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E. coli Engineered to Grow on CO₂ and Formic Acid as Sole Carbon Sources
- An E. coli strain that can grow to a relatively high cell density solely on CO₂ and formic acid was developed by employing metabolic engineering. - Most biorefinery processes have relied on the use of biomass as a raw material for the production of chemicals and materials. Even though the use of CO₂ as a carbon source in biorefineries is desirable, it has not been possible to make common microbial strains such as E. coli grow on CO₂. Now, a metabolic engineering research group at KAIST has developed a strategy to grow an E. coli strain to higher cell density solely on CO₂ and formic acid. Formic acid is a one carbon carboxylic acid, and can be easily produced from CO₂ using a variety of methods. Since it is easier to store and transport than CO₂, formic acid can be considered a good liquid-form alternative of CO₂. With support from the C1 Gas Refinery R&D Center and the Ministry of Science and ICT, a research team led by Distinguished Professor Sang Yup Lee stepped up their work to develop an engineered E. coli strain capable of growing up to 11-fold higher cell density than those previously reported, using CO₂ and formic acid as sole carbon sources. This work was published in Nature Microbiology on September 28. Despite the recent reports by several research groups on the development of E. coli strains capable of growing on CO₂ and formic acid, the maximum cell growth remained too low (optical density of around 1) and thus the production of chemicals from CO₂ and formic acid has been far from realized. The team previously reported the reconstruction of the tetrahydrofolate cycle and reverse glycine cleavage pathway to construct an engineered E. coli strain that can sustain growth on CO₂ and formic acid. To further enhance the growth, the research team introduced the previously designed synthetic CO₂ and formic acid assimilation pathway, and two formate dehydrogenases. Metabolic fluxes were also fine-tuned, the gluconeogenic flux enhanced, and the levels of cytochrome bo3 and bd-I ubiquinol oxidase for ATP generation were optimized. This engineered E. coli strain was able to grow to a relatively high OD600 of 7~11, showing promise as a platform strain growing solely on CO₂ and formic acid. Professor Lee said, “We engineered E. coli that can grow to a higher cell density only using CO₂ and formic acid. We think that this is an important step forward, but this is not the end. The engineered strain we developed still needs further engineering so that it can grow faster to a much higher density.” Professor Lee’s team is continuing to develop such a strain. “In the future, we would be delighted to see the production of chemicals from an engineered E. coli strain using CO₂ and formic acid as sole carbon sources,” he added. Profile: Distinguished Professor Sang Yup Lee email@example.com http://mbel.kaist.ac.kr Department of Chemical and Biomolecular Engineering KAIST
Universal Virus Detection Platform to Expedite Viral Diagnosis
Reactive polymer-based tester pre-screens dsRNAs of a wide range of viruses without their genome sequences The prompt, precise, and massive detection of a virus is the key to combat infectious diseases such as Covid-19. A new viral diagnostic strategy using reactive polymer-grafted, double-stranded RNAs will serve as a pre-screening tester for a wide range of viruses with enhanced sensitivity. Currently, the most widely using viral detection methodology is polymerase chain reaction (PCR) diagnosis, which amplifies and detects a piece of the viral genome. Prior knowledge of the relevant primer nucleic acids of the virus is quintessential for this test. The detection platform developed by KAIST researchers identifies viral activities without amplifying specific nucleic acid targets. The research team, co-led by Professor Sheng Li and Professor Yoosik Kim from the Department of Chemical and Biomolecular Engineering, constructed a universal virus detection platform by utilizing the distinct features of the PPFPA-grafted surface and double-stranded RNAs. The key principle of this platform is utilizing the distinct feature of reactive polymer-grafted surfaces, which serve as a versatile platform for the immobilization of functional molecules. These activated surfaces can be used in a wide range of applications including separation, delivery, and detection. As long double-stranded RNAs are common byproducts of viral transcription and replication, these PPFPA-grafted surfaces can detect the presence of different kinds of viruses without prior knowledge of their genomic sequences. “We employed the PPFPA-grafted silicon surface to develop a universal virus detection platform by immobilizing antibodies that recognize double-stranded RNAs,” said Professor Kim. To increase detection sensitivity, the research team devised two-step detection process analogues to sandwich enzyme-linked