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1. Identification of Genes Encoding Enzymes Catalyzing the Early Steps of Carrot Polyacetylene Biosynthesis

   https://doi.org/https://doi.org/10.1104/pp.18.01195  

   Lucas Busta, Won Cheol ( December 2018 , Plant physiology)

   Polyacetylenic lipids accumulate in various Apiaceae species after pathogen attack, suggesting that these compounds are naturally occurring pesticides and potentially valuable resources for crop improvement. These compounds also promote human health and slow tumor growth. Even though polyacetylenic lipids were discovered decades ago, the biosynthetic pathway underlying their production is largely unknown. To begin filling this gap and ultimately enable polyacetylene engineering, we studied polyacetylenes and their biosynthesis in the major Apiaceae crop carrot (Daucus carota subsp. sativus). Using gas chromatography and mass spectrometry, we identified three known polyacetylenes and assigned provisional structures to two novel polyacetylenes. We also quantified these compounds in carrot leaf, petiole, root xylem, root phloem, and root periderm extracts. Falcarindiol and falcarinol predominated and accumulated primarily in the root periderm. Since the multiple double and triple carbon-carbon bonds that distinguish polyacetylenes from ubiquitous fatty acids are often introduced by Δ12 oleic acid desaturase (FAD2)-type enzymes, we mined the carrot genome for FAD2 genes. We identified a FAD2 family with an unprecedented 24 members and analyzed public, tissue-specific carrot RNA-Seq data to identify coexpressed members with root periderm-enhanced expression. Six candidate genes were heterologously expressed individually and in combination in yeast and Arabidopsis (Arabidopsis thaliana), resultingmore »in the identification of one canonical FAD2 that converts oleic to linoleic acid, three divergent FAD2-like acetylenases that convert linoleic into crepenynic acid, and two bifunctional FAD2s with Δ12 and Δ14 desaturase activity that convert crepenynic into the further desaturated dehydrocrepenynic acid, a polyacetylene pathway intermediate. These genes can now be used as a basis for discovering other steps of falcarin-type polyacetylene biosynthesis, to modulate polyacetylene levels in plants, and to test the in planta function of these molecules. Many organisms implement specialized biochemical pathways to convert ubiquitous metabolites into bioactive chemical compounds. Since plants comprise the majority of the human diet, specialized plant metabolites play crucial roles not only in crop biology but also in human nutrition. Some asterids produce lipid compounds called polyacetylenes (for review, see Negri, 2015) that exhibit antifungal activity (Garrod et al., 1978; Kemp, 1978; Harding and Heale, 1980, 1981; Olsson and Svensson, 1996) and accumulate in response to fungal phytopathogen attack (De Wit and Kodde, 1981; Elgersma and Liem, 1989). These observations have led to the longstanding hypothesis that polyacetylenes are natural pesticides. These same lipid compounds exhibit cytotoxic activity against human cancer cell lines and slow tumor growth (Fujimoto and Satoh, 1988; Matsunaga et al., 1989, 1990; Cunsolo et al., 1993; Bernart et al., 1996; Kobaek-Larsen et al., 2005; Zidorn et al., 2005), making them important nutritional compounds. The major source of polyacetylenes in the human diet is carrot (Daucus carota L.). Carrot is one of the most important crop species in the Apiaceae, with rapidly increasing worldwide cultivation (Rubatzky et al., 1999; Dawid et al., 2015). The most common carrot polyacetylenes are C17 linear aliphatic compounds containing two conjugated carbon-carbon triple bonds, one or two carbon-carbon double bonds, and a diversity of additional in-chain oxygen-containing functional groups. In carrot, the most abundant of these compounds are falcarinol and falcarindiol (Dawid et al., 