DEAttentionDTA: protein-ligand binding affinity prediction based on dynamic embedding and self-attention.

MOTIVATION: Predicting protein-ligand binding affinity is crucial in new drug discovery and development. However, most existing models rely on acquiring 3D structures of elusive proteins. Combining amino acid sequences with ligand sequences and better highlighting active sites are also significant challenges. RESULTS: We propose an innovative neural network model called DEAttentionDTA, based on dynamic word embeddings and a self-attention mechanism, for predicting protein-ligand binding affinity. DEAttentionDTA takes the 1D sequence information of proteins as input, including the global sequence features of amino acids, local features of the active pocket site, and linear representation information of the ligand molecule in the SMILE format. These three linear sequences are fed into a dynamic word-embedding layer based on a 1D convolutional neural network for embedding encoding and are correlated through a self-attention mechanism. The output affinity prediction values are generated using a linear layer. We compared DEAttentionDTA with various mainstream tools and achieved significantly superior results on the same dataset. We then assessed the performance of this model in the p38 protein family. AVAILABILITY AND IMPLEMENTATION: The resource codes are available at https://github.com/whatamazing1/DEAttentionDTA.

Quantitative estimates of the regulatory influence of long non-coding RNAs on global gene expression variation using TCGA breast cancer transcriptomic data.

Long non-coding RNAs (lncRNAs) have received attention in recent years for their regulatory roles in diverse biological contexts including cancer, yet large gaps remain in our understanding of their mechanisms and global maps of their targets. In this work, we investigated a basic unanswered question of lncRNA systems biology: to what extent can gene expression variation across individuals be attributed to lncRNA-driven regulation? To answer this, we analyzed RNA-seq data from a cohort of breast cancer patients, explaining each gene's expression variation using a small set of automatically selected lncRNA regulators. A key aspect of this analysis is that it accounts for confounding effects of transcription factors (TFs) as common regulators of a lncRNA-mRNA pair, to enrich the explained gene expression for lncRNA-mediated regulation. We found that for 16% of analyzed genes, lncRNAs can explain more than 20% of expression variation. We observed 25-50% of the putative regulator lncRNAs to be in 'cis' to, i.e., overlapping or located proximally to the target gene. This led us to quantify the global regulatory impact of such cis-located lncRNAs, which was found to be substantially greater than that of trans-located lncRNAs. Additionally, by including statistical interaction terms involving lncRNA-protein pairs as predictors in our regression models, we identified cases where a lncRNA's regulatory effect depends on the presence of a TF or RNA-binding protein. Finally, we created a high-confidence lncRNA-gene regulatory network whose edges are supported by co-expression as well as a plausible mechanism such as cis-action, protein scaffolding or competing endogenous RNAs. Our work is a first attempt to quantify the extent of gene expression control exerted globally by lncRNAs, especially those located proximally to their regulatory targets, in a specific biological (breast cancer) context. It also marks a first step towards systematic reconstruction of lncRNA regulatory networks, going beyond the current paradigm of co-expression networks, and motivates future analyses assessing the generalizability of our findings to additional biological contexts.

Transcriptome analyses of Acer Truncatum Bunge seeds to delineate the genes involved in fatty acid metabolism.

BACKGROUND: Acer truncatum Bunge is an economic, ecological, oil, and medicinal tree, and its kernel oil is rich in nervonic acid. It is crucial to explore the transcriptional expression patterns of genes affecting fatty acid synthesis to improve the quality of Acer truncatum oil. RESULTS: This study used the seeds from high fatty acid strain YQC and those from low fatty acid strain Y38 as the test materials. Specifically, we performed a comparative transcriptome analysis of Y38 seeds and YQC to identify differentially expressed genes (DEGs) at two time points (seeds 30 days after the blooming period and 90 days after the blooming period). Compared with YQC_1 (YQC seeds at 30 days after the blooming period), a total of 3,618 DEGs were identified, including 2,333 up-regulated and 1,285 downregulated DEGs in Y38_1 (Y38 seeds at 30 days after blooming period). In the Y38_2 (Y38 seeds at 90 days after the blooming period) versus YQC_2 (YQC seeds at 90 days after the blooming period) comparison group, 9,340 genes were differentially expressed, including 5,422 up-regulated and 3,918 down-regulated genes. The number of DEGs in Y38 compared to YQC was significantly higher in the late stages of seed development. Gene functional enrichment analyses showed that the DEGs were mainly involved in the fatty acid biosynthesis pathway. And two fatty acid synthesis-related genes and seven nervonic acid synthesis-related genes were validated by qRT-PCR. CONCLUSIONS: This study provides a basis for further research on biosynthesizing fatty acids and nervonic acidnervonic acids in A. truncatum seeds.

