Heterologous Expression and Rational Design of l-asparaginase from Rhizomucor miehei to Improve Thermostability

l-asparaginase (EC 3.5.1.1) hydrolyzes l-asparagine to produce l-aspartate and ammonia and is widely found in microorganisms, plants, and some rodent sera. l-asparaginase used for industrial production should have good thermostability. We heterologously expressed l-asparaginase from Rhizomucor miehei, selected nine loci for site-directed mutagenesis by rational design, and obtained two mutants with significantly improved thermostability. The optimal temperature of mutants S302I and S302M was 50 °C. After incubating the mutant and wild-type enzymes at 45 °C for 35 h, the residual activity of the wild-type enzyme (WT) was only about 10%. In contrast, the residual activity of S302I and S302M was more than 50%. After combination mutagenesis, Bacillus subtilis 168-pMA5-A344E/S302I was constructed using the food-safe host strain B. subtilis 168. Additionally, a 5′ untranslated region (UTR) modification strategy was adopted to enhance the expression level of R. miehei-derived l-asparaginase in B. subtilis. In a 5-L fermenter scale-up experiment, the enzyme activity of recombinant B. subtilis 168-pMA5-UTR-A344E/S302I reached 521.9 U·mL−1 by fed-batch fermentation.

Categorization of prophage genes in Bacillus subtilis 168 and assessing their relative importance through RNA-seq gene expression analysis

Bacteriophage evolves to control the population of fast-growing bacterial cells, without which explosion in bacterial population may induce unimaginable harm to diverse ecosystems. But, bacteriophage also hide in bacterial genomes when nutritional and environmental circumstances are unfavourable. This involves the integration of phage genome into the host genome at appropriate genomic loci in a process known as lysogeny. This work sought to delineate the prophages present in the annotated genome of Bacillus subtilis 168, and assess their relative importance through RNA-seq expression analysis. Firstly, examination of the annotated genome of the model Gram-positive bacterium revealed five distinct prophage regions: SPBeta, prophage 6, PBSX, prophage 3 region, and prophage 1 region. All prophage regions contain host genes, which suggests that host transposase activity have swapped in host genes for phage genes in the prophage genome. Given the significant number of phage genes that have been swapped into each of the prophage genome, all prophage regions are deemed to be defective. BLAST analysis further highlighted that many of the prophages in B. subtilis are extinct given that they do not have ancestral or daughter brethren. However, RNA-seq transcriptome analysis of B. subtilis turned out an interesting paradox indicative of the important role that host transposase have in swapping in host promoters for prophage genes. Specifically, a significant number of prophage genes are highly expressed, which is implausible given that phage genes should be transcriptionally silent. The result and phenomenon further suggests the relative facile nature in which host promoters could be swapped in for phage genes, which is indicative of presence of genomic motifs in prophage genome recognizable by host transposase. Existence of such sequence motifs is thus indicative of possible co-evolution of transposase and phages where transposases were originally a part of the phage genome, which latter jumped out into the host genome to aid the swapping in of host genes into the prophage genome for augmenting prophage genetic repertoire in the face of changing environmental conditions. Overall, it is not uncommon for bacterial species to harbour multiple prophages. But, lysogeny may not be a viable option for long-term preservation of prophage genetic repertoire given that host transposase would inevitable swap in host genes at random locations in the prophage genome.

Спектральные проявления молекулярных механизмов образования наночастиц сульфида серебра методом бактериального синтеза

Одним из перспективных для биофотоники и медицины материалов, используемых для диагностики и таргетной терапии онкологических заболеваний, являются наночастицы сульфида серебра. Спектральные проявления молекулярных механизмов взаимодействия белковых структур с солями металлов в ходе бактериального синтеза этих наночастиц исследованы с помощью молекулярного моделирования методами теории функционала плотности. Особенностью получения наночастиц сульфида серебра методом биосинтеза с помощью бактерий Bacillus subtilis 168 является то, что единственным белком, участвующим в процессе синтеза и адсорбирующимся на поверхности частиц, является белок флагеллин. В качестве исследуемых объектов рассматривались соли --- нитрат серебра и тиосульфат натрия, участвующие в процессе синтеза, а также нестандартная аминокислота метиллизин, входящая в состав флагеллина. Моделирование проводилось на основе расчета образующихся молекулярных структур и их ИК спектров при помощи программного комплекса Gaussian 09. В ходе анализа параметров образующихся водородных связей было обнаружено, что метиллизин образует достаточно устойчивые молекулярные комплексы с нитратом серебра и тиосульфатом натрия. Это говорит о существенной роли метиллизина в образовании биогенных наночастиц сульфида серебра и проясняет механизм его функционирования в составе флагеллина. Ключевые слова: ИК спектры, наночастицы, биосинтез, сульфид серебра, флагеллин, метиллизин, молекулярное моделирование, водородные связи, теория функционала плотности.

Evaluation of agro-industrial residues produced in Costa Rica for a low-cost culture medium using Bacillus subtilis 168

Agro-industrial residues correspond to all the materials generated from activities that involve the transformation of both crops and livestock to obtain processed or semi-finished products. In Costa Rica, the primary sector of economy generates more than 6.3 trillion tons of organic residues per year. The daily generation of this residues pose environmental and economic problems. In recent years, biotechnological-based alternatives have emerged with the purpose of taking advantage of the high nutritional content of these residues to cultivate microorganisms capable of producing compounds with high demand at a commercial level. The present study evaluates six agro-industrial residues produced in Costa Rica, in order to growth Bacillus subtilis 168. An optimization of the culture medium was carried out under a complete factorial design 23, where the variables evaluated were carbon, nitrogen and phosphorus sources. Molasses at 10% m/v, wheat bran at 0.5% m/v, and K2HPO4 at 0.01%, as a carbon, nitrogen, and phosphorus sources, respectively, were identified as optimal for the growth of Bacillus subtilis 168.