To beat the heat – engineering of the most thermostable pyruvate decarboxylase to date

Pyruvate decarboxylase (PDC) is a key enzyme for the production of ethanol at high temperatures and for cell-free butanol synthesis. Thermostable, organic solvent stable PDC was evolved from bacterial PDCs. The new variant shows >1500-fold-improved half-life at 75 °C and >5000-fold-increased half-life in the presence of 9 vol% butanol at 50 °C.

Pyruvate decarboxylase (PDC) is a key enzyme for the production of ethanol at high temperatures and for cell-free butanol synthesis.

For many years, bioethanol has been used widely as an additive in gasoline or as a fuel by itself.¹ To avoid competition with food, production of ethanol from plant-derived starch or sugar (first generation) has shifted to lignocellulosic biomass (second generation). To date, most of the bioethanol produced industrially still utilises yeasts as the main fermentation organism.^1–3 However, in recent years, several efforts have been made to use thermophilic microorganisms as the new framework.^1,3 The main motivating forces behind this are the volatility of ethanol at higher temperatures, which facilitates easy product removal, reduced risk of contamination and feasibility of consolidated bioprocessing (CBP). In this process, cellulolytic ethanologenic thermophilic microorganisms are applied, thus reducing the energy consumption required for heat exchange between biomass pre-treatment, fermentation and product separation and to avoid cellulase inhibition by accumulating glucose.^3,4There are two major pathways for producing ethanol from pyruvate, the central metabolite in glycolysis: non-oxidative decarboxylation via pyruvate decarboxylase (PDC) and oxidative decarboxylation via pyruvate dehydrogenase (PDH) or pyruvate ferredoxin oxidoreductase (PFOR). Non-oxidative ethanol productions are commonly found in yeasts and other ethanologenic bacteria, while PDH and PFOR routes are described in thermophilic microorganisms.^3,4 PDC mode may offer advantages compared with PDH and PFOR, as it is thermodynamically more favourable and will not lead to any other organic acid by-products.^3–5 In fact, all industrially relevant ethanol producing organisms use PDC. Thermostable PDCs are, therefore, suggested to play an important role in creating homo-ethanologenic, thermophilic microorganisms.^5,6Until recently, there were only limited efforts described in literature for improving the thermostability of PDC. Ancestral sequence reconstruction, supposedly a powerful method of resurrecting ancient thermostable enzymes, did not yield more thermostable PDC.⁷ A computational Rosetta-based design resulted in several variants with improved stability, determined by differential interference contrast (DIC) microscopy; unfortunately, the work was not corroborated by kinetic activity and stability data.⁸ Another effort to redirect the specificity of a thermostable, acetolactate synthase to perform a PDC-like reaction by conventional directed evolution did not yield variants either, with activity similar to that of bacterial PDC.⁹ Thus, creating thermostable PDC variants remains as the main challenge towards second generation ethanol production by thermophilic microorganisms.Besides acting as an important enzyme for ethanol production, PDC has also been applied in the in vitro production of n-butanol (Scheme 1).^10,11 Butanol together with other longer-chain alcohols, such as 1-propanol, isobutanol and isopentanol, is regarded as the next-generation biofuel, due to its closer resemblance to traditional gasoline.¹² Thus, there is an increasing demand to produce longer-chain alcohols sustainably.¹² Industrially, butanol is still produced from petroleum-derived propene utilizing Co-catalyst, H₂, and CO via oxo synthesis and Reppe process.¹³ Albeit its efficiency, production of butanol from petroleum is not sustainable.Open in a separate windowScheme 1Simplified pathway to produce ethanol and 1-butanol from lignocellulosic biomass via pyruvate decarboxylase (PDC). There are two major pathways for producing 1-butanol via pyruvate: CoA-dependent or proline-dependent condensation. ADH is an alcohol dehydrogenase.In a more sustainable way, butanol can be produced via an acetone–butanol–ethanol (ABE) process, utilising Clostridium acetobutylicum.^12,14 Recent work on metabolic engineering has demonstrated the possibility of producing butanol in other microorganisms.^15,16 However, one of the major challenges in butanol fermentation is still the toxicity of butanol to many microorganisms at relatively low concentrations (<2 vol%).^17,18 This effect, however, is less pronounced for the enzymes catalysing butanol production in vitro.^10,19 Many enzymes can already tolerate higher organic solvent concentrations than the corresponding microorganisms.²⁰ Furthermore, engineering enzyme stability towards organic solvents is less challenging than evolving microorganisms to withstand the same concentration of organic solvents.^17,21 Therefore, in vitro butanol production emerges as a very promising alternative to traditional butanol fermentation. Thus, in this work, we focused on the engineering of PDC towards higher thermal stability for prospective ethanol production in thermophilic microorganisms and improved stability in butanol for applications in in vitro, artificial butanol synthesis.Prior to engineering a PDC, we characterised two new yeast PDCs from Candida glabrata (CgPDC) and Zygosaccharomyces rouxii (ZrPDC), as well as other PDCs reported in the literature, to determine the most suitable template. As presented in 22 Characterisation of bacterial PDCs showed similar findings to the previous report, with PDC from Acetobacter pasteurianus (ApPDC) demonstrating the highest kinetic stability (T₅₀^{1 h}) and melting temperature (T_m).²²T₅₀^{1 h} is defined as the temperature at which 50% of the enzymes remained active after 1 h incubation, while T_m is defined as the temperature at which 50% of the enzymes are in an unfolded state.²³ In general, bacterial PDCs showed higher activity at 25 °C and overall stability (T₅₀^{1 h} and T_m) than newly characterised CgPDC and ZrPDC.Summary of kinetic characterisations and thermostability of WT PDCs and variants

