Engineering the synthetic ß-alanine pathway in Komagataella phaffii for conversion of methanol into 3-hydroxypropionic acid.

BACKGROUND: Methanol is increasingly gaining attraction as renewable carbon source to produce specialty and commodity chemicals, as it can be generated from renewable sources such as carbon dioxide (CO2). In this context, native methylotrophs such as the yeast Komagataella phaffii (syn Pichia pastoris) are potentially attractive cell factories to produce a wide range of products from this highly reduced substrate. However, studies addressing the potential of this yeast to produce bulk chemicals from methanol are still scarce. 3-Hydroxypropionic acid (3-HP) is a platform chemical which can be converted into acrylic acid and other commodity chemicals and biopolymers. 3-HP can be naturally produced by several bacteria through different metabolic pathways. RESULTS: In this study, production of 3-HP via the synthetic ß-alanine pathway has been established in K. phaffii for the first time by expressing three heterologous genes, namely panD from Tribolium castaneum, yhxA from Bacillus cereus, and ydfG from Escherichia coli K-12. The expression of these key enzymes allowed a production of 1.0 g l-1 of 3-HP in small-scale cultivations using methanol as substrate. The addition of a second copy of the panD gene and selection of a weak promoter to drive expression of the ydfG gene in the PpCß21 strain resulted in an additional increase in the final 3-HP titer (1.2 g l-1). The 3-HP-producing strains were further tested in fed-batch cultures. The best strain (PpCß21) achieved a final 3-HP concentration of 21.4 g l-1 after 39 h of methanol feeding, a product yield of 0.15 g g-1, and a volumetric productivity of 0.48 g l-1 h-1. Further engineering of this strain aiming at increasing NADPH availability led to a 16% increase in the methanol consumption rate and 10% higher specific productivity compared to the reference strain PpCß21. CONCLUSIONS: Our results show the potential of K. phaffii as platform cell factory to produce organic acids such as 3-HP from renewable one-carbon feedstocks, achieving the highest volumetric productivities reported so far for a 3-HP production process through the ß-alanine pathway.

Systems metabolic engineering of Corynebacterium glutamicum for the efficient production of ß-alanine.

ß-Alanine is an important ß-amino acid with a growing demand in a wide range of applications in chemical and food industries. However, current industrial production of ß-alanine relies on chemical synthesis, which usually involves harmful raw materials and harsh production conditions. Thus, there has been increasing demand for more sustainable, yet efficient production process of ß-alanine. In this study, we constructed Corynebacterium glutamicum strains for the highly efficient production of ß-alanine through systems metabolic engineering. First, aspartate 1-decarboxylases (ADCs) from seven different bacteria were screened, and the Bacillus subtilis ADC showing the most efficient ß-alanine biosynthesis was used to construct a ß-alanine-producing base strain. Next, genome-scale metabolic simulations were conducted to optimize multiple metabolic pathways in the base strain, including phosphotransferase system (PTS)-independent glucose uptake system and the biosynthesis of key precursors, including oxaloacetate and L-aspartate. TCA cycle was further engineered for the streamlined supply of key precursors. Finally, a putative ß-alanine exporter was newly identified, and its overexpression further improved the ß-alanine production. Fed-batch fermentation of the final engineered strain BAL10 (pBA2_tr18) produced 166.6 g/L of ß-alanine with the yield and productivity of 0.28 g/g glucose and 1.74 g/L/h, respectively. To our knowledge, this production performance corresponds to the highest titer, yield and productivity reported to date for the microbial fermentation.

Metabolic engineering of E. coli for ß-alanine production using a multi-biosensor enabled approach.

ß-alanine is an important biomolecule used in nutraceuticals, pharmaceuticals, and chemical synthesis. The relatively eco-friendly bioproduction of ß-alanine has recently attracted more interest than petroleum-based chemical synthesis. In this work, we developed two types of in vivo high-throughput screening platforms, wherein one was utilized to identify a novel target ribonuclease E (encoded by rne) as well as a redox-cofactor balancing module that can enhance de novo ß-alanine biosynthesis from glucose, and the other was employed for screening fermentation conditions. When combining these approaches with rational upstream and downstream module engineering, an engineered E. coli producer was developed that exhibited 3.4- and 6.6-fold improvement in ß-alanine yield (0.85 mol ß-alanine/mole glucose) and specific ß-alanine production (0.74 g/L/OD600), respectively, compared to the parental strain in a minimal medium. Across all of the strains constructed, the best yielding strain exhibited 1.08 mol ß-alanine/mole glucose (equivalent to 81.2% of theoretic yield). The final engineered strain produced 6.98 g/L ß-alanine in a batch-mode bioreactor and 34.8 g/L through a whole-cell catalysis. This approach demonstrates the utility of biosensor-enabled high-throughput screening for the production of ß-alanine.

