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Noncanonical redox cofactors are attractive low-cost alternatives to nicotinamide adenine dinucleotide (phosphate) (NAD(P)+) in biotransformation. However, engineering enzymes to utilize them is challenging. Here, we present a high-throughput directed evolution platform which couples cell growth to the in vivo cycling of a noncanonical cofactor, nicotinamide mononucleotide (NMN+). We achieve this by engineering the life-essential glutathione reductase in Escherichia coli to exclusively rely on the reduced NMN+ (NMNH). Using this system, we develop a phosphite dehydrogenase (PTDH) to cycle NMN+ with ~147-fold improved catalytic efficiency, which translates to an industrially viable total turnover number of ~45,000 in cell-free biotransformation without requiring high cofactor concentrations. Moreover, the PTDH variants also exhibit improved activity with another structurally deviant noncanonical cofactor, 1-benzylnicotinamide (BNA+), showcasing their broad applications. Structural modeling prediction reveals a general design principle where the mutations and the smaller, noncanonical cofactors together mimic the steric interactions of the larger, natural cofactors NAD(P)+.

BackgroundNoncanonical redox cofactors are emerging as important tools in cell-free biosynthesis to increase the economic viability, to enable exquisite control, and to expand the range of chemistries accessible. However, these noncanonical redox cofactors need to be biologically synthesized to achieve full integration with renewable biomanufacturing processes.ResultsIn this work, we engineered Escherichia coli cells to biosynthesize the noncanonical cofactor nicotinamide mononucleotide (NMN+), which has been efficiently used in cell-free biosynthesis. First, we developed a growth-based screening platform to identify effective NMN+ biosynthetic pathways in E. coli. Second, we explored various pathway combinations and host gene disruption to achieve an intracellular level of ~ 1.5 mM NMN+, a 130-fold increase over the cell's basal level, in the best strain, which features a previously uncharacterized nicotinamide phosphoribosyltransferase (NadV) from Ralstonia solanacearum. Last, we revealed mechanisms through which NMN+ accumulation impacts E. coli cell fitness, which sheds light on future work aiming to improve the production of this noncanonical redox cofactor.ConclusionThese results further the understanding of effective production and integration of NMN+ into E. coli. This may enable the implementation of NMN+-directed biocatalysis without the need for exogenous cofactor supply.

It is challenging to biosynthesize industrially important aldehydes, which are readily consumed by the numerous alcohol dehydrogenases (ADHs) in cells. In this work, we demonstrate that a nicotinamide mononucleotide (NMN+)-dependent redox cofactor cycling system enables aldehyde accumulation in Escherichia coli crude lysates and whole cells. By specifically delivering reducing power to a recombinant enoate reductase, but not to endogenous ADHs, we convert citral to citronellal with minimal byproduct formation (97-100% and 83% product purity in crude lysate- and whole cell-based biotransformation, respectively). We envision the system's universal application to lowering the noise in biomanufacturing by silencing the host's metabolic background.

BackgroundNoncanonical redox cofactors are emerging as important tools in cell-free biosynthesis to increase the economic viability, to enable exquisite control, and to expand the range of chemistries accessible. However, these noncanonical redox cofactors need to be biologically synthesized to achieve full integration with renewable biomanufacturing processes.ResultsIn this work, we engineered Escherichia coli cells to biosynthesize the noncanonical cofactor nicotinamide mononucleotide (NMN+), which has been efficiently used in cell-free biosynthesis. First, we developed a growth-based screening platform to identify effective NMN+ biosynthetic pathways in E. coli. Second, we explored various pathway combinations and host gene disruption to achieve an intracellular level of ~ 1.5 mM NMN+, a 130-fold increase over the cell's basal level, in the best strain, which features a previously uncharacterized nicotinamide phosphoribosyltransferase (NadV) from Ralstonia solanacearum. Last, we revealed mechanisms through which NMN+ accumulation impacts E. coli cell fitness, which sheds light on future work aiming to improve the production of this noncanonical redox cofactor.ConclusionThese results further the understanding of effective production and integration of NMN+ into E. coli. This may enable the implementation of NMN+-directed biocatalysis without the need for exogenous cofactor supply.

