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3D Visualization and Quantification of Bioplastic PHA in a Living Bacterial Cell
3D holographic microscopy leads to in-depth analysis of bacterial cells accumulating the bacterial bioplastic, polyhydroxyalkanoate (PHA)
A research team at KAIST has observed how bioplastic granule is being accumulated in living bacteria cells through 3D holographic microscopy. Their 3D imaging and quantitative analysis of the bioplastic ‘polyhydroxyalkanoate’ (PHA) via optical diffraction tomography provides insights into biosynthesizing sustainable substitutes for petroleum-based plastics.
The bio-degradable polyester polyhydroxyalkanoate (PHA) is being touted as an eco-friendly bioplastic to replace existing synthetic plastics. While carrying similar properties to general-purpose plastics such as polyethylene and polypropylene, PHA can be used in various industrial applications such as container packaging and disposable products.
PHA is synthesized by numerous bacteria as an energy and carbon storage material under unbalanced growth conditions in the presence of excess carbon sources. PHA exists in the form of insoluble granules in the cytoplasm. Previous studies on investigating in vivo PHA granules have been performed by using fluorescence microscopy, transmission electron microscopy (TEM), and electron cryotomography.
These techniques have generally relied on the statistical analysis of multiple 2D snapshots of fixed cells or the short-time monitoring of the cells. For the TEM analysis, cells need to be fixed and sectioned, and thus the investigation of living cells was not possible. Fluorescence-based techniques require fluorescence labeling or dye staining. Thus, indirect imaging with the use of reporter proteins cannot show the native state of PHAs or cells, and invasive exogenous dyes can affect the physiology and viability of the cells. Therefore, it was difficult to fully understand the formation of PHA granules in cells due to the technical limitations, and thus several mechanism models based on the observations have been only proposed.
The team of metabolic engineering researchers led by Distinguished Professor Sang Yup Lee and Physics Professor YongKeun Park, who established the startup Tomocube with his 3D holographic microscopy, reported the results of 3D quantitative label-free analysis of PHA granules in individual live bacterial cells by measuring the refractive index distributions using optical diffraction tomography. The formation and growth of PHA granules in the cells of Cupriavidus necator, the most-studied native PHA (specifically, poly(3-hydroxybutyrate), also known as PHB) producer, and recombinant Escherichia coli harboring C. necator PHB biosynthesis pathway were comparatively examined.
From the reconstructed 3D refractive index distribution of the cells, the team succeeded in the 3D visualization and quantitative analysis of cells and intracellular PHA granules at a single-cell level. In particular, the team newly presented the concept of “in vivo PHA granule density.” Through the statistical analysis of hundreds of single cells accumulating PHA granules, the distinctive differences of density and localization of PHA granules in the two micro-organisms were found. Furthermore, the team identified the key protein that plays a major role in making the difference that enabled the characteristics of PHA granules in the recombinant E. coli to become similar to those of C. necator.
The research team also presented 3D time-lapse movies showing the actual processes of PHA granule formation combined with cell growth and division. Movies showing the living cells synthesizing and accumulating PHA granules in their native state had never been reported before.
Professor Lee said, “This study provides insights into the morphological and physical characteristics of in vivo PHA as well as the unique mechanisms of PHA granule formation that undergo the phase transition from soluble monomers into the insoluble polymer, followed by granule formation. Through this study, a deeper understanding of PHA granule formation within the bacterial cells is now possible, which has great significance in that a convergence study of biology and physics was achieved. This study will help develop various bioplastics production processes in the future.”
This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (Grants NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557) and the Bio & Medical Technology Development Program (Grant No. 2021M3A9I4022740) from the Ministry of Science and ICT (MSIT) through the National Research Foundation (NRF) of Korea to S.Y.L. This work was also supported by the KAIST Cross-Generation Collaborative Laboratory project.
-PublicationSo Young Choi, Jeonghun Oh, JaeHwang Jung, YongKeun Park, and Sang Yup Lee. Three-dimensional label-free visualization and quantification of polyhydroxyalkanoates in individualbacterial cell in its native state. PNAS(https://doi.org./10.1073/pnas.2103956118)
-ProfileDistinguished Professor Sang Yup LeeMetabolic Engineering and Synthetic Biologyhttp://mbel.kaist.ac.kr/
Department of Chemical and Biomolecular Engineering KAIST
Endowed Chair Professor YongKeun ParkBiomedical Optics Laboratoryhttps://bmokaist.wordpress.com/
Department of PhysicsKAIST
2021.07.28 View 20134 -
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 (ehukim@kaist.ac.kr)
https://sites.google.com/view/ehukim
Department of Chemical and Biomolecular Engineering
-Distinguished Professor Sang Yup Lee (leesy@kaist.ac.kr)
Department of Chemical and Biomolecular Engineering
http://mbel.kaist.ac.kr
2019.07.09 View 45379 -
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.)
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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
leesy@kaist.ac.kr
Distinguished Professor at the Department of Chemical and Biomolecular Engineering
KAIST
2019.06.19 View 56658 -
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.)
