Cell-free prototyping of limonene biosynthesis using cell-free protein synthesis

Abstract

Metabolic engineering of microorganisms to produce sustainable chemicals has emerged as an important part of the global bioeconomy. Unfortunately, efforts to design and engineer microbial cell factories are challenging because design-built-test cycles, iterations of re-engineering organisms to test and optimize new sets of enzymes, are slow. To alleviate this challenge, we demonstrate a cell-free approach termed in vitro Prototyping and Rapid Optimization of Biosynthetic Enzymes (or iPROBE). In iPROBE, a large number of pathway combinations can be rapidly built and optimized. The key idea is to use cell-free protein synthesis (CFPS) to manufacture pathway enzymes in separate reactions that are then mixed to modularly assemble multiple, distinct biosynthetic pathways. As a model, we apply our approach to the 9-step heterologous enzyme pathway to limonene in extracts from Escherichia coli. In iterative cycles of design, we studied the impact of 54 enzyme homologs, multiple enzyme levels, and cofactor concentrations on pathway performance. In total, we screened over 150 unique sets of enzymes in 580 unique pathway conditions to increase limonene production in 24 hours from 0.2 to 4.5 mM (23 to 610 mg/L). Finally, to demonstrate the modularity of this pathway, we also synthesized the biofuel precursors pinene and bisabolene. We anticipate that iPROBE will accelerate design-build-test cycles for metabolic engineering, enabling data-driven multiplexed cell-free methods for testing large combinations of biosynthetic enzymes to inform cellular design. Dudley, et al. -Cell-free prototyping of limonene biosynthesis 3 TOC Figure Highlights • Applied the iPROBE framework to build the nine-enzyme pathway to produce limonene • Assessed the impact of cofactors and 54 enzyme homologs on cell-free enzyme performance • Iteratively optimized the cell-free production of limonene by exploring more than 580 unique reactions • Extended pathway to biofuel precursors pinene and bisabolene Dudley, et al. -Cell-free prototyping of limonene biosynthesis 4

Modular cell-free expression plasmids to accelerate biological design in cells

“…Utilizing JGI’s gene synthesis program, the GG system was expanded to create recipient and donor vectors with varying promoters, such as P fdx ( 17 ), P pta ( 18 ), P pfor ( 19 ) and P wl ( 18 ). Additionally, GG sites were varied in recipient vectors to allow assembly of anywhere between two to six parts with varying promoters, resulting in a total of 58 modular vectors ( Supplementary Figure S1D and Table S3 ).…”

Modular cell-free expression plasmids to accelerate biological design in cells

“…Utilizing JGI’s gene synthesis program, the GG system was expanded to create recipient and donor vectors with varying promoters, such as P fdx 17 , P pta 18 , P pfor 19 , and P wl 18 . Additionally, GG sites were varied in recipient vectors to allow assembly of anywhere between two to six parts with varying promoters, resulting in a total of 58 modular vectors ( Supplementary Figure 1D ; Supplementary Table 3 ).…”

Industrial Biotechnology—An Industry at an Inflection Point

“…14 Third, datadriven, multiplexed cell-free systems are also accelerating biodiscovery and design. [15][16][17][18] Beyond systems biodesign tools, traditional catalysts continue to improve, further expanding the opportunities for bioconversion. All of these improvements tend to work together to both increase product range while simultaneously driving down cost.…”