Functional differentiation of olive PLP_deC genes: insights into metabolite biosynthesis and genetic improvement at the whole-genome level.

KEY MESSAGE: This study identified 16 pyridoxal phosphate-dependent decarboxylases in olive at the whole-genome level, conducted analyses on their physicochemical properties, evolutionary relationships and characterized their activity. Group II pyridoxal phosphate-dependent decarboxylases (PLP_deC II) mediate the biosynthesis of characteristic olive metabolites, such as oleuropein and hydroxytyrosol. However, there have been no report on the functional differentiation of this gene family at the whole-genome level. This study conducted an exploration of the family members of PLP_deC II at the whole-genome level, identified 16 PLP_deC II genes, and analyzed their gene structure, physicochemical properties, cis-acting elements, phylogenetic evolution, and gene expression patterns. Prokaryotic expression and enzyme activity assays revealed that OeAAD2 and OeAAD4 could catalyze the decarboxylation reaction of tyrosine and dopa, resulting in the formation of their respective amine compounds, but it did not catalyze phenylalanine and tryptophan. Which is an important step in the synthetic pathway of hydroxytyrosol and oleuropein. This finding established the foundational data at the molecular level for studying the functional aspects of the olive PLP_deC II gene family and provided essential gene information for genetic improvement of olive.

ACOD1, rather than itaconate, facilitates p62-mediated activation of Nrf2 in microglia post spinal cord contusion.

BACKGROUND: Spinal cord injury (SCI)-induced neuroinflammation and oxidative stress (OS) are crucial events causing neurological dysfunction. Aconitate decarboxylase 1 (ACOD1) and its metabolite itaconate (Ita) inhibit inflammation and OS by promoting alkylation of Keap1 to induce Nrf2 expression; however, it is unclear whether there is another pathway regulating their effects in inflammation-activated microglia after SCI. METHODS: Adult male C57BL/6 ACOD1-/- mice and their wild-type (WT) littermates were subjected to a moderate thoracic spinal cord contusion. The degree of neuroinflammation and OS in the injured spinal cord were assessed using qPCR, western blot, flow cytometry, immunofluorescence, and trans-well assay. We then employed immunoprecipitation-western blot, chromatin immunoprecipitation (ChIP)-PCR, dual-luciferase assay, and immunofluorescence-confocal imaging to examine the molecular mechanisms of ACOD1. Finally, the locomotor function was evaluated with the Basso Mouse Scale and footprint assay. RESULTS: Both in vitro and in vivo, microglia with transcriptional blockage of ACOD1 exhibited more severe levels of neuroinflammation and OS, in which the expression of p62/Keap1/Nrf2 was down-regulated. Furthermore, silencing ACOD1 exacerbated neurological dysfunction in SCI mice. Administration of exogenous Ita or 4-octyl itaconate reduced p62 phosphorylation. Besides, ACOD1 was capable of interacting with phosphorylated p62 to enhance Nrf2 activation, which in turn further promoted transcription of ACOD1. CONCLUSIONS: Here, we identified an unreported ACOD1-p62-Nrf2-ACOD1 feedback loop exerting anti-inflammatory and anti-OS in inflammatory microglia, and demonstrated the neuroprotective role of ACOD1 after SCI, which was different from that of endogenous and exogenous Ita. The present study extends the functions of ACOD1 and uncovers marked property differences between endogenous and exogenous Ita. KEY POINTS: ACOD1 attenuated neuroinflammation and oxidative stress after spinal cord injury. ACOD1, not itaconate, interacted with p-p62 to facilitate Nrf2 expression and nuclear translocation. Nrf2 was capable of promoting ACOD1 transcription in microglia.

Engineering styrene biosynthesis: designing a functional trans-cinnamic acid decarboxylase in Pseudomonas.

