polyhydroxyalkanoate
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KAIST finds ways for Bacteria to produce PET-like materials
Among various eco-friendly polymers, polyhydroxyalkanoates (PHA) stand out for their excellent biodegradability and biocompatibility. They decompose naturally in soil and marine environments and are used in applications such as food packaging and medical products. However, natural PHA produced to date has faced challenges meeting various physical property requirements, such as durability and thermal stability, and has been limited in its commercial application due to low production concentrations. In light of this, KAIST researchers have recently developed a technology that could play a crucial role in solving the environmental pollution problem caused by plastics.
KAIST (represented by President Kwang-Hyung Lee) announced on August 26th that a research team led by Distinguished Professor Sang Yup Lee from the Department of Chemical and Biomolecular Engineering, including Dr. Youngjoon Lee and master's student Minju Kang, has successfully developed a microbial strain that efficiently produces aromatic polyester* using systems metabolic engineering.
※ Aromatic polyester: A polymer containing aromatic compounds (specific carbon ring structures like benzene) and ester bonds.
In this study, the research team used metabolic engineering to enhance the metabolic flux of the biosynthetic pathway for the aromatic monomer phenyllactate (PhLA) in E. coli. They manipulated the metabolic pathway to increase the polymer fraction accumulated within the cells and employed computer simulations to predict the structure of PHA synthase and improve the enzyme based on the structure-function relationship.
Through subsequent fermentation optimization, the team achieved the world’s highest concentration (12.3±0.1 g/L) for the efficient production of poly (PhLA) and successfully produced polyester through a 30L scale fed-batch fermentation, demonstrating the possibility of industrial-level production. The produced aromatic polyesters showed enhanced thermal properties, improved mechanical properties, and potential for use as drug delivery carriers.
< Figure 1. Development schematics of aromatic polyester producing microorganisms >
The research team also demonstrated that an exogenous phasin protein* plays a crucial role in increasing the intracellular polymer accumulation fraction, which is directly related to the economic feasibility and efficiency of non-natural PHA production. They improved PHA synthase using a rational enzyme design approach, predicting the three-dimensional structure of the enzyme through homology modeling (a method of predicting the three-dimensional structure of a new protein based on the structure of similar proteins) followed by molecular docking simulations (simulations that predict how well a monomer can bind to an enzyme) and molecular dynamics simulations (simulations that predict how molecules move and interact over time) to upgrade the enzyme into a mutant enzyme with enhanced monomer polymerization efficiency.
※ Exogenous phasin protein: Phasin is a protein related to PHA production, interacting with the cytoplasmic environment on the surface of granules of PHA, and playing a role in polymer accumulation and controlling the number and size of granules. In this study, genes encoding phasin proteins derived from various natural PHA-producing microorganisms were selected and introduced.
Dr. Youngjoon Lee, co-first author of the paper, explained, "The significance of this study lies in the fact that we have achieved the world's highest concentration of microbial-based aromatic polyester production using eco-friendly materials and methods. This technology is expected to play a crucial role in addressing the environmental pollution caused by plastics." Distinguished Professor Sang Yup Lee added, "This study, which presents various strategies for the high-efficiency production of useful polymers via systems metabolic engineering, is expected to make a significant contribution to solving climate change issues, particularly the recent plastic problem."
< Figure 2. Detailed development strategy for aromatic polyester producing microorganisms >
The research findings were published on August 21st in Trends in Biotechnology, published by Cell, an international academic journal.
※ Paper Title: “Microbial production of an aromatic homopolyester”
※ Author Information: Youngjoon Lee (KAIST, co-first author), Minju Kang (KAIST, co-first author), Woo Dae Jang (KAIST, second author), So Young Choi (KAIST, third author), Jung Eun Yang (KAIST, fourth author), Sang Yup Lee (KAIST, corresponding author), totaling six authors.
