About
79
Publications
19,869
Reads
How we measure 'reads'
A 'read' is counted each time someone views a publication summary (such as the title, abstract, and list of authors), clicks on a figure, or views or downloads the full-text. Learn more
1,654
Citations
Introduction
Biotechnology, Synthetic biology, Cell-free protein synthesis
Current institution
Additional affiliations
December 2013 - August 2017
Education
October 2010 - September 2013
Publications
Publications (79)
The increasing demand for advanced biosystems necessitates innovative approaches to store and process genetic information. DNA, as a high‐density storage medium, offers a promising solution for creating genetic memory systems that can provide state‐dependent responses to various stimuli. To date, numerous studies have reported on genetic memory sys...
Cell-free gene expression (CFE) systems enable transcription and translation using crude cellular extracts, offering a versatile platform for synthetic biology by eliminating the need to maintain living cells. However, Such systems are constrained by cumbersome composition, high costs, and limited yields due to numerous additional components requir...
Amaryllidaceae alkaloids (AAs) are plant‐derived natural compounds with diverse bioactivities. To biosynthesize AAs, 4′‐O‐methylnorbelladine (4′‐MB) is a key precursor that is first formed by C–N coupling between tyramine and 3,4‐dihydroxybenzaldehyde catalyzed by a NADPH‐dependent reductase. However, 3,4‐dihydroxybenzaldehyde is oxygen sensitive a...
Pickering interfacial biocatalysis (PIB), where biocatalysts stabilize emulsions through carrier coupling or polymer grafting, has emerged as a powerful platform for organic synthesis due to its ability to accommodate water‐insoluble substrates within enzymatic cascade reactions. PIB provides a large interfacial area for two‐phase reactions, reduci...
Cell-free protein synthesis (CFPS) has proven invaluable for expressing a wide array of proteins and enzymes, boasting significant advantages, such as facile manipulation, rapid mass transfer, and high productivity. However, traditional cell-free expression systems typically operate in batch format reactions, posing challenges for enzyme reuse in b...
Synthetic BioBricks introduce novel capabilities to manipulate genetic information, direct transcription‐translation processes, and program cellular behaviors in living organisms. To maintain the stability and functionality of synthetic BioBricks, assembled DNA fragments should be mutually compatible without inducing negative effects such as metabo...
Enzyme catalysis is prominent in chemical synthesis due to its specificity, selectivity, and efficiency. However, enzymes often face challenges, such as inactivation and inhibition in practical applications, and they are also limited by narrow reaction scopes in complex catalytic scenarios like cascade reactions. These persistent issues have driven...
Cell extract-based cell-free gene expression (CFE) systems break cellular boundaries and create emerging platforms for synthetic biology applications. However, these systems lack intrinsic DNA replication capability, thereby limiting sustained transcription and translation in a cell-free environment. Here, we introduce a LoopReX system based on E....
Anthraquinone-fused enediynes are anticancer natural products featuring a DNA-intercalating anthraquinone moiety. Despite recent insights into anthraquinone-fused enediyne (AQE) biosynthesis, the enzymatic steps involved in anthraquinone biogenesis remain to be elucidated. Through a combination of in vitro and in vivo studies, we demonstrated that...
Cell-free gene expression (CFE) systems enable transcription and translation using crude cellular extracts, offering a versatile platform for synthetic biology by eliminating the need to maintain living cells. This allows direct manipulation of molecular components and the focused synthesis of specific products. However, the optimization of CFE sys...
Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural products with diverse chemical structures and potent biological activities. A vast majority of RiPP gene clusters remain unexplored in microbial genomes, which is partially due to the lack of rapid and efficient heterologous expression systems fo...
Molecular cloning facilitates the assembly of heterologous DNA fragments with vectors, resulting in the generation of plasmids that can steadily replicate in host cells. To efficiently and accurately screen out the expected plasmid candidates, various methods, such as blue‐white screening, have been developed for visualization. However, these metho...
Chemical modifications of enzymes excel in the realm of enzyme engineering due to its directness, robustness, and efficiency; however, challenges persist in devising versatile and effective strategies. In this study, we introduce a supramolecular modification methodology that amalgamates a supramolecular polymer with Candida antarctica lipase B (Ca...
Chemical modifications of enzymes excel in the realm of enzyme engineering due to its directness, robustness, and efficiency; however, challenges persist in devising versatile and effective strategies. In this study, we introduce a supramolecular modification methodology that amalgamates a supramolecular polymer with Candida antarctica lipase B (Ca...
Serine integrases are emerging as one of the powerful tools for synthetic biology. They have been widely developed across genome engineering, biological part construction, genetic circuit design, and in vitro DNA assembly. However, the strategy of in vivo DNA assembly by serine integrases has not yet been reported. To address this opportunity, here...