2019.05.15 View 62275 -
Distinguished Professor Lee Receives 2018 George Washington Carver Award
(Distinguished Professor Lee)
Distinguished Professor Sang Yup Lee from the Department of Chemical and Biomolecular Engineering will become the 11th recipient of the George Washington Carver Award. The award ceremony will be held during the 2018 Biotechnology Innovation Organization (BIO) World Congress on Industrial Biotechnology from July 16 through 19 at the Pennsylvania Convention Center in Philadelphia.
The annual Carver award recognizes an individual who has made a significant contribution to building the bio-based economy by applying industrial biotechnology to create environmentally sustainable products. It serves as a lasting memorial to the original vision of George Washington Carver who, over a century ago, pioneered bio-based products, materials, and energy derived from renewable agricultural feedstock. Previous recipients include the founder and CEO of POET Jeff Broin, the CEO of DuPont Ellen Kullman, and Professor Gregory Stephanopoulos at MIT.
Professor Lee is a pioneering scholar of systems metabolic engineering, leveraging technology to develop microbial bioprocesses for the sustainable and environment-friendly production of chemicals, fuels, and materials from non-food renewable biomass. He also serves as the dean of the multi-and interdisciplinary research center hub, KAIST Institute.Through his work, Professor Lee has garnered countless achievements, including being one of only 13 people in the world elected as a foreign member of both the National Academy of Sciences USA and the National Academy of Engineering USA.
He has actively promoted the importance of industrial biotechnology through engagement with the public, policymakers, and decision makers around the world. He currently serves as the co-chairman of the Global Future Council on Biotechnology for the World Economic Forum and served as the Chairman of the Emerging Technologies Council and Biotechnology Council for the World Economic Forum.
Upon the award announcement, Dr. Brent Erickson, executive vice president of BIO’s Industrial & Environmental Section lauded Professor Lee’s achievement, saying “Dr. Lee has advanced the bio-based economy by developing innovative products and processes that are sustainable and environmentally friendly. In doing so, he has become a leader in advocating on the importance of industrial biotechnology. His contributions to the advancement of the industry are a continuation of the legacy left behind by George Washington Carver.”
Professor Lee thanked his research team who has worked together for the past few decades, adding, “Industrial biotechnology is becoming increasingly important to help achieve the UN’s Sustainable Development Goals. We should continue to work together to advance the field and establish a solid foundation for the sustainable future.”
The George Washington Carver Award is sponsored by the Iowa Biotechnology Association. Joe Hrdlicka, executive director of the Iowa Biotechnology Association, said, “Dr. Sang Yup Lee’s significant contributions to the advancement of industrial biotechnology make him the perfect recipient for the George Washington Carver Award. Having published more than 575 peer-reviewed papers, contributed to 82 books, and holding 636 patents, the culmination of Dr. Lee’s work has led to the establishment of sustainable systems for bio-based production of chemicals, fuels, and materials, thus reducing environmental impact and improving quality of life for all.”
2018.07.12 View 18100 -
Distinguished Professor Lee Named International Fellow of the CAS
Distinguished Professor Sang Yup Lee from the Department of Chemical and Biomolecular Engineering at KAIST was awarded the title of distinguished professor and international fellow from the Chinese Academy of Sciences (CAS), and honorary professor from its affiliated organization the Tianjin Institute of Industrial Biotechnology (TIB).
The CAS recognized Distinguished Professor Lee for his significant contributions to biotechnology. He has made significant pioneering academic achievements in the area of systems metabolic engineering, which produces useful chemicals from microorganisms. Not only did he develop the first and best source technology in that field, but also came out with processes for the production of biofuel and environmentally-friendly chemicals.”
As a global leader in systems metabolic engineering, Distinguished Professor Lee has also been appointed as an honorary professor at Jiangnan University in Wuxi, China.
Distinguished Professor Lee was listed in the ‘Top 20 Translational Researchers of 2014’ selected by the renowned international journal Nature Biotechnology. Moreover, he was the first Asian recipient of the James E. Bailey Award in 2016 and Marvin J. Johnson Award in 2012, which are given to scholars in the field of biotechnology.
He is also one of 13 global scientists who are foreign members of the renowned academic societies the National Academy of Engineering and the National Academy of Sciences in the US. Furthermore, he received the ‘2017 Korea Best Scientist Award’ from the president of Korea in July. Finally, his founding field, systems metabolic engineering, was chosen as one of the ‘Top 10 Emerging Technologies of 2016’ by the World Economic Forum.
The Chinese Academy of Sciences, established in November 1949, is an academic organization that carries out research on basic sciences and natural sciences in China. It defined its science and technology system to include the fields of basic sciences, natural sciences, and high technology. While having a base in Beijing, its branch academies are located in 12 main cities along with 117 affiliates and 100 national key labs.
2017.10.26 View 17550 -
Distinguished Professor Lee Elected to the NAS
Distinguished Professor Sang Yup Lee of the Department of Chemical and Biomolecular Engineering was elected as a foreign associate to the US National Academy of Sciences (NAS) on May 2. The National Academy of Sciences elected 84 new members and 21 foreign associates in recognition of their distinguished and continuing achievements in their original research. Election to the Academy is widely regarded as one of the highest honors that a scientist can receive.
