CN116904382B - 联产二羟基丙酮和波色因的宿主细胞及方法 - Google Patents
联产二羟基丙酮和波色因的宿主细胞及方法 Download PDFInfo
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Abstract
本发明公开了一种联产二羟基丙酮和波色因的宿主细胞,其中所述宿主细胞进行了如下工程改造:引入了编码NADPH依赖性甲基乙二醛还原酶GRE2的基因,以及编码甘油脱氢酶GDH的基因。还公开了联产二羟基丙酮和波色因的方法。本发明的宿主菌共表达甘油脱氢酶、波色因还原酶和辅因子转化酶,并敲除sthA基因,从而实现辅因子NADPH和NAD+的循环利用。以甘油和1‑C‑(β‑D‑吡喃木糖基)‑丙酮为底物,通过控制甘油的量,最终达到同时高产二羟基丙酮和波色因的目的。这种方法合成玻色因和二羟基丙酮具有产量高、效率快、成本低和安全性好等优势,适用于玻色因和二羟基丙酮的工业化生产。
Description
技术领域
本发明涉及一种联产二羟基丙酮和波色因的宿主细胞及方法,涉及生物合成技术领域。
背景技术
近年来,随着人们的物质生活越来越丰富,对美的追求也成为日常消费的主流。因此,美妆护肤市场在整个市场中占据了越来越重要的地位。二羟基丙酮和玻色因是美妆市场的两大重要原材料,其生物酶法的合成技术具有效率高、成本低、无毒无害、经济价值高、产品质量高等一系列的优点,因此具有重要的市场价值。
二羟基丙酮(dihydroxyacetone,DHA)是一种有机化合物,无色至浅黄色的液体,具有甜味。在美容行业中,二羟基丙酮被广泛地用于制作自然的晒黑效果,因为它能够与皮肤表面的角质层反应,从而使皮肤看起来更显棕褐色。此外,它还被用于食品、医药和化学制品中。甘油脱氢酶是一种酶,它可以催化甘油的氧化反应。在这个反应中,甘油被氧化为二羟基丙酮以及NADH(还原型辅酶)。通过这个反应,甘油脱氢酶可以合成二羟基丙酮。需要注意的是,甘油脱氢酶合成二羟基丙酮的反应需要有NAD+参与,而NAD+是一种辅酶,需要从其他代谢过程中得到。因此,甘油脱氢酶合成二羟基丙酮的速率和NAD+的浓度有关。
玻色因(Pro-Xylane),又名羟丙基四氢吡喃醇,常用于制备护肤品。它是一种天然多糖,主要来源于植物,如桦树、橡树和云杉等。玻色因可以促进胶原蛋白和弹性纤维的生成,从而改善皮肤的弹性和紧致度,同时还有助于提高皮肤的保湿能力。市面上的玻色因主要来源于植物提取或者化学合成,这些方法成本高、纯度低、操作复杂,且化学合成会导致非理想产物和分离困难等问题。目前玻色因的生物合成主要基于醛酮还原酶催化1-C-(β-D-吡喃木糖基)-丙酮生成,但是这种方法对辅酶的依赖性极强,且成本极高,限制了玻色因的产量和生产成本。
发明内容
本发明公开了一种联产二羟基丙酮和波色因的宿主细胞,其中宿主细胞进行了如下工程改造:引入了编码NADPH依赖性甲基乙二醛还原酶GRE2的基因,以及编码甘油脱氢酶GDH的基因。
优选的,所述宿主细胞还进行了如下工程改造:过表达pntAB基因。
优选的,所述宿主细胞还进行了如下工程改造:抑制sthA基因的表达。
优选的,所述NADPH依赖性甲基乙二醛还原酶GRE2的氨基酸序列如SEQ ID NO:1所示。
优选的,所述甘油脱氢酶GDH的氨基酸序列如SEQ ID NO:3所示。
优选的,pntAB基因的序列如SEQ ID NO:5所示。
优选的,通过CRISPR转座和crRNA的方式敲除sthA基因。
优选的,所述宿主细胞为大肠杆菌,尤其是大肠杆菌BL21、Rossetta、W3110或MG1655。
本发明还公开了一种质粒,构建有启动子介导的GRE2基因、GDH基因以及pntAB基因。质粒载体可以是Pet32a载体,启动子为T7-LacO启动子。
一种联产二羟基丙酮和波色因的方法,其特征在于其步骤包括:
(1)构建上述的宿主细胞;
(2)挑取宿主细胞单克隆,在培养基中进行培养;
