Optimized biotransformation of acid-treated water melon peel hydrolyzate into ethanol / Biotransformação otimizada de hidrolisado de casca de melancia tratada com ácido em etanol

Abstract Today, global focus of research is to explore the solution of energy crisis and environmental pollution. Like other agricultural countries, bulk quantities of watermelon peels (WMP) are disposed-off in environment as waste in Pakistan and appropriate management of this waste is the need of hour to save environment from pollution. The work emphasizes the role of ethanologenic yeasts to utilize significant sugars present in WMP for low-cost bioethanol fermentation. Dilute hydrochloric acid hydrolysis of WMP was carried out on optimized conditions employing RSM (response surface methodology) following central composite design (CCD). This experimental design is based on optimization of ethanologenesis involving some key independent parameters such as WMP hydrolysate and synthetic media ratio (X1), incubation temperature (X2) and incubation temperature (X3) for maximal ethanol yield exploiting standard (Saccharomyces cerevisiae K7) as well as experimental (Metchnikowia cibodasensisY34) yeasts. The results revealed that maximal ethanol yields obtained from S. cerevisiae K7 was 0.36±0.02 g/g of reducing sugars whereas M. cibodasensisY34, yielded 0.40±0.01 g ethanol/g of reducing sugars. The yeast isolate M. cibodasensisY34 appeared as promising ethanologen and embodies prospective potential for fermentative valorization of WMP-to-bioethanol.

Resumo Hoje, o foco global da pesquisa é explorar a solução da crise energética e da poluição ambiental. Como em outros países agrícolas, grandes quantidades de cascas de melancia (WMP) são descartadas como resíduos no meio ambiente no Paquistão, mas a gestão adequada desses resíduos é a mais recente solução para salvar o meio ambiente da poluição. O trabalho enfatiza o papel das leveduras etanologênicas para utilizar açúcares significativos presentes no WMP para fermentação de bioetanol de baixo custo. A hidrólise de ácido clorídrico diluído de WMP foi realizada em condições otimizadas empregando RSM (metodologia de superfície de resposta) e seguindo o projeto de composto central (CCD). Este projeto experimental é baseado na otimização da etanologenesis envolvendo alguns parâmetros independentes importantes, como hidrolisado de WMP e razão de meio sintético (X1), temperatura de incubação (X2) e temperatura de incubação (X3) para rendimento máximo de etanol explorando o padrão (Saccharomyces cerevisiae K7) também como leveduras experimentais (Metchnikowia cibodasensis Y34). Os resultados revelaram que os rendimentos máximos de etanol obtidos a partir de S. cerevisiae K7 foi de 0,36 ± 0,02 g / g de açúcares redutores, enquanto M. cibodasensis Y34 rendeu 0,40 ± 0,01 g de etanol / g de açúcares redutores. O isolado de levedura M. cibodasensis Y34 apareceu como um etanologeno promissor e incorpora um potencial prospectivo para a valorização fermentativa de WMP em bioetanol.

Homeopathy on cultures of Saccharomyces cerevisiaeand impact on fermentation

Studies have shownthat homeopathy modulates the activity of both single-and multi-celled organisms;therefore, we propose a study into the action of Arnica Montanaand S. cerevisiae fungus nosode on growth "in vitro", and on the fermentation of S. cerevisiaeon brewer's wort. Methods:250 µL of medication in 30% alcohol were placed in 5 mL of Sabouraud Broth (SB) or wort, with 20 µL of fungus ata McFarland standard of 0.5 and in a dilution of 1:100. Fungal growth was evaluated via spectrophotometry at 600 nm or a cell count in a Neubauer chamber in a kinetic of 1 to 5 days' incubation at 25ºC. The production of alcohol by the fungus was evaluated using the BRIX index in the samekinetic. 1x107fungi/mL were previously incubated with medication for 5 days and, afterwards, placed in 20 mL of fresh wort, incubated at 25ºC for 7 days and evaluated for growth and sugar consumption. Resultsand Discussion: The SB results revealed that after 2days incubation with Arnica30CH, an increase in fungal growth was observed (p<0.0001), whilewith nosode 6 and 30CH there was a reduction in growth after 2 and 5 days incubation (p<0.001). The fungi incubated with Arnica30CH exhibited increased sugar consumption after 2 and5 days incubation (p<0.05), while the nosode 30CH resulted in lower sugar consumption after 2 and 3 days incubation (p<0.05). The results for fungal growth and sugar consumption with the wort were similar to those using SB.The fungalcultures previously incubated with homeopathic medication and subsequent incubation with fresh wortindicated a loss of distinction, bothin terms of fungal growth and sugar consumption. This piece of data may suggest action by the homeopathic medication only when in contact with the cells. Conclusion: The treatment of the S. cerevisiae fungus using Arnica and the S. cerevisiae nosode produced a significant modulation in fungal growth and sugar consumption.

Caracterização do papel de Nop53 na montagem da subunidade ribossomal maior em Saccharomyces cerevisiae / Characterization of the role of Nop53 in the assembly of the large ribosomal subunit in Saccharomyces cerevisiae