This research was supported by the "Development of Next-Generation Biorefinery Platform Technologies for Leading the Bio-based Chemicals Industry" project led by Distinguished Professor Sang Yup Lee at KAIST, under the eco-friendly chemical technology development project aimed at substituting petroleum, funded by the Ministry of Science and ICT. It was also supported by the "Development of Platform Technology for the Production of Novel Aromatic Bioplastic Using Microbial Cell Factories" project (Project Leader: Si Jae Park, Ewha Woman’s University).
2024.08.28 View 1920 -
A biohybrid system to extract 20 times more bioplastic from CO2 developed by KAIST researchers
As the issues surrounding global climate change intensify, more attention and determined efforts are required to re-grasp the issue as a state of “crisis” and respond to it properly. Among the various methods of recycling CO2, the electrochemical CO2 conversion technology is a technology that can convert CO2 into useful chemical substances using electrical energy. Since it is easy to operate facilities and can use the electricity from renewable sources like the solar cells or the wind power, it has received a lot of attention as an eco-friendly technology can contribute to reducing greenhouse gases and achieve carbon neutrality.
KAIST (President Kwang Hyung Lee) announced on the 30th that the joint research team led by Professor Hyunjoo Lee and Distinguished Professor Sang Yup Lee of the Department of Chemical and Biomolecular Engineering succeeded in developing a technology that produces bioplastics from CO2 with high efficiency by developing a hybrid system that interlinked the electrochemical CO2 conversion and microbial bio conversion methods together. The results of the research, which showed the world's highest productivity by more than 20 times compared to similar systems, were published online on March 27th in the "Proceedings of the National Academy of Sciences (PNAS)".
※ Paper title: Biohybrid CO2 electrolysis for the direct synthesis of polyesters from CO2
※ Author information: Jinkyu Lim (currently at Stanford Linear Accelerator Center, co-first author), So Young Choi (KAIST, co-first author), Jae Won Lee (KAIST, co-first author), Hyunjoo Lee (KAIST, corresponding author), Sang Yup Lee (KAIST, corresponding author)
For the efficient conversion of CO2, high-efficiency electrode catalysts and systems are actively being developed. As conversion products, only compounds containing one or up to three carbon atoms are produced on a limited basis. Compounds of one carbon, such as CO, formic acid, and ethylene, are produced with relatively high efficiency. Liquid compounds of several carbons, such as ethanol, acetic acid, and propanol, can also be produced by these systems, but due to the nature of the chemical reaction that requires more electrons, there are limitations involving the conversion efficiency and the product selection.
Accordingly, a joint research team led by Professor Hyunjoo Lee and Distinguished Professor Sang Yup Lee of the Department of Chemical and Biomolecular Engineering at KAIST developed a technology to produce bioplastics from CO2 by linking electrochemical conversion technology with bioconversion method that uses microorganisms.
This electrochemical-bio hybrid system is in the form of having an electrolyzer, in which electrochemical conversion reactions occur, connected to a fermenter, in which microorganisms are cultured. When CO2 is converted to formic acid in the electrolyzer, and it is fed into the fermenter in which the microbes like the Cupriavidus necator, in this case, consumes the carbon source to produce polyhydroxyalkanoate (PHA), a microbial-derived bioplastic.
According to the research results of the existing hybrid concepts, there was a disadvantage of having low productivity or stopping at a non-continuous process due to problems of low efficiency of the electrolysis and irregular results arising from the culturing conditions of the microbes.
In order to overcome these problems, the joint research team made formic acid with a gas diffusion electrode using gaseous CO2. In addition, the team developed a 'physiologically compatible catholyte' that can be used as a culture medium for microorganisms as well as an electrolyte that allows the electrolysis to occur sufficiently without inhibiting the growth of microorganisms, without having to have a additional separation and purification process, which allowed the acide to be supplied directly to microorganisms.
