loader image





Funding: European Regional Development Fund (ERDF) “On Implementation of Activity “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: “Combination of system and molecular biology approaches for discovery and characterization of plant enzymes for industrial processes”

Project No.

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

Project costs: 133 806,00 EUR

Project implementer: Dr. Paulius Lukas Tamošiūnas

The color was recognized to be the third most important aspect of food purchase choice, and there is a substantial lack of natural blue color pigments in the market. Several artificial colorants, including Blue No. 1 and Blue No. 2, were suggested to be causing hyperactivity in children and allergic reactions (Kobylewski, S., & Jacobson, M. F. (2010)). In recent years, the market of the food colors industry has rapidly increased and it is expected to continue growing 10% to 15% annually (Institute of Food Technologists. 2016) Therefore there is a considerable commercial interest in achieving strong blue food grade preparations and to improve the stability of anthocyanins such that they can be used as industrially-reliable, stable natural colorants. Anthocyanins have been approved for use in foods, in Europe with the label E163 (Cortez, R., Luna-Vital, D. A., Margulis, D., & Gonzalez de Mejia, E. (2017)).

Of particular importance for the use of anthocyanins as colorants in food, additives are the nature of the side-chain modification. Since glycosylation of C3 occurs on all naturally-accumulating anthocyanins and increases the stability of the pigment. Additionally, the sugar residues of anthocyanins are often acylated with aromatic acids (p-coumaric, caffeic, ferulic, sinapic, gallic or p-hydroxybenzoic acids) or aliphatic acids (malonic, acetic, malic, succinic, tartaric and oxalic acids). Aromatic acylation of anthocyanins increases their stability in solution by promoting self-association and/or the intramolecular stacking of the acyl groups onto the chromophore (intramolecular copigmentation). In some plants, the acyl groups are themselves glycosylated. Some complex anthocyanins have alternating glycosyl and\ acyl groups, which lead to further increased color stability in solution. Aromatic acylation additionally shifts the color of the pigments towards the blue (bathochromic shift in λmax), and some of the most intense blue colors of flowers, such as those found in morning glory and lobelia, are conferred by polyacylated (aromatic) anthocyanins. Anthocyanins having a dihydroxy substitution on their B-ring (cyanidin, delphinidin and petunidin derivatives) bind metal ions such as Fe3+, Mg2+, Ca2+, and Al3+. Finally, co-pigmentation and metal binding can combine to produce stable blue color. In the so-called metalloanthocyanins, three anthocyanin chromophores are bound to the metal ion and three flavone or flavonol molecules are inserted between them by p-stacking interactions.

Information published 02.01.2019.

Progress of the project:

1 January 2019 – 31 March 2019

During the initiation phase of the project current state of the biotechnological production of the anthocyanine and other blue-colored natural colorants was reviewed. A putative metabolic pathway to establish an efficient production of core anthocyanine species in yeast S. cerevisiae was designed with four phenolic acids as possible precursors. The stability, inhibition on the growth of various yeast strains, as well as the uptake to the cell of these four precursors were evaluated in MS, spectroscopical and growth assays. The vectors for recombinant production of plantal origin 4-coumaric acid-CoA ligase, chalcone synthase 1, chalcone isomerase, and dihydroflavonol 4-reductase in yeast were prepared.

Information published 29.03.2019.

Progress of the project:

1 April 2019 – 30 June 2019

The expression of recombinant 4-coumaric acid-CoA ligase, chalcone synthase 1, chalcone isomerase, and dihydroflavonol 4-reductase genes in S. cerevisiae was evaluated and the stability of the proteins was confirmed. The vectors for recombinant production of plantal origin dihydroflavonol 4-reductase and UDP-glucose flavonoid 3-O-glucosyltransferase in yeast were prepared. The platform for multiple gene expression in yeast was designed for metabolic pathway construction in yeast S. cerevisiae.

Information published 28.06.2019.

Progress of the project:

1 July 2019 – 30 September 2019

Overexpression of plant-origin dihydroflavonol 4-reductase and UDP-glucose flavonoid 3-O-glucosyltransferase in yeast was performed and the stability of these enzyme was confirmed. Yeast S. cerevisiae strains coexpressing 4-coumaric acid-CoA ligase and chalcone synthase 1 were created for testing the polycistronic enzyme expression approach. Analysis of potential gene candidates for anthocyanine decoration enzymes (acyl- and glucosyltransferases) was performed.

Information published 30.09.2019.

Progress of the project:

1 October 2019 – 31 December 2019

Analysis of the expression levels of some recombinant enzymes revealed that 4-coumaric acid-CoA ligase 2 and chalcone synthase 1 from N. tabacum, but not A. thaliana, is overexpressed in yeast S. cerevisiae. Analysis of the latest strategies for enhancing the malonyl-CoA pool in the cell, which is a bottleneck for the flavonoid synthesis, was performed. Molecular tools for simultaneous genomic integration were tested in yeast and the integration sites were selected for metabolic engineering. A reportage about the project on national television was prepared.

