New Genomic Techniques applied to food cultures: a powerful contribution to innovative, safe, and sustainable food products

Abstract Nontransgenic New Genomic Techniques (NGTs) have emerged as a promising tool for food industries, allowing food cultures to contribute to an innovative, safe, and more sustainable food system. NGTs have the potential to be applied to microorganisms, delivering on challenging performance traits like texture, flavour, and an increase of nutritional value. This paper brings insights on how nontransgenic NGTs applied to food cultures could be beneficial to the sector, enabling food industries to generate innovative, safe, and sustainable products for European consumers. Microorganisms derived from NGTs have the potentials of becoming an important contribution to achieve the ambitious targets set by the European ‘Green Deal’ and ‘Farm to Fork’ policies. To encourage the development of NGT-derived microorganisms, the current EU regulatory framework should be adapted. These technologies allow the introduction of a precise, minimal DNA modification in microbial genomes resulting in optimized products carrying features that could also be achieved by spontaneous natural genetic evolution. The possibility to use NGTs as a tool to improve food safety, sustainability, and quality is the bottleneck in food culture developments, as it currently relies on lengthy natural evolution strategies or on untargeted random mutagenesis.


Introduction
The European 'Green Deal' and 'Farm to Fork' policies aim to make food systems more sustainable, contributing to carbon neutrality and circular economy.This involves significant adv ancements in a gricultur al and food pr oduction to addr ess climate change, sustainable production demands, decarbonization, and reducing food waste .T he increasing market requirements for food diversity , safety , and sustainability are driving the food industry to innovate rapidly, especially in developing plantbased alternatives like dairy and meat substitutes, which must meet high standards in texture , fla vour, and nutritional value (Elhalis et al. 2023 ).
Food cultures as described in this paper are used in food processing and/or as food ingredients, as defined in the Article 2 of Regulation (EC) No 1169/2011 (e.g.dairy products, meats, be v era ges, br eads, v egetables and other plant-based products).They contribute to se v er al food pr operties, suc h as flavour, textur e, digestibility , microbial quality , pr eserv ation/extension of shelf life, nutritional, and health benefits as probiotics .T hrough fermentation, micr obial cultur es play a k e y role in food production.Fer-mentation utilizes the growth and metabolic activities of microorganisms to transform food materials (Terefe 2016 ).Food cultures are defined by the European Food & Fermentation Cultures Association ( EFFCA ) as live bacteria, yeasts, or filamentous fungi (moulds) used in food pr oduction wher e they are food ingredients.They usually have minimum viable cells count of 10 8 CFU/g or ml for bacteria and yeasts, and 10 7 CFU/g for filamentous fungi.Food cultur e pr epar ations can be composed of one or se v er al micr oorganism species with or without specific components (e.g.organic acids , minerals , vitamins) required for their survival, storage, or to facilitate their application in the final food.
Early Summer 2023, the European Commission issued a regulatory proposal on plants obtained by certain New Genomic Techniques (NGTs) and their food and feed.This proposal distinguishes some of these tec hniques fr om geneticall y modified or ganisms (GMOs) obtained by transgenesis and facilitates their placing on the market when they could also occur natur all y or be pr oduced by conv entional br eeding tec hniques .T his proposal does not appl y to deliber atel y r eleased micr oor ganisms, including food cultures.As confirmed by the EU Court of Justice ruling from 2018, most if not all micr oor ganisms obtained through the use of NGT would still classify as GMO as defined by Dir ectiv e 2001/18/EC .
The current EU regulatory framework, which analytically struggles to differentiate between products of classical techniques and of NGTs, places the EU food biotechnology industry at a disadv anta ge, especiall y compar ed to countries with more facilitating regulations.An adapted, more flexible regulatory framework for NGTs could significantly benefit the consumer, industry, and align with the EU policy ambitions.This paper will demonstrate how NGTs applied to microorganisms could be beneficial to the sector, allowing food cultures to contribute to an innov ativ e, safe, and sustainable food system, providing similar attempt for modernization of the legislation as for plants.