Metabolic Blockade-Based Genome Mining Reveals Lipochain-Linked Dihydro-ß-alanine Synthetases Involved in Autucedine Biosynthesis.

Continuously mining the Streptomyces olivaceus SCSIO T05 genome leads to the identification of new lipopeptides (autucedines A-C), constituting members of the 10th skeleton isolated from this strain. The corresponding biosynthetic gene cluster (aut) was verified by heterogeneous expression, and another two analogues (autucedines D and E) were isolated from the heterogeneous expression strain. Gene inactivation experiments revealed that construction of the unique "lipochain-linked dihydro-ß-alanine" unit takes place prior to the NRPS assembly line.

Determination of three sites involved in the divergence of L-aspartate-α-decarboxylase self-cleavage in bacteria.

L-aspartate-α-decarboxylase (PanD) is an essential enzyme catalysing the decarboxylation of L-aspartate to ß-alanine in organisms. To perform the catalytic functions, PanD pro-proteins need to be self-cleaved to form two subunits: active α-subunit and ß-subunit. However, the processes of self-cleavage have diverged in different organisms for unknown reasons. To reveal the possible divergence mechanisms, the molecular evolution, selection pressures and site-directed mutagenesis of the panD gene family were explored in this study. The evolution analysis revealed that the panD genes in bacteria have diverged into three clades: Class I, Class II and Class III. Furthermore, 9 positive selection sites (A13, T14, V23, L32, V44, N49, L55, L78, and V85 in BsupanD) were detected. As shown by SDS-PAGE assay and catalytic activity determination in the mutants of BsupanD and EcoPanD, three of those sites (T14, V44 and V85) affect the PanD activities and are involved in the divergence of panD self-cleavage, while the other 6 sites only influenced the enzymatic activities of PanD. Furthermore, the structure analysis indicated that the structural mechanisms of the 9 sites affecting the catalysis were various. In all, three sites contributing to the divergence of PanD self-cleavage were revealed, and the results also provide foundation for the industrial application of PanD in ß-alanine synthesis.

Engineering precursor and co-factor supply to enhance D-pantothenic acid production in Bacillus megaterium.

High-yielding chemical and chemo-enzymatic methods of D-pantothenic acid (DPA) synthesis are limited by using poisonous chemicals and DL-pantolactone racemic mixture formation. Alternatively, the safe microbial fermentative route of DPA production was found promising but suffered from low productivity and precursor supplementation. In this study, Bacillus megaterium was metabolically engineered to produce DPA without precursor supplementation. In order to provide a higher supply of precursor D-pantoic acid, key genes involved in its synthesis are overexpressed, resulting strain was produced 0.53 ± 0.08 g/L DPA was attained in shake flasks. Cofactor CH2-THF was found to be vital for DPA biosynthesis and was regenerated through the serine-glycine degradation pathway. Enhanced supply of another precursor, ß-alanine was achieved by codon optimization and dosing of the limiting L-asparate-1-decarboxylase (ADC). Co-expression of Pantoate-ß-alanine ligase, ADC, phosphoenolpyruvate carboxylase, aspartate aminotransferase and aspartate ammonia-lyase enhanced DPA concentration to 2.56 ± 0.05 g/L at shake flasks level. Fed-batch fermentation in a bioreactor with and without the supplementation of ß-alanine increased DPA concentration to 19.52 ± 0.26 and 4.78 ± 0.53 g/L, respectively. This present study successfully demonstrated a rational approach combining precursor supply engineering with cofactor regeneration for the enhancement of DPA titer in recombinant B. megaterium.

ß-Ureidopropionase deficiency due to novel and rare UPB1 mutations affecting pre-mRNA splicing and protein structural integrity and catalytic activity.