immunosorbent assay where the bound double-stranded RNAs are then visualized using fluorophore-tagged antibodies that also recognize the RNAs’ double-stranded secondary structure. By utilizing the developed platform, long double-stranded RNAs can be detected and visualized from an RNA mixture as well as from total cell lysates, which contain a mixture of various abundant contaminants such as DNAs and proteins. The research team successfully detected elevated levels of hepatitis C and A viruses with this tool. “This new technology allows us to take on virus detection from a new perspective. By targeting a common biomarker, viral double-stranded RNAs, we can develop a pre-screening platform that can quickly differentiate infected populations from non-infected ones,” said Professor Li. “This detection platform provides new perspectives for diagnosing infectious diseases. This will provide fast and accurate diagnoses for an infected population and prevent the influx of massive outbreaks,” said Professor Kim. This work is featured in Biomacromolecules. This work was supported by the Agency for Defense Development (Grant UD170039ID), the Ministry of Science and ICT (NRF-2017R1D1A1B03034660, NRF-2019R1C1C1006672), and the KAIST Future Systems Healthcare Project from the Ministry of Science and ICT (KAISTHEALTHCARE42). Profile:-Professor Yoosik KimDepartment of Chemical and Biomolecular Engineeringhttps://qcbio.kaist.ac.kr KAIST-Professor Sheng LiDepartment of Chemical and Biomolecular Engineeringhttps://bcpolymer.kaist.ac.kr KAIST Publication:Ku et al., 2020. Reactive Polymer Targeting dsRNA as Universal Virus Detection Platform with Enhanced Sensitivity. Biomacromolecules (https://doi.org/10.1021/acs.biomac.0c00379).
A Single Biological Factor Predicts Distinct Cortical Organizations across Mammalian Species
-A KAIST team’s mathematical sampling model shows that retino-cortical mapping is a prime determinant in the topography of cortical organization.- Researchers have explained how visual cortexes develop uniquely across the brains of different mammalian species. A KAIST research team led by Professor Se-Bum Paik from the Department of Bio and Brain Engineering has identified a single biological factor, the retino-cortical mapping ratio, that predicts distinct cortical organizations across mammalian species. This new finding has resolved a long-standing puzzle in understanding visual neuroscience regarding the origin of functional architectures in the visual cortex. The study published in Cell Reports on March 10 demonstrates that the evolutionary variation of biological parameters may induce the development of distinct functional circuits in the visual cortex, even without species-specific developmental mechanisms. In the primary visual cortex (V1) of mammals, neural tuning to visual stimulus orientation is organized into one of two distinct topographic patterns across species. While primates have columnar orientation maps, a salt-and-pepper type organization is observed in rodents. For decades, this sharp contrast between cortical organizations has spawned fundamental questions about the origin of functional architectures in the V1. However, it remained unknown whether these patterns reflect disparate developmental mechanisms across mammalian taxa, or simply originate from variations in biological parameters under a universal development process. To identify a determinant predicting distinct cortical organizations, Professor Paik and his researchers Jaeson Jang and Min Song examined the exact condition that generates columnar and salt-and-pepper organizations, respectively. Next, they applied a mathematical model to investigate how the topographic information of the underlying retinal mosaics pattern could be differently mapped onto a cortical space, depending on the mapping condition. The research team proved that the retino-cortical feedforwarding mapping ratio appeared to be correlated to the cortical organization of each species. In the model simulations, the team found that distinct cortical circuitries can arise from different V1 areas and retinal ganglion cell (RGC) mosaic sizes. The team’s mathematical sampling model shows that retino-cortical mapping is a prime determinant in the topography of cortical organization, and this prediction was confirmed by neural parameter analysis of the data from eight phylogenetically distinct mammalian species. Furthermore, the researchers proved that the Nyquist sampling theorem explains this parametric division of cortical organization with high accuracy. They showed that a mathematical model predicts that the organization of cortical orientation tuning makes a sharp transition around the Nyquist sampling frequency, explaining why cortical organizations can be observed in either columnar or salt-and-pepper organizations, but not in intermediates between these two stages. Professor Paik said, “Our findings make a significant impact for understanding the origin of functional