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Increased carvone production in Escherichia coli by balancing limonene conversion enzyme expression via targeted quantification concatamer proteome analysis.

ABSTRACT: (-)-Carvone is a monoterpenoid with a spearmint flavor. A sustainable biotechnological production process for (-)-carvone is desirable. Although all enzymes in (-)-carvone biosynthesis have been functionally expressed in Escherichia coli independently, the yield was low in previous studies. When cytochrome P450 limonene-6-hydroxylase (P450)/cytochrome P450 reductase (CPR) and carveol dehydrogenase (CDH) were expressed in a single strain, by-product formation (dihydrocarveol and dihydrocarvone) was detected. We hypothesized that P450 and CDH expression levels differ in E. coli. Thus, two strains independently expressing P450/CPR and CDH were mixed with different ratios, confirming increased carvone production and decreased by-product formation when CDH input was reduced. The optimum ratio of enzyme expression to maximize (-)-carvone production was determined using the proteome analysis quantification concatamer (QconCAT) method. Thereafter, a single strain expressing both P450/CPR and CDH was constructed to imitate the optimum expression ratio. The upgraded strain showed a 15-fold improvement compared to the initial strain, showing a 44 ± 6.3 mg/L (-)-carvone production from 100 mg/L (-)-limonene. Our study showed the usefulness of the QconCAT proteome analysis method for strain development in the industrial biotechnology field.

SUBMITTER: Yoshida E

PROVIDER: S-EPMC8586248 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Increased carvone production in Escherichia coli by balancing limonene conversion enzyme expression via targeted quantification concatamer proteome analysis

Project description:The cytochrome P450 limonene-6-hydroxylase (P450), cytochrome P450 reductase (CPR) and carveol dehydrogenase (CDH) were expressed in Escherichia coli for (−)-carvone production from (−)-limonene. The optimum ratio of enzyme expression to maximize (−)-carvone production was determined using the proteome analysis quantification concatamer (QconCAT) method.

2022-08-24 | PXD028077 | JPOST Repository

Cell-free biosynthesis of limonene using enzyme-enriched Escherichia coli lysates.

Project description:Isoprenoids are an attractive class of metabolites for enzymatic synthesis from renewable substrates. However, metabolic engineering of microorganisms for monoterpenoid production is limited by the need for time-consuming, and often non-intuitive, combinatorial tuning of biosynthetic pathway variations to meet design criteria. Towards alleviating this limitation, the goal of this work was to build a modular, cell-free platform for construction and testing of monoterpenoid pathways, using the fragrance and flavoring molecule limonene as a model. In this platform, multiple Escherichia coli lysates, each enriched with a single overexpressed pathway enzyme, are mixed to construct the full biosynthetic pathway. First, we show the ability to synthesize limonene from six enriched lysates with mevalonate substrate, an adenosine triphosphate (ATP) source, and cofactors. Next, we extend the pathway to use glucose as a substrate, which relies on native metabolism in the extract to convert glucose to acetyl-CoA along with three additional enzymes to convert acetyl-CoA to mevalonate. We find that the native E. coli farnesyl diphosphate synthase (IspA) is active in the lysate and diverts flux from the pathway intermediate geranyl pyrophospahte to farnesyl pyrophsophate and the byproduct farnesol. By adjusting the relative levels of cofactors NAD+, ATP and CoA, the system can synthesize 0.66?mM (90.2?mg?l-1) limonene over 24?h, a productivity of 3.8?mg?l-1 h-1. Our results highlight the flexibility of crude lysates to sustain complex metabolism and, by activating a glucose-to-limonene pathway with 9 heterologous enzymes encompassing 20 biosynthetic steps, expands an approach of using enzyme-enriched lysates for constructing, characterizing and prototyping enzymatic pathways.

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Targeted Quantification of the Lysosomal Proteome in Complex Samples.

