Drosophila melanogaster Uncoupling Protein-4A (UCP4A) Catalyzes a Unidirectional Transport of Aspartate.

Uncoupling proteins (UCPs) form a distinct subfamily of the mitochondrial carrier family (MCF) SLC25. Four UCPs, DmUCP4A-C and DmUCP5, have been identified in Drosophila melanogaster on the basis of their sequence homology with mammalian UCP4 and UCP5. In a Parkinson's disease model, DmUCP4A showed a protective role against mitochondrial dysfunction, by increasing mitochondrial membrane potential and ATP synthesis. To date, DmUCP4A is still an orphan of a biochemical function, although its possible involvement in mitochondrial uncoupling has been ruled out. Here, we show that DmUCP4A expressed in bacteria and reconstituted in phospholipid vesicles catalyzes a unidirectional transport of aspartate, which is saturable and inhibited by mercurials and other mitochondrial carrier inhibitors to various degrees. Swelling experiments carried out in yeast mitochondria have demonstrated that the unidirectional transport of aspartate catalyzed by DmUCP4 is not proton-coupled. The biochemical function of DmUCP4A has been further confirmed in a yeast cell model, in which growth has required an efflux of aspartate from mitochondria. Notably, DmUCP4A is the first UCP4 homolog from any species to be biochemically characterized. In Drosophila melanogaster, DmUCP4A could be involved in the transport of aspartate from mitochondria to the cytosol, in which it could be used for protein and nucleotide synthesis, as well as in the biosynthesis of ß-alanine and N-acetylaspartate, which play key roles in signal transmission in the central nervous system.

Systematic engineering of the rate-limiting step of b-alanine biosynthesis in Escherichia coli

BACKGROUND: Large amounts of b-alanine are required in fine chemical and pharmaceutical synthesis and other fields. Profitable and green methods are required for the industrial production of b-alanine. RESULTS: Replacing endogenous panD of Escherichia coli with heterologous CgpanD from Corynebacterium glutamicum enabled b-alanine synthesis of 0.67 g/L by strain B0016-082BB. Overexpressing CgpanD on both plasmids and chromosomes to enhance the rate-limiting step improved the b-alanine titer to 4.25 g/L in strain B0016-083BB/pPL451-panD with a slighter metabolic burden. Growth factors were introduced by addition of yeast extract, and 6.65 g/L of b-alanine was synthesized by strain B0016- 083BB/pPL451-panD in the M9-3Y medium. CONCLUSIONS: Enhancement of the rate-limiting steps in the b-alanine biosynthetic pathway, recruitment of the temperature-sensitive inducible pL promoter, and optimization of the fermentation process could efficiently increase b-alanine production in E. coli.

Advances in biotechnological production of ß-alanine.

ß-Alanine (3-aminopropionic acid) is the only naturally occurring ß-type amino acid. Although it is not incorporated into proteins, it has important physiological functions in the metabolism of animals, plants and microorganisms. Furthermore, it has attracted great interest due to its wide usage as a precursor of many significant industrial chemicals for medicine, feed, food, environmental applications and other fields. With the depletion of fossil fuels and concerns regarding environmental issues, biological production of ß-alanine has attracted more attention relative to chemical methods. In this review, we first summarize the pathways through which natural microorganisms synthesize ß-alanine. Then, the current research progress in the biological synthesis of ß-alanine is also elaborated. Finally, we discuss the main problems and challenges in optimizing the biological pathways, offering perspectives on promising new biological approaches.

Development of a dual-fluorescence reporter system for high-throughput screening of L-aspartate-α-decarboxylase.

ß-Alanine (3-aminopropionic acid) holds great potential in industrial application. It can be obtained through a chemical synthesis route, which is hazardous to the environment. It is well known that l-aspartate-α-decarboxylase (ADC) can convert l-aspartate to ß-alanine in bacteria. However, due to the low activity of ADC, industrial production of ß-alanine through the green biological route remains unclear. Thus, improving the activity of ADC is critical to reduce the cost of ß-alanine production. In this study, we established a dual-fluorescence high-throughput system for efficient ADC screening. By measuring the amount of ß-alanine and the expression level of ADC using two different fluorescence markers, we can rapidly quantify the relative activity of ADC variants. From a mutagenesis library containing 2000 ADC variants, we obtained a mutant with 33% increased activity. Further analysis revealed that mutations of K43R and P103Q in ADC significantly improved the yield of ß-alanine produced by the whole-cell biocatalysis. Compared with the previous single-fluorescence method, our system can not only quantify the amount of ß-alanine but also measure the expression level of ADC with different fluorescence, making it able to effectively screen out ADC variants with improved relative activity. The dual-fluorescence high-throughput system for rapid screening of ADC provides a good strategy for industrial production of ß-alanine via the biological conversion route in the future.

