Methionine inducing carbohydrate esterase secretion of Trichoderma harzianum enhances the accessibility of substrate glycosidic bonds.

BACKGROUND: The conversion of plant biomass into biochemicals is a promising way to alleviate energy shortage, which depends on efficient microbial saccharification and cellular metabolism. Trichoderma spp. have plentiful CAZymes systems that can utilize all-components of lignocellulose. Acetylation of polysaccharides causes nanostructure densification and hydrophobicity enhancement, which is an obstacle for glycoside hydrolases to hydrolyze glycosidic bonds. The improvement of deacetylation ability can effectively release the potential for polysaccharide degradation. RESULTS: Ammonium sulfate addition facilitated the deacetylation of xylan by inducing the up-regulation of multiple carbohydrate esterases (CE3/CE4/CE15/CE16) of Trichoderma harzianum. Mainly, the pathway of ammonium-sulfate's cellular assimilates inducing up-regulation of the deacetylase gene (Thce3) was revealed. The intracellular metabolite changes were revealed through metabonomic analysis. Whole genome bisulfite sequencing identified a novel differentially methylated region (DMR) that existed in the ThgsfR2 promoter, and the DMR was closely related to lignocellulolytic response. ThGsfR2 was identified as a negative regulatory factor of Thce3, and methylation in ThgsfR2 promoter released the expression of Thce3. The up-regulation of CEs facilitated the substrate deacetylation. CONCLUSION: Ammonium sulfate increased the polysaccharide deacetylation capacity by inducing the up-regulation of multiple carbohydrate esterases of T. harzianum, which removed the spatial barrier of the glycosidic bond and improved hydrophilicity, and ultimately increased the accessibility of glycosidic bond to glycoside hydrolases.

Searching for new plastic-degrading enzymes from the plastisphere of alpine soils using a metagenomic mining approach.

Plastic materials, including microplastics, accumulate in all types of ecosystems, even in remote and cold environments such as the European Alps. This pollution poses a risk for the environment and humans and needs to be addressed. Using shotgun DNA metagenomics of soils collected in the eastern Swiss Alps at about 3,000 m a.s.l., we identified genes and their proteins that potentially can degrade plastics. We screened the metagenomes of the plastisphere and the bulk soil with a differential abundance analysis, conducted similarity-based screening with specific databases dedicated to putative plastic-degrading genes, and selected those genes with a high probability of signal peptides for extracellular export and a high confidence for functional domains. This procedure resulted in a final list of nine candidate genes. The lengths of the predicted proteins were between 425 and 845 amino acids, and the predicted genera producing these proteins belonged mainly to Caballeronia and Bradyrhizobium. We applied functional validation, using heterologous expression followed by enzymatic assays of the supernatant. Five of the nine proteins tested showed significantly increased activities when we used an esterase assay, and one of these five proteins from candidate genes, a hydrolase-type esterase, clearly had the highest activity, by more than double. We performed the fluorescence assays for plastic degradation of the plastic types BI-OPL and ecovio® only with proteins from the five candidate genes that were positively active in the esterase assay, but like the negative controls, these did not show any significantly increased activity. In contrast, the activity of the positive control, which contained a PLA-degrading gene insert known from the literature, was more than 20 times higher than that of the negative controls. These findings suggest that in silico screening followed by functional validation is suitable for finding new plastic-degrading enzymes. Although we only found one new esterase enzyme, our approach has the potential to be applied to any type of soil and to plastics in various ecosystems to search rapidly and efficiently for new plastic-degrading enzymes.

Production of acetic acid from wheat bran by catalysis of an acetoxylan esterase.

In this study, a gene encoding for acetylxylan esterase was cloned and expressed in E. coli. A single uniform band with molecular weight of 31.2 kDa was observed in SDS-PAGE electrophoresis. Served as the substrate, p-nitrophenol butyrate was employed to detect the recombinant enzyme activity. It exhibited activity at a wide temperature range (30-100 °C) and pH (5.0-9.0) with the optimal temperature of 70 °C and pH 8.0. Acetylxylan esterase showed two substrates' specificities with the highest Vmax of 177.2 U/mg and Km of 20.98 mM against p-nitrophenol butyrate. Meanwhile, the Vmax of p-nitrophenol acetate was 137.0 U/mg and Km 12.16 mM. The acetic acid yield of 0.39 g/g was obtained (70 °C and pH 8.0) from wheat bran pretreated using amylase and papain. This study showed the highest yield up to date and developed a promising strategy for acetic acid production using wheat bran.

