Ichiro Matsumura

Natural evolution has been creating new complex systems for billions of years. The process is spontaneous and requires neither intelligence nor moral purpose but is nevertheless difficult to understand. The late Dan Tawfik spent years studying enzymes as they adapted to recognize new substrates. Much of his work focused on gaining fundamental insights, so the practical utility of his experiments may not be obvious even to accomplished protein engineers. Here we focus on two questions fundamental to any directed evolution experiment. Which proteins are the best starting points for such experiments? Which trait(s) of the chosen parental protein should be evolved to achieve the desired outcome? We summarize Tawfik's contributions to our understanding of these problems, to honor his memory and encourage those unfamiliar with his ideas to read his publications.

Aims: New methods of DNA recombination that capture the principal advantages of the BioBrick standard (ease of design) and Golden Gate assembly (decreased labor) are demonstrated here. Methods & ma... Method summary Plasmids that comply with the BioBrick standard are used to transform Escherichia coli cells carrying DNA methyltransferase expression vectors. The methylated plasmids are purified a...

The BioBrick standard makes possible iterated pairwise assembly of cloned parts without any depletion of unique restriction sites. Every part that conforms to the standard is compatible with every other part, thereby fostering a worldwide user community. The assembly methods, however, are labor intensive or inefficient compared to some newer ones so the standard may be falling out of favor. An easier way to assemble BioBricks is described herein. Plasmids encoding BioBrick parts are purified from Escherichia coli cells that express a foreign site-specific DNA methyltransferase, so that each is subsequently protected in vitro from the activity of a particular restriction endonuclease. Each plasmid is double-digested and all resulting restriction fragments are ligated together without gel purification. The ligation products are subsequently double-digested with another pair of restriction endonucleases so only the desired insert-recipient vector construct retains the capacity to transform E. coli. This 4R/2M BioBrick assembly protocol is more efficient and accurate than established workflows including 3A assembly. It is also much easier than gel purification to miniaturize, automate and perform more assembly reactions in parallel. As such, it should streamline DNA assembly for the existing community of BioBrick users, and possibly encourage others to join.

The phenotypes conferred by recombinant plasmids upon host cells often exhibit variability between replicate populations. This statistical noise is mostly a consequence of adaptive evolution in response to fitness burdens imposed by the plasmids themselves. We developed a novel strategy, ‘ribosome pegging’, to exclude common unwanted mutations that benefit host cells at the expense of heterologous gene expression. Plasmids that constitutively co-expressed the fluorescent reporter tagRFP and ribosomal protein L23 (rplW) were used to transform Escherichia coli cells that lacked the essential chromosomal rplW gene. Cells within the population that expressed too little L23, or too much, were evidently inviable. Ribosome pegging obviates the need for antibiotics, thus facilitating the deployment of recombinant bacteria in uncontrolled environments. We show that ribosome-pegged E. coli carrying a plasmid that constitutively expresses L23 and an artificially evolved enzyme protects fruit flies from otherwise toxic doses of the insecticide malathion.

Fruit flies recognize hundreds of ecologically relevant odors and respond appropriately to them. The complexity, redundancy and interconnectedness of the olfactory machinery complicate efforts to pinpoint the functional contributions of any component neuron or receptor to behavior. Some contributions can only be elucidated in flies that carry multiple mutations and transgenes, but the production of such flies is currently labor-intensive and time-consuming. Here, we describe a set of transgenic flies that express the Saccharomyces cerevisiae GAL80 in specific olfactory sensory neurons (OrX-GAL80s). The GAL80s effectively and specifically subtract the activities of GAL4-driven transgenes that impart anatomical and physiological phenotypes. OrX-GAL80s can allow researchers to efficiently activate only one or a few types of functional neurons in an otherwise nonfunctional olfactory background. Such experiments will improve our understanding of the mechanistic connections between odorant inputs and behavioral outputs at the resolution of only a few functional neurons.

Fruit flies recognize hundreds of ecologically relevant odors and respond appropriately to them. The complexity, redundancy and interconnectedness of the olfactory machinery complicate efforts to pinpoint the functional contributions of any component neuron or receptor to behavior. Some contributions can only be elucidated in flies that carry multiple mutations and transgenes, but the production of such flies is currently labor-intensive and time-consuming. Here, we describe a set of transgenic flies that express the Saccharomyces cerevisiae GAL80 in specific olfactory sensory neurons ( OrX-GAL80s ). The GAL80s effectively and specifically subtract the activities of GAL4-driven transgenes that impart anatomical and physiological phenotypes. OrX-GAL80s can allow researchers to efficiently activate only one or a few types of functional neurons in an otherwise nonfunctional olfactory background. Such experiments will improve our understanding of the mechanistic connections between odorant inputs and behavioral outputs at the resolution of only a few functional neurons.

