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Detecting alterations in plasmid structures is often performed using conventional molecular biology. However, these methods are laborious and time-consuming for studying the conditions inducing these mutations, which prevent real-time access to cell heterogeneity during bioproduction. In this work, we propose combining both flow cytometry and fluorescence-activated cell sorting, integrated with mechanistic modelling to study conditions that lead to plasmid recombination using a limonene-producing microbial system as a case study. A gene encoding GFP was introduced downstream of the key enzymes involved in limonene biosynthesis to enable real-time kinetics monitoring and the identification of cell heterogeneity according to microscopic and flow cytometric analyses. Three different plasmid configurations (one correct and two incorrect) were identified through cell sorting based on subpopulations expressing different levels of GFP at 10 and 50 µM IPTG. Higher limonene production (530 mg/L) and lower subpopulation proportion carrying the incorrect plasmid (12%) were observed for 10 µM IPTG compared to 50 µM IPTG (96 mg/L limonene and more than 70% of cell population carrying the incorrect plasmid, respectively) in 100 mL production culture. We also managed to derive exploratory hypotheses regarding the plasmid recombination region using the model and successfully validated them experimentally. Additionally, the results also showed that limonene production was proportional to GFP fluorescence intensity. This correlation could serve as an alternative to using biosensors for a high-throughput screening process. The developed method enables rapid identification of plasmid recombination at single-cell level and correlates the heterogeneity with bioproduction performance. KEY POINTS: • Strategy to study plasmid recombination during bioproduction. • Different plasmid structures can be identified and monitored by flow cytometry. • Mathematical modelling suggests specific alterations in plasmid structures., Competing Interests: Declarations. This article does not contain any studies with animals performed by any of the authors. Conflict of interest: The authors declare no competing interests., (© 2025. The Author(s).)

Benzylisoquinoline alkaloids (BIAs) are a class of natural compounds found in plants of the Ranunculaceae family, known for their diverse pharmacological activities. However, the extraction yields of BIAs from plants are limited, and the cost of chemical synthesis is prohibitively high. Recent advancements in systems metabolic engineering and genomics have made it feasible to use microbes as bioreactors for BIAs production. This review explores recent progress in enhancing the production and yields of BIAs in two microbial systems: Escherichia coli and Saccharomyces cerevisiae . It covers various BIAs, including ( S )-reticuline, morphinane, protoberberine, and aporphine alkaloids. The review provides strategies and technologies for BIAs synthesis, analyzes current challenges in BIAs research, and offers recommendations for future research directions.

This study introduces a synthetic biology approach that reprograms the yeast mating-type switching mechanism for tunable cell differentiation, facilitating synthetic microbial consortia formation and cooperativity. The underlying mechanism was engineered into a genetic logic gate capable of inducing asymmetric sexual differentiation within a haploid yeast population, resulting in a consortium characterized by mating-type heterogeneity and tunable population composition. The utility of this approach in microbial consortia cooperativity was demonstrated through the sequential conversion of xylan into xylose, employing haploids of opposite mating types each expressing a different enzyme of the xylanolytic pathway. This strategy provides a versatile framework for producing and fine-tuning functionally heterogeneous yet isogenic yeast consortia, furthering the advancement of microbial consortia cooperativity and offering additional avenues for biotechnological applications., (© 2024. The Author(s).)

Addressing urgent environmental challenges, this commentary emphasizes the need for green, bio-based solutions in chemical production from renewable feedstocks. It highlights advanced metabolic engineering of microbial strains and the use of microbial consortia as innovative approaches for efficient resource recovery. These strategies aim to enhance the conversion of diverse renewable feedstocks, including agricultural residues, industrial by-products, and greenhouse gases, into value-added chemicals. This article discusses cutting-edge techniques in renewable feedstock upcycling, utilizing both engineered unicellular and multicellular systems. It advocates a paradigm shift in sustainable biomanufacturing, focusing on transforming renewable resources into value-added products. This approach is crucial for developing a circular bioeconomy, aligning with global efforts to mitigate environmental impacts., Competing Interests: The authors declare no conflict of interest. Matthew Wook Chang is the Editor-in-Chief for Biotechnology Notes and was not involved in the editorial review or the decision to publish this article., (© 2024 The Authors.)

Chromosome-level design-build-test-learn cycles (chrDBTLs) allow systematic combinatorial reconfiguration of chromosomes with ease. Here, we established chrDBTL with a redesigned synthetic Saccharomyces cerevisiae chromosome XV , synXV . We designed and built synXV to harbor strategically inserted features, modified elements, and synonymously recoded genes throughout the chromosome. Based on the recoded chromosome, we developed a method to enable chrDBTL: CRISPR-Cas9-mediated mitotic recombination with endoreduplication (CRIMiRE). CRIMiRE allowed the creation of customized wild-type/synthetic combinations, accelerating genotype-phenotype mapping and synthetic chromosome redesign. We also leveraged synXV as a "build-to-learn" model organism for translation studies by ribosome profiling. We conducted a locus-to-locus comparison of ribosome occupancy between synXV and the wild-type chromosome, providing insight into the effects of codon changes and redesigned features on translation dynamics in vivo . Overall, we established synXV as a versatile reconfigurable system that advances chrDBTL for understanding biological mechanisms and engineering strains., Competing Interests: J.D.B. is a founder and director of CDI Labs, Inc.; a founder of and consultant to Neochromosome, Inc.; and a founder of, scientific advisory board member of, and consultant to ReOpen Diagnostics, LLC and serves or served on the scientific advisory boards of the following: Sangamo, Inc.; Logomix, Inc.; Modern Meadow, Inc.; Rome Therapeutics, Inc.; Sample6, Inc.; Tessera Therapeutics, Inc.; and the Wyss Institute., (© 2023 The Authors.)