The need to expand our toolbox beyond natural evolution of microbial genomes
Natural selection and laboratory evolution techniques have been used for many decades to improve microbial strains for use as food cultur es, tar geting tr aits like acid toler ance in yogurt and metabolite production for texture and aroma in food (Johansen 2018 ).Streptococcus thermophilus , a k e y or ganism in man y dairy pr oducts, has under gone substantial genetic adaptation for milkbased gr owth, e videnced by major metabolic changes and gene tr ansfer r elated to str ess toler ance, exopol ysacc harides pr oduction, and bacteriophage immunity (Hols et al. 2005, Eng et al. 2011 ).
Str ain exposur e to infection by bacteriopha ges leading to CRISPR (Cluster ed Regularl y Interspaced Short P alindr omic Repeats)-based immunization of the surviving strains against those phages is an even more active form of microbial genomic e volution (Barr angou et al. 2007 ).Barr angou et al. discov er ed that bacterial genomes pick up sequences over time from phages to which they were exposed, passing on these so-called CRISPR spacers to subsequent generations, then using these spacer sequences to recognize viruses that later invade their cells.The immediate pr actical a pplication of this discov ery w as the av oidance of fermented food spoilage from phage infection.In addition, this discovery laid the foundation for the discovery of the CRISPR-Cas toolbox for genome editing (see NGTs available for deployment).
Beyond these, str ain impr ov ement tec hniques include natur al competence pr ocesses like tr ansformation and conjugation.Natur al competence, contr olled by a pher omone quorum-sensing system (Fontaine et al. 2010 ) allows for exchange of genetic material between related strains based on sufficient homology of the DNA being exchanged for recombination to occur, i.e. homologous recombination.Although this technique is more efficient than natural selection, it tends to require extensive screening after transformation to make sure that only the desired allele is exchanged between strains.
Tec hniques that r el y on natur al competence, e v en when performed in the laboratory, can lead to strain impro vements .Such impr ov ements ar e not classified as genetic modifications under Dir ectiv e 2001/18/EC on the deliberate release of GMOs into the en vironment.T his applies as long as these techniques do not involve the use of recombinant nucleic acid molecules or genetically modified organisms, except for those methods excluded by Annex IB of the Dir ectiv e, like r andom m uta genesis.
Natur al selection, ada ptiv e e volution, and tec hniques based on natur al competence ar e tec hniques of str ain impr ov ement that explore and deploy natural variation.Other techniques that do gener ate ne w genetic v ariation ar e also av ailable, suc h as r andom m uta genesis.Random m uta genesis depends on UV irr adiation or m uta genic c hemicals to induce m utations in the bacterial genome, follo w ed b y screening for the desired phenotype and/or genotype.As this method generates new genetic variation, it is considered a method of genetic modification.Ho w ever, it is listed in Annex IB of Dir ectiv e 2001/18/EC as a method of genetic modification used before 2001 that is exempted from GMO requirements and labelling, as it does not involve the insertion of nucleic acids.
To date, food industries have screened and selected in the natur al envir onment the pr oper micr obial candidates to fulfill market demands.When necessary, natur al str ain selection strategies hav e successfull y been a pplied to deliv er on impr ov ement of performance traits and safety standards (Derkx et al. 2014 ).Howe v er, the constantl y incr easing market r equir ements necessitate a br oad div ersification of foods due to specific dietary r equir ements, sustainability challenges, and higher safety standards, seeking effective solutions in a shorter time frame, all while maintaining high demands on product performance of the solutions provided by the food industries.Natural selection and classical impr ov ement of micr oor ganisms (e.g.r andom m uta genesis, hybridization, ada ptiv e labor atory e volution) r equir e laborious pr ocedur es and long time, particularly when a combination of multiple traits needs to be obtained from the product solution.To ac hie v e the ambitious market demands, the industry needs to accelerate their innov ation and br oaden the scope of their a pplication.To enable operators in the EU's food sectors to meet this challenge, it will be necessary to equip them with modern tools.
