如下:终浓度为1mg/ml的所述DERA蛋白;终浓度为0.5mg/mL的所述yqhD蛋白;终浓度为50mM的甲醛;终浓度为20mM的乙醛;终浓度为25mM的葡萄糖-6-磷酸,终浓度为1mM的NADPH;含量为5U的葡萄糖-6-磷酸脱氢酶;余量为pH为7-9的缓冲液(具体如pH为8.0的50mM磷酸缓冲液)。
在本发明中,所述方法II具体包括如下步骤:配制含有乙醇、甲醛、所述DERA蛋白和所述yqhD蛋白的反应体系,将所述反应体系于20℃-37℃(具体如37℃)反应0.5小时以上(具体如4h),然后从反应产物中获得1,3-丙二醇。
其中,将所述反应体系于20℃-37℃(具体如37℃)反应0.5小时以上(具体如4h),还包括60-100℃(具体如70℃)加热2min以上(具体如3min)使蛋白沉淀,后离心(如12000rpm离心25min)除去蛋白沉淀的步骤。
另外,所述方法II的反应体系中还可含有NADPH。
在所述方法中,所述反应可在pH为7-9的缓冲液中进行。
在本发明中,所述反应具体是在pH为8.0的50mM磷酸缓冲液中进行的。
更加具体地,所述反应体系组成如下:终浓度为1mg/ml的所述DERA蛋白;终浓度为0.5mg/mL的所述yqhD蛋白;终浓度为90mM的乙醇;终浓度为20mM的甲醛;终浓度为5mM的NADPH;余量为pH为7-9的缓冲液(具体如pH为8.0的50mM磷酸缓冲液)。
在本发明中,所述能够表达DERA蛋白的核酸分子为所述DERA蛋白的编码基因,具体可为如下任一:
(C1)SEQ ID No.3所示的DNA分子;
(C2)SEQ ID No.1所示的DNA分子;
(C3)在严格条件下与(C1)或(C2)限定的DNA分子杂交且编码所述DERA蛋白的DNA分子;
(C4)与(C1)-(C3)中任一限定的DNA序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同源性且编码所述DERA蛋白的DNA分子。
所述能够表达yqhD蛋白的核酸分子为所述yqhD蛋白的编码基因,具体可为如下任一:
(D1)SEQ ID No.4所示的DNA分子;
(D2)在严格条件下与(D1)限定的DNA分子杂交且编码所述yqhD蛋白的DNA分子;
(D3)与(D1)或(D2)限定的DNA序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同源性且编码所述yqhD蛋白的DNA分子。
本发明构建并实现了1,3-丙二醇的新途径,此途径不需要辅酶B12的参与,有潜力成为1,3-丙二醇工业生产的新方法。
附图说明
图1为pET-28a-Dera的质粒图谱。
图2为体外纯酶反应GC-MS检测结果。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
pET-28a由苏州鸿讯公司提供。
E.coli BL21(DE3)菌株为全式金公式产品,货号为CD601-03。
实施例1、DERA蛋白和yqhD蛋白的获得
一、DERA蛋白的获得
1、Dera基因获得
2-deoxyribose-5-phosphate aldolase简称DERA酶,在多种物种中存在,在短乳杆菌中发现Dera基因,其核苷酸序列为SEQ ID No.1,该基因可编码DERA蛋白,其氨基酸序列为SEQ ID No.2,在不改变DERA氨基酸序列的前提下,将上述野生型Dera基因的密码子替换为大肠杆菌偏好(高频使用)的密码子,经密码子优化后,得到优化后Dera基因序列,具有大肠杆菌偏爱密码子,其基因序列为SEQ ID No.3。
2、表达载体的构建
将SEQ ID No.3所示的优化后Dera基因克隆到pET-28a载体(Novagen,Kan+,)酶切位点NdeI和XhoI之间的DNA片段,得到重组质粒,命名为pET-28a-Dera(图1)。
pET-28a-Dera的结构描述:将pET-28a载体酶切位点NdeI和XhoI之间的小片段替换为SEQ ID No.3所示DNA片段后得到的重组质粒。
3、基因的表达
为了体外检测DERA酶活性,在大肠杆菌中对该酶进行外源表达及纯化。
