Promoting Cell Growth And Poly-Y-Glutamic Acid Production By Boosting The Synthesis of Alanine And D-Alanyl-D-Alanine In Bacillus Licheniformis

Objective: The production of some bio-chemicals affected by the cell growth. This study aimed at promoting the cell growth by overexpressing the synthesis of peptidoglycans tetrapeptide tail components to improve poly-γ-glutamic acid (γ-PGA) production. Results: L-alanine, D-alanine and D-alanyl-D-alanine are primary precursors for the synthesis of peptidoglycans. The addition of L-alanine and D-alanine significantly increased both the cell growth and production of γ-PGA. Then, several genes encoding key enzymes for L/D-alanine and D-alanyl-D-alanine biosynthesis were overexpressed respectively, including ald (encoding alanine dehydrogenase), dal (encoding alanine racemase) and ddl (encoding D-alanine ligase). The results showed that the overexpression of genes ald , dal and ddl increased the production of γ-PGA by 19.72%, 15.91% and 60.90%, and increased the microbial biomass by 15.58%, 18.34% and 49.85%, respectively. Moreover, we demonstrated that the overexpression of genes ald , dal and ddl increased γ-PGA production mainly by enhancing cell growth rather than providing more precursors. Conclusions: This work illustrated the importance of the L/D-alanine and D-alanyl-D-alanine synthesis to the cell growth and the high yield of γ-PGA, and provided an effective strategy for producing γ-PGA .

Production of d-Tagatose by Whole-Cell Conversion of Recombinant Bacillus subtilis in the Absence of Antibiotics

d-tagatose is a popular functional monosaccharide produced from lactose by β-galactosidase and arabinose isomerase. In this study, two d-alanine-deficient heterologous gene expression systems were constructed, B. subtilis 168 D1 and B. subtilis 168 D2, using overlapping extension PCR and the CRE/loxP system. The lacZ gene for β-galactosidase was integrated into a specific locus of the chassis B. subtilis 168 D2. A mutually complementary plasmid pMA5 with the alanine racemase gene alrA attached to it was constructed and used to assemble recombinant plasmids overexpressing β-galactosidase and arabinose isomerase. Afterward, an integrated recombinant was constructed by the plasmid expressing the arabinose isomerase gene araA of E. coli transform-competent B. subtilis 168 D2 cells. The co-expressing plasmids were introduced into alanine racemase knockout B. subtilis 168 D1. Whole-cell bioconversion was performed using the integrated recombinant with a maximum yield of 96.8 g/L d-tagatose from 500 g/L lactose, and the highest molar conversions were 57.2%. B. subtilis 168 D1/pMA5-alrA-araA-lacZ is capable of single-cell one-step production of d-tagatose. This study provides a new approach to the production of functional sugars.

Immunogenicity of Recombinant-Deficient Lactobacillus casei with Complementary Plasmid Expressing Alanine Racemase Gene and Core Neutralizing Epitope Antigen against Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea (PED), which is caused by the porcine epidemic diarrhea virus (PEDV), has occurred worldwide and poses a serious threat to the pig industry. Intestine is the main function site of PEDV; therefore, it is important to develop an oral mucosal immunity vaccine against this virus infection. Most traditional plasmid delivery vectors use antibiotic genes as a selective marker, easily leading to antibiotic accumulation and gene contamination. In this study, to explore whether the alanine racemase gene (Alr) could be used as a screening marker and develop an efficient oral vaccine against PEDV infection, a recombinant strain was constructed using Lactobacillus casei with Alr deletion (L. casei ΔAlr W56) to deliver the Alr gene and a core-neutralizing epitope (COE) antigen. This recombinant bacterium efficiently induced secretory immunoglobulin A (SIgA)-based mucosal and immunoglobulin G (IgG)-based humoral immune responses via oral vaccination in mice. Compared to the other strains, the recombinant bacteria were able to grow without the addition of D-alanine, revealing that Alr in the plasmid could function normally in defective bacteria. This oral mucosal vaccine would provide a useful strategy to substitute the application of antibiotics in the future and induce efficient immune responses against PEDV infection.

Enzymatic determination of D-alanine with L-alanine dehydrogenase and alanine racemase

An enzymatic assay system of D-Ala, which is reported to affect the taste, was constructed using alanine racemase and L-alanine dehydrogenase. D-Ala is converted to L-Ala by alanine racemase and then deaminated by L-alanine dehydrogenase with the reduction of NAD+ to NADH, which is determined with water-soluble tetrazolium. Using the assay system, the D-Ala contents of seven crustaceans were determined.

Metabolic Engineering of Bacteria for Food Ingredients

Bacteria produces metabolites and their metabolic pathway can be manipulated to produce good food ingredients. These ingredients includes different amino acids, vitamins, carbohydrates, organic acids aroma compounds and bacteriocins. Different genes in different strains of bacteria are identified and harnessed to attain food ingredients and results of manipulation were satisfying. The metabolic pathway of lactic acid bacteria is manipulated to produce amino acids such as L-alanine was produced by silencing the gene that encodes alanine racemase. Moreover, low calorie sugars such as mannitol and tagatose were produced. Lac-lactis is engineered by silencing lacC and lacD genes for the production of tagatose.

