# Engineered E. coli reaches 102.18 g/L of 3′-sialyllactose in a 5-L fed-batch bioreactor

- Published: 2026-11-14 18:54 UTC
- Section: Life & Health › Biotechnology
- Tags: 3′-sialyllactose production, human milk oligosaccharide, engineered Escherichia coli, plasmid-free strain, antibiotic-free production strain, multicopy chromosomal integration, α-2,3-sialyltransferase, fed-batch bioreactor, CMP-Neu5Ac biosynthesis
- URL: https://realtech.media/news/rt-635d2eeb0908de1b8fddeb8b_engineered_e_coli/

**A plasmid-free, antibiotic marker-free E. coli strain produced 3′-sialyllactose, an oligosaccharide of human milk used in infant formula, at what the authors call the highest reported level.**

![Engineered E. coli reaches 102.18 g/L of 3′-sialyllactose in a 5-L fed-batch bioreactor](https://realtech.media/media/2026/11/5557498b62ddfc1a.jpg)
*Peter grotzinger, Wikimedia Commons, CC BY-SA 3.0*

A research team described in Bioresources and Bioprocessing an engineered Escherichia coli BL21(DE3) strain that produced 3′-sialyllactose (3′-SL) at 102.18 g/L in a 5-L fed-batch bioreactor and at 7.75 g/L in shake flasks. By the authors' knowledge, this is the highest level reported to date [1]. The strain contains no plasmids and no antibiotic marker [1].

3′-SL is the simplest sialylated human milk oligosaccharide. It is widely added to infant formula because of its roles in shaping the gut microbiota, inhibiting pathogen adhesion and supporting neurodevelopment [1].

## Sialyltransferase screening and carbon flux

The starting host, the mutant SLEB01, carried deletions in lacZ, nanA, nanKE and nagABE, which block lactose degradation, sialic acid catabolism and amino sugar degradation. The pathway needs neuC, neuB, neuA and an α-2,3-sialyltransferase. In a 48-hour shake-flask comparison, the enzyme from Bibersteinia trehalosi gave 1.78 g/L, against 1.45 g/L for the Neisseria meningitidis reference. The two enzymes share 14.87 percent amino acid identity.

Deleting pfkA with CRISPR-Cas9 reduced cell biomass by 8.5 percent but raised the titer by 58.4 percent, to 2.82 g/L. Overexpressing the lactose permease gene lacY gave 3.20 g/L, and the combination of both changes in strain S11 gave 3.64 g/L. Co-overexpression of fbaA and fructose-1,6-bisphosphatase raised the titer to 4.11 g/L without reducing biomass [1].

## Promoter tuning and multicopy integration

The team also optimized the expression of key enzymes in the biosynthesis of UDP-GlcNAc and CMP-Neu5Ac to strengthen the precursor pool. Promoter engineering was then used to balance the expression of four genes: neuA, neuB, neuC and the sialyltransferase gene ST [1].

The optimized multigene cassette was integrated into the chromosome in a single step with a multicopy integration strategy. This produced the plasmid-free, antibiotic marker-free strain that reached the shake-flask and bioreactor titers above [1].

The authors describe the approach, which combines metabolic rewiring, adaptive tuning of gene expression and multicopy genome integration, as a platform for producing 3′-SL and other high-value human milk oligosaccharides [1]. The results come from laboratory-scale cultivation.

## Sources

1. [Bioresources and Bioprocessing](https://doi.org/10.1186/s40643-026-01110-8)
2. [bioengineer.org](https://bioengineer.org/engineered-escherichia-coli-achieves-record-yields-of-human-milk-oligosaccharide-3-sialyllactose)

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Source: RealTech, realtech.media — https://realtech.media/news/rt-635d2eeb0908de1b8fddeb8b_engineered_e_coli/