Project description:In eukaryotic cells, lysosomes play a crucial role in the breakdown of a variety of components ranging from small molecules to complex structures, ascertaining the continuous turnover of cellular building blocks. Furthermore, they act as a regulatory hub for metabolism, being crucially involved in the regulation of major signaling pathways. Currently, ~450 lysosomal proteins can be reproducibly identified in a single cell line by mass spectrometry, most of which are low-abundant, restricting their unbiased proteomic analysis to lysosome-enriched fractions. In the current study, we applied two strategies for the targeted investigation of the lysosomal proteome in complex samples: data-independent acquisition (DIA) and parallel reaction monitoring (PRM). Using a lysosome-enriched fraction, mouse embryonic fibroblast whole cell lysate, and mouse liver whole tissue lysate, we investigated the capabilities of DIA and PRM to investigate the lysosomal proteome. While both approaches identified and quantified lysosomal proteins in all sample types, and their data largely correlated, DIA identified on average more proteins, especially for lower complex samples and longer chromatographic gradients. For the highly complex tissue sample and shorter gradients, however, PRM delivered a better performance regarding both identification and quantification of lysosomal proteins. All data are available via ProteomeXchange with identifier PXDD023278.

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Interference and Mechanism of Dill Seed Essential Oil and Contribution of Carvone and Limonene in Preventing Sclerotinia Rot of Rapeseed.

Project description:This study aimed to evaluate the inhibitory effects of dill (Anethum graveolens L.) seed essential oil against Sclerotinia sclerotiorum and its mechanism of action. The antifungal activities of the two main constituents, namely carvone and limonene, were also measured. Mycelial growth and sclerotial germination were thoroughly inhibited by dill seed essential oil at the 1.00 ?L/mL under contact condition and 0.125?L/mL air under vapor condition. Carvone also contributed more than limonene in inhibiting the growth of S. sclerotiorum. Carvone and limonene synergistically inhibited the growth of the fungus. In vivo experiments, the essential oil remarkably suppressed S. sclerotiorum, and considerable morphological alterations were observed in the hyphae and sclerotia. Inhibition of ergosterol synthesis, malate dehydrogenase, succinate dehydrogenase activities, and external medium acidification were investigated to elucidate the antifungal mechanism of the essential oil. The seed essential oil of A. graveolens can be extensively used in agriculture for preventing the oilseed crops fungal disease.

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Quantification of enterohemorrhagic Escherichia coli O157:H7 protein abundance by high-throughput proteome.

Project description:Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a human pathogen responsible for diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). To promote a comprehensive insight into the molecular basis of EHEC O157:H7 physiology and pathogenesis, the combined proteome of EHEC O157:H7 strains, Clade 8 and Clade 6 isolated from cattle in Argentina, and the standard EDL933 (clade 3) strain has been analyzed. From shotgun proteomic analysis a total of 2,644 non-redundant proteins of EHEC O157:H7 were identified, which correspond approximately 47% of the predicted proteome of this pathogen. Normalized spectrum abundance factor analysis was performed to estimate the protein abundance. According this analysis, 50 proteins were detected as the most abundant of EHEC O157:H7 proteome. COG analysis showed that the majority of the most abundant proteins are associated with translation processes. A KEGG enrichment analysis revealed that Glycolysis / Gluconeogenesis was the most significant pathway. On the other hand, the less abundant detected proteins are those related to DNA processes, cell respiration and prophage. Among the proteins that composed the Type III Secretion System, the most abundant protein was EspA. Altogether, the results show a subset of important proteins that contribute to physiology and pathogenicity of EHEC O157:H7.

| S-EPMC6312284 | biostudies-literature

Acute Limonene Toxicity in Escherichia coli Is Caused by Limonene Hydroperoxide and Alleviated by a Point Mutation in Alkyl Hydroperoxidase AhpC.

Project description:Limonene, a major component of citrus peel oil, has a number of applications related to microbiology. The antimicrobial properties of limonene make it a popular disinfectant and food preservative, while its potential as a biofuel component has made it the target of renewable production efforts through microbial metabolic engineering. For both applications, an understanding of microbial sensitivity or tolerance to limonene is crucial, but the mechanism of limonene toxicity remains enigmatic. In this study, we characterized a limonene-tolerant strain of Escherichia coli and found a mutation in ahpC, encoding alkyl hydroperoxidase, which alleviated limonene toxicity. We show that the acute toxicity previously attributed to limonene is largely due to the common oxidation product limonene hydroperoxide, which forms spontaneously in aerobic environments. The mutant AhpC protein with an L-to-Q change at position 177 (AhpC(L177Q)) was able to alleviate this toxicity by reducing the hydroperoxide to a more benign compound. We show that the degree of limonene toxicity is a function of its oxidation level and that nonoxidized limonene has relatively little toxicity to wild-type E. coli cells. Our results have implications for both the renewable production of limonene and the applications of limonene as an antimicrobial.

