One-Pot Biosynthesis of l-Aspartate from Maleic Anhydride via a Thermostable Dual-Enzyme System under High Temperature.

l-Aspartate is an important chemical in the food and pharmaceutical industries. Herein, a dual-enzyme system was constructed to synthesize l-aspartate from maleic anhydride at 50 °C, which can reduce the byproduct production. Maleate transformed from maleic anhydride in the solution was converted into l-aspartate via fumarate catalyzed by maleate isomerase (MaiA) and thermostable aspartase (AspB), respectively. Because MaiA is a rate-limiting enzyme, enzyme activities of various MaiAs were compared, and the efficient and thermostable maleate isomerase AaMaiA from Alicyclobacillus acidoterrestris was chosen. The Kcat/Km value of AaMaiA was 264.4 mM-1 min-1. AaMaiA and AspB were coexpressed in E. coli to produce l-aspartate. To improve the l-aspartate production rate, the ribosome binding site (RBS) sequence located upstream of AaMaiA was optimized and the Tat signal peptide was fused with AaMaiA. The conversion rate was 96% within 60 min, and the intermediate was not detected, the possible reason of which is that high temperature inhibits the activity of bacterial endogenous enzymes, but functional enzymes remain active. Cells from fermentation produced 243.6 g/L (1.83 M) of l-aspartate with a 2 M substrate. Our study revealed an effective method to produce l-aspartate without using gene knockout and provided a strategy for l-aspartate production in the industrial field.

C4-dicarboxylate metabolons: interaction of C4-dicarboxylate transporters of Escherichia coli with cytosolic enzymes.

Metabolons represent the structural organization of proteins for metabolic or regulatory pathways. Here, the interaction of fumarase FumB, aspartase AspA, and L-tartrate dehydratase TtdAB with the C4-dicarboxylate (C4-DC) transporters DcuA, DcuB, DcuC, and the L-tartrate transporter TtdT of Escherichia coli was tested by a bacterial two-hybrid (BACTH) assay in situ, or by co-chromatography using mSPINE (membrane Streptavidin protein interaction experiment). From the general C4-DC transporters, DcuB interacted with FumB and AspA, DcuA with AspA, whereas DcuC interacted with neither FumB nor AspA. Moreover, TtdT did not interact with TtdAB. The fumB-dcuB, the dcuA-aspA, and the ttdAB-ttdT genes encoding the respective proteins colocalize on the genome and each pair of genes forms cotranscripts, whereas the dcuC gene lies alone. The data suggest the formation of DcuB/FumB and DcuB/AspA metabolons for the uptake of L-malate, or L-aspartate, and their conversion to fumarate for fumarate respiration and excretion of the product succinate. The DcuA/AspA metabolon catalyzes uptake and conversion of L-aspartate to fumarate coupled to succinate excretion. The DcuA/AspA metabolon provides ammonia at the same time for nitrogen assimilation (ammonia shuttle). On the other hand, TtdT and TtdAB are not organized in a metabolon. Reasons for the formation (DcuA/AspA, DcuB/FumB, and DcuB/AspA) or nonformation (DcuC, TtdT, and TtdAB) of metabolons are discussed based on their metabolic roles.

Development of a Glycerol-Inducible Expression System for High-Yield Heterologous Protein Production in Bacillus subtilis.