AMWEst, a new thermostable and detergent-tolerant esterase retrieved from the Albian aquifer.

A fosmid library was constructed with the metagenomic DNA from the high-temperature sediment-rich water of the Albian aquifer (Algeria). Functional screening of this library was subsequently done looking for genes encoding lipolytic enzymes. We identified a novel gene named AMWEst (1209 base pairs) encoding a protein of 402 amino acids with a predicted molecular weight of 43.44 kDa and conferring esterase activity. AMWEst was successfully overexpressed in the yeast mesophilic host Saccharomyces cerevisiae, and the expression system used proved to be efficient and produced sufficient activity for its biochemical characterization. Multiple sequence alignment indicated that AMWEst contained a conserved pentapeptide motif (Gly120-His121-Ser122-Gln123-Gly124). The optimum pH and temperature of the recombinant esterase AMWEst were 8 and 80 °C, respectively. Additionally, AMWEst showed higher activity towards short carbon substrates and showed maximum activity for p-nitrophenyl hexanoate (C6). Notably, AMWEst has a remarkable thermostability, and the enzyme retains almost maximum activity at 70 °C after incubation for 1 h. Moreover, enzyme activity was enhanced by high concentrations of SDS and Triton X-100 detergents. KEY POINTS: • A novel thermostable esterase has been retrieved through functional metagenomics • The esterase is detergent-tolerant, which is attractive for some applications • The esterase can be expressed in a yeast mesophilic host to enhance its yield.

Enhancing the Enantioselectivity and Catalytic Efficiency of Esterase from Bacillus subtilis for Kinetic Resolution of l-Menthol through Semirational Design.

Enzymatic kinetic resolution is a promising way to produce l-menthol. However, the properties of the reported biocatalysts are still unsatisfactory and far from being ready for industrial application. Herein, a para-nitrobenzylesterase (pnbA) gene from Bacillus subtilis was cloned and expressed to produce l-menthol from d,l-menthyl acetate. The highest enantiomeric excess (ee) value of the product generated by pnbA was only approximately 80%, with a high conversion rate (47.8%) of d,l-menthyl acetate with the help of a cosolvent, indicating high catalytic activity but low enantioselectivity (E = 19.95). To enhance the enantioselectivity and catalytic efficiency of pnbA to d,l-menthyl acetate in an organic solvent-free system, site-directed mutagenesis was performed based on the results of molecular docking. The F314E/F315T mutant showed the best catalytic properties (E = 36.25) for d,l-menthyl acetate, with 92.11% ee and 30.58% conversion of d,l-menthyl acetate. To further improve the properties of pnbA, additional mutants were constructed based on the structure-guided triple-code saturation mutagenesis strategy. Finally, four mutants were screened for the best enantioselectivity (ee > 99%, E > 300) and catalytic efficiency at a high substrate concentration (200 g/L) without a cosolvent. This work provides several generally applicable biocatalysts for the industrial production of l-menthol.

The metagenome-derived esterase PET40 is highly promiscuous and hydrolyses polyethylene terephthalate (PET).

Polyethylene terephthalate (PET) is a widely used synthetic polymer and known to contaminate marine and terrestrial ecosystems. Only few PET-active microorganisms and enzymes (PETases) are currently known, and it is debated whether degradation activity for PET originates from promiscuous enzymes with broad substrate spectra that primarily act on natural polymers or other bulky substrates, or whether microorganisms evolved their genetic makeup to accepting PET as a carbon source. Here, we present a predicted diene lactone hydrolase designated PET40, which acts on a broad spectrum of substrates, including PET. It is the first esterase with activity on PET from a GC-rich Gram-positive Amycolatopsis species belonging to the Pseudonocardiaceae (Actinobacteria). It is highly conserved within the genera Amycolatopsis and Streptomyces. PET40 was identified by sequence-based metagenome search using a PETase-specific hidden Markov model. Besides acting on PET, PET40 has a versatile substrate spectrum, hydrolyzing δ-lactones, ß-lactam antibiotics, the polyester-polyurethane Impranil® DLN, and various para-nitrophenyl ester substrates. Molecular docking suggests that the PET degradative activity is likely a result of the promiscuity of PET40, as potential binding modes were found for substrates encompassing mono(2-hydroxyethyl) terephthalate, bis(2-hydroxyethyl) terephthalate, and a PET trimer. We also solved the crystal structure of the inactive PET40 variant S178A to 1.60 Å resolution. PET40 is active throughout a wide pH (pH 4-10) and temperature range (4-65 °C) and remarkably stable in the presence of 5% SDS, making it a promising enzyme as a starting point for further investigations and optimization approaches.

