Olefine

Safe replacements for insecticides enabled by biotechnology



Banner image displaying corn field

What is Olefine?

Nowadays everybody is aware of the toxic effects of the conventional insecticides that are used to protect our plants from pest insects. The insecticides are harmful for the farmer, who sprays them on the field, for the pollinating insects, e.g., bees, and insecticide residues on fruits and vegetables are harmful for the consumer. But are there other safer ways to protect the plants from pest insects?

Yes, one of most promising methods is mating disruption, where small amounts of insect sex pheromones are released in the field to prevent the males from finding the insect females. This way females do not get fertilized and can not lay eggs that develop into larvae eating the plants. The technology is simple and effective, but until now quite expensive.

The EU-funded project OLEFINE will solve this problem, making pheromones so cheap that they will become an affordable alternative to insecticides. Currently, pheromones are produced by chemical synthesis, which is an expensive and polluting process. The scientists in OLEFINE will use biotechnology instead to produce pheromones at low cost by brewing, in the same way how insulin is made for diabetes treatment and enzymes are produced for washing powders.

News and Social

  • Olefine draws to an end, and it is going out with a bang! (June 22, 2022)

    As the OLEFINE project is drawing to a close, its partners can take pride in having established a truly market-disrupting technology. Read about it all in our newsletter.


  • Pheromones, mulch and wildflowers – how to control pests without pesticides (March 17, 2021)

    Check out the new article publishe in HORIZON - The EU Research and Innovation Magazine. Please visit the website for more information -> HORIZON - The EU Research and Innovation Mahazine


  • Insect sex pheromones as safe replacements for insecticides (October 28, 2019)

  • Irina Borodina wins 2019 EU Prize for Women Innovators (May 16, 2019)

    BioPhero’s Founder and CTO, Irina Borodina, is awarded the 2019 EU Prize for Women Innovators as announced by Carlos Moedas, Commissioner for Research, Science and Innovation, at this year’s VivaTech conference in Paris.


  • Dr. Leoni Wenning joins BioPhero on Marie Skłodowska-Curie Individual Fellowship (April 01, 2019)

    Dr. Leonie Wenning receives a prestigious Marie Skłodowska-Curie Individual Fellowship to join BioPhero as a Research Scientist and develop new pheromone products.


  • 2019 Equinor Prize awarded to Irina Borodina, Founder and CTO of Biophero (March 15, 2019)

    BioPhero’s Founder and CTO Irina Borodina was awarded the prestigious Equinor Prize 2019 at an official ceremony at The National Museum of Denmark. -> DTU Biosustain Press Release


  • Danks Kemi features BioPhero's technology and origin story (March 01, 2019)

    See the whole story here (in Danish only) -> Dansk Kemi


  • We can revolutionise pest management (January 22, 2019)

    BioPhero has been featured in Technologist! The Technologist magazine publishes exclusive articles every week that explain issues related to scientific research and innovation. Access is completely free: www.Technologist.eu Technologist is an initiative from EuroTech alliance, a strategic partnership of five leading European universities of science & technology: Technical University of Denmark (DTU), Ecole Polytechnique Fédérale de Lausanne (EPFL), Ecole Polytechnique (L’X), Eindhoven University of Technology (TU/e) and Technical University of Munich (TUM).


  • Biological Pheromones in fight against pests (December 13, 2018)

    OLEFINE newsletter “Biological Pheromones in fight against pests” motivating the core principles of using pheromones and their benefits versus insecticides.


  • BioPhero ApS has announced the closing of a seed investment of €3 million by Novo Seeds, Syngenta Ventures and Syddansk Innovation (September 17, 2018)

    BioPhero ApS has announced the closing of a seed investment of €3 million by Novo Seeds, Syngenta Ventures and Syddansk Innovation. As part of the investment, Novo Seeds, Syngenta Ventures and Syddansk Innovation have joined the board, and Mr. Hadyn Parry has joined BioPhero’s board as the new chairman. Kristian Ebbensgaard, has been appointed as the CEO of BioPhero, while Dr. Irina Borodina has been appointed as the Research Director.