The development of efficient, low-cost, and robust expression systems is important for the mass production of proteins and natural products in large amounts using cell factories. Glycerol is an ideal carbon source for large-scale fermentation due to its low cost and favorable maintenance of the fermentation process. Here, we used the antiterminator protein GlpP and its target promoter PglpD to construct a highly efficient glycerol-inducible expression system (GIES) in Bacillus subtilis. This system was able to express heterologous genes in an autoinducible manner based on the sequential utilization of glucose and glycerol under the regulation of carbon catabolite repression. In such a system, the concentration of glycerol regulated the strength of gene expression, and the concentration of glucose affected both the timing of induction and the strength of gene expression. By enhancing GlpP, the GIES was further strengthened for high-level intracellular expression of aspartase and secretory expression of nattokinase. High yields of nattokinase in a 5-L fermenter through batch and fed-batch fermentation demonstrated the potential to apply the GIES for large-scale enzyme production. Through the evolution of the -10 box of PglpD, mutants with gradient activities were obtained. In addition, hybrid glycerol-inducible promoters were successfully constructed by combining the constitutive promoters and the 5' untranslated region of PglpD. Collectively, this study developed a GIES to obtain high-value products from inexpensive glycerol. More importantly, the great potential of the pair of inherent terminator and antiterminator protein as a portable biological tool for various purposes in synthetic biology is proposed. IMPORTANCE In this study, a GIES was constructed in B. subtilis by employing the antiterminator protein GlpP and the GlpP-regulated promoter PglpD. Based on the sequential utilization of glucose and glycerol by B. subtilis, the GIES was able to express genes in an autoinducible manner. The amounts and ratio of glucose and glycerol can regulate the gene induction timing and expression strength. The GIES was further applied for high yields of nattokinase, and its robustness in production scale-up was confirmed in a 5-L fermenter. The high-level expression of heterologous proteins demonstrated the huge application potential of the GIES. Furthermore, mutants of PglpD with gradient activities and hybrid glycerol-inducible promoters were obtained through the evolution of the -10 box of PglpD and the combination of the constitutive promoters and the 5' untranslated region of PglpD, respectively. These results demonstrated the use of the antiterminator protein as a regulator for various purposes in synthetic biology.

Genomic rearrangements in the aspA-dcuA locus of Propionibacterium freudenreichii are associated with aspartase activity.

Propionibacterium freudenreichii is crucial in Swiss-type cheese manufacture. Classic propionic acid fermentation yields the nutty flavor and the typical eyes. Co-metabolism of aspartate pronounces the flavor of the cheese; however, it also increases the size of the eyes, which can induce splitting and reduce the cheese quality. Aspartase (EC 4.3.1.1) catalyzes the deamination of aspartate, yielding fumarate and ammonia. The aspartase activity varies considerably among P. freudenreichii strains. Here, the correlation between aspartase activity and the locus of aspartase-encoding genes (aspA ) and dcuA encoding the C4-dicarboxylate transporter was investigated in 46 strains to facilitate strain selection for cheese culture. Low aspartase activity was correlated with a particular genomic rearrangement: low in vitro aspartase activity always occurred in strains with gene clusters aspA - dcuA where the dcuA was frameshifted, producing a stop codon or was disrupted by an ISL3-like element. The low aspartase activity could be due to the protein sequence of the aspartase or a dysfunctional DcuA. The highest values of aspartase activity were detected in strains with aspA1 - aspA2-dcuA with a DcuA sequence sharing 99.07 - 100% identity with the DcuA sequence of strain DSM 20271 T and an additional C4-dicarboxylate transporter belonging to the DcuAB family.

Metabolic engineering of Bacillus subtilis for high-level production of uridine from glucose.

As an intermediate in drug synthesis, uridine has practical applications in the pharmaceutical field. Bacillus subtilis is used as a host to boost uridine yield by manipulating its uridine biosynthesis pathway. In this study, we engineered a high-uridine-producing strain of B. subtilis by modifying its metabolic pathways in vivo. Overexpression of the aspartate ammonia-lyase (ansB) gene increased the relative transcriptional level of ansB in B. subtilis TD320 by 13·18 times and improved uridine production to 15·13 g l-1 after 72-h fermentation. Overexpression of the putative 6-phosphogluconolactonase (ykgB) gene increased uridine production by the derivative strain TD325 to 15·43 g l-1 . Reducing the translation of the amido phosphoribosyl transferase (purF) gene and inducing expression of the subtilisin E (aprE) gene resulted in a 1·99-fold increase in uridine production after 24 h shaking. Finally, uridine production in the optimal strain B. subtilis TD335, which exhibited reduced urease expression, reached 17·9 g l-1 with a yield of 314 mg of uridine g-1 glucose. To our knowledge, this is the first study to obtain high-yield uridine-producing B. subtilis in a medium containing only three components (80 g l-1 glucose, 20 g l-1 yeast powder, and 20 g l-1 urea).

