Recent Advances in Systems Metabolic Engineering Strategies for the Production of Biopolymers

손유정,김희택,조서영,송혜민,Kei-Anne Baritugo,표지원,최종일,주정찬,박시재 한국생물공학회 2020 Biotechnology and Bioprocess Engineering Vol.25 No.6

Biopolymers consisting of at least one monomer, which are produced from renewable carbon sources, are being highly sought out since ubiquitous plastics are mainly produced from petrochemical processes causing severe environmental pollution. Therefore, the development of microbial cell factories, which can efficiently synthesize diverse types of monomers and polymers, is also becoming increasingly important. The applicability of traditional metabolic engineering strategies has extended with the combination of systems biology, synthetic biology, and evolutionary engineering in a systemic and versatile manner, and are collectively termed as systems metabolic engineering. Accordingly, recent advances in biotechnology have paved the way for enabling the production of an increasing number of monomers and polymers by providing several tools and strategies associated with systems metabolic engineering. In this review, we have focused on the substantial efforts made on the development of different approaches of systems metabolic engineering, particularly based on synthetic biology and evolutionary engineering, for the efficient production of monomers and polymers.

Recent Advances in Microbial Cell Growth Regulation Strategies for Metabolic Engineering

노명현,차상학,김민선,정규열 한국생물공학회 2020 Biotechnology and Bioprocess Engineering Vol.25 No.6

Programming a new function into cells and balancing new functions with native cellular metabolism or program related to cell growth have long been a challenge in metabolic engineering. In this regard, advances in synthetic biology and systems biology have broadly expanded the cell growth control strategies, empowering the strategies to be applied in metabolic engineering more effectively. In this review, we describe the advances and perspectives in cell growth control strategies. First, the strategies to utilize the environmental stress was described. Second, the strategies to precisely regulate the central metabolism and to exploit the cellular programs were summarized. Third, strategies related to evolutionary engineering, which have evolved significantly, including the emergence of new types of screening devices, have been summarized. Lastly, we described the strategies through exploiting new systems that are being actively applied in metabolic engineering. We hope that our review can provide enough information and perspectives in growth control strategies.

Evolutionary metabolic engineering of the 3-hydroxypropionic acid production pathway using a synthetic screening device

석주연,양진아,최상진,최운종,유태현,정규열 한국공업화학회 2019 한국공업화학회 연구논문 초록집 Vol.2019 No.0

Evolutionary metabolic engineering has great progress for improving the metabolite-producing strains. In this case, it is important to develop an efficient screening method because of the low probability of incidence of positive mutants in a library. Therefore, in this study, a synthetic selection device using a transcription regulator was constructed and optimized for the efficient accomplishment of the evolutionary metabolic engineering. As the target metabolic pathway, the production pathway of 3-hydroxypropionic acid (3-HP), one of the important platform chemicals, has been adopted. The results indicate the successful applications to the 3-HP production pathway engineering, especially for enzyme engineering. Therefore, the strategy used in this study could expedite the strain improvement in the field of metabolic engineering by developing a high-throughput screening method.

Recent advances in genetic engineering tools based on synthetic biology

Jun Ren,Jingyu Lee,Dokyun Na 한국미생물학회 2020 The journal of microbiology Vol.58 No.1

Genome-scale engineering is a crucial methodology to rationally regulate microbiological system operations, leading to expected biological behaviors or enhanced bioproduct yields. Over the past decade, innovative genome modification technologies have been developed for effectively regulating and manipulating genes at the genome level. Here, we discuss the current genome-scale engineering technologies used for microbial engineering. Recently developed strategies, such as clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9, multiplex automated genome engineering (MAGE), promoter engineering, CRISPR-based regulations, and synthetic small regulatory RNA (sRNA)-based knockdown, are considered as powerful tools for genome-scale engineering in microbiological systems. MAGE, which modifies specific nucleotides of the genome sequence, is utilized as a genome-editing tool. Contrastingly, synthetic sRNA, CRISPRi, and CRISPRa are mainly used to regulate gene expression without modifying the genome sequence. This review introduces the recent genome-scale editing and regulating technologies and their applications in metabolic engineering.

