Bacterial indole-3-lactic acid affects epithelium-macrophage crosstalk to regulate intestinal homeostasis.

Tryptophan and its derivatives perform a variety of biological functions; however, the role and specific mechanism of many tryptophan derivatives in intestinal inflammation remain largely unclear. Here, we identified that an Escherichia coli strain (Ec-TMU) isolated from the feces of tinidazole-treated individuals, and indole-3-lactic acid (ILA) in its supernatant, decreased the susceptibility of mice to dextran sulfate sodium-induced colitis. Ec-TMU and ILA contribute to the relief of colitis by inhibiting the production of epithelial CCL2/7, thereby reducing the accumulation of inflammatory macrophages in vitro and in vivo. Mechanistically, ILA downregulates glycolysis, NF-κB, and HIF signaling pathways via the aryl hydrocarbon receptor, resulting in decreased CCL2/7 production in epithelial cells. Clinical evidence suggests that the fecal ILA level is negatively correlated with the progression indicator of inflammatory bowel diseases. These results demonstrate that ILA has the potential to regulate intestinal homeostasis by modulating epithelium-macrophage interactions.

Graphene oxide-enhanced colorimetric detection of Mec A gene based on toehold-mediated strand displacement.

Mec A, as a representative gene mediating resistance to ß-lactam antibiotics in methicillin-resistant Staphylococcus aureus (MRSA), allows a new genetic analysis for the detection of MRSA. Here, a sensitive, prompt, and visual colorimetry is reported to detect the Mec A gene based on toehold-mediated strand displacement (TMSD) and the enrichment effect of graphene oxide (GO). The Mec A triggers to generate the profuse amount of signal units of single-stranded DNA (SG) composed of a long single-stranded base tail and a base head: the tail can be adsorbed and enriched on the surface of GO; the head can form a G quadruplex structure to exert catalytic function towards 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid). Therefore, through the enrichment effect of GO, the signal units SG reflects different degrees of signal amplification on different substrates (such as aqueous solution or filter membrane). This strategy demonstrates a broad linear working range from 100 pM to 1.5 nM (solution) and 1 pM to 1 nM (filter membrane), with a low detection limit of 39.53 pM (solution) and 333 fM (filter membrane). Analytical performance in real samples suggests that this developed colorimetry is endowed with immense potential for clinical detection applications.

Genomic population structure associated with repeated escape of Salmonella enterica ATCC14028s from the laboratory into nature.

Salmonella enterica serovar Typhimurium strain ATCC14028s is commercially available from multiple national type culture collections, and has been widely used since 1960 for quality control of growth media and experiments on fitness ("laboratory evolution"). ATCC14028s has been implicated in multiple cross-contaminations in the laboratory, and has also caused multiple laboratory infections and one known attempt at bioterrorism. According to hierarchical clustering of 3002 core gene sequences, ATCC14028s belongs to HierCC cluster HC20_373 in which most internal branch lengths are only one to three SNPs long. Many natural Typhimurium isolates from humans, domesticated animals and the environment also belong to HC20_373, and their core genomes are almost indistinguishable from those of laboratory strains. These natural isolates have infected humans in Ireland and Taiwan for decades, and are common in the British Isles as well as the Americas. The isolation history of some of the natural isolates confirms the conclusion that they do not represent recent contamination by the laboratory strain, and 10% carry plasmids or bacteriophages which have been acquired in nature by HGT from unrelated bacteria. We propose that ATCC14028s has repeatedly escaped from the laboratory environment into nature via laboratory accidents or infections, but the escaped micro-lineages have only a limited life span. As a result, there is a genetic gap separating HC20_373 from its closest natural relatives due to a divergence between them in the late 19th century followed by repeated extinction events of escaped HC20_373.

Helicobacter pylori's historical journey through Siberia and the Americas.

The gastric bacterium Helicobacter pylori shares a coevolutionary history with humans that predates the out-of-Africa diaspora, and the geographical specificities of H. pylori populations reflect multiple well-known human migrations. We extensively sampled H. pylori from 16 ethnically diverse human populations across Siberia to help resolve whether ancient northern Eurasian populations persisted at high latitudes through the last glacial maximum and the relationships between present-day Siberians and Native Americans. A total of 556 strains were cultivated and genotyped by multilocus sequence typing, and 54 representative draft genomes were sequenced. The genetic diversity across Eurasia and the Americas was structured into three populations: hpAsia2, hpEastAsia, and hpNorthAsia. hpNorthAsia is closely related to the subpopulation hspIndigenousAmericas from Native Americans. Siberian bacteria were structured into five other subpopulations, two of which evolved through a divergence from hpAsia2 and hpNorthAsia, while three originated though Holocene admixture. The presence of both anciently diverged and recently admixed strains across Siberia support both Pleistocene persistence and Holocene recolonization. We also show that hspIndigenousAmericas is endemic in human populations across northern Eurasia. The evolutionary history of hspIndigenousAmericas was reconstructed using approximate Bayesian computation, which showed that it colonized the New World in a single migration event associated with a severe demographic bottleneck followed by low levels of recent admixture across the Bering Strait.

