RESUMO
Production of polyhydroxyalkanoates (PHAs) with high concentration of carboxylate, that was accumulated from solid state fermentation (SSF) of food waste (FW), was tested using Pseudomonas putida strain KT2440. Mixed-culture SSF of FW supplied in a high concentration of carboxylate, which caused a high PHA production of 0.56 g PHA/g CDM under nutrients control. Interestingly, this high PHA fraction in CDM was almost constant at 0.55 g PHA/g CDM even under high nutrients concentration (25 mM NH4+), probably due to high reducing power maintained by high carboxylate concentration. PHA characterization indicated that the dominant PHA building block produced was 3-hydroxybutyrate, followed by 3-hydroxy-2-methylvalerate and 3-hydroxyhenxanoate. Carboxylate profiles before and after PHA production suggested that acetate, butyrate, and propionate were the main precursors to PHA via several metabolic pathways. Our result support that mixed culture SSF of FW for high concentration carboxylate and P. putida for PHA production enables sustainable production of PHA in cost-effective manners.
Assuntos
Poli-Hidroxialcanoatos , Pseudomonas putida , Eliminação de Resíduos , Pseudomonas putida/metabolismo , Poli-Hidroxialcanoatos/metabolismo , Alimentos , Fermentação , Ácidos CarboxílicosRESUMO
S-layers are paracrystalline proteinaceous lattices that surround prokaryotic cells, forming a critical interface between the cells and their extracellular environment. Here, we report the discovery of a novel S-layer protein present in the Gram-negative marine organism, Pseudoalteromonas tunicata D2. An uncharacterized protein (EAR28894) was identified as the most abundant protein in planktonic cultures and biofilms. Bioinformatic methods predicted a beta-helical structure for EAR28894 similar to the Caulobacter S-layer protein, RsaA, despite sharing less than 20% sequence identity. Transmission electron microscopy revealed that purified EAR28894 protein assembled into paracrystalline sheets with a unique square lattice symmetry and a unit cell spacing of ~9.1 nm. An S-layer was found surrounding the outer membrane in wild-type cells and completely removed from cells in an EAR28894 deletion mutant. S-layer material also appeared to be "shed" from wild-type cells and was highly abundant in the extracellular matrix where it is associated with outer membrane vesicles and other matrix components. EAR28894 and its homologs form a new family of S-layer proteins that are widely distributed in Gammaproteobacteria including species of Pseudoalteromonas and Vibrio, and found exclusively in marine metagenomes. We propose the name Slr4 for this novel protein family.
Assuntos
Biofilmes , Glicoproteínas de Membrana/genética , Pseudoalteromonas/genética , Organismos Aquáticos/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Matriz Extracelular de Substâncias Poliméricas/metabolismo , Glicoproteínas de Membrana/isolamento & purificação , Glicoproteínas de Membrana/ultraestrutura , Filogenia , Conformação ProteicaRESUMO
Promoter engineering has been employed as a strategy to enhance and optimize the production of bio-products. Availability of promoters with predictable activities is needed for downstream application. However, whether promoter activity remains the same in different gene contexts remains unknown. Six consecutive promoters that have previously been determined to have different activity levels were used to construct six different versions of plasmid backbone pTH1227, followed by inserted genes encoding two polymer-producing enzymes. In some cases, promoter activity in the presence of inserted genes did not correspond to the reported activity levels in a previous study. After removing the inserted genes, the activity of these promoters returned to their previously reported level. These changes were further confirmed to occur at the transcriptional level. Polymer production using our newly constructed plasmids showed polymer accumulation levels corresponding to the promoter activity reported in our study. Our study demonstrated the importance of re-assessing promoter activity levels with regard to gene context, which could influence promoter activity, leading to different outcomes in downstream applications.