In this study, we addressed the limitations of conventional enzyme‐polymer‐conjugate‐based Pickering emulsions for interfacial biocatalysis, which traditionally suffer from nonspecific and uncontrollable conjugation positions that can impede catalytic performance. By introducing a non‐canonical amino acid (ncAA) at a specific site on target enzymes...
In this study, we addressed the limitations of conventional enzyme–polymer‐conjugate‐based Pickering emulsions for interfacial biocatalysis, which traditionally suffer from non‐specific and uncontrollable conjugation positions that can impede catalytic performance. By introducing a non‐canonical amino acid (ncAA) at a specific site on target enzyme...
Escherichia coli Nissle 1917 (EcN) is a probiotic microbe that has the potential to be developed as a promising chassis for synthetic biology applications. However, the molecular tools and techniques for utilizing EcN remain to be further explored. To address this opportunity, we systematically expanded the EcN‐based toolbox, enabling EcN as a powe...
Serine integrases are emerging as one of the most powerful biological tools for biotechnology. Over the past decade, many research papers have been published on the use of serine integrases in synthetic biology. In this review, we aim to systematically summarize the various studies ranging from structure and the catalytic mechanism to genetic desig...
Plant-originated natural products are important drug sources. However, total biosynthesis of these compounds is often not achievable due to their uncharacterized, lengthy biosynthetic pathways. In nature, phenethylisoquinoline alkaloids (PIAs) such as colchicine are biosynthesized via a common precursor 6,7-dihydroxy-1-(4-hydroxyphenylethyl)-1,2,3,...
Escherichia coli Nissle 1917 (EcN) is a probiotic microbe that has the potential to be developed as a promising chassis for synthetic biology applications. However, the molecular tools and techniques for utilizing EcN have not been fully explored. To address this opportunity, we systematically expanded the EcN-based toolbox, enabling EcN as a power...
Cell-free expression systems have emerged as a potent and promising platform for the biosynthesis of chemicals by reconstituting in vitro expressed enzymes. Here, we report cell-free biosynthesis of cinnamyl alcohol (cinOH) with enhanced productivity by using the Plackett-Burman experimental design for multifactor optimization. Initially, four enzy...
Crude extract-based cell-free expression systems have been used to produce natural products by reconstitution of their biosynthetic pathways in vitro. However, the chemical scope of cell-free synthesized natural compounds is still limited, which is partially due to the length of biosynthetic gene clusters. To expand the product scope, here, we repo...
Serratiopeptidase is a clinical therapeutic protein for the treatment of human diseases such as arthritis, bronchitis, and thrombosis. Yet production of this protein in a heterologous host (e.g., Escherichia coli) is difficult due to the issue of protein insolubility and the requirement of laborious refolding procedures. Cell-free protein synthesis...
Erythritol, one of the natural sugar alcohols, is widely used as a sugar substitute sweetener in food industries. Humans themselves are not able to catabolize erythritol and their gut microbes lack related catabolic pathways either to metabolize erythritol. Here, Escherichia coli (E. coli) is engineered to utilize erythritol as sole carbon source a...
Ribosomes cannot synthesize peptides using hydroxy acids to replace canonical amino acids as no codons encode hydroxy acid building blocks. Now, this challenge has been addressed by rewriting the genetic code, enabling the direct cellular biosynthesis of non-natural depsipeptides containing non-canonical amino acids.
Nonribosomal peptides (NRPs) are a large family of secondary metabolites with notable bioactivities, which distribute widely in natural resources across microbes and plants. To obtain these molecules, heterologous production of NRPs in robust surrogate hosts like Escherichia coli represent a feasible approach. However, reconstitution of the full bi...
Permeability‐Engineered Compartmentalization Strategy The permeability‐engineered compartmentalization strategy (PeCS) is represented by a magical bubble configurable by a wand. The bubble's outer layer serves not only as a simple physical barrier but a sieving structure that is semi‐permeable to biomolecules. Nucleic acids, proteins, and bacteria...
Fatty acid-derived products such as alkanes, fatty aldehydes, and fatty alcohols have many applications in the chemical industry. These products are predominately produced from fossil resources, but their production processes are often not environmentally friendly. While microbes like Escherichia coli have been engineered to convert fatty acids to...
Streptomyces bacteria are a major microbial source of natural products, which are encoded within so-called biosynthetic gene clusters (BGCs). This highlight discusses the emergence of native Streptomyces cell-free systems as a new tool to accelerate the study of the fundamental chemistry and biology of natural product biosynthesis from these bacter...
