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Metabolic Engineering

Liliya Horbal, Filipe Marques, Suvd Nadmid, Marta V Mendes, Andriy Luzhetskyy
We present a random rational approach enabling the construction of overproducing strains in two steps. The approach first involves creating a library of clusters of interest, in which native promoters are substituted with randomly generated constitutive synthetic promoters, and then expressing this library in an appropriate host strain. This strategy is fast, easy to use, accounts for the architecture of a cluster and completely decouples the expression of a gene cluster from complex native regulatory networks...
September 18, 2018: Metabolic Engineering
Cyrielle Calmels, Andréa McCann, Laetitia Malphettes, Mikael Rørdam Andersen
CHO cells have become the favorite expression system for large scale production of complex biopharmaceuticals. However, industrial strategies for upstream process development are based on empirical results, due to a lack of fundamental understanding of intracellular activities. Genome scale models of CHO cells have been reconstructed to provide an economical way of analyzing and interpreting large-omics datasets, since they add cellular context to the data. Here the most recently available CHO-DG44 genome-scale specific model was manually curated and tailored to the metabolic profile of cell lines used for industrial protein production, by modifying 601 reactions...
September 15, 2018: Metabolic Engineering
Chenglin Zhang, Yanjun Li, Jie Ma, Yuan Liu, Jilong He, Yingzi Li, Fuzhou Zhu, Jing Meng, Junjie Zhan, Zhixiang Li, Lei Zhao, Qian Ma, Xiaoguang Fan, Qingyang Xu, Xixian Xie, Ning Chen
4-Hydroxyisoleucine (4-HIL) exhibits a unique glucose-dependent insulinotropic activity and is a promising candidate for the treatment of diabetes. Direct fermentation of 4-HIL has been recently studied; however, the expected titre and yield were not achieved. In this study, we initially developed a pathway for the synthesis of 4-HIL in an L-isoleucine producer, C. glutamicum YI, but insufficient supply of α-ketoglutarate was a bottleneck for a strong production. Six genes involved in oxaloacetate and α-ketoglutarate branches were overexpressed or deleted, which increased the production of 4-HIL to 5...
September 14, 2018: Metabolic Engineering
Chen Ling, Guan-Qing Qiao, Bo-Wen Shuai, Karel Olavarria, Jin Yin, Rui-Juan Xiang, Kun-Nan Song, Yun-Hao Shen, Yingying Guo, Guo-Qiang Chen
Halomonas bluephagenesis has been developed as a platform strain for the next generation industrial biotechnology (NGIB) with advantages of resistances to microbial contamination and high cell density growth (HCD), especially for production of polyhydroxyalkanoates (PHA) including poly(3-hydroxybutyrate) (PHB), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). However, little is known about the mechanism behind PHA accumulation under oxygen limitation...
September 13, 2018: Metabolic Engineering
Jingxuan Cui, Daniel G Olson, Lee R Lynd
The thermophilic anaerobes Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are good candidates for lignocellulosic ethanol production. T. saccharolyticum has been successfully engineered to produce ethanol at high titer (70g/L). The maximum ethanol titer of engineered strains of C. thermocellum is only 25g/L. We hypothesize that one or more of the enzymes in the ethanol production pathway in C. thermocellum is not adequate for ethanol production at high titer. In this study, we focused on the enzymes responsible for the part of the ethanol production pathway from pyruvate to ethanol...
September 12, 2018: Metabolic Engineering
Carly M Bond, Yi Tang
Simvastatin is a semisynthetic cholesterol-lowering medication and one of the top-selling statins in the world. Currently, industrial production of simvastatin acid (SVA) is a multistep process starting from the natural product lovastatin. For this reason, there is significant interest in direct production of simvastatin from a microbial host. In this study, six heterologous biosynthetic genes were introduced into Saccharomyces cerevisiae and the acyl-donor dimethylbutyryl-S-methyl mercaptopropionate (DMB-SMMP) was added, resulting in initial production of 0...
September 10, 2018: Metabolic Engineering
Sergio Garcia, Cong T Trinh
Diversity of cellular metabolism can be harnessed to produce a large space of molecules. However, development of optimal strains with high product titers, rates, and yields required for industrial production is laborious and expensive. To accelerate the strain engineering process, we have recently introduced a modular cell design concept that enables rapid generation of optimal production strains by systematically assembling a modular cell with an exchangeable production module(s) to produce target molecules efficiently...
September 7, 2018: Metabolic Engineering
Maksym Myronovskyi, Birgit Rosenkränzer, Suvd Nadmid, Petar Pujic, Philippe Normand, Andriy Luzhetskyy
Natural products are a rich source of potential drugs for many applications. Discovery of natural products through the activation of cryptic gene clusters encoding their biosynthetic pathways, engineering of those biosynthetic pathways and optimization of production yields often rely on the expression of these gene clusters in suitable heterologous host strains. Streptomyces albus J1074 provides high success rates of heterologous cluster expression with high levels of metabolite production, rapid growth and amenability to genetic manipulations...