Accurate reconstruction of bacterial pan- and core genomes with PEPPAN.

Bacterial genomes can contain traces of a complex evolutionary history, including extensive homologous recombination, gene loss, gene duplications, and horizontal gene transfer. To reconstruct the phylogenetic and population history of a set of multiple bacteria, it is necessary to examine their pangenome, the composite of all the genes in the set. Here we introduce PEPPAN, a novel pipeline that can reliably construct pangenomes from thousands of genetically diverse bacterial genomes that represent the diversity of an entire genus. PEPPAN outperforms existing pangenome methods by providing consistent gene and pseudogene annotations extended by similarity-based gene predictions, and identifying and excluding paralogs by combining tree- and synteny-based approaches. The PEPPAN package additionally includes PEPPAN_parser, which implements additional downstream analyses, including the calculation of trees based on accessory gene content or allelic differences between core genes. To test the accuracy of PEPPAN, we implemented SimPan, a novel pipeline for simulating the evolution of bacterial pangenomes. We compared the accuracy and speed of PEPPAN with four state-of-the-art pangenome pipelines using both empirical and simulated data sets. PEPPAN was more accurate and more specific than any of the other pipelines and was almost as fast as any of them. As a case study, we used PEPPAN to construct a pangenome of approximately 40,000 genes from 3052 representative genomes spanning at least 80 species of Streptococcus The resulting gene and allelic trees provide an unprecedented overview of the genomic diversity of the entire Streptococcus genus.

The EnteroBase user's guide, with case studies on Salmonella transmissions, Yersinia pestis phylogeny, and Escherichia core genomic diversity.

EnteroBase is an integrated software environment that supports the identification of global population structures within several bacterial genera that include pathogens. Here, we provide an overview of how EnteroBase works, what it can do, and its future prospects. EnteroBase has currently assembled more than 300,000 genomes from Illumina short reads from Salmonella, Escherichia, Yersinia, Clostridioides, Helicobacter, Vibrio, and Moraxella and genotyped those assemblies by core genome multilocus sequence typing (cgMLST). Hierarchical clustering of cgMLST sequence types allows mapping a new bacterial strain to predefined population structures at multiple levels of resolution within a few hours after uploading its short reads. Case Study 1 illustrates this process for local transmissions of Salmonella enterica serovar Agama between neighboring social groups of badgers and humans. EnteroBase also supports single nucleotide polymorphism (SNP) calls from both genomic assemblies and after extraction from metagenomic sequences, as illustrated by Case Study 2 which summarizes the microevolution of Yersinia pestis over the last 5000 years of pandemic plague. EnteroBase can also provide a global overview of the genomic diversity within an entire genus, as illustrated by Case Study 3, which presents a novel, global overview of the population structure of all of the species, subspecies, and clades within Escherichia.

Genome-wide dissection reveals diverse pathogenic roles of bacterial Tc toxins.

Tc toxins were originally identified in entomopathogenic bacteria, which are important as biological pest control agents. Tc toxins are heteromeric exotoxins composed of three subunit types, TcA, TcB, and TcC. The C-terminal portion of the TcC protein encodes the actual toxic domain, which is translocated into host cells by an injectosome nanomachine comprising the other subunits. Currently the pathogenic roles and distribution of Tc toxins among different bacterial genera remain unclear. Here we have performed a comprehensive genome-wide analysis, and established a database that includes 1,608 identified Tc loci containing 2,528 TcC proteins in 1,421 Gram-negative and positive bacterial genomes. Our findings indicate that TcCs conform to the architecture of typical polymorphic toxins, with C-terminal hypervariable regions (HVR) encoding more than 100 different classes of putative toxic domains, most of which have not been previously recognized. Based on further analysis of Tc loci in the genomes of all Salmonella and Yersinia strains in EnteroBase, a "two-level" evolutionary dynamics scenario is proposed for TcC homologues. This scenario implies that the conserved TcC RHS core domain plays a critical role in the taxonomical specific distribution of TcC HVRs. This study provides an extensive resource for the future development of Tc toxins as valuable agrochemical tools. It furthermore implies that Tc proteins, which are encoded by a wide range of pathogens, represent an important versatile toxin superfamily with diverse pathogenic mechanisms.

