Engineering and application of synthetic nar promoter for fine-tuning the expression of metabolic pathway genes in Escherichia coli

Sang Yup Lee,Hee Jin Hwang,Pyung Cheon Lee

doi:10.1186/s13068-018-1104-1

Abstract

BackgroundPromoters regulate the expression of metabolic pathway genes to control the flux of metabolism. Therefore, fine-tuning of metabolic pathway gene expression requires an applicable promoter system. In this study, a dissolved oxygen-dependent nar promoter was engineered for fine-tuning the expression levels of biosynthetic pathway enzymes in Escherichia coli. To demonstrate the feasibility of using the synthetic nar promoters in production of biochemicals in E. coli, the d-lactate pathway consisting of one enzyme and the 2,3-butanediol (BDO) pathway consisting of three enzymes were investigated.ResultsThe spacer sequence of 15 bp between the − 35 and − 10 elements of the upstream region of the wild-type nar promoter was randomized, fused to the GFP gene, transduced into E. coli, and screened by flow cytometry. The sorted synthetic nar promoters were divided into three groups according to fluorescence intensity levels: strong, intermediate, and weak. The selected three representative nar promoters of strong, intermediate, and weak intensities were used to control the expression level of the d-lactate and 2,3-BDO biosynthetic pathway enzymes in E. coli. When the ldhD gene encoding d-lactate dehydrogenase was expressed under the control of the strong synthetic nar promoter in fed-batch cultures of E. coli, the d-lactate titers were 105.6 g/L, 34% higher than those using the wild-type promoter (79.0 g/L). When the three 2,3-BDO pathway genes (ilvBN, aldB, and bdh1) were expressed under the control of combinational synthetic nar promoters (strong–weak–strong) in fed-batch cultures of E. coli, the titers of 2,3-BDO were 88.0 g/L, 72% higher than those using the wild-type promoter (51.1 g/L).ConclusionsThe synthetic nar promoters, which were engineered to have strong, intermediate, and weak intensities, were successfully applied to metabolic engineering of d-lactate and 2,3-BDO pathways in E. coli. By controlling expression levels of d-lactate and 2,3-BDO pathway enzymes using the synthetic nar promoters, the production of d-lactate and 2,3-BDO was increased over that using the wild-type promoter by 34 and 72%, respectively. Thus, this synthetic promoter module system will support the improved production of biochemicals and biofuels through fine-tuning of gene expression levels.

Highlights

Promoters regulate the expression of metabolic pathway genes to control the flux of metabolism
We demonstrated that the production of d-lactate and 2,3-BDO was improved by tuning the expression of their pathway genes under three different strengths of the synthetic nar promoters in E. coli
The randomized synthetic promoters were fused by PCR to a DNA fragment consisting of a Shine–Dalgarno sequence, spacer, and His6-tagged GFPm as a reporter protein for screening based on the fluorescence intensity of the expressed GFPm (Fig. 1a)

Summary

Introduction

Promoters regulate the expression of metabolic pathway genes to control the flux of metabolism. Fine-tuning of metabolic pathway gene expression requires an applicable promoter system. A dissolved oxygen-dependent nar promoter was engineered for fine-tuning the expression levels of biosynthetic pathway enzymes in Escherichia coli. To demonstrate the feasibility of using the synthetic nar promoters in production of biochemicals in E. coli, the d-lactate pathway consisting of one enzyme and the 2,3-butanediol (BDO) pathway consisting of three enzymes were investigated. A dissolved oxygen (DO)-dependent nar promoter was successfully applied to express the d-lactate, 2,3-butanediol (BDO), and 1,3-propanediol (PDO) pathway enzymes in E. coli [13]. Even single-enzyme metabolic pathways should be considered for finetuning of expression, because expression level frequently affects end-product formation due to inclusion body formation [16]

Methods

Results

Discussion

Conclusion