Application of an oxygen‐inducible nar promoter system in metabolic engineering for production of biochemicals in Escherichia coli

Abstract

The nar promoter, a dissolved oxygen (DO)-dependent promoter in Escherichia coli, is simply induced and functional in any cell growth phase, which are advantageous for producing biochemicals/fuels on an industrial scale. To demonstrate the feasibility of using the nar promoter in the metabolic engineering of biochemicals/biofuels in E. coli, three target pathways were examined: the d-lactate, 2,3-butandiol (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 in metabolic engineering, the nar promoter can serve as a controlled expression tool for developing a microbial system to efficiently produce biochemicals and biofuels. Biotechnol. Bioeng. 2017;114: 468-473. © 2016 Wiley Periodicals, Inc.

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

“…lpdE354 K Δmdh ΔarcA gltAR164L [ 13 , 21 ] Lactobacillus citreum Source for ldhD KCTC3721 Lactococcus lactis subsp. lactis Source for aldB KCTC3899 Saccharomyces cerevisiae S288c Source for bdh1 ATCC 204508 Plasmids pUCM Cloning vector modified from pUC19; constitutive lac promoter, Ap R [ 23 ] pUCM- gfpm Constitutive expressed gfpm gene with lac promoter This study pUCN Cloning/expression vector having inducible wild-type nar promoter, Amp R [ 13 ] pUCN- gfpm Inducible expressed gfpm gene with nar promoter This study pQE- gfpm Inducible expressed gfpm gene with T5 promoter [ 22 ] pUCNr Cloning/expression vector having rop gene and wild-type nar promoter, Amp R This study pUCNrS Cloning/expression vector having rop gene and strong nar promoter (S3-2-64), Amp R This study pUCNrI Cloning/expression vector having rop gene and intermediate nar promoter (W2U-30), Amp R This study pUCNrW Cloning/expression vector having rop gene and weak nar promoter (W2L-29), Amp R …”

“…The ldhD gene encoding d -lactate dehydrogenase from L. citreum was cloned into pUCNrS, pUCNrI, pUCNrW, and pUCN (Table 1 ), to be expressed under the control of the four nar promoters: strong, intermediate, weak, and wild-type. The four ldhD gene-expression plasmids (NrSL, strong; NrIL, intermediate; NrWL, weak; NrL, wild-type) were transduced into E. coli , and then the four recombinant E. coli strains were microaerobically grown in flasks containing 20 g/L glucose as a carbon source [ 13 ]. After a 20-h cultivation, d -lactate titers were measured to be 18.6 ± 0.6 in E. coli having NrL, 18.7 ± 0.4 in NrSL, 18.5 ± 0.4 in NrIL, and 18.3 ± 0.1 in NrWL (Fig.…”

“…Because d -lactate titers were similar in all four E. coli strains, two E. coli strains having NrL and NrSL were chosen and fed-batch fermentation with glucose as a carbon source was carried out to investigate the effect of the strength of nar promoters (strong vs. wild-type) on d -lactate production. When the DO-controlled fed-batch fermentation of E. coli strain having NrSL was carried out as described in our previous study [ 13 ], 105.6 g/L of d -lactate was obtained after a 23-h cultivation. The d -lactate yield and productivity were 0.71 g/g-glucose and 4.59 g/L/h, respectively (Fig.…”

“…Recently, 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 ]. However, when a multienzyme biosynthetic pathway was reconstructed in heterologous host cells, individual expression of each pathway enzyme needed to be finely controlled; assembly or organization of multienzyme systems could significantly influence metabolic channeling, and thus suboptimal assembly or organization would cause accumulation of unwanted metabolic intermediates in multi-step enzyme reactions [ 14 , 15 ].…”

“…Compared to other commonly used strong promoters such as lac or araBAD , the intensity of the wild-type nar promoter is relatively weak [ 13 ]; therefore, engineering of the wild-type nar promoter was required for fine control of target pathway gene expression. In this study, in order to generate synthetic nar promoters of diverse strengths, a synthetic nar promoter library was constructed by randomization of the spacer region sequence (15 bp) located between the consensus sequence − 35 and − 10 elements of the wild-type nar promoter (Fig.…”