Stepwise genetic modification for efficient expression of heterologous proteins in Aspergillus nidulans.

Filamentous fungi are widely used in food fermentation and therapeutic protein production due to their prominent protein secretion and post-translational modification system. Aspergillus nidulans is an important model strain of filamentous fungi, but not a fully developed cell factory for heterologous protein expression. One of the limitations is its relatively low capacity of protein secretion. To alleviate this limitation, in this study, the protein secretory pathway and mycelium morphology were stepwise modified. With eGFP as a reporter protein, protein secretion was significantly enhanced through reducing the degradation of heterologous proteins by endoplasmic reticulum-associated protein degradation (ERAD) and vacuoles in the secretory pathway. Elimination of mycelial aggregation resulted in a 1.5-fold and 1.3-fold increase in secretory expression of eGFP in typical constitutive and inducible expression systems, respectively. Combined with these modifications, high secretory expression of human interleukin-6 (HuIL-6) was achieved. Consequently, a higher yield of secretory HuIL-6 was realized by further disruption of extracellular proteases. Overall, a superior chassis cell of A. nidulans suitable for efficient secretory expression of heterologous proteins was successfully obtained, providing a promising platform for biosynthesis using filamentous fungi as hosts. KEY POINTS: • Elimination of mycelial aggregation and decreasing the degradation of heterologous protein are effective strategies for improving the heterologous protein expression. • The work provides a high-performance chassis host △agsB-derA for heterologous protein secretory expression. • Human interleukin-6 (HuIL-6) was expressed efficiently in the high-performance chassis host △agsB-derA.

Development of a base editor for protein evolution via in situ mutation in vivo.

Protein evolution has significantly enhanced the development of life science. However, it is difficult to achieve in vitro evolution of some special proteins because of difficulties with heterologous expression, purification, and function detection. To achieve protein evolution via in situ mutation in vivo, we developed a base editor by fusing nCas with a cytidine deaminase in Bacillus subtilis through genome integration. The base editor introduced a cytidine-to-thymidine mutation of approximately 100% across a 5 nt editable window, which was much higher than those of other base editors. The editable window was expanded to 8 nt by extending the length of sgRNA, and conversion efficiency could be regulated by changing culture conditions, which was suitable for constructing a mutant protein library efficiently in vivo. As proof-of-concept, the Sec-translocase complex and bacitracin-resistance-related protein BceB were successfully evolved in vivo using the base editor. A Sec mutant with 3.6-fold translocation efficiency and the BceB mutants with different sensitivity to bacitracin were obtained. As the construction of the base editor does not rely on any additional or host-dependent factors, such base editors (BEs) may be readily constructed and applicable to a wide range of bacteria for protein evolution via in situ mutation.

Discovery of the ATPase Activity of a Cobalt-Type Nitrile Hydratase Activator and Its Promoting Effect on Enzyme Maturation.

An activator protein and a metal ion are two factors known to be indispensable for the maturation of nitrile hydratase (NHase). Here, the third key factor, adenosine triphosphate (ATP), was identified to play an important role in the activation of Co-type NHase. Free phosphate measurements revealed that the Co-type activator protein can hydrolyze ATP/GTP with appreciable performance and that such catalytic performance is related to NHase activity. Computational analysis and site-directed mutagenesis identified several potential hot spot residues involved in the binding of ATP to Co-type activator protein, and an E60A/W61A/D62A/I139A/T141A combinatorial variant reduced the ATPase activity to 18% of its original level. Further NHase activation studies using the combinatorial variant demonstrated that although the ATPase activity of the Co-type activator protein correlated with NHase activity, a low ATP concentration of 0.5 mmol/L was optimal for NHase activation, with higher ATP concentrations potentially inhibiting NHase activity.

HierCC: a multi-level clustering scheme for population assignments based on core genome MLST.