(3)将1-C-(β-D-吡喃木糖基)-丙酮和甘油加入培养基中,继续培养,获得二羟基丙酮和波色因。
其中1-C-(β-D-吡喃木糖基)-丙酮可以使用木糖和氯丙酮聚合获得。
本发明构建了联产二羟基丙酮和波色因的宿主菌。在宿主菌中共表达甘油脱氢酶、波色因还原酶和辅因子转化酶,并敲除sthA基因,从而实现辅因子NADPH和NAD+的循环利用。以甘油和1-C-(β-D-吡喃木糖基)-丙酮为底物,通过控制甘油的量,最终达到同时高产二羟基丙酮和波色因的目的。这种方法合成玻色因和二羟基丙酮具有产量高、效率快、成本低和安全性好等优势,适用于玻色因和二羟基丙酮的工业化生产。
附图说明
图1玻色因还原酶Gre2催化模型。
图2 1-C-(β-D-吡喃木糖基)-丙酮合成模型。
图3玻色因还原酶Gre2催化生成玻色因结果。
图4甘油脱氢酶GDH催化模型。
图5Gre2-GDH催化生成玻色因和二羟基丙酮结果。
图6NAD+和NADPH再生体系模型。
图7NAD+和NADPH再生体系模型在Gre2-GDH联产玻色因和二羟基丙酮中的模型。
图8Donor序列的模型。
图9crRNA序列的模型。
具体实施方式
以下结合附图,通过实施例进一步说明本发明,但不作为对本发明的限制。以下提供了本发明实施方案中所使用的具体材料及其来源。但是,应当理解的是,这些仅仅是示例性的,并不意图限制本发明,与如下试剂和仪器的类型、型号、品质、性质或功能相同或相似的材料均可以用于实施本发明。下述实施例中所使用的实验方法如无特别说明,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。
实施例1
为了实现玻色因的合成,我们在大肠杆菌中引入了羰基还原酶AKR,来自毕赤酵母的NADPH依赖性甲基乙二醛还原酶GRE2广泛应用在各类醛酮类化合物的羰基还原上,因此我们尝试使用Gre2来实现1-C-(β-D-吡喃木糖基)-丙酮到玻色因的催化(见图1)。
玻色因还原酶Gre2的氨基酸序列见SEQ ID NO:1,经大肠杆菌密码子优化后得到SEQ ID NO:2。序列合成于广州艾基生物,并构建到pET28a上。构建好的质粒转染到BL21(DE3)中,涂布在含50mg/L卡那霉素的LB培养基上,37℃过夜培养挑取单克隆,于含50mg/L卡那霉素的LB液体培养基中培养至OD600值达到0.6,加入终浓度为0.05、0.1、0.2、0.5和1mM的IPTG,28℃诱导过夜。
1-C-(β-D-吡喃木糖基)-丙酮使用木糖和氯丙酮进行合成(见图2)。饱和木糖的DMF水溶液(含1M氢氧化钠)中加入1/10体积氯丙酮,在90℃反应过夜。1-C-(β-D-吡喃木糖基)-丙酮产量大概在88%。
将获得的1-C-(β-D-吡喃木糖基)-丙酮按照10%溶于PBS溶液中,加入到上述获得的菌株中,30℃反应12h,HPLC测定玻色因含量。结果见图3,玻色因还原酶Gre2能够将1-C-(β-D-吡喃木糖基)-丙酮还原成玻色因。尽管玻色因还原酶Gre2的表达量逐渐升高,玻色因的产量上升并不明显。我们推测在全细胞催化合成玻色因的过程中,NADPH的浓度不够导致了玻色因产量升高不明显。
实施例2
为了提升玻色因的产量,同时生产二羟基丙酮。我们在大肠杆菌中构建了甘油脱氢酶体系,催化甘油产二羟基丙酮,同时生产NADH(见图4)。我们使用的是大肠杆菌中NADH依赖性的甘油脱氢酶GDH,氨基酸序列见SEQ ID NO:3,经大肠杆菌密码子优化后得到SEQID NO:4。序列合成于广州艾基生物,并构建到pET32a上。构建好的质粒转染到实施例1中BL21(DE3)中,涂布在含50mg/L卡那霉素和氨苄霉素的LB培养基上,37℃过夜培养挑取单克隆,于含50mg/L卡那霉素和氨苄霉素的LB液体培养基中培养至OD600值达到0.6,加入终浓度为0.2mM的IPTG,28℃诱导过夜。将获得的1-C-(β-D-吡喃木糖基)-丙酮和甘油按照10%溶于PBS溶液中,加入到上述获得的菌株中,30℃反应12h,HPLC测定玻色因含量。结果见图5,加入甘油脱氢酶后,甘油可以被催化成二羟基丙酮,玻色因的产量有明显升高。
