Metabolic Engineering Communications最新文献

{"title":"De novo biosynthesis of diverse plant-derived styrylpyrones in Saccharomyces cerevisiae","authors":"Yinan Wu ,&nbsp;Maple N. Chen ,&nbsp;Sijin Li","doi":"10.1016/j.mec.2022.e00195","DOIUrl":"10.1016/j.mec.2022.e00195","url":null,"abstract":"<div><p>Plant styrylpyrones exerting well-established neuroprotective properties have attracted increasing attention in recent years. The ability to synthesize each individual styrylpyrone in engineered microorganisms is important to understanding the biological activity of medicinal plants and the complex mixtures they produce. Microbial biomanufacturing of diverse plant-derived styrylpyrones also provides a sustainable and efficient approach for the production of valuable plant styrylpyrones as daily supplements or potential drugs complementary to the prevalent agriculture-based approach. In this study, we firstly demonstrated the heterogenous biosynthesis of two 7,8-saturated styrylpyrones (7,8-dihydro-5,6-dehydrokavain (DDK) and 7,8-dihydroyangonin (DHY)) and two 7,8-unsaturated styrylpyrones (desmethoxyyangonin (DMY) and yangonin (Y)), in <em>Saccharomyces cerevisiae</em>. Although plant styrylpyrone biosynthetic pathways have not been fully elucidated, we functionally reconstructed the recently discovered kava styrylpyrone biosynthetic pathway that has high substrate promiscuity in yeast, and combined it with upstream hydroxycinnamic acid biosynthetic pathways to produce diverse plant-derived styrylpyrones without the native plant enzymes. We optimized the <em>de novo</em> pathways by engineering yeast endogenous aromatic amino acid metabolism and endogenous double bond reductases and by CRISPR-mediated <em>δ</em>-integration to overexpress the rate-limiting pathway genes. These combinatorial engineering efforts led to the first three yeast strains that can produce diverse plant-derived styrylpyrones <em>de novo</em>, with the titers of DDK, DMY and Y at 4.40 μM, 1.28 μM and 0.10 μM, respectively. This work has laid the foundation for larger-scale styrylpyrone biomanufacturing and the complete biosynthesis of more complicated plant styrylpyrones.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"14 ","pages":"Article e00195"},"PeriodicalIF":5.2,"publicationDate":"2022-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2214030122000049/pdfft?md5=274933e5057ee58d0dded5f97aaf4138&pid=1-s2.0-S2214030122000049-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45576575","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Elimination of aromatic fusel alcohols as by-products of Saccharomyces cerevisiae strains engineered for phenylpropanoid production by 2-oxo-acid decarboxylase replacement","authors":"Else-Jasmijn Hassing,&nbsp;Joran Buijs,&nbsp;Nikki Blankerts,&nbsp;Marijke A. Luttik,&nbsp;Erik A.de Hulster,&nbsp;Jack T. Pronk,&nbsp;Jean-Marc Daran","doi":"10.1016/j.mec.2021.e00183","DOIUrl":"10.1016/j.mec.2021.e00183","url":null,"abstract":"<div><p>Engineered strains of the yeast <em>Saccharomyces cerevisiae</em> are intensively studied as production platforms for aromatic compounds such as hydroxycinnamic acids, stilbenoids and flavonoids. Heterologous pathways for production of these compounds use <span>l</span>-phenylalanine and/or <span>l</span>-tyrosine, generated by the yeast shikimate pathway, as aromatic precursors. The Ehrlich pathway converts these precursors to aromatic fusel alcohols and acids, which are undesirable by-products of yeast strains engineered for production of high-value aromatic compounds. Activity of the Ehrlich pathway requires any of four <em>S. cerevisiae</em> 2-oxo-acid decarboxylases (2-OADCs): Aro10 or the pyruvate-decarboxylase isoenzymes Pdc1, Pdc5, and Pdc6. Elimination of pyruvate-decarboxylase activity from <em>S. cerevisiae</em> is not straightforward as it plays a key role in cytosolic acetyl-CoA biosynthesis during growth on glucose. In a search for pyruvate decarboxylases that do not decarboxylate aromatic 2-oxo acids, eleven yeast and bacterial 2-OADC-encoding genes were investigated. Homologs from <em>Kluyveromyces lactis</em> (<em>KlPDC1</em>), <em>Kluyveromyces marxianus</em> (<em>KmPDC1</em>), <em>Yarrowia lipolytica</em> (<em>YlPDC1</em>), <em>Zymomonas mobilis</em> (<em>Zmpdc1</em>) and <em>Gluconacetobacter diazotrophicus</em> (<em>Gdpdc1.2</em> and <em>Gdpdc1.3</em>) complemented a Pdc⁻ strain of <em>S. cerevisiae</em> for growth on glucose. Enzyme-activity assays in cell extracts showed that these genes encoded active pyruvate decarboxylases with different substrate specificities. In these <em>in vitro</em> assays, <em>Zm</em>Pdc1, <em>Gd</em>Pdc1.2 or <em>Gd</em>Pdc1.3 had no substrate specificity towards phenylpyruvate. Replacing Aro10 and Pdc1,5,6 by these bacterial decarboxylases completely eliminated aromatic fusel-alcohol production in glucose-grown batch cultures of an engineered coumaric acid-producing <em>S. cerevisiae</em> strain. These results outline a strategy to prevent formation of an important class of by-products in ‘chassis’ yeast strains for production of non-native aromatic compounds.