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Engineering Escherichia coli for increased productivity of serine-rich proteins based on proteome profiling.

Project description:Variations in proteome profiles of Escherichia coli in response to the overproduction of human leptin, a serine-rich (11.6% of total amino acids) protein, were examined by two-dimensional gel electrophoresis. The levels of heat shock proteins increased, while those of protein elongation factors, 30S ribosomal protein, and some enzymes involved in amino acid biosynthesis decreased, after leptin overproduction. Most notably, the levels of enzymes involved in the biosynthesis of serine family amino acids significantly decreased. Based on this information, we designed a strategy to enhance the leptin productivity by manipulating the cysK gene, encoding cysteine synthase A. By coexpression of the cysK gene, we were able to increase the cell growth rate by approximately twofold. Also, the specific leptin productivity could be increased by fourfold. In addition, we found that cysK coexpression can improve the production of another serine-rich protein, interleukin-12 beta chain, suggesting that this strategy may be useful for the production of other serine-rich proteins as well. The approach taken in this study should be useful in designing a strategy for improving recombinant protein production.

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Strategic approach of multifaceted antibacterial mechanism of limonene traced in Escherichia coli.

Project description:Antibacterial potential of Limonene against Multi Drug Resistant (MDR) pathogens was studied and mechanism explored. Microscopic techniques viz. Fluorescent Microscopy (FM), Scanning Electron Microscopy (SEM), and Transmission Electron Microscopy (TEM) indicated membrane disruption, cellular leakage and cell death of Escherichia coli (E. coli) cells when treated with limonene. Leakage of intracellular proteins, lipids and nucleic acid confirmed membrane damage and disruption of cell permeability barrier. Further, release of intracellular ATP, also suggested disruption of membrane barrier. Interaction of limonene with DNA revealed its capability in unwinding of plasmid, which could eventually inhibit DNA transcription and translation. Differential expression of various proteins and enzymes involved in transport, respiration, metabolism, chemotaxis, protein synthesis confirmed the mechanistic role of limonene on their functions. Limonene thus can be a potential candidate in drug development.

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Escherichia coli Small Proteome.

Project description:Escherichia coli was one of the first species to have its genome sequenced and remains one of the best-characterized model organisms. Thus, it is perhaps surprising that recent studies have shown that a substantial number of genes have been overlooked. Genes encoding more than 140 small proteins, defined as those containing 50 or fewer amino acids, have been identified in E. coli in the past 10 years, and there is substantial evidence indicating that many more remain to be discovered. This review covers the methods that have been successful in identifying small proteins and the short open reading frames that encode them. The small proteins that have been functionally characterized to date in this model organism are also discussed. It is hoped that the review, along with the associated databases of known as well as predicted but undetected small proteins, will aid in and provide a roadmap for the continued identification and characterization of these proteins in E. coli as well as other bacteria.

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Downregulated d-limonene transgenic orange trees with increased linalool content showed resistance to Phyllosticta citricarpa

Project description:Terpene volatiles play an important role in the interactions among specialized pathogens and fruits. Citrus Black Spot (CBS), caused by the fungus Phyllosticta citricarpa, is associated with losses in different citrus-growing areas worldwide. The pathogen may infect the fruit for 20-24 weeks after petal fall but the typical hard spot symptoms appear when the fruit become almost mature, as consequence of fungal colonization and induction of cell lyses around essential oil cavities. D-limonene represents about 95% of total oil gland content in mature orange fruit. We have used here d-limonene synthase downregulated orange fruit generated via an antisense (AS) approach to investigate whether d-limonene decrease content in peel oil glands may affect fruit interaction with P. citricarpa compared to that of empty-vector (EV) controls. AS fruit showed enhanced resistance to the fungus compared to EV ones. Because of d-limonene decreased content, an over-accumulation of linalool and other monoterpene alcohols was found in AS compared to EV fruit. A global gene expression analysis at 2h and 8d after inoculation with P. citricarpa revealed activation of defence responses in AS fruit, via the upregulation of different PR genes, likely due to the enhanced constitutive accumulation of linalool and other alcohols. When assayed in vitro and in vivo, the main altered terpene volatile compounds including linalool at concentrations emulating those present in AS fruit, showed strong antifungal activity. We show here that terpene engineering in fruit peels could be a promising method for developing new strategies to obtain resistance to fruit diseases.

2018-04-13 | GSE99295 | GEO

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