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Salimicrobium sp. PL1-032A was isolated from Pearse Lakes, Western Australia. The sequenced genome consists of a single chromosome (2,705,688 bp) with a GC content of 47.2%. The isolation of Salimicrobium sp. PL1-032A contributes to the collection of culturable extremophiles and offers potential insight into the Pearse Lakes biome.
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In synthetic biology, microbial chassis including yeast Saccharomyces cerevisiae are iteratively engineered with increasing complexity and scale. Wet-lab genetic engineering tools are developed and optimised to facilitate strain construction but are often incompatible with each other due to shared regulatory elements, such as the galactose-inducible (GAL) promoter in S. cerevisiae. Here, we prototyped the cyanamide-induced I- SceI expression, which triggered double-strand DNA breaks (DSBs) for selectable marker removal. We further combined cyanamide-induced I- SceI-mediated DSB and maltose-induced MazF-mediated negative selection for plasmid-free in situ promoter substitution, which simplified the molecular cloning procedure for promoter characterisation. We then characterised three tetracycline-inducible promoters showing differential strength, a non-leaky ß-estradiol-inducible promoter, cyanamide-inducible DDI2 promoter, bidirectional MAL32/MAL31 promoters, and five pairs of bidirectional GAL1/GAL10 promoters. Overall, alternative regulatory controls for genome engineering tools can be developed to facilitate genomic engineering for synthetic biology and metabolic engineering applications.
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Marinococcus sp. PL1-022 was isolated from Pearse Lakes, Western Australia. The sequenced genome consists of a chromosome (3,140,198 bp; 48.2% GC) and two plasmids (58,083 bp and 19,399 bp; 41.4 and 50.7% GC-content, respectively). Isolation of Marinococcus sp. PL1-022 adds to the increasing repertoire of culturable extremophiles.
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Idiomarina sp. PL1-037 was isolated from Pearse Lakes, Rottnest Island, Western Australia. The sequenced completed genome for PL1-037 is composed of a single chromosome (2,804,934 bp) with a GC content of 47.1%. Isolation of Idiomarina sp. PL1-037 provides insights about culturable extremophiles from the Pearse lakes microbiome.
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BACKGROUND: Stroke affects long-term physical and cognitive function; many survivors report unmet health needs, such as pain or depression. A hospital-led follow-up service designed to address ongoing health problems may avoid unplanned readmissions and improve quality of life. METHODS: This paper outlines the protocol for a registry-based, randomised controlled trial with allocation concealment of participants and outcome assessors. Based on an intention-to-treat analysis, we will evaluate the feasibility, acceptability, potential effectiveness and cost implications of a new tailored, codesigned, hospital-led follow-up service for people within 6-12 months of stroke. Participants (n = 100) from the Australian Stroke Clinical Registry who report extreme health problems on the EuroQol EQ-5D-3L survey between 90 and 180 days after stroke will be randomly assigned (1:1) to intervention (follow-up service) or control (usual care) groups. All participants will be independently assessed at baseline and 12-14-week post-randomisation. Primary outcomes for feasibility are the proportion of participants completing the trial and for intervention participants the proportion that received follow-up services. Acceptability is satisfaction of clinicians and participants involved in the intervention. Secondary outcomes include effectiveness: change in extreme health problems (EQ-5D-3L), unmet needs (Longer-term Unmet Needs questionnaire), unplanned presentations and hospital readmission, functional independence (modified Rankin Scale) and cost implications estimated from self-reported health service utilisation and productivity (e.g. workforce participation). To inform future research or implementation, the design contains a process evaluation including clinical protocol fidelity and an economic evaluation. DISCUSSION: The results of this study will provide improved knowledge of service design and implementation barriers and facilitators and associated costs and resource implications to inform a future fully powered effectiveness trial of the intervention. TRIAL REGISTRATION: ACTRN12622001015730pr. TRIAL SPONSOR: Florey Institute of Neuroscience and Mental Health, 245 Burgundy Street, Heidelberg, VIC, 3084, PH: +61 3 9035 7032.
