Self-Assembling Metabolon Enables the Cell Free Conversion of Glycerol to 1,3-Propanediol

Qi Xu,Hui Wei,Michael E Himmel,Yannick J Bomble,Ashutosh Mittal,Neal N Hengge,Nicholas S Sarai,Markus Alahuhta,Patrick Hewitt

doi:10.3389/fenrg.2021.680313

Abstract

Cell free biocatalysis is showing promise as a replacement or complement to conventional microbial biocatalysts due to the potential for achieving high yields, titers, and productivities. However, there exist several challenges that need to be addressed before its broader industrial adoption is achieved. New paradigms and innovative solutions are needed to overcome these challenges. In this study we demonstrate high levels of glycerol conversion to 1,3-propanediol using a self-assembling metabolic pathway leveraging the arraying strategy (protein scaffolds) used by thermophilic cellulolytic bacteria to assemble their biomass degrading enzymes. These synthetic metabolons were capable of producing 1,3-PDO at a yield more than 95% at lower glycerol concentration and close to 70% at higher concentrations at a higher productivity rate than the equivalent microbial strain. One of the benefits of our approach is the fact that no enzyme purification is required, and that the assembly of the complex is accomplished in vivo before immobilization, while product formation is conducted in vitro. We also report the recovery of enzymatic activity upon fusion enzymes binding to these protein scaffolds, which could have broader applications when assembling arrayed protein complexes.

Highlights

Microbial biocatalysts have been successfully used for the production of many high-value chemicals, they often suffer from low product yields due to competing metabolic pathways, low productivities, difficult optimization of metabolic pathways, cellular product toxicity, and expensive isolation of target products from cell cultures (Stephanopoulos, 2007; Chen and Liao, 2016; Chubukov et al, 2016; Chae et al, 2017)
In this study we focused on the production of 1,3-PDO from glycerol using two metabolic steps with 3-hydroxypropionic aldehyde (3-HPA) as an intermediate
There are several challenges that need to be overcome for cell-free biocatalysis to become a reality especially for the production of commodity fuels and chemicals–high on that list are 1) enzyme cost, (2) ease of enzyme purification, (3) enzyme stability/solubility, and (4) efficient cofactor recycling schemes

Summary

Introduction

Microbial biocatalysts have been successfully used for the production of many high-value chemicals, they often suffer from low product yields due to competing metabolic pathways, low productivities, difficult optimization of metabolic pathways, cellular product toxicity, and expensive isolation of target products from cell cultures (Stephanopoulos, 2007; Chen and Liao, 2016; Chubukov et al, 2016; Chae et al, 2017). Alternatives to production using microbial biocatalysts include cell-free approaches wherein reactions occur in vitro, in isolation, rather than in cells (Kay and Jewett, 2015; Korman et al, 2017; Petroll et al, 2019; Bergquist et al, 2020; Bowie et al, 2020). Cell-free biocatalysis relies either on purified enzyme systems or lysates with both having advantages and disadvantages (Kay and Jewett, 2015; Rollin et al, 2021). Product yields and titers in cell-free biocatalysis can suffer from diffusion of intermediates and lack of enzyme stability.

Methods

Results

Conclusion