Improving methanol assimilation in Yarrowia lipolytica via systematic metabolic engineering combined with compartmentalization

Abstract

Renewable methanol is an attractive non-food feedstock that can replace sugar-based raw materials for biochemicals production. However, the metabolic construction of synthetic methylotrophy to utilize methanol for cell growth and...

Highly efficient biosynthesis of 2,4-dihydroxybutyric acid by a methanol assimilation pathway in engineered Escherichia coli

“…As shown in Fig. 2, the ratios of the peaks at around m/z 103 and m/z 104 are almost the same with the unlabeled standard sample and the 13 C-HCHO samples. This finding indicated that formaldehyde is an essential precursor in the synthesis of 2,4-DHB, and the coupling of formaldehyde with endogenous pyruvate derived from glycolysis provides an excellent opportunity for the synthesis of the valuable chemical 2,4-DHB.…”

“…Then, the fermentation broth was transferred (1% inoculum) into a 250 mL flask containing 50 mL of M9 medium supplemented with 100 μg mL −1 ampicillin and incubated at 37 °C. When the OD 600 reached about 0.8, 0.5 mM IPTG and 5 mM formaldehyde or 13 C-HCHO were added to induce gene expression. After induction at 30 °C for 12 h, 50 mM formaldehyde, 13 C-HCHO was added as a substrate.…”

“…When the OD 600 reached about 0.8, 0.5 mM IPTG and 5 mM formaldehyde or 13 C-HCHO were added to induce gene expression. After induction at 30 °C for 12 h, 50 mM formaldehyde, 13 C-HCHO was added as a substrate. Batch cultivation was performed at 30 °C and 200 rpm for 48 h. The entire shake flask fermentation was carried out in two independent experiments.…”

“…In order to demonstrate that the exogenously added formaldehyde was condensed into 2,4-DHB, 13 C-HCHO was used as the substrate for performing the biosynthesis. As shown in Fig.…”

“…12 Methanol is first converted into formaldehyde in vivo by enzymatic catalysis and then further oxidized to CO 2 through anabolic pathways or converted into energy and substances required for the vital activities of living organisms through certain assimilation pathways. 4,13 It is difficult to construct a new methanol metabolic pathway in methylotrophic bacteria for the production of commercial chemicals or biofuels using techniques such as genetic engineering because most natural methylotrophic bacteria lack well-established genetic tools. At present, bioavailability platforms such as E. coli have been widely used for industrial production of bulk chemicals, 11 so we constructed a methanol utilization pathway in E. coli with a view to realizing the bioavailability of methanol.…”