Enzymatic and Structural Characterization of Alanine Racemase from Enterococcus faecium by Kinetic and Computational Studies

The surge in vancomycin-resistant enterococci (VRE) strains poses a serious threat to public and clinical health. VRE strains are the leading cause of multi-drug resistant enterococcal infections and are commonly acquired from medical devices. Therefore, it is essential to discover new antibacterial targets and drugs for this pathogen. Alanine racemase could be a valuable drug target due to its crucial role in E. faecium survival. Alr from E. faecium (EF_Alr) was heterologously produced and purified from E. coli., and the steady-state kinetic constants were determined at different pH values. Using a coupled reaction with L-alanine dehydrogenase, rate of production of NADH was measured at 340 nm to observe EF_Alr activity in the D- to L-alanine direction. The highest catalytic efficiency, 8.61 ± 0.5 s-1 mM-1, was found at pH 9. Additionally, the tentative active site residues, Lys40 and Tyr268, for the alanine racemization reaction were assigned by homology modeling and sequence comparison studies. Using UCSF Chimera, the structure of the EF_Alr homology model was superimposed and compared to the crystal structure of Alr from E. faecalis. KEYWORDS: Alanine Racemase; Enterococcus faecium; Vancomycin-Resistant Enterococci; Homology Modeling; pH Optimum; Kinetic Characterization; SWISS-MODEL Server; Steady-State Kinetics

Three-dimensional biofilm colony growth supports a mutualism involving matrix and nutrient sharing

Life in a three-dimensional biofilm is typical for many bacteria, yet little is known about how strains interact in this context. Here, we created essential gene CRISPR interference knockdown libraries in biofilm-forming Bacillus subtilis and measured competitive fitness during colony co-culture with wild type. Partial knockdown of some translation-related genes reduced growth rates and led to out-competition. Media composition led some knockdowns to compete differentially as biofilm versus non-biofilm colonies. Cells depleted for the alanine racemase AlrA died in monoculture but survived in a biofilm colony co-culture via nutrient sharing. Rescue was enhanced in biofilm colony co-culture with a matrix-deficient parent due to a mutualism involving nutrient and matrix sharing. We identified several examples of mutualism involving matrix sharing that occurred in three-dimensional biofilm colonies but not when cultured in two dimensions. Thus, growth in a three-dimensional colony can promote genetic diversity through sharing of secreted factors and may drive evolution of mutualistic behavior.

Interactions of Bacillus subtilis Basement Spore Coat Layer Proteins

Bacillus subtilis endospores are exceptionally resistant cells encircled by two protective layers: a petidoglycan layer, termed the cortex, and the spore coat, a proteinaceous layer. The formation of both structures depends upon the proper assembly of a basement coat layer, which is composed of two proteins, SpoIVA and SpoVM. The present work examines the interactions of SpoIVA and SpoVM with coat proteins recruited to the spore surface during the early stages of coat assembly. We showed that the alanine racemase YncD associates with two morphogenetic proteins, SpoIVA and CotE. Mutant spores lacking the yncD gene were less resistant against wet heat and germinated to a greater extent than wild-type spores in the presence of micromolar concentrations of l-alanine. In seeking a link between the coat and cortex formation, we investigated the interactions between SpoVM and SpoIVA and the proteins essential for cortex synthesis and found that SpoVM interacts with a penicillin-binding protein, SpoVD, and we also demonstrated that SpoVM is crucial for the proper localization of SpoVD. This study shows that direct contacts between coat morphogenetic proteins with a complex of cortex-synthesizing proteins could be one of the tools by which bacteria couple cortex and coat formation.

Inhibition of Campylobacter jejuni Biofilm Formation by D-Amino Acids

The ability of bacterial pathogens to form biofilms is an important virulence mechanism in relation to their pathogenesis and transmission. Biofilms play a crucial role in survival in unfavorable environmental conditions, acting as reservoirs of microbial contamination and antibiotic resistance. For intestinal pathogen Campylobacter jejuni, biofilms are considered to be a contributing factor in transmission through the food chain and currently, there are no known methods for intervention. Here, we present an unconventional approach to reducing biofilm formation by C. jejuni by the application of D-amino acids (DAs), and L-amino acids (LAs). We found that DAs and not LAs, except L-alanine, reduced biofilm formation by up to 70%. The treatment of C. jejuni cells with DAs changed the biofilm architecture and reduced the appearance of amyloid-like fibrils. In addition, a mixture of DAs enhanced antimicrobial efficacy of D-Cycloserine (DCS) up to 32% as compared with DCS treatment alone. Unexpectedly, D-alanine was able to reverse the inhibitory effect of other DAs as well as that of DCS. Furthermore, L-alanine and D-tryptophan decreased transcript levels of peptidoglycan biosynthesis enzymes alanine racemase (alr) and D-alanine-D-alanine ligase (ddlA) while D-serine was only able to decrease the transcript levels of alr. Our findings suggest that a combination of DAs could reduce biofilm formation, viability and persistence of C. jejuni through dysregulation of alr and ddlA.