Assuntos
Plasmídeos , Plasmídeos/genética , Regiões Promotoras GenéticasRESUMO
Despite the risks that hydrocarbon contamination from pipeline leaks or train derailments impose on the health of peatlands in hydrocarbon production areas and transportation corridors, assessing the effect of such contaminations on the health and sustainability of peatlands has received little attention. This study investigates the impacts of hydrocarbons on peat microbial communities. Column experiments were conducted on non-aqueous phase liquid (NAPL) contaminated undisturbed peat core (0-35 cm) under static and fluctuating water table conditions. Water table fluctuations reduced residual NAPL saturation from 8.1-11.3% to 7.7-9.5%. Biodegradation of n-C8 and n-C12 along with oxidation of CH4 together produced high CO2 concentrations in the headspace. Clear patterns in dynamics in the microbial community structure were observed, with a more pronounced population growth. However, a significant loss of microbial richness was observed in contaminated columns. The result indicates that the phylum Proteobacteria benefited most from NAPL; however, their families differed between static and fluctuating water table conditions. This study established strong evidence that peat microbes and water table fluctuation can be an excellent tool for hydrocarbon removal and its control in peatlands.
Assuntos
Água Subterrânea , Microbiota , Poluição Ambiental , Humanos , Hidrocarbonetos , SoloRESUMO
Gibberellins (GAs) are crucial phytohormones involved in many aspects of plant growth and development, including plant-microbe interactions, which has led to GA production by plant-associated fungi and bacteria as well. While the GA biosynthetic pathways in plants and fungi have been elucidated and found to have arisen independently through convergent evolution, little has been uncovered about GA biosynthesis in bacteria. Some nitrogen-fixing, symbiotic, legume-associated rhizobia, including Bradyrhizobium japonicum-the symbiont of soybean-and Sinorhizobium fredii-a broad-host-nodulating species-contain a putative GA biosynthetic operon, or gene cluster. Through functional characterization of five unknown genes, we demonstrate that this operon encodes the enzymes necessary to produce GA9, thereby elucidating bacterial GA biosynthesis. The distinct nature of these enzymes indicates that bacteria have independently evolved a third biosynthetic pathway for GA production. Furthermore, our results also reveal a central biochemical logic that is followed in all three convergently evolved GA biosynthetic pathways.
Assuntos
Bradyrhizobium/metabolismo , Evolução Molecular , Giberelinas/biossíntese , Sinorhizobium fredii/metabolismo , Giberelinas/química , Conformação MolecularRESUMO
Presented here are two engineered bacterial biosensors for detecting and quantifying silver and copper ions. The biosensors contain a silver/copper resistance operon and a Green Fluorescent Protein gene that is strictly regulated through silver activated promoter regions normally found on a silver resistance gene (sil operon). The two biosensors efficiently detected silver and copper concentrations of 40 µM-300 µM and 20 µM-600 µM respectively. A strong correlation (R2 = 0.90 or above) between silver/copper and GFP signal makes it possible to quantify the ions using a linear regression. At room temperature incubation, the GFP signal of the biosensors in Ag+ saturated after 13 h. However, a detectable GFP signal was seen in 4 h.
Assuntos
Técnicas Biossensoriais , Cobre/análise , Escherichia coli/química , Fluorescência , Proteínas de Fluorescência Verde/química , Engenharia de Proteínas , Prata/análise , Clonagem Molecular , Proteínas de Fluorescência Verde/genética , Íons/análise , Regiões Promotoras Genéticas/genéticaRESUMO
The utilization of metagenomic functional interactions represents a key technique for metagenomic functional annotation efforts. By definition, metagenomic operons represent such interactions, but many operon predictions protocols rely on information about orthology and/or gene function that is frequently unavailable for metagenomic genes. Recently, the concept of the metagenomic proximon was proposed for use in metagenomic scenarios where supplemental information is sparse. In this paper, we examine the validity and utility of the proximon proposition by measuring the extent to which proximons emulate actual operons. Using the Escherichia coli K-12 genome, we compare proximons and operons from the same genome and observe the configurations and cardinalities among their corresponding mappings. The results demonstrate that the vast majority of proximons map discretely to a single operon in a conservative fashion where a typical proximon is synonymous to an equivalent or truncated operon. However, a large proportion of operons had no corresponding mappings to any proximon. Various perspectives of operon and proximon intersection are discussed, along with the potential limitations for proximon detection and usage.