We are interested in converting second generation feedstocks into styrene, a valuable chemical compound, using the solvent-tolerant Pseudomonas putida DOT-T1E as a chassis. Styrene biosynthesis takes place from L-phenylalanine in two steps: firstly, L-phenylalanine is converted into trans-cinnamic acid (tCA) by PAL enzymes and secondly, a decarboxylase yields styrene. This study focuses on designing and synthesizing a functional trans-cinnamic acid decarboxylase in Pseudomonas putida. To achieve this, we utilized the "wholesale" method, involving deriving two consensus sequences from multi-alignments of homologous yeast ferulate decarboxylase FDC1 sequences with > 60% and > 50% identity, respectively. These consensus sequences were used to design Pseudomonas codon-optimized genes named psc1 and psd1 and assays were conducted to test the activity in P. putida. Our results show that the PSC1 enzyme effectively decarboxylates tCA into styrene, whilst the PSD1 enzyme does not. The optimal conditions for the PSC1 enzyme, including pH and temperature were determined. The L-phenylalanine DOT-T1E derivative Pseudomonas putida CM12-5 that overproduces L-phenylalanine was used as the host for expression of pal/psc1 genes to efficiently convert L-phenylalanine into tCA, and the aromatic carboxylic acid into styrene. The highest styrene production was achieved when the pal and psc1 genes were co-expressed as an operon in P. putida CM12-5. This construction yielded styrene production exceeding 220 mg L-1. This study serves as a successful demonstration of our strategy to tailor functional enzymes for novel host organisms, thereby broadening their metabolic capabilities. This breakthrough opens the doors to the synthesis of aromatic hydrocarbons using Pseudomonas putida as a versatile biofactory.

The prFMNH2-binding chaperone LpdD assists UbiD decarboxylase activation.

The UbiD enzyme family of prenylated flavin (prFMN)-dependent reversible decarboxylases is near ubiquitously present in microbes. For some UbiD family members, enzyme activation through prFMNH2 binding and subsequent oxidative maturation of the cofactor readily occurs, both in vivo in a heterologous host and through in vitro reconstitution. However, isolation of the active holo-enzyme has proven intractable for others, notably the canonical Escherichia coli UbiD. We show that E. coli heterologous expression of the small protein LpdD-associated with the UbiD-like gallate decarboxylase LpdC from Lactobacillus plantarum-unexpectedly leads to 3,4-dihydroxybenzoic acid decarboxylation whole-cell activity. This activity was shown to be linked to endogenous E. coli ubiD expression levels. The crystal structure of the purified LpdD reveals a dimeric protein with structural similarity to the eukaryotic heterodimeric proteasome assembly chaperone Pba3/4. Solution studies demonstrate that LpdD protein specifically binds to reduced prFMN species only. The addition of the LpdD-prFMNH2 complex supports reconstitution and activation of the purified E. coli apo-UbiD in vitro, leading to modest 3,4-dihydroxybenzoic acid decarboxylation. These observations suggest that LpdD acts as a prFMNH2-binding chaperone, enabling apo-UbiD activation through enhanced prFMNH2 incorporation and subsequent oxidative maturation. Hence, while a single highly conserved flavin prenyltransferase UbiX is found associated with UbiD enzymes, our observations suggest considerable diversity in UbiD maturation, ranging from robust autocatalytic to chaperone-mediated processes. Unlocking the full (de)carboxylation scope of the UbiD-enzyme family will thus require more than UbiX coexpression.

Detection of oxalyl-CoA decarboxylase (oxc) and formyl-CoA transferase (frc) genes in novel probiotic isolates capable of oxalate degradation in vitro.

Oxalate degradation is one of lactic acid bacteria's desirable activities. It is achieved by two enzymes, formyl coenzyme A transferase (frc) and oxalyl coenzyme A decarboxylase (oxc). The current study aimed to screen 15 locally isolated lactic acid bacteria to select those with the highest oxalate degradation ability. It also aimed to amplify the genes involved in degradation. MRS broth supplemented with 20 mM sodium oxalate was used to culture the tested isolates for 72 h. This was followed by an enzymatic assay to detect remaining oxalate. All isolates showed oxalate degradation activity to variable degrees. Five isolates demonstrated high oxalate degradation, 78 to 88%. To investigate the oxalate-degradation potential of the selected isolates, they have been further tested for the presence of genes that encode for enzymes involved in oxalate catabolism, formyl coenzyme A transferase (frc) and oxalyl coenzyme A decarboxylase (oxc). Three strains showed bands with the specific OXC and FRC forward and reverse primers designated as (SA-5, 9 and 37). Species-level identification revealed Loigolactobacillus bifermentans, Lacticaseibacillus paracasei, and Lactiplantibacillus plantarum. Preliminary results revealed that the tested probiotic strains harbored both oxc and frc whose products are putatively involved in oxalate catabolism. The probiotic potential of the selected strains was evaluated, and they showed high survival rates to both simulated gastric and intestinal fluids and variable degrees of antagonism against the tested Gram-positive and negative pathogens and were sensitive to clarithromycin but resistant to both metronidazole and ceftazidime. Finally, these strains could be exploited as an innovative approach to establish oxalate homeostasis in humans and prevent kidney stone formation.