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"13 ","pages":"Article e00183"},"PeriodicalIF":5.2,"publicationDate":"2021-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.mec.2021.e00183","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39467729","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Biosensor-based isolation of amino acid-producing Vibrio natriegens strains","authors":"Roberto Giuseppe Stella ,&nbsp;Philipp Baumann ,&nbsp;Sophia Lorke ,&nbsp;Felix Münstermann ,&nbsp;Astrid Wirtz ,&nbsp;Johanna Wiechert ,&nbsp;Jan Marienhagen ,&nbsp;Julia Frunzke","doi":"10.1016/j.mec.2021.e00187","DOIUrl":"10.1016/j.mec.2021.e00187","url":null,"abstract":"<div><p>The marine bacterium <em>Vibrio natriegens</em> has recently been demonstrated to be a promising new host for molecular biology and next generation bioprocesses. <em>V. natriegens</em> is a Gram-negative, non-pathogenic slight-halophilic bacterium, with a high nutrient versatility and a reported doubling time of under 10 min. However, <em>V. natriegens</em> is not an established model organism yet, and further research is required to promote its transformation into a microbial workhorse.</p><p>In this work, the potential of <em>V. natriegens</em> as an amino acid producer was investigated. First, the transcription factor-based biosensor LysG, from <em>Corynebacterium glutamicum</em>, was adapted for expression in <em>V. natriegens</em> to facilitate the detection of positively charged amino acids. A set of different biosensor variants were constructed and characterized, using the expression of a fluorescent protein as sensor output. After random mutagenesis, one of the LysG-based sensors was used to screen for amino acid producer strains. Here, fluorescence-activated cell sorting enabled the selective sorting of highly fluorescent cells, <em>i.e.</em> potential producer cells. Using this approach, individual L-lysine, L-arginine and L-histidine producers could be obtained producing up to 1 mM of the effector amino acid, extracellularly. Genome sequencing of the producer strains provided insight into the amino acid production metabolism of <em>V. natriegens</em>.</p><p>This work demonstrates the successful expression and application of transcription factor-based biosensors in <em>V. natriegens</em> and provides insight into the underlying physiology, forming a solid basis for further development of this promising microbe.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"13 ","pages":"Article e00187"},"PeriodicalIF":5.2,"publicationDate":"2021-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/ca/05/main.PMC8605253.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39660647","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Overcoming glutamate auxotrophy in Escherichia coli itaconate overproducer by the Weimberg pathway","authors":"Ken W. Lu,&nbsp;Chris T. Wang,&nbsp;Hengray Chang,&nbsp;Ryan S. Wang,&nbsp;Claire R. Shen","doi":"10.1016/j.mec.2021.e00190","DOIUrl":"10.1016/j.mec.2021.e00190","url":null,"abstract":"<div><p>Biosynthesis of itaconic acid occurs through decarboxylation of the TCA cycle intermediate cis-aconitate. Engineering of efficient itaconate producers often requires elimination of the highly active isocitrate dehydrogenase to conserve cis-aconitate, leading to 2-ketoglutarate auxotrophy in the producing strains. Supplementation of glutamate or complex protein hydrolysate then becomes necessary, often in large quantities, to support the high cell density desired during itaconate fermentation and adds to the production cost. Here, we present an alternative approach to overcome the glutamate auxotrophy in itaconate producers by synthetically introducing the Weimberg pathway from <em>Burkholderia xenovorans</em> for 2-ketoglutarate biosynthesis. Because of its independence from natural carbohydrate assimilation pathways in <em>Escherichia coli</em>, the Weimberg pathway is able to provide 2-ketoglutarate using xylose without compromising the carbon flux toward itaconate. With xylose concentration carefully tuned to minimize excess 2-ketoglutarate flux in the stationary phase, the final strain accumulated 20 g/L of itaconate in minimal medium from 18 g/L of xylose and 45 g/L of glycerol. Necessity of the recombinant Weimberg pathway for growth also allowed us to maintain multi-copy plasmids carrying in operon the itaconate-producing genes without addition of antibiotics. Use of the Weimberg pathway for growth restoration is applicable to other production systems with disrupted 2-ketoglutarate synthesis.