ß-Ureidopropionase is the third enzyme of the pyrimidine degradation pathway and catalyses the conversion of N-carbamyl-ß-alanine and N-carbamyl-ß-aminoisobutyric acid to ß-alanine and ß-aminoisobutyric acid, ammonia and CO2. To date, only a limited number of genetically confirmed patients with a complete ß-ureidopropionase deficiency have been reported. Here, we report on the clinical, biochemical and molecular findings of 10 newly identified ß-ureidopropionase deficient individuals. Patients presented mainly with neurological abnormalities and markedly elevated levels of N-carbamyl-ß-alanine and N-carbamyl-ß-aminoisobutyric acid in urine. Analysis of UPB1, encoding ß-ureidopropionase, showed 5 novel missense variants and two novel splice-site variants. Functional expression of the UPB1 variants in mammalian cells showed that recombinant ß-ureidopropionase carrying the p.Ala120Ser, p.Thr129Met, p.Ser300Leu and p.Asn345Ile variant yielded no or significantly decreased ß-ureidopropionase activity. Analysis of the crystal structure of human ß-ureidopropionase indicated that the point mutations affect substrate binding or prevent the proper subunit association to larger oligomers and thus a fully functional ß-ureidopropionase. A minigene approach showed that the intronic variants c.[364 + 6 T > G] and c.[916 + 1_916 + 2dup] led to skipping of exon 3 and 8, respectively, in the process of UPB1 pre-mRNA splicing. The c.[899C > T] (p.Ser300Leu) variant was identified in two unrelated Swedish ß-ureidopropionase patients, indicating that ß-ureidopropionase deficiency may be more common than anticipated.

The dihydropyrimidine dehydrogenase gene contributes to heritable differences in sleep in mice.

Many aspects of sleep are heritable, but only a few sleep-regulating genes have been reported. Here, we leverage mouse models to identify and confirm a previously unreported gene affecting sleep duration-dihydropyrimidine dehydrogenase (Dpyd). Using activity patterns to quantify sleep in 325 Diversity Outbred (DO) mice-a population with high genetic and phenotypic heterogeneity-a linkage peak for total sleep in the active lights off period was identified on chromosome 3 (LOD score = 7.14). Mice with the PWK/PhJ ancestral haplotype at this location demonstrated markedly reduced sleep. Among the genes within the linkage region, available RNA sequencing data in an independent sample of DO mice supported a highly significant expression quantitative trait locus for Dpyd, wherein reduced expression was associated with the PWK/PhJ allele. Validation studies were performed using activity monitoring and EEG/EMG recording in Collaborative Cross mouse strains with and without the PWK/PhJ haplotype at this location, as well as EEG and EMG recording of sleep and wake in Dpyd knockout mice and wild-type littermate controls. Mice lacking Dpyd had 78.4 min less sleep during the lights-off period than wild-type mice (p = 0.007; Cohen's d = -0.94). There was no difference in other measured behaviors in knockout mice, including assays evaluating cognitive-, social-, and affective-disorder-related behaviors. Dpyd encodes the rate-limiting enzyme in the metabolic pathway that catabolizes uracil and thymidine to ß-alanine, an inhibitory neurotransmitter. Thus, data support ß-alanine as a neurotransmitter that promotes sleep in mice.

The role of alanine glyoxylate transaminase-2 (agxt2) in ß-alanine and carnosine metabolism of healthy mice and humans.

PURPOSE: Chronic ß-alanine supplementation leads to increased levels of muscle histidine-containing dipeptides. However, the majority of ingested ß-alanine is, most likely, degraded by two transaminases: GABA-T and AGXT2. In contrast to GABA-T, the in vivo role of AGXT2 with respect to ß-alanine metabolism is unknown. The purpose of the present work is to investigate if AGXT2 is functionally involved in ß-alanine homeostasis. METHODS: Muscle histidine-containing dipeptides levels were determined in AGXT2 overexpressing or knock-out mice and in human subjects with different rs37369 genotypes which is known to affect AGXT2 activity. Further, plasma ß-alanine kinetic was measured and urine was obtained from subjects with different rs37369 genotypes following ingestion of 1400 mg ß-alanine. RESULT: Overexpression of AGXT2 decreased circulating and muscle histidine-containing dipeptides (> 70% decrease; p < 0.05), while AGXT2 KO did not result in altered histidine-containing dipeptides levels. In both models, ß-alanine remained unaffected in the circulation and in muscle (p > 0.05). In humans, the results support the evidence that decreased AGXT2 activity is not associated with altered histidine-containing dipeptides levels (p > 0.05). Additionally, following an acute dose of ß-alanine, no differences in pharmacokinetic response were measured between subjects with different rs37369 genotypes (p > 0.05). Interestingly, urinary ß-alanine excretion was 103% higher in subjects associated with lower AGXT2 activity, compared to subjects associated with normal AGXT2 activity (p < 0.05). CONCLUSION: The data suggest that in vivo, ß-alanine is a substrate of AGXT2; however, its importance in the metabolism of ß-alanine and histidine-containing dipeptides seems small.