Os ribossomos são complexos ribonucleoproteicos conservados formados por duas subunidades assimétricas (40S e 60S em eucariotos) responsáveis pela tradução da informação genética e catálise da síntese proteica. A montagem destes complexos em eucariotos é mais bem descrita em S. cerevisiae, constituindo um processo celular energeticamente dispendioso e com múltiplas etapas. Ela tem origem no nucléolo com a transcrição do pré-rRNA 35S e requer o recrutamento hierárquico e transiente de cerca de 200 fatores de montagem para garantir a formação correta dos centros funcionais aptos à tradução. Neste processo, que se estende no núcleo e citoplasma, 79 proteínas ribossomais associam-se gradativamente à medida que o prérRNA é dobrado, modificado e processado. O processamento do pré-rRNA 35S consiste na remoção progressiva de espaçadores internos (ITS1 e ITS2) e externos (5ETS e 3ETS), que separam e flanqueiam os rRNAs maduros componentes de ambas subunidades ribossomais. A clivagem do ITS1 separa as vias de maturação do pré-60S e do pré-40S. O ITS2, que, em associação a fatores de montagem, forma uma estrutura denominada ITS2-foot, é o último espaçador do pré-60S a ser removido. A composição do ITS2-foot permanece inalterada no nucléolo até a transição entre o estado E nucleolar e a formação da partícula Nog2 nuclear. Nesta etapa, a liberação do fator Erb1 permite o recrutamento do fator de montagem conservado e essencial Nop53. Na base do ITS2-foot, Nop53 recruta o exossomo via RNA helicase Mtr4 para a clivagem 3-5 exonucleolítica de parte do ITS2 levando à desmontagem do ITS2-foot. O fato de Nop53 atuar como ponte entre dois grandes complexos e apresentar uma estrutura flexível e estendida nos levou a aprofundar a caracterização de seu papel durante a maturação do pré60S. Neste trabalho, usando análise proteômica quantitativa label-free baseada em espectrometria de massas, caracterizou-se o interactoma de Nop53, e avaliou-se o impacto da depleção de Nop53 no interactoma da subunidade catalítica do exossomo Rrp6 e na composição de pré-ribossomos representativos de quase todas as etapas de maturação do pré-60S. Em paralelo, foram caracterizados mutantes truncados de Nop53 e avaliada por pull-down a interação de Nop53 com componentes do exossomo. Os resultados obtidos mostraram que Nop53 é capaz de interagir com o cofator do exossomo Mpp6, sugerindo pontos adicionais de interação durante o recrutamento do exossomo ao pré-60S. A análise do interactoma de Rrp6 mostrou uma associação precoce do exossomo aos intermediários pré-ribossomais nucleolares mais iniciais, anteriores aos previamente descritos. Mudanças na composição dos intermediários pré-60S revelaram que a depleção de Nop53 afeta a transição entre o estado E e a partícula Nog2, afetando eventos tardios de maturação como o recrutamento de Yvh1. Comparando-se o efeito da depleção de Nop53 com o de mutantes nop53 desprovidos da região de recrutamento do exossomo, obtivemos evidências bioquímicas do papel estrutural de Nop53 na base do ITS2- foot. Em conjunto, estas observações, à luz de estruturas de intermediários pré-ribossomais recentemente descritas, nos permitiram concluir que o recrutamento de Nop53 ao pré-60S contribui para a estabilização de eventos de remodelamento do rRNA que antecedem a formação da partícula Nog2

Ribosomes are conserved ribonucleoprotein complexes formed by two asymmetric subunits (the 40S and the 60S in eukaryotes) responsible for translating the genetic information and catalyzing protein synthesis. The assembly of these complexes in eukaryotes is best described in S. cerevisiae. It is an energetically demanding, multi-step cellular process, that starts in the nucleolus with the transcription of the 35S pre-rRNA. It requires the hierarchical and transient recruitment of about 200 assembly factors to ensure the correct formation of the functional centers suitable for translation. In this process, which extends into the nucleus and cytoplasm, 79 ribosomal proteins gradually associate as the pre-rRNA is folded, modified, and processed. The 35S pre-rRNA processing happens with the progressive removal of internal (ITS1 and ITS2) and external (5'ETS and 3'ETS) transcribed spacers, which separate and flank the mature rRNA components of both ribosomal subunits. The cleavage at the ITS1 separates the pre-60S and pre40S maturation pathways. The ITS2, which in association with assembly factors constitutes a structure called ITS2-foot, is the last pre-60S spacer to be removed. The composition of the ITS2- foot remains unchanged in the nucleolus until the transition between the nucleolar state E and the nuclear Nog2 particle. At this stage, the release of Erb1 allows the recruitment of the conserved and essential assembly factor Nop53. At the base of the ITS2-foot, Nop53 recruits the exosome via the RNA helicase Mtr4 for the ITS2 3'-5' exonucleolytic cleavage leading to the ITS2-foot disassembly. The fact that Nop53 acts as a bridge between these two large complexes and exhibits a flexible and extended structure led us to further characterize its role in the pre-60S maturation. In this work, using mass spectrometry-based label-free quantitative proteomics, we characterized the interactome of Nop53, as well as the impact of the depletion of Nop53 on the interactome of the exosome catalytic subunit Rrp6 and on the composition of pre-ribosomes representative of almost all pre-60S maturation stages. In parallel, we characterized nop53 truncated mutants and evaluated the interaction of Nop53 with exosome components by pulldown assays. The results showed that Nop53 can interact with the exosome cofactor Mpp6, suggesting the contribution of additional points of interaction during the exosome recruitment to the pre-60S. The analysis of the Rrp6 interactome revealed an early association of the exosome with pre-ribosomal intermediates at very early nucleolar stages, before those previously described. Changes in the composition of pre-60S intermediates revealed that Nop53 depletion affects the transition between the state E and the Nog2 particle, affecting late pre-60S maturation events, such as the Yvh1 recruitment. Comparing the effect of Nop53 depletion with that of nop53 mutants lacking the exosome interacting region, we obtained biochemical evidence of the structural role of Nop53 at the base of the ITS2-foot. Altogether, and in light of recently described structures of pre-ribosomal intermediates, these observations allowed us to conclude that the recruitment of Nop53 to the pre-60S contributes to the stabilization of rRNA remodeling events that precede the formation of the Nog2 particle

Dynamic control of ERG20 expression to improve production of monoterpenes by engineering Saccharomyces cerevisiae / 中国中药杂志