An oxalate decarboxylase-like cupin domain containing protein is involved in imparting acid stress tolerance in Bacillus amyloliquefaciens MBNC.

We report here the structural and functional properties of an oxalate decarboxylase (OxDC)-like cupin domain-containing protein of Bacillus amyloliquefaciens MBNC and its role in imparting tolerance to acid stress conditions. Quantitative real-time PCR (qPCR) analysis revealed 32-fold and 20-fold upregulation of the target gene [(OxDC')cupin] under acetic acid stress and hydrochloric acid stress, respectively, indicating its association with the acid stress response. Bacterial cells with targeted inactivation of the (OxDC')cupin gene using the pMUTIN4 vector system showed decreased growth and survival rate in acidic pH, with drastically reduced exopolysaccharide production. In Silico protein-protein interaction studies revealed seven genes (viz. glmS, nagA, nagB, tuaF, tuaF, gcvT, and ykgA) related to cell wall biosynthesis and biofilm production to interact with OxDC-like cupin domain containing protein. While all these seven genes were upregulated in B. amyloliquefaciens MBNC after 6 h of exposure to pH 4.5, the mutant cells containing the inactivated (OxDC')cupin gene displayed significantly lower expression (RQ: 0.001-0.02) (compared to the wild-type cells) in both neutral and acidic pH. Our results indicate that the OxDC-like cupin domain containing protein is necessary for cell wall biosynthesis and biofilm production in Bacillus amyloliquefaciens MBNC for survival in acid-stress conditions.

Bacillus subtilis YisK possesses oxaloacetate decarboxylase activity and exhibits Mbl-dependent localization.

YisK is an uncharacterized protein in Bacillus subtilis previously shown to interact genetically with the elongasome protein Mbl. YisK overexpression leads to cell widening and lysis, phenotypes that are dependent on mbl and suppressed by mbl mutations. In the present work, we characterize YisK's localization, structure, and enzymatic activity. We show that YisK localizes as puncta that depend on Mbl. YisK belongs to the fumarylacetoacetate hydrolase (FAH) superfamily, and crystal structures revealed close structural similarity to two oxaloacetate (OAA) decarboxylases: human mitochondrial FAHD1 and Corynebacterium glutamicum Cg1458. We demonstrate that YisK can also catalyze the decarboxylation of OAA (K m = 134 µM, K cat = 31 min-1). A catalytic dead variant (YisK E148A, E150A) retains wild-type localization and still widens cells following overexpression, indicating these activities are not dependent on YisK catalysis. Conversely, a non-localizing variant (YisK E30A) retains wild-type enzymatic activity in vitro but localizes diffusely and no longer widens cells following overexpression. Together, these results suggest that YisK may be subject to spatial regulation that depends on the cell envelope synthesis machinery. IMPORTANCE The elongasome is a multiprotein complex that guides lengthwise growth in some bacteria. We previously showed that, in B. subtilis, overexpression of an uncharacterized putative enzyme (YisK) perturbed function of the actin-like elongasome protein Mbl. Here, we show that YisK exhibits Mbl-dependent localization. Through biochemical and structural characterization, we demonstrate that, like its mitochondrial homolog FAHD1, YisK can catalyze the decarboxylation of the oxaloacetate to pyruvate and CO2. YisK is the first example of an enzyme implicated in central carbon metabolism with subcellular localization that depends on Mbl.

Structure-guided engineering of branched-chain α-keto acid decarboxylase for improved 1,2,4-butanetriol production by in vitro synthetic enzymatic biosystem.