实施例3
为了提升玻色因和二羟基丙酮的产量。我们在大肠杆菌中构建了NAD+和NADPH再生的体系。首先,通过过表达pntAB(核苷酸序列见SEQ ID NO:5)来消耗NADH再生NADPH,同时消耗NADP+再生NAD+,同时来敲除可溶型吡啶核苷酸转氢酶sthA基因来抑制NADPH转化成NADH,以及抑制NAD+转化成NADP+(见图6)。这样可以在大肠杆菌体内提高NAD+和NADPH的浓度,来实现玻色因和二羟基丙酮底物浓度的提升(见图7)。为了解决这一问题,我们将T7-LacO启动子介导的GRE2、GDH、pntAB构建到Pet32a载体上(见图8)。同时在PSL1142载体(addgene)上插入crRNA-sthA和crRNA-IS168(见图9)(方法参考文献Zhang Y,Sun X,WangQ,Xu J,Dong F,Yang S,Yang J,Zhang Z,Qian Y,Chen J,Zhang J,Liu Y,Tao R,JiangY,Yang J,Yang S.Multicopy Chromosomal Integration Using CRISPR-AssociatedTransposases.ACS Synth Biol.2020Aug 21;9(8):1998-2008.doi:10.1021/acssynbio.0c00073.Epub 2020Jul 2.Erratumin:ACS Synth Biol.2020Oct 16;9(10):2860.PMID:32551502.)。将两个质粒转化到Rosetta(DE3)菌株中,涂布在含50mg/L卡那霉素和氨苄霉素的的LB培养基上,37℃过夜培养挑取单克隆,于含50mg/L卡那霉素和氨苄霉素的LB液体培养基中培养至OD600值达到0.6,加入终浓度为0.2mM的IPTG,28℃诱导过夜。菌株离心后,将获得的1-C-(β-D-吡喃木糖基)-丙酮和甘油按照10%V/V溶于PBS溶液中,加入到上述获得的菌株沉淀中,30℃反应12h,HPLC测定玻色因含量。插入NAD+和NADPH再生体系后,二羟基丙酮和玻色因的产量分别达到37.2g/L和55.1g/L。
Claims (7)
1.一种联产二羟基丙酮和波色因的宿主细胞,其中所述宿主细胞进行了如下工程改造:引入了编码NADPH依赖性甲基乙二醛还原酶GRE2的基因,以及编码甘油脱氢酶GDH的基因,其中GRE2的氨基酸序列如SEQ ID NO:1所示,GDH的氨基酸序列如SEQ ID NO:3所示,所述宿主细胞为大肠杆菌。
2.根据权利要求1所述的宿主细胞,其特征在于所述宿主细胞还进行了如下工程改造:过表达pntAB基因,其中pntAB基因的序列如SEQ ID NO:5所示。
3.根据权利要求2所述的宿主细胞,其特征在于所述宿主细胞还进行了如下工程改造:抑制sthA基因的表达。
4.根据权利要求3所述的宿主细胞,其特征在于通过CRISPR转座和crRNA的方式敲除sthA基因。
5.根据权利要求1-4中任一项所述的宿主细胞,其特征在于所述宿主细胞为大肠杆菌BL21、Rossetta、W3110或MG1655。
6.一种质粒,其特征在于构建有启动子介导的GRE2基因、GDH基因以及pntAB基因,其中GRE2的氨基酸序列如SEQ ID NO:1所示,GDH的氨基酸序列如SEQ ID NO:3所示,pntAB基因的序列如SEQ ID NO:5所示。
7.一种联产二羟基丙酮和波色因的方法,其特征在于其步骤包括:
(1)构建权利要求1-5中任一项所述的宿主细胞;
(2)挑取宿主细胞单克隆,在培养基中进行培养;
(3)将1-C-(β-D-吡喃木糖基)-丙酮和甘油加入培养基中,继续培养,获得二羟基丙酮和波色因。
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