Genomic divergence and demographic history of Quercus aliena populations.

BACKGROUND: Quercus aliena is a major montane tree species of subtropical and temperate forests in China, with important ecological and economic value. In order to reveal the species' population dynamics, genetic diversity, genetic structure, and association with mountain habitats during the evolutionary process, we re-sequenced the genomes of 72 Q. aliena individuals. RESULTS: The whole chloroplast and nuclear genomes were used for this study. Phylogenetic analysis using the chloroplast genome dataset supported four clades of Q. aliena, while the nuclear dataset supported three major clades. Sex-biased dispersal had a critical role in causing discordance between the chloroplast and nuclear genomes. Population structure analysis showed two groups in Q. aliena. The effective population size sharply declined 1 Mya, coinciding with the Poyang Glaciation in Eastern China. Using genotype-climate association analyses, we found a positive correlation between allele frequency variation in SNPs and temperature, suggesting the species has the capacity to adapt to changing temperatures. CONCLUSION: Overall, this study illustrates the genetic divergence, genomic variation, and evolutionary processes behind the demographic history of Q. aliena.

Comprehensive analysis of the complete mitochondrial genome of Lilium tsingtauense reveals a novel multichromosome structure.

KEY MESSAGE: Lilium tsingtauense mitogenome comprises 27 independent chromosome molecules, it undergoes frequent genomic recombination, and the rate of recombination and mutation between different repetitive sequences affects the formation of multichromosomal structures. Given the extremely large genome of Lily, which likely harbors additional genetic resources, it serves as an ideal material for studying the phylogenetic evolution of organisms. Although the Lilium chloroplast genome has been documented, the sequence of its mitochondrial genome (mitogenome) remains uncharted. Using BGI short reads and Nanopore long reads, we sequenced, assembled, and annotated the mitogenome of Lilium tsingtauense. This effort culminated in the characterization of Lilium's first complete mitogenome. Comparative analysis with other angiosperms revealed the unique multichromosomal structure of the L. tsingtauense mitogenome, spanning 1,125,108 bp and comprising 27 independent circular chromosomes. It contains 36 protein-coding genes, 12 tRNA genes, and 3 rRNA genes, with a GC content of 44.90%. Notably, three chromosomes in the L. tsingtauense mitogenome lack identifiable genes, hinting at the potential existence of novel genes and noncoding elements. The high degree of observed genome fragmentation implies frequent reorganization, with recombination and mutation rates among diverse repetitive sequences likely driving the formation of multichromosomal structures. Our comprehensive analysis, covering genome size, coding genes, structure, RNA editing, repetitive sequences, and sequence migration, sheds light on the evolutionary and molecular biology of multichromosomal mitochondria in Lilium. This high-quality mitogenome of L. tsingtauense not only enriches our understanding of multichromosomal mitogenomes but also establishes a solid foundation for future genome breeding and germplasm innovation in Lilium.

Resequencing of Rosa rugosa accessions revealed the history of population dynamics, breed origin, and domestication pathways.