2015). Based on their structures, it has been hypothesized that these compounds (alias falcarin-type polyacetylenes) are derived from ubiquitous fatty acids. Indeed, biochemical investigations (Haigh et al., 1968; Bohlman, 1988), radio-chemical tracer studies (Barley et al., 1988), and the discovery of pathway intermediates (Jones et al., 1966; Kawazu et al., 1973) implicate a diversion of flux away from linolenate biosynthesis as the entry point into falcarin-type polyacetylene biosynthesis (for review, see Minto and Blacklock, 2008). The final steps of linolenate biosynthesis are the conversion of oleate to linoleate, mediated by fatty acid desaturase 2 (FAD2), and linoleate to linolenate, catalyzed by FAD3. Some plant species contain divergent forms of FAD2 that, instead of or in addition to converting oleate to linoleate, catalyze the installation of unusual in-chain functional groups such as hydroxyl groups, epoxy groups, conjugated double bonds, or carbon-carbon triple bonds into the acyl chain (Badami and Patil, 1980) and thus divert flux from linolenate production into the accumulation of unusual fatty acids. Previous work in parsley (Petroselinum crispum; Apiaceae) identified a divergent form of FAD2 that (1) was up-regulated in response to pathogen treatment and (2) when expressed in soybean embryos resulted in production of the monoyne crepenynate and, by the action of an unassigned enzyme, dehydrocrepenynate (Kirsch et al., 1997; Cahoon et al., 2003). The results of the parsley studies are consistent with a pathogen-responsive, divergent FAD2-mediated pathway that leads to acetylenic fatty acids. However, information regarding the branch point into acetylenic fatty acid production in agriculturally relevant carrot is still largely missing, in particular, the identification and functional characterization of enzymes that can divert carbon flux away from linolenate biosynthesis into the production of dehydrocrepenynate and ultimately falcarin-type polyacetylenes. Such genes, once identified, could be used in the future design of transgenic carrot lines with altered polyacetylene content, enabling direct testing of in planta polyacetylene function and potentially the engineering of pathogen-resistant, more nutritious carrots. These genes could also provide the foundation for further investigations of more basic aspects of plant biology, including the evolution of fatty acid-derived natural product biosynthesis pathways across the Asterid clade, as well as the role of these pathways and compounds in plant ecology and plant defense. Recently, a high-quality carrot genome assembly was released (Iorizzo et al., 2016), providing a foundation for genome-enabled studies of Apiaceous species. This study also provided publicly accessible RNA sequencing (RNA-Seq) data from diverse carrot tissues. Using these resources, this study aimed to provide a detailed gas chromatography-based quantification of polyacetylenes in carrot tissues for which RNA-Seq data are available, then combine this information with bioinformatics analysis and heterologous expression to identify and characterize biosynthetic genes that underlie the major entry point into carrot polyacetylene biosynthesis. To achieve these goals, thin-layer chromatography (TLC) was combined with gas chromatography-mass spectrometry (GC-MS) and gas chromatography-flame ionization detection to identify and quantify polyacetylenic metabolites in five different carrot tissues. Then the sequences and tissue expression profiles of potential FAD2 and FAD2-like genes annotated in the D. carota genome were compared with the metabolite data to identify candidate pathway genes, followed by biochemical functionality tests using yeast (Saccharomyces cerevisae) and Arabidopsis (Arabidopsis thaliana) as heterologous expression systems.« less
2. Engineering acetyl-CoA supply and ERG9 repression to enhance mevalonate production in Saccharomyces cerevisiae

