Explore the vast range of titles available.

Recent attempts to create synthetic Escherichia coli methylotrophs identified that de novo biosynthesis of amino acids, in the presence of methanol, presents significant challenges in achieving autonomous methylotrophic growth. Previously engineered methanol-dependent strains required co-utilization of stoichiometric amounts of co-substrates and methanol. As such, these strains could not be evolved to grow on methanol alone. In this work, we have explored an alternative approach to enable biosynthesis of all amino acids from methanol-derived carbon in minimal media without stoichiometric coupling. First, we identified that biosynthesis of threonine was limiting the growth of our methylotrophic E. coli. To address this, we performed adaptive laboratory evolution to generate a strain that grew efficiently in minimal medium with methanol and threonine. Methanol assimilation and growth of the evolved strain were analyzed, and, interestingly, we found that the evolved strain synthesized all amino acids, including threonine, from methanol-derived carbon. The evolved strain was then further engineered through overexpression of an optimized threonine biosynthetic pathway. We show that the resulting methylotrophic E. coli strain has a methanol-dependent growth phenotype with homoserine as co-substrate. In contrast to previous methanol-dependent strains, co-utilization of homoserine is not stoichiometrically linked to methanol assimilation. As such, future engineering of this strain and successive adaptive evolution could enable autonomous growth on methanol as the sole carbon source.Key points• Adaptive evolution of E. coli enables biosynthesis of all amino acids from methanol.• Overexpression of threonine biosynthesis pathway improves methanol assimilation.• Methanol-dependent growth is seen in minimal media with homoserine as co-substrate.

Educational experiences rooted in community engagement offer a powerful and effective method by which to prepare students as the next generation of active citizens. This study critically analyzes and illustrates the potential of using community engagement as the focus of an informal curriculum in an Asian university’s living-and-learning undergraduate residential college program designed to prepare the next generation of active citizens. Grounded in empirical evidence from four academic years (2013/14–2016/17) and using the active citizenship and community-based learning theoretical frameworks, this research study systematically evaluates the contribution of hands-on community engagement in undergraduate learning and development. Specifically, conceptual codes were derived from the stated informal learning outcomes related to citizenship competencies, to map the extent of achievement of the target outcomes and objectives of the engagement activities over time. Results from this quasi-quantitative content analysis of 89 programs, involving more than 80% of the College students during each academic year, confirm and complicate our understanding of how critical citizenship competencies of awareness, empathy, deeper understanding and hard and soft skills are achieved. Moreover, findings also highlight how perceptions on learning through active community engagement differ between activities/events in shaping the development of active citizenship competencies. The study findings have ramifications for policies related to community-engagement-based learning in higher education.

Patients with chronic kidney disease (CKD) face elevated fracture incidences, but mechanisms underlying CKD-related bone loss remain unclear. Using the adenine-induced chronic kidney injury (AdKI) murine model, we identified that AdKI induces dysregulated glucose metabolism in bones and kidneys via and metabolic tracing. C-metabolic tracing of osteocyte-enriched femora revealed accelerated citrate production from [1,2- C]-glucose and [U- C]-glutamine in AdKI mice. These metabolic changes were observed together with increased circulating citrate and overexpression in bones from AdKI mice. Thus, to explore the role of citrate in AdKI, we utilized mice harboring a loss of function mutation in the citrate importer SLC13A5 ( ). Mutant mice displayed elevated osteocytic citrate production, and elevated circulating citrate, without significantly worsened AdKI-related bone loss. Coincident with this, mutant mice were significantly protected from loss of kidney function with attenuated AdKI-induced nephrolithiasis. We also confirmed that is highly expressed in cortical bone compared to the kidney, suggesting the effect of the mutation is mediated by SLC13A5's function outside the kidney. Altogether, this study finds that accelerated osteocytic citrate production in CKD is associated with protection of kidney function, and modulation of citrate handling may be a site for therapeutic intervention in CKD.

The emergence of various omics techniques in biotechnology research has enabled investigators to study altered phenotypes of microorganisms, including the workhorse microbe, Escherichia coli, at the molecular level. This includes the development of 13C-isotopic tracing methods and 13C-metabolic flux analysis (13C-MFA), which facilitates the interrogation of in vivo metabolic fluxes. Additionally, tools have been developed to more easily genetically engineer E. coli strains with diverse metabolic phenotypes, and adaptive laboratory evolution (ALE) has been increasingly employed as part of these bioengineering strategies. Yet, the principles guiding ALE remain poorly articulated and further basic studies into how metabolic changes take place over the course of ALE are needed. This Thesis seeks to investigate and describe metabolic flux rewiring following ALE in various E. coli strains. First, ALE was applied with the goal of engineering methylotrophic E. coli strains with improved methanol-utilization capabilities. This was undertaken to develop E. coli synthetic methylotrophs that can convert methanol or other reduced C1 compounds to useful platform chemicals. In the first case, a methanol-auxotrophic E. coli methylotroph that requires glucose as a co-substrate was designed and optimized through ALE. Another synthetic E. coli methylotroph was adaptively evolved on threonine and methanol to tune its ability to employ methanol in biosynthesis. 13C-tracing methods were employed to quantitatively interrogate changes in methanol metabolism following ALE in both engineered E. coli methylotrophs. Next, in order to improve basic understanding of how E. coli metabolism is regulated, 13C-MFA was performed on 21 E. coli knockout strains – 6 strains which contain one transcription factor knockout each and 15 strains containing two transcription factor knockouts – all grown at the exponential phase in aerobic conditions and abundance of glucose. These 6 transcription factors exert significant regulatory effect on the central carbon metabolism of E. coli and were hence selected for this study. The data generated here adds to a set of metabolic fluxes previously generated from 45 E. coli central carbon metabolism knockout strains (CCK strains). To better understand the dynamics of how metabolic fluxes are rewired after ALE, additional multi-omics analyses were performed on 5 CCK strains that were subjected to ALE by growing them at the exponential phase in aerobic conditions and excess glucose. These 5 CCK strains, ∆pfkA, ∆rpe, ∆aceEF, ∆acnB, and ∆sucB, were specifically selected as the introduction of these knockouts dramatically altered the metabolic phenotype of these strains with respect to wild-type E. coli. Following ALE, all strains demonstrated an improvement in growth rate. 13C-MFA revealed significant changes in metabolism following ALE. The associations between changes in metabolic phenotypes and mutations accumulated over the course of ALE were revealed by evaluating genomic sequencing data of multiple strains evolved in parallel. The comprehensive fluxomic data sets from this study were then harmonized together with a previous data set involving evolved ∆pgi strains to reveal broad characteristics of E. coli metabolism that emerge when data from multiple CCK strains are analyzed together. It is envisioned that further systematic studies, with even larger data sets, will build toward a more complete understanding of E. coli metabolism and the impact of ALE. Knowledge from such endeavors will benefit future E. coli strain designs. Additionally, similar analytical methods and approaches can also be translated to other organisms of interest.