Assuntos
Algoritmos , Escherichia coli K12/genética , Genoma Bacteriano , Metagenômica , Óperon , Mapeamento Cromossômico , Modelos Genéticos , SoftwareRESUMO
Bacterially produced biodegradable polyhydroxyalkanoates (PHAs) with versatile properties can be achieved using different PHA synthases (PhaCs). This work aims to expand the diversity of known PhaCs via functional metagenomics and demonstrates the use of these novel enzymes in PHA production. Complementation of a PHA synthesis-deficient Pseudomonas putida strain with a soil metagenomic cosmid library retrieved 27 clones expressing either class I, class II, or unclassified PHA synthases, and many did not have close sequence matches to known PhaCs. The composition of PHA produced by these clones was dependent on both the supplied growth substrates and the nature of the PHA synthase, with various combinations of short-chain-length (SCL) and medium-chain-length (MCL) PHA. These data demonstrate the ability to isolate diverse genes for PHA synthesis by functional metagenomics and their use for the production of a variety of PHA polymer and copolymer mixtures.
Assuntos
Aciltransferases/genética , Aciltransferases/metabolismo , Poli-Hidroxialcanoatos/metabolismo , Pseudomonas putida/enzimologia , Pseudomonas putida/genética , Sequência de Bases , Clonagem Molecular , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Biblioteca Gênica , Metagenômica , Pseudomonas putida/metabolismo , Análise de Sequência de DNARESUMO
Economically competitive commercial production of biodegradable bioplastics with desirable properties is an important goal. In this study, we demonstrate the use of chromosome engineering of an alternative bacterial host, Sinorhizobium meliloti, for production of the copolymer, poly(lactate-co-3-hydroxybutyrate). Codon-optimized genes for 2 previously engineered enzymes, Clostridium propionicum propionate CoA transferase (Pct532Cp) and Pseudomonas sp. strain MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1400Ps6-19), were introduced into S. meliloti Rm1021 by chromosome integration, replacing the native phbC gene. On the basis of phenotypic analysis and detection of polymer product by gas chromatography analysis, synthesis and accumulation of the copolymer was confirmed. The chromosome integrant strain, with the introduced genes under the control of the native phbC promoter, is able to produce over 15% cell dry mass of poly(lactate-co-3-hydroxybutyrate), containing 30 mol% lactate, from growth on mannitol. We were also able to purify the polymer from the culture and confirm the structure by NMR and GC-MS. To our knowledge, this is the first demonstration of production of this copolymer in the Alphaproteobacteria. Further optimization of this system may eventually yield strains that are able to produce economically viable commercial product.
Assuntos
Aciltransferases/genética , Coenzima A-Transferases/genética , Poliésteres/metabolismo , Sinorhizobium meliloti/genética , Engenharia Genética , Hidroxibutiratos/metabolismoRESUMO
The vast majority of plants harbor endophytic bacteria that colonize a portion of the plant's interior tissues without harming the plant. Like plant pathogens, endophytes gain entry into their plants hosts through various mechanisms. Bacterial endophytes display a broad range of symbiotic interactions with their host plants. The molecular bases of these plant-endophyte interactions are currently not fully understood. In the present study, a set of genes possibly responsible for endophytic behavior for genus Burkholderia was predicted and then compared and contrasted with a number (nine endophytes from different genera) of endophytes by comparative genome analysis. The nine endophytes included Burkholderia phytofirmans PsJN, Burkholderia spp. strain JK006, Azospirillum lipoferum 4B, Enterobacter cloacae ENHKU01, Klebsiella pneumoniae 342, Pseudomonas putida W619, Enterobacter spp. 638, Azoarcus spp. BH72, and Serratia proteamaculans 568. From the genomes of the analyzed bacterial strains, a set of bacterial genes orthologs was identified that are predicted to be involved in determining the endophytic behavior of Burkholderia spp. The genes and their possible functions were then investigated to establish a potential connection between their presence and the role they play in bacterial endophytic behavior. Nearly all of the genes identified by this bioinformatics procedure encode function previously suggested in other studies to be involved in endophytic behavior.