MOTIVATION: Routine infectious disease surveillance is increasingly based on large-scale whole-genome sequencing databases. Real-time surveillance would benefit from immediate assignments of each genome assembly to hierarchical population structures. Here we present pHierCC, a pipeline that defines a scalable clustering scheme, HierCC, based on core genome multi-locus typing that allows incremental, static, multi-level cluster assignments of genomes. We also present HCCeval, which identifies optimal thresholds for assigning genomes to cohesive HierCC clusters. HierCC was implemented in EnteroBase in 2018 and has since genotyped >530 000 genomes from Salmonella, Escherichia/Shigella, Streptococcus, Clostridioides, Vibrio and Yersinia. AVAILABILITY AND IMPLEMENTATION: https://enterobase.warwick.ac.uk/ and Source code and instructions: https://github.com/zheminzhou/pHierCC. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Correction: Multilocus Sequence Typing as a Replacement for Serotyping in Salmonella enterica.

[This corrects the article DOI: 10.1371/journal.ppat.1002776.].

Evaluation of Metabolic Engineering Strategies on 2-Ketoisovalerate Production by Escherichia coli.

As an important metabolic intermediate, 2-ketoisovalerate has significant potential in the pharmaceutical and biofuel industries. However, a low output through microbial fermentation inhibits its industrial application. The microbial production of 2-ketoisovalerate is representative whereby redox imbalance is generated with two molecules of NADH accumulated and an extra NADPH required to produce one 2-ketoisovalerate from glucose. To achieve efficient 2-ketoisovalerate production, metabolic engineering strategies were evaluated in Escherichia coli. After deleting the competing routes, overexpressing the key enzymes for 2-ketoisovalerate production, tuning the supply of NADPH, and recycling the excess NADH through enhancing aerobic respiration, a 2-ketoisovalerate titer and yield of 46.4 g/L and 0.644 mol/mol glucose, respectively, were achieved. To reduce the main by-product of isobutanol, the activity and expression of acetolactate synthase were modified. Additionally, a protein degradation tag was fused to pyruvate dehydrogenase (PDH) to curtail the conversion of pyruvate precursor into acetyl-CoA and the generation of NADH. The resulting strain, 050TY/pCTSDTQ487S-RBS55, was initially incubated under aerobic conditions to attain sufficient cell mass and then transferred to a microaerobic condition to degrade PDH and inhibit the remaining activity of PDH. Intracellular redox imbalance was relieved with titer, productivity and yield of 2-ketoisovalerate improved to 55.8 g/L, 2.14 g/L h and 0.852 mol/mol glucose. These results revealed metabolic engineering strategies for the production of a redox-imbalanced fermentative metabolite with high titer, productivity, and yield. IMPORTANCE An efficient microbial strain was constructed for 2-ketoisovalerate synthesis. The positive effect of the leuA deletion on 2-ketoisovalerate production was found. An optimal combination of overexpressing the target genes was obtained by adjusting the positions of the multiple enzymes on the plasmid frame and the presence of terminators, which could also be useful for the production of downstream products such as isobutanol and l-valine. Reducing the isobutanol by-product by engineering the acetolactate synthase called for special attention to decreasing the promiscuous activity of the enzymes involved. Redox-balancing strategies such as tuning the expression of the chromosomal pyridine nucleotide transhydrogenase, recycling NADH under aerobic cultivation, switching off PDH by degradation, and inhibiting the expression and activity under microaerobic conditions were proven effective for improving 2-ketoisovalerate production. The degradation of PDH and inhibiting this enzyme's expression would serve as a means to generate a wide range of products from pyruvate.

Characteristics of population structure, antimicrobial resistance, virulence factors, and morphology of methicillin-resistant Macrococcus caseolyticus in global clades.

Macrococcus caseolyticus is an opportunistic pathogen that is frequently isolated from dairy products and veterinary infections. Recent studies have reported the possibility of methicillin resistance that be transferred among staphylococcal species in foods. The present study examined the population structure, antimicrobial resistance, virulence factors, and morphology of methicillin-resistant M. caseolyticus by investigation of 94 genomes derived from both isolates in beef (n = 7) and pork (n = 2) at Shanghai and those deposited in public domain (n = 85). Phylogenetically, M. caseolyticus were divided into four clades, which each consisted of genomes isolated from continent of European countries (82.4%, n = 78), Asian countries (11.3%, n = 10), United States (4.1%, n = 4), Australia (1%, n = 1), and Sudan (1%, n = 1). The M. caseolyticus isolated from present study formed a genetically distinguished clade, which was characterized by novel alleles in the traditional 7-gene MLST scheme. Furthermore, we identified 24 AMR genes that were associated with 10 classes of antimicrobial agents in M. caseolyticus. Most AMR genes were carried by dominant plasmids such as rep7a, rep22 and repUS56. The genomes in the global clades carried significantly less AMR genes (p < 0.05) and more virulence factors (p < 0.001) than present clade. Virulence factors were detected in methicillin resistant M. caseolyticus including genes coding hemolysin, adherence, biofilm formation, exotoxin, and capsule that associated to human health and infection. Finally, as the close relative of the genus Staphylococcus, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were performed for morphological comparison that M. caseolyticus has a larger diameter and thicker cell wall compared with S. aureus ATCC 25,923. Taken together, our study suggested that M. caseolyticus mediating divergent antimicrobial resistance and virulence factors could serve as the vector for methicillin resistance habitats in foodborne microorganisms.

