Thermostable proteases from thermophilic microorganisms

Missing pages 240-290, 295-302Two metallo-proteases, EA1 and YP-T proteases from Bacillus st. EA1 and B. caldolyticus st. YP-T, respectively, differ in amino acid sequence by only one residue (Val61 = Gly61 in EA1 and YP-T respectively). Yet EA1 protease has a higher thermostability than YP-T protease. An analysis of the half lives of the two proteases at 85°C at different Ca²⁺ concentrations shows that at low Ca²⁺ levels, autolysis is the most significant cause of loss of activity, while at higher Ca²⁺ levels denaturation is most significant. A comparison of the sequences of the two proteases and thermolysin shows that this amino acid difference is located within the third Ca²⁺ binding site (based on the known structure of thermolysin). The residues are not directly involved in binding to the Ca²⁺, but are located within this pocket. Molecular modelling studies of EA1 and YP-T proteases, based on the structure of thermolysin, propose that the stabilisation of EA1 over YP-T protease could be due to extra interaction/s of the side chain of Val 61 of EA1 protease with the benzene ring of Tyr 27. YP-T protease has Gly in this position. As the “side chain” of Gly is only a hydrogen, there is unlikely to be any such interaction with Tyr 27. This possible extra interaction/s could increase the bonding of the amino acids within the calcium binding pocket, which may stabilise the pocket. Bacillus st. Ak.1 produces a thermostable serine protease (subtilisin). The 16S rRNA analysis closely groups Bacillus st. Ak.1 and B. thermoglucosidasius. However, the organisms have different growth requirements. Bacillus st. Ak.1 has a requirement for glutamate, while B. thermoglucosidasius has a requirement for a compound found in yeast extract and tryptone, but not glutamate (possibly maltose). RAPD-PCR analysis of twelve organisms show that the banding patterns of the organisms are all unique, though the closest two were between Bacillus st. EA1 and B. caldolyticus. Escherichia coli clone PB5517 produces Ak.1 protease constitutively at 35°C. a 10 1 fermentor run was conducted, and 51 mg of the protease was purified to homogeneity. Ak.1 protease is dramatically stabilised by Ca²⁺ ions. The half life at 70°C increases by four orders of magnitude in the presence of 5 mM Ca²⁺, as compared to the thermostability in the absence of Ca²⁺. As the concentration of Ca²⁺ ions increased, the degree of denaturation decreased. At high Ca²⁺ concentrations, the major cause of loss of activity was due to autolysis. Based on the structural and enzymatic data, the extra degree of stabilisation of Ak.1 protease by Ca²⁺ above that of thermitase-Ca²⁺ could be due to the presence of an extra Ca²⁺-binding site in Ak.1 protease. This new site is located close to Ca(1), and could therefore change the binding properties of this site also. Another possibility is that the binding constants of one or more of the Ca²⁺-binding sites could be higher for Ak.1 protease, as compared to thermitase. Lanthanide ions stabilised the protease, though to a much smaller degree than Ca²⁺. Like Ca²⁺, they stabilised the protease by the prevention of denaturation. Other cations stabilised the protease to a small degree, especially Sr²⁺. Different cations had different effects on the stability of the enzyme. Other significant stabilisers were 90% solutions of sorbitol, trehalose and glycerol. At 105°C, 90% sorbitol increased the thermostability of the protease from <<1 minute to 104 minutes. It did so by the prevention of autolysis. The protease has a limited substrate specificity, preferring to cleave substrates containing neutral or hydrophobic amino acids, such as valine, alanine or phenylalanine, at the P₁ site. It has a preference for proline at the P₂ site, and alanine at the P₁-P₄ sites. It also has esterase activity, being able to cleave methyl, ethyl and p-nitrophenyl esters. Studies with Suc-Alaₙ-pNA substrates (n=2-5) shows that the specific activity of the protease increases with increasing chain length, though such a substrate containing 5 alanine residues appears to be being cleaved significantly at more than one site. A comparison of the Kₘ and Vₘₐₓ of the protease to the substrates Suc-Ala-Ala-Pro-Xaa-pNA, where Xaa = Phe, Leu or Ala, shows that the larger and more hydrophobic the P₁ amino acid is, the higher the specificity of the protease for that substrate. An analysis of the effect of temperature on the Kₘ and Vₘₐₓ of Ak.1 protease with several substrates revealed that the specificity of the protease (Vₘₐₓ/Kₘ) changes with temperature. The Kₘ and Vₘₐₓ decreased with decreasing temperature, but not to the same degree with all substrates. If the protease is assayed with substrates in the presence of 50% methanol, the Kₘ tends to increase dramatically. This is due to hydrophobic partitioning of the solvent. The Vₘₐₓ of the protease decreases under these conditions. The active site is a cleft, composed of hydrophobic amino acids m the substrate-binding cleft. It is similar to other subtilisins, but differs in the presence of a disulphide bond. The space-filling model of the protease with the substrate SAAPFpNA in the active site shows the cleft ‘bends’ at the P₂ site. This is easily accomidated for by proline at this position, as proline causes a bend in the substrate. This can explain the preference for proline at the P₂ site. The sequence of Ak.1 protease indicates the presence of two cysteine residues, separated by only one amino acid. The 3D structure showed that these cysteine residues exist in a disulphide bond. Tests (e.g. Ellman assay) confirmed the presence of not only two cysteine residues, but that in native conditions (i.e. in solution) these residues exist as a disulphide bond. Reductants, such as dithiothreitol (DTI) typically reduce disulphide bonds into their constituent cysteine residues. The lower thermostability in the presence of DTT indicates that it appears to have opened a disulphide bond as disulphide bonds are known to increase the thermostability of proteins. It is proposed that the reduction of the disulphide bond causes a localised opening of the substrate binding cleft at the P₄ site, due to the location of the disulphide bond in this position. This was supported by the results that showed that the larger the substrate, the greater the effect of DTT on the Kₘ of the protease. With that substrate. For example, the Kₘ was unchanged with DTT with a substrate occupying only sites P₂-P₁', while a substrate which occupies sites P₅-P₁' showed a significant increase of the Kₘ, suggesting it binds weaker to the binding cleft. Heavy metals such as Hg²⁺ and Pb²⁺ bind to Ak.1 protease, causing a decrease in the specific activity of the protease. The effects of the heavy metals on activity is much smaller than with DTT. In general, the Kₘ and Vₘₐₓ were only changed to a small degree. Fluorescein mercuric acetate (FMA) binds to Ak.1 protease, causing the inherent fluorescence of FMA to increase. The presence of DTT caused a decrease in the thermostability of Ak.1 protease of 9.3 fold at 85°C. In conclusion, the disulphide bond has a dual role, that of maintaining the integrity of the substrate-binding cleft and increasing the thermostability of the protease

