The Constructive Black Queen hypothesis: new functions can evolve under conditions favouring gene loss.

Duplication is a major route for the emergence of new gene functions. However, the emergence of new gene functions via this route may be reduced in prokaryotes, as redundant genes are often rapidly purged. In lineages with compact, streamlined genomes, it thus appears challenging for novel function to emerge via duplication and divergence. A further pressure contributing to gene loss occurs under Black Queen dynamics, as cheaters that lose the capacity to produce a public good can instead acquire it from neighbouring producers. We propose that Black Queen dynamics can favour the emergence of new function because, under an emerging Black Queen dynamic, there is high gene redundancy spread across a community of interacting cells. Using computational modelling, we demonstrate that new gene functions can emerge under Black Queen dynamics. This result holds even if there is deletion bias due to low duplication rates and selection against redundant gene copies resulting from the high cost associated with carrying a locus. However, when the public good production costs are high, Black Queen dynamics impede the fixation of new functions. Our results expand the mechanisms by which new gene functions can emerge in prokaryotic systems.

An Engineered Glycerol Dehydratase With Improved Activity for the Conversion of meso-2,3-butanediol to Butanone.

There is substantial interest in engineering microorganisms to produce industrial chemicals that are currently derived from petroleum. One of these petrochemicals is butanone, which could be produced from microbially synthesized 2,3-butanediol through the action of a suitable dehydratase enzyme. Unfortunately, however, there are no known enzymes that natively catalyze this reaction. In this work, the authors set out to engineer the B12 -dependent glycerol dehydratase from Klebsiella pneumoniae (KpGDHt), in order to increase its activity for the conversion of meso-2,3-butanediol into butanone. The authors began by fusing the α and ß subunits of the enzyme, to simplify downstream high-throughput screening protocols. Serendipitously, the fusion protein showed a 20°C increase in its temperature optimum. Using this stabilized scaffold as a starting point, the authors employed the combinatorial active site saturation test and consensus-guided mutagenesis to randomize 28 residues within 12 Å of the KpGDHt active site. By screening over 5500 variants, the authors discovered a single point mutation (T200S) that increased the catalytic efficiency of meso-2,3-butanediol dehydration by four-fold, to a value of kcat /KM = 5.1 × 103 M-1 s-1 . Thus the authors report what is, to date, the most comprehensive mutagenesis and the largest engineered increase in catalytic efficiency on the B12 -dependent glycerol dehydratase scaffold.

Substitutions at the cofactor phosphate-binding site of a clostridial alcohol dehydrogenase lead to unexpected changes in substrate specificity.

Changing the cofactor specificity of an enzyme from nicotinamide adenine dinucleotide 2'-phosphate (NADPH) to the more abundant NADH is a common strategy for increasing overall enzyme efficiency in microbial metabolic engineering. The aim of this study was to switch the cofactor specificity of the primary-secondary alcohol dehydrogenase from Clostridium autoethanogenum, a bacterium with considerable promise for the bio-manufacturing of fuels and other petrochemicals, from strictly NADPH-dependent to NADH-dependent. We used insights from a homology model to build a site-saturation library focussed on residue S199, the position deemed most likely to disrupt binding of the 2'-phosphate of NADPH. Although the CaADH(S199X) library did not yield any NADH-dependent enzymes, it did reveal that substitutions at the cofactor phosphate-binding site can cause unanticipated changes in the substrate specificity of the enzyme. Using consensus-guided site-directed mutagenesis, we were able to create an enzyme that was stringently NADH-dependent, albeit with a concomitant reduction in activity. This study highlights the role that distal residues play in substrate specificity and the complexity of enzyme-cofactor interactions.

Reconstruction of an acetogenic 2,3-butanediol pathway involving a novel NADPH-dependent primary-secondary alcohol dehydrogenase.

Acetogenic bacteria use CO and/or CO2 plus H2 as their sole carbon and energy sources. Fermentation processes with these organisms hold promise for producing chemicals and biofuels from abundant waste gas feedstocks while simultaneously reducing industrial greenhouse gas emissions. The acetogen Clostridium autoethanogenum is known to synthesize the pyruvate-derived metabolites lactate and 2,3-butanediol during gas fermentation. Industrially, 2,3-butanediol is valuable for chemical production. Here we identify and characterize the C. autoethanogenum enzymes for lactate and 2,3-butanediol biosynthesis. The putative C. autoethanogenum lactate dehydrogenase was active when expressed in Escherichia coli. The 2,3-butanediol pathway was reconstituted in E. coli by cloning and expressing the candidate genes for acetolactate synthase, acetolactate decarboxylase, and 2,3-butanediol dehydrogenase. Under anaerobic conditions, the resulting E. coli strain produced 1.1 ± 0.2 mM 2R,3R-butanediol (23 µM h(-1) optical density unit(-1)), which is comparable to the level produced by C. autoethanogenum during growth on CO-containing waste gases. In addition to the 2,3-butanediol dehydrogenase, we identified a strictly NADPH-dependent primary-secondary alcohol dehydrogenase (CaADH) that could reduce acetoin to 2,3-butanediol. Detailed kinetic analysis revealed that CaADH accepts a range of 2-, 3-, and 4-carbon substrates, including the nonphysiological ketones acetone and butanone. The high activity of CaADH toward acetone led us to predict, and confirm experimentally, that C. autoethanogenum can act as a whole-cell biocatalyst for converting exogenous acetone to isopropanol. Together, our results functionally validate the 2,3-butanediol pathway from C. autoethanogenum, identify CaADH as a target for further engineering, and demonstrate the potential of C. autoethanogenum as a platform for sustainable chemical production.