3’-Sialyllactose (3’-SL), the simplest sialylated human milk oligosaccharide (HMO), is widely incorporated into infant formula due to its roles in shaping the gut microbiota, inhibiting pathogen adhesion, and supporting neurodevelopment. In this study, we developed an efficient microbial cell factory for 3’-SL production by enhancing carbon utilization and CTP regeneration. Following the identification of a highly active α-2,3-sialyltransferase, metabolic flux was redirected through pfkA deletion and fbaA overexpression. The precursor pool was strengthened by optimizing the expression of key enzymes involved in UDP-GlcNAc and CMP-Neu5Ac biosynthesis. To further improve pathway balance and stability, promoter engineering was applied to optimize the expression of four key genes (neuA, neuB, neuC and ST). The optimized multigene cassette was subsequently integrated into the chromosome using a one-step multicopy integration strategy, generating a plasmid-free and antibiotic marker-free production strain. The resulting strain achieved 3’-SL titers of 7.75 g L−1 in shake flasks and 102.18 g L−1 in a 5-L fed-batch bioreactor, representing, to the best of our knowledge, the highest reported level to date. This work demonstrates a robust strategy combining metabolic rewiring, adaptive gene expression tuning and multicopy genome integration, providing a versatile platform for the sustainable production of 3’-SL and other high-value HMOs.
Yan Wu, Taoling Min, Zhenfeng Ma et al.· Bioresources and Bioprocessi...· 0 citations
Polyketides are a structurally diverse class of natural products with immense therapeutic potential. However, the biosynthetic output of discrete polyketide synthases (PKSs) has been constrained by a fundamental functional limitation: unlike modular Type I systems, discrete PKS systems typically lack integrated enoyl reductase (ER) activity. This constraint restricts their chemical repertoire primarily to unsaturated polyenes or aromatic scaffolds. Here, we characterize PbrC16, a FabV-family ER from a manumycin-type biosynthetic gene cluster (BGC) in Peterkaempfera bronchialis. This enzyme represents the first experimentally validated ER capable of functioning within discrete PKS architectures. In vitro biochemical reconstitution demonstrates that PbrC16 along with its homologue ScFabV catalyze iterative enoyl reductions in both β-ketoacyl–acyl carrier protein synthase III (KAS III)-dependent and highly reducing (HR) Type II PKS contexts, enabling the complete saturation of long-chain polyketide intermediates. Structural and computational analyses reveal the molecular basis for its exceptional substrate promiscuity and versatile acyl carrier protein (ACP) recognition. These findings resolve a long-standing “reductive gap” in discrete PKS biology and provide a “plug-and-play” module for the rational engineering of saturated polyketide scaffolds.
Yan Gao, Kai Jiang, Yuhan Dai et al.· Journal of the American Chem...· 0 citations