Development and standardization of spectrophotometric assay for quantification of thermal hydrolysis-origin melanoidins and its implication in antioxidant activity evaluation.

Melanoidins are brown recalcitrant polymers originating from the thermal hydrolysis pretreatment (THP) of organic solid waste (OSW). Owing to their various formation pathways and complex structures, there is currently no reliable method to quantify melanoidins. In this study, a spectrophotometric method was developed to determine melanoidins concentration in different OSW. Three typical model Maillard reaction systems (glucose-glycine, glucose/fructose-20 amino acids, and dextran-bovine serum albumin) were used to acquire the characteristic peaks and establish standard curves. The results showed that a standard curve using glucose/fructose-20 amino acids model melanoidins at 280 nm was the optimal quantification method, because it had the best correlation with the physicochemical indicators of melanoidins and semi-quantification results calculated by excitation-emission matrix fluorescence. In addition, the applicability of the proposed method was evaluated using multiple real melanoidins samples extracted from thermally pretreated OSW under different THP conditions and food-derived melanoidins as well, demonstrating its validity and advantages. This study is the first to provide a simple, effective, and accurate method for quantifying THP-origin melanoidins from different sources. Remarkably, as a specific and important application scenario, the proposed quantification method was employed to investigate the concentration dependence of melanoidins antioxidation in thermally pretreated OSW.

Genome-wide DNA mutations in Arabidopsis plants after multigenerational exposure to high temperatures.

BACKGROUND: Elevated temperatures can cause physiological, biochemical, and molecular responses in plants that can greatly affect their growth and development. Mutations are the most fundamental force driving biological evolution. However, how long-term elevations in temperature influence the accumulation of mutations in plants remains unknown. RESULTS: Multigenerational exposure of Arabidopsis MA (mutation accumulation) lines and MA populations to extreme heat and moderate warming results in significantly increased mutation rates in single-nucleotide variants (SNVs) and small indels. We observe distinctive mutational spectra under extreme and moderately elevated temperatures, with significant increases in transition and transversion frequencies. Mutation occurs more frequently in intergenic regions, coding regions, and transposable elements in plants grown under elevated temperatures. At elevated temperatures, more mutations accumulate in genes associated with defense responses, DNA repair, and signaling. Notably, the distribution patterns of mutations among all progeny differ between MA populations and MA lines, suggesting that stronger selection effects occurred in populations. Methylation is observed more frequently at mutation sites, indicating its contribution to the mutation process at elevated temperatures. Mutations occurring within the same genome under elevated temperatures are significantly biased toward low gene density regions, special trinucleotides, tandem repeats, and adjacent simple repeats. Additionally, mutations found in all progeny overlap significantly with genetic variations reported in 1001 Genomes, suggesting non-uniform distribution of de novo mutations through the genome. CONCLUSION: Collectively, our results suggest that elevated temperatures can accelerate the accumulation, and alter the molecular profiles, of DNA mutations in plants, thus providing significant insight into how environmental temperatures fuel plant evolution.

Whole-genome resequencing of Osmanthus fragrans provides insights into flower color evolution.

Osmanthus fragrans is a well-known ornamental plant that has been domesticated in China for 2500 years. More than 160 cultivars have been found during this long period of domestication, and they have subsequently been divided into four cultivar groups, including the Yingui, Jingui, Dangui, and Sijigui groups. These groups provide a set of materials to study genetic evolution and variability. Here, we constructed a reference genome of O. fragrans 'Liuyejingui' in the Jingui group and investigated its floral color traits and domestication history by resequencing a total of 122 samples, including 119 O. fragrans accessions and three other Osmanthus species, at an average sequencing depth of 15×. The population structure analysis showed that these 119 accessions formed an apparent regional cluster. The results of linkage disequilibrium (LD) decay analysis suggested that varieties with orange/red flower color in the Dangui group had undergone more artificial directional selection; these varieties had the highest LD values among the four groups, followed by the Sijigui, Jingui, and Yingui groups. Through a genome-wide association study, we further identified significant quantitative trait loci and genomic regions containing several genes, such as ethylene-responsive transcription factor 2 and Arabidopsis pseudoresponse regulator 2, that are positively associated with petal color. Moreover, we found a frameshift mutation with a 34-bp deletion in the first coding region of the carotenoid cleavage dioxygenase 4 gene. This frameshift mutation existed in at least one site on both alleles in all varieties of the Dangui group. The results from this study shed light on the genetic basis of domestication in woody plants, such as O. fragrans.