Efficient synthetic routes for biomanufacturing chemicals often require the overcoming of pathway bottlenecks by tailoring enzymes to improve the catalytic efficiency or even implement non-native activities. 1,2,4-butanetriol (BTO), a valuable commodity chemical, is currently biosynthesized from D-xylose via a four-enzyme reaction cascade, with the ThDP-dependent α-keto acid decarboxylase (KdcA) identified as the potential bottleneck. Here, to further enhance the catalytic activity of KdcA toward the non-native substrate α-keto-3-deoxy-xylonate (KDX), in silico screening and structure-guided evolution were performed. The best mutants, S286L/G402P and V461K, exhibited a 1.8- and 2.5-fold higher enzymatic activity in the conversion of KDX to 3,4-dihydroxybutanal when compared to KdcA, respectively. MD simulations revealed that the two sets of mutations reshaped the substrate binding pocket, thereby increasing the binding affinity for KDX and promoting interactions between KDX and cofactor ThDP. Then, when the V461K mutant instead of wild type KdcA was integrated into the enzyme cascade, a 1.9-fold increase in BTO titer was observed. After optimization of the reaction conditions, the enzyme cocktail contained V461K converted 60 g/L D-xylose to 22.1 g/L BTO with a yield of 52.1 %. This work illustrated that protein engineering is a powerful tool for modifying the output of metabolic pathway.

THI3 contributes to isoamyl alcohol biosynthesis through thiamine diphosphate homeostasis.

Isoamyl alcohol is a precursor of isoamyl acetate, an aromatic compound that imparts the ginjo aroma to sake. The isoamyl alcohol biosynthesis pathway in yeasts involves the genes PDC1, PDC5, PDC6, ARO10, and THI3 encoding enzymes that decarboxylate α-ketoisocaproic acid to isovaleraldehyde. Among these genes, THI3 is the main gene involved in isoamyl alcohol biosynthesis. Decreased production of isoamyl alcohol has been reported in yeast strains with disrupted THI3 (Δthi3). However, it has also been reported that high THI3 expression did not enhance decarboxylase activity. Therefore, the involvement of THI3 in isoamyl alcohol biosynthesis remains unclear. In this study, we investigated the role of THI3 in isoamyl alcohol biosynthesis. While reproducing previous reports of reduced isoamyl alcohol production by the Δthi3 strain, we observed that the decrease in isoamyl alcohol production occurred only at low yeast nitrogen base concentrations in the medium. Upon investigating individual yeast nitrogen base components, we found that the isoamyl alcohol production by the Δthi3 strain reduced when thiamine concentrations in the medium were low. Under low-thiamine conditions, both thiamine and thiamine diphosphate (TPP) levels decreased in Δthi3 cells. We also found that the decarboxylase activity of cell-free extracts of the Δthi3 strain cultured in a low-thiamine medium was lower than that of the wild-type strain, but was restored to the level of the wild-type strain when TPP was added. These results indicate that the loss of THI3 lowers the supply of TPP, a cofactor for decarboxylases, resulting in decreased isoamyl alcohol production.

Arabidopsis 3ß-Hydroxysteroid Dehydrogenases/C4-Decarboxylases Are Essential for the Pollen and Embryonic Development.

The biosynthesis of C27-29 sterols from their C30 precursor squalene involves C24-alkylation and the removal of three methyl groups, including two at the C4 position. The two C4 demethylation reactions require a bifunctional enzyme known as 3ß-hydroxysteroid dehydrogenase/C4-decarboxylase (3ßHSD/D), which removes an oxidized methyl (carboxylic) group at C4 while simultaneously catalyzing the 3ß-hydroxyl→3-keto oxidation. Its loss-of-function mutations cause ergosterol-dependent growth in yeast and congenital hemidysplasia with ichthyosiform erythroderma and limb defect (CHILD) syndrome in humans. Although plant 3ßHSD/D enzymes were well studied enzymatically, their developmental functions remain unknown. Here we employed a CRISPR/Cas9-based genome-editing approach to generate knockout mutants for two Arabidopsis 3ßHSD/D genes, HSD1 and HSD2, and discovered the male gametophytic lethality for the hsd1 hsd2 double mutation. Pollen-specific expression of HSD2 in the heterozygous hsd1 hsd2/+ mutant not only rescued the pollen lethality but also revealed the critical roles of the two HSD genes in embryogenesis. Our study thus demonstrated the essential functions of the two Arabidopsis 3ßHSD/D genes in male gametogenesis and embryogenesis.