Erythritol, one of the natural sugar alcohols, is widely used as a sugar substitute sweetener in food industries. Humans and their native gut microbiota lack erythritol catabolic microorganisms to utilize erythritol as carbon source, which inspires us to engineer a model chassis that can respond and utilize erythritol for establishing wide applicat...
In nature, biological compartments such as cells rely on dynamically controlled permeability for matter exchange and complex cellular activities. Likewise, the ability to engineer compartment permeability is crucial for in vitro systems to gain sustainability, robustness, and complexity. However, rendering in vitro compartments such a capability is...
Cell-free protein synthesis (CFPS) systems are emerging as powerful platforms for in vitro protein production, which leads to the development of new CFPS systems for different applications. To expand the current CFPS toolkit, here we develop a novel CFPS system derived from a chassis microorganism Klebsiella pneumoniae, an important industrial host...
The natural bacterial spores have inspired the development of artificial spores, through coating cells with protective materials, for durable whole-cell catalysis. Despite attractiveness, artificial spores developed to date are generally limited to a few microorganisms with their natural endogenous enzymes, and they have never been explored as a ge...
Natural products have complex chemical structures and exhibit diverse bioactivities. To harness these high-value natural compounds, robust approaches are required for their rapid and sustainable production. Recently, cell-free systems as one promising solution have been applied to produce natural products by reconstitution of their entire biosynthe...
Cell-free system has emerged as a powerful platform with a wide range of in vitro applications and recently has contributed to express metabolic pathways for biosynthesis. Here we report in vitro construction of a native biosynthetic pathway for L-4-nitrotryptophan (L-4-nitro-Trp) synthesis using an Escherichia coli-based cell-free protein synthesi...
The cell-wall recycling process is important for bacterial survival in nutrient-limited conditions and, in certain cases, is directly involved in antibiotic resistance. In the sophisticated cell-wall recycling process in Escherichia coli, the transcriptional repressor MurR controls the expression of murP and murQ, which are involved in transporting...
The compartmentalized and communicative nature of biological cells contributes to the complexity and endurance of living organisms. We present a general compartmentalization strategy for in vitro systems that inherits the passive transport phenomenon of biology. The strategy incorporates layered, micrometer-sized, hydrogel-based compartments featur...
Serine integrases are emerging as one of the most powerful biological tools for synthetic biology. They have been widely used across genome engineering and genetic circuit design. However, developing serine integrase-based tools for directly/precisely manipulating synthetic biobricks is still missing. Here, we report SYMBIOSIS, a versatile method t...
Streptomyces-based cell-free expression systems have been developed to meet the demand for synthetic biology applications. However, protein yields from the previous Streptomyces systems are relatively low, and there is a serious limitation of available genetic tools such as plasmids for gene (co)expression. Here, we sought to expand the plasmid too...
With the rapid development of cell-free biotechnology, more and more cell-free protein synthesis (CFPS) systems have been established and optimized for protein expression in vitro. Here, we aim to improve the productivity of a newly developed Streptomyces-based CFPS system. Protein translation in CFPS systems depends on the entire endogenous transl...
Serine integrases are emerging as one of the most powerful biological tools for synthetic biology. They have been widely used across genome engineering and genetic circuit design. However, developing serine integrase-based tools for directly/precisely manipulating synthetic biobricks is still missing. Here, we report SYMBIOSIS, a versatile method t...
Cell-free systems have been used to synthesize chemicals by reconstitution of in vitro expressed enzymes. However, coexpression of multiple enzymes to reconstitute long enzymatic pathways is often problematic due to resource limitation/competition (e.g., energy) in the one-pot cell-free reactions. To address this limitation, here we aim to design a...
Valinomycin is a nonribosomal peptide that was discovered from Streptomyces in 1955. Over the past more than six decades, it has received continuous attention due to its special chemical structure and broad biological activities. Although many research papers have been published on valinomycin, there has not yet been a comprehensive review that sum...
l-Theanine, as an active component of the leaves of the tea plant, possesses many health benefits and broad applications. Chemical synthesis of l-theanine is possible; however, this method generates chiral compounds and needs further isolation of the pure l-isoform. Heterologous biosynthesis is an alternative strategy, but one main limitation is th...
Biocatalytic cascade reactions have become increasingly important and useful for chemical synthesis. However, biocatalysts are often incompatible with organic solvents, which prohibits many cascade reactions involving nonpolar substrates. In this study, we used cell‐free protein synthesis (CFPS) to express enzymes in an aqueous‐organic biphasic sys...
In recent years, cell-free protein synthesis (CFPS) systems have been used to synthesize proteins, prototype genetic elements, manufacture chemicals, and diagnose diseases. These exciting, novel applications lead to a new wave of interest in the development of new CFPS systems that are derived from prokaryotic and eukaryotic organisms. The eukaryot...
