Genome editing of lactic acid bacteria: opportunities for food, feed, pharma and biotech

Börner, Rosa Aragão; Kandasamy, Vijayalakshmi; Axelsen, Amalie M; Nielsen, Alex Toftgaard; Bosma, Elleke Fenna

doi:10.1093/femsle/fny291

Cited by 77 publications

(72 citation statements)

References 129 publications

(158 reference statements)

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“…Currently, whole-genome sequencing is a common tool for LAB identification in the laboratory [6,8]. Species identification, using sequencing, provides essential information to conduct genome editing of food-grade LAB, i.e., using clustered regularly interspaced short palindromic repeats (CRISPR) tools [13,14]. Potential applications of these technologies include the development of therapeutic probiotics and strains with resistance against viruses [14,15].…”

Section: Introductionmentioning

confidence: 99%

Multi-Product Lactic Acid Bacteria Fermentations: A Review

Mora-Villalobos¹,

Montero-Zamora²,

Barboza

et al. 2020

Fermentation

123

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Industrial biotechnology is a continuously expanding field focused on the application of microorganisms to produce chemicals using renewable sources as substrates. Currently, an increasing interest in new versatile processes, able to utilize a variety of substrates to obtain diverse products, can be observed. A robust microbial strain is critical in the creation of such processes. Lactic acid bacteria (LAB) are used to produce a wide variety of chemicals with high commercial interest. Lactic acid (LA) is the most predominant industrial product obtained from LAB fermentations, and its production is forecasted to rise as the result of the increasing demand of polylactic acid. Hence, the creation of new ways to revalorize LA production processes is of high interest and could further enhance its economic value. Therefore, this review explores some co-products of LA fermentations, derived from LAB, with special focus on bacteriocins, lipoteichoic acid, and probiotics. Finally, a multi-product process involving LA and the other compounds of interest is proposed.Fermentation 2020, 6, 23 2 of 21 by-products, were proposed to improve industrial single-product biotechnological processes [2]. One example is the use of cellulose and hemicellulose from sugarcane bagasse (a residue obtained during the bioethanol production and usually used for energy generation) for the production of value-added chemicals [3]. Likewise, glycerol, a by-product of the biodiesel industry with low value in the market, is targeted as a molecule of interest in fermentation processes. Such multi-product processes, based on the conversion of renewable materials into biobased products, are known as biorefineries [4].The study, development, and application of robust microbial strains, able to utilize a variety of substrates and to produce a wide range of products, is considered a milestone in the development of biorefineries [4]. Lactic acid bacteria (LAB) are a diverse group with recognized potential for the development of integrated biorefineries [5]. LAB are non-sporulating, non-motile, acid-tolerant, non-respiring but aerotolerant, catalase-negative, Gram-positive cocci or rods. They are characterized by the production of lactic acid (LA) as the major end metabolic product of carbohydrate fermentation [6][7][8][9]. Given the lack of a functional respiratory system, LAB obtain energy through substrate-level phosphorylation following two metabolic pathways for hexose fermentation, i.e., homofermentative and heterofermentative. As shown in Figure 1, the first pathway is based on glycolysis with the production of mainly LA, whereas the second one, known as the pentose phosphate pathway, is characterized for the production of CO 2 and ethanol, or acetate in addition to LA [6].

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Section: Introductionmentioning

confidence: 99%

Multi-Product Lactic Acid Bacteria Fermentations: A Review

Mora-Villalobos¹,

Montero-Zamora²,

Barboza

et al. 2020

Fermentation

123

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“…As the strategy described here avoids the use of foreign selection markers, the described genetic toolset further enables so-called "self-cloning." Strains engineered this way are not considered genetically modified organisms (GMO) in some countries (29,30). For this, only endogenously encoded DOIs can be used; examples include the increase of gene dosage or engineering of sequences such as promoter swapping.…”

Section: Discussionmentioning

confidence: 99%

Multiplex Genetic Engineering Exploiting Pyrimidine Salvage Pathway-Based Endogenous Counterselectable Markers