Through this, the electrolyte solution containing formic acid made from CO2 enters the fermentation tank, is used for microbial culture, and enters the electrolyzer to be circulated, maximizing the utilization of the electrolyte solution and remaining formic acid. In addition, a filter was installed to ensure that only the electrolyte solution with any and all microorganisms that can affect the electrosis filtered out is supplied back to the electrolyzer, and that the microorganisms exist only in the fermenter, designing the two system to work well together with utmost efficiency.
Through the developed hybrid system, the produced bioplastic, poly-3-hydroxybutyrate (PHB), of up to 83% of the cell dry weight was produced from CO2, which produced 1.38g of PHB from a 4 cm2 electrode, which is the world's first gram(g) level production and is more than 20 times more productive than previous research. In addition, the hybrid system is expected to be applied to various industrial processes in the future as it shows promises of the continuous culture system.
The corresponding authors, Professor Hyunjoo Lee and Distinguished Professor Sang Yup Lee noted that “The results of this research are technologies that can be applied to the production of various chemical substances as well as bioplastics, and are expected to be used as key parts needed in achieving carbon neutrality in the future.”
This research was received and performed with the supports from the CO2 Reduction Catalyst and Energy Device Technology Development Project, the Heterogeneous Atomic Catalyst Control Project, and the Next-generation Biorefinery Source Technology Development Project to lead the Biochemical Industry of the Oil-replacement Eco-friendly Chemical Technology Development Program by the Ministry of Science and ICT.
Figure 1. Schematic diagram and photo of the biohybrid CO2 electrolysis system.
(A) A conceptual scheme and (B) a photograph of the biohybrid CO2 electrolysis system. (C) A detailed scheme of reaction inside the system. Gaseous CO2 was converted to formate in the electrolyzer, and the formate was converted to PHB by the cells in the fermenter. The catholyte was developed so that it is compatible with both CO2 electrolysis and fermentation and was continuously circulated.
2023.03.30 View 6620 -
Mystery of Biological Plastic Synthesis Machinery Unveiled
Plastics and other polymers are used every day. These polymers are mostly made from fossil resources by refining petrochemicals. On the other hand, many microorganisms naturally synthesize polyesters known as polyhydroxyalkanoates (PHAs) as distinct granules inside cells.
PHAs are a family of microbial polyesters that have attracted much attention as biodegradable and biocompatible plastics and elastomers that can substitute petrochemical counterparts. There have been numerous papers and patents on gene cloning and metabolic engineering of PHA biosynthetic machineries, biochemical studies, and production of PHAs; simple Google search with “polyhydroxyalkanoates” yielded returns of 223,000 document pages. PHAs have always been considered amazing examples of biological polymer synthesis. It is astounding to see PHAs of 500 kDa to sometimes as high as 10,000 kDa can be synthesized in vivo by PHA synthase, the key polymerizing enzyme in PHA biosynthesis. They have attracted great interest in determining the crystal structure of PHA synthase over the last 30 years, but unfortunately without success. Thus, the characteristics and molecular mechanisms of PHA synthase were under a dark veil.
In two papers published back-to-back in Biotechnology Journal online on November 30, 2016, a Korean research team led by Professor Kyung-Jin Kim at Kyungpook National University and Distinguished Professor Sang Yup Lee at the Korea Advanced Institute of Science and Technology (KAIST) described the crystal structure of PHA synthase from Ralstonia eutropha, the best studied bacterium for PHA production, and reported the structural basis for the detailed molecular mechanisms of PHA biosynthesis. The crystal structure has been deposited to Protein Data Bank in February 2016. After deciphering the crystal structure of the catalytic domain of PHA synthase, in addition to other structural studies on whole enzyme and related proteins, the research team also performed experiments to elucidate the mechanisms of the enzyme reaction, validating detailed structures, enzyme engineering, and also N-terminal domain studies among others.
