Manipulation of IME4 expression, a global regulation strategy for metabolic engineering in Saccharomyces cerevisiae

Metabolic engineering has been widely used for production of natural medicinal molecules. However, engineering high-yield platforms is hindered in large part by limited knowledge of complex regulatory machinery of metabolic network. N6-Methyladenosine (m6A) modification of RNA plays critical roles in regulation of gene expression. Herein, we identify 1470 putatively m6A peaks within 1151 genes from the haploid Saccharomyces cerevisiae strain. Among them, the transcript levels of 94 genes falling into the pathways which are frequently optimized for chemical production, are remarkably altered upon overexpression of IME4 (the yeast m6A methyltransferase). In particular, IME4 overexpression elevates the mRNA levels of the methylated genes in the glycolysis, acetyl-CoA synthesis and shikimate/aromatic amino acid synthesis modules. Furthermore, ACS1 and ADH2, two key genes responsible for acetyl-CoA synthesis, are induced by IME4 overexpression in a transcription factor-mediated manner. Finally, we show IME4 overexpression can significantly increase the titers of isoprenoids and aromatic compounds. Manipulation of m6A therefore adds a new layer of metabolic regulatory machinery and may be broadly used in bioproduction of various medicinal molecules of terpenoid and phenol classes.

Integrating balanced mevalonate pathway into chromosome for improving lycopene production in Escherichia coli / 生物工程学报

Isoprenoids are all derived from two five-carbon building blocks called isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are synthesized either by the mevalonate (MVA) pathway or 2-C-methyld-D-erythritol-4-phosphate (MEP) pathway. In this study, the MVA pathway genes were integrated into the chromosome of LYC101, in which the expression of key genes in the MEP synthesis pathway and lycopene synthesis pathway were optimized by artificial regulatory parts, to further improve the production of isoprenoids in Escherichia coli. The plasmids pALV23 and pALV145 were screened from a plasmid library that constructed by using the RBS library to link the genes of the MVA pathway, which greatly increased the production of β-carotene. The effects of plasmids pALV23 and pALV145 on the lycopene production in low and high lycopene production strain, LYC001 and LYC101, were compared, respectively. The production of lycopene was increased by plasmids pALV23 and pALV145 in both strains. In high lycopene production strain LYC101, pALV23 produced more lycopene than pALV145. Then, the MVA gene together of promoter of pALV23 was integrated into the chromosome of LYC101 at poxB site using method of homologous recombination helped by CRISPR-Cas9 system, resulted in genetically stable strain, LYC102. The yield of lycopene of LYC102 was 40.9 mg/g DCW, 1.19-folds higher than that of LYC101, and 20% more than that of LYC101 with pALV23. Simultaneous expression of MVA pathway and MEP pathway in recombinant E. coli can effectively increase the yield of terpenoids. In this study, a plasmid-free, genetically stable, high-yielding lycopene strain was constructed, which could be used for industrialization. Also, the platform strain can be used for the synthesis of other terpenoids.

Molecular cloning and characterization of the 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase gene from Artemisia annua L / 药学学报

The plastidial methylerythritol phosphate (MEP) pathway provides 5-carbon precursors to the biosynthesis of isoprenoid (including artemisinin). 2-C-Methyl-D-erythritol-4-phosphate cytidylyltransferase (MCT) is the third enzyme of the MEP pathway, which catalyzes 2-C-methyl-D-erythritol-4-phosphate to form 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol. The full-length MCT cDNA sequence (AaMCT) was cloned and characterized for the first time from Artemisia annua L. Analysis of tissue expression pattern revealed that AaMCT was highly expressed in glandular secretory trichome and poorly expressed in leaf, flower, root and stem. AaMCT was found to be a methyl jasmonate (MeJA)-induced genes, the expression of AaMCT was significantly increased after MeJA treatment. Subcellular localization indicated that the GFP protein fused with AaMCT was targeted specifically in chloroplasts. The transgenic plants of Arabidopsis thaliana with AaMCT overexpression exhibited a significantly increase in the content of chlorophyll a, chlorophyll b and carotenoids, demonstrating that AaMCT kinase plays an influential role in isoprenoid biosynthesis.