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"13 ","pages":"Article e00190"},"PeriodicalIF":5.2,"publicationDate":"2021-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8661531/pdf/main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39834432","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Engineering Acinetobacter baylyi ADP1 for mevalonate production from lignin-derived aromatic compounds","authors":"Erika Arvay ,&nbsp;Bradley W. Biggs ,&nbsp;Laura Guerrero ,&nbsp;Virginia Jiang ,&nbsp;Keith Tyo","doi":"10.1016/j.mec.2021.e00173","DOIUrl":"10.1016/j.mec.2021.e00173","url":null,"abstract":"<div><p>Utilization of lignin, an abundant renewable resource, is limited by its heterogenous composition and complex structure. Biological valorization of lignin provides advantages over traditional chemical processing as it occurs at ambient temperature and pressure and does not use harsh chemicals. Furthermore, the ability to biologically funnel heterogenous substrates to products eliminates the need for costly downstream processing and separation of feedstocks. However, lack of relevant metabolic networks and low tolerance to degradation products of lignin limits the application of traditional engineered model organisms. To circumvent this obstacle, we employed <em>Acinetobacter baylyi</em> ADP1, which natively catabolizes lignin-derived aromatic substrates through the β-ketoadipate pathway, to produce mevalonate from lignin-derived compounds. We enabled expression of the mevalonate pathway in ADP1 and validated activity in the presence of multiple lignin-derived aromatic substrates. Furthermore, by knocking out wax ester synthesis and utilizing fed-batch cultivation, we improved mevalonate titers 7.5-fold to 1014 mg/L (6.8 mM). This work establishes a foundation and provides groundwork for future efforts to engineer improved production of mevalonate and derivatives from lignin-derived aromatics using ADP1.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"13 ","pages":"Article e00173"},"PeriodicalIF":5.2,"publicationDate":"2021-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.mec.2021.e00173","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39355097","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Production of raspberry ketone by redirecting the metabolic flux to the phenylpropanoid pathway in tobacco plants","authors":"Takao Koeduka ,&nbsp;Sachiho Takarada ,&nbsp;Koya Fujii ,&nbsp;Akifumi Sugiyama ,&nbsp;Kazufumi Yazaki ,&nbsp;Masahiro Nishihara ,&nbsp;Kenji Matsui","doi":"10.1016/j.mec.2021.e00180","DOIUrl":"10.1016/j.mec.2021.e00180","url":null,"abstract":"<div><p>Raspberry ketone is one of the characteristic flavors of raspberry fruits, and it is an important and expensive ingredient in the flavor and fragrance industries. It is present at low levels in plant tissues, and its occurrence is limited to a few taxa. In this context, the stable production of nature-identical raspberry ketone using heterologous synthesis in plants hosts has recently garnered the attention of plant biochemists. In this study, we demonstrate the rational switching of the metabolic flow from anthocyanin pigments to volatile phenylbutanoid production via the phenylpropanoid pathway. This shift led to the efficient and stable production of raspberry ketone and its glycosides via heterologous expression of the biosynthetic enzymes benzalacetone synthase (BAS) and raspberry ketone/zingerone synthase 1 (RZS1) in the transgenic tobacco (<em>Nicotiana tabacum</em> ‘Petit Havana SR-1’). Additionally, we achieved improved product titers by activating the phenylpropanoid pathway with the transcriptional factor, production of anthocyanin pigment 1 (PAP1), from <em>Arabidopsis thaliana</em>. We further demonstrated another metabolic-flow switching by RNA interference (RNAi)-mediated silencing of chalcone synthase (CHS) to increase pathway-intermediate <em>p</em>-coumaroyl-CoA in transgenic tobacco for raspberry-ketone production. The redirection of metabolic flux resulted in transgenic lines producing 0.45 μg/g of raspberry ketone and 4.5 μg/g, on the fresh weight basis, of its glycosides in the flowers. These results suggest that the intracellular enforcement of endogenous substrate supply is an important factor while engineering the phenylpropanoid pathway. This strategy might be useful for the production of other phenylpropanoids/polyketides that are produced via the pathway-intermediate <em>p</em>-coumaroyl-CoA, in tobacco plants.