Application of an Oxygen-Inducible nar Promoter System in Metabolic Engineering for Production of Biochemicals in Escherichia coli

Keywords: nar promoter, lactate, 1,3-propandiol, 2,3-butanediol

Abstract

The nar promoter, a dissolved oxygen (DO)-dependent promoter in Escherichia coli, is simply induced and functional in any cell growth phase, which is advantageous for producing biochemicals and fuels on an industrial scale. To demonstrate the feasibility of using the nar promoter in metabolic engineering for biochemicals and biofuels in E. coli, three target pathways were examined: the D-lactate, 2,3-butanediol (2,3-BDO), and 1,3-propanediol (1,3-PDO) pathways, consisting of one, three, and six genes, respectively. Each pathway gene was expressed under the control of the nar promoter. When the ldhD gene was expressed in fed-batch culture, the titer, yield, and productivity of D-lactate were 113.12 ± 2.37 g/L, 0.91 ± 0.07 g/g-glucose, and 4.19 ± 0.09 g/L/h, respectively. When three 2,3-BDO pathway genes (ilvBN, aldB, bdh1) were expressed in fed-batch culture, the titer, yield, and productivity of (R,R)-2,3-BDO were 48.0 ± 8.48 g/L, 0.43 ± 0.07 g/g-glucose, and 0.76 ± 0.13 g/L/h, respectively. When six 1,3-PDO pathway genes (dhaB1B2B3, yqhD, gdrA, and gdrB) were expressed in fed-batch culture, the titer, yield, and productivity of 1,3-PDO were 15.8 ± 0.62 g/L, 0.35 ± 0.01 g/g-glycerol, and 0.25 ± 0.01 g/L/h, respectively. Based on the reasonable performance, comparable to that observed in previous studies using different promoters, the nar promoter can serve as a controlled expression tool for developing microbial systems to efficiently produce biochemicals and biofuels.

Introduction

Promoters regulate the transcription and translation of genes, controlling metabolic pathway flux. In metabolic engineering, several inducible promoters such as araBAD, tac, lac, trp, T7, λPL, and λPR are widely used to tightly control protein expression in E. coli. However, these promoters have limitations. Chemically induced promoters, including araBAD, lac, and T7, are costly, can cause toxicity due to overexpression, and may suppress glucose concentration. Thermally induced promoters, such as λPL and λPR, can be difficult to control precisely and may cause adverse cellular responses due to temperature shifts.

The DO-dependent nar promoter is an alternative, offering simple, cost-effective induction and functionality in any cell growth phase. The nar promoter, located upstream of the narGHIJ operon encoding nitrate reductase, is induced by both anaerobic conditions and the presence of nitrate. This operon is regulated by two proteins: FNR (fumarate and nitrate reductase regulator) and NarL (nitrate/nitrite response regulator). FNR binds upstream of the transcription start site, and NarL increases expression in the presence of nitrate. A modified nar promoter, with altered -10 and -35 sequences, has been constructed for maximal induction under anaerobic conditions without nitrate.

Previous work utilized the nar promoter to express various proteins, but not in metabolic engineering pathways for biochemical or biofuel production. Since many such products are generated under anaerobic or microaerobic conditions, the nar promoter system’s feasibility for these applications warranted investigation.

Results and Discussion
Induction and Functionality of the nar Promoter

The nar promoter’s induction depends on both DO level and nitrate concentration. Using GFPm as a reporter, induction was significantly higher under low DO conditions, with or without nitrate. In bioreactor cultivation, lowering DO from >80% to 1% induced GFPm expression within 1 hour, which persisted through the stationary phase. This long-lasting, growth-phase-independent expression is a key advantage for multi-step pathway control.

Application to D-Lactate Production

The ldhD gene encoding D-lactate dehydrogenase from Leuconostoc citreum was expressed under the nar promoter in E. coli W023. In flask culture with 20 g/L glucose, D-lactate titer, yield, and productivity were 17.1 ± 0.4 g/L, 0.86 ± 0.02 g/g-glucose, and 0.57 ± 0.02 g/L/h, respectively. In DO-controlled batch bioreactor, the titer reached 19.09 ± 2.36 g/L at 10 h, with a yield of 0.90 ± 0.05 g/g-glucose and productivity of 1.91 ± 0.24 g/L/h. In fed-batch fermentation, 113.1 ± 2.37 g/L D-lactate was obtained at 27 h, with a yield of 0.91 ± 0.07 g/g-glucose and productivity of 4.19 ± 0.09 g/L/h. Acetate was the main by-product.

