De Novo Biosynthesis of Trigonelline in Engineered Escherichia coli

Trigonelline, a naturally occurring pyridine alkaloid found in fenugreek (Trigonella foenum-graecum) seeds and other legumes, exerts diverse pharmacological effects, including anti-inflammatory and neuroprotective effects. Currently, it is predominantly produced through plant extraction and chemical synthesis. Here, we report trigonelline production from a de novo pathway in Escherichia coli for the first time. Identification and expression of the nicotinate N-methyltransferase Aco_009_01073 resulted in an initial trigonelline titer of 13.59 mg/L. Trigonelline titers were subsequently improved through systematic pathway engineering. Supply of the key precursor nicotinate was increased via heterologous expression of sdt1 (encoding NMN/NaMN 5'-nucleotidase) and urh1 (encoding nicotinate riboside hydrolase) from Saccharomyces cerevisiae and overexpression of nadB (encoding aspartate oxidase) and nadA (encoding quinolinate synthase). Nicotinate consumption was reduced by deleting pncB (encoding nicotinate phosphoribosyltransferase). In addition, the NadB-NadA module was assembled into an enzyme complex, the Sdt1-Urh1 module was constructed as a fusion protein, and the quinolinate phosphoribosyltransferase gene nadC was overexpressed. These modifications collectively enhanced trigonelline yield of 29.95 mg/L in shake-flask cultures. Fed-batch fermentation in a 5 L bioreactor led to a final trigonelline yield of 128.38 mg/L. Overall, this study reports the successful engineering of a microbial platform for trigonelline biosynthesis and provides a general framework for nicotinate-based N-methyl alkaloids.