Hypofibrinolysis in type 2 diabetes: the role of the inflammatory pathway and complement C3

Aims/hypothesis

Plasminogen activator inhibitor-1 (PAI-1) has been regarded as the main antifibrinolytic protein in diabetes, but recent work indicates that complement C3 (C3), an inflammatory protein, directly compromises fibrinolysis in type 1 diabetes. The aim of the current project was to investigate associations between C3 and fibrinolysis in a large cohort of individuals with type 2 diabetes.

Methods

Plasma levels of C3, C-reactive protein (CRP), PAI-1 and fibrinogen were analysed by ELISA in 837 patients enrolled in the Edinburgh Type 2 Diabetes Study. Fibrin clot lysis was analysed using a validated turbidimetric assay.

Results

Clot lysis time correlated with C3 and PAI-1 plasma levels (r?=?0.24, p?<?0.001 and r?=?0.22, p?<?0.001, respectively). In a multivariable regression model involving age, sex, BMI, C3, PAI-1, CRP and fibrinogen, and using log-transformed data as appropriate, C3 was associated with clot lysis time (regression coefficient 0.227 [95% CI 0.161, 0.292], p?<?0.001), as was PAI-1 (regression coefficient 0.033 [95% CI 0.020, 0.064], p?<?0.05) but not fibrinogen (regression coefficient 0.003 [95% CI ?0.046, 0.051], p?=?0.92) or CRP (regression coefficient 0.024 [95% CI ?0.008, 0.056], p?=?0.14). No correlation was demonstrated between plasma levels of C3 and PAI-1 (r?=??0.03, p?=?0.44), consistent with previous observations that the two proteins affect different pathways in the fibrinolytic system.

Conclusions/interpretation

Similarly to PAI-1, C3 plasma levels are independently associated with fibrin clot lysis in individuals with type 2 diabetes. Therefore, future studies should analyse C3 plasma levels as a surrogate marker of fibrinolysis potential in this population.     

Targeted profiling of atherogenic phospholipids in human plasma and lipoproteins of hyperlipidemic patients using MALDI-QIT-TOF-MS/MS

ObjectivesPhospholipids (PLs) are increasingly recognized as key molecules with potential diagnostic value in acute inflammation, CVD and atherosclerosis. We introduce a pioneer mass spectrometry (MS)-based approach aiming to investigate the relationship of specific plasma PL-subsets with atherogenic blood parameters in young patients with familial hyperlipidemia representing high-CVD-risk groups.MethodsPlasma of carefully phenotyped FH and FCH patients as well as normolipidemic subjects (age 13 ± 5 years, n = 20) was used. Clinical parameters were assessed using standard laboratory techniques and lipids were subjected to a direct targeted monitoring using LC-ESI-SRM- and MALDI-QIT-TOF-MS/MS, respectively. Statistical analysis was performed to evaluate correlations between PL data and the clinical parameters.ResultsMost characteristically significant differences of SM/PC and PC/LPC ratios and positive correlations between SM vs. LDL-C (r = 0.946; p = 0.004) and LPC vs. VLDL-C (r = 0.669; p = 0.218) were observed in FH in contrast to the other study groups. OxPC levels were found in the range of ～2–20 μmol/L with predominance of short-chain aldehydic species (e.g. SOVPC). A positive correlation of OxPCs with IMT (r = 0.952; p = 0.052) and HDL-C (r = 0.893; p = 0.016) but negative correlation with OxLDL (r = ?0.910; p = 0.096) was observed.ConclusionsOur study was a first attempt to use a MALDI-QIT-TOF-MS/MS based clinical lipidomics approach to investigate atherogenic dyslipidemia in young patients with familial hyperlipidemia. This technique represents a promising platform for clinical screening of lipid biomarkers in the future.     