September 6, 2018: Metabolic Engineering
Sun-Ki Kim, Janet Westpheling
Polyamines are low molecular weight aliphatic nitrogen compounds found ubiquitously in microorganisms, plants, and animals. Spermidine is a common polyamine that plays a role in stabilizing chromatin, DNA replication, transcription, translation, as well as the regulation of cell growth and apoptosis in eukaryotes. Amines are also associated with defense to a number of environmental stresses including elevated temperature and have been shown to be involved in tolerance to fermentation inhibitors such as furan derivatives and acetic acid in Saccharomyces cerevisiae...
September 5, 2018: Metabolic Engineering
Heyun Wu, Yanjun Li, Qian Ma, Qiang Li, Zifan Jia, Bo Yang, Qingyang Xu, Xiaoguang Fan, Chenglin Zhang, Ning Chen, Xixian Xie
Uridine is a kind of pyrimidine nucleoside that has been widely applied in the pharmaceutical industry. Although microbial fermentation is a promising method for industrial production of uridine, an efficient microbial cell factory is still lacking. In this study, we constructed a metabolically engineered Escherichia coli capable of high-yield uridine production. First, we developed a CRISPR/Cas9-mediated chromosomal integration strategy to integrate large DNA into the E. coli chromosome, and a 9.7 kb DNA fragment including eight genes in the pyrimidine operon of Bacillus subtilis F126 was integrated into the yghX locus of E...
September 4, 2018: Metabolic Engineering
Eric Wolfsberg, Christopher P Long, Maciek R Antoniewicz
In this study, we have investigated for the first time the metabolism of E. coli grown on agar using 13 C metabolic flux analysis (13 C-MFA). To date, all 13 C-MFA studies on microbes have been performed with cells grown in liquid culture. Here, we extend the scope of 13 C-MFA to biological systems where cells are grown in dense microbial colonies. First, we identified new optimal 13 C tracers to quantify fluxes in systems where the acetate yield cannot be easily measured. We determined that three parallel labeling experiments with the tracers [1,2-13 C]glucose, [1,6-13 C]glucose, and [4,5,6-13 C]glucose permit precise estimation of not only intracellular fluxes, but also of the amount of acetate produced from glucose...
September 1, 2018: Metabolic Engineering
Yaokang Wu, Taichi Chen, Yanfeng Liu, Xueqin Lv, Jianghua Li, Guocheng Du, Rodrigo Ledesma-Amaro, Long Liu
Glucose and xylose are the two most abundant sugars in renewable lignocellulose sources; however, typically they cannot be simultaneously utilized due to carbon catabolite repression. N-acetylglucosamine (GlcNAc) is a typical nutraceutical and has many applications in the field of healthcare. Here, we have developed a gene repressor system based on xylose-induced CRISPR interference (CRISPRi) in Bacillus subtilis, aimed at downregulating the expression of three genes (zwf, pfkA, glmM) that control the major competing reactions of GlcNAc synthesis (pentose phosphate pathway (HMP), glycolysis, and peptidoglycan synthesis pathway (PSP)), with the potential to relieve glucose repression and allow the co-utilization of both glucose and xylose...
August 31, 2018: Metabolic Engineering
Nobuyuki Okahashi, Kousuke Maeda, Shuichi Kawana, Junko Iida, Hiroshi Shimizu, Fumio Matsuda
Precise measurement of sugar phosphates in glycolysis and the pentose phosphate (PP) pathway for 13 C-metabolic flux analysis (13 C-MFA) is needed to understand cancer-specific metabolism. Although various analytical methods have been proposed, analysis of sugar phosphates is challenging because of the structural similarity of various isomers and low intracellular abundance. In this study, gas chromatography-negative chemical ionization-mass spectrometry (GC-NCI-MS) is applied to sugar phosphate analysis with o-(2,3,4,5,6-pentafluorobenzyl) oxime (PFBO) and trimethylsilyl (TMS) derivatization...
August 31, 2018: Metabolic Engineering
Chang-Ting Chen, Frederic Y-H Chen, Igor W Bogorad, Tung-Yun Wu, Ruoxi Zhang, Abraxa S Lee, James C Liao
Methanol is a potentially attractive substrate for bioproduction of chemicals because of the abundance of natural gas and biogas-derived methane. To move towards utilizing methanol as a sole carbon source, here we engineer an Escherichia coli strain to couple methanol utilization with growth on five-carbon (C5) sugars. By deleting essential genes in the pentose phosphate pathway for pentose utilization and expressing heterologous enzymes from the ribulose-monophosphate (RuMP) pathway, we constructed a strain that cannot grow on xylose or ribose minimal media unless methanol is utilized, creating a phenotype termed "synthetic methanol auxotrophy"...