(1)将大肠杆菌表达型重组质粒pET-28a-Dera转入E.coli BL21(DE3)中,获得重组菌。采用卡那霉素抗性平板进行阳性克隆筛选(Kan+,100mg/mL),37℃过夜培养;
(2)挑取单克隆至5mL LB液体培养基中(Kan+,100mg/mL),37℃、220r/min培养至OD600为0.6-0.8。将5mL LB培养基中菌液转接至800mL 2YT培养基中(Kan+,100mg/mL),37℃、220rpm培养至OD600为0.6-0.8时,降温至16℃,加IPTG至终浓度0.5mM,诱导表达16h;
(3)将上述培养菌液收集到收菌瓶中,5500r/min离心10min;
(4)弃上清,用35mL蛋白缓冲液(pH为8.0的50mM磷酸缓冲液)将所得菌体沉淀悬起,倒入50mL离心管中,-80℃冰箱保存。
4、蛋白纯化
(1)破菌:采用高压低温破碎仪,在压力1200bar,4℃条件下对于上述步骤3得到的菌体沉淀破菌2次。4℃、10000r/min离心45min,取离心后的沉淀、上清,制样。
(2)纯化:上清液经0.45μm微孔滤膜抽滤,进行镍亲和层析纯化,具体步骤如下:
a:柱平衡:挂上清前,先用ddH2O洗2个柱体积,再用蛋白缓冲液(配方同上)平衡Ni亲和层析柱1个柱体积。
b:上样:将上清按0.5mL/min流速缓慢经过Ni亲和层析柱,再重复一次。
c:洗脱杂蛋白:采用蛋白缓冲液(配方同上)冲洗1个柱体积,再用50mL含50mM咪唑的蛋白缓冲液(配方同上)去洗脱结合较强的杂蛋白,取前几滴流穿样品,制样。
d:洗脱目的蛋白:分别用20mL含100mM,200mM,300mM咪唑的蛋白缓冲液(配方同上)将目的蛋白洗脱下来,取前几滴流穿样品,制样,12%SDS-PAGE检测。
(3)浓缩换液:将收集到的目的蛋白用50mL Amicon超滤管(10kDa,Millipore公司)离心浓缩(4℃、3500r/min),浓缩至1mL。加15mL蛋白缓冲液,浓缩至1mL,重复该过程1次,得到纯化的蛋白DERA。
(4)用BCA蛋白浓度检测试剂盒检测浓缩后蛋白浓度,为20mg/mL。即得到纯化浓缩的DERA蛋白,其氨基酸序列为SEQ ID No.2。
二、yqhD蛋白的获得
本发明实验中用到的1.3-丙二醇氧化还原酶基因yqhD来源于大肠杆菌。采用PCR技术以Escherichia coli str.K-12substr.MG1655菌株基因组为模板扩增得到yqhD基因,其核苷酸序列为SEQ ID No.4,将该基因克隆到pET-28a载体上,该基因编码的蛋白为yqhD蛋白,其氨基酸序列为SEQ ID No.5,其表达载体构建及基因表达和蛋白纯化均同步骤一。此处不再赘述。
实施例2、DERA蛋白和yqhD蛋白在合成1.3-丙二醇中的应用
一、DERA参与的催化甲醛及乙醇缩合合成1.3-丙二醇
反应方程式如下:
二、体外DERA纯酶功能验证
空白对照反应体系(500μL):50mM甲醛及20mM乙醛,25mM葡糖糖-6-磷酸,1mMNADPH,5U葡糖糖-6-磷酸脱氢酶,和蛋白缓冲液(50mM磷酸盐,pH=8)。
DERA反应体系(500μL):1mg/ml实施例1制备纯化的DERA蛋白及0.5mg/mL实施例1制备纯化的yqhD蛋白,50mM甲醛及20mM乙醛,25mM葡糖糖-6-磷酸,1mM NADPH,5U葡糖糖-6-磷酸脱氢酶,和蛋白缓冲液(50mM磷酸盐,pH=8)。
将上述各组反应体系混匀后于37℃反应4h。反应结束后,70℃加热3min使蛋白沉淀,后于12000rpm下离心25min除去蛋白沉淀,取出上清进行液相检测。检测到1.3丙二醇。
检测方法如下:
采用Aminex HPX-87H色谱柱;
进样量:10μl;
柱温:40℃;
流动相:5mM稀硫酸;
流速:0.6mL/min。
然后将上清样品冷冻干燥除水衍生后进行GC-MS检测。结果如图2所示。图2中,A为无DERA蛋白对照气相色谱图;B为实验组气相色谱图;C为实验组二级质谱图。由图2可见,在DERA蛋白和yqhD蛋白存在的情况下,甲醛和乙醛能够转化为1.3-丙二醇。
气相色谱色谱条件:
进样量:1μL;
