Abstract
BACKGROUND: High-quality glycine is widely used in the cosmetics and pharmaceutical industries. Conventional chemical synthesis of glycine is disfavored because it relies on toxic reagents and complex downstream purification. RESULTS: Herein, we established an efficient two-step biosynthetic process for producing glycine from glucose. First, an l-threonine−overproducing strain was used to synthesis l-threonine via 50 L fed-batch fermentation, achieving a titer of 119.3 g/L. Then, a highly active l-threonine aldolase (PpLTA) was screened to convert l-threonine into glycine. To overcome thermodynamic and acetaldehyde inhibition constraints, an acetaldehyde elimination system was constructed via introducing ScADH1 and ScCR, which increased the molar conversion yield of glycine increased by 3.9-fold. Whole-cell catalysis gave a significantly higher conversion yield than crude enzyme preparations. A whole-cell co-expressing PpLTA, ScADH1, and ScCR was subsequently constructed. Finally, at 50 L scale, direct conversion of l-threonine fermentation broth afforded a glycine titer of 65.4 g/L within 24 h, with a molar conversion yield of 87.2%, In addition, 99.8% pure glycine was obtained with a yield of 76.6% via separation. CONCLUSIONS: An efficient two-step process for converting glucose to glycine was developed and successfully validated. Evaluation at the 50 L scale demonstrated record-high glycine production performance. This work provides a promising technological route for the industrial manufacture of high-quality glycine. GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-026-02966-3.