BACKGROUND: Rosa rugosa is a shrub that originated in China and has economic and ecological value. However, during the development of R. rugosa, the genetic background was chaotic, and the genetic structure among different wild populations was unclear, as well as wild and cultivated accessions. Here, we report whole-genome resequencing of wild and cultivated R. rugosa accessions. RESULTS: A total of 19,041,284 SNPs were identified in 188 R. rugosa accessions and 3 R. chinensis accessions by resequencing. Population genetic analysis revealed that cultivated and wild groups were separated very early. All R. rugosa accessions were divided into 8 categories based on genetic structure: (1) Weihai, Yantai, and Liaoning category, (2) Jilin category, and (3) Hammonasset category (above three are wild); (4) traditional varieties, (5) hybrids between R. rugosa and R. chinensis, (6) Zizhi Rose, (7) Kushui Rose, (8) hybrids between R. rugosa and R. multiflora. We found that the heterozygosity and genetic diversity of wild accessions were generally lower than those of cultivated individuals. The genes that were selected during cultivation were identified, and it was found that these genes were mainly related to environmental adaptation and growth. CONCLUSIONS: The Jilin population was the oldest population and later migrated to Liaoning and then migrated to Yantai and Weihai by sea regression in the Bohai Basin. The Hammonasset naturalized population probably originated from the Jilin population and then experienced separate differentiation. The long-term asexual reproduction pattern of R. rugosa decreased genetic diversity in the wild population. During R. rugosa cultivation, the ancestors of the Jilin population were involved in breeding traditional varieties, after which almost no wild individuals were engaged in breeding. However, in recent decades, cross breeding of R. rugosa started the utilization of wild germplasms. In comparison, some other species play important roles in variety formation. Few genes related to economic traits were selected, suggesting no directional domestication in the R. rugosa cultivation process.

VarSAn: associating pathways with a set of genomic variants using network analysis.

There is a pressing need today to mechanistically interpret sets of genomic variants associated with diseases. Here we present a tool called 'VarSAn' that uses a network analysis algorithm to identify pathways relevant to a given set of variants. VarSAn analyzes a configurable network whose nodes represent variants, genes and pathways, using a Random Walk with Restarts algorithm to rank pathways for relevance to the given variants, and reports P-values for pathway relevance. It treats non-coding and coding variants differently, properly accounts for the number of pathways impacted by each variant and identifies relevant pathways even if many variants do not directly impact genes of the pathway. We use VarSAn to identify pathways relevant to variants related to cancer and several other diseases, as well as drug response variation. We find VarSAn's pathway ranking to be complementary to the standard approach of enrichment tests on genes related to the query set. We adopt a novel benchmarking strategy to quantify its advantage over this baseline approach. Finally, we use VarSAn to discover key pathways, including the VEGFA-VEGFR2 pathway, related to de novo variants in patients of Hypoplastic Left Heart Syndrome, a rare and severe congenital heart defect.

Characterization of a New Thermostable and Organic Solution-Tolerant Lipase from Pseudomonas fluorescens and Its Application in the Enrichment of Polyunsaturated Fatty Acids.

The search for and characterization of new lipases with excellent properties has always been urgent and is of great importance to meet industrial needs. In this study, a new lipase, lipB, from Pseudomonas fluorescens SBW25, belonging to the lipase subfamily I.3, was cloned and expressed in Bacillus subtilis WB800N. Enzymatic properties studies of recombinant LipB found that it exhibited the highest activity towards p-nitrophenyl caprylate at 40 °C and pH 8.0, retaining 73% of its original activity after incubation at 70 °C for 6 h. In addition, Ca2+, Mg2+, and Ba2+ strongly enhanced the activity of LipB, while Cu2+, Zn2+, Mn2+, and CTAB showed an inhibiting effect. The LipB also displayed noticeable tolerance to organic solvents, especially acetonitrile, isopropanol, acetone, and DMSO. Moreover, LipB was applied to the enrichment of polyunsaturated fatty acids from fish oil. After hydrolyzing for 24 h, it could increase the contents of polyunsaturated fatty acids from 43.16% to 72.18%, consisting of 5.75% eicosapentaenoic acid, 19.57% docosapentaenoic acid, and 46.86% docosahexaenoic acid, respectively. The properties of LipB render it great potential in industrial applications, especially in health food production.

De Novo Computational Design of a Lipase with Hydrolysis Activity towards Middle-Chained Fatty Acid Esters.

