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. TOC Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=50 SRC="FIGDIR/small/057737v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@19aa729org.highwire.dtl.DTLVardef@547698org.highwire.dtl.DTLVardef@13604b6org.highwire.dtl.DTLVardef@16f6718_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIApplied the iPROBE framework to build the nine-enzyme pathway to produce limonene C_LIO_LIAssessed the impact of cofactors and 54 enzyme homologs on cell-free enzyme performance C_LIO_LIIteratively optimized the cell-free production of limonene by exploring more than 580 unique reactions C_LIO_LIExtended pathway to biofuel precursors pinene and bisabolene C_LI