architectures in the visual cortex of the brain, and will provide a broad conceptual advancement as well as advanced insights into the mechanism underlying neural development in evolutionarily divergent species.” He continued, “We believe that our findings will be of great interest to scientists working in a wide range of fields such as neuroscience, vision science, and developmental biology.” This work was supported by the National Research Foundation of Korea (NRF). Image credit: Professor Se-Bum Paik, KAIST Image usage restrictions: News organizations may use or redistribute this image, with proper attribution, as part of news coverage of this paper only. Publication: Jaeson Jang, Min Song, and Se-Bum Paik. (2020). Retino-cortical mapping ratio predicts columnar and salt-and-pepper organization in mammalian visual cortex. Cell Reports. Volume 30. Issue 10. pp. 3270-3279. Available online at https://doi.org/10.1016/j.celrep.2020.02.038 Profile: Se-Bum Paik Assistant Professor firstname.lastname@example.org http://vs.kaist.ac.kr/ VSNN Laboratory Department of Bio and Brain Engineering Program of Brain and Cognitive Engineering http://kaist.ac.kr Korea Advanced Institute of Science and Technology (KAIST) Daejeon, Republic of Korea Profile: Jaeson Jang Ph.D. Candidate email@example.com Department of Bio and Brain Engineering, KAIST Profile: Min Song Ph.D. Candidate firstname.lastname@example.org Program of Brain and Cognitive Engineering, KAIST (END)
Professor Jong Chul Ye Appointed as Distinguished Lecturer of IEEE EMBS
Professor Jong Chul Ye from the Department of Bio and Brain Engineering was appointed as a distinguished lecturer by the International Association of Electrical and Electronic Engineers (IEEE) Engineering in Medicine and Biology Society (EMBS). Professor Ye was invited to deliver a lecture on his leading research on artificial intelligence (AI) technology in medical video restoration. He will serve a term of two years beginning in 2020. IEEE EMBS's distinguished lecturer program is designed to educate researchers around the world on the latest trends and technology in biomedical engineering. Sponsored by IEEE, its members can attend lectures on the distinguished professor's research subject. Professor Ye said, "We are at a time where the importance of AI in medical imaging is increasing.” He added, “I am proud to be appointed as a distinguished lecturer of the IEEE EMBS in recognition of my contributions to this field.” (END)
Scientists Discover the Mechanism of DNA High-Order Structure Formation
(Molecular structures of Abo1 in different energy states (left), Demonstration of an Abo1-assisted histone loading onto DNA by the DNA curtain assay. ) The genetic material of our cells—DNA—exists in a high-order structure called “chromatin”. Chromatin consists of DNA wrapped around histone proteins and efficiently packs DNA into a small volume. Moreover, using a spool and thread analogy, chromatin allows DNA to be locally wound or unwound, thus enabling genes to be enclosed or exposed. The misregulation of chromatin structures results in aberrant gene expression and can ultimately lead to developmental disorders or cancers. Despite the importance of DNA high-order structures, the complexity of the underlying machinery has circumvented molecular dissection. For the first time, molecular biologists have uncovered how one particular mechanism uses energy to ensure proper histone placement onto DNA to form chromatin. They published their results on Dec. 17 in Nature Communications. The study focused on proteins called histone chaperones. Histone chaperones are responsible for adding and removing specific histones at specific times during the DNA packaging process. The wrong histone at the wrong time and place could result in the misregulation of gene expression or aberrant DNA replication. Thus, histone chaperones are key players in the assembly and disassembly of chromatin. “In order to carefully control the assembly and disassembly of chromatin units, histone chaperones act as molecular escorts that prevent histone aggregation and undesired interactions,” said Professor Ji-Joon Song in the Department of Biological Sciences at KAIST. “We set out to understand how a unique histone chaperone uses chemical energy to assemble or disassemble chromatin.” Song and his team looked to Abo1, the only known histone chaperone that utilizes cellular energy (ATP). While Abo1 is found in yeast, it has an analogous partner in other organisms, including humans, called ATAD2. Both use ATP, which is produced through a cellular process where enzymes break down a molecule’s phosphate bond. ATP energy is typically used to power other cellular processes, but it is a rare partner for histone chaperones. “This was an interesting problem in the field because all other histone chaperones studied to date do not use ATP,” Song said. By imaging Abo1 with a single-molecule fluorescence imaging technique known as the DNA curtain assay, the