Development of an auto-inducible expression system by nitrogen sources switching based on the nitrogen catabolite repression regulation.

BACKGROUND: The construction of protein expression systems is mainly focused on carbon catabolite repression and quorum-sensing systems. However, each of these regulatory modes has an inherent flaw, which is difficult to overcome. Organisms also prioritize using different nitrogen sources, which is called nitrogen catabolite repression. To date, few gene regulatory systems based on nitrogen catabolite repression have been reported. RESULTS: In this study, we constructed a nitrogen switching auto-inducible expression system (NSAES) based on nitrogen catabolite regulation and nitrogen utilization in Aspergillus nidulans. The PniaD promoter that is highly induced by nitrate and inhibition by ammonia was used as the promoter. Glucuronidase was the reporter protein. Glucuronidase expression occurred after ammonium was consumed in an ammonium and nitrate compounding medium, achieving stage auto-switching for cell growth and gene expression. This system maintained a balance between cell growth and protein production to maximize stress products. Expressions of glycosylated and secretory proteins were successfully achieved using this auto-inducible system. CONCLUSIONS: We described an efficient auto-inducible protein expression system based on nitrogen catabolite regulation. The system could be useful for protein production in the laboratory and industrial applications. Simultaneously, NSAES provides a new auto-inducible expression regulation mode for other filamentous fungi.

Exploration of key residues and conformational change of anti-terminator protein GlpP for ligand and RNA binding.

Anti-terminator protein GlpP regulates gene expression of glycerol uptake operon at post-transcriptional level in a number of bacteria. By now, the molecular dynamics details of ligand and RNA binding by GlpP are still obscure. In this study, we employed the molecular dynamic (MD) simulation and constructed a functional verification platform of GlpP to resolve these puzzles. By combining molecular docking, MD simulation and alanine scanning mutagenesis, a ligand binding pocket consisting of R14, R104 and R157 was identified. Among these residues with positive charge, R14 was dominant for binding glycerol-3-phosphate (G3P). Moreover, the "parallel to crossed" conformational change of the predicted RNA binding region was observed in MD simulation. In this process, the interaction between R104 and E129 was crucial to trigger the conformational change. To further verify this speculation, three ligand independent mutants were obtained by error-prone PCR. The MD simulation indicated that the conformational change happened in all the three mutants, confirming the "parallel to crossed" conformational change endowed GlpP the activity of binding RNA. In recent years, as a potable biological part, anti-terminator was more and more widely used to regulate gene expression in metabolic engineering and synthetic biology. The work in this study deepened our understanding to the typical anti-terminator GlpP, contributing to the further engineering and application of this type of regulator.

Substrate access tunnel engineering for improving the catalytic activity of a thermophilic nitrile hydratase toward pyridine and pyrazine nitriles.

Nitrile hydratase (NHase) is able to bio-transform nitriles into amides. As nitrile hydration being an exothermic reaction, a NHase with high activity and stability is needed for amide production. However, the widespread use of NHase for amide bio-production is limited by an activity-stability trade-off. In this study, through the combination of substrate access tunnel calculation, residue conservative analysis and site-saturation mutagenesis, a residue located at the substrate access tunnel entrance of the thermophilic NHase from extremophile Caldalkalibacillus thermarum TA2. A1, ßLeu48, was semi-rationally identified as a potential gating residue that directs the enzymatic activity toward various pyridine and pyrazine nitriles. The specific activity of the corresponding mutant ßL48H towards 3-cyanopyridine, 2-cyanopyridine and cyanopyrazine were 2.4-fold, 2.8-fold and 3.1-fold higher than that of its parent enzyme, showing a great potential in the industrial production of high-value pyridine and pyrazine carboxamides. Further structural analysis demonstrated that the ßHis48 could form a long-lasting hydrogen bond with αGlu166, which contributes to the expansion of the entrance of substrate access tunnel and accelerate substrate migration.