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.

Protein Engineering of a Pyridoxal-5'-Phosphate-Dependent l-Aspartate-α-Decarboxylase from Tribolium castaneum for ß-Alanine Production.

In the present study, a pyridoxal-5'-phosphate (PLP)-dependent L-aspartate-α-decarboxylase from Tribolium castaneum (TcPanD) was selected for protein engineering to efficiently produce ß-alanine. A mutant TcPanD-R98H/K305S with a 2.45-fold higher activity than the wide type was selected through error-prone PCR, site-saturation mutagenesis, and 96-well plate screening technologies. The characterization of purified enzyme TcPanD-R98H/K305S showed that the optimal cofactor PLP concentration, temperature, and pH were 0.04% (m/v), 50 °C, and 7.0, respectively. The 1mM of Na+, Ni2+, Co2+, K+, and Ca2+ stimulated the activity of TcPanD-R98H/K305S, while only 5 mM of Ni2+ and Na+ could increase its activity. The kinetic analysis indicated that TcPanD-R98H/K305S had a higher substrate affinity and enzymatic reaction rate than the wild enzyme. A total of 267 g/L substrate l-aspartic acid was consumed and 170.5 g/L of ß-alanine with a molar conversion of 95.5% was obtained under the optimal condition and 5-L reactor fermentation.

A novel way to synthesize pantothenate in bacteria involves ß-alanine synthase present in uracil degradation pathway.

Pantothenate is an indispensable vitamin precursor of the synthesis of coenzyme A (CoA), a key metabolite required in over 100 metabolic reactions. ß-Alanine (ß-ala) is an indispensable component of pantothenate. Due to the metabolic relevance of this pathway, we assumed that orthologous genes for ß-alanine synthesis would be present in the genomes of bacteria, archaea, and eukaryotes. However, comparative genomic studies revealed that orthologous gene replacement and loss of synteny occur at high frequency in panD genes. We have previously reported the atypical plasmid-encoded location of the pantothenate pathway genes panC and panB (two copies) in R. etli CFN42. This study also revealed the unexpected absence of a panD gene encoding the aspartate decarboxylase enzyme (ADC), required for the synthesis of ß-ala. The aim of this study was to identify the source of ß-alanine in Rhizobium etli CFN42. In this study, we present a bioinformatic analysis and an experimental validation demonstrating that the source of ß-ala in this R. etli comes from ß-alanine synthase, the last enzyme of the uracil degradation pathway.

Pathway construction and metabolic engineering for fermentative production of ß-alanine in Escherichia coli.

ß-Alanine is a naturally occurring ß-amino acid that has been widely applied in the life and health field. Although microbial fermentation is a promising method for industrial production of ß-alanine, an efficient microbial cell factory is still lacking. In this study, a new metabolically engineered Escherichia coli strain for ß-alanine production was developed through a series of introduction, deletion, and overexpression of genes involved in its biosynthesis pathway. First, the L-aspartate a-decarboxylase gene, BtADC, from Bacillus tequilensis, with higher catalytic activity to produce ß-alanine from aspartate, was constitutively expressed in E. coli, leading to an increased production of ß-alanine up to 2.76 g/L. Second, three native aspartate kinase genes, akI, akII, and akIII, were knocked out to promote the production of ß-alanine to a higher concentration of 4.43 g/L by preventing from bypass loss of aspartate. To increase the amount of aspartate, the native AspC gene was replaced with PaeAspDH, a L-aspartate dehydrogenase gene from Pseudomonas aeruginosa, accompanied with the overexpression of the native AspA gene, to further improve the production level of ß-alanine to 9.27 g/L. Last, increased biosynthesis of oxaloacetic acid (OAA) was achieved by a combination of overexpression of the native PPC, introduction of CgPC, a pyruvate decarboxylase from Corynebacterium glutamicum, and deletion of ldhA, pflB, pta, and adhE in E. coli, to further enhance the production of ß-alanine. Finally, the engineered E. coli strain produced 43.12 g/L ß-alanine in fed-batch fermentation. Our study will lay a solid foundation for the promising application of ß-alanine in the life and health field. KEY POINTS: • Overexpression of BtADC resulted in substantial accumulation of ß-alanine. • The native AspC was replaced with PaeAspDH to catalyze the transamination of OAA. • Deletion of gluDH prevented from losing carbon flux in TCA recycle. • A 43.12-g/L ß-alanine production in fed-batch fermentation was achieved. Graphical abstract.