Since its inception, synthetic biology has overcome many technical barriers but is at a crossroads for high-precision biological design. Devising ways to fully utilize big biological data may be the key to achieving greater heights in synthetic biology., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Kitano et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Nucleic acid detection is crucial for monitoring diseases for which rapid, sensitive, and easy-to-deploy diagnostic tools are needed. CRISPR-based technologies can potentially fulfill this need for nucleic acid detection. However, their widespread use has been restricted by the requirement of a protospacer adjacent motif in the target and extensive guide RNA optimization. In this study, we developed FELICX, a technique that can overcome these limitations and provide a useful alternative to existing technologies. FELICX comprises flap endonuclease, Taq ligase and CRISPR-Cas for diagnostics (X) and can be used for detecting nucleic acids and single-nucleotide polymorphisms. This method can be deployed as a point-of-care test, as only two temperatures are needed without thermocycling for its functionality, with the result generated on lateral flow strips. As a proof-of-concept, we showed that up to 0.6 copies/μL of DNA and RNA could be detected by FELICX in 60 min and 90 min, respectively, using simulated samples. Additionally, FELICX could be used to probe any base pair, unlike other CRISPR-based technologies. Finally, we demonstrated the versatility of FELICX by employing it for virus detection in infected human cells, the identification of antibiotic-resistant bacteria, and cancer diagnostics using simulated samples. Based on its unique advantages, we envision the use of FELICX as a next-generation CRISPR-based technology in nucleic acid diagnostics., Competing Interests: Declaration of competing interest N.A., J.L.F., H.L., I.Y.H. and M.W.C have filed a patent application through the National University of Singapore based on the work described in this manuscript. The remaining authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Fluorescent proteins are widely used molecular reporters in studying gene expression and subcellular protein localization. To enable the monitoring of transient cellular events in the model yeast Saccharomyces cerevisiae , destabilized green and cyan fluorescent proteins have been constructed. However, their co-utilization is limited by an overlap in their excitation and emission spectra. Although red fluorescent protein is compatible with both green and cyan fluorescent proteins with respect to spectra resolution, a destabilized red fluorescent protein is yet to be constructed for applications in S. cerevisiae . To realize this, we adopted a degron-fusion strategy to prompt destabilization of red fluorescent protein. Specifically, we fused two degrons derived from Cln2, a G 1 -specific cyclin that mediates cell cycle transition, to the N- or C-terminus of mCherry to generate four destabilized fluorescent proteins that are soluble and functional in S . cerevisiae . Importantly, the four mCherry fluorescent proteins are highly differential with regards to fluorescence half-life and intensity, which provides a greater choice of tools available for the study of dynamic gene expression and transient cellular processes in the model yeast., Competing Interests: The authors declare no conflict of interest., (© 2022 The Authors.)

Biosensors can be used for real-time monitoring of metabolites and high-throughput screening of producer strains. Use of biosensors has facilitated strain engineering to efficiently produce value-added compounds. Following our recent work on the production of short branched-chain fatty acids (SBCFAs) in engineered Saccharomyces cerevisiae , here we harnessed a weak organic acid transporter Pdr12p, engineered a whole-cell biosensor to detect exogenous and intracellular SBCFAs and optimized the biosensor's performance by varying PDR12 expression. We firstly constructed the biosensor and evaluated its response to a range of short-chain carboxylic acids. Next, we optimized its sensitivity and operational range by deletion and overexpression of PDR12 . We found that the biosensor responded to exogenous SBCFAs including isovaleric acid, isobutyric acid and 2-methylbutanoic acid. PDR12 deletion enhanced the biosensor's sensitivity to isovaleric acid at a low concentration and PDR12 overexpression shifted the operational range towards a higher concentration. Lastly, the deletion of PDR12 improved the biosensor's sensitivity to the SBCFAs produced in our previously engineered SBCFA-overproducing strain. To our knowledge, our work represents the first study on employing an ATP-binding-cassette transporter to engineer a transcription-factor-based genetic biosensor for sensing SBCFAs in S. cerevisiae . Our findings provide useful insights into SBCFA detection by a genetic biosensor that will facilitate the screening of SBCFA-overproducing strains., Competing Interests: Authors RM and NF were employed by company Mitsubishi Chemical Corporation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Miyake, Ling, Foo, Fugono and Chang.)

Monoterpenoids are an important class of natural products that are derived from the condensation of two five‑carbon isoprene subunits. They are widely used for flavouring, fragrances, colourants, cosmetics, fuels, chemicals, and pharmaceuticals in various industries. They can also serve as precursors for the production of many industrially important products. Currently, monoterpenoids are produced predominantly through extraction from plant sources. However, the small quantity of monoterpenoids in nature renders this method of isolation non-economically viable. Similarly impractical is the chemical synthesis of these compounds as they suffer from high energy consumption and pollutant discharge. Microbial biosynthesis, however, exists as a potential solution to these hindrances, but the transformation of cells into efficient factories remains a major impediment. Here, we critically review the recent advances in engineering microbes for monoterpenoid production, with an emphasis on categorized strategies, and discuss the challenges and perspectives to offer guidance for future engineering., (Copyright © 2021. Published by Elsevier Inc.)

