Metabolic engineering strategies for producing oleochemicals in bacteria

Finding a sustainable alternative for today’s petrochemical industry is a major challenge facing chemical engineers and society at large. To be sustainable, routes for converting carbon dioxide and light into organic compounds for use as both fuels and chemical building blocks must be identified, understood, and engineered. Advances in metabolic engineering, synthetic biology, and other bioengineering disciplines have expanded the scope of what can be produced in a living organism. As in other engineering disciplines, synthetic biologists want to apply a general understanding of science (e.g. microbiology and biochemistry) to construct complex systems from well-characterized parts (e.g. DNA and protein). Once novel synthetic biological systems (e.g. enzymes for biofuel synthesis) are constructed, they must be engineered to function inside evolving cells without negatively impacting the host’s physiology. In this talk, I will describe pathways for producing high-value commodity chemicals derived from fatty-acids and how my group and others have combined synthetic biology and systems biology to improve oleochemical production in bacteria using sustainable feedstocks. The talk will describe the critical regulatory points in native fatty acid metabolism, strategies for deregulating the pathway, and alternatives that by-pass it altogether. I will highlight the use of heterologous plant and bacterial enzymes to alter the chain length distribution of products from common long-chain molecules to higher-value medium-chain analogs. I will also highlight strategies that we have used to produce medium-chain fatty alcohols, the highest value compounds in the class, through engineering of thioesterase and thiolase driven pathways. I will conclude with commentary on the remaining barriers to commercializing these technologies and areas where further research investment could prove fruitful

Survey of the Endogonaceae in Minnesota With Synoptic Keys to Genera and Species

Sixteen species in the Endogonaceae (Zygomycotina) were identified from 22 different plant species from a native prairie, an intensively cultivated vegetable field, a reclaimed iron ore tailings basin, an undisturbed site adjacent to the iron ore tailings basin, and from a Pinus resinosa plantation. Seven species of Endogonaceae identified in this study are new records for Minnesota. Synoptic keys to genera and species are presented

Computational redesign of acyl-ACP thioesterase with improved selectivity towards medium chain fatty acids at high production levels

Enzyme and metabolic engineering offer the potential to develop biocatalysts for converting natural resources into a wide range of chemicals. To broaden the scope of potential products beyond natural metabolites, methods of engineering enzymes to accept alternative substrates and/or perform novel chemistries must be developed. DNA synthesis can create large libraries of enzyme-coding sequences, but most biochemistries lack a simple assay to screen for promising enzyme variants. Our solution to this challenge is structure-guided mutagenesis in which optimization algorithms select the best sequences from libraries based on specified criteria (i.e. binding selectivity). Our computational procedure was demonstrated by tuning substrate binding of the highly-active ‘TesA thioesterase in Escherichia coli in favor of medium-chain (C6-C12) lengths. Specifically, the Iterative Protein Redesign & Optimization procedure (IPRO) was used to design ‘TesA variants with enhanced C12- or C8specificity while maintaining high activity. After four rounds of structure-guided mutagenesis, we identified three thioesterases with enhanced production of dodecanoic acid (C12) and twenty-seven thioesterases with enhanced production of octanoic acid (C8), the fatty acid products of thioesterase-mediated catalysis. The top variants reached up to 49% C12 and 50% C8 while exceeding native levels of total free fatty acids. A similar sized library created through random mutagenesis failed to identify medium-chain specific, highly-active variants. The chain length-preference of ‘TesA and the best mutant were confirmed in vitro using acyl-CoA substrates. Molecular dynamics simulations, confirmed by resolved crystal structures, of ‘TesA variants suggest that hydrophobic forces govern ‘TesA substrate specificity. In this work, we not only successfully modified ‘TesA substrate preference but in doing so, we identified the third most C12-specific and tenth most C8-specific thioesterase characterized to date. These results are significant because medium-chain fatty acids are limited in natural abundance relative to long-chain fatty acids. This limited supply leads to high costs of C6-C10 oleochemicals such as fatty alcohols, amines, and esters. We expect that the new thioesterase variants will be useful to metabolic engineering projects aimed at sustainable production of medium-chain oleochemicals. Furthermore, we anticipate that the lessons learned from both successful and failed computational designs can guide algorithmic advancements aiding with future enzyme engineering endeavors

Getting the right clones in an automated manner : an alternative to sophisticated colony-picking robotics

In recent years, the design-build-test-learn (DBTL) cycle has become a key concept in strain engineering. Modern biofoundries enable automated DBTL cycling using robotic devices. However, both highly automated facilities and semi-automated facilities encounter bottlenecks in clone selection and screening. While fully automated biofoundries can take advantage of expensive commercially available colony pickers, semi-automated facilities have to fall back on affordable alternatives. Therefore, our clone selection method is particularly well-suited for academic settings, requiring only the basic infrastructure of a biofoundry. The automated liquid clone selection (ALCS) method represents a straightforward approach for clone selection. Similar to sophisticated colony-picking robots, the ALCS approach aims to achieve high selectivity. Investigating the time analogue of five generations, the model-based set-up reached a selectivity of 98 ± 0.2% for correctly transformed cells. Moreover, the method is robust to variations in cell numbers at the start of ALCS. Beside Escherichia coli , promising chassis organisms, such as Pseudomonas putida and Corynebacterium glutamicum , were successfully applied. In all cases, ALCS enables the immediate use of the selected strains in follow-up applications. In essence, our ALCS approach provides a ‘low-tech’ method to be implemented in biofoundry settings without requiring additional devices.German Research Foundation (DFG