Peroxisome-targeted and tandem repeat multimer expressions of human antimicrobial peptide LL37 in Pichia pastoris.

Although the human antimicrobial peptide LL37 has a broad spectrum of antimicrobial activities, it easily damages host cells following heterologous expressions. This study attempted two strategies to alleviate its damage to host cells when expressed in Pichia pastoris using the AOX1 promoter. Tandem repeat multimers of LL37 were first designed, and secretion expression strains GS115-9K-(DPLL37DP)n (n = 2, 4, 6 and 8) containing different copies of the LL37 gene were constructed. However, LL37 tandems still killed the cells after 96 hr of induction. Subsequently, peroxisome-targeted expression was performed by adding a peroxisomal targeting signal 1 (SKL) at the C-terminus of LL37. The LL37 expression strain GS115-3.5K-LL37-SKL showed no significant inhibition in the cells after induction. Antibacterial activity assays showed that the recombinant LL37 expressed in peroxisomes had good antimicrobial activities. Then, a strain GS115-3.5K-LL37-GFP-SKL producing LL37, green fluorescent protein, and SKL fusion proteins was constructed, and the fusion protein was confirmed to be targeting the peroxisomes. However, protein extraction analysis indicated that most of the fusion proteins were still located in the cell debris after cell disruption, and further studies are required to extract more proteins from the peroxisome membrane.

Engineered fungal polyketide biosynthesis in Pichia pastoris: a potential excellent host for polyketide production.

BACKGROUND: Polyketides are one of the most important classes of secondary metabolites and usually make good drugs. Currently, heterologous production of fungal polyketides for developing a high potential industrial application system with high production capacity and pharmaceutical feasibility was still at its infancy. Pichia pastoris is a highly successful system for the high production of a variety of heterologous proteins. In this work, we aim to develop a P. pastoris based in vivo fungal polyketide production system for first time and evaluate its feasibility for future industrial application. RESULTS: A recombinant P. pastoris GS115-NpgA-ATX with Aspergillus nidulans phosphopantetheinyl transferase (PPtase) gene npgA and Aspergillus terrus 6-methylsalicylic acid (6-MSA) synthase (6-MSAS) gene atX was constructed. A specific compound was isolated and identified as 6-MSA by HPLC, LC-MS and NMR. Transcription of both genes were detected. In 5-L bioreactor, the GS115-NpgA-ATX grew well and produced 6-MSA quickly until reached a high value of 2.2 g/L by methanol induction for 20 hours. Thereafter, the cells turned to death ascribing to high concentration of antimicrobial 6-MSA. The distribution of 6-MSA changed that during early and late induction phase it existed more in supernatant while during intermediate stage it mainly located intracellular. Different from 6-MSA production strain, recombinant M. purpureus pksCT expression strains for citrinin intermediate production, no matter PksCT located in cytoplasm or in peroxisomes, did not produce any specific compound. However, both npgA and pksCT transcripted effectively in cells and western blot analysis proved the expression of PPtase. Then the PPTase was expressed and purified, marked by fluorescent probes, and reacted with purified ACP domain and its mutant ACPm of PksCT. Fluoresence was only observed in ACP but not ACPm, indicating that the PPTase worked well with ACP to make it bioactive holo-ACP. Thus, some other factors may affect polyketide synthesis that include activities of the individual catalytic domains and release of the product from the synthase of PksCT. CONCLUSIONS: An efficient P. pastoris expression system of fungal polyketides was successfully constructed. It produced a high production of 6-MSA and holds potential for future industrial application of 6-MSA and other fungal polyketides.