Metabolic engineering of Escherichia coli for efficient ectoine production

Abstract

Ectoine is a high-value stabilizer and protective agent with various applications in enzyme industry, cosmetics, and biomedicine. In this study, rational engineering strategies have been implemented in Escherichia coli to efficiently produce ectoine. First, the synthetic pathway of ectoine was constructed in E. coli MG1655 by introducing an artificial thermal switch system harboring the ectABC cluster from Halomonas elongate, and the resulting strain produced 1.95 g/L ectoine. Second, crr encoding the glucose-specific enzyme II domain A of phosphotransferase system and iclR encoding the glyoxylate shunt transcriptional repressor were deleted in E. coli for enhancing the oxaloacetate supply, leading to the increasement of the ectoine titer to 9.09 g/L. Third, thrA encoding the bifunctional aspartokinase/homoserine dehydrogenase was removed from the genome to weaken the competitive pathway; simultaneously, an endogenous feedback-resistant lysC was overexpressed to complement the enzymatic activity deficiency of the aspartate kinase, leading to 30.36% increase of ectoine titer. Next, the expression of phosphoenolpyruvate carboxylase was modulated with varying gradient strength promoters to accelerate the biosynthesis efficiency of ectoine. Finally, aspDH encoding aspartate dehydrogenase from Pseudomonas aeruginosa PAO1 was overexpressed to further improve the biosynthesis of ectoine. The final strain MWZ003/pFT28-ectABC-EclysC*-aspDH-ppc3 produced 30.37 g/L ectoine after 36-h fed-batch fermentation with a yield of 0.132 g/g glucose and a productivity of 0.844 g/(L h).

Perspectives in growth production trade-off in microbial bioproduction

“…23 The transition from one phase to another is induced by several different strategies. Examples include glucose starvation and carbon source switching for the production of PHB in E. coli in rich and minimal media, 24 nitrogen starvation for fatty alcohol production in E. coli, 25 phosphate limitation for linalool production in Pantoea ananatis, 26 oxygen supply limitation, 27 biotin limitation for proline production in Corynebacterium glutamicum, 28 pH control for propionic acid production in Propionibacterium jensenii 19 and temperature shi 29 to regulate isocitrate dehydrogenase activity to increase itaconate production in E. coli. 30 Extreme examples are growth in one carbon source and conversion of a different source to the product 21,23 or use of whole cells as biocatalysts for the production of acetoin, gamma-amino-butyric acid (GABA) and 3-caprolactone using engineered E. coli.…”

Exploring the De Novo NMN Biosynthesis as an Alternative Pathway to Enhance NMN Production

“…Biological synthetic routes, specifically enzyme-catalyzed in vitro, whole-cell catalysis, and fermentative synthesis routes, are emerging as the preferred methods for NMN production due to their high stereoselectivity. However, enzyme-catalyzed in vitro routes require expensive substrates and enzymes, while scaling up whole-cell catalytic processes for industrial production remains challenging. − Therefore, the development of an economical and effective route for NMN production, such as fermentative synthesis, becomes necessary, where NMN can be synthesized using inexpensive materials. − …”

Enhanced production of ectoine from methane using metabolically engineered Methylomicrobium alcaliphilum 20Z

“…Commercially, Halomonas elongata , a moderately halophilic bacterium, is a commonly used ectoine producer that has an established biosynthetic pathway for ectoine metabolism using glucose as a carbon source [ 19 , 20 ]. Several reports considered to the heterologous synthesis of ectoine in a well-established industrial host such as Escherichia coli and Corynebacterium glutamicum [ 21 , 22 ].…”