August 30, 2018: Metabolic Engineering
Dauenpen Meesapyodsuk, Shengjian Ye, Yan Chen, Yi Chen, Robert G Chapman, Xiao Qiu
Very long chain polyunsaturated fatty acids (VLCPUFAs) are well recognized for their health benefits in humans and animals. Here we report that identification and characterization of a gene (EhELO1) encoding the first functional ELO type elongase (3-ketoacyl-CoA synthase) in higher plants that is involved in the biosynthesis of two VLCPUFAs docosadienoic acid (DDA, 22:2n-6) and docosatrienoic acid (DTA, 22:3n-3) that possess potential health-promoting properties. Functional analysis of the gene in yeast indicated that this novel enzyme could elongate a wide range of polyunsaturated fatty acids with 18-22 carbons and effectively catalyze the biosynthesis of DDA and DTA by the sequential elongations of linoleic acid and alpha-linolenic acid, respectively...
August 24, 2018: Metabolic Engineering
Ian Sofian Yunus, Julian Wichmann, Robin Wördenweber, Kyle J Lauersen, Olaf Kruse, Patrik R Jones
Liquid fuels sourced from fossil sources are the dominant energy form for mobile transport today. The consumption of fossil fuels is still increasing, resulting in a continued search for more sustainable methods to renew our supply of liquid fuel. Photosynthetic microorganisms naturally accumulate hydrocarbons that could serve as a replacement for fossil fuel, however productivities remain low. We report successful introduction of five synthetic metabolic pathways in two green cell factories, prokaryotic cyanobacteria and eukaryotic algae...
August 23, 2018: Metabolic Engineering
Hee Taek Kim, Tae Uk Khang, Kei-Anne Baritugo, Sung Min Hyun, Kyoung Hee Kang, Sol Hee Jung, Bong Keun Song, Kyungmoon Park, Min-Kyu Oh, Gi Bae Kim, Hyun Uk Kim, Sang Yup Lee, Si Jae Park, Jeong Chan Joo
Corynebacterium glutamicum was metabolically engineered for the production of glutaric acid, a C5 dicarboxylic acid that can be used as platform building block chemicals including nylon and plasticizer. C. glutamicum gabT and gabD genes and Pseudomonas putida davT and davD genes encoding 5-aminovalerate transaminase and glutarate semialdehyde dehydrogenase, respectively, were examined in C. glutamicum for the construction of a glutaric acid bio-synthesis pathway along with P. putida davB and davA genes encoding lysine 2-monooxygenase and delta-aminovaleramidase, respectively...
August 22, 2018: Metabolic Engineering
Cristian Varela, Simon A Schmidt, Anthony R Borneman, Chi Nam Ignatius Pang, Jens O Krömerx, Alamgir Khan, Xiaomin Song, Mark P Hodson, Mark Solomon, Christine M Mayr, Wade Hines, Isak S Pretorius, Mark S Baker, Ute Roessner, Meagan Mercurio, Paul A Henschke, Marc R Wilkins, Paul J Chambers
Metabolic engineering has been vital to the development of industrial microbes such as the yeast Saccharomyces cerevisiae. However, sequential rounds of modification are often needed to achieve particular industrial design targets. Systems biology approaches can aid in identifying genetic targets for modification through providing an integrated view of cellular physiology. Recently, research into the generation of commercial yeasts that can produce reduced-ethanol wines has resulted in metabolically-engineered strains of S...
August 20, 2018: Metabolic Engineering
Shuang Zheng, Jin Hou, Yi Zhou, Hao Fang, Ting-Ting Wang, Fei Liu, Feng-Shan Wang, Ju-Zheng Sheng
The development of D-glucaric acid (GA) production in recombinant cells has leapt forward in recent years, and higher throughput screening and selection of better-performing recombinant cells or biocatalysts is in current demand. A biosensor system which converts GA concentration into fluorescence signal in Escherichia coli was developed in 2016, but its application has rarely been reported. Herein, an effective high-throughput screening approach independent of special-purpose devices such as microfluidic platforms was established and tentatively applied...
August 18, 2018: Metabolic Engineering
Sammy Pontrelli, Riley C B Fricke, Sana Subhan Memon Sakurai, Sastia Prama Putri, Sorel Fitz-Gibbon, Matthew Chung, Hsin-Yi Wu, Yu-Ju Chen, Matteo Pellegrini, Eiichiro Fukusaki, James C Liao
Engineering a microbial strain for production sometimes entails metabolic modifications that impair essential physiological processes for growth or production. Restoring these functions may require amending a variety of non-obvious physiological networks, and thus, rational design strategies may not be practical. Here we demonstrate that growth and production may be restored by evolution that repairs impaired metabolic function. Furthermore, we use genomics, metabolomics and proteomics to identify several underlying mutations and metabolic perturbations that allow metabolism to repair...
August 11, 2018: Metabolic Engineering
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