Professor Lee was also elected in 2010 as a member of the US National Academy of Engineering (NAE) for his leadership in microbial biotechnology and metabolic engineering, including the development of fermentation processes for biodegradable polymers and organic acids. Until 2016, there are only 12 people worldwide who are foreign associates of both NAS and NAE.
He is the first Korean elected to both prestigious academies, the NAS and the NAE in the US. Professor Lee is currently the dean of KAIST Institutes, the world leading institute for multi-and interdisciplinary research. He is also serving as co-chair of the Global Council on Biotechnology and member of the Global Future Council on the Fourth Industrial Revolution, the World Economic Forum.
2017.05.16 View 14864 -
Nature Biotechnology Nominates Sang Yup Lee of KAIST for Top 20 Translational Researchers of 2014
Nature Biotechnology, recognized as the most prestigious journal in the field of biotechnology, has released today its list of the Top 20 Translational Researchers of 2014. Distinguished Professor Sang Yup Lee of the Department of Chemical and Biomolecular Engineering at KAIST (Korea Advanced Institute of Science and Technology) ranked seventh in the list. He is the only Asian researcher listed.
The journal, in partnership with IP Checkups, a patent analytics firm, presents an annual ranking of researchers based on their paper and patent output. The list includes, among others, each researcher’s most-cited patent in the past five years and their H index, a measurement to evaluate the impact of a researcher’s published work utilizing citation analysis. (More details can be found at http://www.nature.com/bioent/2015/150801/full/bioe.2015.9.html.)
American institutions made up the majority of the list, with 18 universities and research institutes, and the remainder was filled by KAIST in Korea and the Commonwealth Scientific and Industrial Research Organization (CSIRO) in Australia.
Globally known as a leading researcher in systems metabolic engineering, Professor Lee has published more than 500 journal papers and 580 patents. He has received many awards, including the Citation Classic Award, Elmer Gaden Award, Merck Metabolic Engineering Award, ACS Marvin Johnson Award, SIMB Charles Thom Award, POSCO TJ Park Prize, Amgen Biochemical Engineering Award, and the Ho Am Prize in Engineering.
2015.08.27 View 16055 -
Professor Sang-Yup Lee Receives the Order of Service Merit Red Stripes from the Korean Government
The government of the Republic of Korea named Professor Sang-Yup Lee of the Department of Chemical and Bio-molecular Engineering at KAIST as the fiftieth recipient of the Order of Service Merit Red Stripes on May 19, 2015.
This medal is awarded to government employees, officials, and teachers in recognition of their contributions to public services including education.
Professor Lee is regarded as a leading scientist in the field of metabolic engineering, genomics, proteomics, metabolomics, and bioinformatics on microorganism producing various primary and secondary metabolites. He contributed significantly to the advancement of bio-based engineering research in Korea.
In addition, his research in microorganism metabolic engineering propelled him to the front of his field, making him the world’s founder of systems metabolic engineering, inventing numerous technologies in strain development.
Professor Lee has received many patent rights in bioprocess engineering. While at KAIST, he applied for 585 patents and registered 227 patents. In particular, he has applied for 135 patents and registered 99 patents in the past five years, successfully turning research results into commercial applications.
Professor Lee said, “I’m glad to contribute to the development of Korean science and technology as a researcher and teacher. I would like to share this honor with my students, master’s and doctoral students in particular, because without their support, it wouldn’t have been possible to pull off the highest level of research results recognized by this medal.”
2015.05.21 View 12548 -
KAIST Professor Sang-Yup Lee Chair of International Metabolic Engineering Conference Due Next Year
KAIST distinguished professor Sang-Yup Lee was named to chair the 17th Metabolic Engineering Conference which will convene on Jeju Island, Korea, next year, under the theme of "Metabolic Engineering for Green Growth." It was decided at the 16th Biochemical Engineering Conference held in Burlington, Vermont, on July 5-9.
Metabolic Engineering Conference in 2010 will not only involve presentations and discussions about metabolic engineering, but will inaugurate the “World Council on Industrial Biotechnology,” which will bring together global corporations and the world’s experts in industrial biochemical engineering, according to sources at KAIST.
A KAIST official commented, “The fact that the Metabolic Engineering Conference is to be held here [in Korea] proves that Korea is being acknowledged as a key player in this field.”
As the world faces the depletion of fossil fuels and environmental pollution, nations are showing increasing interest in industrial biochemical alternatives, such as microscopic organisms or new chemicals, to solve their problems. In addition, efficient production of biochemical materials and bio-fuels using microbes is deemed vital for the future.
“The Korean government has become a model to other countries thanks to its leadership in carrying out the ‘Green Growth’ policy,” Professor Sang-Yup Lee said. He stated that KAIST is recognized for its research in advanced biochemical material and fuel production methods.
“Green Growth,” a concept first developed by ESCAP, the UN agency working for social and economic cooperation in Asia and the Pacific, aims to achieve sustainable economic growth without destroying the environment.
Ref. Department of Biochemical Engineering, Metabolic and BioMolecular Engineering Lab, KAIST
2009.07.17 View 18341