Immobilization of the Aspartate Ammonia-Lyase from Pseudomonas fluorescens R124 on Magnetic Nanoparticles: Characterization and Kinetics.

Aspartate ammonia-lyases (AALs) catalyze the non-oxidative elimination of ammonia from l-aspartate to give fumarate and ammonia. In this work the AAL coding gene from Pseudomonas fluorescens R124 was identified, isolated, and cloned into the pET-15b expression vector and expressed in E. coli. The purified enzyme (PfAAL) showed optimal activity at pH 8.8, Michaelis-Menten kinetics in the ammonia elimination from l-aspartate, and no strong dependence on divalent metal ions for its activity. The purified PfAAL was covalently immobilized on epoxy-functionalized magnetic nanoparticles (MNP), and effective kinetics of the immobilized PfAAL-MNP was compared to the native solution form. Glycerol addition significantly enhanced the storability of PfAAL-MNP. Inhibiting effect of the growing viscosity (modulated by addition of glycerol or glucose) on the enzymatic activity was observed for the native and immobilized form of PfAAL, as previously described for other free enzymes. The storage stability and recyclability of PfAAL-MNP is promising for further biocatalytic applications.

One-Pot Biosynthesis of 3-Aminopropionic Acid from Fumaric Acid Using Recombinant Bacillus megaterium Containing a Linear Dual-Enzyme Cascade.

3-Aminopropionic acid (3-APA) has wide applications in food, cosmetics, pharmaceuticals, chemical, and polymer industries. This present study aimed to develop an eco-friendly whole-cell biocatalytic process for the bio-production of 3-APA from fumaric acid (FA) using Bacillus megaterium. A dual-enzyme cascade route with aspartate-1-decarboxylases (ADC) from Bacillus subtilis and native aspartate ammonia-lyase (AspA) was developed. Divergent catalytic efficiencies between these two enzymes led to an imbalance between both enzyme reactions. In order to coordinate AspA and ADC expression levels, gene mining, optimization, and duplication strategies were employed. Additionally, culture cultivation conditions and biocatalysis process parameters were optimized. A maximum 3-APA titer was obtained (11.68 ± 0.26 g/L) with a yield of 0.78 g/g under the following optimal conditions: 45 °C, pH 6.0, and 15 g/L FA. This study established a biocatalysis process for the production of 3-APA from FA using the whole cells of the recombinant B. megaterium.

L-Aspartate as a high-quality nitrogen source in Escherichia coli: Regulation of L-aspartase by the nitrogen regulatory system and interaction of L-aspartase with GlnB.

Escherichia coli uses the C4-dicarboxylate transporter DcuA for L-aspartate/fumarate antiport, which results in the exploitation of L-aspartate for fumarate respiration under anaerobic conditions and for nitrogen assimilation under aerobic and anaerobic conditions. L-Aspartate represents a high-quality nitrogen source for assimilation. Nitrogen assimilation from L-aspartate required DcuA, and aspartase AspA to release ammonia. Ammonia is able to provide by established pathways the complete set of intracellular precursors (ammonia, L-aspartate, L-glutamate, and L-glutamine) for synthesizing amino acids, nucleotides, and amino sugars. AspA was regulated by a central regulator of nitrogen metabolism, GlnB. GlnB interacted with AspA and stimulated its L-aspartate deaminase activity (NH3 -forming), but not the reverse amination reaction. GlnB stimulation required 2-oxoglutarate and ATP, or uridylylated GlnB-UMP, consistent with the activation of nitrogen assimilation under nitrogen limitation. Binding to AspA was lost in the GlnB(Y51F) mutant of the uridylylation site. AspA, therefore, represents a new type of GlnB target that binds GlnB (with ATP and 2-oxoglutarate), or GlnB-UMP (with or without effectors), and both situations stimulate AspA deamination activity. Thus, AspA represents the central enzyme for nitrogen assimilation from L-aspartate, and AspA is integrated into the nitrogen assimilation network by the regulator GlnB.