researchers could examine the protein interactions at the single-molecule level. The technique allows scientists to arrange the DNA molecules and proteins on a single layer of a microfluidic chamber and examine the layer with fluorescence microscopy. The researchers found through real-time observation that Abo1 is ring-shaped and changes its structure to accommodate a specific histone and deposit it on DNA. Moreover, they found that the accommodating structural changes are powered by ADP. “We discovered a mechanism by which Abo1 accommodates histone substrates, ultimately allowing it to function as a unique energy-dependent histone chaperone,” Song said. “We also found that despite looking like a protein disassembly machine, Abo1 actually loads histone substrates onto DNA to facilitate chromatin assembly.” The researchers plan to continue exploring how energy-dependent histone chaperones bind and release histones, with the ultimate goal of developing therapeutics that can target cancer-causing misbehavior by Abo1’s analogous human counterpart, ATAD2. Profile -Professor Ji-Joon Song ( www.song-kaist.org) Associate Professor Department of Biological Sciences Email:email@example.com KI for the BioCentury (https://kis.kaist.ac.kr/index.php?mid=KIB_O) KAIST -Dr. Carol Cho Department of Biological Sciences The Research Center for Natural Sciences KI for the BioCentury (https://kis.kaist.ac.kr/index.php?mid=KIB_O) KAIST
A Single, Master Switch for Sugar Levels?
When a fly eats sugar, a single brain cell sends simultaneous messages to stimulate one hormone and inhibit another to control glucose levels in the body. Further research into this control system with remarkable precision could shed light on the neural mechanisms of diabetes and obesity in humans . A single neuron appears to monitor and control sugar levels in the fly body, according to research published this week in Nature. This new insight into the mechanisms in the fly brain that maintain a balance of two key hormones controlling glucose levels, insulin and glucagon, can provide a framework for understanding diabetes and obesity in humans. Neurons that sense and respond to glucose were identified more than 50 years ago, but what they do in our body has remained unclear. Researchers at the Korea Advanced Institute of Science and Technology (KAIST) and New York University School of Medicine have now found a single “glucose-sensing neuron” that appears to be the master controller in Drosophila, the vinegar fly, for maintaining an ideal glucose balance, called homeostasis. Professor Greg Seong-Bae Suh, Dr. Yangkyun Oh and colleagues identified a key neuron that is excited by glucose, which they called CN neuron. This CN neuron has a unique shape – it has an axon (which is used to transmit information to downstream cells) that is bifurcated. One branch projects to insulin-producing cells, and sends a signal triggering the secretion of the insulin equivalent in flies. The other branch projects to glucagon-producing cells and sends a signal inhibiting the secretion of the glucagon equivalent. When flies consume food, the levels of glucose in their body increase; this excites the CN neuron, which fires the simultaneous signals to stimulate insulin and inhibit glucagon secretion, thereby maintaining the appropriate balance between the hormones and sugar in the blood. The researchers were able to see this happening in the brain in real time by using a combination of cutting-edge fluorescent calcium imaging technology, as well as measuring hormone and sugar levels and applying highly sophisticated molecular genetic techniques. When flies were not fed, however, the researchers observed a reduction in the activity of CN neuron, a reduction in insulin secretion and an increase in glucagon secretion. These findings indicate that these key hormones are under the direct control of the glucose-sensing neuron. Furthermore, when they silenced the CN neuron rendering dysfunctional CN neuron in flies, these animals experienced an imbalance, resulting in hyperglycemia – high levels of sugars in the blood, similar to what is observed in diabetes in humans. This further suggests that the CN neuron is critical to maintaining glucose homeostasis in animals. While further research is required to investigate this process in humans, Suh notes this is a significant step forward in the fields of both neurobiology and endocrinology. “This work lays the foundation for translational research to better understand how this delicate regulatory process is affected by diabetes, obesity, excessive nutrition and diets high in sugar,” Suh said. Profile: Greg Seong-Bae Suh firstname.lastname@example.org Professor Department of Biological Sciences KAIST (Figure: A single glucose-excited CN neuron extends bifurcated axonal branches, one of which innervates insulin producing cells and stimulates their activity an the other axonal branch projects to glucagon producing cells and inhibits their activity.)