Birštonas

Dallemulle

López‐Berges

et al. 2020

mBio

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Selectable markers are indispensable for genetic engineering, yet their number and variety are limited. Most selection procedures for prototrophic cells rely on the introduction of antibiotic resistance genes. New minimally invasive tools are needed to facilitate sophisticated genetic manipulations. Here, we characterized three endogenous genes in the human fungal pathogen Aspergillus fumigatus for their potential as markers for targeted genomic insertions of DNAs of interest (DOIs). Since these genes are involved in uptake and metabolization of pyrimidines, resistance to the toxic effects of prodrugs 5-fluorocytosine and 5-fluorouracil can be used to select successfully integrated DOIs. We show that DOI integration, resulting in the inactivation of these genes, caused no adverse effects with respect to nutrient requirements, stress resistance, or virulence. Beside the individual use of markers for site-directed integration of reporter cassettes, including the 17-kb penicillin biosynthetic cluster, we demonstrate their sequential use by inserting three genes encoding fluorescent proteins into a single strain for simultaneous multicolor localization microscopy. In addition to A. fumigatus, we validated the applicability of this novel toolbox in Penicillium chrysogenum and Fusarium oxysporum. Enabling multiple targeted insertions of DOIs without the necessity for exogenous markers, this technology has the potential to significantly advance genetic engineering. IMPORTANCE This work reports the discovery of a novel genetic toolbox comprising multiple, endogenous selectable markers for targeted genomic insertions of DNAs of interest (DOIs). Marker genes encode proteins involved in 5-fluorocytosine uptake and pyrimidine salvage activities mediating 5-fluorocytosine deamination as well as 5-fluorouracil phosphoribosylation. The requirement for their genomic replacement by DOIs to confer 5-fluorocytosine or 5-fluorouracil resistance for transformation selection enforces site-specific integrations. Due to the fact that the described markers are endogenously encoded, there is no necessity for the exogenous introduction of commonly employed markers such as auxotrophy-complementing genes or antibiotic resistance cassettes. Importantly, inactivation of the described marker genes had no adverse effects on nutrient requirements, growth, or virulence of the human pathogen Aspergillus fumigatus. Given the limited number and distinct types of selectable markers available for the genetic manipulation of prototrophic strains such as wild-type strains, we anticipate that the proposed methodology will significantly advance genetic as well as metabolic engineering of fungal species.

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“…As the described strategy avoids the use of foreign selection markers, the described genetic toolset further enables so called ‘self-cloning’. Strains engineered this way are not considered genetically modified organisms (GMO) in some countries (Steensels et al 2014; Borner et al 2019). For this, only endogenously encoded DOIs can be used; examples include the increase of gene dosage or engineering of sequences such as promoter swapping.…”

Section: Discussionmentioning

confidence: 99%

Multiplex genetic engineering exploiting pyrimidine salvage pathway-based self-encoded selectable markers

Birštonas

Dallemulle

López‐Berges

et al. 2020

Preprint

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28Selectable markers are indispensable for genetic engineering, yet their number and variety is 29 limited. Most selection procedures for prototrophic cells rely on the introduction of antibiotic 30 resistance genes. New minimally invasive tools are needed to facilitate sophisticated genetic 31 manipulations. Here, we characterized three endogenous genes in the human fungal pathogen 32Aspergillus fumigatus for their potential as markers for targeted genomic insertions of DNAs-of-33 interest (DOIs). Since these genes are involved in uptake and metabolization of pyrimidines, 34 resistance to the toxic effects of prodrugs 5-fluorocytosine and 5-fluorouracil can be used to 35 select successfully integrated DOIs. We show that DOI integration, resulting in the inactivation of 36 these genes, caused no adverse effects with respect to nutrient requirements, growth or 37 virulence. Beside the individual use of markers for site-directed integration of reporter cassettes 38 including the 17-kb penicillin biosynthetic cluster, we demonstrate their sequential use 39 inserting three fluorescent protein encoding genes into a single strain for simultaneous 40 multicolor localization microscopy. In addition to A. fumigatus, we validated the applicability of 41 this novel toolbox in Penicillium chrysogenum and Fusarium oxysporum.

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Genome editing of lactic acid bacteria: opportunities for food, feed, pharma and biotech

Cited by 77 publications

References 129 publications

Multi-Product Lactic Acid Bacteria Fermentations: A Review

Multi-Product Lactic Acid Bacteria Fermentations: A Review

Multiplex Genetic Engineering Exploiting Pyrimidine Salvage Pathway-Based Endogenous Counterselectable Markers

Multiplex genetic engineering exploiting pyrimidine salvage pathway-based self-encoded selectable markers

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