Riboflavin is a crucial micronutrient that is a precursor to coenzymes flavin mononucleotide and flavin adenine dinucleotide, and it is required for biochemical reactions in all living cells. For decades, one of the most important applications of riboflavin has been its global use as an animal and human nutritional supplement. Being well-informed of the latest research on riboflavin production via the fermentation process is necessary for the development of new and improved microbial strains using biotechnology and metabolic engineering techniques to increase vitamin B2 yield. In this review, we describe well-known industrial microbial producers, namely, Ashbya gossypii , Bacillus subtilis , and Candida spp. and summarize their biosynthetic pathway optimizations through genetic and metabolic engineering, combined with random chemical mutagenesis and rational medium components to increase riboflavin production.
This review focuses on the emerging role of site-specific mutagenesis and chimeragenesis for the functional improvement of proteins in areas where traditional protein engineering methods have been extensively used and practically exhausted. The novel path for the creation of the novel proteins has been created on the farther development of the new structure and sequence optimization algorithms for generating and designing the accurate structure models in result of x-ray crystallography studies of a lot of proteins and their mutant forms. Artificial genetic modifications aim to expand nature's repertoire of biomolecules. One of the most exciting potential results of mutagenesis or chimeragenesis finding could be design of effective diagnostics, bio-therapeutics and biocatalysts. A sampling of recent examples is listed below for the in vivo and in vitro genetically improvement of various binding protein and enzyme functions, with references for more in-depth study provided for the reader's benefit.
Data is presented in support of functionality of hyper-diverse protein families encoded by the Cobetia amphilecti KMM 296 (formerly Cobetia marina KMM 296) genome (“The genome of the marine bacterium Cobetia marina KMM 296 isolated from the mussel Crenomytilus grayanus (Dunker, 1853)” [1]) providing its nutritional versatility, adaptability and biocontrol that could be the basis of the marine bacterium evolutionary and application potential. Presented data include the information of growth and biofilm-forming properties of the food-associated isolates of Pseudomonas, Bacillus, Listeria, Salmonella and Staphylococcus under the conditions of their co-culturing with C. amphilecti KMM 296 to confirm its high inter-species communication and anti-microbial activity. Also included are the experiments on the crude petroleum consumption by C. amphilecti KMM 296 as the sole source of carbon in the presence of sulfate or nitrate to ensure its bioremediation capacity. The multifunctional C. amphilecti KMM 296 genome is a promising source for the beneficial psychrophilic enzymes and essential secondary metabolites.
The biofilm-producing strains of P. aeruginosa colonize various surfaces, including food products and industry equipment that can cause serious human and animal health problems. The biofilms enable microorganisms to evolve the resistance to antibiotics and disinfectants. Analysis of the P. aeruginosa strain (serotype O6, sequence type 2502), isolated from an environment of meat processing (PAEM) during a ready-to-cook product storage (−20 °C), showed both the mosaic similarity and differences between free-living and clinical strains by their coding DNA sequences. Therefore, a cold shock protein (CspA) has been suggested for consideration of the evolution probability of the cold-adapted P. aeruginosa strains. In addition, the study of the action of cold-active enzymes from marine bacteria against the food-derived pathogen could contribute to the methods for controlling P. aeruginosa biofilms. The genes responsible for bacterial biofilm regulation are predominantly controlled by quorum sensing, and they directly or indirectly participate in the synthesis of extracellular polysaccharides, which are the main element of the intercellular matrix. The levels of expression for 14 biofilm-associated genes of the food-derived P. aeruginosa strain PAEM in the presence of different concentrations of the glycoside hydrolase of family 36, α-galactosidase α-PsGal, from the marine bacterium Pseudoalteromonas sp. KMM 701 were determined. The real-time PCR data clustered these genes into five groups according to the pattern of positive or negative regulation of their expression in response to the action of α-galactosidase. The results revealed a dose-dependent mechanism of the enzymatic effect on the PAEM biofilm synthesis and dispersal genes.
An efficient Agrobacterium-mediated genetic transformation based on the plant binary vector pPZP-RCS2 was carried out for the multiple heterologous protein production in filamentous fungus Thermothelomyces thermophilus F-859 (formerly Myceliophthora thermophila F-859). The engineered fungus Th. thermophilus was able to produce plant storage proteins of Zea mays (α-zein Z19) and Amaranthus hypochondriacus (albumin A1) to enrich fungal biomass by valuable nutritional proteins and improved amino acid content. The mRNA levels of z19 and a1 genes were significantly dependent on their driving promoters: the promoter of tryptophan synthase (PtrpC) was more efficient to express a1, while the promoter of translation elongation factor (Ptef) provided much higher levels of z19 transcript abundance. In general, the total recombinant proteins and amino acid contents were higher in the Ptef-containing clones. This work describes a new strategy to improve mycoprotein nutritive value by overexpression of plant storage proteins.
A plasmid based on pET-40b was constructed to synthesize recombinant α-N-acetylgalactosaminidase of the marine bacterium Arenibacter latericius KMM 426T (α-AlNaGal) in Escherichia coli cells. The yield of α-Al- NaGal attains 10 mg/ml with activity of 49.7 ± 1.3 U at 16°C, concentration of inductor 2 mM, and cultivation for 12 h. Techniques such as anion exchange, metal affinity and gel filtration chromatography to purify α-AlNaGal were applied. α-AlNaGal is a homodimer with a molecular weight of 164 kDa. This enzyme is stable at up to 50°C with a temperature range optimum activity of 20–37°C. Furthermore, its activity is independent of the presence of metal ions in the incubation medium. 1H NMR spectroscopy revealed that α-AlNaGal catalyzes the hydrolysis of the O-glycosidic bond with retention of anomeric stereochemistry and possesses a mechanism of action identical to that of other glycoside hydrolases of the 109 family. α-AlNaGal reduces the serological activity of A erythrocytes at pH 7.3. This property of α-AlNaGal can potentially be used for enzymatic conversion of A and AB erythrocytes to blood group O erythrocytes.
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