A Mathematical Model Reveals Long-Distance Cell Communication Mechanism
How can tens of thousands of people in a large football stadium all clap together with the same beat even though they can only hear the people near them clapping? A combination of a partial differential equation and a synthetic circuit in microbes answers this question. An interdisciplinary collaborative team of Professor Jae Kyoung Kim at KAIST, Professor Krešimir Josić at the University of Houston, and Professor Matt Bennett at Rice University has identified how a large community can communicate with each other almost simultaneously even with very short distance signaling. The research was reported at Nature Chemical Biology. Cells often communicate using signaling molecules, which can travel only a short distance. Nevertheless, the cells can also communicate over large distances to spur collective action. The team revealed a cell communication mechanism that quickly forms a network of local interactions to spur collective action, even in large communities. The research team used an engineered transcriptional circuit of combined positive and negative feedback loops in E. coli, which can periodically release two types of signaling molecules: activator and repressor. As the signaling molecules travel over a short distance, cells can only talk to their nearest neighbors. However, cell communities synchronize oscillatory gene expression in spatially extended systems as long as the transcriptional circuit contains a positive feedback loop for the activator. Professor Kim said that analyzing and understanding such high-dimensional dynamics was extremely difficult. He explained, “That’s why we used high-dimensional partial differential equation to describe the system based on the interactions among various types of molecules.” Surprisingly, the mathematical model accurately simulates the synthesis of the signaling molecules in the cell and their spatial diffusion throughout the chamber and their effect on neighboring cells. The team simplified the high-dimensional system into a one-dimensional orbit, noting that the system repeats periodically. This allowed them to discover that cells can make one voice when they lowered their own voice and listened to the others. “It turns out the positive feedback loop reduces the distance between moving points and finally makes them move all together. That’s why you clap louder when you hear applause from nearby neighbors and everyone eventually claps together at almost the same time,” said Professor Kim. Professor Kim added, “Math is a powerful as it simplifies complex thing so that we can find an essential underlying property. This finding would not have been possible without the simplification of complex systems using mathematics." The National Institutes of Health, the National Science Foundation, the Robert A. Welch Foundation, the Hamill Foundation, the National Research Foundation of Korea, and the T.J. Park Science Fellowship of POSCO supported the research. (Figure: Complex molecular interactions among microbial consortia is simplified as interactions among points on a limit cycle (right).)
Accurate Detection of Low-Level Somatic Mutation in Intractable Epilepsy
KAIST medical scientists have developed an advanced method for perfectly detecting low-level somatic mutation in patients with intractable epilepsy. Their study showed that deep sequencing replicates of major focal epilepsy genes accurately and efficiently identified low-level somatic mutations in intractable epilepsy. According to the study, their diagnostic method could increase the accuracy up to 100%, unlike the conventional sequencing analysis, which stands at about 30% accuracy. This work was published in Acta Neuropathologica. Epilepsy is a neurological disorder common in children. Approximately one third of child patients are diagnosed with intractable epilepsy despite adequate anti-epileptic medication treatment. Somatic mutations in mTOR pathway genes, SLC35A2, and BRAF are the major genetic causes of intractable epilepsies. A clinical trial to target Focal Cortical Dysplasia type II (FCDII), the mTOR inhibitor is underway at Severance Hospital, their collaborator in Seoul, Korea. However, it is difficult to detect such somatic mutations causing intractable epilepsy because their mutational burden is less than 5%, which is similar to the level of sequencing artifacts. In the clinical field, this has remained a standing challenge for the genetic diagnosis of somatic mutations in intractable epilepsy. Professor Jeong Ho Lee’s team at the Graduate School of Medical Science and Engineering analyzed paired brain and peripheral tissues from 232 intractable epilepsy patients with various brain pathologies at Severance Hospital using deep sequencing and extracted the major focal epilepsy genes. They narrowed down target genes to eight major focal epilepsy genes, eliminating almost all of the false positive calls using deep targeted sequencing. As a