In vitro prototyping and rapid optimization of biosynthetic enzymes for cell design.

The design and optimization of biosynthetic pathways for industrially relevant, non-model organisms is challenging due to transformation idiosyncrasies, reduced numbers of validated genetic parts and a lack of high-throughput workflows. Here we describe a platform for in vitro prototyping and rapid optimization of biosynthetic enzymes (iPROBE) to accelerate this process. In iPROBE, cell lysates are enriched with biosynthetic enzymes by cell-free protein synthesis and then metabolic pathways are assembled in a mix-and-match fashion to assess pathway performance. We demonstrate iPROBE by screening 54 different cell-free pathways for 3-hydroxybutyrate production and optimizing a six-step butanol pathway across 205 permutations using data-driven design. Observing a strong correlation (r = 0.79) between cell-free and cellular performance, we then scaled up our highest-performing pathway, which improved in vivo 3-HB production in Clostridium by 20-fold to 14.63 ± 0.48 g l-1. We expect iPROBE to accelerate design-build-test cycles for industrial biotechnology.

First Biochemical Characterization of a Methylcitric Acid Cycle from Bacillus subtilis Strain 168.

The genome of Bacillus subtilis strain 168 contains the mother cell metabolic gene (mmg) operon that encodes homologues from the methylcitric acid cycle. We showed that the three genes, mmgDE and yqiQ(mmgF), provide three of the five steps of the methylcitric acid cycle. We also showed that the fourth step can be supplied by citB (aconitase), and we suggest that the fifth missing step, the propionyl-CoA synthetase, is probably skipped because the ß-oxidation of methyl-branched fatty acids by the enzymes encoded by mmgABC should produce propionyl-CoA. We also noted interesting enzymology for MmgD and MmgE. First, MmgD is a bifunctional citrate synthase/2-methylcitrate synthase with 2.3-fold higher activity as a 2-methylcitrate synthase. This enzyme catalyzes the formation of either (2S,3R)- or (2R,3S)-2-methylcitrate, but reports of 2-methylcitrate synthases from other species indicated that they produced the (2S,3S) isomer. However, we showed that MmgD and PrpC (from Escherichia coli) in fact produce the same stereoisomer. Second, the MmgE enzyme is not a stereospecific 2-methylcitrate dehydratase because it can dehydrate at least two of the four diastereomers of 2-methylcitrate to yield either (E)-2-methylaconitate or (Z)-2-methylaconitate. We also showed for the first time that the E. coli homologue PrpD exhibited the same lack of stereospecificity. However, the physiological pathways proceed via (Z)-2-methylaconitate, which served as the substrate for the citB enzyme in the synthesis of 2-methylisocitrate. We completed our characterization of this pathway by showing that the 2-methylisocitrate produced by CitB is converted to pyruvate and succinate by the enzyme YqiQ(MmgF).