Multiple Gene Expression in Cell-Free Protein Synthesis Systems for Reconstructing Bacteriophages and Metabolic Pathways

Abstract

As a fast and reliable technology with applications in diverse biological studies, cell-free protein synthesis has become popular in recent decades. The cell-free protein synthesis system can be considered a complex chemical reaction system that is also open to exogenous manipulation, including that which could otherwise potentially harm the cell's viability. On the other hand, since the technology depends on the cell lysates by which genetic information is transformed into active proteins, the whole system resembles the cell to some extent. These features make cell-free protein synthesis a valuable addition to synthetic biology technologies, expediting the design-build-test-learn cycle of synthetic biology routines. While the system has traditionally been used to synthesize one protein product from one gene addition, recent studies have employed multiple gene products in order to, for example, develop novel bacteriophages, viral particles, or synthetic metabolisms. Thus, we would like to review recent advancements in applying cell-free protein synthesis technology to synthetic biology, with an emphasis on multiple gene expressions.

Keywords: VLPs; cell-free protein synthesis; metabolic pathways.

Figure 1

Cell-free multiple gene expression for viral particle synthesis or novel metabolic pathway construction. The one gene-one tube strategy is employed in typical CFPS approaches (Right). Genes for isoenzymes are shown in different shades in the same color. Even though this figure indicates that plasmids were used, amplified gene fragments can be used to prime the CFPS reaction. In this strategy, each product is mixed in varying ratios in search for an optimized metabolic pathway or successful viral particle reconstitutions. Large circles and thicker arrows are used to describe strengthened steps in the imaginary metabolic pathway. On the other hand, multiple genes are added to a single tube to accomplish the construction of a metabolic pathway or viral particles (Left). In this presentation, the concentration of each gene is manipulated to achieve varied ratios of all synthesized proteins, and, thus, optimal results. By completing the metabolic pathway in both strategies, endogenous metabolisms in the CFPS system can be utilized.

Figure 2

Typical cell-free protein synthesis system showing major components.

Figure 3

Metabolic pathways constructed by cell-free multiple gene expression. Single enzyme reactions are presented as solid arrows, with the enzyme’s name inside, and multiple step reactions involving multiple enzymes are shown as broken arrows. Key substrates of each reaction are shown beneath the arrow. Desired end products of the constructed pathways are shown in blue boxes, while unwanted side products and related reactions are shown in grey boxes. For simplicity, not all names of the intermediates are described. (A) Schematic diagram of the synthesis of UDP-N-acetylmuramyl pentapeptide from N-acetylglucosamine (GlcNAc). Pi, phosphate; PEP, phosphoenolpyruvate; m-DAP, meso-diaminopimelic acid. (B) Schematic diagram of a violacein biosynthetic pathway from tryptophan. IPAI, indole-3-pyrivic acid imine. (C) Schematic representation of a biosynthetic pathway to n-butanol from glucose using a hybrid pathway that consists of five cell-free expressed enzymes (indicated by solid arrows) and endogenous glycolytic pathway enzymes (shown in a box with a dashed line). (D) Schematic representation of a lycopene biosynthesis pathway from acetyl-CoA. The endogenous metabolic pathway, including the mevalonate pathway, is shown in a box with a dashed line. IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl diphosphate; GGPP, geranylgeranyl pyrophosphate. (E) The biosynthetic pathway to 1,4-butanediol (BDO) synthesis from succinyl-CoA. AcCoA, acetyl-CoA.

Figure 3

Metabolic pathways constructed by cell-free multiple gene expression. Single enzyme reactions are presented as solid arrows, with the enzyme’s name inside, and multiple step reactions involving multiple enzymes are shown as broken arrows. Key substrates of each reaction are shown beneath the arrow. Desired end products of the constructed pathways are shown in blue boxes, while unwanted side products and related reactions are shown in grey boxes. For simplicity, not all names of the intermediates are described. (A) Schematic diagram of the synthesis of UDP-N-acetylmuramyl pentapeptide from N-acetylglucosamine (GlcNAc). Pi, phosphate; PEP, phosphoenolpyruvate; m-DAP, meso-diaminopimelic acid. (B) Schematic diagram of a violacein biosynthetic pathway from tryptophan. IPAI, indole-3-pyrivic acid imine. (C) Schematic representation of a biosynthetic pathway to n-butanol from glucose using a hybrid pathway that consists of five cell-free expressed enzymes (indicated by solid arrows) and endogenous glycolytic pathway enzymes (shown in a box with a dashed line). (D) Schematic representation of a lycopene biosynthesis pathway from acetyl-CoA. The endogenous metabolic pathway, including the mevalonate pathway, is shown in a box with a dashed line. IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl diphosphate; GGPP, geranylgeranyl pyrophosphate. (E) The biosynthetic pathway to 1,4-butanediol (BDO) synthesis from succinyl-CoA. AcCoA, acetyl-CoA.