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Metabolic Engineering

Lemeng Dong, Jacob Pollier, Jean-Etienne Bassard, Georgios Ntallas, Aldo Almeida, Eleni Lazaridi, Bekzod Khakimov, Philipp Arendt, Louisi Souza de Oliveira, Frédéric Lota, Alain Goossens, Franck Michoux, Søren Bak
Triterpene cyclases catalyze the first committed step in triterpene biosynthesis, by forming mono- to pentacyclic backbone structures from oxygenated C30 isoprenoid precursors. Squalene epoxidase precedes this cyclization by providing the oxygenated and activated substrate for triterpene biosynthesis. Three squalene epoxidases from Cucurbita pepo (CpSEs) were isolated and shown to have evolved under purifying selection with signs of sites under positive selection in their N- and C-termini. They all localize to the Endoplasmic Reticulum (ER) and produce 2,3-oxidosqualene and 2,3:22,23-dioxidosqualene when expressed in a yeast erg1 (squalene epoxidase) erg7 (lanosterol synthase) double mutant...
July 14, 2018: Metabolic Engineering
Tianyi Li, Xiuqi Chen, Yizhi Cai, Junbiao Dai
Scaffold proteins influence cellular signaling by orchestrating multiple enzymes, receptors or ion channels, and could be tailored to enhance the efficiency of biochemical reactions by positioning related enzymes physically together. However, the number of applicable domains remains small, and the construction of scaffold proteins with optimal domain ratio could be tedious and time-consuming. In this study, we outlined a modular design to quickly assemble scaffold proteins using protein interaction domains, which have been constructed into a standardized vector...
July 13, 2018: Metabolic Engineering
Kyle J Lauersen, Julian Wichmann, Thomas Baier, Sotirios C Kampranis, Irini Pateraki, Birger Lindberg Møller, Olaf Kruse
Photosynthetic microalgae harbor enormous potential as light-driven green-cell factories for sustainable bio-production of a range of natural and heterologous products such as isoprenoids. Their capacity for photosynthesis and rapid low-input growth with (sun)light and CO2 is coupled to a robust metabolic architecture structured toward the generation of isoprenoid pigments and compounds involved in light capture, electron transfer, and radical scavenging. Metabolic engineering approaches using eukaryotic green microalgae have previously been hampered mainly by low-levels of nuclear transgene expression...
July 12, 2018: Metabolic Engineering
Tadas Jakočiūnas, Lasse E Pedersen, Alicia V Lis, Michael K Jensen, Jay D Keasling
Here we describe a method for robust directed evolution using mutagenesis of large sequence spaces in their genomic contexts. The method employs error-prone PCR and Cas9-mediated genome integration of mutant libraries of large-sized donor variants into single or multiple genomic sites with efficiencies reaching 98-99%. From sequencing of genome integrants, we determined that the mutation frequency along the donor fragments is maintained evenly and successfully integrated into the genomic target loci, indicating that there is no bias of mutational load towards the proximity of the double strand break...
July 5, 2018: Metabolic Engineering
Adelfia Talà, Fabrizio Damiano, Giuseppe Gallo, Eva Pinatel, Matteo Calcagnile, Mariangela Testini, Daniela Fico, Daniela Rizzo, Alberto Sutera, Giovanni Renzone, Andrea Scaloni, Gianluca De Bellis, Luisa Siculella, Giuseppe Egidio De Benedetto, Anna Maria Puglia, Clelia Peano, Pietro Alifano
Pirins are evolutionarily conserved iron-containing proteins that are found in all kingdoms of life, and have been implicated in diverse molecular processes, mostly associated with cellular stress. In the present study, we started from the evidence that the insertional inactivation of pirin-like gene SAM23877_RS18305 (pirA) by ΦC31 Att/Int system-based vectors in spiramycin-producing strain Streptomyces ambofaciens ATCC 23877 resulted in marked effects on central carbon and energy metabolism gene expression, high sensitivity to oxidative injury and repression of polyketide antibiotic production...
June 23, 2018: Metabolic Engineering
Haijiao Wang, Wenqian Liu, Feng Shi, Lei Huang, Jiazhang Lian, Liang Qu, Jin Cai, Zhinan Xu
Cellular metabolic networks should be carefully balanced using metabolic engineering to produce the desired products at the industrial scale. As the precursor for the biosynthesis of the neurotransmitter serotonin, 5-hydroxytryptophan (5-HTP) is effective in treating a variety of diseases, such as depression, fibromyalgia, obesity, and cerebellar ataxia. Due to the lack of an efficient synthetic method, commercial production of 5-HTP is only achieved by extracting from the seeds of Griffonia Smplicifolia. This study reports efficient microbial production of 5-HTP via metabolically engineered Escherichia coli...