Innovations in biocatalysts provide great prospects for intolerant environments or novel reactions. Due to the limited catalytic capacity and the long-term and labor-intensive characteristics of mining enzymes with the desired functions, de novo enzyme design was developed to obtain industrial application candidates in a rapid and convenient way. Here, based on the catalytic mechanisms and the known structures of proteins, we proposed a computational protein design strategy combining de novo enzyme design and laboratory-directed evolution. Starting with the theozyme constructed using a quantum-mechanical approach, the theoretical enzyme-skeleton combinations were assembled and optimized via the Rosetta "inside-out" protocol. A small number of designed sequences were experimentally screened using SDS-PAGE, mass spectrometry and a qualitative activity assay in which the designed enzyme 1a8uD1 exhibited a measurable hydrolysis activity of 24.25 ± 0.57 U/g towards p-nitrophenyl octanoate. To improve the activity of the designed enzyme, molecular dynamics simulations and the RosettaDesign application were utilized to further optimize the substrate binding mode and amino acid sequence, thus keeping the residues of theozyme intact. The redesigned lipase 1a8uD1-M8 displayed enhanced hydrolysis activity towards p-nitrophenyl octanoate-3.34 times higher than that of 1a8uD1. Meanwhile, the natural skeleton protein (PDB entry 1a8u) did not display any hydrolysis activity, confirming that the hydrolysis abilities of the designed 1a8uD1 and the redesigned 1a8uD1-M8 were devised from scratch. More importantly, the designed 1a8uD1-M8 was also able to hydrolyze the natural middle-chained substrate (glycerol trioctanoate), for which the activity was 27.67 ± 0.69 U/g. This study indicates that the strategy employed here has great potential to generate novel enzymes exhibiting the desired reactions.

Exposure to diisononyl phthalate promotes atopic march by activating of NF-κB and p38 MAPK.

What factors and underlying mechanisms influence the occurrence of the atopic march remain unclear. Recent studies suggest that exposure to diisononyl phthalate (DINP) might be associated with the occurrence of atopic dermatitis (AD) and asthma. However, little is known about the role of DINP exposure in the atopic march. In this study, we investigated the effect of DINP exposure on the progression from AD to asthma, and explored the potential mechanisms. We built an atopic march mouse model from AD to asthma, by exposure to DINP and sensitization with OVA. Pyrrolidine dithiocarbamate and SB203580 were used to block NF-κB and p38 MAPK respectively, to explore the possible molecular mechanisms. The data showed that DINP aggravated airway remodeling and airway hyperresponsiveness (AhR) in the progression from AD to asthma, induced a sharp increase in IL-33, IgE, Th2 and Th17 cytokines, and resulted in an increase in the expression of thymic stromal lymphopoietin (TSLP) and in the number of inflammatory cells. Blocking NF-κB inhibited AD-like lesions, and the production of IL-33 and TSLP in the progression of AD, while alleviating airway remodeling, AhR, and the expression of Th2 and Th17 cytokines in both the progression of AD and the asthmatic phenotype. Blocking p38 MAPK in the progression of asthma, inhibited airway remodeling, AhR, and the expression of Th2 and Th17 cytokines. The results demonstrated that exposure to DINP enhanced the immune response to memory CD4+ T helper cells through the NF-κB and p38 MAPK signaling pathways, leading to an aggravation of the atopic march.

Exposure to polystyrene microplastics causes reproductive toxicity through oxidative stress and activation of the p38 MAPK signaling pathway.