  • The OLEFINE EU Horizon 2020 project is featured in The Guardian (June 12, 2018)

    To see the whole text click here -> The Guardian


  • The OLEFINE EU Horizon 2020 project introduced at the National Center for Scientific Research "Demokritos" (June 12, 2018)

    ChThe Laboratory of Chemical Ecology and Natural Products of the Institute of Biosciences and Applications, NCSR “Demokritos”, led by Dr. Maria Konstantopoulou, participates in the European OLEFINE (OLEFINE yeast platforms for FINE chemicals) HORIZON2020 (Grant Agreement No. 760798). The program, which lasts 48 months, involves 5 academics and 4 industrial partners. Read more here -> Demokritos


  • BioPhero will participate at the Metabolic Engineering Conference in Munich in June 2018 (April 25, 2018)

    BioPhero will present the results on biological production of pheromones at Metabolic Engineering Conference in Munich, Germany on the 27th June 2018. Please visit the website for more information -> Metabolic Engineering Conference


  • Next Olefine Project Meeting at DTU, 4th of May 2018 (April 03, 2018)

    The next project meeting will be held at DTU on 4th of May 2018. All partners are invited to join the next Half-Annual Meeting at DTU, Denmark.


  • Kick-off meeting (January 16, 2018)

    All partners gathered in Brussels for the official start of OLEFINE project.


Jobs

Publications

  • Carina Holkenbrink, Marie I. Dam, Kanchana R. Kildegaard, Johannes Beder, Jonathan Dahlin, David Doménech Belda, Irina Borodina, EasyCloneYALI: CRISPR/Cas9‐Based Synthetic Toolbox for Engineering of the Yeast Yarrowia lipolytica, Biotechnology, 10.1002/biot.201700543
    April 26, 2018

    The oleaginous yeast Yarrowia lipolytica is an emerging host for production of fatty acid‐derived chemicals. To enable rapid iterative metabolic engineering of this yeast, there is a need for well‐characterized genetic parts and convenient and reliable methods for their incorporation into yeast. Here, the EasyCloneYALI genetic toolbox, which allows streamlined strain construction with high genome editing efficiencies in Y. lipolytica via the CRISPR/Cas9 technology is presented. The toolbox allows marker‐free integration of gene expression vectors into characterized genome sites as well as marker‐free deletion of genes with the help of CRISPR/Cas9. Genome editing efficiencies above 80% were achieved with transformation protocols using non‐replicating DNA repair fragments (such as DNA oligos). Furthermore, the toolbox includes a set of integrative gene expression vectors with prototrophic markers conferring resistance to hygromycin and nourseothricin.


  • Dahlin J, Holkenbrink C, Marella ER, Wang G, Liebal U, Lieven C, Weber D, McCloskey D, Ebert BE, Herrgård MJ, Blank LM, Borodina I, Multi-Omics Analysis of Fatty Alcohol Production in Engineered Yeasts Saccharomyces cerevisiae and Yarrowia lipolytica, Frontiers in genetics, 10, 747, 10.3389/fgene.2019.00747
    Fatty alcohols are widely used in various applications within a diverse set of industries, such as the soap and detergent industry, the personal care, and cosmetics industry, as well as the food industry. The total world production of fatty alcohols is over 2 million tons with approximately equal parts derived from fossil oil and from plant oils or animal fats. Due to the environmental impact of these production methods, there is an interest in alternative methods for fatty alcohol production via microbial fermentation using cheap renewable feedstocks. In this study, we aimed to obtain a better understanding of how fatty alcohol biosynthesis impacts the host organism, baker’s yeast Saccharomyces cerevisiae or oleaginous yeast Yarrowia lipolytica. Producing and non-producing strains were compared in growth and nitrogen-depletion cultivation phases. The multi-omics analysis included physiological characterization, transcriptome analysis by RNAseq, 13Cmetabolic flux analysis, and intracellular metabolomics. Both species accumulated fatty alcohols under nitrogen-depletion conditions but not during growth. The fatty alcohol–producing Y. lipolytica strain had a higher fatty alcohol production rate than an analogous S. cerevisiae strain. Nitrogen-depletion phase was associated with lower glucose uptake rates and a decrease in the intracellular concentration of acetyl–CoA in both yeast species, as well as increased organic acid secretion rates in Y. lipolytica. Expression of the fatty alcohol–producing enzyme fatty acyl–CoA reductase alleviated the growth defect caused by deletion of hexadecenal dehydrogenase encoding genes (HFD1 and HFD4) in Y. lipolytica. RNAseq analysis showed that fatty alcohol production triggered a cell wall stress response in S. cerevisiae. RNAseq analysis also showed that both nitrogen-depletion and fatty alcohol production have substantial effects on the expression of transporter encoding genes in Y. lipolytica. In conclusion, through this multi-omics study, we uncovered some effects of fatty alcohol production on the host metabolism. This knowledge can be used as guidance for further strain improvement towards the production of fatty alcohols.