Identification, evolution, and expression of GDSL-type Esterase/Lipase (GELP) gene family in three cotton species: a bioinformatic analysis.

BACKGROUND: GDSL esterase/lipases (GELPs) play important roles in plant growth, development, and response to biotic and abiotic stresses. Presently, an extensive and in-depth analysis of GELP family genes in cotton is still not clear enough, which greatly limits the further understanding of cotton GELP function and regulatory mechanism. RESULTS: A total of 389 GELP family genes were identified in three cotton species of Gossypium hirsutum (193), G. arboreum (97), and G. raimondii (99). These GELPs could be classified into three groups and eight subgroups, with the GELPs in same group to have similar gene structures and conserved motifs. Evolutionary event analysis showed that the GELP family genes tend to be diversified at the spatial dimension and certain conservative at the time dimension, with a trend of potential continuous expansion in the future. The orthologous or paralogous GELPs among different genomes/subgenomes indicated the inheritance from genome-wide duplication during polyploidization, and the paralogous GELPs were derived from chromosomal segment duplication or tandem replication. GELP genes in the A/D subgenome underwent at least three large-scale replication events in the evolutionary process during the period of 0.6-3.2 MYA, with two large-scale evolutionary events between 0.6-1.8 MYA that were associated with tetraploidization, and the large-scale duplication between 2.6-9.1 MYA that occurred during diploidization. The cotton GELPs indicated diverse expression patterns in tissue development, ovule and fiber growth, and in response to biotic and abiotic stresses, combining the existing cis-elements in the promoter regions, suggesting the GELPs involvements of functions to be diversification and of the mechanisms to be a hormone-mediated manner. CONCLUSIONS: Our results provide a systematic and comprehensive understanding the function and regulatory mechanism of cotton GELP family, and offer an effective reference for in-depth genetic improvement utilization of cotton GELPs.

Understanding the resistance mechanisms of Rhipicephalus microplus ticks to synthetic pyrethroids and organophosphates in south-west regions of Haryana, North India.

Chemical control of tick infestation on dairy farms in India strongly relies upon the use of synthetic pyrethroids (deltamethrin) and organophosphate (coumaphos) drugs. Therefore, the present manuscript aims to investigate the resistance status of Rhipicephalus microplus ticks against these acaricides. Fully engorged adult R. microplus ticks were randomly collected from 8 dairy farms in North India and evaluated for acaricide resistance by using the Larval Packet Test (LPT). Of these, ticks collected from one and three farms showed the emergence of Level I acaricide resistance against deltamethrin and coumaphos, respectively. Significant positive correlations were found in the enzymatic activity (α-esterase, ß-esterase, glutathione-S-transferase, and mono-oxygenase) of R. microplus tick resistant against coumaphos. Native electrophoretogram analysis showed six different types of esterase activity in R. microplus (EST-1b to EST-6b), and EST-5b activity was more predominantly expressed in resistant ticks. Further, inhibitor studies using various esterase inhibitors suggested that EST-5b is a putative acetylcholine-esterase (AchE), and increased expression of one of the AchE might be responsible for the emergence of acaricide resistance. Further, no mutations were detected in the carboxylesterase (G1120A) and domain II S4-5 linker region (C190A) of the sodium channel genes of resistant R. microplus ticks, indicating that increased expression of detoxification enzymes was the probable mechanism for the development of acaricide resistance in the resistant ticks.

Overexpression of cytochrome P450 and esterase genes involved in permethrin resistance in larvae and adults of Culex quinquefasciatus.