Microplastics (MP) are receiving increased attention as a harmful environmental pollutant, however information on the reproduction toxicity of MP in terrestrial animals, especially mammals, is limited. In this experiment, we investigated the impact of polystyrene microplastics (micro-PS) on the reproductive system of male mice. Healthy Balb/c mice were exposed to saline or to different doses of micro-PS for 6 weeks. The results showed that micro-PS exposure resulted in a significant decrease in the number and motility of sperm, and a significant increase in sperm deformity rate. We also detected a decrease in the activity of the sperm metabolism-related enzymes, succinate dehydrogenase (SDH) and lactate dehydrogenase (LDH), and a decrease in the serum testosterone content in the micro-PS exposure group. We found that micro-PS exposure caused oxidative stress and activated JNK and p38 MAPK. In addition, we found that when N-acetylcysteine (NAC) scavenges ROS, and when the p38 MAPK-specific inhibitor SB203580 inhibits p38MAPK, the micro-PS-induced sperm damage is alleviated and testosterone secretion improves. In conclusion, our findings suggest that micro-PS induces reproductive toxicity in mice through oxidative stress and activation of the p38 MAPK signaling pathways.

Mechanistic interpretation of non-coding variants for discovering transcriptional regulators of drug response.

BACKGROUND: Identification of functional non-coding variants and their mechanistic interpretation is a major challenge of modern genomics, especially for precision medicine. Transcription factor (TF) binding profiles and epigenomic landscapes in reference samples allow functional annotation of the genome, but do not provide ready answers regarding the effects of non-coding variants on phenotypes. A promising computational approach is to build models that predict TF-DNA binding from sequence, and use such models to score a variant's impact on TF binding strength. Here, we asked if this mechanistic approach to variant interpretation can be combined with information on genotype-phenotype associations to discover transcription factors regulating phenotypic variation among individuals. RESULTS: We developed a statistical approach that integrates phenotype, genotype, gene expression, TF ChIP-seq, and Hi-C chromatin interaction data to answer this question. Using drug sensitivity of lymphoblastoid cell lines as the phenotype of interest, we tested if non-coding variants statistically linked to the phenotype are enriched for strong predicted impact on DNA binding strength of a TF and thus identified TFs regulating individual differences in the phenotype. Our approach relies on a new method for predicting variant impact on TF-DNA binding that uses a combination of biophysical modeling and machine learning. We report statistical and literature-based support for many of the TFs discovered here as regulators of drug response variation. We show that the use of mechanistically driven variant impact predictors can identify TF-drug associations that would otherwise be missed. We examined in depth one reported association-that of the transcription factor ELF1 with the drug doxorubicin-and identified several genes that may mediate this regulatory relationship. CONCLUSION: Our work represents initial steps in utilizing predictions of variant impact on TF binding sites for discovery of regulatory mechanisms underlying phenotypic variation. Future advances on this topic will be greatly beneficial to the reconstruction of phenotype-associated gene regulatory networks.

Comparing the effects of diethylhexyl phthalate and dibutyl phthalate exposure on hypertension in mice.

Epidemiological studies have shown that high molecular weight phthalates (HMW) such as diethylhexyl phthalate (DEHP), are associated with hypertension in humans, while low molecular weight phthalates (LMW) such as dibutyl phthalate (DBP), have hardly any impact on the elevation of blood pressure. However, the molecular mechanisms responsible for this difference are not completely understood. In this experiment, mice were exposed to 0.1/1/10 mg/kg/day DEHP and 0.1/1/10 mg/kg/day DBP for 6 weeks, and their blood pressure was monitored using the tail pressure method. The results showed that exposure to DEHP dosages of 1 or 10 mg/kg/day resulted in a sharp increase in blood pressure, while exposure to DBP did not induce any significant changes in blood pressure. Investigating the renin-angiotensin-aldosterone system (RAAS) and NO pathway in mice exposed to DEHP, we found that levels of angiotensin-converting enzyme (ACE) and angiotensin II (AngII) increased with increasing exposure to DEHP, and the expression of nitric oxide synthase (eNOS) and the level of NO decreased. Treatment with ACE inhibitor (ACEI) to block the ACE pathway inhibited the enhancement of RAAS expression, inhibited the increase in blood pressure, and inhibited the decrease in NO levels induced by DEHP. However, the expression of ACE, AngII, AT1R, and eNOS in the DBP treatment groups showed no significant changes. When examining estradiol in vivo, we found that exposure to DBP resulted in a significant increase in the level of estradiol, while exposure to DEHP did not lead to a significant change. When ICI182780 was used to block the estradiol receptors, any increase in the level of NO induced by DBP exposure, was inhibited. These results indicate that exposure to DEHP induces an increase in mouse blood pressure through RAAS, and the different effects of DEHP and DBP on blood pressure are partly due to the different estradiol levels induced by DEHP and DBP.