Bioproduction of Linalool From Paper Mill Waste

A key challenge in chemicals biomanufacturing is the maintenance of stable, highly productive microbial strains to enable cost-effective fermentation at scale. A “cookie-cutter” approach to microbial engineering is often used to optimize host stability and productivity. This can involve identifying potential limitations in strain characteristics followed by attempts to systematically optimize production strains by targeted engineering. Such targeted approaches however do not always lead to the desired traits. Here, we demonstrate both ‘hit and miss’ outcomes of targeted approaches in attempts to generate a stable Escherichia coli strain for the bioproduction of the monoterpenoid linalool, a fragrance molecule of industrial interest. First, we stabilized linalool production strains by eliminating repetitive sequences responsible for excision of pathway components in plasmid constructs that encode the pathway for linalool production. These optimized pathway constructs were then integrated within the genome of E. coli in three parts to eliminate a need for antibiotics to maintain linalool production. Additional strategies were also employed including: reduction in cytotoxicity of linalool by adaptive laboratory evolution and modification or homologous gene replacement of key bottleneck enzymes GPPS/LinS. Our study highlights that a major factor influencing linalool titres in E. coli is the stability of the genetic construct against excision or similar recombination events. Other factors, such as decreasing linalool cytotoxicity and changing pathway genes, did not lead to improvements in the stability or titres obtained. With the objective of reducing fermentation costs at scale, the use of minimal base medium containing paper mill wastewater secondary paper fiber as sole carbon source was also investigated. This involved simultaneous saccharification and fermentation using either supplemental cellulase blends or by co-expressing secretable cellulases in E. coli containing the stabilized linalool production pathway. Combined, this study has demonstrated a stable method for linalool production using an abundant and low-cost feedstock and improved production strains, providing an important proof-of-concept for chemicals production from paper mill waste streams. For scaled production, optimization will be required, using more holistic approaches that involve further rounds of microbial engineering and fermentation process development