Background: In biological cells, promoters drive gene expression by specific binding of RNA polymerase. They determine the starting position, timing and level of gene expression. Therefore, rational fine-tuning of promoters to regulate the expression levels of target genes for optimizing biosynthetic pathways in metabolic engineering has recently become an active area of research., Results: In this study, we systematically detected and characterized the common promoter elements in the unconventional yeast Yarrowia lipolytica, and constructed an artificial hybrid promoter library that covers a wide range of promoter strength. The results indicate that the hybrid promoter strength can be fine-tuned by promoter elements, namely, upstream activation sequences (UAS), TATA box and core promoter. Notably, the UASs of Saccharomyces cerevisiae promoters were reported for the first time to be functionally transferred to Y. lipolytica. Subsequently, using the production of a versatile platform chemical isoamyl alcohol as a test study, the hybrid promoter library was applied to optimize the biosynthesis pathway expression in Y. lipolytica. By expressing the key pathway gene, ScARO10, with the promoter library, 1.1-30.3 folds increase in the isoamyl alcohol titer over that of the control strain Y. lipolytica Po1g KU70∆ was achieved. Interestingly, the highest titer increase was attained with a weak promoter P UAS1B4-EXPm to express ScARO10. These results suggest that our hybrid promoter library can be a powerful toolkit for identifying optimum promoters for expressing metabolic pathways in Y. lipolytica., Conclusion: We envision that this promoter engineering strategy and the rationally engineered promoters constructed in this study could also be extended to other non-model fungi for strain improvement.

Limonene is an important plant natural product widely used in food and cosmetics production as well as in the pharmaceutical and chemical industries. However, low efficiency of plant extraction and high energy consumption in chemical synthesis limit the sustainability of industrial limonene production. Recently, the advancement of metabolic engineering and synthetic biology has facilitated the engineering of microbes into microbial cell factories for producing limonene. However, the deleterious effects on cellular activity by the toxicity of limonene is the major obstacle in achieving high-titer production of limonene in engineered microbes. In this study, by using transcriptomics, we identified 82 genes from the nonconventional yeast Yarrowia lipolytica that were up-regulated when exposed to limonene. When overexpressed, 8 of the gene candidates improved tolerance of this yeast to exogenously added limonene. To determine whether overexpression of these genes could also improve limonene production, we individually coexpressed the tolerance-enhancing genes with a limonene synthase gene. Indeed, expression of 5 of the 8 candidate genes enhanced limonene production in Y. lipolytica . Particularly, overexpressing YALI0F19492p led to an 8-fold improvement in product titer. Furthermore, through short-term adaptive laboratory evolution strategy, in combination with morphological and cytoplasmic membrane integrity analysis, we shed light on the underlying mechanism of limonene cytotoxicity to Y. lipolytica . This study demonstrated an effective strategy for improving limonene tolerance of Y. lipolytica and limonene titer in the host strain through the combinatorial use of tolerance engineering and evolutionary engineering.

Carboxylic acids contain carboxyl groups that can undergo a wide range of chemical transformation. Therefore, they serve as key platform chemicals for the production of high value-added industrial products. Currently, the majority of carboxylic acid platform chemicals is produced predominantly through traditional chemical synthesis. However, these chemical conversion processes are heavily dependent on fossil fuels and often lead to serious environmental pollution. Recently, the rapid development in metabolic engineering of microbes provide a new and promising alternative route for producing carboxylic acids as platform chemicals. We envision that these bio-based manufacturing processes using microbial cell factories will help move the industrial production of carboxylic acid platform chemicals towards a more sustainable, environmentally friendly and economically competitive direction. While Escherichia coli and Saccharomyces cerevisiae have been the workhorses for biochemical production through metabolic engineering, non-conventional microbes are emerging as suitable hosts for producing carboxylic acids to meet the needs of the industries. Here, we review the employment of metabolic engineering strategies on non-conventional microbes to serve as microbial cell factories for the production of industrially important carboxylic acid platform chemicals., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Aldehydes are a class of highly versatile chemicals that can undergo a wide range of chemical reactions and are in high demand as starting materials for chemical manufacturing. Biologically, fatty aldehydes can be produced from fatty acyl-CoA by the action of fatty acyl-CoA reductases. The aldehydes produced can be further converted enzymatically to other valuable derivatives. Thus, metabolic engineering of microorganisms for biosynthesizing aldehydes and their derivatives could provide an economical and sustainable platform for key aldehyde precursor production and subsequent conversion to various value-added chemicals. Saccharomyces cerevisiae is an excellent host for this purpose because it is a robust organism that has been used extensively for industrial biochemical production. However, fatty acyl-CoA-dependent aldehyde-forming enzymes expressed in S. cerevisiae thus far have extremely low activities, hence limiting direct utilization of fatty acyl-CoA as substrate for aldehyde biosynthesis. Toward overcoming this challenge, we successfully engineered an alcohol-forming fatty acyl-CoA reductase for aldehyde production through rational design. We further improved aldehyde production through strain engineering by deleting competing pathways and increasing substrate availability. Subsequently, we demonstrated alkane and alkene production as one of the many possible applications of the aldehyde-producing strain. Overall, by protein engineering of a fatty acyl-CoA reductase to alter its activity and metabolic engineering of S. cerevisiae , we generated strains with the highest reported cytosolic aliphatic aldehyde and alkane/alkene production to date in S. cerevisiae from fatty acyl-CoA., (Copyright © 2020 Foo, Rasouliha, Susanto, Leong and Chang.)

