Construction of a synthetic pathway for the production of 1,3-propanediol from glucose

In this work, we describe the construction of a synthetic metabolic pathway enabling direct biosynthesis of 1,3-propanediol (PDO) from glucose via the Krebs cycle intermediate malate. This non-natural pathway extends a previously published synthetic pathway for the synthesis of (L)-2,4-dihydroxybutyrate (L-DHB) from malate by three additional reaction steps catalyzed respectively, by a DHB dehydrogenase, a 2-keto-4-hydroxybutyrate (OHB) dehydrogenase and a PDO oxidoreductase. Screening and structure-guided protein engineering provided a (L)-DHB dehydrogenase from the membrane-associated (L)-lactate dehydrogenase of E. coli and OHB decarboxylase variants derived from the branched-chain keto-acid decarboxylase encoded by kdcA from Lactococcus lactis or pyruvate decarboxylase from Zymomonas mobilis. The simultaneous overexpression of the genes encoding these enzymes together with the endogenous ydhD-encoded aldehyde reductase enabled PDO biosynthesis from (L)-DHB. While the simultaneous expression of the six enzymatic activities in a single engineered E. coli strain resulted in a low production of 0.1 mM PDO from 110 mM glucose, a 40-fold increased PDO titer was obtained by co-cultivation of an E. coli strain expressing the malate-DHB pathway with another strain harboring the DHB-to-PDO pathway.

. Primers used in strain construction.

Primer DNA sequences (5' -3')
Plasmid construction pen286 (fw-IldD) The standard Gibbs free energy for the formation of the DHB pathway intermediates (ΔfG 0' ) was calculated based on the group contribution theory 1 using the dataset published by Jankowski 2 (Supplementary Table S1).
The standard Gibbs free energy of a reaction (ΔrG 0' ) can be estimated according to Equation S8 from the standard Gibbs free energy of the formation of the participating compounds (ΔfG 0' ), with νi being the stoichiometric coefficients.
The complete PDO pathway has a standard Gibbs free energy of -60.9 kJ mol -1 .

Note 2: Calculation of the theoretical PDO yield
A previously published stoichiometric model of the central carbon metabolism in E. coli 3 was extended by 6 reactions steps enabling PDO production from malate. The theoretical yield was calculated based on elementary mode analysis using the CellNetAnalyzer software package 4 . The flux map showing one of the predicted carbon flux distributions that provide maximum yield was determined (data not shown). In the absence of cell growth, the maximum PDO yield is 1.5 mol per mol glucose. Figure S1. Optimal carbon flux distribution for the production of 1,3-propanediol (PDO) in the central metabolic network of E. coli. Figure S2. Influence of (D/L)-DHB feeding on PDO biosynthesis.
Production of PDO from 50 mM (D/L)-DHB after 24 h of culture of Pen913 strain (E. coli MG1655 pACT3-llDdV108C-kdcAV461I-yqhD). The error bars represent the standard deviation (SD) from the mean of two biological replicate experiments. All cell cultivation was carried out at 37 ºC on a rotary shaker (Infors HT, France) running at 200 rpm. Pre-cultures were made in 5 mL of LB in 50 mL falcon tubes. After ~10 h, 500 µL was used to inoculate a second pre-culture (10 mL of 90 % v/v M9 mineral medium supplemented with 20 g L -1 glucose and 10 % v/v LB in 50 mL falcon tubes) that was cultivated overnight. The biomass needed to start main cultures with a starting OD600 of 0.2 was transferred to 250 mL baffled shake flasks containing 25 mL of 90 % v/v M9 mineral medium supplemented with 20 g L -1 glucose and 10 % v/v LB, and 1 mM IPTG and 50 mM (D/L)-DHB were added when OD600 reached ~0.6. The antibiotic chloramphenicol was added when required at 25 mg L -1 . Figure S3. Influence of host strain on bioconversion of (D/L)-DHB into PDO.
Production of PDO from 50 mM (D/L)-DHB after 48 h of culture of various E. coli host strains harboring DHB downstream pathway encoded from the medium-copy plasmid pACT3-llDdV108C-kdcAV461I-yqhD. The error bars represent the standard deviation (SD) from the mean of two biological replicate experiments. All host strains originated from the parent strain E. coli MG1655. Conditions of cell cultivation are as in Figure S1 For each condition PDO yields on (D/L)-DHB were as follows, wt: 0.55 ±0.24 mol mol -1 , glcA proD : 0.44 ±0.13 mol mol -1 , and kgtP proD : 0.46 ±0.19 mol mol -1 .