Consolidated Bioprocessing: Synthetic Biology Routes to Fuels and Fine Chemicals

From MDPI via Jisc Publications RouterHistory: accepted 2021-05-14, pub-electronic 2021-05-18Publication status: PublishedFunder: Biotechnology and Biological Sciences Research Council; Grant(s): BB/S011684/1Funder: Engineering and Physical Sciences Research Council; Grant(s): EP/S01778X/1Funder: Office of Naval Research Global; Grant(s): N62909-18-1-2137The long road from emerging biotechnologies to commercial “green” biosynthetic routes for chemical production relies in part on efficient microbial use of sustainable and renewable waste biomass feedstocks. One solution is to apply the consolidated bioprocessing approach, whereby microorganisms convert lignocellulose waste into advanced fuels and other chemicals. As lignocellulose is a highly complex network of polymers, enzymatic degradation or “saccharification” requires a range of cellulolytic enzymes acting synergistically to release the abundant sugars contained within. Complications arise from the need for extracellular localisation of cellulolytic enzymes, whether they be free or cell-associated. This review highlights the current progress in the consolidated bioprocessing approach, whereby microbial chassis are engineered to grow on lignocellulose as sole carbon sources whilst generating commercially useful chemicals. Future perspectives in the emerging biofoundry approach with bacterial hosts are discussed, where solutions to existing bottlenecks could potentially be overcome though the application of high throughput and iterative Design-Build-Test-Learn methodologies. These rapid automated pathway building infrastructures could be adapted for addressing the challenges of increasing cellulolytic capabilities of microorganisms to commercially viable levels

Chemoautotrophic production of gaseous hydrocarbons, bioplastics and osmolytes by a novel <i>Halomonas</i> species

BackgroundProduction of relatively low value, bulk commodity chemicals and fuels by microbial species requires a step-change in approach to decrease the capital and operational costs associated with scaled fermentation. The utilisation of the robust and halophilic industrial host organisms of the genus Halomonas could dramatically decrease biomanufacturing costs owing to their ability to grow in seawater, using waste biogenic feedstocks, under non-sterile conditions.ResultsWe describe the isolation of Halomonas rowanensis, a novel facultative chemoautotrophic species of Halomonas from a natural brine spring. We investigated the ability of this species to produce ectoine, a compound of considerable industrial interest, under heterotrophic conditions. Fixation of radiolabelled NaH14CO3 by H. rowanensis was confirmed in mineral medium supplied with thiosulfate as an energy source. Genome sequencing suggested carbon fixation proceeds via a reductive tricarboxylic acid cycle, and not the Calvin-Bensen-Bassham cycle. The mechanism of energy generation to support chemoautotrophy is unknown owing to the absence of an annotated SOX-based thiosulfate-mediated energy conversion system. We investigated further the biotechnological potential of the isolated H. rowanensis by demonstrating production of the gaseous hydrocarbon (bio-propane), bioplastics (poly-3-hydroxybutyrate) and osmolytes (ectoine) under heterotrophic and autotrophic CO2 fixation growth conditions.ConclusionsThis proof-of-concept study illustrates the value of recruiting environmental isolates as industrial hosts for chemicals biomanufacturing, where CO2 utilisation could replace, or augment, the use of biogenic feedstocks in non-sterile, industrialised bioreactors

Are quasars accreting at super-Eddington rates?