Assuntos
Burkholderia/genética , Biologia Computacional/métodos , Endófitos/genética , Genes Bacterianos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/genética , Biopolímeros/metabolismo , Plantas/microbiologiaRESUMO
Tomato Brown Rugose Fruit Virus (ToBRFV) is a plant pathogen that infects important Solanaceae crop species and can dramatically reduce tomato crop yields. The ToBRFV has rapidly spread around the globe due to its ability to escape detection by antiviral host genes which confer resistance to other tobamoviruses in tomato plants. The development of robust and reproducible methods for detecting viruses in the environment aids in the tracking and reduction of pathogen transmission. We detected ToBRFV in municipal wastewater influent (WWI) samples, likely due to its presence in human waste, demonstrating a widespread distribution of ToBRFV in WWI throughout Ontario, Canada. To aid in global ToBRFV surveillance efforts, we developed a tiled amplicon approach to sequence and track the evolution of ToBRFV genomes in municipal WWI. Our assay recovers 95.7% of the 6393 bp ToBRFV RefSeq genome, omitting the terminal 5' and 3' ends. We demonstrate that our sequencing assay is a robust, sensitive, and highly specific method for recovering ToBRFV genomes. Our ToBRFV assay was developed using existing ARTIC Network resources, including primer design, sequencing library prep, and read analysis. Additionally, we adapted our lineage abundance estimation tool, Alcov, to estimate the abundance of ToBRFV clades in samples.
Assuntos
Solanum lycopersicum , Tobamovirus , Purificação da Água , Humanos , Ontário , Frutas , Tobamovirus/genéticaRESUMO
Wastewater surveillance of SARS-CoV-2 has emerged as a critical tool for tracking the spread of COVID-19. In addition to estimating the relative case numbers using quantitative PCR, SARS-CoV-2 genomic RNA can be extracted from wastewater and sequenced. There are many existing techniques for using the sequenced RNA to determine the relative abundance of known lineages in a sample. However, it is very challenging to predict novel lineages from wastewater data due to its mixed composition and unreliable genomic coverage. In this work, we present a novel technique based on non-negative matrix factorization which is able to reconstruct lineage definitions by analyzing data from across different samples. We test the method both on synthetic and real wastewater sequencing data. We show that the technique is able to determine major lineages such as Omicron and Delta as well as sub-lineages such as BA.5.2.1. We provide a method for determining emerging lineages in wastewater without the need for genomic data from clinical samples. This could be used for routine monitoring of SARS-CoV-2 as well as other emerging viral pathogens in wastewater. Additionally, it may be used to determine more full-genome sequences for viruses with fewer available genomes.
Assuntos
COVID-19 , Genoma Viral , Filogenia , RNA Viral , SARS-CoV-2 , Águas Residuárias , Águas Residuárias/virologia , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , COVID-19/virologia , Humanos , RNA Viral/genéticaRESUMO
Wastewater surveillance of coronavirus disease 2019 (COVID-19) commonly applies reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to quantify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA concentrations in wastewater over time. In most applications worldwide, maximal sensitivity and specificity of RT-qPCR has been achieved, in part, by monitoring two or more genomic loci of SARS-CoV-2. In Ontario, Canada, the provincial Wastewater Surveillance Initiative reports the average copies of the CDC N1 and N2 loci normalized to the fecal biomarker pepper mild mottle virus. In November 2021, the emergence of the Omicron variant of concern, harboring a C28311T mutation within the CDC N1 probe region, challenged the accuracy of the consensus between the RT-qPCR measurements of the N1 and N2 loci of SARS-CoV-2. In this study, we developed and applied a novel real-time dual loci quality assurance and control framework based on the relative difference between the loci measurements to the City of Ottawa dataset to identify a loss of sensitivity of the N1 assay in the period from July 10, 2022 to January 31, 2023. Further analysis via sequencing and allele-specific RT-qPCR revealed a high proportion of mutations C28312T and A28330G during the study period, both in the City of Ottawa and across the province. It is hypothesized that nucleotide mutations in the probe region, especially A28330G, led to inefficient annealing, resulting in reduction in sensitivity and accuracy of the N1 assay. This study highlights the importance of implementing quality assurance and control criteria to continually evaluate, in near real-time, the accuracy of the signal produced in wastewater surveillance applications that rely on detection of pathogens whose genomes undergo high rates of mutation.