进样口温度:250℃;
分流比10:1;
载气流量:1.2mL/min;
程序升温条件:起始温度60℃,维持一分钟,以5℃/min升到100℃,以25℃/min升到300℃,维持5min。
质谱条件为:
离子源:EI,离子源温度:250℃,发射电流:9.6μA,电离能:70ev,溶剂延迟:3min,质量范围:50-450amu。
三、DERA联合yqhD参与的催化甲醛及乙醇缩合合成1.3-丙二醇
空白对照反应体系(500μL):20mM甲醛及90mM乙醇,5mM NADPH,和蛋白缓冲液(50mM磷酸盐,pH=8)。
混合酶反应体系(500μL):1mg/ml实施例1制备纯化的DERA蛋白及0.5mg/mL实施例1制备纯化的yqhD蛋白,20mM甲醛及90mM乙醇,5mM NADPH,和蛋白缓冲液(50mM磷酸盐,pH=8)。
将上述各组反应体系混匀后于37℃反应4h。反应结束后,70℃加热3min使蛋白沉淀,后于12000rpm下离心25min除去蛋白沉淀,取出上清进行液相检测,检测到1.3丙二醇。
检测方法如下:
采用Aminex HPX-87H色谱柱;
进样量:10μl;
柱温:40℃;
流动相:5mM稀硫酸;
流速:0.6mL/min。
<110> 中国科学院天津工业生物技术研究所
<120> 一种利用生物酶合成1,3-丙二醇的方法
<130> GNCLN172325
<160> 5
<170> PatentIn version 3.5
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<213> 短乳杆菌(Lactobacillus breris)
<400> 1
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cggaatccaa taggagttta cgcagaccga agccgtgtta aatttcttag cttcgtcaca 600
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gaagctacca ccgctatcga ccagggtgct gaagaaatcg acatggttct gaacgttggt 300
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Claims (17)
1.DERA蛋白或所述DERA蛋白相关生物材料在合成1,3-丙二醇中的应用;
所述DERA蛋白为如下任一所示蛋白质:
(A1)氨基酸序列为SEQ ID No.2的蛋白质;
(A2)在(A1)所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白;
所述DERA蛋白相关生物材料为能够表达所述DERA蛋白的核酸分子,或含有所述核酸分子的表达盒、重组载体、重组菌或转基因细胞系。
2.(1)和(2)在合成1,3-丙二醇中的应用;
(1)DERA蛋白或所述DERA蛋白相关生物材料;
(2)yqhD蛋白或所述yqhD蛋白相关生物材料;
所述DERA蛋白为如下任一所示蛋白质:
(A1)氨基酸序列为SEQ ID No.2的蛋白质;
(A2)在(A1)所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白;
所述DERA蛋白相关生物材料为能够表达所述DERA蛋白的核酸分子,或含有所述核酸分子的表达盒、重组载体、重组菌或转基因细胞系;
所述yqhD蛋白为如下任一所示蛋白质:
(B1)氨基酸序列为SEQ ID No.5的蛋白质;
(B2)在(B1)所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白;
所述yqhD蛋白相关生物材料为能够表达所述yqhD蛋白的核酸分子,或含有所述核酸分子的表达盒、重组载体、重组菌或转基因细胞系。
3.一种合成的1,3-丙二醇方法,包括如下步骤:
(a1)以甲醛和乙醛作为底物,经DERA蛋白催化反应生成3-羟基丙醛;
(a2)以3-羟基丙醛作为底物,经yqhD蛋白催化反应生成1,3-丙二醇;