result, the advanced method robustly increased the accuracy and enabled them to detect low-level somatic mutations in unmatched Formalin Fixed Paraffin Embedded (FFPE) brain samples, the most clinically relevant samples. Professor Lee conducted this study in collaboration with Professor Dong Suk Kim and Hoon-Chul Kang at Severance Hospital of Yonsei University. He said, “This advanced method of genetic analysis will improve overall patient care by providing more comprehensive genetic counseling and informing decisions on alternative treatments.” Professor Lee has investigated low-level somatic mutations arising in the brain for a decade. He is developing innovative diagnostics and therapeutics for untreatable brain disorders including intractable epilepsy and glioblastoma at a tech-startup called SoVarGen. “All of the technologies we used during the research were transferred to the company. This research gave us very good momentum to reach the next phase of our startup,” he remarked. The work was supported by grants from the Suh Kyungbae Foundation, a National Research Foundation of Korea grant funded by the Ministry of Science and ICT, the Korean Health Technology R&D Project from the Ministry of Health & Welfare, and the Netherlands Organization for Health Research and Development. (Figure: Landscape of somatic and germline mutations identified in intractable epilepsy patients. a Signaling pathways for all of the mutated genes identified in this study. Bold: somatic mutation, Regular: germline mutation. b The distribution of variant allelic frequencies (VAFs) of identified somatic mutations. c The detecting rate and types of identified mutations according to histopathology. Yellow: somatic mutations, green: two-hit mutations, grey: germline mutations.)
Deciphering Brain Somatic Mutations Associated with Alzheimer's Disease
Researchers have found a potential link between non-inherited somatic mutations in the brain and the progression of Alzheimer’s disease Researchers have identified somatic mutations in the brain that could contribute to the development of Alzheimer’s disease (AD). Their findings were published in the journal Nature Communications last week. Decades worth of research has identified inherited mutations that lead to early-onset familial AD. Inherited mutations, however, are behind at most half the cases of late onset sporadic AD, in which there is no family history of the disease. But the genetic factors causing the other half of these sporadic cases have been unclear. Professor Jeong Ho Lee at the Graduate School of Medical Science and Engineering and colleagues analysed the DNA present in post-mortem hippocampal formations and in blood samples from people aged 70 to 96 with AD and age-matched controls. They specifically looked for non-inherited somatic mutations in their brains using high-depth whole exome sequencing. The team developed a bioinformatics pipeline that enabled them to detect low-level brain somatic single nucleotide variations (SNVs) – mutations that involve the substitution of a single nucleotide with another nucleotide. Brain somatic SNVs have been reported on and accumulate throughout our lives and can sometimes be associated with a range of neurological diseases. The number of somatic SNVs did not differ between individuals with AD and non-demented controls. Interestingly, somatic SNVs in AD brains arise about 4.8 times more slowly than in blood. When the team performed gene-set enrichment tests, 26.9 percent of the AD brain samples had pathogenic brain somatic SNVs known to be linked to hyperphosphorylation of tau proteins, which is one of major hallmarks of AD. Then, they pinpointed a pathogenic SNV in the PIN1 gene, a cis/trans isomerase that balances phosphorylation in tau proteins, found in one AD patient’s brain. They found the mutation was 4.9 time more abundant in AT8-positive – a marker for hyper-phosphorylated tau proteins– neurons in the entorhinal cortex than the bulk hippocampal tissue. Furthermore, in a series of functional assays, they observed the mutation causing a loss of function in PIN1 and such haploinsufficiency increased the phosphorylation and aggregation of tau proteins. “Our study provides new insights into the molecular genetic factors behind Alzheimer’s disease and other neurodegenerative diseases potentially linked to somatic mutations in the brain,” said Professor Lee. The team is planning to expand their study to a larger cohort in order to establish stronger links between these brain somatic mutations and the pathogenesis of Alzheimer’s disease. (Figure 1. Bioinformatic pipeline for detecting low-level brain somatic mutations in AD and non-AD.) (Figure 2. Pathogenic brain somatic mutations associated with tau phosphorylation are significantly enriched in AD brains.) (Figure 3. A pathogenic brain somatic mutation in PIN1 (c. 477 C>T) is a loss-of-function and related functional assays show its haploinsufficiency increases phosphorylation and aggregation of tau.)