Comprehensive analysis of the UDP-glucuronate decarboxylase (UXS) gene family in tobacco and functional characterization of NtUXS16 in Golgi apparatus in Arabidopsis.

BACKGROUND: UDP-glucuronate decarboxylase (also named UXS) converts UDP-glucuronic acid (UDP-GlcA) to UDP-xylose (UDP-Xyl) by decarboxylation of the C6-carboxylic acid of glucuronic acid. UDP-Xyl is an important sugar donor that is required for the synthesis of plant cell wall polysaccharides. RESULTS: In this study, we first carried out the genome-wide identification of NtUXS genes in tobacco. A total of 17 NtUXS genes were identified, which could be divided into two groups (Group I and II), and the Group II UXSs can be further divided into two subgroups (Group IIa and IIb). Furthermore, the protein structures, intrachromosomal distributions and gene structures were thoroughly analyzed. To experimentally verify the subcellular localization of NtUXS16 protein, we transformed tobacco BY-2 cells with NtUXS16 fused to the monomeric red fluorescence protein (mRFP) at the C terminus under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The fluorescent signals of NtUXS16-mRFP were localized to the medial-Golgi apparatus. Contrary to previous predictions, protease digestion analysis revealed that NtUXS16 is not a type II membrane protein. Overexpression of NtUXS16 in Arabidopsis seedling in darkness led to a significant increase in hypocotyl length and a reduction in root length compared with the wild type. In summary, these results suggest Golgi apparatus localized-NtUXS16 plays an important role in hypocotyl and root growth in the dark. CONCLUSION: Our findings facilitate our understanding of the novel functions of NtUXS16 and provide insights for further exploration of the biological roles of NtUXS genes in tobacco.

Construction of an Escherichia coli cell factory for de novo synthesis of tyrosol through semi-rational design based on phenylpyruvate decarboxylase ARO10 engineering.

Tyrosol (2-(4-hydroxyphenyl) ethanol) is extensively used in the pharmaceutical industry as an important natural product from plants. In previous research, we constructed a recombinant Escherichia coli strain capable of de novo synthesis of tyrosol by integrating the phenylpyruvate decarboxylase ARO10 derived from Saccharomyces cerevisiae. Nevertheless, the insufficient catalytic efficiency of ARO10 required the insertion of multiple gene copies into the genome to attain enhanced tyrosol production. In this study, we constructed a mutation library of ARO10 based on a computer-aided semi-rational design strategy and developed a high-throughput screening method for selecting high-yield tyrosol mutants by introducing the heterologous hydroxylase complex HpaBC. Through multiple rounds of screening and site-saturation mutagenesis, we ultimately identified the two optimal ARO10 mutants, ARO10D331V and ARO10D331C, which respectively achieved a tyrosol titer of 2.02 g/L and 2.04 g/L in shake flasks, both representing more than 50 % improvement compared to the wild-type. Our study demonstrates the great potential of computer-based semi-rational enzyme design strategy in metabolic engineering. The high-throughput screening method for target compound derivative possesses a certain level of generality. Ultimately, we obtained promising mutants capable of achieving industrial-scale production of tyrosol, which also lays a solid foundation for the efficient synthesis of tyrosol derivatives.

Identification and expression analysis of transcripts involved in taurine biosynthesis during early ontogeny of tropical gar Atractosteus tropicus.