   https://doi.org/10.1093/jimb/kuab050  

   Wegner, Scott A. ; Chen, Jhong-Min ; Ip, Samantha S. ; Zhang, Yanfei ; Dugar, Deepak ; Avalos, José L. ( August 2021 , Journal of Industrial Microbiology and Biotechnology)

   Mevalonate is a key precursor in isoprenoid biosynthesis and a promising commodity chemical. Although mevalonate is a native metabolite in Saccharomyces cerevisiae, its production is challenged by the relatively low flux toward acetyl-CoA in this yeast. In this study we explore different approaches to increase acetyl-CoA supply in S. cerevisiae to boost mevalonate production. Stable integration of a feedback-insensitive acetyl-CoA synthetase (Se-acsL641P) from Salmonella enterica and the mevalonate pathway from Enterococcus faecalis results in the production of 1,390 ± 10 mg/l of mevalonate from glucose. While bifid shunt enzymes failed to improve titers in high-producing strains, inhibition of squalene synthase (ERG9) results in a significant enhancement. Finally, increasing coenzyme A (CoA) biosynthesis by overexpression of pantothenate kinase (CAB1) and pantothenate supplementation further increased production to 3,830 ± 120 mg/l. Using strains that combine these strategies in lab-scale bioreactors results in the production of 13.3 ± 0.5 g/l, which is ∼360-fold higher than previously reported mevalonate titers in yeast. This study demonstrates the feasibility of engineering S. cerevisiae for high-level mevalonate production.
3. Improving therapeutic protein secretion in the probiotic yeast Saccharomyces boulardii using a multifactorial engineering approach

   https://doi.org/10.1186/s12934-023-02117-y  

   Durmusoglu, Deniz ; Al’Abri, Ibrahim ; Li, Zidan ; Islam Williams, Taufika ; Collins, Leonard B. ; Martínez, José L. ; Crook, Nathan ( June 2023 , Microbial Cell Factories)

   The probiotic yeastSaccharomyces boulardii(Sb) is a promising chassis to deliver therapeutic proteins to the gut due toSb’s innate therapeutic properties, resistance to phage and antibiotics, and high protein secretion capacity. To maintain therapeutic efficacy in the context of challenges such as washout, low rates of diffusion, weak target binding, and/or high rates of proteolysis, it is desirable to engineerSbstrains with enhanced levels of protein secretion. In this work, we explored genetic modifications in bothcis-(i.e. to the expression cassette of the secreted protein) andtrans-(i.e. to theSbgenome) that enhanceSb’s ability to secrete proteins, taking aClostridioides difficileToxin A neutralizing peptide (NPA) as our model therapeutic. First, by modulating the copy number of the NPA expression cassette, we found NPA concentrations in the supernatant could be varied by sixfold (76–458 mg/L) in microbioreactor fermentations. In the context of high NPA copy number, we found a previously-developed collection of native and synthetic secretion signals could further tune NPA secretion between 121 and 463 mg/L. Then, guided by prior knowledge ofS. cerevisiae’s secretion mechanisms, we generated a library of homozygous single gene deletion strains, the most productive of which achieved 2297 mg/L secretory production of NPA. We then expanded on this library by performing combinatorial gene deletions, supplementedmore »by proteomics experiments. We ultimately constructed a quadruple protease-deficientSbstrain that produces 5045 mg/L secretory NPA, an improvement of > tenfold over wild-typeSb. Overall, this work systematically explores a broad collection of engineering strategies to improve protein secretion inSband highlights the ability of proteomics to highlight under-explored mediators of this process. In doing so, we created a set of probiotic strains that are capable of delivering a wide range of protein titers and therefore furthers the ability ofSbto deliver therapeutics to the gut and other settings to which it is adapted.

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4. Beyond the Biosynthetic Gene Cluster Paradigm: Genome-Wide Coexpression Networks Connect Clustered and Unclustered Transcription Factors to Secondary Metabolic Pathways

   https://doi.org/10.1128/Spectrum.00898-21  

   Kwon, Min Jin ; Steiniger, Charlotte ; Cairns, Timothy C. ; Wisecaver, Jennifer H. ; Lind, Abigail L. ; Pohl, Carsten ; Regner, Carmen ; Rokas, Antonis ; Meyer, Vera ( October 2021 , Microbiology Spectrum)

   Goldman, Gustavo H. (Ed.)