Role of aspartate ammonia-lyase in Pasteurella multocida.

BACKGROUND: Pasteurella multocida is responsible for a highly infectious and contagious disease in birds, leading to heavy economic losses in the chicken industry. However, the pathogenesis of this disease is poorly understood. We recently identified an aspartate ammonia-lyase (aspA) in P. multocida that was significantly upregulated under iron-restricted conditions, the protein of which could effectively protect chicken flocks against P. multocida. However, the functions of this gene remain unclear. In the present study, we constructed aspA mutant strain △aspA::kan and complementary strain C△aspA::kan to investigate the function of aspA in detail. RESULT: Deletion of the aspA gene in P. multocida resulted in a significant reduction in bacterial growth in LB (Luria-Bertani) and MH (Mueller-Hinton) media, which was rescued by supplementation with 20 mM fumarate. The mutant strain △aspA::kan showed significantly growth defects in anaerobic conditions and acid medium, compared with the wild-type strain. Moreover, growth of △aspA::kan was more seriously impaired than that of the wild-type strain under iron-restricted conditions, and this growth recovered after supplementation with iron ions. AspA transcription was negatively regulated by iron conditions, as demonstrated by quantitative reverse transcription-polymerase chain reaction. Although competitive index assay showed the wild-type strain outcompetes the aspA mutant strain and △aspA::kan was significantly more efficient at producing biofilms than the wild-type strain, there was no significant difference in virulence between the mutant and the wild-type strains. CONCLUSION: These results demonstrate that aspA is required for bacterial growth in complex medium, and under anaerobic, acid, and iron-limited conditions.

Surface charge-based rational design of aspartase modifies the optimal pH for efficient ß-aminobutyric acid production.

ß-Aminobutyric acid (BABA) can be widely used in the preparation of anti-tumor drugs, AIDS drugs, penicillin antibiotics, and plant initiators. However, the efficient, economical, and environmentally friendly production of BABA still faces challenges. Its important production enzyme, aspartase, catalyzes the substrate crotonic acid, and depends on harsh conditions, such as high temperatures and the presence of strong alkali. Here, we modified the surface charge of the enzyme to enable it to become more negatively charged (K19E, N87E, N125D, S133D, Q262E, and N451E; from -60 to -80), reducing its optimal pH from 9.0 to 8.0. The M20 enzyme showed improved specific activity (400.21 mU/mg at pH 8.0; 2.47-fold that of aspartase), and at pH 7.0, its activity increased 3-fold. The thermal stability of the enzyme was also improved. For the production of BABA, a 500 g/L whole-cell transformation was obtained with a 1.41-fold increase in yield, and the final production of BABA reached 556.1 g/L within 12 h. Our method provides a new strategy for modifying the characteristics of the enzyme through the modification of its surface charge, which also represents the first modification of the optimal pH for aspartase.

Enhanced production of ß-alanine through co-expressing two different subtypes of L-aspartate-α-decarboxylase.