Interspecific chloroplast genome sequence diversity and genomic resources in Diospyros.

BACKGROUND: Fruits of persimmon plants are traditional healthy food in China, Korea and Japan. However, due to the shortage of morphological and DNA markers, the development of persimmon industry has been heavily inhibited. RESULTS: Chloroplast genomes of Diospyros cathayensis, D. virginiana, D. rhombifolia and D. deyangensis were newly sequenced. Comparative analyses of ten chloroplast genomes including six previously published chloroplast genomes of Diospyros provided new insights into the genome sequence diversity and genomic resources of the genus. Eight hyper-variable regions, trnH-psbA, rps16-trnQ, rpoB-trnC, rps4-trnT-trnL, ndhF, ndhF-rpl32-trnL, ycf1a, and ycf1b, were discovered and can be used as chloroplast DNA markers at/above species levels. The complete chloroplast genome sequences provided the best resolution at inter-specific level in comparison with different chloroplast DNA sequence datasets. CONCLUSION: Diospyros oleifera, D. deyangensis, D. virginiana, D. glaucifolia, D. lotus and D. jinzaoshi are important wild species closely related to the cultivated persimmon D. kaki. The hyper-variable regions can be used as DNA markers for global genetic diversity detection of Diospyros. Deeper study on these taxa would be helpful for elucidating the origin of D. kaki.

Enzymes involved in the anaerobic degradation of phenol by the sulfate-reducing bacterium Desulfatiglans anilini.

BACKGROUND: The sulfate-reducing bacterium Desulfatiglans anilini can grow with phenol as sole source of carbon and energy under strictly anaerobic, sulfate-reducing conditions. In the nitrate-reducing bacterium Thauera aromatica, the enzymes involved in phenol degradation have been well elucidated, whereas the anaerobic phenol degradation pathway by D. anilini was not studied in detail yet. RESULTS: The pathway of anaerobic phenol degradation by the sulfate-reducing bacterium Desulfatiglans anilini was studied by identification of genes coding for phenylphosphate synthase (encoded by pps genes) and phenylphosphate carboxylase (encoded by ppc genes) in the genome of D. anilini, by analysis of the transcription and translation of pps-ppc genes, and by measurement of phenylphosphate synthase activity in cell-free extracts of phenol-grown cells. The majority of genes involved in phenol degradation were found to be organized in one gene cluster. The gene cluster contained genes ppsα (phenylphosphate synthase alpha subunit), ppsß (phenylphosphate synthase beta subunit), ppcß (phenylphosphate carboxylase beta subunit), as well as 4-hydroxybenzoyl-CoA ligase and 4-hydroxylbenzoyl-CoA reductase-encoding genes. The genes ppsγ (phenylphosphate synthase gamma subunit), ppcα (phenylphosphate carboxylase alpha subunit) and ppcδ (phenylphosphate carboxylase delta subunit) were located elsewhere in the genome of D. anilini, and no obvious homologue of ppcγ (phenylphosphate carboxylase gamma subunit) was found in the genome. Induction of genes pps and ppc during growth on phenol was confirmed by reverse transcription polymerase chain reaction. Total proteome analysis revealed that the abundance of enzymes encoded by the gene cluster under study was much higher in phenol-grown cells than that in benzoate-grown cells. In in-vitro enzyme assays with cell-free extracts of phenol-grown cells, phenylphosphate was formed from phenol in the presence of ATP, Mg2+, Mn2+, K+ as co-factors. CONCLUSIONS: The genes coding for enzymes involved in the anaerobic phenol degradation pathway were identified in the sulfate-reducing bacterium D. anilini. The results indicate that the first steps of anaerobic phenol degradation in D. anilini are phosphorylation of phenol to phenylphosphate by phenylphosphate synthase and carboxylation of phenylphosphate by phenylphosphate carboxylase.