Application to 2,3-Butanediol Production

Three genes (ilvBN from E. coli, aldB from L. lactis, bdh1 from S. cerevisiae) were placed under individual nar promoters. In flask culture, (R,R)-2,3-BDO titer, yield, and productivity were 5.13 ± 0.43 g/L, 0.37 ± 0.05 g/g-glucose, and 0.21 ± 0.02 g/L/h, respectively. In batch bioreactor, titer reached 9.73 ± 0.28 g/L at 17 h, with a yield of 0.48 ± 0.01 g/g-glucose and productivity of 0.48 ± 0.01 g/L/h. In fed-batch, the highest titer was 48.0 ± 8.48 g/L, with a yield of 0.43 ± 0.07 g/g-glucose and productivity of 0.76 ± 0.13 g/L/h. Succinate and acetate were detected as by-products.

Application to 1,3-Propanediol Production

Six genes (dhaB1, dhaB2, dhaB3, yqhD, gdrA, gdrB) were expressed under the nar promoter (with dhaB2 and dhaB3 as an operon). In flask culture with 10 g/L glycerol, 1,3-PDO titer and yield were 1.02 ± 0.05 g/L and 0.10 ± 0.01 g/g-glycerol, respectively. In DO-controlled fed-batch fermentation, 15.8 ± 0.62 g/L 1,3-PDO was produced at 63 h, with a yield of 0.35 ± 0.01 g/g-glycerol and productivity of 0.25 ± 0.01 g/L/h. Succinate and acetate were also produced.

Summary

The nar promoter enables cost-effective, growth-phase-independent, and long-lasting induction for the controlled expression of single- and multi-gene pathways in E. coli. The system achieved high titers, yields, and productivities for D-lactate, 2,3-BDO, and 1,3-PDO, comparable to or exceeding those obtained with other commonly used promoters. Further improvements could be achieved by optimizing host strains and engineering nar promoters of varying strengths.

Materials and Methods
Strains and Plasmids

E. coli TOP10 was used for cloning and plasmid maintenance.W023 strain was used as the host for biochemical production.Plasmids with nar promoter sequences were constructed by replacing the lac promoter in pUCM with a synthetic nar promoter. Reporter (gfpm) and pathway genes were cloned downstream of the nar promoter.Genes for D-lactate, 2,3-BDO, and 1,3-PDO pathways were PCR-amplified from various sources and cloned into appropriate vectors, some using the USER cloning method.

Media and Cultivation

Seed cultures were grown in LB medium with antibiotics.Flask cultivations for biochemical production were performed in LB with 20 g/L glucose and appropriate antibiotics. For D-lactate, 10 g/L CaCO₃ was added for pH control.Cultures were grown aerobically to OD600 of 1.0 at 30°C, then shifted to microaerobic conditions (reduced shaking) to induce the nar promoter.Bioreactor fermentations were conducted in modified R (MR) medium with glucose, yeast extract, and antibiotics. DO was reduced from ~80% to 1-2% to induce the nar promoter. Fed-batch fermentations involved feeding concentrated solutions when substrate levels dropped.

Protein and Metabolite Analysis

Proteins were extracted, quantified, and analyzed by SDS-PAGE.GFPm fluorescence was measured by plate reader and flow cytometry.Metabolites (glucose, glycerol, D-lactate, acetate, succinate, 1,3-PDO, 2,3-BDO) were quantified by HPLC. Optical purity and stereoisomer composition were determined by enzymatic assay and GC-MS, respectively.

Conclusions

The nar promoter is a robust, oxygen-inducible system for controlled gene expression in E. coli, suitable for metabolic engineering of both single- and multi-gene pathways. Its simple induction, cost-effectiveness,3BDO and sustained expression make it a valuable tool for industrial-scale production of biochemicals and biofuels.