Through several biochemical studies based on crystal structure, the authors show that PHA synthase exists as a dimer and is divided into two distinct domains, the N-terminal domain (RePhaC1ND) and the C-terminal domain (RePhaC1CD). The RePhaC1CD catalyzes the polymerization reaction via a non-processive ping-pong mechanism using a Cys-His-Asp catalytic triad. The two catalytic sites of the RePhaC1CD dimer are positioned 33.4 Å apart, suggesting that the polymerization reaction occurs independently at each site. This study also presents the structure-based mechanisms for substrate specificities of various PHA synthases from different classes.
Professor Sang Yup Lee, who has worked on this topic for more than 20 years, said,
“The results and information presented in these two papers have long been awaited not only in the PHA community, but also metabolic engineering, bacteriology/microbiology, and in general biological sciences communities. The structural information on PHA synthase together with the recently deciphered reaction mechanisms will be valuable for understanding the detailed mechanisms of biosynthesizing this important energy/redox storage material, and also for the rational engineering of PHA synthases to produce designer bioplastics from various monomers more efficiently.”
Indeed, these two papers published in Biotechnology Journal finally reveal the 30-year mystery of machinery of biological polyester synthesis, and will serve as the essential compass in creating designer and more efficient bioplastic machineries.
References:
Jieun Kim, Yeo-Jin Kim, So Young Choi, Sang Yup Lee and Kyung-Jin Kim. “Crystal structure of Ralstonia eutropha polyhydroxyalkanoate synthase C-terminal domain and reaction mechanisms” Biotechnology Journal DOI: 10.1002/biot.201600648
http://onlinelibrary.wiley.com/doi/10.1002/biot.201600648/abstract
Yeo-Jin Kim, So Young Choi, Jieun Kim, Kyeong Sik Jin, Sang Yup Lee and Kyung-Jin Kim. “Structure and function of the N-terminal domain of Ralstonia eutropha polyhydroxyalkanoate synthase, and the proposed structure and mechanisms of the whole enzyme” Biotechnology Journal DOI: 10.1002/biot.201600649
http://onlinelibrary.wiley.com/doi/10.1002/biot.201600649/abstract
2016.12.02 View 9027 -
Professor Sang-Yup Lee publishes a requested paper in Nature Biotechnology
Professor Sang-Yup Lee publishes a requested paper in Nature Biotechnology
“The era of commercialized bioplastic is coming”
Disclose an opinion as specialist at a requested paper in Nature Biotechnology, October 2006
A team led by Barbel Friedrich, Professor of Humboldt-Universitat zu Berlin, and Alexander Steinbuchel, Professor of West falische Wilhelms-Universitat Munster, found out the entire genome sequence of the typical bioplastic-producing microorganism ‘Ralstonia eutropha’ and published a paper on it in Nature Biotechnology, October 2006. As the entire genome sequence of the typical bioplactic-producing microorganism has been discovered, it is expected that the efficient production of bioplastic will be available through strain improvement at a more systematic level.
Regarding this paper, Nature Biotechnology requested world-renowned scholar Sang-Yup Lee, LG Chemical Chair-Professor of KAIST Chemical and Biomolecular Engineering Department, an expert analysis on the future of bioplastic production as a result of the deciphering of the genome sequence, and Professor Lee revealed his opinion at ‘News and Views’ in Nature Biotechnology, October 2006, issued on October 10. In the analysis, he insisted, “The deciphering of the genomes of Ralstonia means to pave the way for the improvement of strains at a system level by combining simulation through various omics and imaginary cells and engineering at a genome level. It will be possible to produce plastic with desired properties by altering the components of plastic as desired and produce bioplastic, more efficient and economical than have been reported so far, through the optimization of metabolic flow.”
Professor Lee is a world-renowned scholar in the bioplastic field, who has presented about 70 SCI papers in the field. He created a word ‘Plastic Bacteria’ at Trends in Biotechnology in 1996 and published an expert paper regarding E.Coli Plastic at Nature Biotechnology in 1997. He is now performing a research concerning the improvement of bioplastic-producing strains as an example of a research employing a systematic method for the system biological research and development project of the Ministry of Science and Technology.