GrapeTree: visualization of core genomic relationships among 100,000 bacterial pathogens.

Current methods struggle to reconstruct and visualize the genomic relationships of large numbers of bacterial genomes. GrapeTree facilitates the analyses of large numbers of allelic profiles by a static "GrapeTree Layout" algorithm that supports interactive visualizations of large trees within a web browser window. GrapeTree also implements a novel minimum spanning tree algorithm (MSTree V2) to reconstruct genetic relationships despite high levels of missing data. GrapeTree is a stand-alone package for investigating phylogenetic trees plus associated metadata and is also integrated into EnteroBase to facilitate cutting edge navigation of genomic relationships among bacterial pathogens.

Nitroreductase Increases Menadione-Mediated Oxidative Stress in Aspergillus nidulans.

Nitroreductases (NTRs) catalyze the reduction of a wide range of nitro-compounds and quinones using NAD(P)H. Although the physiological functions of these enzymes remain obscure, a tentative function of resistance to reactive oxygen species (ROS) via the detoxification of menadione has been proposed. This suggestion is based primarily on the transcriptional or translational induction of an NTR response to menadione rather than on convincing experimental evidence. We investigated the performance of a fungal NTR from Aspergillus nidulans (AnNTR) exposed to menadione to address the question of whether NTR is really an ROS defense enzyme. We confirmed that AnNTR was transcriptionally induced by external menadione. We observed that menadione treatment generated cytotoxic levels of O2•-, which requires well-known antioxidant enzymes such as superoxide dismutase, catalase, and peroxiredoxin to protect A. nidulans against menadione-derived ROS stress. However, AnNTR was counterproductive for ROS defense, since knocking out AnNTR decreased the intracellular O2•- levels, resulting in fungal viability higher than that of the wild type. This observation implies that AnNTR may accelerate the generation of O2•- from menadione. Our in vitro experiments indicated that AnNTR uses NADPH to reduce menadione in a single-electron reaction, and the subsequent semiquinone-quinone redox cycling resulted in O2•- generation. We demonstrated that A. nidulans nitroreductase should be an ROS generator, but not an ROS scavenger, in the presence of menadione. Our results clarified the relationship between nitroreductase and menadione-derived ROS stress, which has long been ambiguous. IMPORTANCE Menadione is commonly used as an O2•- generator in studies of oxidative stress responses. However, the precise mechanism through which menadione mediates cellular O2•- generation, as well as the way in which cells respond, remains unclear. Elucidating these events will have important implications for the use of menadione in biological and medical studies. Our results show that the production of Aspergillus nidulans nitroreductase (AnNTR) was induced by menadione. However, the accumulated AnNTR did not protect cells but instead increased the cytotoxic effect of menadione through a single-electron reduction reaction. Our finding that nitroreductase is involved in the menadione-mediated O2•- generation pathway has clarified the relationship between nitroreductase and menadione-derived ROS stress, which has long been ambiguous.

Characterization and rational modification of aspartate 4-decarboxylase from Acinetobacter radioresistens for the production of l-alanine.

Enzymatic synthesis of l-alanine has the advantages of less byproducts, strong stereoselectivity, and high catalytic efficiency. Aspartate 4-decarboxylase (ASD) is used industrially in DL-aspartic acid resolution and l-alanine production because it catalyzes the decarboxylation of l-aspartic acid. In this study, the ASD gene from Acinetobacter radioresistens (ArASD) was cloned, and its enzymatic properties were analyzed. ArASD is a dodecamer and has the highest enzyme activity ever reported to date. The optimal conditions for ArASD catalysis are 50°C and pH 4.5. Site-directed mutagenesis was used to improve ArASD stability under acidic conditions to compensate for its weak acid resistance, and the variant N35D with higher catalytic ability was obtained. The conversion by N35 recombinant cells of l-aspartic acid to l-alanine was 92.5% at pH 4.5% and 99.9% at pH 6.0, whereas that of the wild-type recombinant cells was 29.7% and 31.4%, respectively. Aspartase from Escherichia coli (AspA) was employed with ArASD to construct a dual-enzyme system that catalyzes fumaric acid to l-alanine, and the conversion reached 97.1% using recombinant cells harboring the dual-enzyme system. This study explored the enzymatic properties of ArASD and an effective strategy for the acidic resistance modification of ASD. Moreover, the strain expressing the ArASD variant and AspA engineered in this study has great potential application for the l-alanine production industry, especially in the case of high optical purity requirements.