  • Larissa Tramontin, Kanchana Kildegaard,Suresh Sudarsan, Irina Borodina, Enhancement of Astaxanthin Biosynthesis in Oleaginous Yeast Yarrowia lipolytica via Microalgal Pathway, Microorganisms, 7(10) , 472, 10.3390/microorganisms7100472
    Astaxanthin is a high-value red pigment and antioxidant used by pharmaceutical, cosmetics, and food industries. The astaxanthin produced chemically is costly and is not approved for human consumption due to the presence of by-products. The astaxanthin production by natural microalgae requires large open areas and specialized equipment, the process takes a long time, and results in low titers. Recombinant microbial cell factories can be engineered to produce astaxanthin by fermentation in standard equipment. In this work, an oleaginous yeast Yarrowia lipolytica was engineered to produce astaxanthin at high titers in submerged fermentation. First, a platform strain was created with an optimised pathway towards β-carotene. The platform strain produced 331 ± 66 mg/L of β-carotene in small-scale cultivation, with the cellular content of 2.25% of dry cell weight. Next, the genes encoding β-ketolase and β-hydroxylase of bacterial (Paracoccus sp. and Pantoea ananatis) and algal (Haematococcus pluvialis) origins were introduced into the platform strain in different copy numbers. The resulting strains were screened for astaxanthin production, and the best strain, containing algal β-ketolase and β-hydroxylase, resulted in astaxanthin titer of 44 ± 1 mg/L. The same strain was cultivated in controlled bioreactors, and a titer of 285 ± 19 mg/L of astaxanthin was obtained after seven days of fermentation on complex medium with glucose. Our study shows the potential of Y. lipolytica as the cell factory for astaxanthin production.

  • Jonathan Asmund Arnesen, Kanchana Rueksomtawin Kildegaard,Marc Cernuda Pastor, Sidharth Jayachandran, Mette Kristensen and Irina Borodina, Yarrowia lipolytica Strains Engineered for the Production of Terpenoids, Frontiers Bioengineering and Biotechnology, 8, 10.3389/fbioe.2020.00945
    Terpenoids are a diverse group of over 55,000 compounds with potential applications as advanced fuels, bulk and fine chemicals, pharmaceutical ingredients, agricultural chemicals, etc. To facilitate their bio-based production, there is a need for plug-and-play hosts, capable of high-level production of different terpenoids. Here we engineer Yarrowia lipolytica platform strains for the overproduction of mono-, sesqui-, di-, tri-, and tetraterpenoids. The monoterpene platform strain was evaluated by expressing Perilla frutescens limonene synthase, which resulted in limonene titer of 35.9 mg/L and was 100-fold higher than when the same enzyme was expressed in the strain without mevalonate pathway improvement. Expression of Callitropsis nootkatensis valencene synthase in the sesquiterpene platform strain resulted in 113.9 mg/L valencene, an 8.4-fold increase over the control strain. Platform strains for production of squalene, complex triterpenes, or diterpenes and carotenoids were also constructed and resulted in the production of 402.4 mg/L squalene, 22 mg/L 2,3-oxidosqualene, or 164 mg/L β-carotene, respectively. The presented terpenoid platform strains can facilitate the evaluation of terpenoid biosynthetic pathways and are a convenient starting point for constructing efficient cell factories for the production of various terpenoids. The platform strains and exemplary terpenoid strains can be obtained from Euroscarf.

