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Project Title: „The development of an efficient pilot-scale leghemoglobine production technology, based on recombinant Pichia pastoris and Kluyveromyceslactis fed-batch fermentations (BioHeme)”

Funding: European Regional Development Fund (ERDF), Measure “Support for applied research”

Project No.:

Period: 1 January 2022 – 30 November 2023

Project costs: 540 540,00 EUR

Project implementer: Latvian State Institute of Wood Chemistry

Cooperation partner: Latvian Biomedical Research and Study Centre

Principle Investigator BMC: Dr.biol. A. Kazāks

Project summary:

An alternative to livestock production can be plant-based meat. Plant-based meat is defined as a meat-like substance made from vegetarian friendly ingredients. Plant-based meat reproduces the flavour and texture of real meat without using products of animal origin. In plant-based meats, the leghemoglobin protein (LegH) is essential to comprise the unique flavour and aroma of traditional meat. As of now, the most perspective process of manufacturing LegH in appropriate amounts, is through fermentation using recombinant microorganism strains defined as methylotrophic yeast (for example, Pichiapastoris), which utilize methanol as substrate, and in their recombinant form can produce LegH via methanol-inducible promoter element. The mentioned research is driven by two problems, which are: (1) the use of volatile and flammable substances during the production process and (2) relatively high cost of applicable substrates. One of biological platforms, which was not studied earlier for the production of LegH and can potentially outperform P.pastoris is Kluyveromyces lactis. The mentioned organism can grow on cheap substrates, such as cheese whey, and does not require the use of methanol for protein synthesis induction. Therefore, the objective of the BioHeme project: is to develop effective fed-batch fermentation and scale-up techniques for the production of plant-based meat substitutes, utilizing a recombinant LegH producing strain of Pichia pastoris and Kluyveromyceslactis. The main activities of the BioHeme project are: (i) obtainment of recombinant (LegH producing) strains of P. Pastoris and K. lactis, with a subsequent evaluation of heme production efficiency and technological potential, (ii) optimization of fermentation medium composition for biomass growth and hemeprotein production phases for the two mentioned recombinant strains, (iii) the application of Model-Predictive control systems for the control of biomass growth rates and hemeprotein synthesis, (iv) fermentation process transfer to pilot scale and evaluation of applications sustainability.

Information published 03.01.2022.

Progress of the project:

1 January 2022 – 31 March 2022

We have designed genes and constructed appropriate vectors for expression of soy leghemoglobin (LegHb) intracellularly and secretory in yeast P. pastoris and K. lactis systems. In case of K. lactis no detectable LegHb synthesis has been demonstrated in both expression variants. In contrast, for P. pastoris well-detectable high level protein synthesis was observed intracellularly.  However, attempts to improve expression by lowering temperature, addition of iron ions and increasing cultivation temperature did not give expected results. In addition, for P. pastoris secretion of LegHb was also detected, but expression medium dis not have characteristic reddish brown color. It has been decided that secreted protein does not contain heme group and therefore is not useful for set goals. We consider selection of other alternative yeast hosts as S. cerevisiae in nearest future.

Information published 31.03.2022.

Progress of the project:

1 April 2022 – 30 June 2022

We have developed fast and efficient purification method for soy leghemoglobin (LegHb) from Pichia pastoris cells. It was detected that in current conditions ~1 mg LegHb can be obtained from 1 g wet cells. This amount could be estimated as medium high.

We continued attempts to obtain LegHb synthesis in yeast Kluyveromyces lactis. It was believed that in these cells production can not be detected, but performing purification by developed method a small amount of target protein was recognized. Outcome, however, is significantly lower than in P. pastoris.

As alternative to K. lactis, novel genes were designed and vectors constructed for intracellular expression of LegHb in yeasts Saccharomyces cerevisiae and Hansenula polymorpha. Currently no LegHb synthesis has been confirmed in both cases. A further optimization of expression conditions is in progress.

Information published 30.06.2022.