In fishes, the availability of taurine is regulated during ontogenetic development, where its endogenous synthesis capacity is species dependent. Thus, different pathways and involved enzymes have been described: pathway I (cysteine sulfinate-dependent pathway), cysteine dioxygenase type 1 (cdo1) and cysteine sulfinic acid decarboxylase (csad); pathway II (cysteic acid pathway), cdo1 and glutamic acid decarboxylase (gad); and pathway III (cysteamine pathway), 2-aminoethanethiol dioxygenase (ado); whereas taurine transporter (taut) is responsible for taurine entry into cells on the cell membrane and the mitochondria. This study determined if the tropical gar (Atractosteus tropicus), an ancient holostean fish model, has the molecular mechanism to synthesize taurine through the identification and analysis expression of transcripts coding for proteins involved in its biosynthesis and transportation, at different embryo-larvae stages and in different organs of juveniles (31 dah). We observed a fluctuating expression of all transcripts involved in the three pathways at all analyzed stages. All transcripts are expressed during the beginning of larval development; however, ado and taut show a peak expression at 9 dah, and all transcripts but csad decreased at 23 dah, when the organism ended the larval period. Furthermore, at 31 dah, we observed taut expression in all examined organs. The transcripts involved in pathways I and III are expressed differently across all organs, whereas pathway II was only observed in the brain, eye, and skin. The results suggested that taurine biosynthesis in tropical gar is regulated during its early development before first feeding, and the pathway might also be organ-type dependent.

Neofunctionalization of S-adenosylmethionine decarboxylase into pyruvoyl-dependent L-ornithine and L-arginine decarboxylases is widespread in bacteria and archaea.

S-adenosylmethionine decarboxylase (AdoMetDC/SpeD) is a key polyamine biosynthetic enzyme required for conversion of putrescine to spermidine. Autocatalytic self-processing of the AdoMetDC/SpeD proenzyme generates a pyruvoyl cofactor from an internal serine. Recently, we discovered that diverse bacteriophages encode AdoMetDC/SpeD homologs that lack AdoMetDC activity and instead decarboxylate L-ornithine or L-arginine. We reasoned that neofunctionalized AdoMetDC/SpeD homologs were unlikely to have emerged in bacteriophages and were probably acquired from ancestral bacterial hosts. To test this hypothesis, we sought to identify candidate AdoMetDC/SpeD homologs encoding L-ornithine and L-arginine decarboxylases in bacteria and archaea. We searched for the anomalous presence of AdoMetDC/SpeD homologs in the absence of its obligatory partner enzyme spermidine synthase, or the presence of two AdoMetDC/SpeD homologs encoded in the same genome. Biochemical characterization of candidate neofunctionalized genes confirmed lack of AdoMetDC activity, and functional presence of L-ornithine or L-arginine decarboxylase activity in proteins from phyla Actinomycetota, Armatimonadota, Planctomycetota, Melainabacteria, Perigrinibacteria, Atribacteria, Chloroflexota, Sumerlaeota, Omnitrophota, Lentisphaerota, and Euryarchaeota, the bacterial candidate phyla radiation and DPANN archaea, and the δ-Proteobacteria class. Phylogenetic analysis indicated that L-arginine decarboxylases emerged at least three times from AdoMetDC/SpeD, whereas L-ornithine decarboxylases arose only once, potentially from the AdoMetDC/SpeD-derived L-arginine decarboxylases, revealing unsuspected polyamine metabolic plasticity. Horizontal transfer of the neofunctionalized genes appears to be the more prevalent mode of dissemination. We identified fusion proteins of bona fide AdoMetDC/SpeD with homologous L-ornithine decarboxylases that possess two, unprecedented internal protein-derived pyruvoyl cofactors. These fusion proteins suggest a plausible model for the evolution of the eukaryotic AdoMetDC.

Pyruvate decarboxylase and thiamine biosynthetic genes are regulated differently by Pdc2 in S. cerevisiae and C. glabrata.