Biological production of chemicals often requires the use of cellular cofactors, such as nicotinamide adenine dinucleotide phosphate (NADP+). These cofactors are expensive to use in vitro and difficult to control in vivo. We demonstrate the development of a noncanonical redox cofactor system based on nicotinamide mononucleotide (NMN+). The key enzyme in the system is a computationally designed glucose dehydrogenase with a 107-fold cofactor specificity switch toward NMN+ over NADP+ based on apparent enzymatic activity. We demonstrate that this system can be used to support diverse redox chemistries in vitro with high total turnover number (~39,000), to channel reducing power in Escherichia coli whole cells specifically from glucose to a pharmaceutical intermediate, levodione, and to sustain the high metabolic flux required for the central carbon metabolism to support growth. Overall, this work demonstrates efficient use of a noncanonical cofactor in biocatalysis and metabolic pathway design.

Biological production of chemicals often requires the use of cellular cofactors, such as nicotinamide adenine dinucleotide phosphate (NADP+). These cofactors are expensive to use in vitro and difficult to control in vivo. We demonstrate the development of a noncanonical redox cofactor system based on nicotinamide mononucleotide (NMN+). The key enzyme in the system is a computationally designed glucose dehydrogenase with a 107-fold cofactor specificity switch toward NMN+ over NADP+ based on apparent enzymatic activity. We demonstrate that this system can be used to support diverse redox chemistries in vitro with high total turnover number (~39,000), to channel reducing power in Escherichia coli whole cells specifically from glucose to a pharmaceutical intermediate, levodione, and to sustain the high metabolic flux required for the central carbon metabolism to support growth. Overall, this work demonstrates efficient use of a noncanonical cofactor in biocatalysis and metabolic pathway design.

The Ehrlich pathway is a major route for the renewable production of higher alcohols. However, the product scope of the Ehrlich pathway is restricted, and the product selectivity is suboptimal. Here, we demonstrate that a Coenzyme A (CoA) detour, which involves conversion of the 2-keto acids into acyl-CoAs, expands the biological toolkit of reaction chemistries available in the Ehrlich pathway to include the gamut of CoA-dependent enzymes. As a proof-of-concept, we demonstrated the first biosynthesis of a tertiary branched-alcohol, pivalcohol, at a level of ∼10 mg/L from glucose in Escherichia coli, using a pivalyl-CoA mutase from Xanthobacter autotrophicus. Furthermore, engineering an enzyme in the CoA detour, the Lactobacillus brevis CoA-acylating aldehyde dehydrogenase, allowed stringent product selectivity. Targeted production of 3-methyl-1-butanol (3-MB) in E. coli mediated by the CoA detour showed a 3-MB:side-product (isobutanol) ratio of >20, an increase over the ratios previously achieved using the conventional Ehrlich pathway.

The Ehrlich pathway is a major route for the renewable production of higher alcohols. However, the product scope of the Ehrlich pathway is restricted, and the product selectivity is suboptimal. Here, we demonstrate that a Coenzyme A (CoA) detour, which involves conversion of the 2-keto acids into acyl-CoAs, expands the biological toolkit of reaction chemistries available in the Ehrlich pathway to include the gamut of CoA-dependent enzymes. As a proof-of-concept, we demonstrated the first biosynthesis of a tertiary branched-alcohol, pivalcohol, at a level of ∼10 mg/L from glucose in Escherichia coli, using a pivalyl-CoA mutase from Xanthobacter autotrophicus. Furthermore, engineering an enzyme in the CoA detour, the Lactobacillus brevis CoA-acylating aldehyde dehydrogenase, allowed stringent product selectivity. Targeted production of 3-methyl-1-butanol (3-MB) in E. coli mediated by the CoA detour showed a 3-MB:side-product (isobutanol) ratio of >20, an increase over the ratios previously achieved using the conventional Ehrlich pathway.

Digital-tablets distribute cognition through visual, auditoryand haptic interactivity. We designed a tutor-game thatexplored how narratives ((S)trong/(W)eak) and gestures((I)conic/(D)eictic) could be combined to situate embodiedlearning. Students played seven levels of a fractions gamedesigned to teach them how to create and compare fractions.One hundred thirty-one students (N=131, age x̄ =8.78 yrs,52.6% Female) were randomly assigned to one of four groups(SI, SD, WI, WD) in a 2x2 factorial experiment. Studentscompleted pre/post direct and transfer assessments and tutor-game log data was mined to explore characteristics ofstudents learning. Results revealed a significant interactionbetween narrative and gesture moderated by studentproficiency. In effect, students new to fractions performedbetter in an abstract environment using deictic (pointing)gestures. However, as students' proficiencies improved, theylearned better using iconically enactive gestures in strongnarrative with setting, characters and a plot. This hasimportant implications for designing adaptive learningplatforms and curricula for teaching fractions.