Effect of lovastatin on in vitro methane production and dry matter digestibility of kikuyu grass (Cenchrus clandestinus) / Efecto de la lovastatina sobre la producción de metano y la digestibilidad de la materia seca in vitro del pasto kikuyo (Cenchrus clandestinus) / Efeito da lovastatina na produção de metano e digestibilidade da matéria seca in vitro do capim kikuyu (Cenchrus clandestinus)

Emissions of methane (CH4), from cattle rumen fermentation, have a negative impact on the environment and energy balance of animals, increasing global warming and reducing the energy retained, respectively. There is evidence that lovastatin inhibits methanogens growth without affecting other rumen microbial populations. The aim of this study was to determine the effect of increasing levels of lovastatin on in vitro CH4 production and dry matter digestibility (DMD) of kikuyu grass (Cenchrus clandestinus). It was used the in vitro gas production technique, incubing kikuyu grass of 45 days old with four levels of lovastatin (0, 0.01, 0.05, 0.10 mg/ml). At the end of 24 hours, gas and CH4 productions, DMD and fermentation profile was evaluated. A completely randomized design was used for data analysis, through the PROC MIXED SAS procedure. The highest level of lovastatin significantly reduced (p <0.05) the gas and CH4 productions and the DMD. The fermentation profile showed a tendency not defined in terms of levels evaluated, finding a higher concentration of VFA in the treatment 0.05 mg/ml. Although the lovastatin significantly reduces CH4 production, without affecting the DMD equally, this effect was evidenced with higher level than the reported in the literature. Perhaps the answer is conditioned to use of rumen fluid, contrasting with pure cultures of methanogens used in other studies.

Las emisiones de metano (CH4), provenientes de la fermentación ruminal del ganado, tienen un impacto negativo sobre el ambiente y el balance energético de los animales, aumentando el calentamiento global y disminuyendo la energía retenida, respectivamente. Existe evidencia que la lovastatina inhibe el crecimiento de metanógenos sin afectar otras poblaciones microbianas del rumen. El objetivo de este trabajo fue determinar el efecto de niveles crecientes de lovastatina sobre la producción de CH4 y la digestibilidad de la materia seca (DMS) in vitro del pasto kikuyo (Cenchrus clandestinus). Fue empleada la técnica in vitro de producción de gas, incubando pasto kikuyo de 45 días de edad con cuatro niveles de lovastatina (0, 0.01, 0.05, 0.10 mg/ml). Al término de 24 horas se evaluó la producción de gas y de CH4, la DMS y el perfil de fermentación. Un diseño completamente aleatorizado fue utilizado para el análisis de los datos, a través del procedimiento PROC MIXED de SAS. El mayor nivel de lovastatina redujo significativamente (p<0,05) la DMS y la producción de gas y CH4. El perfil de fermentación no presentó una tendencia definida en función de los niveles evaluados, encontrando una mayor concentración de AGV en el tratamiento 0.05 mg/ml. Aunque se confirma que la utilización de lovastatina reduce significativamente la producción de CH4, sin afectar en igual medida la DMS, este efecto se evidenció con un nivel mayor al reportado por la literatura. Posiblemente la respuesta está condicionada a la utilización de líquido ruminal, contrastando con los cultivos puros de metanógenos empleados en otros estudios.

