An Aldolase-Based New Pathway for Bioconversion of Formaldehyde and Ethanol into 1,3-Propanediol in Escherichia coli

Abstract

Formaldehyde (HCHO) is a reactive one-carbon compound that is interesting for biosynthesis. The assimilation of HCHO depends on the catalysis of aldolase. Here, we present a novel synthetic pathway in E. coli to convert HCHO and ethanol into 1,3-propanediol (PDO) using a deoxyribose-5-phosphate aldolase (DERA). DERA condenses HCHO and acetaldehyde to form 3-hydroxypropionaldehyde, the direct precursor of PDO formation. This new pathway opens up the possibility to synthesize an appealing C3 compound from a C1 compound and a C2 compound without carbon loss in contrast to all the other known PDO synthetic pathways where typically 30–50% of the carbons are lost as CO2 and other byproducts. The pathway is successfully demonstrated by elaborating three metabolic modules. First, DERA from Thermotoga maritima was found to be efficient for the aldol condensation and PDO production module. For the module of acetaldehyde supply from ethanol, an alcohol dehydrogenase from Hansenula polymorpha was selected. For the HCHO supply module, the control of HCHO concentration and its utilization were shown to be important for achieving the assimilation of HCHO in recombinant E. coli cells. By deleting the gene frmA for endogenous conversion of HCHO to formate and controlling HCHO at a level of about 0.6 mM, the concentration and yield of PDO were increased from initially 5.67 mM (0.43 g/L) and 0.057 mol/mol to 17.35 mM (1.32 g/L) and 0.096 mol/mol in bioconversion of ethanol and HCHO with resting E. coli cells. Further engineering of DERA and the HCHO supply module is necessary to realize the potential of this promising metabolic pathway.

In Vitro One-Pot 3-Hydroxypropanal Production from Cheap C1 and C2 Compounds

“…The catalytic efficiency ( k cat / K m , min −1 mM) of DERA Tma for formaldehyde was 2.8-fold higher than that for acetaldehyde, suggesting that DERA Tma prefers formaldehyde (C1) than acetaldehyde (C2). Zeng et al investigated the K m of DERA Tma toward formaldehyde and acetaldehyde, which were 2.54 and <1 mM (at pH 7.0 and 30 °C), respectively [ 25 ]. However, the K m for formaldehyde was similar to that reported previously, but the K m value for acetaldehyde was 7-fold higher than that reported previously (at pH 7.0 and 40 °C).…”

“…Till date, 3-HPA has been produced as an intermediate by glucose or glycerol fermentation using wild-type strain cells and metabolically engineered cells such as E . coli [ 15 , 21 , 22 , 24 , 25 , 41 ], Lactobacillus sp. [ 42 , 43 , 44 , 45 , 46 , 47 , 48 ], and Klebsiella pneumoniae [ 49 , 50 , 51 , 52 , 53 ].…”

“…To date, they have been produced from glucose or glycerol using metabolically engineered cells, including those producing glycerol dehydrogenase and aldehyde dehydrogenase enzymes [ 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 ]. Recently, deoxyribose-5-phosphate aldolase (DERA) from Thermotoga maritima (DERA Tma , E.C 4.1.2.4) has been introduced as an intermediate enzyme to condense formaldehyde and acetaldehyde to 3-HPA for PDO biosynthesis in engineered Escherichia coli [ 25 ]. In this metabolic pathway, aldol condensation between aldehydes is the key reaction point; however, the typical limitations of metabolic engineering include a lack of enzyme engineering or pathway design optimization.…”

Recent advances in biological production of 1,3-propanediol: new routes and engineering strategies

“…However, since methanol dehydrogenase and 2-keto-4-hydroxybutyrate aldolase have very high K m values for methanol and formaldehyde (>500 mM), the engineered E. coli overexpressing khb, kdc encoding α-keto acid decarboxylase, and dhaT genes only accumulated 32.7 mg L −1 1,3-PDO using glucose and methanol as co-substrates. 87 The second biological route tried to use methanol (or formaldehyde) and ethanol as co-substrates for 1,3-PDO production 88 (Fig. 2, the blue line).…”

“…78 Principally, microorganisms which can naturally utilize methanol, starch, lignocellulose, CO 2 , or syngas can be engineered to produce 1,3-PDO with the introduction of this pathway although the efficiency of the pathway should be further increased by protein engineering and metabolic engineering. 78,84,88,89 Development of new processes to efficiently utilize these cheap feedstocks may provide potentially sustainable routes for large-scale production of 1,3-PDO. In particular, the utilization of CO 2 for 1,3-PDO is promising in the future considering the extremely low price and high abundance of CO 2 .…”