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"13 ","pages":"Article e00180"},"PeriodicalIF":5.2,"publicationDate":"2021-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.mec.2021.e00180","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39306449","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cell line development for continuous high cell density biomanufacturing: Exploiting hypoxia for improved productivity","authors":"Nikolas Zeh,&nbsp;Patrick Schlossbauer,&nbsp;Nadja Raab,&nbsp;Florian Klingler,&nbsp;René Handrick,&nbsp;Kerstin Otte","doi":"10.1016/j.mec.2021.e00181","DOIUrl":"10.1016/j.mec.2021.e00181","url":null,"abstract":"<div><p>Oxygen deficiency (hypoxia) induces adverse effects during biotherapeutic protein production leading to reduced productivity and cell growth. Hypoxic conditions occur during classical batch fermentations using high cell densities or perfusion processes. Here we present an effort to create novel engineered Chinese hamster ovary (CHO) cell lines by exploiting encountered hypoxic bioprocess conditions to reinforce cellular production capacities. After verifying the conservation of the hypoxia-responsive pathway in CHO cell lines by analyzing oxygen sensing proteins HIF1a, HIF1β and VDL, hypoxia-response-elements (HREs) were functionally analyzed and used to create hypoxia-responsive expression vectors. Subsequently engineered hypoxia sensitive CHO cell lines significantly induced protein expression (SEAP) during adverse oxygen limitation encountered during batch fermentations as well as high cell density perfusion processes (2.7 fold). We also exploited this novel cell system to establish a highly effective oxygen shift as innovative bioprocessing strategy using hypoxia induction to improve production titers. Thus, substantial improvements can be made to optimize CHO cell productivity for novel bioprocessing challenges as oxygen limitation, providing an avenue to establish better cell systems by exploiting adverse process conditions for optimized biotherapeutic production.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"13 ","pages":"Article e00181"},"PeriodicalIF":5.2,"publicationDate":"2021-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.mec.2021.e00181","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39317753","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Identification of a lichen depside polyketide synthase gene by heterologous expression in Saccharomyces cerevisiae","authors":"James T. Kealey,&nbsp;James P. Craig,&nbsp;Philip J. Barr","doi":"10.1016/j.mec.2021.e00172","DOIUrl":"10.1016/j.mec.2021.e00172","url":null,"abstract":"<div><p>Lichen-forming fungi produce a variety of secondary metabolites including bioactive polyketides. Advances in DNA and RNA sequencing have led to a growing database of new lichen gene clusters encoding polyketide synthases (PKS) and associated ancillary activities. Definitive assignment of a PKS gene to a metabolic product has been challenging in the lichen field due to a lack of established gene knockout or heterologous gene expression systems. Here, we report the reconstitution of a non-reducing PKS gene from the lichen <em>Pseudevernia furfuracea</em> and successful heterologous expression of the synthetic lichen PKS gene in engineered <em>Saccharomyces cerevisiae</em>. We show that <em>P. furfuracea</em> PFUR17_02294 produces lecanoric acid, the depside dimer of orsellinic acid, at 360 mg/L in small-scale yeast cultures. Our results unequivocally identify PFUR17_02294 as a lecanoric acid synthase and establish that a single lichen PKS synthesizes two phenolic rings and joins them by an ester linkage to form the depside product.