Monoterpenes are widely used in cosmetics, food, medicine, agriculture and other fields. With the development of synthetic biology, it is considered as a potential way to create microbial cell factories to produce monoterpenes. Engineering Saccharomyces cerevisiae to produce monoterpenes has been a research hotspot in synthetic biology. In S. cerevisiae, the production of geranyl pyrophosphate(GPP) and farnesyl pyrophosphate(FPP) is catalyzed by a bifunctional enzyme farnesyl pyrophosphate synthetase(encoded by ERG20 gene) which is inclined to synthesize FPP essential for yeast growth. Therefore, reasonable control of FPP synthesis is the basis for efficient monoterpene synthesis in yeast cell factories. In order to achieve dynamic control from GPP to FPP biosynthesis in S. cerevisiae, we obtained a novel chassis strain HP001-pERG1-ERG20 by replacing the ERG20 promoter of the chassis strain HP001 with the promoter of cyclosqualene cyclase(ERG1) gene. Further, we reconstructed the metabolic pathway by using GPP and neryl diphosphate(NPP), cis-GPP as substrates in HP001-pERG1-ERG20. The yield of GPP-derived linalool increased by 42.5% to 7.6 mg·L~(-1), and that of NPP-derived nerol increased by 1 436.4% to 8.3 mg·L~(-1). This study provides a basis for the production of monoterpenes by microbial fermentation.

Construction of cell factories for high production of ginsenoside Rh_2 in Saccharomyces cerevisiae / 中国中药杂志

Ginsenoside Rh_2 is a rare active ingredient in precious Chinese medicinal materials such as Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma, and Panacis Quinquefolii Radix. It has important pharmacological activities such as anti-cancer and improving human immunity. However, due to the extremely low content of ginsenoside Rh_2 in the source plants, the traditional way of obtaining it has limitations. This study intended to apply synthetic biological technology to develop a cell factory of Saccharomyces cerevisiae to produce Rh_2 by low-cost fermentation. First, we used the high protopanaxadiol(PPD)-yielding strain LPTA as the chassis strain, and inserted the Panax notoginseng enzyme gene Pn1-31, together with yeast UDP-glucose supply module genes[phosphoglucose mutase 1(PGM1), α-phosphoglucose mutase(PGM2), and uridine diphosphate glucose pyrophosphorylase(UGP1)], into the EGH1 locus of yeast chromosome. The engineered strain LPTA-RH2 produced 17.10 mg·g~(-1) ginsenoside Rh_2. This strain had low yield of Rh_2 while accumulated much precursor PPD, which severely restricted the application of this strain. In order to further improve the production of ginsenoside Rh_2, we strengthened the UDP glucose supply module and ginsenoside Rh_2 synthesis module by engineered strain LPTA-RH2-T. The shaking flask yield of ginsenoside Rh_2 was increased to 36.26 mg·g~(-1), which accounted for 3.63% of the dry weight of yeast cells. Compared with those of the original strain LPTA-RH2, the final production and the conversion efficiency of Rh_2 increased by 112.11% and 65.14%, respectively. This study provides an important basis for further obtaining the industrial-grade cell factory for the production of ginsenoside Rh_2.

Identification and verification of key salt-tolerant amino acid sites of banana MaNHX5 / 生物工程学报

In order to improve the salt tolerance of banana NHX genes, we cloned a MaNHX5 gene from Musa acuminata L. AAA group and predicted the key salt-tolerant amino acid sites and mutant protein structure changes of MaNHX5 by using bioinformatics tools. The 276-position serine (S) of MaNHX5 protein was successfully mutated to aspartic acid (D) by site-directed mutagenesis, and the AXT3 salt-sensitive mutant yeast was used for a functional complementation test. The results showed that after the mutated MaNHX5 gene was transferred to AXT3 salt-sensitive mutant yeast, the salt tolerance of the mutant yeast was significantly improved under 200 mmol/L NaCl treatment. It is hypothesized that Ser276 of MaNHX5 protein plays an important role in the transport of Na+ across the tonoplast.

N-terminal truncation of prenyltransferase enhances the biosynthesis of prenylnaringenin / 生物工程学报

8-prenylnaringenin (8-PN) is a potent estrogen with high medicinal values. It also serves as an important precursor for many prenylated flavonoids. Microbial synthesis of 8-PN is mainly hindered by the low catalytic activity of prenyltransferases (PTS) and insufficient supply of precursors. In this work, a SfN8DT-1 from Sophora flavescens was used to improve the efficiency of (2S)-naringenin prenylation. The predicted structure of SfN8DT-1 showed that its main body is comprised of 9 α-helices and 8 loops, along with a long side chain formed by nearly 120 amino acids. SfN8DT-1 mutants with different side-chain truncated were tested in Saccharomyces cerevisiae. A mutant expressing the truncated enzyme at K62 site, designated as SfND8T-1-t62, produced the highest 8-PN titer. Molecular docking of SfN8DT-1-t62 with (2S)-naringenin and dimethylallyl diphosphate (DMAPP) showed that K185 was a potentially crucial residue. Alanine scanning within a range of 0.5 nm around these two substrates showed that the mutant K185A may decrease its affinity to substrates, which also indicated K185 was a potentially critical residue. Besides, the mutant K185W enhanced the affinity to ligands implied by the simulated saturation mutation, while the saturated mutation of K185 showed a great decrease in 8-PN production, indicating K185 is vital for the activity of SfN8DT-1. Subsequently, overexpressing the key genes of Mevalonate (MVA) pathway further improved the titer of 8-PN to 31.31 mg/L, which indicated that DMAPP supply is also a limiting factor for 8-PN synthesis. Finally, 44.92 mg/L of 8-PN was produced in a 5 L bioreactor after 120 h, which is the highest 8-PN titer reported to date.