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Ethylene and ethylene oxide are widely used in the chemical industry, and ethylene is also important for its role in fruit ripening. Better sensing systems would assist risk management of these chemicals. Here, we characterise the ethylene regulatory system in Mycobacterium strain NBB4 and use these genetic parts to create a biosensor. The regulatory genes etnR1 and etnR2 and cognate promoter Petn were combined with a fluorescent reporter gene (fuGFP) in a Mycobacterium shuttle vector to create plasmid pUS301-EtnR12P. Cultures of M. smegmatis mc2-155(pUS301-EtnR12P) gave a fluorescent signal in response to ethylene oxide with a detection limit of 0.2 µM (9 ppb). By combining the epoxide biosensor cells with another culture expressing the ethylene monooxygenase, the system was converted into an ethylene biosensor. The co-culture was capable of detecting ethylene emission from banana fruit. These are the first examples of whole-cell biosensors for epoxides or aliphatic alkenes. This work also resolves long-standing questions concerning the regulation of ethylene catabolism in bacteria.
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Técnicas Biossensoriais , Óxido de Etileno , Etilenos , Técnicas Biossensoriais/métodos , Etilenos/metabolismo , Óxido de Etileno/metabolismo , Mycobacterium/genética , Mycobacterium/metabolismo , Musa/microbiologia , Genes Reporter , Plasmídeos/genéticaRESUMO
Rare earth elements (REEs) are critical for our modern lifestyles and the transition to a low-carbon economy. Recent advances in our understanding of the role of REEs in biology, particularly methylotrophy, have provided opportunities to explore biotechnological innovations to improve REE mining and recycling. In addition to bacterial accumulation and concentration of REEs, biological REE binders, including proteins (lanmodulin, lanpepsy) and small molecules (metallophores and cofactors) have been identified that enable REE concentration and separation. REE-binding proteins have also been used in several mechanistically distinct REE biosensors, which have potential application in mining and medicine. Notably, the role of REEs in biology has only been known for a decade, suggesting their considerable scope for developing new understanding and novel applications.
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Bactérias , Metais Terras Raras , Metais Terras Raras/metabolismo , Metais Terras Raras/química , Bactérias/metabolismo , Bactérias/genética , Bactérias/química , Biotecnologia/métodosRESUMO
Understanding the mechanism of interfacial enzyme kinetics is critical to the development of synthetic biological systems for the production of value-added chemicals. Here, the interfacial kinetics of the catalysis of ß-nicotinamide adenine dinucleotide (NAD+)-dependent enzymes acting on NAD+ tethered to the surface of silica nanoparticles (SiNPs) has been investigated using two complementary and supporting kinetic approaches: enzyme excess and reactant (NAD+) excess. Kinetic models developed for these two approaches characterize several critical reaction steps including reversible enzyme adsorption, complexation, decomplexation, and catalysis of the surface-bound enzyme/NAD+ complex. The analysis reveals a concentrating effect resulting in a very high local concentration of enzyme and cofactor on the particle surface, in which the enzyme is saturated by surface-bound NAD, facilitating a rate enhancement of enzyme/NAD+ complexation and catalysis. This resulted in high enzyme efficiency within the tethered NAD+ system compared to that of the free enzyme/NAD+ system, which increases with decreasing enzyme concentration. The role of enzyme adsorption onto solid substrates with a tethered catalyst (such as NAD+) has potential for creating highly efficient flow biocatalytic systems.
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NAD , NAD/química , Biocatálise , Catálise , Cinética , AdsorçãoRESUMO
Per- and polyfluoroalkyl substances (PFAS) are highly fluorinated synthetic organic compounds that have been used extensively in various industries owing to their unique properties. The PFAS family encompasses diverse classes, with only a fraction being commercially relevant. These substances are found in the environment, including in water sources, soil, and wildlife, leading to human exposure and fueling concerns about potential human health impacts. Although PFAS degradation is challenging, biodegradation offers a promising, eco-friendly solution. Biodegradation has been effective for a variety of organic contaminants but is yet to be successful for PFAS due to a paucity of identified microbial species capable of transforming these compounds. Recent studies have investigated PFAS biotransformation and fluoride release; however, the number of specific microorganisms and enzymes with demonstrable activity with PFAS remains limited. This review discusses enzymes that could be used in PFAS metabolism, including haloacid dehalogenases, reductive dehalogenases, cytochromes P450, alkane and butane monooxygenases, peroxidases, laccases, desulfonases, and the mechanisms of microbial resistance to intracellular fluoride. Finally, we emphasize the potential of enzyme and microbial engineering to advance PFAS degradation strategies and provide insights for future research in this field.