Lipoic acid is a valuable organosulfur compound used as an antioxidant for dietary supplementation, and potentially anti-diabetic and anti-cancer. Currently, lipoic acid is obtained mainly through chemical synthesis, which requires toxic reagents and organic solvents, thus causing environmental issues. Moreover, chemically synthesized lipoic acid is conventionally a racemic mixture. To obtain enantiomerically pure R -lipoic acid, which has superior bioactivity than the S form, chiral resolution and asymmetric synthesis methods require additional reagents and solvents, and often lead to wastage of S -lipoic acid or precursors with undesired chirality. Toward sustainable production of R -lipoic acid, we aim to develop a synthetic biology-based method using engineered yeast. Here, we deepened mechanistic understanding of lipoic acid biosynthesis and protein lipoylation in the model yeast Saccharomyces cerevisiae to facilitate metabolic engineering of the microbe for producing free R -lipoic acid. In brief, we studied the biosynthesis and confirmed the availability of protein-bound lipoate in yeast cells through LC-MS/MS. We then characterized in vitro the activity of a lipoamidase from Enterococcus faecalis for releasing free R -lipoic acid from lipoate-modified yeast proteins. Overexpression of the lipoamidase in yeast mitochondria enabled de novo free R -lipoic acid production in vivo. By overexpressing pathway enzymes and regenerating the cofactor, the production titer was increased ∼2.9-fold. This study represents the first report of free R -lipoic acid biosynthesis in S. cerevisiae . We envision that these results could provide insights into lipoic acid biosynthesis in eukaryotic cells and drive development of sustainable R -lipoic acid production., (Copyright © 2020 Chen, Foo, Ling and Chang.)

Living organisms evolve complex genetic networks to interact with the environment. Due to the rapid development of synthetic biology, various modularized genetic parts and units have been identified from these networks. They have been employed to construct synthetic genetic circuits, including toggle switches, oscillators, feedback loops and Boolean logic gates. Building on these circuits, complex genetic machines with capabilities in programmable decision-making could be created. Consequently, these accomplishments have led to novel applications, such as dynamic and autonomous modulation of metabolic networks, directed evolution of biological units, remote and targeted diagnostics and therapies, as well as biological containment methods to prevent release of engineered microorganisms and genetic materials. Herein, we outline the principles in genetic circuit design that have initiated a new chapter in transforming concepts to realistic applications. The features of modularized building blocks and circuit architecture that facilitate realization of circuits for a variety of novel applications are discussed. Furthermore, recent advances and challenges in employing genetic circuits to impart microorganisms with distinct and programmable functionalities are highlighted. We envision that this review gives new insights into the design of synthetic genetic circuits and offers a guideline for the implementation of different circuits in various aspects of biotechnology and bioengineering., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Cyanobacteria are of great importance to Earth's ecology. Due to their capability in photosynthesis and C1 metabolism, they are ideal microbial chassis that can be engineered for direct conversion of carbon dioxide and solar energy into biofuels and biochemicals. Facilitated by the elucidation of the basic biology of the photoautotrophic microbes and rapid advances in synthetic biology, genetic toolkits have been developed to enable implementation of nonnatural functionalities in engineered cyanobacteria. Hence, cyanobacteria are fast becoming an emerging platform in synthetic biology and metabolic engineering. Herein, the progress made in the synthetic biology toolkits for cyanobacteria and their utilization for transforming cyanobacteria into microbial cell factories for sustainable production of biofuels and biochemicals is outlined. Current techniques in heterologous gene expression, strategies in genome editing, and development of programmable regulatory parts and modules for engineering cyanobacteria towards biochemical production are discussed and prospected. As cyanobacteria synthetic biology is still in its infancy, apart from the achievements made, the difficulties and challenges in applying and developing genetic toolkits in cyanobacteria for biochemical production are also evaluated., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Synthetic biologists construct biological components and systems to look into biological phenomena and drive a myriad of practical applications that aim to tackle current global challenges in energy, healthcare and the environment. While most tools have been established in bacteria, particularly Escherichia coli, recent years have seen parallel developments in the model yeast strain Saccharomyces cerevisiae, one of the most well-understood eukaryotic biological system. Here, we outline the latest advances in yeast synthetic biology tools based on a framework of abstraction hierarchies of parts, circuits and genomes. In brief, the creation and characterization of biological parts are explored at the transcriptional, translational and post-translational levels. Using characterized parts as building block units, the designing of functional circuits is elaborated with examples. In addition, the status and potential applications of synthetic genomes as a genome level platform for biological system construction are also discussed. In addition to the development of a toolkit, we describe how those tools have been applied in the areas of drug production and screening, study of disease mechanisms, pollutant sensing and bioremediation. Finally, we provide a future outlook of yeast as a workhorse of eukaryotic genetics and a chosen chassis in this field., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Living organisms have evolved over millions of years to fine tune their metabolism to create efficient pathways for producing metabolites necessary for their survival. Advancement in the field of synthetic biology has enabled the exploitation of these metabolic pathways for the production of desired compounds by creating microbial cell factories through metabolic engineering, thus providing sustainable routes to obtain value-added chemicals. Following the past success in metabolic engineering, there is increasing interest in diversifying natural metabolic pathways to construct non-natural biosynthesis routes, thereby creating possibilities for producing novel valuable compounds that are non-natural or without elucidated biosynthesis pathways. Thus, the range of chemicals that can be produced by biological systems can be expanded to meet the demands of industries for compounds such as plastic precursors and new antibiotics, most of which can only be obtained through chemical synthesis currently. Herein, we review and discuss novel strategies that have been developed to rewrite natural metabolic blueprints in a bid to broaden the chemical repertoire achievable in microorganisms. This review aims to provide insights on recent approaches taken to open new avenues for achieving biochemical production that are beyond currently available inventions.