Biomanufacturing driven by engineered microbes / 生物工程学报

This article summarized the reviews and research articles published in Chinese Journal of Biotechnology in the field of biomanufacturing in 2021. The article covered major chassis cells such as Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae, filamentous fungi, non-model bacteria and non-conventional yeasts. Moreover, this article summarized the advances in the production of amino acids, organic acids, vitamins, higher alcohols, natural compounds (terpenoids, flavonoids, alkaloids), antibiotics, enzymes and enzyme-catalyzed products, biopolymers, as well as the utilization of biomass and one-carbon materials. The key technologies used in the construction of cell factories, such as regulation, evolution, and high-throughput screening, were also included. This article may help the readers better understand the R & D trend in biomanufacturing driven by engineered microbes.

URA3 affects artemisinic acid production by an engineered Saccharomyces cerevisiae in pilot-scale fermentation / 生物工程学报

CRISPR/Cas9 has been widely used in engineering Saccharomyces cerevisiae for gene insertion, replacement and deletion due to its simplicity and high efficiency. The selectable markers of CRISPR/Cas9 systems are particularly useful for genome editing and Cas9-plasmids removing in yeast. In our previous research, GAL80 gene has been deleted by the plasmid pML104-mediated CRISPR/Cas9 system in an engineered yeast, in order to eliminate the requirement of galactose supplementation for induction. The maximum artemisinic acid production by engineered S. cerevisiae 1211-2 (740 mg/L) was comparable to that of the parental strain 1211 without galactose induction. Unfortunately, S. cerevisiae 1211-2 was inefficient in the utilization of the carbon source ethanol in the subsequent 50 L pilot fermentation experiment. The artemisinic acid yield in the engineered S. cerevisiae 1211-2 was only 20%-25% compared with that of S. cerevisiae 1211. The mutation of the selection marker URA3 was supposed to affect the growth and artemisinic acid production. A ura3 mutant was successfully restored by a recombinant plasmid pML104-KanMx4-u along with a 90 bp donor DNA, resulting in S. cerevisiae 1211-3. This mutant could grow normally in a fed-batch fermentor with mixed glucose and ethanol feeding, and the final artemisinic acid yield (> 20 g/L) was comparable to that of the parental strain S. cerevisiae 1211. In this study, an engineered yeast strain producing artemisinic acid without galactose induction was obtained. More importantly, it was the first report showing that the auxotrophic marker URA3 significantly affected artemisinic acid production in a pilot-scale fermentation with ethanol feeding, which provides a reference for the production of other natural products in yeast chassis.

Engineering Saccharomyces cerevisiae for efficient production of glucaric acid / 生物工程学报

As an important dicarboxylic acids existing in nature, glucaric acid has been widely used in medical, health, and polymer materials industry, therefore it is considered as one of the "top value-added chemicals from biomass". In this study, using Saccharomyces cerevisiae as a chassis microorganism, the effects of overexpression of myo-inositol transporter Itr1, fusional expression of inositol oxygenase MIOX4 and uronate dehydrogenase Udh, and down-expression of glucose-6-phosphate dehydrogenase gene ZWF1 on the glucaric acid production were investigated. The results showed that the yield of glucaric acid was increased by 26% compared with the original strain Bga-3 under shake flask fermentation after overexpressing myo-inositol transporter Itr1. The yield of glucaric acid was increased by 40% compared with Bga-3 strain by expressing the MIOX4-Udh fusion protein. On these basis, the production of glucaric acid reached 5.5 g/L, which was 60% higher than that of Bga-3 strain. In a 5 L fermenter, the highest yield of glucaric acid was 10.85 g/L, which was increased 80% compared with that of Bga-3 strain. The application of the above metabolic engineering strategy improved the pathway efficiency and the yield of glucaric acid, which may serve as a reference for engineering S. cerevisiae to produce other chemicals.

Effect of key enzymes ubiquitination sites on the biosynthesis of naringenin / 生物工程学报

Flavonoids have a variety of biological activities and have important applications in food, medicine, cosmetics, and many other fields. Naringenin is a platform chemical for the biosynthesis of many important flavonoids. Ubiquitination plays a pivotal role in the post-translational modification of proteins and participates in the regulation of cellular activities. Ubiquitinated proteins can be degraded by the ubiquitin-protease system, which is important for maintaining the physiological activities of cells, and may also exert a significant impact on the expression of exogenous proteins. In this study, a real-time in-situ detection system for ubiquitination modification has been established in Saccharomyces cerevisiae by using a fluorescence bimolecular complementation approach. The ubiquitination level of protein was characterized by fluorescence intensity. By using the approach, the potential ubiquitination sites of proteins involved in the naringenin biosynthesis pathway have been obtained. The lysine residues of the relevant ubiquitination sites were mutated to arginine to reduce the ubiquitination level. The mutants of tyrosine ammonia-lyase (FjTAL) and chalcone synthase (SjCHS, SmCHS) showed decreased fluorescence, suggested that a decreased ubiquitination level. After fermentation verification, the S. cerevisiae expressing tyrosine ammonia-lyase FjTAL mutant FjTAL-K487R accumulated 74.2 mg/L p-coumaric acid at 72 h, which was 32.3% higher than that of the original FjTAL. The strains expressing chalcone synthase mutants showed no significant change in the titer of naringenin. The results showed that mutation of the potential ubiquitination sites of proteins involved in the naringenin biosynthesis pathway could increase the titer of p-coumaric acid and have positive effect on naringenin biosynthesis.

