Funding: European Regional Development Fund (ERDF) “On Implementation of Activity 1.1.1.2 “Post-doctoral Research Aid” of the Specific Aid Objective 1.1.1 “To increase the research and innovative capacity of scientific institutions of Latvia and the ability to attract external financing, investing in human resources and infrastructure” of the Operational Programme “Growth and Employment”
Project Title: “Optimization of novel plant derived enzyme expression in microorganisms for biotechnological application”
Project No.: 1.1.1.2/VIAA/2/18/286
Period: 1st January 2019 – 31st December 2022
Project costs: 133 806,00 EUR
Project implementer: Dr.PhD. Kaia Kukk
Based on the current genetic, phytochemical and food processing knowledge on anthocyanins, project will apply and validate the following strategies for production of natural colorants. Genes encoding proteins from the anthocyanin synthesis pathway derived from the species Lupinus angustifolius (narrow-leaved blue lupine) with a spectrum of blue anthocyanins will be explored. The genes will be expressed in different yeasts (Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Kluyveromyces lactis) to identify the most suitable host for heterologous expression. Both S. cerevisae and P. pastoris are established host systems, which allow cell-surface protein display for rapid enzyme characterization, omitting the costly protein purification procedures. The recombinant yeast cells or purified proteins will be used for biosynthesis of anthocyanins. Enzymatic activity of the recombinant proteins will be tested with individual substrates and their natural mixtures (deproteinated plant extracts) used in industry (for the list of suppliers, refer to). The product formation and their stability will be monitored using spectrophotometric, thin-layer chromatography and HPLC methods.
The current project is a complex study for improving the competitiveness of biosynthesis of anthocyanins in microorganisms compared to extraction from plants. The approach is to screen several yeasts for the expression of enzymes from the anthocyanin synthesis pathway and to apply cell surface display to generating whole cell biocatalysts. The know-how generated during implementation of the project will be valuable for relevant industries.
Information published 02.01.2019.
Progress of the project:
1 January 2019 – 31 March 2019
The first months were spent on carrying out extensive literature review and settling into the new work environment. Arabidopsis thaliana and Nicotiana tabacum leaves were subjected to RNA extraction and complementary DNA (cDNA) synthesis. Specific primers were designed and successfully used for amplifying the sequence of 4-coumarate-CoA ligase (one of the enzymes from the early steps of the anthocyanin synthesis pathway) from A. thaliana and N. tabacum cDNA. Several attempts to extract RNA from freeze-dried berries of Vaccinium myrtillus and Vaccinium vitis-idaea were made. Meetings with potential future collaborators at University of Latvia and BIOR were attended.
Information published 29.03.2019.
Progress of the project:
1 April 2019 – 30 June 2019
Both sunflower (Helianthus annuus) and calendula (Calendula officinalis) plants were grown in the laboratory. Leaves from young plants were subjected to RNA extraction and cDNA synthesis. Degenerate primers were designed and successfully used for amplification of fragments of 4-coumarate-CoA ligase, chalcone synthase and flavanone 3-hydroxylase from both plants. Specific primers were designed and successfully used for amplifying chalcone isomerase from Arabidopsis thaliana and Nicotiana tabacum leaves.
Information published 28.06.2019.
Progress of the project:
1 July 2019 – 30 September 2019
Plant samples were collected from July to September and properly stored for further experiments. RNA was extracted from berries of three Vaccinium species (V. uliginosum, V. myrtillus and V. vitis-idaea) and petals of four flowers from the family of Asteraceae (Centaurea cyanus, Cichorium intybus, Calendula officinalis and Helianthus annuus). The RNAs were used for cDNA synthesis. Sequence fragments of three to six proteins (4-coumarate-CoA ligase, chalcone synthase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, anthocyanidin synthase and flavanoid 3’, 5’-hydroxylase) from five plants were obtained.
Information published 30.09.2019.
Progress of the project:
1 October 2019 – 31 December 2019
The work continued with sequencing the fragments encoding the proteins of the anthocyanin synthesis pathway. Specific primers were designed and used for obtaining 3’ sequences of six proteins from four plants having blue flowers or berries (Centaurea cyanus, Cichorium intybus, Vaccinium uliginosum and Vaccinium myrtillus). Preliminary experiments for obtaining 5’ sequences were carried out. Specific primers were designed and successfully used for amplifying chalcone isomerase from strawberry (Fragaria ananassa). The work was introduced to a wide audience in the TV show “Izziņas impulss” at LTV1 (05.12.2019).
Information published 30.12.2019.