Biomolecular engineering : a new frontier in biotechnology 생명공학의 새로운 개척분야

Ryu, Dewey D.Y.,Nam, Doo-Hyun 영남대학교 약품개발연구소 2001 영남대학교 약품개발연구소 연구업적집 Vol.11 No.-

The advances in high throughput screening technology for discovery of target molecules and the accumulation of functional genomics and proteomics data at an ever-accelerating rate will enable us to design and discover novel biomolecules and proteins on a rational basis in diverse areas of pharmaceutical, agricultural, industrial, and environmental applications. The biomolecular engineering will no doubt become one of the most important scientific disciplines in that it will enable us to comprehensively analyze gene expression pattems in both normal and diseased cells and to discover many new biologically active molecules rationally and systematically. As an applied molecular evolution technology. DNA shuffling will play play a key role in biomolecular engineering. In contrast to the point mutation techniques. DNA shuffling exchanges large functional domains of sequences to search for the best candidate molecule, thus mimicking and accelerating the process of sexual recombination in the evolution of life. The phage-display system of combinatorial peptide libraries will be extensively exploited to design and create many more novel proteins, due to the relative ease of screening and identifying desirable proteins. Its application will be extended further into the science of protein-receptor or protein-ligand interactions. The bioinformatics including EST-based or SAGE-tag-based functional genomics and proteomics will continue to advance rapidly. Its biological knowledge base will expand the scope of biomolecular engineering, and the impact of well-coordinated biomolecular engineering research will be very significant on our understanding of gene expression, upregulation and downregulation, and posttranslational protein processing in healthy and diseased cells. The bioinformatics for genome and proteome analysis will contribute substantially toward over more accelerated advances in pharmaceutical industry. When the functional genomics database. EST and SAGE techniques, microarray technique, and proteome analysis by 2-dimensional gel electrophoresis or capillary electrophoresis are all put to good use, the biomolecular engineering research will yield new drug discoveries, improved therapies, and new or significantly improved bioprocesses. With the advances in biomotecular engineering, the rate of finding new high-value peptides or proteins including antibodies, vaccines, enzymes, and therapcutic peptides will continue to be accelerated. The targets for rational design of biomolecules will be bery broad, diverse, and complex, but many application goals can be achieved throught the expansion of knowledge base on biomolecules of interest and their roles and functionsl in cells and tissues. In the near future, more therapeutic drugs and high-value biomolecules will be designed and produced for the treatment or prevention of not-so-easily-cured diseases such as cancers, genetic diseases, age-related diseases, and other metabolic diseases. Also anticipated are many more industnal enzymes that will be engineered to confer desirable properties for the process improvement and manufacturing of many high-value biomolecular products. Many more new metabolites including novel antibioties that are active agains resistant strains will be also produced by recombinant organisms having de novo engineered biosynthetic pathwy enzyme systems. The biomolecular engineering era is here and a great deal of benefits can be derived form this field of scientific research for many years to come if we are willing to put it to good use. ⓒ 2000 Elsevier Science B.V All rights reserved.

Impact of the Synthetic Scaffold Strategy on the Metabolic Pathway Engineering

Kim-Ngan T. Tran,쿠마라벨 아쇽 쿠마,홍순호 한국생물공학회 2023 Biotechnology and Bioprocess Engineering Vol.28 No.3

For the development of the efficient bio-refinery process or biochemical producer, metabolic engineering has become an attractive choice recently. However, engineered metabolic pathways often suffer from flux imbalances due to a lack of corresponding regulatory mechanisms associated with natural metabolism. The interaction among different enzymes within a metabolic pathway plays an important role in regulating the efficiency of metabolic processes. Consequently, the creation of protein scaffolds has helped with the spatial co-localization of proteins in metabolic engineering. Research on protein scaffolds indicated scaffold systems may enhance metabolic productivity further. In this review, the specificity, selectivity, and regulatory mechanisms of protein-protein interactions are discussed in the context of the important effects that they exert on various biological processes.