June 19, 2018: Metabolic Engineering
Benjamin M Woolston, David F Emerson, Devin H Currie, Gregory Stephanopoulos
Clostridium ljungdahlii has emerged as an attractive candidate for the bioconversion of synthesis gas (CO, CO2 , H2 ) to a variety of fuels and chemicals through the Wood-Ljungdahl pathway. However, metabolic engineering and pathway elucidation in this microbe is limited by the lack of genetic tools to downregulate target genes. To overcome this obstacle, here we developed an inducible CRISPR interference (CRISPRi) system for C. ljungdahlii that enables efficient (> 94%) transcriptional repression of several target genes, both individually and in tandem...
June 15, 2018: Metabolic Engineering
Douglas McCloskey, Sibei Xu, Troy E Sandberg, Elizabeth Brunk, Ying Hefner, Richard Szubin, Adam M Feist, Bernhard O Palsson
Aromatic metabolites provide the backbone for numerous industrial and pharmaceutical compounds of high value. The Phosphotransferase System (PTS) is common to many bacteria, and is the primary mechanism for glucose uptake by Escherichia coli. The PTS was removed to conserve phosphoenolpyruvate (pep), which is a precursor for aromatic metabolites and consumed by the PTS, for aromatic metabolite production. Replicate adaptive laboratory evolution (ALE) of PTS and detailed omics data sets collected revealed that the PTS bridged the gap between respiration and fermentation, leading to distinct high fermentative and high respiratory rate phenotypes...
June 15, 2018: Metabolic Engineering
Ingo Appelhagen, Anders Keim Wulff-Vester, Micael Wendell, Anne-Kathrine Hvoslef-Eide, Julia Russell, Anne Oertel, Stefan Martens, Hans-Peter Mock, Cathie Martin, Andrea Matros
Anthocyanins are widely distributed, glycosylated, water-soluble plant pigments, which give many fruits and flowers their red, purple or blue colouration. Their beneficial effects in a dietary context have encouraged increasing use of anthocyanins as natural colourants in the food and cosmetic industries. However, the limited availability and diversity of anthocyanins commercially have initiated searches for alternative sources of these natural colourants. In plants, high-level production of secondary metabolites, such as anthocyanins, can be achieved by engineering of regulatory genes as well as genes encoding biosynthetic enzymes...
June 8, 2018: Metabolic Engineering
Mary Ann Franden, Lahiru N Jayakody, Wing-Jin Li, Neil J Wagner, Nicholas S Cleveland, William E Michener, Bernhard Hauer, Lars M Blank, Nick Wierckx, Janosch Klebensberger, Gregg T Beckham
Ethylene glycol is used as a raw material in the production of polyethylene terephthalate, in antifreeze, as a gas hydrate inhibitor in pipelines, and for many other industrial applications. It is metabolized by aerobic microbial processes via the highly toxic intermediates glycolaldehyde and glycolate through C2 metabolic pathways. Pseudomonas putida KT2440, which has been engineered for environmental remediation applications given its high toxicity tolerance and broad substrate specificity, is not able to efficiently metabolize ethylene glycol, despite harboring putative genes for this purpose...
June 7, 2018: Metabolic Engineering
Shubin Li, Tao Sun, Chunxiao Xu, Lei Chen, Weiwen Zhang
The fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 (hereafter Synechococcus 2973) has been considered a good chassis candidate for "microbial cell factory" as it can perform oxygenic photosynthesis and its doubling time can be as short as 1.9 h. However, the limited genetic tools currently restrict its further research and application efforts using synthetic biology approaches. In this study, a series of genetic tools were systematically developed and optimized for Synechococcus 2973...
June 6, 2018: Metabolic Engineering
Shivani Garg, Hao Wu, James M Clomburg, George N Bennett
Methane, the primary component of natural gas, is the second most abundant greenhouse gas (GHG) and contributes significantly to climate change. The conversion of methane to industrial platform chemicals provides an attractive opportunity to decrease GHG emissions and utilize this inexpensive and abundantly available gas as a carbon feedstock. While technologies exist for chemical conversion of methane to liquid fuels, the technical complexity of these processes mandate high capital expenditure, large-scale commercial facilities to leverage economies of scale that cannot be efficiently scaled down...
June 5, 2018: Metabolic Engineering
Jiwon Yu, Jonghyeok Shin, Myungseo Park, Emine Seydametova, Sang-Min Jung, Jin-Ho Seo, Dae-Hyuk Kweon
Fucosyllactoses (FLs), present in human breast milk, have been reported to benefit human health immensely. Especially, 3-fucosyllactose (3-FL) has numerous benefits associated with a healthy gut ecosystem. Metabolic engineering of microorganisms is thought to be currently the only option to provide an economically feasible route for large-scale production of 3-FL. However, engineering principles for α-1,3-fucosyltransferases (1,3-FTs) are not well-known, resulting in the lower productivity of 3-FL than that of 2'-fucosyllactose (2'-FL), although both 2'-FL and 3-FL follow a common pathway to produce GDP-L-fucose...