  • Holkenbrink C, Ding BJ, Wang H-L, Dam MI,Petkevicius K, Rueksomtawin Kildegaard K, Wennig L, Sinkwitz C, Lorántfy B, Koutsoumpeli E, França L, Pires M, Bernard C, Urrutia W, Mafra-Neto A, Sommer Ferreira B, Raptopoulos D, Konstantopoulou M, Löfstedt C, Borodina I., Production of moth sex pheromones for pest control by yeast fermentation, Metabolic Engineering, 62, 4312-321, 10.1016/j.ymben.2020.10.001
    The use of insect sex pheromones is an alternative technology for pest control in agriculture and forestry, which, in contrast to insecticides, does not have adverse effects on human health or environment and is efficient also against insecticide-resistant insect populations. Due to the high cost of chemically synthesized pheromones, mating disruption applications are currently primarily targeting higher value crops, such as fruits. Here we demonstrate a biotechnological method for the production of (Z)-hexadec-11-en-1-ol and (Z)-tetradec-9-en-1-ol, using engineered yeast cell factories. These unsaturated fatty alcohols are pheromone components or the immediate precursors of pheromone components of several economically important moth pests. Biosynthetic pathways towards several pheromones or their precursors were reconstructed in the oleaginous yeast Yarrowia lipolytica, which was further metabolically engineered for improved pheromone biosynthesis by decreasing fatty alcohol degradation and downregulating storage lipid accumulation. The sex pheromone of the cotton bollworm Helicoverpa armigera was produced by oxidation of fermented fatty alcohols into corresponding aldehydes. The resulting yeast-derived pheromone was just as efficient and specific for trapping of H. armigera male moths in cotton fields in Greece as a conventionally produced synthetic pheromone mixture. We further demonstrated the production of (Z)-tetradec-9-en-1-yl acetate, the main pheromone component of the fall armyworm Spodoptera frugiperda. Taken together our work describes a biotech platform for the production of commercially relevant titres of moth pheromones for pest control via yeast fermentation.

  • Karolis Petkevicius, Christer Löfstedt, Irina Borodina, Insect sex pheromone production in yeasts and plants, Current Opinion in Biotechnology, 10.1016/j.copbio.2020.07.011
    Insect infestation is a major problem in agriculture and forestry addressed primarily with insecticide sprays or genetically modified plant breeds. The problem has aggravated in the last decade due to the emergence of resistance among key insect pests and the removal of multiple insecticides from the market due to their toxicity. Pheromone-based methods for pest management have been in use for over 30 years, though primarily for high-value fruits due to the high cost of the chemical synthesis and pheromone application. As biotechnology solutions for pheromone production are emerging, pheromones will become an economically competitive technology for pest management also in low-value row crops. This review describes the advances in the discovery of pheromone biosynthetic pathways and the recent engineering of yeasts and plants for recombinant production of pheromones.

  • Karolis Petkevicius, Eleni Koutsoumpeli,Petri Christina Betsi, Bao-Jian Ding, Kanchana Rueksomtawin Kildegaard, Hilbert Jensen, Nora Mezo, Andrea Mazziotta, Anders Gabrielsson, Christina Sinkwitz, Bettina Lorantfy, Carina Holkenbrink, Christer Löfstedt, Dimitris Raptopoulos, Maria Konstantopoulou, Irina Borodina, Biotechnological production of the European corn borer sex pheromone in the yeast Yarrowia lipolyticay, Biotechnology Journal, 2100004, 10.1002/biot.202100004
    The European corn borer (ECB) Ostrinia nubilalis is a widespread pest of cereals, particularly maize. Mating disruption with the sex pheromone is a potentially attractive method for managing this pest; however, chemical synthesis of pheromones requires expensive starting materials and catalysts and generates hazardous waste. The goal of this study was to develop a biotechnological method for the production of ECB sex pheromone. Our approach was to engineer the oleaginous yeast Yarrowia lipolytica to produce (Z)-11-tetradecenol (Z11-14:OH), which can then be chemically acetylated to (Z)-11-tetradecenyl acetate (Z11-14:OAc), the main pheromone component of the Z-race of O. nubilalis. First, a C14 platform strain with increased biosynthesis of myristoyl-CoA was obtained by introducing a point mutation into the α-subunit of fatty acid synthase, replacing isoleucine 1220 with phenylalanine (Fas2pI1220F). The intracellular accumulation of myristic acid increased 8.4-fold. Next, fatty acyl-CoA desaturases (FAD) and fatty acyl-CoA reductases (FAR) from nine different species of Lepidoptera were screened in the C14 platform strain, individually and in combinations. A titer of 29.2 ± 1.6 mg L-1 Z11-14:OH was reached in small-scale cultivation with an optimal combination of a FAD (Lbo_PPTQ) from Lobesia botrana and FAR (HarFAR) from Helicoverpa armigera. When the second copies of FAD and FAR genes were introduced, the titer improved 2.1-fold. The native FAS1 gene's overexpression led to a further 1.5-fold titer increase, reaching 93.9 ± 11.7 mg L-1 in small-scale cultivation. When the same engineered strain was cultivated in controlled 1 L bioreactors in fed-batch mode, 188.1 ± 13.4 mg L-1 of Z11-14:OH was obtained. Fatty alcohols were extracted from the biomass and chemically acetylated to obtain Z11-14:OAc. Electroantennogram experiments showed that males of the Z-race of O. nubilalis were responsive to biologically-derived pheromone blend. Behavioral bioassays in a wind tunnel revealed attraction of male O. nubilalis, although full precopulatory behavior was observed less often than for the chemically synthesized pheromone blend. The study paves the way for the production of ECB pheromone by fermentation.Astaxanthin is a high-value red pigment and antioxidant used by pharmaceutical, cosmetics, an