Cell-free protein synthesis (CFPS) systems are emerging as effective platforms for the production of recombinant proteins in vitro. To enable the expression of various proteins, different CFPS systems have been developed to better mimic the cellular environment of native hosts. In this context, a Streptomyces-based CFPS system was recently develope...
Biocatalytic cascade reactions have become increasingly important and useful for chemical synthesis. However, biocatalysts are often incompatible with organic solvents, which prohibits many cascade reactions involving nonpolar substrates. In this work, we used cell-free protein synthesis (CFPS) to express enzymes in an aqueous-organic biphasic syst...
Natural products are important because of their significant pharmaceutical properties such as antiviral, antimicrobial, and anticancer activity. Recent breakthroughs in DNA sequencing reveal that a great number of cryptic natural product biosynthetic gene clusters are encoded in microbial genomes, for example, those of Streptomyces species. However...
Cell-free protein synthesis (CFPS) is a technology for the production of proteins in vitro, which has emerged as a robust platform for biotechnology and synthetic biology applications. Recently, the renewed scientific interest in CFPS technology has driven the rapid development of cell-free biotechnology. In this review, we focus on the very recent...
Cell-free protein synthesis (CFPS) systems enable the production of protein without the use of living, intact cells. An emerging area of interest is to use CFPS systems to characterize individual elements for genetic programs [e.g. promoters, ribosome binding sites (RBS)]. To enable this research area, robust CFPS systems must be developed from new...
Centralized facilities for genetic engineering, or “biofoundries,” offer the potential to design organisms to address emerging needs in medicine, agriculture, industry, and defense. The field has seen rapid advances in technology, but it is difficult to gauge current capabilities or identify gaps across projects. To this end, our foundry was assess...
Natural products with significant biological activities continuously act as rich sources for drug discovery and development. To harness the potential of these valuable compounds, robust methods need to be developed for their rapid and sustainable production. Cell-free biosynthesis of pharmaceutical natural products by in vitro reconstruction of the...
Cell-free protein synthesis (CFPS) has emerged as a powerful approach to recombinant protein biosynthesis for applications in biochemical engineering and synthetic biology. To date, CFPS systems have been most commonly derived from a variety of organism sources including microbes (e.g., Escherichia coli and yeast), plants (e.g., wheat germ and toba...
Cell-free protein synthesis (CFPS) has emerged as a powerful platform for applied biotechnology and synthetic biology, with a range of applications in synthesizing proteins, evolving proteins, and prototyping genetic circuits. To expand the current CFPS repertoire, we report here the development and optimization of a Streptomyces-based CFPS system...
Genome sequencing has revealed that a far greater number of natural product biosynthetic pathways exist than there are known natural products. To access these molecules directly and deterministically, a new generation of heterologous expression methods is needed. Cell-free protein synthesis has not previously been used to study nonribosomal peptide...
Multicopper oxidases (MCOs) are broadly distributed in all kingdoms of life and perform a variety of important oxidative reactions. These enzymes have potential biotechnological applications; however, the applications are impeded by low expression yields in traditional recombinant hosts, solubility issues, and poor copper cofactor assembly. As an a...
We report a simple temperature-responsive bioconjugate system comprised of superfolder green fluorescent protein (sfGFP) decorated with poly[(oligo ethylene glycol) methyl ether methacrylate] (PEGMA) polymers. We used amber suppression to site-specifically incorporate the non-natural azide-functional amino acid p-azidophenylalanine (pAzF) into sfGF...
Heterologous production of natural products in Escherichia coli has emerged as an attractive strategy to obtain molecules of interest. Although technically feasible most of them are still constrained to laboratory scale production. Therefore, it is necessary to develop reasonable scale-up strategies for bioprocesses aiming at the overproduction of...
Nonribosomal peptides (NRPs), a large family of natural products, possess numerous pharmaceutically significant bioactivities. However, many native microbial producers of NRPs are not cultivable or have low production yields making mass production infeasible. The recombinant production of natural products in a surrogate host has emerged as a strate...
The structural complexity of nonribosomal peptides (NRPs) impeding economic chemical synthesis and poor cultivability of source organisms limits the development of bioprocesses for novel bioactive compounds. Since nonribosomal peptide synthetases (NRPSs) assemble NRPs from simple amino acid building blocks, heterologous expression of NRPSs in a rob...
Nichtribosomale Peptide (NRPs) gehören zu einer großen Familie von Naturstoffen und werden von multimodularen Nichtribosomalen Peptidsynthetasen (NRPSs) in verschiedenen Mikroorganismen wie Bakterien und Pilzen synthetisiert. Diese natürlich vorkommenden Verbindungen sind von großem Interesse, da sie zahlreiche bedeutende antivirale, antimikrobiell...