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"13 ","pages":"Article e00172"},"PeriodicalIF":5.2,"publicationDate":"2021-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.mec.2021.e00172","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39355096","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Transport engineering for improving the production and secretion of valuable alkaloids in Escherichia coli","authors":"Yasuyuki Yamada ,&nbsp;Miya Urui ,&nbsp;Hidehiro Oki ,&nbsp;Kai Inoue ,&nbsp;Haruyuki Matsui ,&nbsp;Yoshito Ikeda ,&nbsp;Akira Nakagawa ,&nbsp;Fumihiko Sato ,&nbsp;Hiromichi Minami ,&nbsp;Nobukazu Shitan","doi":"10.1016/j.mec.2021.e00184","DOIUrl":"10.1016/j.mec.2021.e00184","url":null,"abstract":"<div><p>Microorganisms can be metabolically engineered to produce specialized plant metabolites. However, these methods are limited by low productivity and intracellular accumulation of metabolites. We sought to use transport engineering for producing reticuline, an important intermediate in the alkaloid biosynthetic pathway. In this study, we established a reticuline-producing <em>Escherichia coli</em> strain into which the multidrug and toxic compound extrusion transporter <em>Arabidopsis</em> AtDTX1 was introduced. AtDTX1 was selected due to its suitable expression in <em>E. coli</em> and its reticuline-transport activity. Expression of AtDTX1 enhanced reticuline production by 11-fold, and the produced reticuline was secreted into the medium. AtDTX1 expression also conferred high plasmid stability and resulted in upregulation or downregulation of several genes associated with biological processes, including metabolic pathways for reticuline biosynthesis, leading to the production and secretion of high levels of reticuline. The successful employment of a transporter for alkaloid production suggests that the proposed transport engineering approach may improve the biosynthesis of specialized metabolites <em>via</em> metabolic engineering.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"13 ","pages":"Article e00184"},"PeriodicalIF":5.2,"publicationDate":"2021-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8449128/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39452761","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Resveratrol production from several types of saccharide sources by a recombinant Scheffersomyces stipitis strain","authors":"Yuma Kobayashi ,&nbsp;Kentaro Inokuma ,&nbsp;Mami Matsuda ,&nbsp;Akihiko Kondo ,&nbsp;Tomohisa Hasunuma","doi":"10.1016/j.mec.2021.e00188","DOIUrl":"10.1016/j.mec.2021.e00188","url":null,"abstract":"<div><p>Resveratrol is a plant-derived aromatic compound with a wide range of beneficial properties including antioxidant and anti-aging effects. The resveratrol currently available on the market is predominantly extracted from certain plants such as grape and the Japanese knotweed <em>Polygonum cuspidatum</em>. Due to the unstable harvest of these plants and the low resveratrol purity obtained, it is necessary to develop a stable production process of high-purity resveratrol from inexpensive feedstocks. Here, we attempted to produce resveratrol from a wide range of sugars as carbon sources by a using the genetically-engineered yeast <em>Scheffersomyces stipitis</em> (formerly known as <em>Pichia stipitis</em>), which possesses a broad sugar utilization capacity. First, we constructed the resveratrol producing strain by introducing genes coding the essential enzymes for resveratrol biosynthesis [tyrosine ammonia-lyase 1 derived from <em>Herpetosiphon aurantiacus</em> (<em>HaTAL1</em>), 4-coumarate: CoA ligase 2 derived from <em>Arabidopsis thaliana</em> (<em>At4CL2</em>), and stilbene synthase 1 derived from <em>Vitis vinifera</em> (<em>VvVST1</em>)]. Subsequently, a feedback-insensitive allele of chorismate mutase was overexpressed in the constructed strain to improve resveratrol production. The constructed strain successfully produced resveratrol from a broad range of biomass-derived sugars [glucose, fructose, xylose, <em>N</em>-acetyl glucosamine (GlcNAc), galactose, cellobiose, maltose, and sucrose] in shake flask cultivation. Significant resveratrol titers were detected in cellobiose and sucrose fermentation (529.8 and 668.6 mg/L after 120 h fermentation, respectively), twice above the amount obtained with glucose (237.6 mg/L). Metabolomic analysis revealed an altered profile of the metabolites involved in the glycolysis and shikimate pathways, and also of cofactors and metabolites of energy metabolisms, depending on the substrate used. The levels of resveratrol precursors such as L-tyrosine increased in cellobiose and sucrose-grown cells. The results indicate that <em>S. stipitis</em> is an attractive microbial platform for resveratrol production from broad types of biomass-derived sugars and the selection of suitable substrates is crucial for improving resveratrol productivity of this yeast.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"13 ","pages":"Article e00188"},"PeriodicalIF":5.2,"publicationDate":"2021-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8637140/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39711568","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}