June 2, 2018: Metabolic Engineering
Pavel Dvořák, Víctor de Lorenzo
Given its capacity to tolerate stress, NAD(P)H/ NAD(P) balance, and increased ATP levels, the platform strain Pseudomonas putida EM42, a genome-edited derivative of the soil bacterium P. putida KT2440, can efficiently host a suite of harsh reactions of biotechnological interest. Because of the lifestyle of the original isolate, however, the nutritional repertoire of P. putida EM42 is centered largely on organic acids, aromatic compounds and some hexoses (glucose and fructose). To enlarge the biochemical network of P...
June 2, 2018: Metabolic Engineering
Guanglu Wang, Ting Shi, Tao Chen, Xiaoyue Wang, Yongcheng Wang, Dingyu Liu, Jiaxin Guo, Jing Fu, Lili Feng, Zhiwen Wang, Xueming Zhao
Commercial riboflavin production with Bacillus subtilis has been developed by combining rational and classical strain development for almost two decades, but how an improved riboflavin producer can be created rationally is still not completely understood. In this study, we demonstrate the combined use of integrated genomic and transcriptomic analysis of the genetic basis for riboflavin over-production in B. subtilis. This methodology succeeded in discerning the positive mutations in the mutagenesis derived riboflavin producer B...
June 2, 2018: Metabolic Engineering
Chae Won Kang, Hyun Gyu Lim, Jina Yang, Myung Hyun Noh, Sang Woo Seo, Gyoo Yeol Jung
Although plasmid-based expression systems have advantages in multi-copy expression of genes, heterogeneity of plasmid copy number (PCN) in individual cells is inevitable even with the addition of antibiotics. Here, we developed a synthetic auxotrophic system for stable and tunable maintenance of the PCN in Escherichia coli without addition of antibiotics. This auxotroph expresses infA, one of the essential genes encoding a translation initiation factor, on a plasmid instead of on the chromosome. With this system, the gene expression was stably maintained for 40 generations with minimized cell-to-cell variation under antibiotic-free conditions...
June 2, 2018: Metabolic Engineering
Fabian Schweizer, Maite Colinas, Jacob Pollier, Alex Van Moerkercke, Robin Vanden Bossche, Rebecca de Clercq, Alain Goossens
To fend off microbial pathogens and herbivores, plants have evolved a wide range of defense strategies such as physical barriers, or the production of anti-digestive proteins or bioactive specialized metabolites. Accumulation of the latter compounds is often regulated by transcriptional activation of the biosynthesis pathway genes by the phytohormone jasmonate-isoleucine. Here, we used our recently developed flower petal transformation method in the medicinal plant Catharanthus roseus to shed light on the complex regulatory mechanisms steering the jasmonate-modulated biosynthesis of monoterpenoid indole alkaloids (MIAs), to which the anti-cancer compounds vinblastine and vincristine belong...
May 28, 2018: Metabolic Engineering
Erik K R Hanko, Charles M Denby, Violeta Sànchez I Nogué, Weiyin Lin, Kelsey J Ramirez, Christine A Singer, Gregg T Beckham, Jay D Keasling
Medium- and long-chain methyl ketones are fatty acid-derived compounds that can be used as biofuel blending agents, flavors and fragrances. However, their large-scale production from sustainable feedstocks is currently limited due to the lack of robust microbial biocatalysts. The oleaginous yeast Yarrowia lipolytica is a promising biorefinery platform strain for the production of methyl ketones from renewable lignocellulosic biomass due to its natively high flux towards fatty acid biosynthesis. In this study, we report the metabolic engineering of Y...
May 28, 2018: Metabolic Engineering
Peter Eisenhut, Gerald Klanert, Marcus Weinguny, Laurenz Baier, Vaibhav Jadhav, Daniel Ivansson, Nicole Borth
Manipulation of multiple genes to engineer Chinese Hamster Ovary (CHO) cells for better performance in production processes of biopharmaceuticals has recently become more and more popular. Yet, identification of useful genes and the unequivocally assessment of their effect alone and in combination(s) on the cellular phenotype is difficult due to high variation between subclones. Here, we present development and proof-of-concept of a novel engineering strategy using multiplexable activation of artificially repressed genes (MAARGE)...
May 28, 2018: Metabolic Engineering
Ran Liu, Zixin Deng, Tiangang Liu
There is considerable interest in mining organisms for new natural products (NPs) and in improving methods to overproduce valuable NPs. Because of the rapid development of tools and strategies for metabolic engineering and the markedly increased knowledge of the biosynthetic pathways and genetics of NP-producing organisms, genome mining and overproduction of NPs can be dramatically accelerated. In particular, Streptomyces species have been proposed as suitable chassis organisms for NP discovery and overproduction because of their many unique characteristics not shared with yeast, Escherichia coli, or other microorganisms...
May 28, 2018: Metabolic Engineering
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