Flavonoid 생합성:생화학과 대사공학적 응용

박종석,박종범,김경환,하선화,한범수,김용환,Park, Jong-Sug,Kim, Jong-Bum,Kim, Kyung-Hwan,Ha, Sun-Hwa,Han, Bum-Soo,Kim, Yong-Hwan 한국식물생명공학회 2002 식물생명공학회지 Vol.29 No.4

주요 농작물에서 건강-방어용 flavonoids 생성, phytoalexin (isoflavonoid, flavanol, proanthocyanidin)의 생성 및 소절을 통한 식물의 저항력 증대, 색소 (flavonol, anthocyanin)의 합성에 의한 자외선 방어, nod 유전자 inducer (flavones, isoflavones)의 대량 발현에 의한 혹 형성 (nodulation) 효율증대 등은 대사공학 적으로 향상 가능한 부분들이다. 파란 꽃을 개화하는 품종이 카네이션, 국화, 장미 등 중요 장식용 화훼작물들에는 결핍되어 있는데,이는 F3'5'H 유전자가 없어서 파란색 delphinidin 색소를 생산할 수 없기 때문으로 추정된다. 따라서 F3'5'H 유전자를 형질전환 하여 이러한 제한을 극복하고 delphinidin 유도체 생산이 가능하게 되면 파란색 꽃의 생산 가능성을 증대시킬 수 있게 된다. 또한 영양학적인 측면에서 이미 중요한 생리적 기능이 밝혀진 catechin을 비롯한 proanthocyanidin 과 anthocyanin은 의약품 및 식품첨가제 등 다양한 분야에서 크게 시장성을 넓히고 있어 상업적 측면에서 대사공학의 유망한 목표가 되고 있다. 최근의 대사공학 분야에서의 많은 성공에도 불구하고, flavonoid에 대한 고도의 대사공학 조절을 이용하여 원하는 flavonoid 화합물을 생성하거나, 원치 않는 flavonoid 화합물을 억제하도록 하는 데는 여전히 기술적 문제점들이 남아있다. 예를 들면 IFS와 FLS 등의 유전자 분리 그리고 조직 및 시기 특이적인 promoter 개발 등이 동시에 이루어져야 하며, co-pigmentation 및 액포 pH와 관련된 메카니즘에 대한 이해, 화훼작물들의 형질전환 기술 개발 등이 이루어져야 원하는 꽃의 착색 조절이 가능하게 될 것이다. 최근 나팔꽃에서 액포의 $Na^{+}$H$^{+}$ exchanger를 파괴하여 화색을 변경시킨 mutants 연구를 통하여 조만간 액포 pH의 조절을 이용한 식물 대사공학이 가능할 것으로 기대되고 있다 (Yamaguchi et al. 2001). 아직 자연계에서 기본적인 골격의 변경만으로 수천 종류의 flavonoid가 생성 가능한가는 여전히 의문점으로 남아 있으나, 분명한 것은 다양한 식물 체계에서의 노력으로 농업, 원예, 그리고 영양분 증대를 위한 flavonoid 대사를 어떻게 조절할 것인가에 대한 정보를 얻을 수 있고, 또한 flavonoid 생합성 연구로부터 얻어진 정보들을 통하여 세포질 대사와 기본적인 생물학적 현상에 대한 이해를 넓힐 수 있게 될 것이다. Flavonoid biosynthesis is one of the most extensively studied areas in the secondary metabolism. Due to the study of flavonoid metabolism in diverse plant system, the pathways become the best characterized secondary metabolites and can be excellent targets for metabolic engineering. These flavonoid-derived secondary metabolites have been considerably divergent functional roles: floral pigment, anticancer, antiviral, antitoxin, and hepatoprotective. Three species have been significant for elucidating the flavonoid metabolism and isolating the genes controlling the flavonoid genes: maize (Zea mays), snapdragon (Antirrhinum majus) and petunia (Prtunia hybrida). Recently, many genes involved in biosynthesis of flavonoid have been isolated and characterized using mutation and recombinant DNA technologies including transposon tagging and T-DNA tagging which are novel approaches for the discovery of uncharacterized genes. Metabolic engineering of flavonoid biosynthesis was approached by sense or antisense manipulation of the genes related with flavonoid pathway, or by modified expression of regulatory genes. So, the use of a variety of experimental tools and metabolic engineering facilitated the characterization of the flavonoid metabolism. Here we review recent progresses in flavonoid metabolism: confirmation of genes, metabolic engineering, and applications in the industrial use.