Mosquitoes are important vectors of several arthropod-borne diseases, which remain a priority for epidemiological research. Mosquito vector control strategies have traditionally relied on chemical insecticides such as synthetic pyrethroids. However, the indiscriminate use of pesticides has resulted in the development of resistance in many mosquito species. In insects, resistance evolves primarily through the overexpression of one or more gene products from the cytochrome P450, carboxylesterase, and glutathione superfamilies. The current study examined the expression of cytochrome P450 CYP6M2, CYP6AA7, CYP6Z2, CYP9J34, α-Esterase, Esterase B1, and neuroactin genes in larvae and adults of a permethrin-resistant (PerRes) and susceptible (Sus) Culex quinquefasciatus strains. The results showed that the CYP6AA7 gene was overexpressed (10-fold) in larvae and adults with PerRes (p < 0.01) followed by CYPJ34 (9.0-fold) and CYP6Z2 (5.0-fold) compared to the Sus, whereas fewer changes in CYP6M gene expression were observed in PerRes adults (p < 0.05), and no expression was found in larvae. The esterase gene was overexpressed in PerRes larvae (9.0-fold) followed by adults (2.5-fold) compared to the susceptible strain. Based on data, the present study suggests that cytochrome P450, CYP6AA7, CYP6Z2, CYP9J34, α-Esterase, Esterase B1, and neuroactin genes were involved in permethrin resistance in larval and adult Cx. quinquefasciatus.

Characterization of caffeoyl shikimate esterase gene family identifies CsCSE5 as a positive regulator of Podosphaera xanthii and Corynespora cassiicola pathogen resistance in cucumber.

KEY MESSAGE: CsCSE genes might be involved in the tolerance of cucumber to pathogens. Silencing of the CsCSE5 gene resulted in attenuated resistance of cucumber to Podosphaera xanthii and Corynespora cassiicola. Caffeoyl shikimate esterase (CSE), a key enzyme in the lignin biosynthetic pathway, has recently been characterized to play a key role in defense against pathogenic infection in plants. However, a systematic analysis of the CSE gene family in cucumber (Cucumis sativus) has not yet been conducted. Here, we identified eight CsCSE genes from the cucumber genome via bioinformatic analyses, and these genes were unevenly distributed on chromosomes 1, 3, 4, and 5. Results from multiple sequence alignment indicated that the CsCSE proteins had domains required for CSE activity. Phylogenetic analysis of gene structure and protein motifs revealed the conservation and diversity of the CsCSE gene family. Collinearity analysis showed that CsCSE genes had high homology with CSE genes in wax gourd (Benincasa hispida). Cis-acting element analysis of the promoters suggested that CsCSE genes might play important roles in growth, development, and stress tolerance. Expression pattern analysis indicated that CsCSE5 might be involved in regulating the resistance of cucumber to pathogens. Functional verification data confirmed that CsCSE5 positively regulates the resistance of cucumber to powdery mildew pathogen Podosphaera xanthii and target leaf spot pathogen Corynespora cassiicola. The results of our study provide information that will aid the genetic improvement of resistant cucumber varieties.

Crystal structure of the GDSL family esterase EstL5 in complex with PMSF reveals a branch channel of the active site pocket.

Esterases/lipases from the GDSL family have potential applications in the hydrolysis and synthesis of important esters of pharmaceutical, food, and biotechnical interests. However, the structural and functional understanding of GDSL enzymes is still limited. Here, we report the crystal structure of the GDSL family esterase EstL5 complexed with PMSF at 2.34 Šresolution. Intriguingly, the PMSF binding site is not located at the active site pocket but is situated in a surface cavity. At the active site, we note that there is a trapped crystallization solvent 1,6-hexanediol, which mimics the bound ester chain, allowing for further definition of the active site pocket of EstL5. The most striking structural feature of EstL5 is the presence of a unique channel, which extends approximately 18.9 Å, with a bottleneck radius of 6.8 Å, connecting the active-site pocket and the surface cavity. Replacement of Ser205 with the bulk aromatic residue Trp or Phe could partially block the channel at one end and perturb its access. Reduced enzymatic activity is found in the EstL5 S205W and EstL5 S205F mutants, suggesting the functional relevance of the channel to enzyme catalysis. Our study provides valuable information regarding the properties of the GDSL-family enzymes for designing more efficient and robust biocatalysts.

Unravelling the hidden power of esterases for biomanufacturing of short-chain esters.