Deep Learning-Powered 'DeepEC' Helps Accurately Understand Enzyme Functions
(Figure: Overall scheme of DeepEC) A deep learning-powered computational framework, ‘DeepEC,’ will allow the high-quality and high-throughput prediction of enzyme commission numbers, which is essential for the accurate understanding of enzyme functions. A team of Dr. Jae Yong Ryu, Professor Hyun Uk Kim, and Distinguished Professor Sang Yup Lee at KAIST reported the computational framework powered by deep learning that predicts enzyme commission (EC) numbers with high precision in a high-throughput manner. DeepEC takes a protein sequence as an input and accurately predicts EC numbers as an output. Enzymes are proteins that catalyze biochemical reactions and EC numbers consisting of four level numbers (i.e., a.b.c.d) indicate biochemical reactions. Thus, the identification of EC numbers is critical for accurately understanding enzyme functions and metabolism. EC numbers are usually given to a protein sequence encoding an enzyme during a genome annotation procedure. Because of the importance of EC numbers, several EC number prediction tools have been developed, but they have room for further improvement with respect to computation time, precision, coverage, and the total size of the files needed for the EC number prediction. DeepEC uses three convolutional neural networks (CNNs) as a major engine for the prediction of EC numbers, and also implements homology analysis for EC numbers if the three CNNs do not produce reliable EC numbers for a given protein sequence. DeepEC was developed by using a gold standard dataset covering 1,388,606 protein sequences and 4,669 EC numbers. In particular, benchmarking studies of DeepEC and five other representative EC number prediction tools showed that DeepEC made the most precise and fastest predictions for EC numbers. DeepEC also required the smallest disk space for implementation, which makes it an ideal third-party software component. Furthermore, DeepEC was the most sensitive in detecting enzymatic function loss as a result of mutations in domains/binding site residue of protein sequences; in this comparative analysis, all the domains or binding site residue were substituted with L-alanine residue in order to remove the protein function, which is known as the L-alanine scanning method. This study was published online in the Proceedings of the National Academy of Sciences of the United States of America (PNAS) on June 20, 2019, entitled “Deep learning enables high-quality and high-throughput prediction of enzyme commission numbers.” “DeepEC can be used as an independent tool and also as a third-party software component in combination with other computational platforms that examine metabolic reactions. DeepEC is freely available online,” said Professor Kim. Distinguished Professor Lee said, “With DeepEC, it has become possible to process ever-increasing volumes of protein sequence data more efficiently and more accurately.” This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science and ICT through the National Research Foundation of Korea. This work was also funded by the Bio & Medical Technology Development Program of the National Research Foundation of Korea funded by the Korean government, the Ministry of Science and ICT. Profile: -Professor Hyun Uk Kim (email@example.com) https://sites.google.com/view/ehukim Department of Chemical and Biomolecular Engineering -Distinguished Professor Sang Yup Lee (firstname.lastname@example.org) Department of Chemical and Biomolecular Engineering http://mbel.kaist.ac.kr
Efficiently Producing Fatty Acids and Biofuels from Glucose
Researchers have presented a new strategy for efficiently producing fatty acids and biofuels that can transform glucose and oleaginous microorganisms into microbial diesel fuel, with one-step direct fermentative production. The newly developed strain, created by Distinguished Professor Sang Yup Lee and his team, showed the highest efficiency in producing fatty acids and biodiesels ever reported. It will be expected to serve as a new platform to sustainably produce a wide array of fatty acid-based products from glucose and other carbon substrates. Fossil fuels, which have long been energy resources for our daily lives, are now facing serious challenges: depletion of their reserves and their role in global warming. The production of sustainable bio-based renewable energy has emerged as an essential alternative and many studies to replace fossil fuels are underway. One of the representative examples is biodiesel. Currently, it is mainly being produced through the transesterification of vegetable oils or animal fats. The research team engineered oleaginous microorganisms, Rhodococcus opacus, to produce fatty acids and their