Design and Construction of a Non-Natural Malate to 1,2,4-Butanetriol Pathway Creates Possibility to Produce 1,2,4-Butanetriol from Glucose

Abstract

1,2,4-butanetriol (BT) is an important bulk chemical mainly used for producing the superior energetic plasticizer (1,2,4-butanetriol trinitrate) in propellant and explosive formulations. BT is commercially produced by chemical synthesis from petroleum-based feedstocks; until recently a costly biosynthetic route from xylose or arabinose was reported. Here we designed a novel biosynthetic pathway for BT from malate, for the purpose of using glucose as an alternative and cheaper substrate in future. This biosynthetic pathway was achieved through six sequential enzymatic reactions. Following tests of several combinations of enzymes for the pathway, five enzymes including malate thiokinase, succinate-semialdehyde dehydrogenase, 4-hydroxybutyrate dehydrogenase, 4-hydroxybutyrate CoA-transferase and bifunctional aldehyde/alcohol dehydrogenase were finally chosen. All enzyme genes were expressed on two compatible plasmids in E. coli, and their functions verified separately. Following assembly of two functional modules, BT was detected in the fermentation broth upon addition of malate, proving BT can be biosynthesized from malate. Furthermore, BT was detected in the fermentation using glucose as the sole carbon source, suggesting that such novel BT biosynthetic pathway has created the possibility for the production of BT from the cheaper substrate glucose.

Engineering microbial pathways for production of bio-based chemicals from lignocellulosic sugars: current status and perspectives

“…Metabolic pathway has also been engineered in E. coli for the production of BTO and 3,4-DHBA or its lactone derivative from glucose [ 101 – 103 ]. As indicated in Fig.…”

Coupled biosynthesis and esterification of 1,2,4‐butanetriol to simplify its separation from fermentation broth

“…And NaBH 4 is required as the reducing agent resulting with plenty of borate salts generated . In recent years, BT was successfully produced from cheap sugars through biological pathways , which possesses mild conditions and can reduce the environmental pollution compared to the traditional petrochemical routes. A patent claimed that 18 g/L BT could be achieved from xylose in a single strain with a yield of 0.55 mol/mol .…”

Synthetic and Systems Biology Approach towards Designing Metabolic Bypass and Identifying Novel Enzymes for Cholesterol Lowering Drug Precursor (BTO) Biosynthesis from Crude Glycerol

“…Afterwards several microbial engineering techniques have been applied but that did not overcome existing production yield (Valdehuesa et al, 2014;Cao et al, 2015;Zhang et al, 2016). Various chaperone proteins (DnaK-DnaJ-GrpE; GroES-GroEL; Trigger factor) have also been co-expressed in pre-established BTO biosynthesizing E.coli chassis, but there is no improvement obtained (Lu et al, 2016). Moreover, these well established synthetic metabolic pathways were implemented in higher plant Arabidopsis thaliana resulting into low production with 20 mg of BTO per gram of soil-grown plants (Abdel-Ghany et al, 2013).…”

“…A synthetic bacterial metabolic network has also been reconstituted for improved synthesis of BTO from malate, a Tricarboxylic acid cycle (TCA) cycle metabolic intermediate (In this case, the production was negligible i.e. 180 ng/L) (Li et al, 2014).…”