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Fluorocarbonos , Poluentes Químicos da Água , Humanos , Animais , Fluoretos , Alcanos , Animais Selvagens , Biodegradação AmbientalRESUMO
Mevalonate kinase is central to the isoprenoid biosynthesis pathway. Here, high-resolution X-ray crystal structures of two mevalonate kinases are presented: a eukaryotic protein from Ramazzottius varieornatus and an archaeal protein from Methanococcoides burtonii. Both enzymes possess the highly conserved motifs of the GHMP enzyme superfamily, with notable differences between the two enzymes in the N-terminal part of the structures. Biochemical characterization of the two enzymes revealed major differences in their sensitivity to geranyl pyrophosphate and farnesyl pyrophosphate, and in their thermal stabilities. This work adds to the understanding of the structural basis of enzyme inhibition and thermostability in mevalonate kinases.
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Archaea , Ácido Mevalônico , Ácido Mevalônico/metabolismo , Archaea/metabolismo , Methanosarcinaceae/química , Methanosarcinaceae/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/químicaRESUMO
Podcasts represent a new source of information for patients and families dealing with prostate cancer, but no studies have been conducted evaluating the quality of information in them. Evaluating for: (1) quality based on the validated DISCERN criteria, (2) understandability and actionability based on the Patient Education Materials Assessment Tool (PEMAT), (3) misinformation, and (4) commercial bias, we concluded that podcasts are currently not good sources of information for lay health consumers.
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Letramento em Saúde , Neoplasias da Próstata , Masculino , Humanos , Materiais de Ensino , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/terapia , CompreensãoRESUMO
Novosphingobium aromaticivorans has the ability to survive in harsh environments by virtue of its suite of iron-containing oxygenases that biodegrade an astonishing array of aromatic compounds. It is also resistant to heavy metals through Atm1, an ATP-binding cassette protein that mediates active efflux of heavy metals conjugated to glutathione. However, Atm1 orthologues in higher organisms have been implicated in the intracellular transport of organic iron complexes. Our hypothesis suggests that the ability of Atm1 to remove heavy metals is related to the need for regulated iron handling in N. aromaticivorans to support high oxygenase activity. Here we provide the first data demonstrating a direct interaction between an iron-porphyrin compound (hemin) and NaAtm1. Hemin displayed considerably higher binding affinity and lower EC50 to stimulate ATP hydrolysis by Atm1 than Ag-GSH, GSSG or GSH, established substrates of the transporter. Co-incubation of NaAtm1 and hemin with Ag-GSH in ATPase assays revealed a non-competitive interaction, indicating distinct binding sites on NaAtm1 and this property was reinforced using molecular docking analysis. Our data suggests that NaAtm1 has considerable versatility in transporting organic conjugates of metals and that this versatility enables it to play roles in detoxification processes for toxic metals and in homeostasis of iron. The ability to play these distinct roles is enabled by the plasticity of the substrate binding site within the central cavity of NaAtm1.
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Hemina , Metais Pesados , Simulação de Acoplamento Molecular , Transportadores de Cassetes de Ligação de ATP/metabolismo , Metais Pesados/metabolismo , Ferro/metabolismo , Proteínas de Membrana Transportadoras , Trifosfato de Adenosina/química , Glutationa/metabolismoRESUMO
Enzyme immobilization offers considerable advantage for biocatalysis in batch and continuous flow reactions. However, many currently available immobilization methods require that the surface of the carrier is chemically modified to allow site specific interactions with their cognate enzymes, which requires specific processing steps and incurs associated costs. Two carriers (cellulose and silica) were investigated here, initially using fluorescent proteins as models to study binding, followed by assessment of industrially relevant enzyme performance (transaminases and an imine reductase/glucose oxidoreductase fusion). Two previously described binding tags, the 17 amino acid long silica-binding peptide from the Bacillus cereus CotB protein and the cellulose binding domain from the Clostridium thermocellum, were fused to a range of proteins without impairing their heterologous expression. When fused to a fluorescent protein both tags conferred high avidity specific binding with their respective carriers (low nanomolar Kd values). The CotB peptide (CotB1p) induced protein aggregation in the transaminase and imine reductase/glucose oxidoreductase fusions when incubated with the silica carrier. The Clostridium thermocellum cellulose binding domain (CBDclos) allowed immobilization of all the proteins tested, but immobilization led to loss of enzymatic activity in the transaminases (< 2-fold) and imine reductase/glucose oxidoreductase fusion (> 80%). A transaminase-CBDclos fusion was then successfully used to demonstrate the application of the binding tag in repetitive batch and a continuous-flow reactor.