Microbiomes exist in all ecosystems and are composed of diverse microbial communities. Perturbation to microbiomes brings about undesirable phenotypes in the hosts, resulting in diseases and disorders, and disturbs the balance of the associated ecosystems. Engineering of microbiomes can be used to modify structures of the microbiota and restore ecological balance. Consequently, microbiome engineering has been employed for improving human health and agricultural productivity. The importance and current applications of microbiome engineering, particularly in humans, animals, plants and soil is reviewed. Furthermore, we explore the challenges in engineering microbiome and the future of this field, thus providing perspectives and outlook of microbiome engineering., (Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Fatty aldehydes and alcohols are valuable precursors used in the industrial manufacturing of a myriad of specialty products. Herein, we demonstrate the de novo production of odd chain-length fatty aldehydes and fatty alcohols in Saccharomyces cerevisiae by expressing a novel biosynthetic pathway involving cytosolic thioesterase, rice α-dioxygenase and endogenous aldehyde reductases. We attained production titers of ∼20 mg/l fatty aldehydes and ∼20 mg/l fatty alcohols in shake flask cultures after 48 and 60 h respectively without extensive fine-tuning of metabolic fluxes. In contrast to prior studies which relied on bi-functional fatty acyl-CoA reductase to produce even chain-length fatty alcohols, our biosynthetic route exploits α-oxidation reaction to produce odd chain-length fatty aldehyde intermediates without using NAD(P)H cofactor, thereby conserving cellular resource during the overall synthesis of odd chain-length fatty alcohols. The biosynthetic pathway presented in this study has the potential to enable sustainable and efficient synthesis of fatty acid-derived chemicals from processed biomass., (© 2015 Wiley Periodicals, Inc.)

Short branched-chain fatty acids (SBCFAs, C4-6) are versatile platform intermediates for the production of value-added products in the chemical industry. Currently, SBCFAs are mainly synthesized chemically, which can be costly and may cause environmental pollution. In order to develop an economical and environmentally friendly route for SBCFA production, we engineered Saccharomyces cerevisiae, a model eukaryotic microorganism of industrial significance, for the overproduction of SBCFAs. In particular, we employed a combinatorial metabolic engineering approach to optimize the native Ehrlich pathway in S. cerevisiae. First, chromosome-based combinatorial gene overexpression led to a 28.7-fold increase in the titer of SBCFAs. Second, deletion of key genes in competing pathways improved the production of SBCFAs to 387.4 mg/L, a 31.2-fold increase compared to the wild-type. Third, overexpression of the ATP-binding cassette (ABC) transporter PDR12 increased the secretion of SBCFAs. Taken together, we demonstrated that the combinatorial metabolic engineering approach used in this study effectively improved SBCFA biosynthesis in S. cerevisiae through the incorporation of a chromosome-based combinatorial gene overexpression strategy, elimination of genes in competitive pathways and overexpression of a native transporter. We envision that this strategy could also be applied to the production of other chemicals in S. cerevisiae and may be extended to other microbes for strain improvement., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Unlabelled: Engineering microbial hosts for the production of fungible fuels requires mitigation of limitations posed on the production capacity. One such limitation arises from the inherent toxicity of solvent-like biofuel compounds to production strains, such as Escherichia coli. Here we show the importance of host engineering for the production of short-chain alcohols by studying the overexpression of genes upregulated in response to exogenous isopentenol. Using systems biology data, we selected 40 genes that were upregulated following isopentenol exposure and subsequently overexpressed them in E. coli. Overexpression of several of these candidates improved tolerance to exogenously added isopentenol. Genes conferring isopentenol tolerance phenotypes belonged to diverse functional groups, such as oxidative stress response (soxS, fpr, and nrdH), general stress response (metR, yqhD, and gidB), heat shock-related response (ibpA), and transport (mdlB). To determine if these genes could also improve isopentenol production, we coexpressed the tolerance-enhancing genes individually with an isopentenol production pathway. Our data show that expression of 6 of the 8 candidates improved the production of isopentenol in E. coli, with the methionine biosynthesis regulator MetR improving the titer for isopentenol production by 55%. Additionally, expression of MdlB, an ABC transporter, facilitated a 12% improvement in isopentenol production. To our knowledge, MdlB is the first example of a transporter that can be used to improve production of a short-chain alcohol and provides a valuable new avenue for host engineering in biogasoline production., Importance: The use of microbial host platforms for the production of bulk commodities, such as chemicals and fuels, is now a focus of many biotechnology efforts. Many of these compounds are inherently toxic to the host microbe, which in turn places a limit on production despite efforts to optimize the bioconversion pathways. In order to achieve economically viable production levels, it is also necessary to engineer production strains with improved tolerance to these compounds. We demonstrate that microbial tolerance engineering using transcriptomics data can also identify targets that improve production. Our results include an exporter and a methionine biosynthesis regulator that improve isopentenol production, providing a starting point to further engineer the host for biogasoline production., (Copyright © 2014 Foo et al.)