Assuntos
Vigilância Epidemiológica Baseada em Águas Residuárias , Águas Residuárias , Alelos , Mutação , Ontário/epidemiologia , SARS-CoV-2/genética , RNA Viral/genéticaRESUMO
During the COVID-19 pandemic, the Province of Ontario, Canada, launched a wastewater surveillance program to monitor SARS-CoV-2, inspired by the early work and successful forecasts of COVID-19 waves in the city of Ottawa, Ontario. This manuscript presents a dataset from January 1, 2021, to March 31, 2023, with RT-qPCR results for SARS-CoV-2 genes and PMMoV from 107 sites across all 34 public health units in Ontario, covering 72% of the province's and 26.2% of Canada's population. Sampling occurred 2-7 times weekly, including geographical coordinates, serviced populations, physico-chemical water characteristics, and flowrates. In doing so, this manuscript ensures data availability and metadata preservation to support future research and epidemic preparedness through detailed analyses and modeling. The dataset has been crucial for public health in tracking disease locally, especially with the rise of the Omicron variant and the decline in clinical testing, highlighting wastewater-based surveillance's role in estimating disease incidence in Ontario.
Assuntos
COVID-19 , SARS-CoV-2 , Águas Residuárias , Ontário/epidemiologia , COVID-19/epidemiologia , Águas Residuárias/virologia , Humanos , Pandemias , Carga ViralRESUMO
Wastewater-based surveillance (WBS) is an important epidemiological and public health tool for tracking pathogens across the scale of a building, neighbourhood, city, or region. WBS gained widespread adoption globally during the SARS-CoV-2 pandemic for estimating community infection levels by qPCR. Sequencing pathogen genes or genomes from wastewater adds information about pathogen genetic diversity, which can be used to identify viral lineages (including variants of concern) that are circulating in a local population. Capturing the genetic diversity by WBS sequencing is not trivial, as wastewater samples often contain a diverse mixture of viral lineages with real mutations and sequencing errors, which must be deconvoluted computationally from short sequencing reads. In this study we assess nine different computational tools that have recently been developed to address this challenge. We simulated 100 wastewater sequence samples consisting of SARS-CoV-2 BA.1, BA.2, and Delta lineages, in various mixtures, as well as a Delta-Omicron recombinant and a synthetic 'novel' lineage. Most tools performed well in identifying the true lineages present and estimating their relative abundances and were generally robust to variation in sequencing depth and read length. While many tools identified lineages present down to 1â% frequency, results were more reliable above a 5â% threshold. The presence of an unknown synthetic lineage, which represents an unclassified SARS-CoV-2 lineage, increases the error in relative abundance estimates of other lineages, but the magnitude of this effect was small for most tools. The tools also varied in how they labelled novel synthetic lineages and recombinants. While our simulated dataset represents just one of many possible use cases for these methods, we hope it helps users understand potential sources of error or bias in wastewater sequencing analysis and to appreciate the commonalities and differences across methods.
Assuntos
COVID-19 , Genoma Viral , SARS-CoV-2 , Águas Residuárias , Águas Residuárias/virologia , SARS-CoV-2/genética , SARS-CoV-2/classificação , COVID-19/virologia , COVID-19/epidemiologia , Humanos , Biologia Computacional/métodos , Genômica/métodos , Vigilância Epidemiológica Baseada em Águas Residuárias , FilogeniaRESUMO
BACKGROUND: The Sinorhizobium meliloti ExoS/ChvI two component regulatory system is required for N2-fixing symbiosis and exopolysaccharide synthesis. Orthologous systems are present in other Alphaproteobacteria, and in many instances have been shown to be necessary for normal interactions with corresponding eukaryotic hosts. Only a few transcriptional regulation targets have been determined, and as a result there is limited understanding of the mechanisms that are controlled by the system. RESULTS: In an attempt to better define the members of the regulon, we have applied a simple in vitro electrophoretic screen for DNA fragments that are bound by the ChvI response regulator protein. Several putative transcriptional targets were identified and three were further examined by reporter gene fusion experiments for transcriptional regulation. Two were confirmed to be repressed by ChvI, while one was activated by ChvI. CONCLUSIONS: Our results suggest a role for ChvI as both a direct activator and repressor of transcription. The identities and functions of many of these genes suggest explanations for some aspects of the pleiotropic phenotype of exoS and chvI mutants. This work paves the way for in depth characterization of the ExoS/ChvI regulon and its potential role in directing bacteria-host relationships.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Regulon , Sinorhizobium meliloti/genética , Fatores de Transcrição/metabolismo , DNA Bacteriano/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Ligação Proteica , Transcrição GênicaRESUMO
The ability to produce high-value products using bacteria will increasingly rely on continued research to make large-scale bacterial fermentation cost-efficient. Engineering bacteria to use alternate carbon sources as feedstock provides an opportunity to reduce production costs. Using inexpensive carbon sources from various forms of waste provides an opportunity to substantially reduce feedstock costs. Functional carbon metabolism pathways can be identified by the introduction of metagenomic libraries into the organism of interest followed by screening for the desired phenotype. We present here a method to transfer metagenomic libraries from E. coli to Pseudomonas alloputida, followed by screening for use of galactose as a sole carbon source.