  • Ding B-J, Xia Y-H, Wang H-L, Andersson F, Hedenström E, Gross J, Löfstedt C., Biosynthesis of the sex pheromone component(E,Z)-7,9-dodecadienyl acetate in the European Grapevine Moth, Lobesia botrana, involving ∆11 desaturation and an elusive ∆7 desaturase, J Chem Ecol, 47(3) , 248-264, 10.1007/s10886-021-01252-3
    The European grapevine moth, Lobesia botrana, uses (E,Z)-7,9-dodecadienyl acetate as its major sex pheromone component. Through in vivo labeling experiments we demonstrated that the doubly unsaturated pheromone component is produced by ∆11 desaturation of tetradecanoic acid, followed by chain shortening of (Z)-11-tetradecenoic acid to (Z)-9-dodecenoic acid, and subsequently introduction of the second double bond by an unknown ∆7 desaturase, before final reduction and acetylation. By sequencing and analyzing the transcriptome of female pheromone glands of L. botrana, we obtained 41 candidate genes that may be involved in sex pheromone production, including the genes encoding 17 fatty acyl desaturases, 13 fatty acyl reductases, 1 fatty acid synthase, 3 acyl-CoA oxidases, 1 acetyl-CoA carboxylase, 4 fatty acid transport proteins and 2 acyl-CoA binding proteins. A functional assay of desaturase and acyl-CoA oxidase gene candidates in yeast and insect cell (Sf9) heterologous expression systems revealed that Lbo_PPTQ encodes a ∆11 desaturase producing (Z)-11-tetradecenoic acid from tetradecanoic acid. Further, Lbo_31670 and Lbo_49602 encode two acyl-CoA oxidases that may produce (Z)-9-dodecenoic acid by chain shortening (Z)-11-tetradecenoic acid. The gene encoding the enzyme introducing the E7 double bond into (Z)-9-dodecenoic acid remains elusive even though we assayed 17 candidate desaturases in the two heterologous systems.

  • Lassance J-M, Ding B-J, Löfstedt C., Evolution of the codling moth pheromone via an ancient gene duplication, BMC Biology, 19, 83, 10.1186/s12915-021-01001-8
    Background Defining the origin of genetic novelty is central to our understanding of the evolution of novel traits. Diversification among fatty acid desaturase (FAD) genes has played a fundamental role in the introduction of structural variation in fatty acyl derivatives. Because of its central role in generating diversity in insect semiochemicals, the FAD gene family has become a model to study how gene family expansions can contribute to the evolution of lineage-specific innovations. Here we used the codling moth (Cydia pomonella) as a study system to decipher the proximate mechanism underlying the production of the ∆8∆10 signature structure of olethreutine moths. Biosynthesis of the codling moth sex pheromone, (E8,E10)-dodecadienol (codlemone), involves two consecutive desaturation steps, the first of which is unusual in that it generates an E9 unsaturation. The second step is also atypical: it generates a conjugated diene system from the E9 monoene C12 intermediate via 1,4-desaturation.