Association between productive parameters and intestinal histomorphological findings in broilers supplemented with probiotics (Saccharomyces cerevisiae, Lactobacillus acidophilus and Bacillus subtilis) / Asociación entre parámetros productivos y hallazgos histomorfológicos en pollos de engorde suplementados con probióticos (Saccharomyces cerevisiae, Lactobacillus acidophilus y Bacillus subtilis)

SUMMARY: This study evaluates the effect of probiotics Saccharomyces cerevisiae, Lactobacillus acidophilus and Bacillus subtilis on production parameters and intestinal histomorphology of broilers of 45 days of age. Eleven 45-day-old Ross 500 broilers were used and classified as control group (CG) (n = 5) or supplemented with probiotics group (n = 8). Histopathological evaluation of duodenum, ileum, and jejunum was performed. The area of the villi height, base and apex were evaluated as well as the size and number of crypts. In addition, mucus production was quantified in different portions of the small intestine. The villi present duodenum of broilers supplemented with probiotics had a greater area (p = 0.0127), a greater basal width (p = 0.0049) and a greater apical width (p = 0.0024), as well as a greater crypt area (p = 0.0189). Significantly higher levels of mucus were noted in the duodenum (p = 0.0480) and jejunum (p = 0.0480) of broilers supplemented with probiotics. We suggest that probiotic supplementation improve the intestinal nutrients absorption.

RESUMEN: Este estudio evalúa el efecto del uso de probióticos como: Saccharomyces cerevisiae, Lactobacillus acidophilus, Bacillus subtilis en parámetros productivos e histomorfologia intestinal de pollos de engorde de 45 días de edad. Fueron usados 11, los cuales fueron clasificados en grupo control (CG) (n = 5) y grupo suplementado con probióticos (PG) (n = 8). Fue realizado análisis histopatológico de secciones de duodeno, íleon y yeyuno. Fue evaluado ancho, altura y área del ápice de la vellosidad, área y número de criptas. Además, fue estimada la producción de moco en los diferentes segmentos del intestino delgado. Fue observada mayor área de la vellosidad en duodeno, PG (p = 0.0127), ancho basal mayor en PG (p = 0.0049) ancho apical mayor en PG (p = 0.0024), así como mayor área de criptas en PG (p = 0.0189). No fueron encontradas diferencias significativas respecto a los segmentos de yeyuno e íleon. PG presentó mayor producción de moco en duodeno (p = 0.0480) y en yeyuno (p = 0.0480). Concluimos que la suplementación con probióticos en pollos de engorde genera cambios en la histomorfologia intestinal, evidenciables en áreas apicales y basales de las vellosidades intestinales. Soporte financiero: Dirección General de Investigaciones - Universidad de los Llanos.

Efecto radioprotector del ácido tánico y vinos de la var vitis vinífera L. cv tannat en Saccharomyces cerevisiae / Radioprotective effect of tannic acid and wines of the vitis vinifera L. cv tannat on Saccharomyces cerevisiae / Proteção contra radiação por vinho tinto var tannat e ácido tanico em Saccharomyces cerevisiae

El vino tinto variedad Vitis vinifera L. cv Tannat en los últimos años ha tomado relevancia por su alta concentración de polifenoles, esto le podría significar un rol protector sobre el genoma disminuyendo la formación de lesiones oxidativas. Los efectos a nivel celular de las radiaciones ionizantes en blancos como el ADN, componentes de cascadas de transducción de señales, resultan en lesiones letales, mutagénicas y recombinogénicas y en retardos en el ciclo celular. Se utilizó como modelo eucariota poblaciones de Saccharomyces cerevisiae en fase exponencial expuestas a radiación gamma (200 Gy) en presencia, o ausencia, de vino Tannat (10 % v/v) o de ácido tánico (60 µg/mL). Se estimaron las probabilidades de sobrevida y frecuencia mutagénica en distintas condiciones. Las muestras celulares expuestas a radiación ionizante presentaron una fracción de sobrevida de 0.21 ± 0.02 mientras que en las muestras irradiadas en presencia de vino Tannat o de ácido tánico la fracción de sobrevida fue de 0.33 ± 0.03 y 0.30 ± 0.03 respectivamente. Se observó en las poblaciones irradiadas un aumento significativo de la probabilidad de mutagénesis. En el caso de los tratamientos combinados se observó que la frecuencia mutagénica fue significativamente menor (gamma Tannat: 33%, gamma ácido tánico: 45% ). Estos resultados preliminares podrían indicar radioprotección moderada por parte de los compuestos estudiados, efecto que podría explicarse por las interacciones redox del ácido tánico y polifenoles contenidos en el vino con los radicales libres formados por las radiaciones ionizantes, además de la activación de vías de reparación genómica.

The red wine variety Vitis vinifera L. cv Tannat in recent years has gained relevance due to its high concentration of polyphenols, this could mean a protective role on the genome, reducing the formation of oxidative lesions. The effects at the cellular level of ionizing radiation on targets such as DNA, components of signal transduction cascades, result in lethal, mutagenic and recombinogenic lesions and delays in the cell cycle. Exponential phase populations of Saccharomyces cerevisiae exposed to gamma radiation (200 Gy) in the presence or absence of Tannat wine (10% v / v) or tannic acid (60 µg / ml) were used as a eukaryotic model. The probabilities of survival and mutagenic frequency in different conditions were estimated. Cellular samples exposed to ionizing radiation presented a survival fraction of 0.21 ± 0.02, while in samples irradiated in the presence of Tannat wine or tannic acid, the survival fraction was 0.33 ± 0.03 and 0.30 ± 0.03 respectively. A significant increase in the probability of mutagenesis was observed in irradiated populations. In the case of the combined treatments, it was observed that the mutagenic frequency was significantly lower (Tannat gamma: 33%, Tannic acid gamma: 45%). These preliminary results could indicate moderate radioprotection by the compounds studied, an effect that could be explained by the redox interactions of tannic acid and polyphenols contained in wine with the free radicals formed by ionizing radiation, in addition to the activation of genomic repair pathways.