所述DERA蛋白为如下任一所示蛋白质:
(A1)氨基酸序列为SEQ ID No.2的蛋白质;
(A2)在(A1)所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白;
所述yqhD蛋白为如下任一所示蛋白质:
(B1)氨基酸序列为SEQ ID No.5的蛋白质;
(B2)在(B1)所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白。
4.一种合成的1,3-丙二醇方法,包括如下步骤:
(b1)以乙醇为底物经yqhD蛋白催化反应生成乙醛;
(b2)以乙醛和甲醛作为底物,经DERA蛋白催化反应生成3-羟基丙醛;
(b3)以3-羟基丙醛作为底物,经yqhD蛋白催化反应生成1,3-丙二醇;
所述DERA蛋白为如下任一所示蛋白质:
(A1)氨基酸序列为SEQ ID No.2的蛋白质;
(A3)在(A1)所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白;
所述yqhD蛋白为如下任一所示蛋白质:
(B1)氨基酸序列为SEQ ID No.5的蛋白质;
(B2)在(B1)所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白。
5.根据权利要求3或4所述的方法,其特征在于:在所述方法中,所述DERA蛋白和所述yqhD蛋白均是以粗酶液、粗酶液冻干粉、纯酶或全细胞的形式发生催化作用的。
6.根据权利要求5所述的方法,其特征在于:所述粗酶液、粗酶液冻干粉和纯酶均按照包括如下步骤的方法制备得到:在宿主细胞中表达所述DERA蛋白和/或所述yqhD蛋白,得到重组细胞;裂解所述重组细胞获得所述粗酶液、粗酶液冻干粉或纯酶。
7.根据权利要求5所述的方法,其特征在于:所述全细胞均按照包括如下步骤的方法制备得到:在宿主细胞中表达所述DERA蛋白和/或所述yqhD蛋白,得到的重组细胞即为所述全细胞。
8.根据权利要求7所述的方法,其特征在于:所述重组细胞是按照包括如下步骤的方法制备获得的:向所述宿主细胞导入能够表达所述DERA蛋白和/或所述yqhD蛋白的核酸分子,经诱导培养后获得表达所述DERA蛋白和/或所述yqhD蛋白的所述重组细胞。
9.根据权利要求8所述的方法,其特征在于:所述“能够表达所述DERA蛋白和/或所述yqhD蛋白的核酸分子”是通过重组载体的形式导入到所述宿主细胞中的;所述重组载体为携带有所述DERA蛋白和/或所述yqhD蛋白的编码基因的细菌质粒、噬菌体、酵母质粒或逆转录病毒包装质粒。
10.根据权利要求7所述的方法,其特征在于:所述宿主细胞为原核细胞或低等真核细胞。
11.根据权利要求10所述的方法,其特征在于:所述原核细胞为细菌;所述低等真核细胞为酵母细胞。
12.根据权利要求11所述的方法,其特征在于:所述细菌为大肠杆菌。
13.根据权利要求3所述的方法,其特征在于:所述方法包括如下步骤:配制含有甲醛、乙醛、所述DERA蛋白和所述yqhD蛋白的反应体系,将所述反应体系于20-37℃反应0.5h以上,然后从反应产物中获得1,3-丙二醇。
14.根据权利要求13所述的方法,其特征在于:所述反应体系中还含有能够为反应外加还原力的物质以及NADPH。
15.根据权利要求4所述的方法,其特征在于:所述方法包括如下步骤:配制含有乙醇、甲醛、所述DERA蛋白和所述yqhD蛋白的反应体系,将所述反应体系于20-37℃反应0.5h以上,然后从反应产物中获得1,3-丙二醇。
16.根据权利要求15所述的方法,其特征在于:所述反应体系中还含有NADPH。
17.根据权利要求3或4所述的方法,其特征在于:所述能够表达DERA蛋白的核酸分子为所述DERA蛋白的编码基因,为如下任一:
(C1)SEQ ID No.3所示的DNA分子;
(C2)SEQ ID No.1所示的DNA分子;
所述能够表达yqhD蛋白的核酸分子为所述yqhD蛋白的编码基因,为SEQ ID No.4所示的DNA分子。
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