Engineering of Synthetic Multiplexed Pathways for High-Level N-Acetylneuraminic Acid Bioproduction

Abstract

N-Acetylneuraminic acid (NeuAc) is widely used as a supplement to promote brain health and enhance immunity. However, the low efficiency of de novo NeuAc synthesis limits its cost-efficient bioproduction. Herein, a synthetic multiplexed pathway engineering (SMPE) strategy is proposed to improve NeuAc synthesis. First, we compare the key enzyme sources and optimize the expression levels of three NeuAc synthesis pathways in Bacillus subtilis; the AGE, NeuC, and NanE pathways, for which NeuAc production reached 3.94, 5.67, and 0.19 g/L, respectively. Next, these synthesis pathways were combined and modularly optimized via the SMPE strategy, with production reaching 7.87 g/L. Finally, fed-batch fermentation in a 5 L fermenter reached 30.10 g/L NeuAc production, the highest reported production using glucose as the sole carbon source. Using a generally regarded as safe strain as a production host, the developed NeuAc-producing approach should be favorable for efficient bioproduction, without the need for plasmids, antibiotics, or chemical inducers.

Efficient Production of a Functional Human Milk Oligosaccharide 3′-Sialyllactose in Genetically Engineered Escherichia coli

“…Due to the lack of knowledge on the complete biosynthesis of 3′-SL in natural microorganisms, we focused on the screening of the requisite pathway enzymes previously reported and then appropriately assembled these enzymes for 3′-SL in vivo production. Literature survey suggested three different ManNAc biosynthetic routes, which generally use GlcNAc 6-phosphate as the precursor, have been verified in engineered strains . Two different Neu5Ac biosynthetic routes starting from ManNAc involved in Neu5Ac synthase or sialic acid aldolase have also been investigated, and one 3′-SL biosynthetic route starting from Neu5Ac by using a two-step successive conversion of Neu5Ac to 3′-SL catalyzed by CMP-Neu5Ac synthetase and α2,3-SiaT has been constructed. , As mentioned above, the 3′-SL biosynthesis route has been introduced in engineered E.…”

“…Literature survey suggested three different ManNAc biosynthetic routes, which generally use GlcNAc 6-phosphate as the precursor, have been verified in engineered strains. 17 Two different Neu5Ac biosynthetic routes starting from ManNAc involved in Neu5Ac synthase or sialic acid aldolase have also been investigated, 18 and one 3′-SL biosynthetic route starting from Neu5Ac by using a two-step successive conversion of Neu5Ac to 3′-SL catalyzed by CMP-Neu5Ac synthetase and α2,3-SiaT has been constructed. 16,15 As mentioned above, the 3′-SL biosynthesis route has been introduced in engineered E. coli using exogenously added Neu5Ac and lactose as substrates 15 or simply exogenously added lactose along with endogenously generated Neu5Ac.…”

High-Level 5-Methyltetrahydrofolate Bioproduction in Bacillus subtilis by Combining Modular Engineering and Transcriptomics-Guided Global Metabolic Regulation

“…This sequence was inserted into the terminator locus, where the yqzH and yqjV genes meet. 25 The DNA fragment of folB overexpression cassette consisted of the sequence of terminator 1 (sequence: GAAATAAGT-…”

“…The recombination of these four fragments was done by fusion PCR. This sequence was inserted into the terminator locus, where the yqzH and yqjV genes meet . The DNA fragment of folB overexpression cassette consisted of the sequence of terminator 1 (sequence: GAAATAAGT­TAATGTC­ACAGAAC­GCCTGCG­TTATTG­CGCAG G­CGTTTTG­TAATAA­AAAAAG­AGCCT), spectinomycin, promoter P10, gene folB , and the sequence of terminator 2 (sequence: CTATTGCAGA­ATAACTT­GTCAGACTG­CCGGGAAAT­CCCGGCAGT­CTTTTT­TCCATTAAA­ACACGGC) in turn, which was transformed into strain A9 to obtain strain B76 .…”

Metabolic Engineering of Escherichia coli for Increased Bioproduction of N-Acetylneuraminic Acid

“…subtilis inducible population quality control system based on inducible promoters and metabolite responsive biosensor gene circuits and developing genetic code expansion-based cell growth and biosynthesis balance engineering, the titer of NeuAc increased to 4.23 and 9.71 g/L in a 3 L bioreactor, respectively. , Zhang et al further proposed a synthetic multiplexed pathway engineering strategy that combined AGE, NeuC, and N -acetylmannosamine-6-phosphate epimerase (NanE) synthesis pathways, demonstrating the high-yielding potential of NeuAc in B. subtilis . In recent years, although various NeuAc production strategies have been developed, they are still lacking in terms of production yield.…”