Characterization of cysteine sulfinic acid decarboxylase from Tribolium castaneum and its application in the production of ß-alanine.

ß-alanine is a precursor for the production of pharmaceuticals and food additives that is produced by chemical methods in industry. As concerns about the environment and energy are increasing, biocatalysis using L-aspartate-α-decarboxylase (ADC) to convert L-aspartate to ß-alanine has great potential. Many studies have focused on the catalytic activity of ADC, but these researches were limited to the prokaryotic enzymes. In this study, the gene encoding cysteine sulfinic acid decarboxylase from Tribolium castaneum (TcCSADC) was synthesized and overexpressed in Escherichia coli, and the enzyme was purified and characterized for the first time. It could use L-aspartate as its substrate, and the specific activity was 4.83 µmol/min/mg, which was much higher than that of ADCs from prokaryotes. A homology modeling assay indicated that TcCSADC had a dimer structure. Based on the evolutionary information from thermophilic bacteria, twenty-three variants were constructed to attempt to improve its abilities that transform L-aspartate to ß-alanine. One mutant, G369A, was screened that had improved thermal stability. An analysis of the suitability of the catalytic process showed that the up to 162 g/L ß-alanine could be produced using cells expressing the recombinant G369A variant, which is the highest yield to date. The CSADC from T. castaneum has important value for studies of the mechanism of ADCs and CSADCs from eukaryotes, and the engineered strain containing the G369A variant has great potential for the industrial production of ß-alanine.

Extracellular Expression of L-Aspartate-α-Decarboxylase from Bacillus tequilensis and Its Application in the Biosynthesis of ß-Alanine.

L-aspartate-α-decarboxylase was extracellularly expressed to enhance its production for ß-alanine biosynthesis. L-aspartate-α-decarboxylase and cutinase were coexpressed in Escherichia coli; more than 40% of the L-aspartate-α-decarboxylase was secreted into the medium. Selection of best conditions among tested variables enhanced L-aspartate-α-decarboxylase production by the recombinant strain. The total L-aspartate-α-decarboxylase activity reached 20.3 U/mL. Analysis of the enzymatic properties showed that the optimum temperature and pH for L-aspartate-α-decarboxylase were 60 °C and 7.5, respectively. Enzyme activity was stable at pH 4.0-8.5 and displayed sufficient thermal stability at temperatures < 50 °C. In addition, enzymatic synthesis of ß-alanine was performed using extracellularly expressed L-aspartate-α-decarboxylase, and a mole conversion rate of > 99% was reached with a substrate concentration of 1.5 M. Extracellular expression of L-aspartate-α-decarboxylase resulted in increased enzyme production, indicating its possible application in the enzymatic synthesis of ß-alanine.

Substrate inactivation of bacterial L-aspartate α-decarboxylase from Corynebacterium jeikeium K411 and improvement of molecular stability by saturation mutagenesis.

Bacterial L-aspartate α-decarboxylase (PanD) is a potential biocatalyst for the green production of ß-alanine, an important block chemical for manufacturing nitrogen-containing chemicals in bio-refinery field. It was reported that the poor catalytic stability caused by substrate inactivation limited the large-scale application. Here, we investigated the characters of inactivation by L-aspartate of PanD from Corynebacterium jeikeium (PDCjei), and found that L-aspartate induced a time-, and concentration-dependent inactivation of PDCjei with the values of KI and kinact being 288.4 mM and 0.235/min, respectively. To improve the catalytic stability of PDCjei, conserved amino acid residues essential to catalytic stability were analyzed by comparing the discrepancy in the observed inactivation rate of various sources. By an efficient colorimetric high-throughput screening method, four mutants with 3.18-24.69% higher activity were obtained from mutant libraries. Among them, the best mutation (R3K) also performed 66.38% higher catalytic stability than the wild type, showing great potential for industrial bio-production of ß-alanine.

Molecular engineering of L-aspartate-α-decarboxylase for improved activity and catalytic stability.

