Plant-Derived Cell-Free Biofactories for the Production of Secondary Metabolites

Abstract

Cell-free expression systems enable the production of proteins and metabolites within a few hours or days. Removing the cellular context while maintaining the protein biosynthesis apparatus provides an open system that allows metabolic pathways to be installed and optimized by expressing different numbers and combinations of enzymes. This facilitates the synthesis of secondary metabolites that are difficult to produce in cell-based systems because they are toxic to the host cell or immediately converted into downstream products. Recently, we developed a cell-free lysate derived from tobacco BY-2 cell suspension cultures for the production of recombinant proteins. This system is remarkably productive, achieving yields of up to 3 mg/mL in a one-pot in vitro transcription–translation reaction and contains highly active energy and cofactor regeneration pathways. Here, we demonstrate for the first time that the BY-2 cell-free lysate also allows the efficient production of several classes of secondary metabolites. As case studies, we synthesized lycopene, indigoidine, betanin, and betaxanthins, which are useful in the food, cosmetic, textile, and pharmaceutical industries. Production was achieved by the co-expression of up to three metabolic enzymes. For all four products, we achieved medium to high yields. However, the yield of betanin (555 μg/mL) was outstanding, exceeding the level reported in yeast cells by a factor of more than 30. Our results show that the BY-2 cell-free lysate is suitable not only for the verification and optimization of metabolic pathways, but also for the efficient production of small to medium quantities of secondary metabolites.

Scaling eukaryotic cell‐free protein synthesis achieved with the versatile and high‐yielding tobacco BY‐2 cell lysate

“…We have shown that BYL can produce functional proteins as diverse as enzymes, multi‐pass transmembrane GPCRs, small, soluble growth factors, and immunologically‐active viral antigens. Moreover, functional expression of various biosynthetic enzymes, Cas9 nuclease and the antibody M12 have previously been reported from BYL (Buntru et al, 2015, 2022; Schiermeyer et al, 2022). Protein function is intrinsically linked to correct folding but further molecular characterization was warranted to prove that the microsomes of this eukaryotic CFPS system are able to produce recombinant proteins at the same quality as a cell and with batch‐to‐batch consistency.…”

“…Beyond proteins as target molecules, cell‐free is also proving useful for prototyping of biosynthetic enzyme pathways for the production of high‐value chemicals (Buntru et al, 2022; Liew et al, 2022). Prototyping is enabled by throughput and whilst microfluidics show increasing potential for CFPS workflows, the equipment is broadly inaccessible in standardized laboratory setups.…”

ALiCE®: A versatile, high yielding and scalable eukaryotic cell-free protein synthesis (CFPS) system

“…We have shown that BYL can produce functional proteins as diverse as enzymes, multi-pass transmembrane GPCRs, small, soluble growth factors and immunologically-active viral antigens. Moreover, functional expression of various biosynthetic enzymes, Cas9 nuclease and the antibody M12 have previously been reported from BYL 10,40,41 . Protein function is intrinsically linked to correct folding but further molecular characterisation was warranted to prove that the microsomes of this eukaryotic CFPS system are able to produce recombinant proteins at the same quality as a cell and with batch-to-batch consistency.…”

“…Beyond proteins as target molecules, cell-free is also proving useful for prototyping of biosynthetic enzyme pathways for the production of high-value chemicals 40,49 . Prototyping is enabled by throughput and whilst microfluidics show increasing potential for CFPS workflows, the equipment is broadly inaccessible in standardized laboratory setups.…”

Rapid screening and scaled manufacture of immunogenic virus-like particles in a tobacco BY-2 cell-free protein synthesis system

“…The BY-2 tobacco cell-free lysate (BYL) is a novel plant-based CFPS system that could serve as an effective alternative to previous eukaryotic platforms. BYL has shown higher recombinant protein production yields than other CFPS systems with the potential to cost-effectively scale both production and reaction volumes ( 26 – 28 ). The high yields, scalability, and possibility to produce complex proteins led to the commercialization of BYL as ALiCE ® ( 29 ).…”