以纤维二糖为底物利用重组大肠杆菌合成海藻糖

以纤维二糖为底物利用重组大肠杆菌合成海藻糖

李新，郑兆娟，岳泰稳，欧阳嘉*

（南京林业大学化学工程学院，江苏南京 210037）

摘 要：探讨以纤维二糖为底物合成海藻糖的可行性。首先克隆来自施氏假单胞菌A1501的otsA/otsB基因，外源导入Escherichia coli BL21（DE3）构建以葡萄糖为底物合成海藻糖的OtsAB途径。继而通过过表达E. coli本身的galU 基因增加海藻糖合成前体物质尿苷二磷酸（uridine diphosphate，UDP）-葡萄糖的含量，使海藻糖产量提高了3 倍。

通过添加井冈霉素抑制海藻糖的降解，进一步提高海藻糖的产量。在此基础上，过表达来自天然纤维素分解细菌Saccharophagus degradans的纤维二糖磷酸化酶基因cepA，使重组大肠杆菌具有利用纤维二糖产海藻糖的能力。利用该重组大肠杆菌全细胞催化生产海藻糖，底物为20 g/L纤维二糖，并添加0.05 mmol/L的井冈霉素抑制海藻糖降解时，48 h后可生成1.3 g/L的海藻糖。本研究利用重组大肠杆菌以纤维二糖为底物合成海藻糖，证实了以纤维二糖为底物合成海藻糖的可行性，为纤维二糖来源精细化学品的生产提供了新的思路。

关键词：纤维二糖；纤维二糖磷酸化酶；海藻糖；UDP-葡萄糖；OtsAB途径

Biosynthesis of Trehalose from Cellobiose by Recombinant Escherichia coli

LI Xin, ZHENG Zhaojuan, YUE Taiwen, OUYANG Jia*

(College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China) Abstract: This study aimed to explore the feasibility of synthesizing trehalose using cellobiose as substrate. Firstly, the otsA/otsB gene from Pseudomonas stutzeri A1501 was cloned and exogenously introduced into Escherichia coli BL21(DE3) to construct an OtsAB pathway to synthesize trehalose using glucose as the substrate. Subsequently, the content of uridine diphosphate (UDP)-glucose, the key precursor for biosynthesis of trehalose was increased by overexpressing the galU gene of E. coli itself, resulting in a 4-fold increase in trehalose production. By adding validamycin, the degradation of trehalose was suppressed to increase its yield. Furthermore, by overexpressing the cellobiose phosphorylase gene (cepA) from the naturally occurring cellulolytic bacterium Saccharophagus degradans, the recombinant E.coli was rendered capable of producing trehalose from cellobiose. After 48 h culture with the addition of 0.05 mmol/L validamycin against trehalose degradation, whole recombinant cells catalyzed the transformation of 20 g/L cellobiose to 1.3 g/L trehalose. This study confirms the feasibility of synthesizing trehalose using cellobiose as substrate and provides new ideas for producing fine chemicals from cellobiose.

Keywords: cellobiose; cellobiose phosphorylase; trehalose; UDP-glucose; OtsAB pathway

DOI:10.7506/spkx1002-6630-20180411-132

中图分类号：Q78 文献标志码：A 文章编号：1002-6630（2019）06-0180-07引文格式：

李新, 郑兆娟, 岳泰稳, 等. 以纤维二糖为底物利用重组大肠杆菌合成海藻糖[J]. 食品科学, 2019, 40(6): 180-186.

DOI:10.7506/spkx1002-6630-20180411-132.

LI Xin, ZHENG Zhaojuan, YUE Taiwen, et al. Biosynthesis of trehalose from cellobiose by recombinant Escherichia coli[J]. Food Science, 2019, 40(6): 180-186. (in Chinese with English abstract) DOI:10.7506/spkx1002-6630-20180411-132.

收稿日期：2018-04-11

基金项目：江苏省高校自然科学研究重大项目（16KJA220004）；江苏省高校优势学科建设工程资助项目（PAPD）

第一作者简介：李新（1994—）（ORCID: 0000-0002-3520-8931），男，硕士研究生，研究方向为生物化工。

E-mail: 1633670510@

*通信作者简介：欧阳嘉（1972—）（ORCID: 0000-0002-3467-253X），女，教授，博士，研究方向为生物化工。

E-mail: hgouyj@