ß-Alanine (ß-Ala) is an important intermediate with numerous applications in food and feed additives, pharmaceuticals, polymeric materials, and electroplating industries. Its biological production routes that employ L-aspartate-α-decarboxylase (ADC) as the key enzyme are attractive. In this study, we developed an efficient and environmentally safe method for ß-Ala production by co-expressing two different subtypes of ADC. A bacterial ADC from Bacillus subtilis (BSADC) and an insect ADC from Tribolium castaneum (TCADC) use pyruvoyl and pyridoxal-5'-phosphate (PLP) as cofactor, respectively. 3050 mM (271.5 g/L) ß-Ala was achieved from L-aspartic acid by using the whole-cell biocatalyst co-expressing BSADC and TCADC, corresponding to a conversion rate of 92.4%. Meanwhile, one-pot synthesis of ß-Ala from fumaric acid through using a tri-enzyme cascade route with two different subtypes of ADC and L-aspartase (AspA) from Escherichia coli was established. 2250 mM (200.3 g/L) ß-Ala was obtained from fumaric acid with a conversion rate of 90.0%. This work proposes a novel strategy that improves ß-Ala production in the decarboxylation pathway of L-aspartic acid.

A Novel Biosynthetic Pathway for the Production of Acrylic Acid through ß-Alanine Route in Escherichia coli.

Acrylic acid (AA) is an important industrial chemical used for several applications including superabsorbent polymers and acrylate esters. Here, we report the development of a new biosynthetic pathway for the production of AA from glucose in metabolically engineered Escherichia coli through the ß-alanine (BA) route. The AA production pathway was partitioned into two modules: an AA forming downstream pathway and a BA forming upstream pathway. We first validated the operation of the downstream pathway in vitro and in vivo, and then constructed the downstream pathway by introducing efficient enzymes (Act, Acl2, and YciA) screened out of various microbial sources and optimizing the expression levels. For the direct fermentative production of AA from glucose, the downstream pathway was introduced into the BA producing E. coli strain. The resulting strain could successfully produce AA from glucose in flask cultivation. AA production was further enhanced by expressing the upstream genes (panD and aspA) under the constitutive BBa_J23100 promoter. Replacement of the native promoter of the acs gene with the BBa_J23100 promoter in the genome increased AA production to 55.7 mg/L in flask. Fed-batch fermentation of the final engineered strain allowed production of 237 mg/L of AA in 57.5 h, representing the highest AA titer reported to date.

Protection of chickens against fowl cholera by supernatant proteins of Pasteurella multocida cultured in an iron-restricted medium.

Pasteurella multocida (P. multocida), a causative agent of fowl cholera, is an important pathogen in the poultry industry. In the present study, we found that the inactivated vaccine of P. multocida grown in an iron-restricted medium provided better protection than that grown in normal medium. Thus, we adopted a comparative proteomics approach, by using two-dimensional gel electrophoresis (2-DE), coupled with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF MS), to profile the supernatant proteins associated with P. multocida under both conditions. Eleven upregulated proteins were identified, including aspartate ammonia-lyase (AspA), diacylglycerol kinase (DgK), 30S ribosomal protein S6 (RpsF), and eight outer membrane proteins (OMPs). To further characterize the three novel supernatant proteins identified under iron-restricted conditions, the AspA, DgK and RpsF proteins were expressed and purified, and used as immunogens to vaccinate chickens. The results showed that AspA, DgK and RpsF proteins induced 80.0%, 66.7%, and 80.0% immunity, respectively. These data indicate that the three novel proteins identified in the supernatant of the culture media might play important roles in the survival of bacteria under iron-restricted conditions, and thus protect chickens against P. multocida. These findings also suggest that the proteins identified can be used as subunit vaccines.

Computational redesign of enzymes for regio- and enantioselective hydroamination.

Introduction of innovative biocatalytic processes offers great promise for applications in green chemistry. However, owing to limited catalytic performance, the enzymes harvested from nature's biodiversity often need to be improved for their desired functions by time-consuming iterative rounds of laboratory evolution. Here we describe the use of structure-based computational enzyme design to convert Bacillus sp. YM55-1 aspartase, an enzyme with a very narrow substrate scope, to a set of complementary hydroamination biocatalysts. The redesigned enzymes catalyze asymmetric addition of ammonia to substituted acrylates, affording enantiopure aliphatic, polar and aromatic ß-amino acids that are valuable building blocks for the synthesis of pharmaceuticals and bioactive compounds. Without a requirement for further optimization by laboratory evolution, the redesigned enzymes exhibit substrate tolerance up to a concentration of 300 g/L, conversion up to 99%, ß-regioselectivity >99% and product enantiomeric excess >99%. The results highlight the use of computational design to rapidly adapt an enzyme to industrially viable reactions.

