Cell-Free Biosynthesis System: Methodology and Perspective of in Vitro Efficient Platform for Pyruvate Biosynthesis and Transformation

Abstract

The modification of intracellular metabolic pathways by metabolic engineering has generated many engineered strains with relatively high yields of various target products in the past few decades. However, the unpredictable accumulation of toxic products, the cell membrane barrier, and competition between the carbon flux of cell growth and product synthesis have severely retarded progress toward the industrial-scale production of many essential chemicals. On the basis of an in-depth understanding of intracellular metabolic pathways, scientists intend to explore more sustainable methods and construct a cell-free biosynthesis system in vitro. In this review, the synthesis and application of pyruvate as a platform compound is used as an example to introduce cell-free biosynthesis systems. We systematically summarize a proposed methodology workflow of cell-free biosynthesis systems, including pathway design, enzyme mining, enzyme modification, multienzyme assembly, and pathway optimization. Some new methods, such as machine learning, are also mentioned in this review.

Core–shell BaPP@BDO-Rh benzimidazole oligomer photocatalyst can effectively promote NADH regeneration and CO2 conversion to fumarate

“…5b). 47,48 Notably, the presence of fumarate and NADH was conrmed by 1H NMR, providing further evidence for the feasibility of this photoenzyme system (Fig. S16 †).…”

Metabolic Engineering of Microorganisms to Produce Pyruvate and Derived Compounds

“…The increase in the number of omics datasets, coupled with advances in ML, has also created tremendous opportunities for metabolic engineering. The increase in the number of omics datasets coupled with advances in ML have allowed insight into intracellular metabolic pathways, thus creating tremendous opportunities for scientists to explore cell-free biosynthesis systems in vitro [ 129 ]. Cell-free biosynthesis includes steps such as pathway design, enzyme mining, enzyme modification, multi-enzyme assembly, and pathway optimization, which will rely on metabolic pathway databases and applications of ML to design components and modules [ 130 ].…”