Microbial production of esters has recently garnered wide attention, but the current production metrics are low. Evidently, the ester precursors (organic acids and alcohols) can be accumulated at higher titers by microbes like Escherichia coli. Hence, we hypothesized that their 'direct esterification' using esterases will be efficient. We engineered esterases from various microorganisms into E. coli, along with overexpression of ethanol and lactate pathway genes. High cell density fermentation exhibited the strains possessing esterase-A (SSL76) and carbohydrate esterase (SSL74) as the potent candidates. Fed-batch fermentation at pH 7 resulted in 80 mg/L of ethyl acetate and 10 mg/L of ethyl lactate accumulation by SSL76. At pH 6, the total ester titer improved by 2.5-fold, with SSL76 producing 225 mg/L of ethyl acetate, and 18.2 mg/L of ethyl lactate, the highest reported titer in E. coli. To our knowledge, this is the first successful demonstration of short-chain ester production by engineering 'esterases' in E. coli.

The Elk-3 target Abhd10 ameliorates hepatotoxic injury and fibrosis in alcoholic liver disease.

Alcoholic liver disease (ALD) and other forms of chronic hepatotoxic injury can lead to transforming growth factor ß1 (TGFß1)-induced hepatic fibrosis and compromised liver function, underscoring the need to develop novel treatments for these conditions. Herein, our analyses of liver tissue samples from severe alcoholic hepatitis (SAH) patients and two murine models of ALD reveals that the ALD phenotype was associated with upregulation of the transcription factor ETS domain-containing protein (ELK-3) and ELK-3 signaling activity coupled with downregulation of α/ß hydrolase domain containing 10 (ABHD10) and upregulation of deactivating S-palmitoylation of the antioxidant protein Peroxiredoxin 5 (PRDX5). In vitro, we further demonstrate that ELK-3 can directly bind to the ABHD10 promoter to inhibit its transactivation. TGFß1 and epidermal growth factor (EGF) signaling induce ABHD10 downregulation and PRDX5 S-palmitoylation via ELK-3. This ELK-3-mediated ABHD10 downregulation drives oxidative stress and disrupts mature hepatocyte function via enhancing S-palmitoylation of PRDX5's Cys100 residue. In vivo, ectopic Abhd10 overexpression ameliorates liver damage in ALD model mice. Overall, these data suggest that the therapeutic targeting of the ABHD10-PRDX5 axis may represent a viable approach to treating ALD and other forms of hepatotoxicity.

GDSL Esterase/Lipase GELP1 Involved in the Defense of Apple Leaves against Colletotrichum gloeosporioides Infection.

GDSL esterases/lipases are a subclass of lipolytic enzymes that play critical roles in plant growth and development, stress response, and pathogen defense. However, the GDSL esterase/lipase genes involved in the pathogen response of apple remain to be identified and characterized. Thus, in this study, we aimed to analyze the phenotypic difference between the resistant variety, Fuji, and susceptible variety, Gala, during infection with C. gloeosporioides, screen for anti-disease-associated proteins in Fuji leaves, and elucidate the underlying mechanisms. The results showed that GDSL esterase/lipase protein GELP1 contributed to C. gloeosporioides infection defense in apple. During C. gloeosporioides infection, GELP1 expression was significantly upregulated in Fuji. Fuji leaves exhibited a highly resistant phenotype compared with Gala leaves. The formation of infection hyphae of C. gloeosporioides was inhibited in Fuji. Moreover, recombinant His:GELP1 protein suppressed hyphal formation during infection in vitro. Transient expression in Nicotiana benthamiana showed that GELP1-eGFP localized to the endoplasmic reticulum and chloroplasts. GELP1 overexpression in GL-3 plants increased resistance to C. gloeosporioides. MdWRKY15 expression was upregulated in the transgenic lines. Notably, GELP1 transcript levels were elevated in GL-3 after salicylic acid treatment. These results suggest that GELP1 increases apple resistance to C. gloeosporioides by indirectly regulating salicylic acid biosynthesis.

Revealing the key gene involved in bioplastic degradation from superior bioplastic degrader Bacillus sp. JY35.

The use of bioplastics, which can alleviate environmental pollution caused by non-degradable bioplastics, has received attention. As there are many types of bioplastics, method that can treat them simultaneously is important. Therefore, Bacillus sp. JY35 which can degrade different types of bioplastics, was screened in previous study. Most types of bioplastics, such as polyhydroxybutyrate (PHB), (P(3HB-co-4HB)), poly(butylene adipate-co-terephthalate) (PBAT), polybutylene succinate (PBS), and polycaprolactone (PCL), can be degraded by esterase family enzymes. To identify the genes that are involved in bioplastic degradation, analysis with whole-genome sequencing was performed. Among the many esterase enzymes, three carboxylesterase and one triacylglycerol lipase were identified and selected based on previous studies. Esterase activity using p-nitrophenyl substrates was measured, and the supernatant of JY35_02679 showed strong emulsion clarification activity compared with others. In addition, when recombinant E. coli was applied to the clear zone test, only the JY35_02679 gene showed activity in the clear zone test with bioplastic containing solid cultures. Further quantitative analysis showed 100 % PCL degradation at 7 days and 45.7 % PBS degradation at 10 days. We identified a gene encoding a bioplastic-degrading enzyme in Bacillus sp. JY35 and successfully expressed the gene in heterologous E. coli, which secreted esterases with broad specificity.