A variedade de vinho tinto Vitis vinifera L. cv Tannat nos últimos anos tem ganhado relevância devido à sua alta concentração de polifenóis, o que pode significar um papel protetor do genoma, reduzindo a formação de lesões oxidativas. Os efeitos no nível celular da radiação ionizante em alvos como o DNA, componentes de cascatas de transdução de sinal, resultam em lesões letais, mutagênicas e recombinogênicas e atrasos no ciclo celular. Populações de fase exponencial de Saccharomyces cerevisiae expostas à radiação gama (200 Gy) na presença ou ausência de vinho Tannat (10% v / v) ou ácido tânico (60 µg / ml) foram utilizadas como modelo eucariótico. Foram estimadas as probabilidades de sobrevivência e frequência mutagênica em diferentes condições. As amostras celulares expostas à radiação ionizante apresentaram uma fração de sobrevivência de 0,21 ± 0,02, enquanto nas amostras irradiadas na presença de vinho Tannat ou ácido tânico, a fração de sobrevivência foi de 0,33 ± 0,03 e 0,30 ± 0,03, respectivamente. Um aumento significativo na probabilidade de mutagênese foi observado nas populações irradiadas. No caso dos tratamentos combinados, observou-se que a frequência mutagênica foi significativamente menor (Tannat gama: 33%, ácido tânico gama: 45%). Esses resultados preliminares podem indicar radioproteção moderada pelos compostos estudados, efeito que pode ser explicado pelas interações redox do ácido tânico e polifenóis contidos no vinho com os radicais livres formados pela radiação ionizante, além da ativação de vias de reparo genômico.

Expression of antimicrobial peptide Cecropin P1 in Saccharomyces cerevisiae and its antibacterial and antiviral activity in vitro

BACKGROUND: Cecropin P1, acting as an antimicrobial, has a broad-spectrum antibacterial activity with some antiviral and antifungal properties. It is a promising natural alternative to antibiotics which is originally isolated from the pig intestinal parasitic nematode Ascaris suum. Many studies have shown that Cecropin P1 is helpful for the prevention or treatment of clinical diseases. Therefore, it is very necessary to establish a safe, nontoxic, and efficient expression method of Cecropin P1. RESULTS: The results indicated that the recombinant protein was about 5.5 kDa showed by Tricine­SDS­ PAGE and Western blot. And Cecropin P1 was efficiently secreted and expressed after 12 h of induction, with an increasing yield over the course of the induction. Its maximum concentration was 7.83 mg/L after concentration and purification. In addition, in vitro experiments demonstrated that Cecropin P1 not only exerted a strong inhibitory effect on Escherichia coli, Salmonella sp., Shigella sp., and Pasteurella sp., but also displayed an antiviral activity against PRRSV NADC30-Like strain. CONCLUSIONS: Collectively, the strategy of expressing Cecropin P1 in Saccharomyces cerevisiae is harmless, efficient, and safe for cells. In addition, the expressed Cecropin P1 has antiviral and antibacterial properties concurrently.

Biological and Oxidative Effect of Ellagic Acid on Saccharomyces cerevisiae: A New Way for Culture Developing

Abstract In this study, the effects of Ellagic acid (EA) on protein expression in yeasts and cellular development were investigated. Four groups were formed. Groups: 1) Control group; yeast only cultivated group; 2) Ellagic Acid (EA) group: EA (10%) given group; 3) Hydrogen peroxide (H2O2) Group: The group given H2O2 (15 mM); 4) EA + H2O2 group: EA (10%) + H2O2 (15 mM) group. After sterilization, EA (10%) and H2O2 (15 mM) were added to the Saccharomyces cerevisiae (S. cerevisiae) cultures and the cultures were grown at 30 °C for 1 hour, 3 hours, 5 hours and 24 hours (overnight). S. cerevisiae cell growth, lipid peroxidation MDA (malondialdehyde) analysis and GSH (glutathione) level were analyzed by spectrophotometer. Total protein changes were determined by SDS-PAGE electrophoresis and measured by the Bradford method. According to the obtained results, compared with the H2O2 group, cell development (1, 3, 5 and 24 hours), GSH level and total protein synthesis (24 hours) were increased with EA, while MDA level (24 hours) decreased. These results show that EA reduces oxidative damage, increases cell growth and it has a protective effect to promote protein synthesis in S. cerevisiae culture.

Efeitos do probiótico Saccharomyces ceveriseae no tratamento da periodontite experimental em ratos submetidos à quimioterapia / Effects of the probiotic Saccharomyces Ceveriseae in the treatment of experimental periodontitis in rats undergoing chemotherapy