In silico design of multipoint mutants for enhanced performance of Thermomyces lanuginosus lipase for efficient biodiesel production.

BACKGROUND: Biodiesel, an emerging sustainable and renewable clean energy, has garnered considerable attention as an alternative to fossil fuels. Although lipases are promising catalysts for biodiesel production, their efficiency in industrial-scale application still requires improvement. RESULTS: In this study, a novel strategy for multi-site mutagenesis in the binding pocket was developed via FuncLib (for mutant enzyme design) and Rosetta Cartesian_ddg (for free energy calculation) to improve the reaction rate and yield of lipase-catalyzed biodiesel production. Thermomyces lanuginosus lipase (TLL) with high activity and thermostability was obtained using the Pichia pastoris expression system. The specific activities of the mutants M11 and M21 (each with 5 and 4 mutations) were 1.50- and 3.10-fold higher, respectively, than those of the wild-type (wt-TLL). Their corresponding melting temperature profiles increased by 10.53 and 6.01 °C, [Formula: see text] (the temperature at which the activity is reduced to 50% after 15 min incubation) increased from 60.88 to 68.46 °C and 66.30 °C, and the optimum temperatures shifted from 45 to 50 °C. After incubation in 60% methanol for 1 h, the mutants M11 and M21 retained more than 60% activity, and 45% higher activity than that of wt-TLL. Molecular dynamics simulations indicated that the increase in thermostability could be explained by reduced atomic fluctuation, and the improved catalytic properties were attributed to a reduced binding free energy and newly formed hydrophobic interaction. Yields of biodiesel production catalyzed by mutants M11 and M21 for 48 h at an elevated temperature (50 °C) were 94.03% and 98.56%, respectively, markedly higher than that of the wt-TLL (88.56%) at its optimal temperature (45 °C) by transesterification of soybean oil. CONCLUSIONS: An integrating strategy was first adopted to realize the co-evolution of catalytic efficiency and thermostability of lipase. Two promising mutants M11 and M21 with excellent properties exhibited great potential for practical applications for in biodiesel production.

In-situ co-immobilization of lipase, lipoxygenase and L-cysteine within a metal-amino acid framework for conversion of soybean oil into higher-value products.

We propose a co-immobilized chemo-enzyme cascade system to mitigate random intermediate diffusion from the mixture of individual immobilized catalysts and achieve a one-pot reaction of multi-enzyme and reductant. Catalyzed by lipase and lipoxygenase, unsaturated lipid hydroperoxides (HPOs) were synthesized. 13(S)-hydroperoxy-9Z, 11E-octadecadienoic acid (13-HPODE), one compound of HPOs, was subsequently reduced to 13(S)-hydroxy-9Z, 11E-octadecadienoic acid (13-HODE) by cysteine. Upon the optimized conditions, 75.28 mg of 13-HPODE and 4.01 mg of 13-HODE were produced from per milliliter of oil. The co-immobilized catalysts exhibited improved yield compared to the mixture of individually immobilized catalysts. Moreover, it demonstrated satisfactory durability and recyclability, maintaining a relative HPOs yield of 78.5% after 5 cycles. This work has achieved the co-immobilization of lipase, lipoxygenase and the reductant cysteine for the first time, successfully applying it to the conversion of soybean oil into 13-HODE. It offers a technological platform for transforming various oils into high-value products.

Engineering Yarrowia lipolytica as a Cellulolytic Cell Factory for Production of p-Coumaric Acid from Cellulose and Hemicellulose.

De novo biosynthesis of high-value added food additive p-coumaric acid (p-CA) direct from cellulose/hemicellulose is a more sustainable route compared to the chemical route, considering the abundant cellulose/hemicellulose resources. In this study, a novel factory was constructed for the production of p-CA in Yarrowia lipolytica using cellulose/hemicellulose as the sole carbon source. Based on multicopy integration of the TAL gene and reprogramming the shikimic acid pathway, the engineered strain produced 1035.5 ± 67.8 mg/L p-CA using glucose as a carbon source. The strains with overexpression of cellulases and hemicellulases produced 84.3 ± 2.4 and 65.3 ± 4.6 mg/L p-CA, using cellulose (carboxymethyl-cellulose) or hemicellulose (xylan from bagasse) as the carbon source, respectively. This research demonstrated the feasibility of conversion of cost-effective cellulose/hemicellulose into a value-added product and provided a sustainable cellulolytic cell factory for the utilization of cellulose/hemicellulose.