Genome-wide analysis of the GDSL esterase/lipase family genes in Physcomitrium patens and the involvement of GELP31 in spore germination.

In plants, the ability to produce hydrophobic substances that would provide protection from dehydration was required for the transition to land. This genome-wide investigation outlines the evolution of GDSL-type esterase/lipase (GELP) proteins in the moss Physcomitrium patens and suggests possible functions of some genes. GELP proteins play roles in the formation of hydrophobic polymers such as cutin and suberin that protect against dehydration and pathogen attack. GELP proteins are also implicated in processes such as pollen development and seed metabolism and germination. The P. patens GELP gene family comprises 48 genes and 14 pseudogenes. Phylogenetic analysis of all P. patens GELP sequences along with vascular plant GELP proteins with reported functions revealed that the P. patens genes clustered within previously identified A, B and C clades. A duplication model predicting the expansion of the GELP gene family within the P. patens lineage was constructed. Expression analysis combined with phylogenetic analysis suggested candidate genes for functions such as defence against pathogens, cutin metabolism, spore development and spore germination. The presence of relatively fewer GELP genes in P. patens may reduce the occurrence of functional redundancy that complicates the characterization of vascular plant GELP genes. Knockout lines of GELP31, which is highly expressed in sporophytes, were constructed. Gelp31 spores contained amorphous oil bodies and germinated late, suggesting (a) role(s) of GELP31 in lipid metabolism in spore development or germination. Future knockout studies of other candidate GELP genes will further elucidate the relationship between expansion of the family and the ability to withstand the harsh land environment.

Substrate-enzyme interactions and catalytic mechanism in a novel family VI esterase with dibutyl phthalate-hydrolyzing activity.

Microbial degradation has been confirmed as effective and environmentally friendly approach to remediate phthalates from the environment, and hydrolase is an effective element for contaminant degradation. In the present study, a novel dibutyl phthalate (DBP)-hydrolyzing carboxylesterase (named PS06828) from Pseudomonas sp. PS1 was heterogeneously expressed in E. coli, which was identified as a new member of the lipolytic family VI. Purified PS06828 could efficiently degrade DBP with a wide range of temperature (25-37 °C) and pH (6.5-9.0). Multi-spectroscopy methods combined with molecular docking were employed to study the interaction of PS06828 with DBP. Fluorescence and UV-visible absorption spectra revealed the simultaneous presence of static and dynamic component in the fluorescence quenching of PS06828 by DBP. Synchronous fluorescence and circular dichroism spectra showed inconspicuous alteration in micro-environmental polarity around amino acid residues but obvious increasing of α-helix and reducing of ß-sheet and random coil in protein conformation. Based on the information on exact binding sites of DBP on PS06828 provided by molecular docking, the catalytic mechanism mediated by key residues (Ser113, Asp166, and His197) was proposed and subsequently confirmed by site-directed mutagenesis. The results can strengthen our mechanistic understanding of family VI esterase involved in hydrolysis of phthalic acid esters, and provide a solid foundation for further enzymatic modification.

Xanthophyll esterases in association with fibrillins control the stable storage of carotenoids in yellow flowers of rapeseed (Brassica juncea).