In a previous paper, Collin & Hur\'e (2001), using a sample of Active Galactic Nuclei (AGN) where the mass has been determined by reverberation studies (Kaspi et al. 2000), have shown that if the optical luminosity is emitted by a steady accretion disc, about half of the objects are accreting close to or higher than the Eddington rate. We conclude here that this result is unavoidable, unless the masses are strongly underestimated by reverberation studies, which does not seem to be the case. There are three issues to the problem: 1. Accretion proceeds at Eddington or super-Eddington rates through thick discs. Several consequences follow: an anti-correlation between the line widths of the lines and the Eddington ratios, and a decrease of the Eddington ratio with an increasing black hole mass. Extrapolated to all quasars, these results imply that the amount of mass locked in massive black holes should be larger than presently thought. 2. The optical luminosity is not produced directly by the gravitational release of energy, and super-Eddington rates are not required. The optical luminosity has to be emitted by a dense and thick medium located at large distances from the center (10$^3$ to $10^4$ gravitational radii). It can be due to reprocessing of the X-ray photons from the central source in a geometrically thin warped disc, or in dense "blobs" forming a geometrically thick system, which can be a part of the accretion flow or the basis of an outflow. 3. Accretion discs are completely "non standard". Presently neither the predictions of models nor the observed spectral distributions are sufficient to help choosing between these solutions.Comment: 16 pages, 11 figures, accepted in A&

Renewable and tuneable bio-LPG blends derived from amino acids

From Springer Nature via Jisc Publications RouterHistory: received 2020-05-21, accepted 2020-07-08, registration 2020-07-08, pub-electronic 2020-07-14, online 2020-07-14, collection 2020-12Publication status: PublishedFunder: C3 Biotechnologies LtdFunder: Engineering and Physical Sciences Research Council; doi: http://dx.doi.org/10.13039/501100000266; Grant(s): EP/S01778X/1, EP/J020192/1Funder: Biotechnology and Biological Sciences Research Council; doi: http://dx.doi.org/10.13039/501100000268; Grant(s): BB/M017702/1, BB/L010798/1Funder: Newton-Mosharafa fundAbstract: Background: Microbial biorefinery approaches are beginning to define renewable and sustainable routes to clean-burning and non-fossil fuel-derived gaseous alkanes (known as ‘bio-LPG’). The most promising strategies have used a terminal fatty acid photodecarboxylase, enabling light-driven propane production from externally fed waste butyric acid. Use of Halomonas (a robust extremophile microbial chassis) with these pathways has enabled bio-LPG production under non-sterile conditions and using waste biomass as the carbon source. Here, we describe new engineering approaches to produce next-generation pathways that use amino acids as fuel precursors for bio-LPG production (propane, butane and isobutane blends). Results: Multiple pathways from the amino acids valine, leucine and isoleucine were designed in E. coli for the production of propane, isobutane and butane, respectively. A branched-chain keto acid decarboxylase-dependent pathway utilising fatty acid photodecarboxylase was the most effective route, generating higher alkane gas titres over alternative routes requiring coenzyme A and/or aldehyde deformylating oxygenase. Isobutane was the major gas produced in standard (mixed amino acid) medium, however valine supplementation led to primarily propane production. Transitioning pathways into Halomonas strain TQ10 enabled fermentative production of mixed alkane gases under non-sterile conditions on simple carbon sources. Chromosomal integration of inducible (~ 180 mg/g cells/day) and constitutive (~ 30 mg/g cells/day) pathways into Halomonas generated production strains shown to be stable for up to 7 days. Conclusions: This study highlights new microbial pathways for the production of clean-burning bio-LPG fuels from amino acids. The use of stable Halomonas production strains could lead to gas production in the field under non-sterile conditions following process optimisation