Tar geted m uta genesis is essentiall y a m uc h mor e pr ecise method of m uta genesis by which specific target nucleotides are deleted or altered using site-directed nuclease (SDN) enzymes and sophisticated repair mechanisms.Collectively, these SDN techniques are also referred to as NGTs, and their applications are discussed in the 'Application examples of nontransgenic NGTs' section.Nontr ansgenic NGTs a pplied to micr oor ganisms ar e among the solutions to ac hie v e these ambitious goals .T hese inno v ativ e techniques allow to introduce a precise, minimal DNA modification in microbial genomes, resulting in optimized products carrying features that could also be ac hie v ed by spontaneous natural genetic evolution, without insertion of heterologous material.
Due to the near identity in the genetic information between nontransgenic NGTs and classical improvement techniques (current and future), analytical methods may not be able to distinguish between the food cultures obtained by different techniques.This implies that the current technique-based GMO regulatory fr ame work is not fit-for-purpose, and it is prone to noncompliance especiall y fr om imports fr om third countries, wher e certain NGT pr oducts benefit fr om a facilitating r egulatory fr ame work for marketing (e.g.USA, Canada, Austr alia, Isr ael and India, wher eas in se v er al other countries, a case-by-case a ppr oac h is implemented).This places the EU food biotechnology industry at a competitive disadv anta ge.
The current EU regulatory framework discourages the development of micr oor ganisms deriv ed fr om NGTs and pr e v ents consumers from benefitting from these products.NGTs bring opportunities to pr ecisel y alter the genetic material of micr oor ganisms, allowing the r a pid de v elopment of ne w micr oor ganisms to face continuousl y e volving market needs and to satisfy the incr easing variability of customers' demands .Ha ving an adapted and facilitating r egulatory fr ame work for NGTs on micr oor ganisms w ould allo w the industry to contribute efficiently to EU policy ambitions.

NGTs available for deployment
In this section, we put the spotlight on the possibility to perform nontr ansgenic pr ecision genome editing in micr oor ganisms via NGTs, suc h as r ecombineering and CRISPR-Cas-mediated editing systems .T hese techniques have significantly expanded the genetic toolbox, allowing to pr ecisel y make genetic c hanges, suc h as point mutations in specific positions of bacterial genomes, without leaving any trace of exogenous DNA.
Recombineering (recombination-mediated genetic engineering) is a genome editing system mediated by bacteriophageencoded recombinase machineries ( λ-Red and RecET).The technique allows for the direct integration of linear DNA fragments (either single-stranded or double-stranded), containing the desired mutation(s) flanked by homologous regions of the target DNA, into a microbial cell expressing the phage-derived recombination enzymes .T hese enzymes recombine the linear DNA at the target site, introducing the designed mutation into the microbial genome (Fig. 1 ).
The dsDNA recombineering approach has been exploited to gener ate str ains carrying clean modifications, such as deletions, insertions, or replacements of large gene fragment, while ssDNA recombineering enables the engineering of point mutations in the genome without the use of selectable markers .T he latter technique has been successfully established in several lactic acid bacteria (LAB) species, such as Lactococcus lactis , Limosilactobacillus reuteri , and Lac.gasseri (van Pijkeren and Britton 2012 ).Recombineering systems need to be optimized for eac h LAB str ain to r eac h r ecombination fr equencies sufficientl y high for the selection of mutant cells without selective pressure.Interestingly, ssDNA recombineering has been successfully combined with CRISPR-Cas to simplify the selection of recombinant cells (van Pijkeren and Britton 2014 ).
CRISPR-Cas systems are widespread in bacteria and constitute their native adaptive immune system, providing defence a gainst for eign nucleic acids, suc h as plasmids or DNA fr om viruses (Barangou et al. 2007, Bahya et al. 2011 ).The molecular F igure 2. Workflo w for r epr ogr amming endogenous CRISPR-Cas system to perform precise genome editing.Created with Biorender.com.mechanism of adaptive immunity is a phenomenon that was first experimentally confirmed in S. thermophilus by Danisco molecular biologists working with cheese and yoghurt cultures, who found that CRISPR sequences (bacteriophage DNA sequences inserted as DNA spacers separated by short palindromic repeats and grouped into clusters in intergenic regions) are naturally incor por ated into the bacterial genome of phage-infected surviving strains and, together with Cas proteins , pro vide acquired r esistance a gainst viruses .T he bacterial CRISPR spacers create a permanent record of phages against which the bacteria have mounted defences.Since the early production of cheese and yoghurt, people across the globe have been consuming these products made with starter cultures immune to phage infection due to this natural mechanism.