ß-Alanine is an important precursor for the production of food additives, pharmaceuticals, and nitrogen-containing chemicals. Compared with the conventional chemical routes for ß-alanine production, the biocatalytic routes using L-aspartate-α-decarboxylase (ADC) are more attractive when energy and environment are concerned. However, ADC's poorly understood properties and its inherent mechanism-based inactivation significantly limited the application of this enzyme. In this study, three genes encoding the ADC enzymes from Escherichia coli, Corynebacterium glutamicum, and Bacillus subtilis were overexpressed in E. coli. Their properties including specific activity, thermostability, and mechanism-based inactivation were characterized. The ADC enzyme from B. subtilis, which had higher specific activity and thermostability than the others, was selected for further study. In order to improve its activity and relieve its mechanism-based inactivation by molecular engineering so as to improve its catalytic stability, a high-throughput fluorometric assay of ß-alanine was developed. From a library of 4000 mutated enzymes, two variants with 18-22% higher specific activity and 29-64% higher catalytic stability were obtained. The best variant showed 50% higher ß-alanine production than the wild type after 8 h of conversion of L-aspartate, showing great potential for industrial biocatalytic production of ß-alanine.

Systematic unravelling of the biosynthesis of poly (L-diaminopropionic acid) in Streptomyces albulus PD-1.

Poly(L-diaminopropionic acid) (PDAP) is one of the four homopoly(amino acid)s that have been discovered in nature. However, the molecular mechanism of PDAP biosynthesis has yet to be described. In this work, the general layout of the PDAP biosynthetic pathway is characterised in Streptomyces albulus PD-1 by genome mining, gene disruption, heterologous expression and in vitro feeding experiments. As a result, L-diaminopropionic acid (L-DAP), which is the monomer of PDAP, is shown to be jointly synthesised by two protein homologues of cysteine synthetase and ornithine cyclodeaminase. Then, L-DAP is assembled into PDAP by a novel nonribosomal peptide synthetase (NRPS) with classical adenylation and peptidyl carrier protein domains. However, instead of the traditional condensation or thioesterase domain of NRPSs, this NRPS has seven transmembrane domains surrounding three tandem soluble domains at the C-terminus. As far as we know, this novel single-module NRPS structure has only been reported in poly(ε-L-lysine) synthetase. The similar NRPS structure of PDAP synthetase and poly(ε-L-lysine) synthetase may be a common characteristic of homopoly(amino acid)s synthetases. In this case, we may discover and/or design more homopoly(amino acid)s by mining this kind of novel NRPS structure in the future.

NAD(+)-aminoaldehyde dehydrogenase candidates for 4-aminobutyrate (GABA) and ß-alanine production during terminal oxidation of polyamines in apple fruit.

The last step of polyamine catabolism involves the oxidation of 3-aminopropanal or 4-aminobutanal via aminoaldehyde dehydrogenase. In this study, two apple (Malus x domestica) AMADH genes were selected (MdAMADH1 and MdAMADH2) as candidates for encoding 4-aminobutanal dehydrogenase activity. Maximal activity and catalytic efficiency were obtained with NAD(+) and 3-aminopropanal, followed by 4-aminobutanal, at pH 9.8. NAD(+) reduction was accompanied by the production of GABA and ß-alanine, respectively, when 4-aminobutanal and 3-aminopropanal were utilized as substrates. MdAMADH2 was peroxisomal and MdAMADH1 cytosolic. These findings shed light on the potential role of apple AMADHs in 4-aminobutyrate and ß-alanine production.

Metabolic engineering of Escherichia coli for the production of 3-aminopropionic acid.

A novel metabolic pathway was designed for the production of 3-aminopropionic acid (3-AP), an important platform chemical for manufacturing acrylamide and acrylonitrile. Using a fumaric acid producing Escherichia coli strain as a host, the Corynebacterium glutamicum panD gene (encoding L-aspartate-α-decarboxylase) was overexpressed and the native promoter of the aspA gene was replaced with the strong trc promoter, which allowed aspartic acid production through the aspartase-catalyzed reaction. Additional overexpression of aspA and ppc genes, and supplementation of ammonium sulfate in the medium allowed production of 3.49 g/L 3-AP. The 3-AP titer was further increased to 3.94 g/L by optimizing the expression level of PPC using synthetic promoters and RBS sequences. Finally, native promoter of the acs gene was replaced with strong trc promoter to reduce acetic acid accumulation. Fed-batch culture of the final strain allowed production of 32.3 g/L 3-AP in 39 h.

