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• Prorocentrum lima can produce polyketides responsible for diarrheic shellfish poisoning (DSP), however no gene has been confirmatively assigned to DSP toxin production. • Both single-domain and type I modular polyketide synthases were found in P. lima through transcriptomic sequencing and bioinformatics analysis. • Some polyketide synthase genes identified from comparative transcriptome sequencing and differential expression analysis were consistent with toxin content changes. • Our study provides valuable genomic resource for future research in order to understand the complex mechanism of toxin production in this dinoflagellate. The benthic dinoflagellate Prorocentrum lima is among the most common toxic morphospecies with a cosmopolitan distribution. P. lima can produce polyketide compounds, such as okadaic acid (OA), dinophysistoxin (DTX) and their analogues, which are responsible for diarrhetic shellfish poisoning (DSP). Studying the molecular mechanism of DSP toxin biosynthesis is crucial for understanding the environmental driver influencing toxin biosynthesis as well as for better monitoring of marine ecosystems. Commonly, polyketides are produced by polyketide synthases (PKS). However, no gene has been confirmatively assigned to DSP toxin production. Here, we assembled a transcriptome from 94,730,858 Illumina RNAseq reads using Trinity, resulting in 147,527 unigenes with average sequence length of 1035 nt. Using bioinformatics analysis methods, we found 210 unigenes encoding single-domain PKS with sequence similarity to type I PKSs, as reported in other dinoflagellates. In addition, 15 transcripts encoding multi-domain PKS (forming typical type I PKSs modules) and 5 transcripts encoding hybrid nonribosomal peptide synthetase (NRPS)/PKS were found. Using comparative transcriptome and differential expression analysis, a total of 16 PKS genes were identified to be up-regulated in phosphorus-limited cultures, which was related to the up regulation of toxin expression. In concert with other recent transcriptome analyses, this study contributes to the building consensus that dinoflagellates may utilize a combination of Type I multi-domain and single-domain PKS proteins, in an as yet undefined manner, to synthesize polyketides. Our study provides valuable genomic resource for future research in order to understand the complex mechanism of toxin production in this dinoflagellate. [Display omitted] [ABSTRACT FROM AUTHOR]

Sesquiterpenes have been identified as promising ingredients for aviation fuels due to their high energy density and combustion heat properties. Despite the characterization of numerous sesquiterpene structures, studies testing their performance properties and feasibility as fuels are scarce. In this study, 122 sesquiterpenoid skeleton compounds, obtained from existing literature reports, are tested using group contribution and gaussian quantum chemistry methods to assess their potential as high‐energy aviation fuels. Seventeen sesquiterpene compounds exhibit good predictive performance and nine compounds are further selected for overproduction in yeast. Through fed‐batch fermentation, all compounds achieve the highest reported titers to date. Subsequently, three representative products, pentalenene, presilphiperfol‐1‐ene, and α‐farnesene, are selected, produced, purified in large quantities, and tested for use as potential fuels. The performance of pentalenene, presilphiperfol‐1‐ene, and their derivatives reveals favorable prospects as high‐energy aviation fuels. [ABSTRACT FROM AUTHOR]

Background: The sesquiterpene germacrene D is a highly promising product due to its wide variety of insecticidal activities and ability to serve as a precursor for many other sesquiterpenes. Biosynthesis of high value compounds through genome mining for synthases and metabolic engineering of microbial factories, especially Saccharomyces cerevisiae, has been proven to be an effective strategy. However, there have been no studies on the de novo synthesis of germacrene D from carbon sources in microbes. Hence, the construction of the S. cerevisiae cell factory to achieve high production of germacrene D is highly desirable. Results: We identified five putative sesquiterpene synthases (AcTPS1 to AcTPS5) from Acremonium chrysogenum and the major product of AcTPS1 characterized by in vivo, in vitro reaction and NMR detection was revealed to be (–)-germacrene D. After systematically comparing twenty-one germacrene D synthases, AcTPS1 was found to generate the highest amount of (–)-germacrene D and was integrated into the terpene precursor-enhancing yeast strain, achieving 376.2 mg/L of (–)-germacrene D. Iterative engineering was performed to improve the production of (–)-germacrene D, including increasing the copy numbers of AcTPS1, tHMG1 and ERG20, and downregulating or knocking out other inhibitory factors (such as erg9, rox1, dpp1). Finally, the optimal strain LSc81 achieved 1.94 g/L (–)-germacrene D in shake-flask fermentation and 7.9 g/L (–)-germacrene D in a 5-L bioreactor, which is the highest reported (–)-germacrene D titer achieved to date. Conclusion: We successfully achieved high production of (–)-germacrene D in S. cerevisiae through terpene synthase mining and metabolic engineering, providing an impressive example of microbial overproduction of high-value compounds. [ABSTRACT FROM AUTHOR]

