First insight into endolysin mediated lysis in the wine microbiota

Project Information

ENDOWINE

Grant agreement ID: 101154771

DOI

10.3030/101154771

Project closed

EC signature date 14 May 2024

Start date 1 June 2024

End date 31 May 2026

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 195 914,88

Coordinated by

UNIVERSITE DE BORDEAUX
France

Periodic Reporting for period 1 - ENDOWINE (First insight into endolysin mediated lysis in the wine microbiota)

Reporting period: 2024-06-01 to 2026-05-31

Summary of the context and overall objectives of the project

Grapes are the largest fruit crop worldwide. Globally, half of the harvested grapes are turned into wine, and the sector is of great economic importance for the European Union. The transformation of grapes into wine is a multi-step process that relies on specific metabolic processes carried out by the resident microbiota. One widely represented group consists of lactic acid bacteria, which includes various species that are feared for their potential to alter the desired organoleptic qualities of wines. Hence, in some wine types, the development of Oenococcus oeni, Lentilactobacillus hilgardii, and Pediococcus spp. needs to be prevented. Currently, the primary method to combat microbial wine spoilage is through the addition of sulphites. However, given that global warming limits the effectiveness of sulphites, and that the use of this preservative no longer meets consumer expectations, initiatives are being developed to find alternative methods.
In ENDOWINE, we examined the potential of phage endolysins as antimicrobials in wine. Endolysins are phage-encoded enzymes that degrade the peptidoglycan layer of the host bacterial cells at the end of the lytic phage replication cycle, and they have been shown to be bactericidal upon direct exogenous application to bacterial cells. To date, little research has been conducted on the application of endolysins in food products, but they are already used in medical and veterinary settings, demonstrating their potential. In addition, the structurally and functionally related egg lysozyme is already permitted as a food additive.
ENDOWINE focused first on determining the diversity of endolysins in phages infecting wine LAB through in silico analysis. Next, candidate endolysins were selected, cloned, overexpressed, and purified in order to test their enzymatic activity and determine their antimicrobial spectra. Ultimately, the results of ENDOWINE should lead to an application that allows the use of endolysins to target spoilage bacteria in wine.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Through extensive bioinformatics, we have compiled a set of genomes from prophages and strictly lytic phages that infect O. oeni (oenophages). Publicly available L. hilgardii genomes were also analysed to extract the prophage sequences. All viral genomes were screened for putative endolysins. This led to the detection of 206 and 143 genes encoding putative endolysins active against O. oeni and L. hilgardii, respectively.
Remarkably, all oenophage endolysins contained a conserved glycosyl hydrolase family 25 (muramidase) motif as the enzymatically active domain (EAD). Greater diversity was found at the level of their cell-wall binding domains (LysM domains, or CBDs that do not resemble any known CBD at the sequence or structural level), which allowed for division into 3 categories based on domain composition and the presence or absence of a signal peptide.
The diversity of endolysins found in the L. hilgardii genomes was significantly larger. A total of 11 different categories were identified, each with a unique combination of EAD and CBD. The EADs found included glycosyl hydrolase family 25, family 43, family 73, lysozyme-like domains, and amidase domains. The CBDs included DUF5776 and multiple currently unknown domains.
Interestingly, all currently isolated strictly lytic oenophages belonging to the Krappator and Vinitor groups contained a rare Category 3 endolysin (representing only 3% of all endolysins). Therefore, two of these endolysins were selected for cloning, overexpression, and purification. Three endolysins from L. hilgardii prophages were also selected to represent the diversity of endolysins found across all genomes.
The five selected endolysins were successfully cloned. Strong overexpression was achieved for the three L. hilgardii prophage endolysins, but they were found to be insoluble. Expression of the oenophage endolysins was modest, but column purification allowed for protein concentrations of up to 3 mg/mL of purified endolysin.
The activity of the two oenophage endolysins was visualised against autoclaved O. oeni cells. Both endolysins were active against members of all four phylogroups of the species. Their activity did not extend to the tested L. hilgardii or Pediococcus spp. strains. No activity was found in the insoluble fractions containing the L. hilgardii prophage endolysins. Attempts to induce the prophages from their host strains using mitomycin C were unsuccessful, suggesting that the prophages (and their endolysins) may be inactive.
The two oenophage endolysins were incubated for one month in a wine-like medium and were found to retain the same level of activity as those incubated in buffer, indicating that the endolysins are resistant to the harsh environment of wine.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

ENDOWINE has revealed the diversity of endolysins found within phages and prophages associated with wine lactic acid bacteria. The diversity of endolysins in oenophages was found to be relatively small compared to that in prophages from L. hilgardii. However, despite the limited domain-level diversity observed among oenophage endolysins, sequential and structural differences occur within the same category that may influence their activity and specificity. Preliminary data suggest that the spectrum of the endolysins is relatively narrow, allowing targeted control of the species to be eliminated. The data generated by ENDOWINE have contributed to the current knowledge of endolysins, particularly in the context of a fermented beverage. Next, the endolysins will be tested in wine itself to determine their impact on the winemaking process. A possible application could also be of interest for other fermented beverages, such as kefir or kombucha, whose microbiota overlaps with that of wine.