The followings are the contents of Professor Lee’s paper concerning microorganism plastic published at ‘News and Views’ in Nature Biotechnology, October 2006.
- Polyhydroxyalkanoate (PHA) is a high molecule that numerous microorganisms accumulate in their own cells as energy storage substance when they are rich in carbonic resources, but poor in the other growth factors. The PHA high molecule is polyester, in which the unit substances (unit chemicals) are ester-bonded, and has been studied worldwide about twenty years before. However, PHA’s worse properties than petrochemical plastic and extremely high production cost have prevented its commercialization. The production cost of PHA was 15 dollars per kg in 1980’s, twenty times higher than the price of polypropylene. Sang-Yup Lee, LG Chemical Chair-Professor of KAIST Chemical & Biomolecular Department’s BK21 Project Group, has performed a research concerning the efficient production of microorganism plastic through the combination of metabolic engineering and fermentation process under the support of the Ministry of Science and Technology, and developed a process that lowers the production cost of PHA to 2-3 dollars per kg. He also has developed PHA-producing bacteria, efficient enough to fill plastic tightly, and named it ‘Plastic Bacteria’.
- The unprecedented rise of oil price for the past two years activated the researches on Bio-based energies and chemical production globally. PHA is also regaining attentions although the researches on it have been withered so far due to its poor economical efficiency and properties. The result of the genome deciphering of the typical plastic-producing microorganism ‘Ralstonia eutropha’ published by a German research team in Nature Biotechnology, October 2006 suggests huge meanings. That is, it will provide a blueprint over the metabolic activities of the bacteria and thus enables more systematic strain improvement.
- Eyeing on these facts, Nature Biotechnology requested Professor Sang-Yup Lee an expert analysis, and Professor Lee analyzed that there would be a dramatic development of microorganism plastic production through the application of the system biological engineering method, which is now being performed actively by Professor Lee at KAIST. In the analysis, Professor Lee revealed, “As the genome sequence has been found out, it becomes possible to establish metabolic network at a genome level, and since simulation becomes available, numberless trial and errors and experiments can be replaced with imaginary experiments rapidly. In addition, It makes the more efficient development of strains possible by fusion-analyzing the omics result such as various transcripts, proteins, metabolites, etc.” He also expected that it would be possible to produce tailor-made PHA having desired properties through metabolic engineering as well as the efficient production of plastic. Besides, he prospected that his research on the production of optically pure hydroxyl carboxyl acid, Professor Lee’s international patent right, would gain driving forces and technical development would be made rapidly at biological hydrogen production, production, dissolution and application of aromatic compounds, etc. by featuring this strain.
- Recently, Metabolic and ADM, U.S. companies, jointly started to produce PHA at a commercialization level, and Brazil having rich natural resources is commercializing PHA, following Bio-ethanol. In addition, Japan and Germany having a bunch of research performance in this field, and Australia having rich biomass are also performing consistent researches on PHA’s commercialization. Professor Lee prospected, “With the finding out of the genome sequence of the typical bioplastic-producing microorganism, competition for commercialization will be fiercer among nations through the development of efficient production systems.”
- Professor Lee prospected that as the efficient production of PHA becomes possible, the production of plastic from various renewable ingredients (cellurose, starch, suger, etc.) through microorganism fermentation would be made practically and the white biotechnologies of existing chemicals would gain more power. He also said, “Korea also will have to try to secure the production technologies and industry of Bio-based chemicals through strategic cooperation with resource powerfuls, etc. on the basis of the technical dominancy in some system metabolic engineering fields.”
- ‘News and Views’ in Nature Biotechnology is a section that publishes analyses of world-renowned specialists in the corresponding fields over the contents of some papers having great influences among papers published in the issue. KAIST Professor Sang-Yup Lee has published his second expert analysis of ‘Deciphering bioplastic production’ in the volume of October 2006, following the first paper ‘Going into the era of E.Coli plastic’.
2006.10.23 View 15008