Sesquiterpene synthases convert farnesyl diphosphate into various sesquiterpenes, which find wide applications in the food, cosmetics and pharmaceutical industries. Although numerous putative sesquiterpene synthases have been identified in fungal genomes, many lack biochemical characterization. In this study, we identified a putative terpene synthase AcTPS3 from Acremonium chrysogenum. Through sequence analysis and in vitro enzyme assay, AcTPS3 was identified as a sesquiterpene synthase. To obtain sufficient product for NMR testing, a metabolic engineered Saccharomyces cerevisiae was constructed to overproduce the product of AcTPS3. The major product of AcTPS3 was identified as (+)-cubenene (55.46%) by GC-MS and NMR. Thus, AcTPS3 was confirmed as (+)-cubenene synthase, which is the first report of (+)-cubenene synthase. The optimized S. cerevisiae strain achieved a biosynthesis titer of 597.3 mg/L, the highest reported for (+)-cubenene synthesis. • A metabolic engineered Saccharomyces cerevisiae was used to overproduce the product of AcTPS3. • A (+)-cubenene synthase was reported for the first time. • (+)-Cubenene was biosynthesized at a titer of 597.3 mg/L, which provides a sustainable synthesis route for this compound. [ABSTRACT FROM AUTHOR]

Confronting the global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), simple, fast and specific non-laboratory SARS-CoV-2 diagnostic tests are urgently required. However, the current nucleic acid assays generally rely on the diagnostic laboratory, trained staff and specialized equipment for execution and analysis, presenting clear limitations in the field detection. Here, we describe a portable and reliable immobilization-based loop-mediated isothermal amplification (LAMP) device which is mobile, without the requirement of any complicated instrument and appropriate for high-throughput testing. This device was constructed by utilizing the interaction between a carboxyl-tagged primer and an amino-tagged substrate, and capable of catching the target sequence in SARS-CoV-2 produced via the immobilization-based LAMP. In this study, the immobilization conditions and immobilized primer structure were explored and optimized. With this proposed device, the analysis result can be obtained rapidly in 30 min with excellent specificity, even if the template is extracted from a complex sample containing pharyngeal swab or human blood. In addition, the device can be applied to detect the nucleic acid of SARS-CoV-2 and various other pathogens, showing attractive potential for rapid and high-throughput detection at airports, railway stations, cold-chain transportations, community hospitals and so on. Therefore, we believe that the immobilization-based LAMP device is an advanced approach to developing a portable, specific, low-cost and high-throughput diagnostic platform. [ABSTRACT FROM AUTHOR]

β -myrcene is a high-value acyclic monoterpene. The low activity of myrcene synthase resulted to low biosynthetic titer of it. Biosensor is a promising tool applied for enzyme directed evolution. In this work, a novel genetically encoded biosensor responding to myrcene was established based on the MyrR regulator from Pseudomonas sp. Through sensing promoter characterization and engineering, the biosensor exhibiting excellent specificity and dynamic range was developed, and applied for directed evolution of myrcene synthase. After high-throughput screening of the myrcene synthase random mutation library, the best mutant R89G/N152S/D517N was obtained. Its catalytic efficiency was 1.47-fold than that of parent. Based on the mutants, the final production of myrcene reached 510.38 mg/L, which is the highest myrcene titer reported to date. This work demonstrates the great potential of whole-cell biosensor for improving enzymatic activity and the production of target metabolite. [ABSTRACT FROM AUTHOR]