Metabolic Engineering of Escherichia coli for Efficient Production of Pseudouridine

Abstract

Pseudouridine-incorporated mRNA vaccines can enhance protein expression and reduce immunogenicity, leading to a high demand for pseudouridine to be used in mRNA drug production. To achieve the low-cost production of pseudouridine, Escherichia coli was systematically modified to utilize inexpensive raw materials to efficiently produce pseudouridine. First, in the pyrimidine biosynthesis pathway, genes related to the precursor competing pathway and the negative regulator were deleted, which increased pseudouridine production. Second, two critical genes, pseudouridine-5′-phosphate glycosidase (psuG) and phosphatase genes from different bacteria, were screened and employed in various genetic constructs, and the pseudouridine yield of the optical strain increased to 599 mg/L. The accumulation of pseudouridine was further increased by the deletion of pseudouridine catabolism-related genes. Ultimately, the pseudouridine titer in a 5 L bioreactor reached 7.9 g/L, and the yield of pseudouridine on glucose was 0.15 g/g. Overall, a cell factory producing pseudouridine was successfully constructed and showed potential for industrial production.

C-Ribosylating Enzymes in the (Bio)Synthesis of C-Nucleosides and C-Glycosylated Natural Products

“…Because of their diverse biological activities, the C -β-riboside products have broad interest in translational medicine, ,,, as antibiotics, and as components of synthetic RNA , therapeutics, for example, as well as in other biotechnological applications ,, Enzyme applications in C -β-riboside synthesis might involve cascade biocatalysis ,, or fermentation done with metabolically reconfigured microbial strains . The example of Ψ ( 2a ) prepared via atom-efficient N1–C5 rearrangement of U ( 1a ) highlights the power of one-pot multienzyme reactions for C -β-riboside production …”

“…ΨMPGs have drawn interest for use in biocatalytic synthesis. ,,,,, The enzymes appear to be convenient to work with in respect to expression in standard hosts (e.g., E. coli BL21), specific activity, and general stability. ,,,, Their substrates [e.g., Rib5P ( 22 ), uracil ( 23 ), and polyketides] are usually available as reagents for small-scale reactions. Aside from divalent metals (Mg 2+ , Mn 2+ ) that are added at low mM concentration, the enzymatic reactions do not require complex supplementation. ,,, This facilitates product recovery.…”

“…Besides isolation from natural sources (e.g., human urine, RNA), − microbial synthesis of Ψ ( 2a ) has also been considered . Recently, Zhou et al reported engineered E. coli for the overproduction of Ψ ( 2a ) from nutrient (glucose, 78 ) . The engineering strategy used is summarized in Figure .…”

“… E. coli cell factory engineered for the fermentative production of Ψ ( 2a ) . The red crosses show knocked-out genes, and green arrows show genes overexpressed from the plasmid vector.…”

Systematic Engineering of Escherichia coli for Efficient Production of Pseudouridine from Glucose and Uracil

“…As an alternative, the synthesis of Ψ using an engineered microbe capable of synthesizing ribose 5-phosphate from cheap glucose was a better route . For example, Zhou et al successfully synthesized Ψ from glucose by fed-batch fermentation of engineered Escherichia coli . However, the highest concentration is just 7.9 g/L, and there is an urgent need to increase the production intensity of Ψ for achieving its scale-up production.…”