A substrate ambiguous enzyme facilitates genome reduction in an intracellular symbiont.

BACKGROUND: Genome evolution in intracellular microbial symbionts is characterized by gene loss, generating some of the smallest and most gene-poor genomes known. As a result of gene loss these genomes commonly contain metabolic pathways that are fragmented relative to their free-living relatives. The evolutionary retention of fragmented metabolic pathways in the gene-poor genomes of endosymbionts suggests that they are functional. However, it is not always clear how they maintain functionality. To date, the fragmented metabolic pathways of endosymbionts have been shown to maintain functionality through complementation by host genes, complementation by genes of another endosymbiont and complementation by genes in host genomes that have been horizontally acquired from a microbial source that is not the endosymbiont. Here, we demonstrate a fourth mechanism. RESULTS: We investigate the evolutionary retention of a fragmented pathway for the essential nutrient pantothenate (vitamin B5) in the pea aphid, Acyrthosiphon pisum endosymbiosis with Buchnera aphidicola. Using quantitative analysis of gene expression we present evidence for complementation of the Buchnera pantothenate biosynthesis pathway by host genes. Further, using complementation assays in an Escherichia coli mutant we demonstrate functional replacement of a pantothenate biosynthesis enzyme, 2-dehydropantoate 2-reductase (E.C. 1.1.1.169), by an endosymbiont gene, ilvC, encoding a substrate ambiguous enzyme. CONCLUSIONS: Earlier studies have speculated that missing enzyme steps in fragmented endosymbiont metabolic pathways are completed by adaptable endosymbiont enzymes from other pathways. Here, we experimentally demonstrate completion of a fragmented endosymbiont vitamin biosynthesis pathway by recruitment of a substrate ambiguous enzyme from another pathway. In addition, this work extends host/symbiont metabolic collaboration in the aphid/Buchnera symbiosis from amino acid metabolism to include vitamin biosynthesis.

Comportamentos e práticas sexuais de homens que fazem sexo com homens / Sexual behaviors and practices of men who have sex with men / Conductas y prácticas sexuales de los hombres que tienen sexo con hombres

Objetivou-se identificar comportamentos e práticas sexuais de homens que fazem sexo com homens no contexto da vulnerabilidade ao HIV/AIDS. Estudo transversal, exploratório descritivo. Foi realizado em um local de sociabilidade gay de Fortaleza, no Estado do Ceará, entre novembro de 2010 e março de 2011, por meio de entrevista com 189 homens que fazem sexo com homens. Encontrou-se uma amostra composta, majoritariamente, por jovens, solteiros e com alto nível educacional. A história sexual demonstrou o início precoce da vida sexual, com prevalência elevada de relação sexual com parceira do sexo oposto. Houve alta frequência de testagem para o HIV. As práticas sexuais revelaram prevalência superior da realização de sexo oral e anal, bem como altos níveis de proteção no sexo anal, apesar de baixa no sexo oral. Constatou-se uma maior incorporação das práticas de prevenção em relação ao panorama nacional do início da epidemia.

The objective was to identify behaviors and sexual practices of men who have sexual relations with other men in the context of vulnerability to HIV/AIDS. This was a cross-sectional, exploratory and descriptive study. It was carried out in a gay sociability place in Fortaleza, Ceará, Brazil, between November 2010 and March 2011, through interviews with 189 men who have sex with men. The ethical aspects were respected. We found a sample consisting mostly by young, single, and highly educated men. The sexual history demonstrated the early onset of sexual activity, with a high prevalence of sexual intercourse with a partner of the opposite sex. There was also a high prevalence of HIV testing. Sexual practices revealed high prevalence of performing oral and anal sex, as well as high levels of protection in anal sex, despite the low protection in oral sex. A greater incorporation of prevention practices was found compared to the national scene in the beginning of the disease outbreak.