Biosynthesis, stabilization, and storage of carotenoids are vital processes in plants that collectively contribute to the vibrant colors observed in flowers and fruits. Despite its importance, the carotenoid storage pathway remains poorly understood and lacks thorough characterization. We identified two homologous genes, BjA02.PC1 and BjB04.PC2, belonging to the esterase/lipase/thioesterase (ELT) family of acyltransferases. We showed that BjPCs in association with fibrillin gene BjFBN1b control the stable storage of carotenoids in yellow flowers of Brassica juncea. Through genetic, high-resolution mass spectrometry and transmission electron microscopy analyses, we demonstrated that both BjA02.PC1 and BjB04.PC2 can promote the accumulation of esterified xanthophylls, facilitating the formation of carotenoid-enriched plastoglobules (PGs) and ultimately producing yellow pigments in flowers. The elimination of BjPCs led to the redirection of metabolic flux from xanthophyll ester biosynthesis to lipid biosynthesis, resulting in white flowers for B. juncea. Moreover, we genetically verified the function of two fibrillin genes, BjA01.FBN1b and BjB05.FBN1b, in mediating PG formation and demonstrated that xanthophyll esters must be deposited in PGs for stable storage. These findings identified a previously unknown carotenoid storage pathway that is regulated by BjPCs and BjFBN1b, while offering unique opportunities for improving the stability, deposition, and bioavailability of carotenoids.

Altered profile of floral volatiles and lignin content by down-regulation of Caffeoyl Shikimate Esterase in Petunia.

BACKGROUND: The floral volatile profile of Petunia x hybrida 'Mitchell diploid' (MD) is dominated by phenylpropanoids, many of which are derived from p-coumaric acid. However, the downstream processes involved in the production of caffeoyl-CoA and feruloyl-CoA from p-coumaric acid are complex, as the genes and biosynthesis steps are associated with flavonoids and lignin synthesis as well as floral volatiles benzenoid/phenylpropanoid (FVBP). Caffeoyl shikimate esterase (CSE) converts caffeoyl shikimate to caffeic acid and is considered one of the essential regulators in lignin production. Moreover, CSE in involved in phenylpropanoid production. To investigate the roles of CSE in FVBP biosynthesis, we used RNAi-mediated CSE down-regulated (ir-PhCSE) petunias. RESULTS: Lowered CSE transcript accumulation in ir-PhCSE plants resulted in reduced lignin layers in the stems and stunted growth, suggesting a positive correlation between lignin layers and lignin content. The altered CSE level influenced the expression of many FVBP genes, including elevated transcripts of p-coumarate-3-hydroxylase (C3H), hydroxycinnamoyl transferase (HCT), and 4-coumaric acid: CoA ligase (4CL). In particular, the expression of C4H in ir-PhCSE plants was more than twice the expression in MD plants. Moreover, the production of volatile compounds was alterend in ir-PhCSE plants. Most floral volatiles decreased, and the amounts of phenylalanine and caffeic acid were significantly lower. CONCLUSIONS: Reduced lignin layers in the stems and stunted growth in ir-PhCSE plants suggest that PhCSE is essential for lignin production and plant growth in petunia. The decreased CSE level influenced the expression of many FVBP genes, and interference of shikimate derivates altered volatile compound production. Significantly decreased caffeic acid, but not ferulic acid, in ir-PhCSE plants suggest that CSE is primarily involved in the reaction of caffeoyl shikimate. Higher C3H and C4H transcripts seem to alleviate accumulated p-coumaric acid resulting from altered CSE. Finally, alteration in C3H, HCT, and 4CL in CSE down-regulated plants suggests an interaction of the FVBP genes, leading to the regulation of floral volatiles of petunia.

AsiteDesign: a Semirational Algorithm for an Automated Enzyme Design.

With advances in protein structure predictions, the number of available high-quality structures has increased dramatically. In light of these advances, structure-based enzyme engineering is expected to become increasingly important for optimizing biocatalysts for industrial processes. Here, we present AsiteDesign, a Monte Carlo-based protocol for structure-based engineering of active sites. AsiteDesign provides a framework for introducing new catalytic residues in a given binding pocket to either create a new catalytic activity or alter the existing one. AsiteDesign is implemented using pyRosetta and incorporates enhanced sampling techniques to efficiently explore the search space. The protocol was tested by designing an alternative catalytic triad in the active site of Pseudomonas fluorescens esterase (PFE). The designed variant was experimentally verified to be active, demonstrating that AsiteDesign can find alternative catalytic triads. Additionally, the AsiteDesign protocol was employed to enhance the hydrolysis of a bulky chiral substrate (1-phenyl-2-pentyl acetate) by PFE. The experimental verification of the designed variants demonstrated that F158L/F198A and F125A/F158L mutations increased the hydrolysis of 1-phenyl-2-pentyl acetate from 8.9 to 66.7 and 23.4%, respectively, and reversed the enantioselectivity of the enzyme from (R) to (S)-enantiopreference, with 32 and 55% enantiomeric excess (ee), respectively.