Metabolic engineering of Escherichia coli for production of L-aspartate and its derivative ß-alanine with high stoichiometric yield.

L-aspartate is an important 4-carbon platform compound that can be used as the precursor of numerous chemical products. The bioproduction of L-aspartate directly from biomass resources is expected to provide a more cost-competitive technique route. Yet little metabolic engineering work on this matter has been carried out. In this study, we designed a shortcut pathway of L-aspartate biosynthesis in Escherichia coli, with a maximized stoichiometric yield of 2 mol/mol glucose. L-aspartate aminotransferase (AspC) was overexpressed for producing L-aspartate and coexpressed with L-aspartate-a-decarboxylase (PanD) for producing L-aspartate's derivative ß-alanine. L-aspartate could only be detected after directing carbon flux towards oxaloacetate and blocking the "futile cycle" with TCA cycle. A cofactor self-sufficient system successfully improved the efficiency of AspC-catalyzing L-aspartate biosynthesis reaction, and the glucose uptake remolding capably decreased byproducts from pyruvate. More targets were modified for relieving the bottleneck during fed-batch bioconversion. As a result, 1.01 mol L-aspartate/mol glucose and 1.52 mol ß-alanine/mol glucose were produced in corresponding strains respectively. Fed-batch bioconversion allowed 249 mM (33.1 g/L) L-aspartate or 424 mM (37.7 g/L) ß-alanine production, respectively. The study provides a novel promising metabolic engineering route for the production of L-aspartate and its derivate chemicals using biomass resources. These results also represent the first report of the efficient bioproduction of L-aspartate directly from glucose in E. coli and the highest yield of ß-alanine reported so far.

Integrated transcriptomic and metabolomic analysis shows that disturbances in metabolism of tumor cells contribute to poor survival of RCC patients.

PURPOSE: Cellular metabolism of renal cell carcinoma (RCC) tumors is disturbed. The clinical significance of these alterations is weakly understood. We aimed to find if changes in metabolic pathways contribute to survival of RCC patients. MATERIAL AND METHODS: 35 RCC tumors and matched controls were used for metabolite profiling using gas chromatography-mass spectrometry and transcriptomic analysis with qPCR-arrays targeting the expression of 93 metabolic genes. The clinical significance of obtained data was validated on independent cohort of 468 RCC patients with median follow-up of 43.22months. RESULTS: The levels of 31 metabolites were statistically significantly changed in RCC tumors compared with controls. The top altered metabolites included beta-alanine (+4.2-fold), glucose (+3.4-fold), succinate (-11.0-fold), myo-inositol (-4.6-fold), adenine (-4.2-fold), uracil (-3.7-fold), and hypoxanthine (-3.0-fold). These disturbances were associated with altered expression of 53 metabolic genes. ROC curve analysis revealed that the top metabolites discriminating between tumor and control samples included succinate (AUC=0.91), adenine (AUC=0.89), myo-inositol (AUC=0.87), hypoxanthine (AUC=0.85), urea (AUC=0.85), and beta-alanine (AUC=0.85). Poor survival of RCC patients correlated (p<0.0001) with altered expression of genes involved in metabolism of succinate (HR=2.7), purines (HR=2.4), glucose (HR=2.4), beta-alanine (HR=2.5), and myo-inositol (HR=1.9). CONCLUSIONS: We found that changes in metabolism of succinate, beta-alanine, purines, glucose and myo-inositol correlate with poor survival of RCC patients.

