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.
Highlights
Secondary metabolites are organic natural products synthesized by plants, microbes, or animals via enzymatic cascades
The bacterial blue pigment synthetase A (bpsA) and sfp sequences were codon optimized for tobacco to facilitate comparison with a previous study in which codon-optimized sequences were used for cell-based production in Rhodosporidium toruloides (Wehrs et al, 2019)
A C-terminal StrepII tag sequence was introduced into the correct size: kDa (CrtE), CrtB, and CrtI constructs since Matthäus et al (2014) have shown that this tag did not interfere with the catalytic activity of the heterologous enzymes
Summary
Secondary metabolites are organic natural products synthesized by plants, microbes, or animals via enzymatic cascades. They are generally not required for normal growth and development and instead facilitate ecological interactions, the benefit to the producer is not always clear. Many secondary metabolites, including alkaloids, terpenoids, polyketides, antibiotics, peptides, and growth hormones, have structures or biological activities that make them useful for applications in the chemical, agri-food, cosmetic, and pharmaceutical industries. Secondary metabolites are extracted from their natural sources using solvents or are produced by chemical synthesis. Both methods tend to be inefficient and harmful to the environment, making them costly and unsustainable.
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