Progress of the project

1 July 2022 – 30 September 2022

We assessed synthesis of soy leghemoglobin (LegHb) in yeasts S. cerevisiae and H. polymorpha. In S. cerevisiae production is detectable although significantly lesser than in P. pastoris. In H. polymorpha, no synthesis of LegHb is detectable. Therefore P. pastoris stands out as a more perspective yeast for LegHb synthesis, and further optimization will be performed in these cells. Lowering cultivation temperature to 24°C did not result in positive effect. Cultivation in presence of glycine led to decrease of specific product as well. We have compared LegHb synthesis in fermentor using rich medium and minimal medium with trace salt additives (BSM). In BSM production was higher, therefore further experiments with addition of different components will be assessed in minimal medium. We have performed further optimization of LegHb purification to obtain product with at least 90% purity which is essential for precise quantification.

Information published 30.09.2022.

Progress of the project

1 October 2022 – 31 December 2022

We performed optimization of purification for soy leghemoglobin (LegH) to obtain product of more than 90% purity. This allows accurate comparison of LegH production in different cultivation processes. A set of fermentations were performed in different minimal media and outcome of LegH was compared 48 hours post methanol induction. The highest level of synthesis was reached in BSM medium. In this medium we performed also a study of LegH synthesis kinetics. Maximum of LegH synthesis was reached 48-72h post methanol induction. These conditions will be set up for next fermentations with optimization of growth conditions.

Information published 30.12.2022.

Progress of the project

1 January 2023 – 31 March 2023

During this time period we have compilated obtained data regarding LegH expression in P. pastoris using 5 L table-top fermenter. A first paper draft was prepared which is planned to be complemented and finalized in next quarter. This paper summarizes LegH expression, purification and outcome in different minimal media as well as in rich medium. We have compared LegH outcome in rich medium when cultivated in flasks and fermenter and found out that in last case it was significantly higher. Acquired data provide an insight and directions on LegH obtainment at preparative amounts in bioreactor.

Information published 31.03.2023.

Progress of the project:

1 April 2022 – 30 June 2022

We have compilated data from previous bioreactor cultivation experiments. Mathematical model was developed describing accumulation dynamics of yeast P. pastoris biomass, substrate, reactor volume and specific product during cultivation process. Based on developed model we initiated development of substrate feeding speed control program in MATLAB medium, with an aim to optimize methanol supply profile for cultivation process to obtain maximum of LegH outcome. The work on scientific publication draft has been continued.  Several experiments were performed to characterize and quantify secretory LegH protein.

Information published 31.03.2022.

Progress of the project

1 July 2023 – 30 September 2023

It was continued work on mathematic model of cultivation process to select optimal model parameters. Operation of mathematic model was verified in real cultivation process, by modelling yeast P. pastoris biomass, substrate, reactor volume and product synthesis dynamics. PID control program has been developed to control methanol feeding rate to ensure cell growth with selected specific growth speed (µ-stat). It was initiated work on artificial neural networks-based process model development using data accumulated in previous cultivation processes. We continued work on paper on LegH synthesis in laboratory-scale bioreactor in different media.

Information published 02.10.2023.

Progress of the project

1 October 2023 – 30 November 2023

During the final two months of the project, data were collected on LegH production in a laboratory-scale bioreactor in different media. The yield of LegH was compared and it was concluded that the highest level of synthesis can be achieved in the BMGY rich medium (1.77 mg/g WCW) and in the BSM minimal medium (1.56 mg/g WCW). A series of experiments carried out during the project with the aim of increasing LegH expression by varying the culture conditions were not successful. Apparently, the acquisition of functional LegH is limited by such intracellular processes as the biosynthesis of the heme group, which are practically unaffected by the expression conditions. From the data of the bioreactor cultivation process, a mathematical sensor based on artificial neural networks was created for determining the wet biomass of cells, depending on the standard bioreactor data – stirrer speed (rpm), dissolved oxygen concentration (%), inlet air enrichment with oxygen (%), amount of supplied base and substrates (glycerol, methanol) (mL), and culture volume (L). The calculated WCW values of the developed sensor showed a good correlation with the experimental data of the processes with an average error value of 3.72 %. The obtained results were processed and published in the open access journal Processes (IF 3.5).

Processes 2023, 11(11), 3215; https://doi.org/10.3390/pr11113215

Information published 30.11.2023.