Research progress on the application of cell-free synthesis systems for enzymatic processes.

Cell-free synthesis systems can complete the transcription and translation process in vitro to produce complex proteins that are difficult to be expressed in traditional cell-based systems. Such systems also can be used for the assembly of efficient localized multienzyme cascades to synthesize products that are toxic to cells. Cell-free synthesis systems provide a simpler and faster engineering solution than living cells, allowing unprecedented design freedom. This paper reviews the latest progress on the application of cell-free synthesis systems in the field of enzymatic catalysis, including cell-free protein synthesis and cell-free metabolic engineering. In cell-free protein synthesis: complex proteins, toxic proteins, membrane proteins, and artificial proteins containing non-natural amino acids can be easily synthesized by directly controlling the reaction conditions in the cell-free system. In cell-free metabolic engineering, the synthesis of desired products can be made more specific and efficient by designing metabolic pathways and screening biocatalysts based on purified enzymes or crude extracts. Through the combination of cell-free synthesis systems and emerging technologies, such as: synthetic biology, microfluidic control, cofactor regeneration, and artificial scaffolds, we will be able to build increasingly complex biomolecule systems. In the next few years, these technologies are expected to mature and reach industrialization, providing innovative platforms for a wide range of biotechnological applications.

Pathway engineering of Saccharomyces cerevisiae for efficient lycopene production.

To construct a Saccharomyces cerevisiae strain for efficient lycopene production, we used a pathway engineering strategy based on expression modules comprising fusion proteins and a strong constitutive promoter. The two recombinant plasmids pEBI encoding the fusion genes with an inducible promoter, as well as pIETB with a constitutive promoter and terminator were introduced into S. cerevisiae YPH499 and BY4741 to obtain the four recombinant strains ypEBI, ypIETB, byEBI and byIETB. The lycopene production and the transcription levels of key genes were higher in the BY4741 chassis than in YPH499. Accordingly, the content of total and unsaturated fatty acids was also higher in BY4741, which also exhibited a decrease of glucose, increase of trehalose, increase of metabolite in citrate cycle, and low levels of amino acids. These changes rerouted metabolic fluxes toward lycopene synthesis, indicating that the BY4741 chassis was more suitable for lycopene synthesis. The lycopene content of bpIETB in SG-Leu medium supplemented with 100 mg/L of linolenic acid reached 10.12 mg/g dry cell weight (DCW), which was 85.7% higher than without the addition of unsaturated fatty acids. The constitutive promoter expression strategy employed in this study achieved efficient lycopene synthesis in S. cerevisiae, and the strain bpIETB was obtained a suitable chassis host for lycopene production, which provides a basis for further optimization of lycopene production in artificial synthetic cells and a reference for the multi-enzyme synthesis of other similar complex terpenoids.