EasyCloneMulti: A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is widely used in the biotechnology industry for production of ethanol, recombinant proteins, food ingredients and other chemicals. In order to generate highly producing and stable strains, genome integration of genes encoding metabolic pathway enzymes is the preferred option. However, integration of pathway genes in single or few copies, especially those encoding rate-controlling steps, is often not sufficient to sustain high metabolic fluxes. By exploiting the sequence diversity in the long terminal repeats (LTR) of Ty retrotransposons, we developed a new set of integrative vectors, EasyCloneMulti, that enables multiple and simultaneous integration of genes in S. cerevisiae. By creating vector backbones that combine consensus sequences that aim at targeting subsets of Ty sequences and a quickly degrading selective marker, integrations at multiple genomic loci and a range of expression levels were obtained, as assessed with the green fluorescent protein (GFP) reporter system. The EasyCloneMulti vector set was applied to balance the expression of the rate-controlling step in the ß-alanine pathway for biosynthesis of 3-hydroxypropionic acid (3HP). The best 3HP producing clone, with 5.45 g.L(-1) of 3HP, produced 11 times more 3HP than the lowest producing clone, which demonstrates the capability of EasyCloneMulti vectors to impact metabolic pathway enzyme activity.

Mitochondrial defects associated with ß-alanine toxicity: relevance to hyper-beta-alaninemia.

Hyper-beta-alaninemia is a rare metabolic condition that results in elevated plasma and urinary ß-alanine levels and is characterized by neurotoxicity, hypotonia, and respiratory distress. It has been proposed that at least some of the symptoms are caused by oxidative stress; however, only limited information is available on the mechanism of reactive oxygen species generation. The present study examines the hypothesis that ß-alanine reduces cellular levels of taurine, which are required for normal respiratory chain function; cellular taurine depletion is known to reduce respiratory function and elevate mitochondrial superoxide generation. To test the taurine hypothesis, isolated neonatal rat cardiomyocytes and mouse embryonic fibroblasts were incubated with medium lacking or containing ß-alanine. ß-alanine treatment led to mitochondrial superoxide accumulation in conjunction with a decrease in oxygen consumption. The defect in ß-alanine-mediated respiratory function was detected in permeabilized cells exposed to glutamate/malate but not in cells utilizing succinate, suggesting that ß-alanine leads to impaired complex I activity. Taurine treatment limited mitochondrial superoxide generation, supporting a role for taurine in maintaining complex I activity. Also affected by taurine is mitochondrial morphology, as ß-alanine-treated fibroblasts undergo fragmentation, a sign of unhealthy mitochondria that is reversed by taurine treatment. If left unaltered, ß-alanine-treated fibroblasts also undergo mitochondrial apoptosis, as evidenced by activation of caspases 3 and 9 and the initiation of the mitochondrial permeability transition. Together, these data show that ß-alanine mediates changes that reduce ATP generation and enhance oxidative stress, factors that contribute to heart failure.

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.

Establishing a synthetic pathway for high-level production of 3-hydroxypropionic acid in Saccharomyces cerevisiae via ß-alanine.

Microbial fermentation of renewable feedstocks into plastic monomers can decrease our fossil dependence and reduce global CO2 emissions. 3-Hydroxypropionic acid (3HP) is a potential chemical building block for sustainable production of superabsorbent polymers and acrylic plastics. With the objective of developing Saccharomyces cerevisiae as an efficient cell factory for high-level production of 3HP, we identified the ß-alanine biosynthetic route as the most economically attractive according to the metabolic modeling. We engineered and optimized a synthetic pathway for de novo biosynthesis of ß-alanine and its subsequent conversion into 3HP using a novel ß-alanine-pyruvate aminotransferase discovered in Bacillus cereus. The final strain produced 3HP at a titer of 13.7±0.3gL(-1) with a 0.14±0.0C-molC-mol(-1) yield on glucose in 80h in controlled fed-batch fermentation in mineral medium at pH 5, and this work therefore lays the basis for developing a process for biological 3HP production.

A genetically encoded fluorescent probe in mammalian cells.

Fluorescent reporters are useful in vitro and in vivo probes of protein structure, function, and localization. Here we report that the fluorescent amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), can be site-specifically incorporated into proteins in mammalian cells in response to the TAG codon with high efficiency using an orthogonal amber suppressor tRNA/aminoacyl-tRNA synthetase (aaRS) pair. We further demonstrate that Anap can be used to image the subcellular localization of proteins in live mammalian cells. The small size of Anap, its environment-sensitive fluorescence, and the ability to introduce Anap at specific sites in the proteome by simple mutagenesis make it a unique and valuable tool in eukaryotic cell biology.