Information published 30.12.2019.

Progress of the project:

1 January 2020 – 31 March 2020

Yeast strains expressing the plant-origin anthocyanin synthase, flavone 3-hydroxylase, flavonoid 3‘5‘ hydroxylase isoenzymes were engineered and the optimal conditions for the expression of these recombinant proteins were determined. Further work on the establishment of the yeast-based platform for the biochemical screening of the anthocyanin pathway gene variants for optimal metabolic flow was done.

Information published 31.03.2020.

Progress of the project:

1 April 2020 – 30 June 2020

Sequences for yeast episomal complementation expression system were designed and constructed. Expression vectors for a panel of acyl- and glycosil-transferase allels were prepared. Target regions for mutational analysis of genes for anthocyanin synthesis pathway have been identified.

Information published 30.06.2020.

Progress of the project:

1 July 2020 – 30 September 2020

Further work on the establishment of the yeast episomal complementation expression system has been performed, including the screening of genomic integrations.

Information published 30.09.2020.

Progress of the project:

1 October 2020 – 31 December 2020

From 23.10.2020 to 23.12.2020 the researcher spent in Vilnius University Life Sciences Centre. The visit focused in learning the novel CRISPR/Cas9-based yeast genome expression modulation techniques. The expression and stability of new variants of CHI, CHS and GT enzymes were tested.

Information published 30.12.2020.

Progress of the project:

1 January 2021 – 31 March 2021

Yeast strains harbouring episomally expressed and genome-integrated genes for production of naringenin from media-supplemented aromatic acids were produced. Cultivation conditions and strain fitness were investigated. Vectors for dicarboxylic acid plasma membrane transporters form Schizosaccharomyces pombe and Fusarium graminearum were prodused in order to increase the citoplasmic pool of precursor malonate.

Information published 31.03.2021.

Progress of the project:

1 April 2021 – 30 June 2021

A collection of yeast strains, harbouring the genome-integrated anthocyanin pathway  genes and the complementing episomal alleles for each of 9 genes was created. Cultivation conditions for these new strains were incestigated. SOPs for the modular construction of multiple gene expression panel, rapid protein expression screening and yeast cultivation were prepared.

Information published 30.06.2021.

Progress of the project:

1 July 2021 – 30 September 2021

Aga2-fused Vaccinium corymbosum and Lupinus angustifolius anthocyanidin 3-O-glucoside-5-O-glucosyltransferase (Vc5OGT, Lang5OGT), L. angustifolius anthocyanidin 3-O-glucoside-6’’-O-coumaroyltransferase (Lang6OCT) and L. albus anthocyanidin 3-O-glucoside-6’’-O-malonyltransferase (Lalb6OMT)  proteins were successfully synthesised in yeast Saccharomyces cerevisiae EBY100 strain and were shown to be exposed to the cell surface. In comparison, the synthesis of only Vc5OGT and Lang6OCT were successful as soluble N’-polyhistidine tagged proteins in S. cerevisiae. Preliminary tests of whole-cell incubation with anthocyanin extract were performed.

 Information published 30.09.2021.

Progress of the project:

1 October 2021 – 31 December 2021

From 16.6.2021 to 31.10.2020 the researcher spent in Vilnius University Life Sciences Centre, where yeast strains for anthocyanin production and modification were characterized. Poster presentation “Evaluation of Yeast Surface Display System for Synthesis of Anthocyanin-Decorating Enzymes” was given on November 2-4, 2021 at the PEGS Europe conference. In silico modelling of activity-enhancing point mutations of Pa.UFGT and Vc.DFR was performed (WP3) and corresponding primers for mutagenesis were designed.

Information published 30.12.2021.

Progress of the project:

1 January 2022 – 31 March 2022

Synthesis of Vc.5OGT, Lang.5OGT, Lang.6OCT, and Lalb.6OMT enzymes were obtained as C’-polyhistidine tagged proteins in S. cerevisiae.  Expression of flavonoid 3′,5′-hydroxylases from Vaccinium corymbosum, Ribes rubrum, and Ribes alpinum was achieved as membrane-bound as C’-polyhistidine tagged proteins. Bioconversion medium-suplemented coumaric acid to naringenin by yeast strains expressing Nt.4CL2, Pa.CHS, and Ath.CHI was achieved and quantified by LC-MS.

Information published 31.03.2022.

Progress of the project:

1 April 2022 – 30 June 2022

Two manuscripts „Yeast-based system for in vivo evaluation of alleles of the anthocyanin production pathway” and „Yeast-expressed acyl and glycosyltransferases for anthocyanin stabilization in fruit juice” were prepared and submitted. The project results disseminated as poster presentations at FEBS3+ (Tallinn) and FEMS (Belgrade) conferences. Educational material of practical work for school children was prepared as a part of outreach activities (https://biomed.lu.lv/jaunumi/laboratorijas-darbu-materiali-skolam/ ).

Information published 30.06.2022.