Background: The depletion of fossil fuels and the rising need to meet global energy demands have led to a growing interest in microbial biofuel synthesis, particularly in Escherichia coli, due to its tractable characteristics. Besides engineering more efficient metabolic pathways for synthesizing biofuels, efforts to improve production yield by engineering efflux systems to overcome toxicity problems is also crucial. This study aims to enhance hydrocarbon efflux capability in E. coli by engineering a native inner membrane transporter, AcrB, using the directed evolution approach., Results: We developed a selection platform based on competitive growth using a toxic substrate surrogate, which allowed rapid selection of AcrB variants showing enhanced efflux of linear and cyclic fuel molecule candidates, n-octane and α-pinene. Two mutants exhibiting increased efflux efficiency for n-octane and α-pinene by up to 47% and 400%, respectively, were isolated. Single-site mutants based on the mutations found in the isolated variants were synthesized and the amino acid substitutions N189H, T678S, Q737L and M844L were identified to have conferred improvement in efflux efficiency. The locations of beneficial mutations in AcrB suggest their contributions in widening the substrate channel, altering the dynamics of substrate efflux and promoting the assembly of AcrB with the outer membrane channel protein TolC for more efficient substrate export. It is interesting to note that three of the four beneficial mutations were located relatively distant from the known substrate channels, thus exemplifying the advantage of directed evolution over rational design., Conclusions: Using directed evolution, we have isolated AcrB mutants with improved efflux efficiency for n-octane and α-pinene. The utilization of such optimized native efflux pumps will increase productivity of biofuels synthesis and alleviate toxicity and difficulties in production scale-up in current microbial platforms.

Visualization of medical data in three-dimensional (3D) or two-dimensional (2D) views is a complex area of research. In many fields 3D views are used to understand the shape of an object, and 2D views are used to understand spatial relationships. It is unclear how 2D/3D views play a role in the medical field. Using 3D views can potentially decrease the learning curve experienced with traditional 2D views by providing a whole representation of the patient's anatomy. However, there are challenges with 3D views compared with 2D. This current study expands on a previous study to evaluate the mental workload associated with both 2D and 3D views. Twenty-five first-year medical students were asked to localize three anatomical structures--gallbladder, celiac trunk, and superior mesenteric artery--in either 2D or 3D environments. Accuracy and time were taken as the objective measures for mental workload. The NASA Task Load Index (NASA-TLX) was used as a subjective measure for mental workload. Results showed that participants viewing in 3D had higher localization accuracy and a lower subjective measure of mental workload, specifically, the mental demand component of the NASA-TLX. Results from this study may prove useful for designing curricula in anatomy education and improving training procedures for surgeons.

Protein engineering has for decades been a powerful tool in biotechnology for generating vast numbers of useful enzymes for industrial applications. Today, protein engineering has a crucial role in advancing the emerging field of synthetic biology, where metabolic engineering efforts alone are insufficient to maximize the full potential of synthetic biology. This article reviews the advancements in protein engineering techniques for improving biocatalytic properties to optimize engineered pathways in host systems, which are instrumental to achieve high titer production of target molecules. We also discuss the specific means by which protein engineering has improved metabolic engineering efforts and provide our assessment on its potential to continue to advance biology engineering as a whole., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Directed evolution was used to enhance the activity of the glycerophosphodiesterase enzyme from Enterobacter aerogenes, GpdQ, toward bis(para-nitrophenol) phosphate (BpNPP), a substrate that is frequently used to assay phosphodiesterases. Native GpdQ has a low level of activity toward BpNPP while the evolved enzymes exhibited k(cat) values that were well over 100 times better while improvements in k(cat)/K(m) of around 500 times were observed along with improved activity we observed a change in the oligomeric structure in the evolved enzymes. The native enzyme is a hexamer with tightly associated dimers related by a 3-fold axis. The stability of the dimer was attributed in part to the cap domain that forms a disulfide bond with its 2-fold-related subunit and in part due to the fact that dimerization results in burying 23.6% of the monomer's accessible surface area. The cap domain also forms the top of the active site and contributes an essential part of the interface between 3-fold-related molecules. The evolved proteins quickly lost one of the cysteine residues that formed the disulfide bond and other mutations that might stabilize the cap domain. The likely effect of these mutations was to open up the active site for the new substrate and to favor the formation of dimeric molecules. The breakdown of the oligomeric structure was accompanied by a reduction in the thermal stability of the protein-as monitored by the residual activity of the native and mutant proteins following pre-incubation at elevated temperatures. A discussion on the evolutionary implications of these studies is presented.

The bacterial phosphotriesterases catalyze hydrolysis of the pesticide paraoxon with very fast turnover rates and are thought to be near to their evolutionary limit for this activity. To test whether the naturally evolved turnover rate could be improved through the incorporation of unnatural amino acids and to probe the role of peripheral active site residues in nonchemical steps of the catalytic cycle (substrate binding and product release), we replaced the naturally occurring tyrosine amino acid at position 309 with unnatural L-(7-hydroxycoumarin-4-yl)ethylglycine (Hco) and L-(7-methylcoumarin-4-yl)ethylglycine amino acids, as well as leucine, phenylalanine, and tryptophan. Kinetic analysis suggests that the 7-hydroxyl group of Hco, particularly in its deprotonated state, contributes to an increase in the rate-limiting product release step of substrate turnover as a result of its electrostatic repulsion of the negatively charged 4-nitrophenolate product of paraoxon hydrolysis. The 8-11-fold improvement of this already highly efficient catalyst through a single rationally designed mutation using an unnatural amino acid stands in contrast to the difficulty in improving this native activity through screening hundreds of thousands of mutants with natural amino acids. These results demonstrate that designer amino acids provide easy access to new and valuable sequence and functional space for the engineering and evolution of existing enzyme functions.