Overexpression of sICAM-1 in the alveolar epithelial space results in an exaggerated inflammatory response and early death in Gram negative pneumonia.

BACKGROUND: A sizeable body of data demonstrates that membrane ICAM-1 (mICAM-1) plays a significant role in host defense in a site-specific fashion. On the pulmonary vascular endothelium, mICAM-1 is necessary for normal leukocyte recruitment during acute inflammation. On alveolar epithelial cells (AECs), we have shown previously that the presence of normal mICAM-1 is essential for optimal alveolar macrophage (AM) function. We have also shown that ICAM-1 is present in the alveolar space as a soluble protein that is likely produced through cleavage of mICAM-1. Soluble intercellular adhesion molecule-1 (sICAM-1) is abundantly present in the alveolar lining fluid of the normal lung and could be generated by proteolytic cleavage of mICAM-1, which is highly expressed on type I AECs. Although a growing body of data suggesting that intravascular sICAM-1 has functional effects, little is known about sICAM-1 in the alveolus. We hypothesized that sICAM-1 in the alveolar space modulates the innate immune response and alters the response to pulmonary infection. METHODS: Using the surfactant protein C (SPC) promoter, we developed a transgenic mouse (SPC-sICAM-1) that constitutively overexpresses sICAM-1 in the distal lung, and compared the responses of wild-type and SPC-sICAM-1 mice following intranasal inoculation with K. pneumoniae. RESULTS: SPC-sICAM-1 mice demonstrated increased mortality and increased systemic dissemination of organisms compared with wild-type mice. We also found that inflammatory responses were significantly increased in SPC-sICAM-1 mice compared with wild-type mice but there were no difference in lung CFU between groups. CONCLUSIONS: We conclude that alveolar sICAM-1 modulates pulmonary inflammation. Manipulating ICAM-1 interactions therapeutically may modulate the host response to Gram negative pulmonary infections.

Alternative tasks of Drosophila tan in neurotransmitter recycling versus cuticle sclerotization disclosed by kinetic properties.

Upon a stimulus of light, histamine is released from Drosophila photoreceptor axonal endings. It is taken up into glia where Ebony converts it into beta-alanyl-histamine (carcinine). Carcinine moves into photoreceptor cells and is there cleaved into beta-alanine and histamine by Tan activity. Tan thus provides a key function in the recycling pathway of the neurotransmitter histamine. It is also involved in the process of cuticle formation. There, it cleaves beta-alanyl-dopamine, a major component in cuticle sclerotization. Active Tan enzyme is generated by a self-processing proteolytic cleavage from a pre-protein at a conserved Gly-Cys sequence motif. We confirmed the dependence on the Gly-Cys motif by in vitro mutagenesis. Processing time delays the rise to full Tan activity up to 3 h behind its putative circadian RNA expression in head. To investigate its pleiotropic functions, we have expressed Tan as a His(6) fusion protein in Escherichia coli and have purified it to homogeneity. We found wild type and mutant His(6)-Tan protein co-migrating in size exclusion chromatography with a molecular weight compatible with homodimer formation. We conclude that dimer formation is preceding pre-protein processing. Drosophila tan(1) null mutant analysis revealed that amino acid Arg(217) is absolutely required for processing. Substitution of Met(256) in tan(5), on the contrary, does not affect processing extensively but renders it prone to degradation. This also leads to a strong tan phenotype although His(6)-Tan(5) retains activity. Kinetic parameters of Tan reveal characteristic differences in K(m) and k(cat) values of carcinine and beta-alanyl-dopamine cleavage, which conclusively illustrate the divergent tasks met by Tan.

Genetic incorporation of a small, environmentally sensitive, fluorescent probe into proteins in Saccharomyces cerevisiae.

Here, we report that the fluorescent amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), can be genetically incorporated into proteins in yeast with excellent selectivity and efficiency by means of an orthogonal tRNA/aminoacyl-tRNA synthetase pair. This small, environmentally sensitive fluorophore was site-specifically incorporated into Escherichia coli glutamine binding protein and used to directly probe local structural changes caused by ligand binding. The small size of Anap and the ability to introduce it by simple mutagenesis at defined sites in the proteome make it a useful local probe of protein structure, molecular interactions, protein folding, and localization.