derivatives that can be used as biodiesel from glucose, one of the most abundant and cheap sugars derived from non-edible biomass. Professor Lee’s team has already engineered Escherichia coli to produce short-chain hydrocarbons, which can be used as gasoline (published in Nature as the cover paper in 2013). However, the production efficiency of the short-chain hydrocarbons using E. coli (0.58 g/L) fell short of the levels required for commercialization. To overcome these issues, the team employed oil-accumulating Rhodococcus opacus as a host strain in this study. First, the team optimized the cultivation conditions of Rhodococcus opacus to maximize the accumulation of oil (triacylglycerol), which serves as a precursor for the biosynthesis of fatty acids and their derivatives. Then, they systematically analyzed the metabolism of the strain and redesigned it to enable higher levels of fatty acids and two kinds of fatty acid-derived biodiesels (fatty acid ethyl esters and long-chain hydrocarbons) to be produced. They found that the resulting strains produced 50.2, 21.3, and 5.2 g/L of fatty acids, fatty acid ethyl esters, and long-chain hydrocarbons, respectively. These are all the highest concentrations ever reported by microbial fermentations. It is expected that these strains can contribute to the future industrialization of microbial-based biodiesel production. “This technology creates fatty acids and biodiesel with high efficiency by utilizing lignocellulose, one of the most abundant resources on the Earth, without depending on fossil fuels and vegetable or animal oils. This will provide new opportunities for oil and petroleum industries, which have long relied on fossil fuels, to turn to sustainable and eco-friendly biotechnologies,” said Professor Lee. This paper titled “Engineering of an oleaginous bacterium for the production of fatty acids and fuels” was published in Nature Chemical Biology on June 17. This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science and ICT through the National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557). (Figure: Metabolic engineering for the production of free fatty acids (FFAs), fatty acid ethyl esters (FAEEs), and long-chain hydrocarbons (LCHCs) in Rhodococcus opacus PD630. Researchers have presented a new strategy for efficiently producing fatty acids and biofuels that can transform glucose and oleaginous microorganisms into microbial diesel fuel, with one-step direct fermentative production.) # # # Source: Hye Mi Kim, Tong Un Chae, So Young Choi, Won Jun Kim and Sang Yup Lee. Engineering of an oleaginous bacterium for the production of fatty acids and fuels. Nature Chemical Biology ( https://www.nature.com/nchembio/ ) DOI: 10.1038/s41589-019-0295-5 Profile Dr. Sang Yup Lee email@example.com Distinguished Professor at the Department of Chemical and Biomolecular Engineering KAIST
Engineered Microbial Production of Grape Flavoring
(Image 1: Engineered bacteria that produce grape flavoring.) Researchers report a microbial method for producing an artificial grape flavor. Methyl anthranilate (MANT) is a common grape flavoring and odorant compound currently produced through a petroleum-based process that uses large volumes of toxic acid catalysts. Professor Sang-Yup Lee’s team at the Department of Chemical and Biomolecular Engineering demonstrated production of MANT, a naturally occurring compound, via engineered bacteria. The authors engineered strains of Escherichia coli and Corynebacetrium glutamicum to produce MANT through a plant-based engineered metabolic pathway. The authors tuned the bacterial metabolic pathway by optimizing the levels of AAMT1, the key enzyme in the process. To maximize production of MANT, the authors tested six strategies, including increasing the supply of a precursor compound and enhancing the availability of a co-substrate. The most productive strategy proved to be a two-phase extractive culture, in which MANT was extracted into a solvent. This strategy produced MANT on the scale of 4.47 to 5.74 grams per liter, a significant amount, considering that engineered microbes produce most natural products at a scale of milligrams or micrograms per liter. According to the authors, the results suggest that MANT and other related molecules produced through industrial processes can be produced at scale by engineered microbes in a manner that would allow them to be marketed as natural one, instead of artificial one. This study, featured at the Proceeding of the National Academy of Sciences of the USA on May 13, was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science and ICT. (Image 2. Overview of the strategies applied for the microbial production of grape flavoring.)
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