Metabolic engineers develop inexpensive enantioselective syntheses of high-value compounds, but their designs are sometimes confounded by the misfolding of heterologously expressed proteins. Geobacillus stearothermophilus NUB3621 is a readily transformable facultative thermophile. It could be used to express and properly fold proteins derived from its many mesophilic or thermophilic Bacillaceae relatives or to direct the evolution of thermophilic variants of mesophilic proteins. Moreover, its capacity for high-temperature growth should accelerate chemical transformation rates in accordance with the Arrhenius equation and reduce the risks of microbial contamination. Its tendency to sporulate in response to nutrient depletion lowers the costs of storage and transportation. Here, we present a draft genome sequence of G. stearothermophilus NUB3621 and describe inducible and constitutive expression plasmids that function in this organism. These tools will help us and others to exploit the natural advantages of this system for metabolic engineering applications.

Metabolic engineers strive to improve the production yields of microbial fermentations, sometimes by mutating the genomes of production strains. Some mutations are detrimental to the health of the organism, so a quantitative and mechanistic understanding of the trade-offs could inform better designs. We employed the bacterial luciferase operon (luxABCDE), which uses ubiquitous energetic cofactors (NADPH, ATP, FMNH₂, acetyl-CoA) from the host cell, as a proxy for a novel anabolic pathway. The strains in the Escherichia coli Keio collection, each of which contains a single deletion of a non-essential gene, represent mutational choices that an engineer might make to optimize fermentation yields. The Keio strains and the parental BW25113 strain were transformed with a luxABCDE expression vector. Each transformant was propagated in defined M9 medium at 37°C for 48 hours; the cell density (optical density at 600 nanometers, OD₆₀₀) and luminescence were measured every 30 minutes. The trade-offs were visualized by plotting the maximum growth rate and luminescence/OD₆₀₀ of each transformant across a “production possibility frontier”. Our results show that some loss-of-function mutations enhance growth in vitro or light production, but that improvement in one trait generally comes at the expense of the other.

The rules that govern adaptive protein evolution remain incompletely understood. Aminoglycoside aminotransferase (3′) type IIIa (hereafter abbreviated APH(3′)-IIIa) is a good model enzyme because it inactivates kanamycin efficiently; it recognizes other aminoglycoside antibiotics, including amikacin, but not nearly as well. Here we direct the evolution of APH(3′)-IIIa variants with increased activity against amikacin. After four rounds of random mutation and selection in Escherichia coli, the minimum inhibitory concentration of amikacin rose from 18 micrograms/mL (wild-type enzyme) to over 1200 micrograms/mL (clone 4.1). The artificially evolved 4.1 APH(3′)-IIIa variant exhibited 19-fold greater catalytic efficiency (k_cat/K_M) than did the wild-type enzyme in reactions with amikacin. E. coli expressing the evolved 4.1 APH(3′)-IIIa also exhibited a four-fold decrease in fitness (as measured by counting colony forming units in liquid cultures with the same optical density) compared with isogenic cells expressing the wild-type protein under non-selective conditions. We speculate that these fitness costs, in combination with the prevalence of other amikacin-modifying enzymes, hinder the evolution of APH(3′)-IIIa in clinical settings.

Many enzymes exhibit some catalytic promiscuity or substrate ambiguity. These weak activities do not affect the fitness of the organism under ordinary circumstances, but can serve as potential evolutionary precursors of new catalytic functions. We wondered whether different proteins with the same substrate ambiguous activity evolve differently under identical selection conditions. Patrick et al. (Patrick WM, Quandt EM, Swartzlander DB, Matsumura I. 2007. Multicopy suppression underpins metabolic evolvability. Mol Biol Evol. 24:2716–2722.) previously showed that three multicopy suppressors, gph, hisB, and ytjC, rescue ΔserB Escherichia coli cells from starvation on minimal media. We directed the evolution of variants of Gph, histidinol phosphatase (HisB), and YtjC that complemented ΔserB more efficiently, and characterized the effects of the amino acid changes, alone and in combination, upon the evolved phosphoserine phosphatase (PSP) activity. Gph and HisB are members of the HAD superfamily of hydrolases, but they adapted through different, kinetically distinguishable, biochemical mechanisms. All of the selected mutations, except N102T in YtjC, proved to be beneficial in isolation. They exhibited a pattern of antagonistic epistasis, as their effects in combination upon the kinetic parameters of the three proteins in reactions with phosphoserine were nonmultiplicative. The N102T mutation exhibited sign epistasis, as it was deleterious in isolation but beneficial in the context of other mutations. We also showed that the D57N mutation in the chromosomal copy of hisB is sufficient to suppress the ΔserB deletion. These results in combination show that proteomes can offer multiple mechanistic solutions to a molecular recognition problem.