Coenzyme M biosynthesis in bacteria involves phosphate elimination by a functionally distinct member of the aspartase/fumarase superfamily.

For nearly 30 years, coenzyme M (CoM) was assumed to be present solely in methanogenic archaea. In the late 1990s, CoM was reported to play a role in bacterial propene metabolism, but no biosynthetic pathway for CoM has yet been identified in bacteria. Here, using bioinformatics and proteomic approaches in the metabolically versatile bacterium Xanthobacter autotrophicus Py2, we identified four putative CoM biosynthetic enzymes encoded by the xcbB1, C1, D1, and E1 genes. Only XcbB1 was homologous to a known CoM biosynthetic enzyme (ComA), indicating that CoM biosynthesis in bacteria involves enzymes different from those in archaea. We verified that the ComA homolog produces phosphosulfolactate from phosphoenolpyruvate (PEP), demonstrating that bacterial CoM biosynthesis is initiated similarly as the phosphoenolpyruvate-dependent methanogenic archaeal pathway. The bioinformatics analysis revealed that XcbC1 and D1 are members of the aspartase/fumarase superfamily (AFS) and that XcbE1 is a pyridoxal 5'-phosphate-containing enzyme with homology to d-cysteine desulfhydrases. Known AFS members catalyze ß-elimination reactions of succinyl-containing substrates, yielding fumarate as the common unsaturated elimination product. Unexpectedly, we found that XcbC1 catalyzes ß-elimination on phosphosulfolactate, yielding inorganic phosphate and a novel metabolite, sulfoacrylic acid. Phosphate-releasing ß-elimination reactions are unprecedented among the AFS, indicating that XcbC1 is an unusual phosphatase. Direct demonstration of phosphosulfolactate synthase activity for XcbB1 and phosphate ß-elimination activity for XcbC1 strengthened their hypothetical assignment to a CoM biosynthetic pathway and suggested functions also for XcbD1 and E1. Our results represent a critical first step toward elucidating the CoM pathway in bacteria.

[Biocatalytic access to ß-alanine by a two-enzyme cascade synthesis].

Enzymatic synthesis is an important way to produce ß-alanine, but the biological method is expensive generally because of the high price of substrate. In this paper, a two-step enzymatic cascade system was developed, combining L-aspartase from Escherichia coli DH5α and L-aspartate α-decarboxylase from Corynebacterium glutamicum. This system catalyzes Fumarate and ammonia to ß-alanine. The optimal ratio of AspA and PanD was 1:80 (W/W), and the concentration of AspA was 10 µg/mL, at 37 ℃ and pH 7.0. When the concentration of Fumarate was 100 mmol/L, the reaction reached its equilibrium after 8 h, and the yield of ß-alanine was 90 mmol/L (7 g/L). The yield of ß-alanine can reach 126 mmol/L (9.8 g/L) when the concentration of Fumarate increased to 200 mmol/L. Extending reaction time, the conversion rate did not change obviously. Using this two-step enzymatic cascade system, ß-alanine from cheaper substrate Fumarate can be obtained.

Bioproduction of L-Aspartic Acid and Cinnamic Acid by L-Aspartate Ammonia Lyase from Pseudomonas aeruginosa PAO1.