  • Xia Y-H, Wang H-L, Ding B-J, Svensson GP, Jarl-Sunesson C, Cahoon EB, Hofvander P, Löfstedt C., Green Chemistry Production of Codlemone, the Sex Pheromone of the Codling Moth (Cydia pomonella), by Metabolic Engineering of the Oilseed Crop Camelina (Camelina sativa)., J Chem Ecol, 47, 950-967, 10.1007/s10886-021-01316-4
    Synthetic pheromones have been used for pest control over several decades. The conventional synthesis of di-unsaturated pheromone compounds is usually complex and costly. Camelina (Camelina sativa) has emerged as an ideal, non-food biotech oilseed platform for production of oils with modified fatty acid compositions. We used Camelina as a plant factory to produce mono- and di-unsaturated C12 chain length moth sex pheromone precursors, (E)-9-dodecenoic acid and (E,E)-8,10-dodecadienoic acid, by introducing a fatty acyl-ACP thioesterase FatB gene UcTE from California bay laurel (Umbellularia californica) and a bifunctional ∆9 desaturase gene Cpo_CPRQ from the codling moth, Cydia pomonella. Different transgene combinations were investigated for increasing pheromone precursor yield. The most productive Camelina line was engineered with a vector that contained one copy of UcTE and the viral suppressor protein encoding P19 transgenes and three copies of Cpo_CPRQ transgene. The T2 generation of this line produced 9.4% of (E)-9-dodecenoic acid and 5.5% of (E,E)-8,10-dodecadienoic acid of the total fatty acids, and seeds were selected to advance top-performing lines to homozygosity. In the T4 generation, production levels of (E)-9-dodecenoic acid and (E,E)-8,10-dodecadienoic acid remained stable. The diene acid together with other seed fatty acids were converted into corresponding alcohols, and the bioactivity of the plant-derived codlemone was confirmed by GC-EAD and a flight tunnel assay. Trapping in orchards and home gardens confirmed significant and specific attraction of C. pomonella males to the plant-derived codlemone.

  • Kanchana R. Kildegaard, Jonathan A. Arnesen, Belén Adiego-Pérez, Daniela Rago, Mette Kristensen, Andreas K. Klitgaard, Esben H. Hansen, Jørgen Hansen, Irina Borodina, Tailored biosynthesis of gibberellin plant hormones in yeast, Metabolic Engineering, 66, 1-11, 10.1016/j.ymben.2021.03.010

    The application of small amounts of natural plant growth hormones, such as gibberellins (GAs), can increase the productivity and quality of many vegetable and fruit crops. However, gibberellin growth hormones usage is limited by the high cost of their production, which is currently based on fermentation of a natural fungal producer Fusarium fujikuroi that produces a mix of several GAs. We explored the potential of the oleaginous yeast Yarrowia lipolytica to produce specific profiles of GAs. Firstly, the production of the GA-precursor ent-kaurenoic acid (KA) at 3.75 mg/L was achieved by expression of biosynthetic enzymes from the plant Arabidopsis thaliana and upregulation of the mevalonate (MVA) pathway.


  • Jonathan Asmund Arnesen, Arian Belmonte Del Ama, Sidharth Jayachandran, Jonathan Dahlin, Daniela Rago, Aaron John Christian Andersen, Irina Borodina, Engineering of Yarrowia lipolytica for the production of plant triterpenoids: asiatic, madecassic, and arjunolic acids., Metabolic Engineering Communications, 14, e00197, 10.1016/j.mec.2022.e00197

    Several plant triterpenoids have valuable pharmaceutical properties, but their production and usage is limited since extraction from plants can burden natural resources, and result in low yields and purity. Here, we engineered oleaginous yeast Yarrowia lipolytica to produce three valuable plant triterpenoids (asiatic, madecassic, and arjunolic acids) by fermentation. First, we established the recombinant production of precursors, ursolic and oleanolic acids, by expressing plant enzymes in free or fused versions in a Y. lipolytica strain previously optimized for squalene production. Engineered strains produced up to 11.6 mg/g DCW ursolic acid or 10.2 mg/g DCW oleanolic acid. The biosynthetic pathway from ursolic acid was extended by expressing the Centella asiatica cytochrome P450 monoxygenases CaCYP716C11p, CaCYP714E19p, and CaCYP716E41p, resulting in the production of trace amounts of asiatic acid and 0.12 mg/g DCW madecassic acid. Expressing the same C. asiatica cytochromes P450 in oleanolic acid-producing strain resulted in the production of oleanane triterpenoids. Expression of CaCYP716C11p in the oleanolic acid-producing strain yielded 8.9 mg/g DCW maslinic acid. Further expression of a codon-optimized CaCYP714E19p resulted in 4.4 mg/g DCW arjunolic acid. Lastly, arjunolic acid production was increased to 9.1 mg/g DCW by swapping the N-terminal domain of CaCYP714E19p with the N-terminal domain from a Kalopanax septemlobus cytochrome P450. In summary, we have demonstrated the production of asiatic, madecassic, and arjunolic acids in a microbial cell factory. The strains and fermentation processes need to be further improved before the production of these molecules by fermentation can be industrialized.


Consortium

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