Automatic segmentation of tumors is a complicated and difficult process as most tumors are rarely clearly delineated from healthy tissues. A new method for probabilistic segmentation to efficiently segment tumors within CT data and to improve the use of digital medical data in diagnosis has been developed. Image data are first enhanced by manually setting the appropriate window center and width, and if needed a sharpening or noise removal filter is applied. To initialize the segmentation process, a user places a seed point within the object of interest and defines a search region for segmentation. Based on the pixels' spatial and intensity properties, a probabilistic selection criterion is used to extract pixels with a high probability of belonging to the object. To facilitate the segmentation of multiple slices, an automatic seed selection algorithm was developed to keep the seeds in the object as its shape and/or location changes between consecutive slices. The seed selection algorithm performs a greedy search by searching for pixels with matching intensity close to the location of the original seed point. A total of ten CT datasets were used as test cases, each with varying difficulty in terms of automatic segmentation. Five test cases had mean false positive error rates less than 10%, and four test cases had mean false negative error rates less than 10% when compared to manual segmentation of those tumors by radiologists., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Graphics technology has extended medical imaging tools to the hands of surgeons and doctors, beyond the radiology suite. However, a common issue in most medical imaging software is the added complexity for non-radiologists. This paper presents the development of a unique software toolset that is highly customizable and targeted at the general physicians as well as the medical specialists. The core functionality includes features such as viewing medical images in two-and three-dimensional representations, clipping, tissue windowing, and coloring. Additional features can be loaded in the form of 'plug-ins' such as tumor segmentation, tissue deformation, and surgical planning. This allows the software to be lightweight and easy to use while still giving the user the flexibility of adding the necessary features, thus catering to a wide range of user population.

The metal ion co-ordination sites of many metalloproteins have been characterized by a variety of spectroscopic techniques and small-molecule model systems, revealing many important insights into the structural determinants of metal ion co-ordination. However, our understanding of this fundamentally and practically important phenomenon remains frustratingly simplistic; in many proteins it is essentially impossible to predict metal ion specificity and the effects of remote 'outer-shell' residues on metal ion co-ordination strength are also poorly defined. This is exemplified by our inability to explain why metalloenzymes with identical metal ion co-ordination spheres, such as the closely related orthologues of bacterial PTE (phosphotriesterase) from Agrobacterium radiobacter and Pseudomonas diminuta, display different metal ion specificity and co-ordination strength. In the present study, we present a series of PTE variants that all possess identical metal ion co-ordination spheres, yet display large differences in their metal ion co-ordination strength. Using measurement of the rates of metal ion dissociation from the active site alongside analysis of structural data obtained through X-ray crystallography, we show that 'outer-shell' residues provide essential support for the metal ion ligands, in effect buttressing them in their optimal orientation. Remote mutations appear to modulate metal ion interactions by increasing or decreasing the stabilizing effects of these networks. The present study therefore provides a description of how the greater protein fold can be modified to 'tune' the strength of metal ion co-ordination and metal ion specificity, as well as reinforcing the concept of proteins as ensembles of conformational states with unique structures and biochemical properties.

To efficiently catalyze a chemical reaction, enzymes are required to maintain fast rates for formation of the Michaelis complex, the chemical reaction and product release. These distinct demands could be satisfied via fluctuation between different conformational substates (CSs) with unique configurations and catalytic properties. However, there is debate as to how these rapid conformational changes, or dynamics, exactly affect catalysis. As a model system, we have studied bacterial phosphotriesterase (PTE), which catalyzes the hydrolysis of the pesticide paraoxon at rates limited by a physical barrier-either substrate diffusion or conformational change. The mechanism of paraoxon hydrolysis is understood in detail and is based on a single, dominant, enzyme conformation. However, the other aspects of substrate turnover (substrate binding and product release), although possibly rate-limiting, have received relatively little attention. This work identifies "open" and "closed" CSs in PTE and dominant structural transition in the enzyme that links them. The closed state is optimally preorganized for paraoxon hydrolysis, but seems to block access to/from the active site. In contrast, the open CS enables access to the active site but is poorly organized for hydrolysis. Analysis of the structural and kinetic effects of mutations distant from the active site suggests that remote mutations affect the turnover rate by altering the conformational landscape.

A new segmentation method using a fuzzy rule based system to segment tumors in a three-dimensional CT data was developed. To initialize the segmentation process, the user selects a region of interest (ROI) within the tumor in the first image of the CT study set. Using the ROI's spatial and intensity properties, fuzzy inputs are generated for use in the fuzzy rules inference system. With a set of predefined fuzzy rules, the system generates a defuzzified output for every pixel in terms of similarity to the object. Pixels with the highest similarity values are selected as tumor. This process is automatically repeated for every subsequent slice in the CT set without further user input, as the segmented region from the previous slice is used as the ROI for the current slice. This creates a propagation of information from the previous slices, used to segment the current slice. The membership functions used during the fuzzification and defuzzification processes are adaptive to the changes in the size and pixel intensities of the current ROI. The method is highly customizable to suit different needs of a user, requiring information from only a single two-dimensional image. Test cases success in segmenting the tumor from seven of the 10 CT datasets with <10% false positive errors and five test cases with <10% false negative errors. The consistency of the segmentation results statistics also showed a high repeatability factor, with low values of inter- and intra-user variability for both methods.