Muscle-derived progenitor cell (myoblast) therapy has promise for the treatment of denervated, weakened, and fibrotic muscle. The best methods for injecting myoblasts to promote fusion and retention have yet to be determined, however. Mesenchymal stem/stromal cells have also been reported to have beneficial effects in restoring damaged tissue, through increasing vascularization and reducing inflammation. The interactions between human primary skeletal myoblasts and bone marrow-derived mesenchymal stem/stromal cells were examined using time-lapse images put into video format. Of interest, there is a high degree of cell-to-cell interaction with microparticles transferring between both cell types, and formation of nanotubules to bridge cytoplasmic contents between the two types of cell. This model provides an in vitro platform for examining mechanisms for cell-to-cell interaction preceding myoblast fusion.

Muscle-derived progenitor cell (myoblast) therapy has promise for the treatment of denervated, weakened, and fibrotic muscle. The best methods for injecting myoblasts to promote fusion and retention have yet to be determined, however. Mesenchymal stem/stromal cells have also been reported to have beneficial effects in restoring damaged tissue, through increasing vascularization and reducing inflammation. The interactions between human primary skeletal myoblasts and bone marrow-derived mesenchymal stem/stromal cells were examined using time-lapse images put into video format. Of interest, there is a high degree of cell-to-cell interaction with microparticles transferring between both cell types, and formation of nanotubules to bridge cytoplasmic contents between the two types of cell. This model provides an in vitro platform for examining mechanisms for cell-to-cell interaction preceding myoblast fusion.

Researchers developed Iteration-1 of a digital tablet tutorgameexploring the impact of narratives (strong (S) vs. weak(W)) and gestural mechanics (conceptual (C) vs. deictic (D))on players’ understanding of mathematical fractions. In atwo-by-two factorial design, 3rd, 4th, and 5th grade elementarystudents at an afterschool program in Harlem, NYC(NTTL=72; x̄AGE=10.31 years [1.64], 67% female) wererandomly assigned to play one of the four tutor-gameenvironments (SC, SD, WC, WD). Pre/post scores on formalfractions assessments showed significant learning for allgroups. Tutor-log data revealed that students usingconceptual gestures were significantly more accurate atestimating and denominating fractions than students usingdeictic gestures. Observational notes, student exit surveys andclinical interviews corroborated that many students used thetutors’ gestures in their explanations of fractions. Thiscollection of data is used to discuss the impact of gesture andnarrative on learning fractions and digital-tutor design.

Students’ beliefs about a subject influence their comprehension and learning of that subject (Ornek et. al., 2008).Many students consider science as a difficult subject to learn. Therefore, this study explored a new way in helping elementarychildren understand abstract science concepts using embodiment, or physically moving their own bodies. Students engagedin activities that helped them learn about abstract science concepts by physically performing tasks related to these scienceconcepts. The purpose of this study was to examine the importance and role of embodiment in students’ understanding andmotivation in elementary science learning. The results provide evidence to suggest that embodiment has remarkable potentialto enhance both children’s understanding and motivation in abstract scientific concepts through the use embodiment.

Lehnert (1980) proposed a model thematic knowledge structures called 'plot units', which are structurally deflned sequences of mental states, positive events, and negative events In a clustering experiment, subjects were asked to sort 36 stories into groups. These groups were labeled by the subjects, and that data used to identify the nature of each mental category. Plot units generally provided a good fit to the clustering patterns in the data, with higher level clusters corresponding to discriminations on the nature of the outcome and judgements about the 'fairness' of the protagonist.

We examined the reasons people give for the actions in routine events and classified them as action enablement, main goal satisfaction, or external. We argue that the external reasons indicate that some actions in events are stored at more general lev- els in memory than the specific event schemas

Two experiments investigated the effects of the number of interesting events in a story on memory for that story. The results differed depending on whether or not the story followed a stereotypical script. For script-based stories, adding interesting events spaced throughout the stories decreased the stories' memorability. However, for non-script-based stories, adding a few interesting events aided memory for the stories, but adding too many decreased memory. These results were interpreted in terms of a limited resource model of story understanding limited resource model of story understanding that focusses processing on interesting events.