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

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.

Biosynthesis of plant-specific stilbene polyketides in metabolically engineered Escherichia coli.

BACKGROUND: Phenylpropanoids are the precursors to a range of important plant metabolites such as the cell wall constituent lignin and the secondary metabolites belonging to the flavonoid/stilbene class of compounds. The latter class of plant natural products has been shown to function in a wide range of biological activities. During the last few years an increasing number of health benefits have been associated with these compounds. In particular, they demonstrate potent antioxidant activity and the ability to selectively inhibit certain tyrosine kinases. Biosynthesis of many medicinally important plant secondary metabolites, including stilbenes, is frequently not very well understood and under tight spatial and temporal control, limiting their availability from plant sources. As an alternative, we sought to develop an approach for the biosynthesis of diverse stilbenes by engineered recombinant microbial cells. RESULTS: A pathway for stilbene biosynthesis was constructed in Escherichia coli with 4-coumaroyl CoA ligase 1 4CL1) from Arabidopsis thaliana and stilbene synthase (STS) cloned from Arachis hypogaea. E. coli cultures expressing these enzymes together converted the phenylpropionic acid precursor 4-coumaric acid, added to the growth medium, to the stilbene resveratrol (>100 mg/L). Caffeic acid, added in the same way, resulted in the production of the expected dihydroxylated stilbene, piceatannol (>10 mg/L). Ferulic acid, however, was not converted to the expected stilbene product, isorhapontigenin. Substitution of 4CL1 with a homologous enzyme, 4CL4, with a preference for ferulic acid over 4-coumaric acid, had no effect on the conversion of ferulic acid. Accumulation of tri- and tetraketide lactones from ferulic acid, regardless of the CoA-ligase expressed in E. coli, suggests that STS cannot properly accommodate and fold the tetraketide intermediate to the corresponding stilbene structure. CONCLUSION: Phenylpropionic acids, such as 4-coumaric acid and caffeic acid, can be efficiently converted to stilbene compounds by recombinant E. coli cells expressing plant biosynthetic genes. Optimization of precursor conversion and cyclization of the bulky ferulic acid precursor by host metabolic engineering and protein engineering may afford the synthesis of even more structurally diverse stilbene compounds.

Generation of structurally novel short carotenoids and study of their biological activity.

Recent research interest in phytochemicals has consistently driven the efforts in the metabolic engineering field toward microbial production of various carotenoids. In spite of systematic studies, the possibility of using C30 carotenoids as biologically functional compounds has not been explored thus far. Here, we generated 13 novel structures of C30 carotenoids and one C35 carotenoid, including acyclic, monocyclic, and bicyclic structures, through directed evolution and combinatorial biosynthesis, in Escherichia coli. Measurement of radical scavenging activity of various C30 carotenoid structures revealed that acyclic C30 carotenoids showed higher radical scavenging activity than did DL-α-tocopherol. We could assume high potential biological activity of the novel structures of C30 carotenoids as well, based on the neuronal differentiation activity observed for the monocyclic C30 carotenoid 4,4'-diapotorulene on rat bone marrow mesenchymal stem cells. Our results demonstrate that a series of structurally novel carotenoids possessing biologically beneficial properties can be synthesized in E. coli.