   ABSTRACT Fungal secondary metabolites are widely used as therapeutics and are vital components of drug discovery programs. A major challenge hindering discovery of novel secondary metabolites is that the underlying pathways involved in their biosynthesis are transcriptionally silent under typical laboratory growth conditions, making it difficult to identify the transcriptional networks that they are embedded in. Furthermore, while the genes participating in secondary metabolic pathways are typically found in contiguous clusters on the genome, known as biosynthetic gene clusters (BGCs), this is not always the case, especially for global and pathway-specific regulators of pathways’ activities. To address these challenges, we used 283 genome-wide gene expression data sets of the ascomycete cell factory Aspergillus niger generated during growth under 155 different conditions to construct two gene coexpression networks based on Spearman’s correlation coefficients (SCCs) and on mutual rank-transformed Pearson’s correlation coefficients (MR-PCCs). By mining these networks, we predicted six transcription factors, named MjkA to MjkF, to regulate secondary metabolism in A. niger . Overexpression of each transcription factor using the Tet-On cassette modulated the production of multiple secondary metabolites. We found that the SCC and MR-PCC approaches complemented each other, enabling the delineation of putative global (SCC) and pathway-specific (MR-PCC) transcriptionmore »factors. These results highlight the potential of coexpression network approaches to identify and activate fungal secondary metabolic pathways and their products. More broadly, we argue that drug discovery programs in fungi should move beyond the BGC paradigm and focus on understanding the global regulatory networks in which secondary metabolic pathways are embedded. IMPORTANCE There is an urgent need for novel bioactive molecules in both agriculture and medicine. The genomes of fungi are thought to contain vast numbers of metabolic pathways involved in the biosynthesis of secondary metabolites with diverse bioactivities. Because these metabolites are biosynthesized only under specific conditions, the vast majority of the fungal pharmacopeia awaits discovery. To discover the genetic networks that regulate the activity of secondary metabolites, we examined the genome-wide profiles of gene activity of the cell factory Aspergillus niger across hundreds of conditions. By constructing global networks that link genes with similar activities across conditions, we identified six putative global and pathway-specific regulators of secondary metabolite biosynthesis. Our study shows that elucidating the behavior of the genetic networks of fungi under diverse conditions harbors enormous promise for understanding fungal secondary metabolism, which ultimately may lead to novel drug candidates.« less
5. The entry reaction of the plant shikimate pathway is subjected to highly complex metabolite-mediated regulation

   https://doi.org/10.1093/plcell/koaa042  

   Yokoyama, Ryo ; de Oliveira, Marcos V ; Kleven, Bailey ; Maeda, Hiroshi A ( January 2021 , The Plant Cell)

   Abstract The plant shikimate pathway directs bulk carbon flow toward biosynthesis of aromatic amino acids (AAAs, i.e. tyrosine, phenylalanine, and tryptophan) and numerous aromatic phytochemicals. The microbial shikimate pathway is feedback inhibited by AAAs at the first enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DHS). However, AAAs generally do not inhibit DHS activities from plant extracts and how plants regulate the shikimate pathway remains elusive. Here, we characterized recombinant Arabidopsis thaliana DHSs (AthDHSs) and found that tyrosine and tryptophan inhibit AthDHS2, but not AthDHS1 or AthDHS3. Mixing AthDHS2 with AthDHS1 or 3 attenuated its inhibition. The AAA and phenylpropanoid pathway intermediates chorismate and caffeate, respectively, strongly inhibited all AthDHSs, while the arogenate intermediate counteracted the AthDHS1 or 3 inhibition by chorismate. AAAs inhibited DHS activity in young seedlings, where AthDHS2 is highly expressed, but not in mature leaves, where AthDHS1 is predominantly expressed. Arabidopsis dhs1 and dhs3 knockout mutants were hypersensitive to tyrosine and tryptophan, respectively, while dhs2 was resistant to tyrosine-mediated growth inhibition. dhs1 and dhs3 also had reduced anthocyanin accumulation under high light stress. These findings reveal the highly complex regulation of the entry reaction of the plant shikimate pathway and lay the foundation for efforts to control the production ofmore »AAAs and diverse aromatic natural products in plants.« less