Cell-Free Biocatalysis for the Production of Platform Chemicals

Abstract

Genetically engineered host bacteria have an extensive history for the production of specific proteins including the synthesis of single enzymes for the modification of compounds produced for industrial purposes by biological or chemical processes. Such processes have been developed largely through the process of discovery. The ability to assemble multiple enzymes into synthetic pathways is a new development aided by the synthetic biology approach of constructing and assembling suitable enzymes into pathways that may or may not occur in Nature to provide a highimpact platform for bio-manufacturing of chemicals, biofuels and pharmaceuticals. Industry has depended on chemical catalysts because of the known constraints experienced frequently with biological catalysts but when high stereochemistry, mild synthesis conditions and environmentally friendly processes are significant, application of enzymes is preferred, especially in the case of drug synthesis where enantioselectivity is important. However, many whole cell production processes are beset by toxicity problems, metabolite competition, the production of side products, sub-optimal enzyme ratios and varying temperature optima. As a result, a cell-free biocatalysis allows the manipulation of substrate ratios, provision of regenerated cofactors and adjustment of high energy flux ratios that are difficult or impossible to control in whole cell synthesis. We discuss here the construction of cell free biocatalytic pathways as added free enzymes or multi-enzyme modules that may contain heterologous catalysts. We examine the status of applications leading to commercialisation, emphasizing the importance of economical cofactor regeneration. Nevertheless, problems remain, particularly protein post-translational modifications including glycosylation, phosphorylation, ubiquitination, acetylation, proteolysis, etc. The National Renewable Energy Laboratory and Pacific North West Laboratory have published lists of top value-added chemicals from biomass that include glucaric acid. There have been few reports on the combination of synthetic biology and cell-free protein synthesis to set up pathways for these valuable intermediate compounds. This observation is despite the existence of at least one large scale but specialized cell-free production of antibody conjugates. This review will provide a description of one successful attempt at the cell-free production of glucaric acid and will evaluate progress for other key intermediate and platform chemicals.

Application of Nucleic Acid Frameworks in the Construction of Nanostructures and Cascade Biocatalysts: Recent Progress and Perspective

“…A multi-enzyme system is composed of a series of enzymes that are well organized in a certain spatial order, which facilitates the transfer of reaction intermediates, and thus significantly enhances the overall catalytic activities (Hwang and Lee, 2019). Inspired by the outstanding catalytic performance of natural multi-enzyme systems, numerous artificial multi-enzyme molecular machines have been designed and applied in a variety of scenarios, ranging from bio-catalysis to biopharmaceutical applications (Zhang et al, 2018;Bergquist et al, 2020). In these artificial multienzyme systems, the scaffolds that were used to immobilize and organize the enzymes were prepared from many different materials, including inorganic materials, organic molecules, proteins, and nucleic acids (Jia et al, 2014;Xu K. et al, 2020).…”

Metabolic Dynamics inEscherichia coli-based Cell-Free Systems

“…Moreover, the lack of cellular membranes removes transport limitations than can hinder biocatalysis and allows for continuous monitoring and manipulation. These advantages have led to the successful implementation of CFE systems for the production of small molecules and proteins in broad application areas ranging from therapeutics and biosensors to biofuels [6][7][8][9] .…”

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

“…Although 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).…”