Research Projects All Projects
Projects

Quick links

Optimization of novel plant derived enzyme expression in microorganisms for biotechnological application

 

 

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 Nr.: 1.1.1.2/VIAA/2/18/286

Period: 36 month (1st January 2019 – 31 st December 2021)

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.




Mājas lapas izstrādi finansēja ERAF 2.1.1.2. aktivitātes projekts Nr. 2010/0196/2DP/2.1.1.2.0/10/APIA/VIAA/004 "Latvijas biomedicīnas pētījumu integrācija Eiropas zinātnes telpā".