Formaldehyde formation in the glycine cleavage system and its use for an aldolase-based biosynthesis of 1,3-propanediol

Yingying Xu,Jie Ren,An-Ping Zeng,Hao Meng

doi:10.1186/s13036-020-00237-2

Abstract

Glycine cleavage system (GCS) occupies a key position in one-carbon (C1) metabolic pathway and receives great attention for the use of C1 carbons like formate and CO2 via synthetic biology. In this work, we demonstrate that formaldehyde exists as a substantial byproduct of the GCS reaction cycle. Three causes are identified for its formation. First, the principal one is the decomposition of N5,N10-methylene-tetrahydrofolate (5,10-CH2-THF) to form formaldehyde and THF. Increasing the rate of glycine cleavage promotes the formation of 5,10-CH2-THF, thereby increasing the formaldehyde release rate. Next, formaldehyde can be produced in the GCS even in the absence of THF. The reason is that T-protein of the GCS can degrade methylamine-loaded H-protein (Hint) to formaldehyde and ammonia, accompanied with the formation of dihydrolipoyl H-protein (Hred), but the reaction rate is less than 0.16% of that in the presence of THF. Increasing T-protein concentration can speed up the release rate of formaldehyde by Hint. Finally, a certain amount of formaldehyde can be formed in the GCS due to oxidative degradation of THF. Based on a formaldehyde-dependent aldolase, we elaborated a glycine-based one carbon metabolic pathway for the biosynthesis of 1,3-propanediol (1,3-PDO) in vitro. This work provides quantitative data and mechanistic understanding of formaldehyde formation in the GCS and a new biosynthetic pathway of 1,3-PDO.

Highlights

Traditional feedstocks for bioproduction processes mainly base on carbohydrates found in food crops, such as simple sugars and starches
We demonstrated that formaldehyde generated from glycine via the glycine cleavage system (GCS) is able to be directly used for the synthesis of 1,3-PDO through our recently proposed aldolase-based metabolic pathway [15]
Formaldehyde as a by-product in the GCS reaction In our initial study of the GCS as a potentially important route for formate-based biosynthesis, a series of experiments at different concentrations of glycine, Hox, NAD+ and THF were carried out and the NADH production was monitored as a measure of the reaction rate of the GCS

Summary

Introduction

Traditional feedstocks for bioproduction processes mainly base on carbohydrates found in food crops, such as simple sugars and starches. Our recent study [15] showed a novel pyruvate-based C1 metabolic pathway to synthesize 1,3-PDO from formaldehyde and glucose It was successfully implemented in E. coli and demonstrated that C1 compounds like methanol can be used to synthesize 1,3-PDO via formaldehyde as a metabolic intermediate. One of the purposes of the present work is to explore the possibility of generating formaldehyde from glycine using the glycine cleavage system (GCS) for 1,3-PDO synthesis. This may help to circumvent critical issues like substrate toxicity and limited conversion rate in the direct use of formaldehyde and/or methanol. It consists of four different component proteins named as P-protein (glycine decarboxylase; EC 1.4.4.2), T-protein (aminomethyltransferase; EC 2.1.2.10), L-protein (dihydrolipoyl dehydrogenase; EC 1.8.1.4) and H-protein (lipoamide-containing aminomethylene carrier) and catalyzes the oxidative decarboxylation and deamination of glycine to yield one molecule each of CO2 and ammonia, in accompany with the transfer of a methylene group to tetrahydrofolate (THF), forming thereby 5,10-CH2-THF (Eq 1): Glycine þ NADþ þ THF↔CO2 þ NH3 þ 5; 10−CH2−THF þ NADH þ Hþ ð1Þ

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Results

Conclusion