T he disco v ery of the r ole of CRISPR in imm unity to pha ges ultimately led to the de v elopment of CRISPR-Cas-based genome editing b y resear ch groups at UC Berk ele y and the Broad Institute at Harvard.CRISPR-Cas systems have since then been perfected and have become one of the most promising NGTs to acceler ate pr ecise and marker-fr ee genetic impr ov ement not onl y of prokaryotes but also of fungi, plants, and animals (Barrangou and Horvath 2017 ).For example, by utilizing the Cas9 enzyme and a guide RN A, resear chers can target specific genomic loci and intr oduce pr ecise point m utations (Komor et al. 2016 ) .Endogenous CRISPR-Cas systems have been repurposed in LAB to introduce point mutations at a specific position of the genome in a precise, pr ogr ammable, and efficient manner (Hidalgo-Cantabrana et al. 2019 ).Minimal CRISPR arrays with self-targeting DNA fragments could be engineered and delivered to the native host in combination with a mutated repair template, resulting in accurate genome editing (Fig. 2 ).This process simply exploits the natural ability of bacteria to over come DN A damage b y homology-directed repair with the provided DNA template (Fig. 2 ).The strain development is ther efor e ac hie v ed by the tr ansient deliv ery of a plasmid that is lost from the cell after the editing (Fig. 2 ).

Application examples of nontransgenic NGTs
Research in food applications is looking for optimized safe food cultures not carrying undesirable traits (e.g.antibiotic resistance, production of d -lactic acid), and/or resulting in food products with impr ov ed nutritional v alue (e.g. higher vitamin content), incr eased or ganoleptic and rheological pr operties as well as extended shelf life.Microbial genetic improvement is the bottleneck in food cultur e de v elopments, as it curr entl y r elies on lengthy natural genetic evolution strategies or on untargeted random mutagenesis.In this scenario, precise microbial genome editing by nontransgenic NGTs may be beneficial to food a pplications, de v eloping strains that could enhance food safety , sustainability , and quality (Fig. 3 ).Nontransgenic NGTs are no w adays exploited as research tools to gain insights into microbial pathways and understand the function of pr edicted genes, r educing the screening burden for the identification of impr ov ed str ains after natur al e volution or r andom m uta genesis campaigns (Derkx et al. 2014 ).These tec hniques hav e e volv ed fr om nativ e micr obial mac hineries and hav e the potentiality to design tailored food cultures having the desired properties with a targeted minimal DNA modification.
The endogenous CRISPR-Cas system was successfull y r epr ogrammed in the probiotic Bifidobacterium animalis subsp.lactis to inactivate the tetW gene encoding resistance to tetracycline (Pan et al. 2022 ).Mor eov er, a tar geted ssDNA r ecombineering mac hinery was de v eloped and tr ansientl y expr essed in a Limosilactobacillus reuteri candidate probiotic strain to create a vancomycinsensitiv e v ariant (v an Pijker en and Britton 2012 ).Importantl y, the introduction of a single amino acid change in the enzymatic target of the antibiotic, the D-ala D-ala ligase, was sufficient to lower the minim um inhibitory concentr ation for v ancomycin fr om > 256 to 1.5 μg/ml, w ell belo w the clinically relevant threshold.The inactivation of genes coding for antibiotic resistance in LAB transiting or colonizing the human gut would be pivotal to pr e v ent the spread of suc h r esistances to intestinal pathobionts, r educing the risk for the insurgence of multidrug-resistant bacteria (Fig. 3 A).This has important implications not only for probiotics, but also for micr oor ganisms used as starter cultures or cultures with protectiv e effects.Nontr ansgenic NGTs may also be exploited to r edir ect LAB metabolic fluxes to w ar ds the pr oduction of high added-v alue molecules.Lactic acid is the primary product of LAB carbohydrate metabolism and is the most valuable product in the food industry, having also important applications in cosmetic, pharmaceutical, and a gricultur al sectors (Castillo Martinez et al. 2013 ) Nontransgenic NGTs can also be used on yeasts to reduce the production of undesirable or toxic substances.Ethyl carbamate may be produced during fermentation of alcoholic be v era ges, fr om the r eaction of ethanol with ur ea, r aising health concerns in humans.CRISPR-Cas system has been used to disrupt the CAR1 gene encoding arginase of the w ell-kno wn Sacc harom yces cerevisiae yeast, to decrease the formation of this undesirable substance during ethanolic fermentation (Chin et al. 2021 ).A double deletion of CAR1 and GZF3 using the same technique allo w ed to r educe e v en mor e the content of ethyl carbamate (Jung et al. 2022 ).
