Metabolic engineering of Saccharomyces cerevisiae for de novo production of dihydrochalcones with known antioxidant,antidiabetic, and sweet tasting properties

Dihydrochalcones are plant secondary metabolites comprising molecules of significant commercial interest as antioxidants, antidiabetics, or sweeteners. To date, their heterologous biosynthesis in microorganisms has been achieved only by precursor feeding or as minor by-products in strains engineered for flavonoid production. Here, the native ScTSC13 was overexpressed in Saccharomyces cerevisiae to increase its side activity in reducing p-coumaroyl-CoA to p-dihydrocoumaroyl-CoA. De novo production of phloretin, the first committed dihydrochalcone, was achieved by co-expression of additional relevant pathway enzymes. Naringenin, a major by-product of the initial pathway, was practically eliminated by using a chalcone synthase from barley with unexpected substrate specificity. By further extension of the pathway from phloretin with decorating enzymes with known specificities for dihydrochalcones, and by exploiting substrate flexibility of enzymes involved in flavonoid biosynthesis, de novo production of the antioxidant molecule nothofagin, the antidiabetic molecule phlorizin, the sweet molecule naringin dihydrochalcone, and 3-hydroxyphloretin was achieved.     

Lysine Succinylation of VBS Contributes to Sclerotia Development and Aflatoxin Biosynthesis in Aspergillus flavus

Aspergillus flavus is a common saprophytic and pathogenic fungus, and its secondary metabolic pathways are one of the most highly characterized owing to its aflatoxin (AF) metabolite affecting global economic crops and human health. Different natural environments can cause significant variations in AF synthesis. Succinylation was recently identified as one of the most critical regulatory post-translational modifications affecting metabolic pathways. It is primarily reported in human cells and bacteria with few studies on fungi. Proteomic quantification of lysine succinylation (Ksuc) exploring its potential involvement in secondary metabolism regulation (including AF production) has not been performed under natural conditions in A. flavus. In this study, a quantification method was performed based on tandem mass tag labeling and antibody-based affinity enrichment of succinylated peptides via high accuracy nano-liquid chromatography with tandem mass spectrometry to explore the succinylation mechanism affecting the pathogenicity of naturally isolated A. flavus strains with varying toxin production. Altogether, 1240 Ksuc sites in 768 proteins were identified with 1103 sites in 685 proteins quantified. Comparing succinylated protein levels between high and low AF-producing A. flavus strains, bioinformatics analysis indicated that most succinylated proteins located in the AF biosynthetic pathway were downregulated, which directly affected AF synthesis. Versicolorin B synthase is a key catalytic enzyme for heterochrome B synthesis during AF synthesis. Site-directed mutagenesis and biochemical studies revealed that versicolorin B synthase succinylation is an important regulatory mechanism affecting sclerotia development and AF biosynthesis in A. flavus. In summary, our quantitative study of the lysine succinylome in high/low AF-producing strains revealed the role of Ksuc in regulating AF biosynthesis. We revealed novel insights into the metabolism of AF biosynthesis using naturally isolated A. flavus strains and identified a rich source of metabolism-related enzymes regulated by succinylation.     

Transmission of killer activity into laboratory and industrial strains of Saccharomyces cerevisiae by electroinjection

The killer character was electrically introduced into protoplasts of three yeast strains. These were the killer-negative variant of the K1 killer strain Saccharomyces cerevisiae T 158 C (his-); the killer-sensitive laboratory strain S. cerevisiae AH 215 (leu-, his-); and the killer-sensitive industrial strain S. cerevisiae AS 4/H2 (rho-). The killer dsRNA used for electroinjection was isolated from the super-killer strain S. cerevisiae T 158 C. Optimum numbers of transformed cells were obtained after regeneration and selection in appropriate media if the protoplasts were exposed to three exponentially decaying field pulses of 18.2 kV/cm strength and 40 microseconds duration at 4 degrees C. In the case of the killer-negative variant of S. cerevisiae T 158 C the majority of the protoplasts were transformed, whereas in the case of the two other strains the yield of transformed clones was much less. This latter result is expected if the expression of the electroinjected dsRNA was diminished in these two strains. Gel electrophoresis of the dsRNA of the clones of the three strains supported the conclusion that the transformed clones exhibited killer activity. The transformed clones of all three species were stable.     

The effects of tunicamycin on the metabolism of acetylated low density lipoproteins

The effect of tunicamycin (TM) on the metabolism of acetylated low-density lipoprotein (AcLDL) was examined to determine whether N-linked glycosylation is required for the proper function of the AcLDL pathway. Proteolytic degradation of [¹²⁵I]-AcLDL was increased twofold in the presence of TM. This did not occur via an increase in total lysosomal enzyme activity or extracellular proteolysis; rather, the rate of uptake of [¹²⁵I]-AcLDL was increased. The enhanced degradation of AcLDL did not lead to a commensurate increase in the rate of synthesis of cholesteryl oleate. Conversely, the rate of cholesterol esterification was reduced in the presence of TM. The uptake of [¹²⁵I]-AcLDL was more sensitive to inhibition by chloroquine in TM-treated cells. However, the presence of TM did not affect the ability of chloroquine to inhibit constitutive recycling of AcLDL binding sites. These results suggest that N-linked glycosylation may be involved in the regulation of AcLDL metabolism in J774 cells.     