As emissões de metano (CH4), produzido na fermentação ruminal do gado, tem um impacto negativo sobre o meio ambiente e o balanço energético dos animais, aumentando o aquecimento global e reduzindo a energia retida, respectivamente. Há evidências do que a lovastatina inibe o crescimento de metanógenos, sem afetar outras populações microbianas ruminais. O objetivo deste estudo foi determinar o efeito de níveis crescentes de lovastatina sobre a produção de CH4 e a digestibilidade da matéria seca (DMS) in vitro do capim kikuyu (Cenchrus clandestinus). Foi empregada a técnica in vitro de produção de gás, incubando capim kikuyu de 45 dias de idade com quatro níveis de lovastatina (0, 0.01, 0.05, 0.10 mg/ml). Depois de 24 horas, a produção de gás e de CH4, a DMS e o perfil de fermentação foi avaliado. Um delineamento inteiramente casualizado foi utilizado para a análise dos dados, através do procedimento PROC MIXED de SAS. O nível mais elevado de lovastatina reduziu significativamente (p<0,05) a DMS e a produção de gás e de CH4. O perfil de fermentação não mostrou uma tendência definida em função dos níveis avaliados, encontrando uma maior concentração de AGV no tratamento 0,05 mg/ml. Embora confirma-se que a utilização de lovastatina reduz significativamente a produção de CH4, sem afectar em igual medida a DMS, este efeito foi evidenciado a um nível maior do que o relatado na literatura. Talvez a resposta seja condicionada à utilização de líquido ruminal, contrastando com as culturas puras de methanogens utilizados em outros estudos.

Isoprenoid biosynthesis in the erythrocytic stages of Plasmodium falciparum

The development of new drugs is one strategy for malaria control. Biochemical pathways localised in the apicoplast of the parasite, such as the synthesis of isoprenic precursors, are excellent targets because they are different or absent in the human host. Isoprenoids are a large and highly diverse group of natural products with many functions and their synthesis is essential for the parasite's survival. During the last few years, the genes, enzymes, intermediates and mechanisms of this biosynthetic route have been elucidated. In this review, we comment on some aspects of the methylerythritol phosphate pathway and discuss the presence of diverse isoprenic products such as dolichol, ubiquinone, carotenoids, menaquinone and isoprenylated proteins, which are biosynthesised during the intraerythrocytic stages of Plasmodium falciparum.

Atividade antibacteriana de plantas úteis e constituintes químicos da raiz de Copernicia prunifera / Antibacterial activity of useful plants and chemical constituents of the roots of Copernicia prunifera

Extratos etanólicos de Qualea grandiflora e Copernicia prunifera e extrato hexânico de Dipteryx lacunifera foram avaliados quanto a atividade antibacteriana, utilizando ensaios de difusão a partir de orifício e concentração inibitória mínima (CIM), frente a cepas Gram positivas e Gram-negativas, incluindo espécies multidroga resistentes. O extrato de Q. grandiflora apresentou atividade moderada para as cepas de Staphylococcus epidermidis (CIM = 500 µg/mL) e atividade fraca sobre as demais bactérias Gram-positivas testadas e inativo sobre bactérias Gram-negativas. Os resultados obtidos com S. epidermidis apesar de moderados são importantes, uma vez que este microorganismo é o principal causador de bacteremias e sepse associada com dispositivos médicos implantados. gama-Tocoferol e a mistura de sitosterol e estigmasterol foram isolados do extrato etanólico de raiz de C. prunifera e as estruturas destes compostos foram identificadas com base na análise dos dados espectrais de RMN e comparação com a literatura.

Ethanol extracts of Qualea grandiflora and Copernicia prunifera and hexane extract of Dipteryx lacunifera were evaluated by diffusion in agar and minimum inhibitory concentration (MIC) against Gram-positive and Gram-negative bacteria including multiresistant drug strains. The extract of Q. grandiflora presented moderate activity for Staphylococcus epidermidis (MIC = 500 µg/mL) and weak activity against other Gram-positive strains and inactive for Gram-negative species. The results obtained for S. epidermidis despite being moderate are important because this pathogen is often recovered from bacteremia and sepsis from infections of implanted devices. gamma-Tocopherol and the mixture of sitosterol and stigmasterol were isolated from the ethanol extract of the roots of C. prunifera. The structures of these compounds were identified by NMR spectroscopy and comparison with literature data.