Aspartase (L-aspartate ammonia lyase, EC 4.3.1.1) catalyses the reversible amination and deamination of L-aspartic acid to fumaric acid which can be used to produce important biochemical. In this study, we have explored the characteristics of aspartase from Pseudomonas aeruginosa PAO1 (PA-AspA). To overproduce PA-AspA, the 1425-bp gene was introduced in Escherichia coli BL21 and purified. A 51.0-kDa protein was observed as a homogenous purified protein on SDS-PAGE. The enzyme was optimally active at pH 8.0 and 35 °C. PA-AspA has retained 56% activity after 7 days of incubation at 35 °C, which displays the hyperthermostablility characteristics of the enzyme. PA-AspA is activated in the presence of metal ions and Mg2+ is found to be most effective. Among the substrates tested for specificity of PA-AspA, L-phenylalanine (38.35 ± 2.68) showed the highest specific activity followed by L-aspartic acid (31.21 ± 3.31) and fumarate (5.42 ± 2.94). K m values for L-phenylalanine, L-aspartic acid and fumarate were 1.71 mM, 0.346 µM and 2 M, respectively. The catalytic efficiency (k cat/K m) for L-aspartic acid (14.18 s-1 mM-1) was higher than that for L-phenylalanine (4.65 s-1 mM-1). For bioconversion, from an initial concentration of 1000 mM of fumarate and 30 mM of L-phenylalanine, PA-AspA was found to convert 395.31 µM L-aspartic acid and 3.47 mM cinnamic acid, respectively.

Taspase1: a 'misunderstood' protease with translational cancer relevance.

Proteolysis is not only a critical requirement for life, but the executing enzymes also play important roles in numerous pathological conditions, including cancer. Therefore, targeting proteases is clearly relevant for improving cancer patient care. However, to effectively control proteases, a profound knowledge of their mechanistic function as well as their regulation and downstream signalling in health and disease is required. The highly conserved protease Threonine Aspartase1 (Taspase1) is overexpressed in numerous liquid and solid malignancies and was characterized as a 'non-oncogene addiction' protease. Although Taspase1 was shown to cleave various regulatory proteins in humans as well as leukaemia provoking mixed lineage leukaemia fusions, our knowledge on its detailed functions and the underlying mechanisms contributing to cancer is still incomplete. Despite superficial similarity to type 2 asparaginases as well as Ntn proteases, such as the proteasome, Taspase1-related research so far gives us the picture of a unique protease exhibiting special features. Moreover, neither effective genetic nor chemical inhibitors for this enzyme are available so far, thus hampering not only to further dissect Taspase1's pathobiological functions but also precluding the assessment of its clinical impact. Based on recent insights, we here critically review the current knowledge of Taspase1's structure-function relationship and its mechanistic relevance for tumorigenesis obtained from in vitro and in vivo cancer models. We provide a comprehensive overview of tumour entities for which Taspase1 might be of predictive and therapeutic value, and present the respective experimental evidence. To stimulate progress in the field, a comprehensive overview of Taspase1 targeting approaches is presented, including coverage of Taspase1-related patents. We conclude by discussing future inhibition strategies and relevant challenges, which need to be resolved by the field.

Efficient aspartic acid production by a psychrophile-based simple biocatalyst.

We previously constructed a Psychrophile-based Simple bioCatalyst (PSCat) reaction system, in which psychrophilic metabolic enzymes are inactivated by heat treatment, and used it here to study the conversion of aspartic acid from fumaric acid mediated by the activity of aspartate ammonia-lyase (aspartase). In Escherichia coli, the biosynthesis of aspartic acid competes with that of L-malic acid produced from fumaric acid by fumarase. In this study, E. coli aspartase was expressed in psychrophilic Shewanella livingstonensis Ac10 heat treated at 50 °C for 15 min. The resultant PSCat could convert fumaric acid to aspartic acid without the formation of L-malic acid because of heat inactivation of psychrophilic fumarase activity. Furthermore, alginate-immobilized PSCat produced high yields of aspartic acid and could be re-used nine times. The results of our study suggest that PSCat can be applied in biotechnological production as a new approach to increase the yield of target compounds.