Assuntos
Carbono , Microbiota , Carbono/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Metagenoma , Metagenômica , FermentaçãoRESUMO
Research on the role of gut microbiota in behavior has grown dramatically. The probiotic L. reuteri can alter social and stress-related behaviors - yet, the underlying mechanisms remain largely unknown. Although traditional laboratory rodents provide a foundation for examining the role of L. reuteri on the gut-brain axis, they do not naturally display a wide variety of social behaviors. Using the highly-social, monogamous prairie vole (Microtus ochrogaster), we examined the effects of L. reuteri administration on behaviors, neurochemical marker expression, and gut-microbiome composition. Females, but not males, treated with live L. reuteri displayed lower levels of social affiliation compared to those treated with heat-killed L. reuteri. Overall, females displayed a lower level of anxiety-like behaviors than males. Live L. reuteri-treated females had lower expression of corticotrophin releasing factor (CRF) and CRF type-2-receptor in the nucleus accumbens, and lower vasopressin 1a-receptor in the paraventricular nucleus of the hypothalamus (PVN), but increased CRF in the PVN. There were both baseline sex differences and sex-by-treatment differences in gut microbiome composition. Live L. reuteri increased the abundance of several taxa, including Enterobacteriaceae, Lachnospiraceae NK4A136, and Treponema. Interestingly, heat-killed L. reuteri increased abundance of the beneficial taxa Bifidobacteriaceae and Blautia. There were significant correlations between changes in microbiota, brain neurochemical markers, and behaviors. Our data indicate that L. reuteri impacts gut microbiota, gut-brain axis and behaviors in a sex-specific manner in socially-monogamous prairie voles. This demonstrates the utility of the prairie vole model for further examining causal impacts of microbiome on brain and behavior.
RESUMO
Microbiomes have highly important roles for ecosystem functioning and carry out key functions that support planetary health, including nutrient cycling, climate regulation, and water filtration. Microbiomes are also intimately associated with complex multicellular organisms such as humans, other animals, plants, and insects and perform crucial roles for the health of their hosts. Although we are starting to understand that microbiomes in different systems are interconnected, there is still a poor understanding of microbiome transfer and connectivity. In this review we show how microbiomes are connected within and transferred between different habitats and discuss the functional consequences of these connections. Microbiome transfer occurs between and within abiotic (e.g., air, soil, and water) and biotic environments, and can either be mediated through different vectors (e.g., insects or food) or direct interactions. Such transfer processes may also include the transmission of pathogens or antibiotic resistance genes. However, here, we highlight the fact that microbiome transmission can have positive effects on planetary and human health, where transmitted microorganisms potentially providing novel functions may be important for the adaptation of ecosystems.
Assuntos
Microbiota , Planetas , Animais , Humanos , Microbiologia do Solo , Microbiota/fisiologia , Solo , ÁguaRESUMO
Bacterial cells are widely used to produce value-added products due to their versatility, ease of manipulation, and the abundance of genome engineering tools. However, the efficiency of producing these desired biomolecules is often hindered by the cells' own metabolism, genetic instability, and the toxicity of the product. To overcome these challenges, genome reductions have been performed, making strains with the potential of serving as chassis for downstream applications. Here we review the current technologies that enable the design and construction of such reduced-genome bacteria as well as the challenges that limit their assembly and applicability. While genomic reductions have shown improvement of many cellular characteristics, a major challenge still exists in constructing these cells efficiently and rapidly. Computational tools have been created in attempts at minimizing the time needed to design these organisms, but gaps still exist in modelling these reductions in silico. Genomic reductions are a promising avenue for improving the production of value-added products, constructing chassis cells, and for uncovering cellular function but are currently limited by their time-consuming construction methods. With improvements to and the creation of novel genome editing tools and in silico models, these approaches could be combined to expedite this process and create more streamlined and efficient cell factories.