El objetivo fue identificar los comportamientos y las prácticas sexuales de los hombres que tienen sexo con hombres en el contexto de la vulnerabilidad al VIH/SIDA. Fue un estudio transversal, descriptivo y exploratorio. Se celebró en una sociabilidad local gay de Fortaleza, Ceará, Brasil, entre noviembre de 2010 y marzo de 2011, a través de entrevistas con 189 hombres que tienen sexo con hombres. Se encontró una muestra compuesta en su mayoría por jóvenes, solteros y con alto nivel de educación. La historia sexual demostró el inicio temprano de la actividad sexual, la alta prevalencia de relaciones sexuales con una pareja del sexo opuesto. Hubo alta prevalencia de la prueba del VIH. Las prácticas sexuales revelaron una alta prevalencia de realizar sexo oral y anal, así como altos niveles de protección en el sexo anal, a pesar de la baja protección en el sexo oral. Se encontró una mayor incorporación de las prácticas de prevención en relación con la escena nacional en el inicio de la epidemia.

ß-alanine biosynthesis in Methanocaldococcus jannaschii.

One efficient approach to assigning function to unannotated genes is to establish the enzymes that are missing in known biosynthetic pathways. One group of such pathways is those involved in coenzyme biosynthesis. In the case of the methanogenic archaeon Methanocaldococcus jannaschii as well as most methanogens, none of the expected enzymes for the biosynthesis of the ß-alanine and pantoic acid moieties required for coenzyme A are annotated. To identify the gene(s) for ß-alanine biosynthesis, we have established the pathway for the formation of ß-alanine in this organism after experimentally eliminating other known and proposed pathways to ß-alanine from malonate semialdehyde, l-alanine, spermine, dihydrouracil, and acryloyl-coenzyme A (CoA). Our data showed that the decarboxylation of aspartate was the only source of ß-alanine in cell extracts of M. jannaschii. Unlike other prokaryotes where the enzyme producing ß-alanine from l-aspartate is a pyruvoyl-containing l-aspartate decarboxylase (PanD), the enzyme in M. jannaschii is a pyridoxal phosphate (PLP)-dependent l-aspartate decarboxylase encoded by MJ0050, the same enzyme that was found to decarboxylate tyrosine for methanofuran biosynthesis. A Km of ∼0.80 mM for l-aspartate with a specific activity of 0.09 µmol min(-1) mg(-1) at 70°C for the decarboxylation of l-aspartate was measured for the recombinant enzyme. The MJ0050 gene was also demonstrated to complement the Escherichia coli panD deletion mutant cells, in which panD encoding aspartate decarboxylase in E. coli had been knocked out, thus confirming the function of this gene in vivo.

Synthesis of ß-alanine from L-aspartate using L-aspartate-α-decarboxylase from Corynebacterium glutamicum.

ß-Alanine is mainly produced by chemical methods in current industrial processes. Here, panD from Corynebacterium glutamicum encoding L-aspartate-α-decarboxylase (ADC) was cloned and expressed in Escherichia coli BL21(DE3). ADC C.g catalyzes the α-decarboxylation of L-aspartate to ß-alanine. The purified ADC C.g was optimal at 55 °C and pH 6 with excellent stability at 16-37 °C and pH 4-7. A pH-stat directed, fed-batch feeding strategy was developed for enzymatic synthesis of ß-alanine to keep the pH value within 6-7.2 and thus attenuate substrate inhibition. A maximum conversion of 97.2 % was obtained with an initial 5 g L-aspartate/l and another three feedings of 0.5 % (w/v) L-aspartate at 8 h intervals. The final ß-alanine concentration was 12.85 g/l after 36 h. This is the first study concerning the enzymatic production of ß-alanine by using ADC.

Synthesis of L-2,3-diaminopropionic acid, a siderophore and antibiotic precursor.

L-2,3-diaminopropionic acid (L-Dap) is an amino acid that is a precursor of antibiotics and staphyloferrin B a siderophore produced by Staphylococcus aureus. SbnA and SbnB are encoded by the staphyloferrin B biosynthetic gene cluster and are implicated in L-Dap biosynthesis. We demonstrate here that SbnA uses PLP and substrates O-phospho-L-serine and L-glutamate to produce a metabolite N-(1-amino-1-carboxyl-2-ethyl)-glutamic acid (ACEGA). SbnB is shown to use NAD(+) to oxidatively hydrolyze ACEGA to yield α-ketoglutarate and L-Dap. Also, we describe crystal structures of SbnB in complex with NADH and ACEGA as well as with NAD(+) and α-ketoglutarate to reveal the residues required for substrate binding, oxidation, and hydrolysis. SbnA and SbnB contribute to the iron sparing response of S. aureus that enables staphyloferrin B biosynthesis in the absence of an active tricarboxylic acid cycle.