Designing Modular Cell-free Systems for Tunable Biotransformation of l-phenylalanine to Aromatic Compounds

Abstract

Cell-free systems have been used to synthesize chemicals by reconstitution of in vitro expressed enzymes. However, coexpression of multiple enzymes to reconstitute long enzymatic pathways is often problematic due to resource limitation/competition (e.g., energy) in the one-pot cell-free reactions. To address this limitation, here we aim to design a modular, cell-free platform to construct long biosynthetic pathways for tunable synthesis of value-added aromatic compounds, using (S)-1-phenyl-1,2-ethanediol ((S)-PED) and 2-phenylethanol (2-PE) as models. Initially, all enzymes involved in the biosynthetic pathways were individually expressed by an E. coli-based cell-free protein synthesis (CFPS) system and their catalytic activities were confirmed. Then, three sets of enzymes were coexpressed in three cell-free modules and each with the ability to complete a partial pathway. Finally, the full biosynthetic pathways were reconstituted by mixing two related modules to synthesize (S)-PED and 2-PE, respectively. After optimization, the final conversion rates for (S)-PED and 2-PE reached 100 and 82.5%, respectively, based on the starting substrate of l-phenylalanine. We anticipate that the modular cell-free approach will make a possible efficient and high-yielding biosynthesis of value-added chemicals.

Cell-free expression of NO synthase and P450 enzyme for the biosynthesis of an unnatural amino acid L-4-nitrotryptophan

“…Further increase of CFPS reaction time did not significantly improve the product yield, perhaps as a result of other limiting factors in the reaction. This result is similar to a previous report that 2-h CFPS reaction was the best for enzyme expression before mixing and the resultant bioconversion [ 30 ].

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“…For example, the reaction format and cofactor supplementation can be readily manipulated to achieve high productivity. Previous studies also have demonstrated the flexibility of cell-free systems by designing modular CFPS reactions, expressing enzymes in separate modules, and eventually mixing cell-free modules for long metabolic pathway construction and product formation [ 30 , 47 ]. Third, cell-free systems without the use of living cells are tolerant to toxic compounds such as the cellular toxic NO if it is highly accumulated in cells.…”

“…Recently, CFPS-based systems have been used in an aqueous-organic biphasic system for cascade biotransformation by construction of an artificial enzymatic pathway [ 29 ]. If the full metabolic pathway is long with multiple steps (e.g., 6–7 enzymes), cell-free systems can also be designed with different reaction modules for enzyme expression, followed by combining the enzyme-enriched modules for efficient biocatalysis [ 30 ]. Moreover, there are no living, intact cells in CFPS reactions.…”

Biotechnology Applications of Cell-Free Expression Systems

“…Cell-free platforms also have numerous advantages over cell-based platforms on an industrial scale, such as faster development times, adaptability to various production schemes, resistance to conditions toxic to cell-based systems, and cost-effectiveness at scale [25]. The increasing demand for low-cost and sustainable products manufactured from low-carbon emission methods can also be addressed using cell-free systems optimized to utilize more sustainable carbon utilization pathways and green energy regeneration modules [20,25,26]. While cell-free platforms have been demonstrated to be adaptable to industrial production schemes, they can also be utilized for smaller-scale on-demand production of target molecules.…”

“…While cell-free platforms have been demonstrated to be adaptable to industrial production schemes, they can also be utilized for smaller-scale on-demand production of target molecules. This is particularly useful for the on-demand production of therapeutics, of which many niche drugs do not have the demand for pharmaceutical companies to warrant building expensive cell-based infrastructure to produce them [26]. Cell-free systems could be used at a scale where the on-demand manufacturing of custom therapeutics for individual customers would be feasible [27].…”