The food industry is facing incr easing pr essur e to cr eate pr oducts with longer shelf life .T his leads to a greater emphasis on utilizing natural microbial processes instead of relying on chemical ad diti ves .For instance , diacetyl is a by-product of fermentation by many LAB strains, responsible for the buttery flavour in many dairy pr oducts, whic h was r ecentl y shown to r educe food spoila ge by its intrinsic antifungal activity (Shi and Maktabdar 2022 ).Redirecting metabolic fluxes to w ar ds the synthesis of higher amount of diacetyl r epr esents an important strategy to contribute to the reduction of food waste .T his could be ac hie v ed by inserting precise point m utations, thr ough nontr ansgenic NGTs, in specific genes belonging to the glucose metabolic pathway (Fig. 3 C).An engineered Lac.lactis strain overproducing diacetyl was developed by sim ultaneousl y inactiv ating the aldB gene, to pr e v ent the enzymatic conversion of the diacetyl precursor α-acetolactate ( α-AL) to acetoin, and ov er pr oducing the NADH oxidase enzyme, to r edirect the metabolic flux a wa y from lactic acid synthesis to w ar ds α-AL production (Hugenholtz et al. 2000 ).
Market demands on food quality have become increasingly critical, with raising consumers attention to w ar ds safe and tasty foods and be v er a ges.Sensory c har acteristics of fermented pr oducts can be impr ov ed by acting pr ecisel y on micr oor ganisms' genes using nontransgenic NGTs.Saccharomyces FDC1 gene is involved in the production of 4-vinyl guaiacol, which gives a spicy and clove-like flavour in beers, so called phenolic c har acter of beers .T hese phenolic off-fla vours ha v e been significantl y r educed by induction of a loss-of-function mutation in the FDC1 gene of yeasts (Mertens et al. 2019 ).Improving sensory quality can also be a matter of increasing desirable flavour compounds.Yeast has a major role in winemaking when it comes to develop wine fla vours .Production of some acetate esters by Sac.cerevisiae induces fruity and flo w er-like aromas (b y producing isoamyl acetate and phenylethyl acetate).Overexpressing ATF1 could increase the synthesis of alcohol acetyltr ansfer ase I, whic h catal yzes the formation of acetate esters from acetyl coenzyme A (Vilela 2021 ).Precise combination of genes ov er expr ession and/or attenuation ac hie v ed via the use of nontransgenic NGTs w ould allo w inhibiting or de v eloping se v er al specific flavours in fermented foods and be v er a ges.
Increasing food nutritional value should also be addressed to r elie v e the pr essur e on food systems, considering the high nutritional r equir ements of the gr owing population.Se v er al micr oorganisms ar e ca pable of pr oducing substances with a high nutritional profile for humans, including essential amino acids, vitamins , minerals , and antioxidants (Graham and Ledesma-Amaro 2023 ).Group B vitamins are normally assimilated with the diet and play important roles in human cells metabolism.Despite its presence in foods, deficiencies in vitamin B2, known also as riboflavin, ar e v ery common in both de v eloping and industrialized countries and are associated with se v er al pathologies (Thakur et al. 2016 ).Riboflavin production has been observed in several LAB used in food culture formulations, and it is related to the presence of an intact riboflavin biosynthetic ( rib ) operon (Thakur et al. 2016 ).Intr oducing point m utations in the pr omoter r egion of the rib operon via nontransgenic NGTs would help boosting riboflavin production in food cultures (Fig. 3 D).In fact, riboflavin production has been enhanced in a Lac.lactis subsp.cremoris strain when all four biosynthetic genes were simultaneously overexpressed in a plasmid (Burgess et al. 2004 ).