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Celulose , Enzimas Imobilizadas , Biocatálise , Enzimas Imobilizadas/metabolismo , Celulose/metabolismo , Oxirredutases/metabolismo , Peptídeos/metabolismo , Transaminases/metabolismo , Dióxido de Silício/química , Glucose Desidrogenase/metabolismoRESUMO
The stereoselective reduction of alkenes conjugated to electron-withdrawing groups by ene-reductases has been extensively applied to the commercial preparation of fine chemicals. Although several different enzyme families are known to possess ene-reductase activity, the old yellow enzyme (OYE) family has been the most thoroughly investigated. Recently, it was shown that a subset of ene-reductases belonging to the flavin/deazaflavin oxidoreductase (FDOR) superfamily exhibit enantioselectivity that is generally complementary to that seen in the OYE family. These enzymes belong to one of several FDOR subgroups that use the unusual deazaflavin cofactor F420. Here, we explore several enzymes of the FDOR-A subgroup, characterizing their substrate range and enantioselectivity with 20 different compounds, identifying enzymes (MSMEG_2027 and MSMEG_2850) that could reduce a wide range of compounds stereoselectively. For example, MSMEG_2027 catalyzed the complete conversion of both isomers of citral to (R)-citronellal with 99% ee, while MSMEG_2850 catalyzed complete conversion of ketoisophorone to (S)-levodione with 99% ee. Protein crystallography combined with computational docking has allowed the observed stereoselectivity to be mechanistically rationalized for two enzymes. These findings add further support for the FDOR and OYE families of ene-reductases displaying general stereocomplementarity to each other and highlight their potential value in asymmetric ene-reduction.
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Mycobacterium smegmatis , Oxirredutases , Oxirredutases/metabolismo , Mycobacterium smegmatis/metabolismo , Oxirredução , NADPH Desidrogenase/química , NADPH Desidrogenase/metabolismoRESUMO
Asymmetric reduction by ene-reductases has received considerable attention in recent decades. While several enzyme families possess ene-reductase activity, the Old Yellow Enzyme (OYE) family has received the most scientific and industrial attention. However, there is a limited substrate range and few stereocomplementary pairs of current ene-reductases, necessitating the development of a complementary class. Flavin/deazaflavin oxidoreductases (FDORs) that use the uncommon cofactor F420 have recently gained attention as ene-reductases for use in biocatalysis due to their stereocomplementarity with OYEs. Although the enzymes of the FDOR-As sub-group have been characterized in this context and reported to catalyse ene-reductions enantioselectively, enzymes from the similarly large, but more diverse, FDOR-B sub-group have not been investigated in this context. In this study, we investigated the activity of eight FDOR-B enzymes distributed across this sub-group, evaluating their specific activity, kinetic properties, and stereoselectivity against α,ß-unsaturated compounds. The stereochemical outcomes of the FDOR-Bs are compared with enzymes of the FDOR-A sub-group and OYE family. Computational modelling and induced-fit docking are used to rationalize the observed catalytic behaviour and proposed a catalytic mechanism.
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Mycobacterium smegmatis , Oxirredutases , Oxirredutases/metabolismo , Riboflavina/metabolismo , NADPH Desidrogenase/química , Biocatálise , OxirreduçãoRESUMO
Bottlenecks in metabolic pathways due to insufficient gene expression levels remain a significant problem for industrial bioproduction using microbial cell factories. Increasing gene dosage can overcome these bottlenecks, but current approaches suffer from numerous drawbacks. Here, we describe HapAmp, a method that uses haploinsufficiency as evolutionary force to drive in vivo gene amplification. HapAmp enables efficient, titratable, and stable integration of heterologous gene copies, delivering up to 47 copies onto the yeast genome. The method is exemplified in metabolic engineering to significantly improve production of the sesquiterpene nerolidol, the monoterpene limonene, and the tetraterpene lycopene. Limonene titre is improved by 20-fold in a single engineering step, delivering â¼1 g L-1 in the flask cultivation. We also show a significant increase in heterologous protein production in yeast. HapAmp is an efficient approach to unlock metabolic bottlenecks rapidly for development of microbial cell factories.
