Formaldehyde (HCHO) is an important intermediate in the metabolism of one-carbon (C1) compounds such as methanol, formate, and methane. The ribulose monophosphate (RuMP) pathway is the most-studied HCHO assimilation route and the 3-hexulose-6-phosphate synthase (Hps) plays an important role for HCHO fixation. In this study, we proposed and selected a pyruvate-dependent aldolase to channel HCHO into 2-keto-4-hydroxybutyrate as an important intermediate for biosynthesis. By combining this reaction with three further enzymes we demonstrated a pyruvate-based C1 metabolic pathway for biosynthesis of the appealing compound 1,3-propanediol (1,3-PDO). This novel pathway is first confirmed in vitro using HCHO and pyruvate as substrates. It is then demonstrated in vivo in E. coli for 1,3-PDO production from HCHO and methanol with glucose as a cosubstrate. This de novo pathway has several decisive advantages over the known metabolic pathways for 1,3-PDO: (1) C1 carbon is directly channeled into a precursor of 1,3-PDO; (2) the use of pyruvate as an acceptor of HCHO is glycerol-independent, circumventing thus the need of coenzyme B 12 as cofactor for glycerol dehydration; (3) the pathway is much shorter and more simple than the recently proposed L-homoserine-dependent pathway, thus avoiding complicated regulations involving precursors for essential amino acids. In addition to proof-of-concept we further improved the host strain by deleting a gene (f rmA) responsible for the conversion of HCHO to formate, thereby increasing the production of 1,3-PDO from 298.3 ± 11.4 mg/L to 508.3 ± 9.1 mg/L and from 3.8 mg/L to 32.7 ± 0.8 mg/L with HCHO and methanol as cosubstrate of glucose fermentation, respectively. This work is the first study demonstrating a genetically engineered E. coli that can directly use HCHO or methanol for the synthesis of 2-keto-4-hydroxybutyrate and its further conversion to 1,3-PDO.

Ornamental kale is a popular decorative plant. We identified a peculiar bicolor leaf double haploid line, with green margins and red centers. The development of bicolor leaves can be divided into three stages: S1, S2, and S3. To probe the reason for bicolor formation, we analyzed the anthocyanin and chlorophyll contents, detected the changes in indole-3-acetic acid (IAA), abscisic acid (ABA), gibberellin 3 (GA3), sugar, and starch contents, and identified the differentially expressed genes (DEGs) using RNA-seq. Results showed that the bicolor leaf phenotype is gradually formed with anthocyanin degrading and chlorophyll accumulation. Anthocyanin content is lower in the green margin (S3_S) than in the red center (S3_C) part at S3. IAA content was positively correlated with anthocyanin content during the bicolor leaf development. During anthocyanin degrading from S1 to S2, cinnamate-4-hydroxylase (C4H) and transport inhibitor response 1 (TIR1) were downregulated, while lateral organ boundaries domain 39 (LBD39) was upregulated. Two peroxidases, two β-glucosidases (BGLU), LBD39, LBD37, detoxifying efflux carrier 35 (DTX35), three no apical meristem (NAC) transcription factors (TFs), and 15 WRKY DNA-binding protein (WRKY) TFs were downregulated in S3_S vs. S3_C. The bicolor phenotype was mainly linked to anthocyanin degrading and chlorophyll accumulation, and that anthocyanin degrading resulted from reduced anthocyanin biosynthesis and increased anthocyanin degradation.

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The design of novel metabolic pathways for efficient biosynthesis of natural products has received much interest, but often lacks systematic approach and chemistry-based guideline. Here we propose carbon skeleton reconstruction based on retrobiosynthetic design as a new approach and chemistry-guideline to solve the problem of properly matching precursors, one of the key issues for efficient biosynthesis. It is demonstrated for the development of an unnatural pathway for efficient biosynthesis of 5-aminolevulinic acid. The new pathway has several advantages compared to the existing natural ones such as high carbon utilization efficiency and orthogonality. It is particularly useful for overcoming the problem of glycine supply. The unnatural pathway is verified in vitro in an enzymatic cascade and in vivo in recombinant E. coli with an exogenous glyoxylate transaminase as a key enzyme.