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.

1,4-Butanediol (1,4-BDO) is currently produced from succinate via six enzymatic reactions in an engineered Escherichia coli strain. Butyraldehyde dehydrogenase (Bld) and butanol dehydrogenase of Clostridium saccharoperbutylacetonicum were selected based on their activities of catalyzing the final two reactions in the 1,4-BDO pathway. To fit Bld into the non-natural 1,4-BDO pathway, we engineered it through random mutagenesis. Five Bld mutants were then isolated using a colorimetric Schiff's reagent-based method. Subsequent site-directed mutagenesis of Bld generated the two best Bld mutants, L273I and L273T, which produced 1,4-BDO titers fourfold greater than those of wild-type Bld. The enhanced 1,4-BDO titers obtained using L273I and L273T clearly correlated with their enhanced activities, which were caused by amino acid mutations at position 273 of Bld. The highest titer of 1,4-BDO (660 ± 40 mg/L) was obtained in a knock-out E. coli strain [ΔldhA ΔpflB ΔadhE ΔlpdA::K. lpd(E354K) Δmdh ΔarcA gltA(R164L)] coexpressing Bld273T+Bdh.

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.

Chemically synthesized retinyl palmitate has been widely used in the cosmetic and biotechnology industry. In this study, we aimed to demonstrate the microbial production of retinyl palmitate and the benefits of microbial retinyl palmitate in skin physiology. A heterologous retinyl palmitate biosynthesis pathway was reconstructed in metabolically engineered Escherichia coli using synthetic expression modules from Pantoea agglomerans, Salinibacter ruber, and Homo sapiens. High production of retinyl palmitate (69.96 ± 2.64 mg/L) was obtained using a fed-batch fermentation process. Moreover, application of purified microbial retinyl palmitate to human foreskin HS68 fibroblasts led to increased cellular retinoic acid-binding protein 2 (CRABP2) mRNA level [1.7-fold (p = 0.001) at 100 μg/mL], acceleration of cell proliferation, and enhancement of procollagen synthesis [111% (p < 0.05) at 100 μg/mL], strongly indicating an anti-ageing-related effect of this substance. These results would pave the way for large-scale production of retinyl palmitate in microbial systems and represent the first evidence for the application of microbial retinyl palmitate as a cosmeceutical.

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