O presente estudo teve a finalidade de avaliar os efeitos do uso do probiótico (PRO) Saccharomyces ceveriseae coadjuvante à raspagem e alisamento radicular (RAR) no tratamento da periodontite experimental (PE) induzida em animais imunossuprimidos com o quimioterápico 5-Fluorouracil (5FU). Cento e oito animais foram submetidos à indução da PE por meio da instalação de um fio de algodão ao redor do primeiro molar inferior esquerdo ou direito. A imunossupressão foi obtida pela injeção intraperitoneal do 5FU aplicada em dois momentos: no ato da instalação da ligadura e após 48 horas. Sete dias após instalação a ligadura foi removida e os animais separados aleatoriamente em seis grupos experimentais que receberam os seguintes tratamentos: PE (n=18) - animais que não sofreram nenhum tratamento sistêmico ou local; SS (n=18) - animais que não sofreram tratamento local, apenas tratamento sistêmico com solução salina (SS); 5FU (n=18) - animais tratados sistemicamente com 5FU que não sofreram nenhum tratamento local; 5FU-PRO (n=18) - animais tratados sistemicamente com 5FU que receberam irrigação local com 0,6 ml de PRO; 5FURAR (n=18) - animais tratados sistemicamente com 5FU que receberam RAR e; 5FURAR-PRO (n=18) - animais tratados sistemicamente com 5FU, que receberam tratamento com RAR e irrigação local com 0,6 ml de PRO. Seis animais de cada período foram submetidos à eutanásia aos 7, 15 e 30 dias após os tratamentos. Os espécimes contendo a área interessada foram obtidos e processados para a análise histopatológica, histométrica da perda óssea alveolar (POA), porcentagem de tecido ósseo (PTO), porcentagem de osso vital (POV) e porcentagem de osso não vital (PONV), e análise imunoistoquímica (TRAP, RANKL e OPG). Os dados obtidos foram submetidos à análise estatística (α=5%). Os resultados histológicos demonstraram que nos espécimes tratados com 5FU houve agravamento da perda óssea na região de furca comparado com os animais sistemicamente saudáveis (grupos PE e SS); que o tratamento com PRO como monoterapia (grupo 5FU-PRO) promoveu redução do processo inflamatório, denotando estabilização da doença periodontal; que o tratamento com RAR (grupo 5FU-RAR) reduziu o volume do infiltrado inflamatório mostrando sinais de reparação e a associação PRO-RAR (grupo 5FU-RAR-PRO) evidenciou nítida reestruturação tecidual aos 15 dias e menor área de reabsorção óssea aos 30 dias. Observou-se na análise histométrica que a POA foi maior no grupo 5FU-PRO em relação ao grupo SS aos 30 dias (p<0,05); a PTO foi maior no grupo 5FU-RAR-PRO aos 30 dias em comparação aos grupos EP, SS, 5FU e 5FU-PRO (p<0,05) e a POV foi maior no grupo 5FU-RAR-PRO em comparação ao grupo 5FU aos 7 dias, e maior aos 30 dias em comparação a todos os grupos experimentais (p<0,05). Não houve diferença entre os grupos e períodos na imunomarcação das células TRAP positivas. A RANKL mostrou alto padrão de imunomarcação nos grupos PE, SS e 5FU em todos períodos, e aos 7 dias no grupo 5FU-PRO, 5FU-RAR e 5FURAR-PRO, moderado padrão de imunomarcação nos grupos 5FU, 5FU-PRO, 5FURAR, e 5FU-RAR-PRO, mantendo-se baixo padrão neste último grupo, no período de 30 dias. Houve baixo padrão de imunomarcação de OPG em todos os grupos e períodos, exceto no grupo 5FU-RAR-PRO que mostrou moderado padrão. Diante dos resultados obtidos pode ser concluído que o uso do quimioterápico 5FU agravou a PE; que o uso do Saccharomyces ceveriseae associado à RAR, minimizou os efeitos do quimioterápico nos tecidos periodontais reduzindo o processo inflamatório, e reduzindo a porcentagem de destruição de tecido ósseo na área de furca em animais imunossuprimidos(AU)

The present study aimed to evaluate the effects of using probiotic (PRO) Saccharomyces ceveriseae as an adjuvant of scaling and root planing (SRP) in the treatment of experimental periodontitis (EP) induced in immunosuppressed animals with chemotherapeutic 5-Fluorouracil (5FU). One hundred and eight animals were submitted to EP induction by installing a cotton thread around the lower left or right first molar. Immunosuppression was obtained by intraperitoneal injection of 5FU applied in two moments: at the time of installing the ligature and after 48 hours. Seven days after installation, the ligature was removed and the animals were randomly separated into six experimental groups that received the following treatments: EP (n = 18) - animals that did not receive any systemic or local treatment; SS (n = 18) - animals that did not receive local treatment, only systemic treatment with saline solution (SS); 5FU (n = 18) - animals treated systemically with 5FU that did not receive any local treatment; 5FUPRO (n = 18) - animals treated systemically with 5FU that received local irrigation with 0.6 ml of PRO; 5FU-SRP (n = 18) - animals treated systemically with 5FU that received RAR and; 5FU-SRP-PRO (n = 18) - animals treated systemically with 5FU, which received treatment with SRP and local irrigation with 0.6 ml of PRO. Six animals from each period were euthanized at 7, 15 and 30 days after treatments. The specimens containing the interested area were obtained and processed for histopathological, histometric analysis of alveolar bone loss (ABL), percentage of bone tissue (PBT), percentage of vital bone (PVB) and percentage of non-vital bone (PNVB), and immunohistochemical analysis (TRAP, RANKL and OPG). The data obtained were subjected to statistical analysis (α = 5%). The histological results showed that in the specimens treated with 5FU there was worsening of bone loss in the furcation region compared to healthy animals (EP and SS groups); that treatment with PRO as monotherapy (group 5FU-PRO) reduced the inflammatory process, denoting stabilization of periodontal disease; that treatment with SRP (5FU-SRP group) reduced the volume of the inflammatory infiltrate showing signs of repair and the PRO-SRP association (5FU-SRP-PRO group) showed a clear tissue restructuring at 15 days and a smaller area of bone resorption at 30 days. It was observed in the histometric analysis that ABL was higher in the 5FU-PRO group compared to SS group at 30 days (p <0.05); PTO was higher in 5FU-SRP-PRO group at 30 days compared to EP, SS, 5FU and 5FU-PRO groups (p <0.05) and PVB was higher in the 5FU-SRP-PRO group compared to 5FU group at 7 days, and greater at 30 days compared to all experimental groups (p <0.05). There was no difference between groups and periods in the immunostaining of TRAP positive cells. RANKL showed a high immunostaining pattern in the EP, SS and 5FU groups in all periods, and at 7 days in 5FU-PRO, 5FU-SRP and 5FU-SRP-PRO groups, a moderate immunostaining pattern in the 5FU, 5FU-PRO, 5FU-SRP and 5FU-SRP-PRO groups, maintaining a low standard in 5FU-SRP-PRO group, in the period of 30 days. There was a low pattern of OPG immunostaining in all groups and periods, except in 5FU-SRP-PRO group, which showed a moderate pattern. In view of the results obtained, it can be concluded that the use of 5FU chemotherapy has worsened the evolution of EP; that the use of Saccharomyces ceveriseae associated with RAR, minimized the effects of chemotherapy on periodontal tissues, reducing the inflammatory process, and reducing the percentage of bone tissue destruction in the furcation area in immunosuppressed animals(AU)