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Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Amplificação de Genes , Limoneno/metabolismo , Engenharia Metabólica/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Anthozoan chromoproteins are highly pigmented, diversely coloured and readily produced in recombinant expression systems. While they are a versatile and powerful building block in synthetic biology for applications such as biosensor development, they are not widely used in comparison to the related fluorescent proteins, partly due to a lack of structural characterization to aid protein engineering. Here, high-resolution X-ray crystal structures of four open-source chromoproteins, gfasPurple, amilCP, spisPink and eforRed, are presented. These proteins are dimers in solution, and mutation at the conserved dimer interface leads to loss of visible colour development in gfasPurple. The chromophores are trans and noncoplanar in gfasPurple, amilCP and spisPink, while that in eforRed is cis and noncoplanar, and also emits fluorescence. Like other characterized chromoproteins, gfasPurple, amilCP and eforRed contain an sp2-hybridized N-acylimine in the peptide bond preceding the chromophore, while spisPink is unusual and demonstrates a true sp3-hybridized trans-peptide bond at this position. It was found that point mutations at the chromophore-binding site in gfasPurple that substitute similar amino acids to those in amilCP and spisPink generate similar colours. These features and observations have implications for the utility of these chromoproteins in protein engineering and synthetic biology applications.
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Peptídeos , Fluorescência , Proteínas Luminescentes/química , Peptídeos/químicaRESUMO
BACKGROUND: Statin treatment is a potent lipid-lowering therapy associated with decreased cardiovascular risk and mortality. Recent studies including the PARADIGM trial have demonstrated the impact of statins on promoting calcified coronary plaque. HYPOTHESIS: The degree of systemic inflammation impacts the amount of increase in coronary plaque calcification over 2 years of statin treatment. METHODS: A subgroup of 142 participants was analyzed from the Risk Stratification with Image Guidance of HMG CoA Reductase Inhibitor Therapy (RIGHT) study (NCT01212900), who were on statin treatment and underwent cardiac computed tomography angiography (CCTA) at baseline and 2-year follow-up. This cohort was stratified by baseline median levels of high-sensitivity hs-CRP and analyzed with linear regressions using Stata-17 (StataCorp). RESULTS: In the high versus low hs-CRP group, patients with higher baseline median hs-CRP had increased BMI (median [IQR]; 29 [27-31] vs. 27 [24-28]; p < .001), hypertension (59% vs. 41%; p = .03), and LDL-C levels (97 [77-113] vs. 87 [75-97] mg/dl; p = .01). After 2 years of statin treatment, the high hs-CRP group had significant increase in dense-calcified coronary burden versus the low hs-CRP group (1.27 vs. 0.32 mm2 [100×]; p = .02), beyond adjustment (ß = .2; p = .03). CONCLUSIONS: Statin treatment over 2 years associated with a significant increase in coronary calcification in patients with higher systemic inflammation, as measured by hs-CRP. These findings suggest that systemic inflammation plays a role in coronary calcification and further studies should be performed to better elucidate these findings.
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Calcinose , Doença da Artéria Coronariana , Inibidores de Hidroximetilglutaril-CoA Redutases , Placa Aterosclerótica , Proteína C-Reativa , Doença da Artéria Coronariana/diagnóstico por imagem , Doença da Artéria Coronariana/tratamento farmacológico , Progressão da Doença , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Inflamação/tratamento farmacológico , Estudos Prospectivos , Medição de RiscoRESUMO
Temporal control of heterologous pathway expression is critical to achieve optimal efficiency in microbial metabolic engineering. The broadly-used GAL promoter system for engineered yeast (Saccharomyces cerevisiae) suffers from several drawbacks; specifically, unintended induction during laboratory development, and unintended repression in industrial production applications, which decreases overall production capacity. Eukaryotic synthetic circuits have not been well examined to address these problems. Here, we explore a modularised engineering method to deploy new genetic circuits applicable for expanding the control of GAL promoter-driven heterologous pathways in S. cerevisiae. Trans- and cis- modules, including eukaryotic trans-activating-and-repressing mechanisms, were characterised to provide new and better tools for circuit design. A eukaryote-like tetracycline-mediated circuit that delivers stringent repression was engineered to minimise metabolic burden during strain development and maintenance. This was combined with a novel 37 °C induction circuit to relief glucose-mediated repression on the GAL promoter during the bioprocess. This delivered a 44% increase in production of the terpenoid nerolidol, to 2.54 g L-1 in flask cultivation. These negative/positive transcriptional regulatory circuits expand global strategies of metabolic control to facilitate laboratory maintenance and for industry applications.