Imputation of missing genotypes from low‐ to high‐density SNP panel in different population designs

Imputation of missing genotypes, in particular from low density to high density, is an important issue in genomic selection and genome‐wide association studies. Given the marker densities, the most important factors affecting imputation accuracy are the size of the reference population and the relationship between individuals in the reference (genotyped with high‐density panel) and study (genotyped with low‐density panel) populations. In this study, we investigated the imputation accuracies when the reference population (genotyped with Illumina BovineSNP50 SNP panel) contained sires, halfsibs, or both sires and halfsibs of the individuals in the study population (genotyped with Illumina BovineLD SNP panel) using three imputation programs (fimpute v2.2, findhap v2, and beagle v3.3.2). Two criteria, correlation between true and imputed genotypes and missing rate after imputation, were used to evaluate the performance of the three programs in different scenarios. Our results showed that fimpute performed the best in all cases, with correlations from 0.921 to 0.978 when imputing from sires to their daughters or between halfsibs. In general, the accuracies of imputing between halfsibs or from sires to their daughters were higher than were those imputing between non‐halfsibs or from sires to non‐daughters. Including both sires and halfsibs in the reference population did not improve the imputation performance in comparison with when only including halfsibs in the reference population for all the three programs.     

非药物治疗极低出生体重儿喂养不耐受的临床观察

目的:将非药物性干预措施与传统药物方法治疗极低出生体重儿喂养不耐受的临床疗效进行观察比较。方法:选取哈尔滨医科大学附属第二医院2011年1月至2013年4月出生的喂养不耐受极低出生体重儿110例。按入院顺序随机分为药物治疗组(MED)55例、非药物治疗组(NON-MED)55例。药物治疗组采用红霉素;非药物治疗组采用腹部按摩法与非营养性吸吮。结果:两组患儿经治疗后,MED组腹胀消失天数(4.4±0.5)、呕吐天数(2.2±0.5)、每日呕吐次数(3.5±0.8)、胃潴留量占每日喂养量(18.3±0.8)、达全肠道喂养天数(8.2±0.7);NON-MED组腹胀消失天数(3.7±0.1)、呕吐天数(1.9±0.6)、每日呕吐次数(2.8±0.1)、胃潴留量占每日喂养量(12.6±0.4)、达全肠道喂养天数(6.1±0.3)。各项治疗指标的对比中,差异均有统计学意义(P0.05)。MED组治疗有效率76.4%;NON-MED组治疗有效率83.6%。结论:非药物性干预措施可有效改善极低出生体重儿喂养不耐受。     

Enteric and serological distribution of serotonin and its precursor tryptophan in perinatal low and normal weight piglets

Perinatal mortality is high among small-for-gestational age (SGA) piglets and continues to be an economic burden and threat to animal welfare. As the physiological role of serotonin (5-hydroxytryptamine, 5-HT) in perinatal development and gastrointestinal function in the pig remains unknown, the aim of this study was to assess the enteric distribution of 5-HT cells and to determine 5-HT together with its precursor tryptophan in the serum of perinatal normal and SGA piglets. For this purpose, proximal and distal parts of the small intestine (SI) were processed for immunohistochemical analysis to assess the presence of 5-HT endocrine cells. Serum 5-HT was measured with ELISA, whereas its precursor, that is, the free fraction of tryptophan (FFT) together with albumin-bound tryptophan and total tryptophan, were analysed with HPLC in postnatal piglets. In addition, the morphological growth patterns of the different intestinal tissue layers of both normal and SGA piglets were stereologically analysed. The stereological volume density of 5-HT enteroendocrine cells showed a significant interaction effect between age and region. Indeed, the amount of 5-HT cells in both the proximal and distal part of the SI tended to decrease according to age, with the lowest values detected at day 3 postpartum. No differences could be observed related to BW. Interestingly, the serum concentration of 5-HT was higher in normal piglets compared with SGA piglets. Moreover, the ratio of FFT to total tryptophan was significantly affected by age and BW. Normal piglets had, on average, a lower FFT/total tryptophan ratio compared with SGA piglets. An approximate linear decrease was observed with increasing age. Finally, the immaturity of the intestinal system of the SGA piglets was not reflected in altered volume densities of the different intestinal layers. To conclude, although no BW effect could be detected in the distribution of enteric 5-HT cells, serum 5-HT and the ratio of FFT to total tryptophan ratio showed significant differences between normal piglets and their SGA littermates.     

Modification of Brassica napus seed oil by expression of the Escherichia coli fabH gene,encoding 3-ketoacyl-acyl carrier protein synthase III

The Escherichia coli fabH gene encoding 3-ketoacyl-acyl carrier protein synthase III (KAS III) was isolated and the effect of overproduction of bacterial KAS III was compared in both E. coli and Brassica napus. The change in fatty acid profile of E. coli was essentially the same as that reported by Tsay et al. (J Biol Chem 267 (1992) 6807–6814), namely higher C14:0 and lower C18:1 levels. In our study, however, an arrest of cell growth was also observed. This and other evidence suggests that in E. coli the accumulation of C14:0 may not be a direct effect of the KAS III overexpression, but a general metabolic consequence of the arrest of cell division. Bacterial KAS III was expressed in a seed- and developmentally specific manner in B. napus in either cytoplasm or plastid. Significant increases in KAS III activities were observed in both these transformation groups, up to 3.7 times the endogenous KAS III activity in mature seeds. Only the expression of the plastid-targeted KAS III gene, however, affected the fatty acid profile of the storage lipids, such that decreased amounts of C18:1 and increased amounts of C18:2 and C18:3 were observed as compared to control plants. Such changes in fatty acid composition reflect changes in the regulation and control of fatty acid biosynthesis. We propose that fatty acid biosynthesis is not controlled by one rate-limiting enzyme, such as acetyl-CoA carboxylase, but rather is shared by a number of component enzymes of the fatty acid biosynthetic machinery.