Conclusion
The possibility to use nontransgenic NGTs would guarantee an impr ov ed scenario for food industries, which are part of the solution to ac hie v e the ambitious targets set by the European 'Green Deal' and 'Farm to Fork' policies in a meaningful time frame widening their opportunities to gener ate innov ativ e, safe, and more sustainable products for European consumers .T his use is hindered by the current process-centric GM regulatory framework that is not fit for this purpose , ha ving been established before the era of NGTs .T he risk assessment and management of the final pr oduct/micr oor ganism should be based on actual genome properties, as it is the case for those than can be ac hie v ed spontaneously in nature or by conventional breeding techniques, and not on the technique itself.This would also ensure the adaptability of the fr ame work to ne w futur e tec hniques ac hie ving the same type of pr oducts, ther eby pr ecluding an y futur e la g between the e volution of the science and market access of the concerned products, ensuring European consumers can quickly benefit from innovative and safe products.
The r e vision of the EU r egulatory fr ame work is necessary as the EU market is missing opportunities for innov ation, whic h is crucial to ac hie v e the ambitious tar gets set by the Eur opean 'Gr een Deal' and 'Farm to Fork' policies in a meaningful timeframe.In other regions of the world, pragmatic regulatory frameworks are already in place.As a result, certain products are exempted from r equir ements of GM (and equivalent) regulatory frameworks in the USA, Canada, Australia, Israel, and India, whereas in several other countries, a case-by-case a ppr oac h is implemented.EU is curr entl y pr one to noncompliance of imports fr om these third countries, where some NGTs products are already being marketed, as analytical methods capable of identifying nontransgenic products obtained by NGTs are missing.
As a first step to w ar ds a product-centric approach, microorganisms obtained by nontransgenic NGTs should be exempted from the obligations imposed by the EU GM r egulatory fr ame work for deliber ate r elease, as is alr eady the case for those obtained by conv entional m uta genesis, wher e 'nontr ansgenic' e v ents ar e identical to those spontaneously happening in nature.

Figure 1 .
Figure 1.Precision genome editing via the recombineering technique.Recombineering DNA fragment can be double-stranded (dsDNA) or single-stranded (ssDN A). dsDN A molecules ar e degr aded in the 5 to 3 dir ection by Exo/RecE exonucleases to gener ate ssDNA intermediates, whic h ar e pr otected fr om the degr adation b y Beta/RecT ssDN A-binding proteins.In addition, Beta/RecT pr omote the annealing of the ssDNA r ecombineering fr a gments to the complementary region on the target bacterial genome, thanks to the homology arms.Recombination occurring between the homology regions can result in wild-type or recombinant genotype, which needs to be checked by sequencing or polymerase chain reaction (in case of large editing).ssDNA molecules must pair with the lagging strand of DNA synthesis to optimize the recombineering efficiency.Created with Biorender.com.
. It exists in two enantiomeric forms, l -and d -lactic acid, which are produced b y tw o different enzymes ( l -or d -lactate dehydrogenase, r espectiv el y) and differs in the tec hnological uses.Micr obial fer-mentation can ther efor e r esult in the pr oduction of an opticall y pure isomer of lactic acid or a racemic mixture, according to the LAB species selected.Food industry prefers the (L + ) isomer, since it is the only isomer that can be efficiently metabolized by humans .Moreo ver, elevated levels of d -lactic acid in the blood or other body tissues may lead to insurgence of the metabolic syndr ome, d -lactic acidosis (Mac k 2004 ).Highl y opticall y pur e d -lactic acid is synthesized by some LAB species, including Lac. delbrueckii and Leuconostoc sp.(Tashiro et al. 2011 , Alexandri et al. 2022 ).Precision genome editing by nontransgenic NGTs could pr e v ent dlactic acid production by generating LAB variants encoding a nonfunctional d -lactate dehydrogenase ( d -LDH; Fig 3 B).A similar appr oac h was alr eady a pplied to de v elop a Lacticaseibacillus paracasei str ain pr oducing opticall y pur e l -lactic acid after the interruption of ldhD gene via plasmid-based homologous recombination (Kuo et al. 2015 ).