Modification of Yarrowia lipolytica via metabolic engineering for effective remediation of heavy metals from wastewater.

With the increasing demand for heavy metals due to the advancement of industrial activities, large proportions of heavy metals have been discharged into aquatic ecosystems, causing serious harm to human health and the environment. Existing physical and chemical methods for recovering heavy metals from wastewater encounter challenges, such as low efficiency, high processing costs, and potential secondary pollution. In this study, we developed a novel approach by engineering the endogenous sulphur metabolic pathway of Yarrowia lipolytica, providing it with the ability to produce approximately 550 ppm of sulphide. Subsequently, sulphide-producing Y. lipolytica was used for the first time in heavy metal remediation. The engineered strain exhibited a high capacity to remove various heavy metals, especially achieving over 90 % for cadmium (Cd), copper (Cu) and lead (Pb). This capacity was consistent when applied to both synthetic and actual wastewater samples. Microscopic analyses revealed that sulphide-mediated biological precipitation of metal sulphides on the cell surface is responsible for their removal. Our findings demonstrate that sulphide-producing yeasts are a robust and effective bioremediation strategy for heavy metals, showing great potential for future heavy metal pollution remediation practices.

Genome-wide analysis of the heat shock transcription factor family reveals saline-alkali stress responses in Xanthoceras sorbifolium.

The heat shock transcription factor (HSF) family is involved in regulating growth, development, and abiotic stress. The characteristics and biological functions of HSF family member in X. sorbifolium, an important oil and ornamental plant, have never been reported. In this study, 21 XsHSF genes were identified from the genome of X. sorbifolium and named XsHSF1-XsHSF21 based on their chromosomal positions. Those genes were divided into three groups, A, B, and C, containing 12, one, and eight genes, respectively. Among them, 20 XsHSF genes are located on 11 chromosomes. Protein structure analysis suggested that XsHSF proteins were conserved, displaying typical DNA binding domains (DBD) and oligomerization domains (OD). Moreover, HSF proteins within the same group contain specific motifs, such as motif 5 in the HSFC group. All XsHSF genes have one intron in the CDS region, except XsHSF1 which has two introns. Promoter analysis revealed that in addition to defense and stress responsiveness elements, some promoters also contained a MYB binding site and elements involved in multiple hormones responsiveness and anaerobic induction. Duplication analysis revealed that XsHSF1 and XsHSF4 genes were segmentally duplicated while XsHSF2, XsHSF9, and XsHSF13 genes might have arisen from transposition. Expression pattern analysis of leaves and roots following salt-alkali treatment using qRT-PCR indicated that five XsHSF genes were upregulated and one XsHSF gene was downregulated in leaves upon NaCl treatment suggesting these genes may play important roles in salt response. Additionally, the expression levels of most XsHSFs were decreased in leaves and roots following alkali-induced stress, indicating that those XsHSFs may function as negative regulators in alkali tolerance. MicroRNA target site prediction indicated that 16 of the XsHSF genes may be regulated by multiple microRNAs, for example XsHSF2 might be regulated by miR156, miR394, miR395, miR408, miR7129, and miR854. And miR164 may effect the mRNA levels of XsHSF3 and XsHSF17, XsHSF9 gene may be regulated by miR172. The expression trends of miR172 and miR164 in leaves and roots on salt treatments were opposite to the expression trend of XsHSF9 and XsHSF3 genes, respectively. Promoter analysis showed that XsHSFs might be involved in light and hormone responses, plant development, as well as abiotic stress responses. Our results thus provide an overview of the HSF family in X. sorbifolium and lay a foundation for future functional studies to reveal its roles in saline-alkali response.