Background & Aims: Diosmectite is a clay used to treat children with acute watery diarrhea. However, its effects on stool output reduction, the key outcome for pediatric antidiarrheal drugs, have not been shown., Methods: Two parallel, double-blind studies of diosmectite efficacy on stool reduction were conducted in children 1 to 36 months old in Peru (n = 300) and Malaysia (n = 302). Inclusion criteria included 3 or more watery stools per day for less than 72 hours and weight/height ratios of 0.8 or greater. Exclusion criteria were the need for intravenous rehydration, gross blood in stools, fever higher than 39 degrees C, or current treatment with antidiarrheal or antibiotic medications. Rotavirus status was determined. Diosmectite dosage was 6 g/day (children 1-12 months old) or 12 g/day (children 13-36 months old), given for at least 3 days, followed by half doses until complete recovery. Patients were assigned randomly to groups given diosmectite or placebo, in addition to oral rehydration solution (World Health Organization)., Results: Children in each study had comparable average ages and weights. The frequencies of rotavirus infection were 22% in Peru and 12% in Malaysia. Similar amounts of oral rehydration solution were given to children in the diosmectite and placebo groups. Stool output was decreased significantly by diosmectite in both studies, especially among rotavirus-positive children. In pooled data, children had a mean stool output of 94.5 +/- 74.4 g/kg of body weight in the diosmectite group versus 104.1 +/- 94.2 g/kg in the placebo group (P = .002). Diarrhea duration was reduced by diosmectite, which was well tolerated., Conclusions: These results show that diosmectite significantly decreased stool output in children with acute watery diarrhea, especially those who were rotavirus-positive.

Visualizing patient data in a three-dimensional (3D) representation can be an effective surgical planning tool.As medical imaging technologies improve with faster and higher resolution scans, the use of virtual reality for interacting with medical images adds another level of realism to a 3D representation. The software framework presented in this paper is designed to load and display any DICOM/PACS-compatible 3D image data for visualization and interaction in an immersive virtual environment. In "examiner" mode, the surgeon can interact with a 3D virtual model of the patient by using an intuitive set of controls designed to allow slicing, coloring,and windowing of the image to show different tissue densities and enhance important structures. In the simulated"endoscopic camera" mode, the surgeon can see through the point of view of a virtual endoscopic camera to navigate inside the patient. These tools allow the surgeon to perform virtual endoscopy on any suitable structure.The software is highly scalable, as it can be used on a single desktop computer to a cluster of computers in an immersive multiprojection virtual environment. By wearing a pair of stereo glasses, a surgeon becomes immersed within the model itself, thus providing a sense of realism, as if the surgeon is "inside" the patient.

The visualization of medical images obtained from scanning techniques such as computed tomography and magnetic resonance imaging is a well-researched field. However, advanced tools and methods to manipulate these data for surgical planning and other tasks have not seen widespread use among medical professionals. Radiologists have begun using more advanced visualization packages on desktop computer systems, but most physicians continue to work with basic two-dimensional grayscale images or not work directly with the data at all. In addition, new display technologies that are in use in other fields have yet to be fully applied in medicine. It is our estimation that usability is the key aspect in keeping this new technology from being more widely used by the medical community at large. Therefore, we have a software and hardware framework that not only make use of advanced visualization techniques, but also feature powerful, yet simple-to-use, interfaces. A virtual reality system was created to display volume-rendered medical models in three dimensions. It was designed to run in many configurations, from a large cluster of machines powering a multiwalled display down to a single desktop computer. An augmented reality system was also created for, literally, hands-on interaction when viewing models of medical data. Last, a desktop application was designed to provide a simple visualization tool, which can be run on nearly any computer at a user's disposal. This research is directed toward improving the capabilities of medical professionals in the tasks of preoperative planning, surgical training, diagnostic assistance, and patient education.

The mechanism by which the binuclear metallophosphotriesterases (PTEs, E.C. 3.1.8.1) catalyse substrate hydrolysis has been extensively studied. The mu-hydroxo bridge between the metal ions has been proposed to be the initiating nucleophile in the hydrolytic reaction. In contrast, analysis of some biomimetic systems has indicated that mu-hydroxo bridges are often not themselves nucleophiles, but act as general bases for freely exchangeable nucleophilic water molecules. Herein, we present crystallographic analyses of a bacterial PTE from Agrobacterium radiobacter, OpdA, capturing the enzyme-substrate complex during hydrolysis. This model of the Michaelis complex suggests the alignment of the substrate will favour attack from a solvent molecule terminally coordinated to the alpha-metal ion. The bridging of both metal ions by the product, without disruption of the mu-hydroxo bridge, is also consistent with nucleophilic attack occurring from the terminal position. When phosphodiesters are soaked into crystals of OpdA, they coordinate bidentately to the beta-metal ion, displacing the mu-hydroxo bridge. Thus, alternative product-binding modes exist for the PTEs, and it is the bridging mode that appears to result from phosphotriester hydrolysis. Kinetic analysis of the PTE and promiscuous phosphodiesterase activities confirms that the presence of a mu-hydroxo bridge during phosphotriester hydrolysis is correlated with a lower pK(a) for the nucleophile, consistent with a general base function during catalysis.