The Drosophila melanogaster Apaf-1 homologue ARK is required for most, but not all, programmed cell death

The Apaf-1 protein is essential for cytochrome c–mediated caspase-9 activation in the intrinsic mammalian pathway of apoptosis. Although Apaf-1 is the only known mammalian homologue of the Caenorhabditis elegans CED-4 protein, the deficiency of apaf-1 in cells or in mice results in a limited cell survival phenotype, suggesting that alternative mechanisms of caspase activation and apoptosis exist in mammals. In Drosophila melanogaster, the only Apaf-1/CED-4 homologue, ARK, is required for the activation of the caspase-9/CED-3–like caspase DRONC. Using specific mutants that are deficient for ark function, we demonstrate that ARK is essential for most programmed cell death (PCD) during D. melanogaster development, as well as for radiation-induced apoptosis. ark mutant embryos have extra cells, and tissues such as brain lobes and wing discs are enlarged. These tissues from ark mutant larvae lack detectable PCD. During metamorphosis, larval salivary gland removal was severely delayed in ark mutants. However, PCD occurred normally in the larval midgut, suggesting that ARK-independent cell death pathways also exist in D. melanogaster

Terminal Ligand and Packing Effects on Slow Relaxation in an Isostructural Set of [Dy(H2_{2}dapp)X2_{2}]+ Single Molecule Magnets**

Three new dysprosium complexes with a pentadentate ligand occupying five equatorial sites differ only in the nature of the axial ligands. These help tune the relaxation properties as judged by an analysis of the AC susceptibility data. More in depth analysis by using two recently suggested fitting equations lead to similar outcomes for all three systems. As a further contribution to the relaxation pathway involving the phonon bath it is concluded that a short nitrate-nitrate interaction between molecules helps dampen the spin phonon coupling. We report three structurally related single ion Dy compounds using the pentadentate ligand 2,6-bis((E)-1-(2-(pyridin-2-yl)-hydrazineylidene)ethyl)pyridine (H$_{2}$dapp) [Dy(H$_{2}$dapp)(NO$_{3}$)$_{2}$]NO$_{3}$ (1), [Dy(H$_{2}$dapp)(OAc)$_{2}$]Cl (2) and [Dy(H$_{2}$dapp)(NO$_{3}$)$_{2}$]Cl$_{0.92}$(NO$_{3}$)$_{0.08}$ (3). The (H$_{2}$dapp) occupies a helical twisted pentagonal equatorial arrangement with two anionic ligands in the axial positions. Further influence on the electronic and magnetic structure is provided by a closely associated counterion interacting with the central N−H group of the (H$_{2}$dapp). The slow relaxation of the magnetisation shows that the anionic acetates give the greatest slowing down of the magnetisation reversal. Further influence on the relaxation properties of compounds1 and 2 is the presence of short nitrate-nitrate intermolecular ligand contact opening further lattice relaxation pathways

Growing Broccoli and Cauliflower in Minnesota

This archival publication may not reflect current scientific knowledge or recommendations. Current information available from the University of Minnesota Extension: https://www.extension.umn.edu

A cross-sectional survey of the access of older people in the Scottish Highlands to general medical practices, community pharmacies and prescription medicines.

Access to medicines and healthcare is more problematic in remote and rural areas. The purpose of this study was to quantify issues of access to general practitioners (GPs), community pharmacies and prescribed medicines in older people resident in the Scottish Highlands. Anonymized questionnaires were mailed to a random sample of 2000 older people (≥60 years) resident in the Scottish Highlands. Questionnaire items were: access and convenience to GP and pharmacy services (10 items); prescribed medicines (13 items); attitudinal statements based on the Theoretical Domains Framework (12 items); quality of life (SF8, 8 items); and demographics (12 items). Results were analysed using descriptive, inferential and spatial statistics, and principal component analysis (PCA) of attitudinal items. With a response rate of 54.2%, the majority reported convenient access to GPs (89.1%) and community pharmacies (84.3%). Older age respondents (p < 0.0001) were more likely to state that their access to GP services was not convenient and those in rural areas to community pharmacies (p < 0.01). For access to prescribed medicines, those in poorer health (p < 0.001) and taking five or more regular prescribed medicines (p = 0.002) were more likely to state access not convenient. PCA identified three components of beliefs of capabilities, emotions and memory. Those with poorer health had more negative scores for all (p < 0.001). Those reporting issues of access to prescribed medicines had more negative scores for beliefs of capabilities (p < 0.001) while those of older age, living alone, and taking five or more regular prescribed medicines (all p < 0.001) had more negative scores for emotions. In conclusion, while the majority of respondents have convenient access to their GP practice, pharmacy and prescribed medicines, there is a need for further review of the pharmaceutical care of those of older age with poorer health, living alone in the more remote and rural areas and taking five or more prescribed medicines

Commercial Vegetable Weed, Insect, and Disease Control Guide: Beets, Carrots, Lettuce, Onions, Parsnips, Radishes, Rutabagas, Turnips, Spinach (Revised 1983)

This archival publication may not reflect current scientific knowledge or recommendations. Current information available from the University of Minnesota Extension: https://www.extension.umn.edu