Active encoding of decisions about stimulus absence in primate prefrontal cortex neurons

Judging the presence or absence of a stimulus is likely the most basic perceptual decision. A fundamental difference of detection tasks in contrast to discrimination tasks is that only the stimulus presence decision can be inferred from sensory evidence, whereas the alternative decision about stimulus absence lacks sensory evidence by definition. Detection decisions have been studied in an intentional, action-based framework, in which decisions were regarded as intentions to pursue particular actions. These studies have found that only stimulus-present decisions are actively encoded by neurons, whereas the decision about the absence of a stimulus does not affect default neuronal responses. We tested whether this processing mechanism also holds for abstract detection decisions that are dissociated from motor preparation. We recorded single-neuron activity from the prefrontal cortex (PFC) of monkeys performing a visual detection task that forced a report-independent decision. We not only found neurons that actively encoded the subjective decision of monkeys about the presence of a stimulus, but also cells responding actively for the decision about the absence of stimuli. These results suggest that abstract detection decisions are processed in a different way compared with the previously reported action-based decisions. In a report-independent framework, neuronal networks seem to generate a second set of neurons actively encoding the absence of sensory stimulation, thus translating decisions into abstract categories. This mechanism may allow the brain to "buffer" a decision in a nonmovement-related framework.     

Mitochondrial hexokinase II (HKII) and phosphoprotein enriched in astrocytes (PEA15) form a molecular switch governing cellular fate depending on the metabolic state

The metabolic state of a cell is a key determinant in the decision to live and proliferate or to die. Consequently, balanced energy metabolism and the regulation of apoptosis are critical for the development and maintenance of differentiated organisms. Hypoxia occurs physiologically during development or exercise and pathologically in vascular disease, tumorigenesis, and inflammation, interfering with homeostatic metabolism. Here, we show that the hypoxia-inducible factor (HIF)-1-regulated glycolytic enzyme hexokinase II (HKII) acts as a molecular switch that determines cellular fate by regulating both cytoprotection and induction of apoptosis based on the metabolic state. We provide evidence for a direct molecular interactor of HKII and show that, together with phosphoprotein enriched in astrocytes (PEA15), HKII inhibits apoptosis after hypoxia. In contrast, HKII accelerates apoptosis in the absence of PEA15 and under glucose deprivation. HKII both protects cells from death during hypoxia and functions as a sensor of glucose availability during normoxia, inducing apoptosis in response to glucose depletion. Thus, HKII-mediated apoptosis may represent an evolutionarily conserved altruistic mechanism to eliminate cells during metabolic stress to the advantage of a multicellular organism.     

Bacterial expression of mutant argininosuccinate lyase reveals imperfect correlation of in-vitro enzyme activity with clinical phenotype in argininosuccinic aciduria

Background

The urea cycle defect argininosuccinate lyase (ASL) deficiency has a large spectrum of presentations from highly severe to asymptomatic. Enzyme activity assays in red blood cells or fibroblasts, although diagnostic of the deficiency, fail to discriminate between severe, mild or asymptomatic cases. Mutation/phenotype correlation studies are needed to characterize the effects of individual mutations on the activity of the enzyme.

Methods

Bacterial in-vitro expression studies allowed the enzyme analysis of purified mutant ASL proteins p.I100T (c.299?T?>?C), p.V178M (c.532?G?>?A), p.E189G (c.566A?>?G), p.Q286R (c.857A?>?G), p.K315E (c.943A?>?G), p.R379C (c.1135?C?>?T) and p.R385C (c.1153?C?>?T) in comparison to the wildtype protein.

Results

In the bacterial in-vitro expression system, ASL wild-type protein was successfully expressed. The known classical p.Q286R, the novel classical p.K315E and the known mutations p.I100T, p.E189G and p.R385C, which all have been linked to a mild phenotype, showed no significant residual activity. There was some enzyme activity detected with the p.V178M (5 % of wild-type) and p.R379C (10 % of wild-type) mutations in which K_m values for argininosuccinic acid differed significantly from the wild-type ASL protein.

Conclusion

The bacterially expressed enzymes proved that the mutations found in patients and studied here indeed are detrimental. However, as in the case of red cell ASL activity assays, some mutations found in genetically homozygous patients with mild presentations resulted in virtual loss of enzyme activity in the bacterial system, suggesting a more protective environment for the mutant enzyme in the liver than in the heterologous expression system and/or in the highly dilute assays utilized here.