Progress of the project:
1 January 2020 – 29 February 2020
The work focused on amplifying 5’ sequences of proteins of the anthocyanin synthesis pathway. Several potential full-length sequences were obtained through combining the 5’ fragments with the previously caught 3’ fragments (mostly dihydroflavonol 4-reductase and anthocyanidin synthase sequences). Specific primers and RNA extracted from cornflower (Centaurea cyanus) leaf were used for obtaining full-length sequence encoding chalcone synthase. A detailed protocol “How to get sequences encoding proteins of plants that have no genome data available” was written
Information published 28.02.2020.
Progress of the project:
1 March 2021 – 31 May 2021
Synthetic sequences of predicted anthocyanin 3-O-glucoside 5-O-glucosyltransferase from Vaccinium corymbosum and Lupinus angustifolius, anthocyanin 3-O-glucoside-6”-O-coumaroyltransferase from Lupinus angustifolius and anthocyanin 3-O-glucoside-6”-O-malonyltransferase from Lupinus albus were ordered and used for transforming Saccharomyces cerevisiae. First part of the sequencing data of the project was published with the DOI 10.15156/BIO/807453. Protocol describing getting sequences encoding proteins of plants that have no genome data available was improved and published at protocols.io (dx.doi.org/10.17504/protocols.io.btxwnppe).
Information published 31.05.2021.
Progress of the project:
1 June 2021 – 31 August 2021
Synthetic sequences of predicted anthocyanin 3-O-glucoside 5-O-glucosyltransferase, anthocyanin 3-O-glucoside-6”-O-coumaroyltransferase and anthocyanin 3-O-glucoside-6”-O-malonyltransferase from Lupinus and Vaccinium species were used for transforming the yeast Pichia pastoris. The proteins were expressed in S. cerevisiae and P. pastoris. Several full-length sequences encoding proteins from the anthocyanin synthesis pathway were obtained and deposited in GenBank.
Information published 31.08.2021.
Progress of the project:
1 September 2021 – 30 November 2021
The project was introduced to non-scientists at the Long night of research. Virtual poster presentation was made at the 13th annual PEGS Europe conference. A four-day FEBS advanced course in Computational approaches to understanding and engineering enzyme catalysis was attended. The work continued with preparations for expressing anthocyanin decorating enzymes in the yeast Kluyveromyces marxianus.
Information published 30.11.2021.
Progress of the project:
1 December 2021 – 28 February 2022
Different induction methods and affinity tag locations were evaluated for expressing anthocyanin decorating enzymes in the yeast Pichia pastoris. Codon-optimized sequences of the proteins were ordered to test the yeast Rhodosporidium toruloides as an expression host. Golden Gate assembly was used to construct expression vectors for Kluyveromyces marxianus.
Information published 28.02.2022.
Progress of the project:
1 March 2022 – 31 May 2022
Golden Gate assembly was used to construct expression vectors for R. toruloides. First attempts to transform R. toruloides were made. The sequences of flavonoid 3′, 5′-hydroxylases (F3’5’H) from three Vaccinium species were fused with an N- or C-terminal polyhistidine-tag and expressed in P. pastoris. Structural models of Vaccinium F3’5’H were predicted and docked with ligands. Several expression promoters were amplified from genomic DNA of K. marxianus.
Information published 31.05.2022.
Progress of the project:
1 June 2022 – 31 August 2022
Truncated versions of Vaccinium F3’5’H proteins were created according to the predicted structural models. These truncated F3’5’Hs were then expressed in the yeast P. pastoris. As F3’5’Hs require cytochrome P450 reductase (CPR) for catalytic activity, a homology search was conducted to identify potential CPRs from available plant material. Three predicted CPR sequences were successfully amplified from the cDNAs of sunflower (Helianthus annuus) and bilberry (Vaccinium myrtillus). Co-expression experiments will follow.
Information published 31.08.2022.
Progress of the project:
1 September 2022 – 30 November 2022
Co-expression strains of P. pastoris were created that expressed both Vaccinium F3’5’Hs and CPRs. Several medium supplements were tested for increasing the production of F3’5’Hs. Activity assays of F3’5’Hs were started. Protocol describing construction of an P. pastoris expression vector encoding hygromycin B resistance marker was prepared for publishing at protocols.io. Expression experiments with K. marxianus were finished.
Information published 30.11.2022.
Progress of the project:
1 December 2022 – 31 December 2022
Protein expression experiments using the yeast Rhodosporidium toruloides were finished. Standard operation protocols, popular science texts and manuscripts of articles were written.
Information published 30.12.2022.