Understanding metabolism in the pathogen Candida glabrata is key to identifying new targets for antifungals. The thiamine biosynthetic (THI) pathway is partially defective in C. glabrata, but the transcription factor CgPdc2 upregulates some thiamine biosynthetic and transport genes. One of these genes encodes a recently evolved thiamine pyrophosphatase (CgPMU3) that is critical for accessing external thiamine. Here, we demonstrate that CgPdc2 primarily regulates THI genes. In Saccharomyces cerevisiae, Pdc2 regulates both THI and pyruvate decarboxylase (PDC) genes, with PDC proteins being a major thiamine sink. Deletion of PDC2 is lethal in S. cerevisiae in standard growth conditions, but not in C. glabrata. We uncover cryptic cis elements in C. glabrata PDC promoters that still allow for regulation by ScPdc2, even when that regulation is not apparent in C. glabrata. C. glabrata lacks Thi2, and it is likely that inclusion of Thi2 into transcriptional regulation in S. cerevisiae allows for a more complex regulation pattern and regulation of THI and PDC genes. We present evidence that Pdc2 functions independent of Thi2 and Thi3 in both species. The C-terminal activation domain of Pdc2 is intrinsically disordered and critical for species differences. Truncation of the disordered domains leads to a gradual loss of activity. Through a series of cross species complementation assays of transcription, we suggest that there are multiple Pdc2-containing complexes, and C. glabrata appears to have the simplest requirement set for THI genes, except for CgPMU3. CgPMU3 has different cis requirements, but still requires Pdc2 and Thi3 to be upregulated by thiamine starvation. We identify the minimal region sufficient for thiamine regulation in CgTHI20, CgPMU3, and ScPDC5 promoters. Defining the cis and trans requirements for THI promoters should lead to an understanding of how to interrupt their upregulation and provide targets in metabolism for antifungals.

EvdS6 is a bifunctional decarboxylase from the everninomicin gene cluster.

The everninomicins are bacterially produced antibiotic octasaccharides characterized by the presence of two interglycosidic spirocyclic ortho-δ-lactone (orthoester) moieties. The terminating G- and H-ring sugars, L-lyxose and C-4 branched sugar ß-D-eurekanate, are proposed to be biosynthetically derived from nucleotide diphosphate pentose sugar pyranosides; however, the identity of these precursors and their biosynthetic origin remain to be determined. Herein we identify a new glucuronic acid decarboxylase from Micromonospora belonging to the superfamily of short-chain dehydrogenase/reductase enzymes, EvdS6. Biochemical characterization demonstrated that EvdS6 is an NAD+-dependent bifunctional enzyme that produces a mixture of two products, differing in the sugar C-4 oxidation state. This product distribution is atypical for glucuronic acid decarboxylating enzymes, most of which favor production of the reduced sugar and a minority of which favor release of the oxidized product. Spectroscopic and stereochemical analysis of reaction products revealed that the first product released is the oxidatively produced 4-keto-D-xylose and the second product is the reduced D-xylose. X-ray crystallographic analysis of EvdS6 at 1.51 Å resolution with bound co-factor and TDP demonstrated that the overall geometry of the EvdS6 active site is conserved with other SDR enzymes and enabled studies probing structural determinants for the reductive half of the net neutral catalytic cycle. Critical active site threonine and aspartate residues were unambiguously identified as essential in the reductive step of the reaction and resulted in enzyme variants producing almost exclusively the keto sugar. This work defines potential precursors for the G-ring L-lyxose and resolves likely origins of the H-ring ß-D-eurekanate sugar precursor.

Purification and characterization of tomato arginine decarboxylase and its inhibition by the bacterial small molecule phevamine A.

Polyamines play essential roles in plant growth and survival. Arginine decarboxylase (ADC), which converts arginine to agmatine, catalyzes the first step in polyamine biosynthesis from arginine. However, few biochemical studies with purified plant ADCs have been conducted to evaluate their catalytic efficiency. Tomato genome encodes two arginine decarboxylases: SlADC1 and SlADC2, which are critical for growth, development, and immune responses against bacterial pathogens. We expressed and purified soluble SlADC1 as a recombinant protein fused with maltose-binding protein tag from E. coli Rosetta 2(DE3) cells. Using the purified fusion protein, we characterized the biochemical properties of SlADC1 in vitro and explored it as a target of the bacterial small molecule phevamine A. We confirmed that the activity of SlADC1 depends on the cofactor pyridoxal 5'-phosphate. SlADC1 is specific toward l-arginine and its kinetic parameters were measured using a liquid chromatography-mass spectrometry method. Phevamine A is a competitive inhibitor of SlADC1 and reduces the activity of SlADC1 at high micromolar concentrations. Our purification and biochemical characterization of SlADC1 sets the stage for inhibition studies of this enzyme.

