a, Schematic representation of the folate and monapterin pathway. Chemical structures and enzyme annotations of these pathway are given in Extended Data Fig. 4a. b, Abolishment of major colipterins 1-4 in monapterin biosynthetic mutants, ΔfolX and ΔfolM. c, Abolishment of 1-4 in two distinct double-mutants of aspC and tyrB in E. coli BW25113. d, Production of colipterins 3-8 via in vitro biomimetic synthesis. Tetrahydropterin (THP) was used as a dihydropterin (DHP) substrate, which reacts with phenylpyruvate (PP) to yield 3-6 (middle) and L-phenylalanine (L-Phe) to yield 7 and 8 (bottom), respectively. e, Pre-incubated phenylpyruvate (PP) and L-phenylalanine (L-Phe) reactions with THP yielded 1 and 2. f, Proposed colipterin pathway in E. coli. Proposed monapterin recycling is boxed. Enzyme annotations (blue colored) are as follows: FolE, GTP cyclohydrolase I; FolX, 7,8-dihydroneopterin triphosphate 2ʹ-epimerase; and FolM, 7,8-dihydromonapterin reductase. Abbreviations are as follows: GTP, guanosine triphosphate; DHNTP, 7,8-dihydroneopterin triphosphate; DHMTP, 7,8-dihydromonapterin triphosphate; DHM, 7,8-dihydromonapterin; THM, Tetrahydromonapterin; and qDHM, quinoid dihydromonapterin. Solid blue and red arrows indicate coupling of PP and L-Phe to DHP, respectively. Dashed arrows represent the formation of PP-Phe conjugate (purple) and coupling of this conjugate to DHP (green), respectively.