Effect of RIM21 gene disruption on flocculation of lager yeast / 生物工程学报

Lager yeast is the most popular yeast strain used for beer production in China. The flocculation of yeast plays an important role in cell separation at the end of fermentation. Therefore, appropriately enhancing the flocculation capability of the lager yeast without affecting its fermentation performance would be desirable for beer industry. Our previous study showed that the defect of gene RIM21 might contribute to the enhanced flocculation capability of a lager yeast G03. To further investigate the role of the RIM21 gene in flocculation of strain G03, this study constructed a RIM21-deleted mutant strain G03-RIM21Δ through homologous recombination. Deletion of RIM21 improved the flocculation capability of strain G03 during wort fermentation at 11 °C without changing its fermentation performance significantly. The expression of FLO5, Lg-FLO1 and some other genes involved in cell wall integrity pathway were up-regulated in strain G03-RIM21Δ. In addition, the disruption of RIM21 enhanced resistance of yeast cells to cell wall inhibitors. These results provide a basis for elucidating the flocculation mechanism of lager yeast under low-temperature fermentation conditions.

Overexpression of a leucine transfer RNA gene tL(CAA)K improves the acetic acid tolerance of Saccharomyces cerevisiae / 生物工程学报

Acetic acid is a common inhibitor present in lignocellulosic hydrolysate. Development of acetic acid tolerant strains may improve the production of biofuels and bio-based chemicals using lignocellulosic biomass as raw materials. Current studies on stress tolerance of yeast Saccharomyces cerevisiae have mainly focused on transcription control, but the role of transfer RNA (tRNA) was rarely investigated. We found that some tRNA genes showed elevated transcription levels in a stress tolerant yeast strain. In this study, we further investigated the effects of overexpressing an arginine transfer RNA gene tR(ACG)D and a leucine transfer RNA gene tL(CAA)K on cell growth and ethanol production of S. cerevisiae BY4741 under acetic acid stress. The tL(CAA)K overexpression strain showed a better growth and a 29.41% higher ethanol productivity than that of the control strain. However, overexpression of tR(ACG)D showed negative influence on cell growth and ethanol production. Further studies revealed that the transcriptional levels of HAA1, MSN2, and MSN4, which encode transcription regulators related to stress tolerance, were up-regulated in tL(CAA)K overexpressed strain. This study provides an alternative strategy to develop robust yeast strains for cellulosic biorefinery, and also provides a basis for investigating how yeast stress tolerance is regulated by tRNA genes.

Reinforcement of sterols production through directed storage and transportation in yeast: a review / 生物工程学报

Sterols, a class of cyclopentane poly-hydrophenanthrene derivatives, are the predominant membrane constituent of eukaryotes. These substances have a variety of biological activities and have been widely used in food and pharmaceutical industries. The presence of endogenous ergosterol biosynthetic pathway in Saccharomyces cerevisiae cells make it an ideal chassis for the de novo synthesis of sterol and its derivatives. Most recently, the rational modification of organelles provides a novel strategy for the directed transportation and storage of target products and the ultimate enhanced product synthesis. This review summarizes the phenotypic responses of S. cerevisiae cells upon different physiological stimulations and the underlying molecular mechanisms. Reinforcement of sterol production through directed storage, transportation, and excretion of sterols offers a novel strategy for breaking the limitation of de novo biosynthesis of sterols in yeast.

High-throughput screening of Saccharomyces cerevisiae efficiently producing tyrosine / 生物工程学报

Tyrosine is an important aromatic amino acid. Besides its nutritional value, tyrosine is also an important precursor for the synthesis of coumarins and flavonoids. Previously, our laboratory constructed a Saccharomyces cerevisiae strain LTH0 (ARO4K229L, ARO7G141S, Δaro10, Δzwf1, Δura3) where tyrosine feedback inhibition was released. In the present study, heterologous expression of betaxanthins synthesis genes DOD (from Mirabilis jalapa) and CYP76AD1 (from sugar beet B. vulgaris) in strain LTH0 enabled production of yellow fluorescence. The engineered strain LTH0-DOD-CYP76AD1 was subjected to UV combined with ARTP mutagenesis, followed by flow cytometry screening. Among the mutants screened, the fluorescence intensity of the mutant strain LTH2-5-DOD-CYP76AD1 at the excitation wavelength of 485 nm and emission wavelength of 505 nm was (5 941±435) AU/OD, which was 8.37 times higher than that of strain LTH0-DOD-CYP76AD1. Fourteen mutant strains were subjected to fermentation to evaluate their tyrosine producing ability. The highest extracellular tyrosine titer reached 26.8 mg/L, which was 3.96 times higher than that of strain LTH0-DOD-CYP76AD1. Heterologous expression of the tyrosine ammonia lyase FjTAL derived from Flavobacterium johnsoniae further increased the titer of coumaric acid to 119.8 mg/L, which was 1.02 times higher than that of the original strain LTH0-FjTAL.