Formation of vinylphenolic pyranoanthocyanins by selected indigenous yeasts displaying high hydroxycinnamate decarboxylase activity during mulberry wine fermentation and aging.

The color of mulberry wine is difficult to maintain since the main chromogenic substances, anthocyanins, are severely degraded during fermentation and aging. This study selected Saccharomyces cerevisiae I34 and Wickerhamomyces anomalus D6, both displaying high hydroxycinnamate decarboxylase (HCDC) activity (78.49% and 78.71%), to enhance the formation of stable vinylphenolic pyranoanthocyanins (VPAs) pigments during mulberry wine fermentation. The HCDC activity of 84 different strains from eight regions in China was primarily screened via the deep well plate micro fermentation method, after which the tolerance and brewing characteristics were evaluated via simulated mulberry juice. The two selected strains and a commercial Saccharomyces cerevisiae were then inoculated individually or sequentially into the fresh mulberry juice, while the anthocyanin precursors and VPAs were identified and quantified via UHPLC-ESI/MS. The results showed that the HCDC-active strains facilitated the synthesis of stable pigments, cyanidin-3-O-glucoside-4-vinylcatechol (VPC3G), and cyanidin-3-O-rutinoside-4-vinylcatechol (VPC3R), highlighting its potential for enhancing color stability.

Identifying Key Residues in Lysine Decarboxylase for Soluble Expression Using Consensus Design Soluble Mutant Screening (ConsenSing).

Although recent advances in deep learning approaches for protein engineering have enabled quick prediction of hot spot residues improving protein solubility, the predictions do not always correspond to an actual increase in solubility under experimental conditions. Therefore, developing methods that rapidly confirm the linkage between computational predictions and empirical results is essential to the success of improving protein solubility of target proteins. Here, we present a simple hybrid approach to computationally predict hot spots possibly improving protein solubility by sequence-based analysis and empirically explore valuable mutants using split GFP as a reporter system. Our approach, Consensus design Soluble Mutant Screening (ConsenSing), utilizes consensus sequence prediction to find hot spots for improvement of protein solubility and constructs a mutant library using Darwin assembly to cover all possible mutations in one pot but still keeps the library as compact as possible. This approach allowed us to identify multiple mutants of Escherichia coli lysine decarboxylase, LdcC, with substantial increases in soluble expression. Further investigation led us to pinpoint a single critical residue for the soluble expression of LdcC and unveiled its mechanism for such improvement. Our approach demonstrated that following a protein's natural evolutionary path provides insights to improve protein solubility and/or increase protein expression by a single residue mutation, which can significantly change the profile of protein solubility.

Acid-tolerant bacteria and prospects in industrial and environmental applications.

Acid-tolerant bacteria such as Streptococcus mutans, Acidobacterium capsulatum, Escherichia coli, and Propionibacterium acidipropionici have developed several survival mechanisms to sustain themselves in various acid stress conditions. Some bacteria survive by minor changes in the environmental pH. In contrast, few others adapt different acid tolerance mechanisms, including amino acid decarboxylase acid resistance systems, mainly glutamate-dependent acid resistance (GDAR) and arginine-dependent acid resistance (ADAR) systems. The cellular mechanisms of acid tolerance include cell membrane alteration in Acidithiobacillus thioxidans, proton elimination by F1-F0-ATPase in Streptococcus pyogenes, biofilm formation in Pseudomonas aeruginosa, cytoplasmic urease activity in Streptococcus mutans, synthesis of the protective cloud of ammonia, and protection or repair of macromolecules in Bacillus caldontenax. Apart from cellular mechanisms, there are several acid-tolerant genes such as gadA, gadB, adiA, adiC, cadA, cadB, cadC, speF, and potE that help the bacteria to tolerate the acidic environment. This acid tolerance behavior provides new and broad prospects for different industrial applications and the bioremediation of environmental pollutants. The development of engineered strains with acid-tolerant genes may improve the efficiency of the transgenic bacteria in the treatment of acidic industrial effluents. KEY POINTS: • Bacteria tolerate the acidic stress by methylating unsaturated phospholipid tail • The activity of decarboxylase systems for acid tolerance depends on pH • Genetic manipulation of acid-tolerant genes improves acid tolerance by the bacteria.