Recent advancements in bioreactions of cellular and cell-free systems: A study of bacterial cellulose as a model

Muhammad Wajid Ullah,마잘울이슬람,Shaukat Khan,Nasrullah Shah,박중곤 한국화학공학회 2017 Korean Journal of Chemical Engineering Vol.34 No.6

Conventional approaches of regulating natural biochemical and biological processes are greatly hampered by the complexity of natural systems. Therefore, current biotechnological research is focused on improving biological systems and processes using advanced technologies such as genetic and metabolic engineering. These technologies, which employ principles of synthetic and systems biology, are greatly motivated by the diversity of living organisms to improve biological processes and allow the manipulation and reprogramming of target bioreactions and cellular systems. This review describes recent developments in cell biology, as well as genetic and metabolic engineering, and their role in enhancing biological processes. In particular, we illustrate recent advancements in genetic and metabolic engineering with respect to the production of bacterial cellulose (BC) using the model systems Gluconacetobacter xylinum and Gluconacetobacter hansenii. Besides, the cell-free enzyme system, representing the latest engineering strategies, has been comprehensively described. The content covered in the current review will lead readers to get an insight into developing novel metabolic pathways and engineering novel strains for enhanced production of BC and other bioproducts formation.

The Role of High-throughput Transcriptome Analysis in Metabolic Engineering

Jewett, Michael C.,Oliveira, Ana Paula,Patil, Kiran Raosaheb,Nielsen, Jens The Korean Society for Biotechnology and Bioengine 2005 Biotechnology and Bioprocess Engineering Vol.10 No.5

The phenotypic response of a cell results from a well orchestrated web of complex interactions which propagate from the genetic architecture through the metabolic flux network. To rationally design cell factories which carry out specific functional objectives by controlling this hierarchical system is a challenge. Transcriptome analysis, the most mature high-throughput measurement technology, has been readily applied In strain improvement programs in an attempt to Identify genes involved in expressing a given phenotype. Unfortunately, while differentially expressed genes may provide targets for metabolic engineering, phenotypic responses are often not directly linked to transcriptional patterns, This limits the application of genome-wide transcriptional analysis for the design of cell factories. However, improved tools for integrating transcriptional data with other high-throughput measurements and known biological interactions are emerging. These tools hold significant promise for providing the framework to comprehensively dissect the regulatory mechanisms that identify the cellular control mechanisms and lead to more effective strategies to rewire the cellular control elements for metabolic engineering.

Directed evolution of the 3-hydroxypropionic acid production pathway by engineering aldehyde dehydrogenase using a synthetic selection device

Seok, Joo Yeon,Yang, Jina,Choi, Sang Jin,Lim, Hyun Gyu,Choi, Un Jong,Kim, Kyung-Jin,Park, Sunghoon,Yoo, Tae Hyeon,Jung, Gyoo Yeol Elsevier 2018 Metabolic engineering Vol.47 No.-

<P><B>Abstract</B></P> <P>3-Hydroxypropionic acid (3-HP) is an important platform chemical, and biological production of 3-HP from glycerol as a carbon source using glycerol dehydratase (GDHt) and aldehyde dehydrogenase (ALDH) has been revealed to be effective because it involves a relatively simple metabolic pathway and exhibits higher yield and productivity than other biosynthetic pathways. Despite the successful attempts of 3-HP production from glycerol, the biological process suffers from problems arising from low activity and inactivation of the two enzymes. To apply the directed evolutionary approach to engineer the 3-HP production system, we constructed a synthetic selection device using a 3-HP-responsive transcription factor and developed a selection approach for screening 3-HP-producing microorganisms. The method was applied to an ALDH library, specifically aldehyde-binding site library of alpha-ketoglutaric semialdehyde dehydrogenase (KGSADH). Only two serial cultures resulted in enrichment of strains showing increased 3-HP production, and an isolated KGSADH variant enzyme exhibited a 2.79-fold higher catalytic efficiency toward its aldehyde substrate than the wild-type one. This approach will provide the simple and efficient tool to engineer the pathway enzymes in metabolic engineering.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A synthetic selection device was designed responding to 3-HP concentration. </LI> <LI> ALDH in the 3-HP pathway was engineered through a simple selection method. </LI> <LI> An ALDH variant exhibited a 2.79-fold improvement in the catalytic efficiency. </LI> <LI> <I>E. coli</I> with ALDH variant showed a 25% higher productivity than the parental strain. </LI> <LI> The developed strategy was highly efficient for engineering the pathway enzymes. </LI> </UL> </P>