Genetically Modified Micro-Organisms for Industrial Food Enzyme Production: An Overview

Abstract

The use of food enzymes (FE) by the industrial food industry is continuously increasing. These FE are mainly obtained by microbial fermentation, for which both wild-type (WT) and genetically modified (GM) strains are used. The FE production yield can be increased by optimizing the fermentation process, either by using genetically modified micro-organism (GMM) strains or by producing recombinant enzymes. This review provides a general overview of the different methods used to produce FE preparations and how the use of GMM can increase the production yield. Additionally, information regarding the construction of these GMM strains is provided. Thereafter, an overview of the different European regulations concerning the authorization of FE preparations on the European market and the use of GMM strains is given. Potential issues related to the authorization and control of FE preparations sold on the European market are then identified and illustrated by a case study. This process highlighted the importance for control of FE preparations and the consequent need for appropriate detection methods targeting the presence of GMM, which is used in fermentation products.

Keywords: European regulations; food enzymes; genetically modified micro-organisms; safety control.

Figure 1

Schematic representation of an expression vector. (A) Expression vectors contain an origin of replication (ORI), followed by the multiple cloning site (MCS) where the gene of interest is integrated and a selection marker (SM). (B) Both an episomal plasmid and integrative plasmid, containing flanked integration sites, can be created. ARS, autonomous replication sequence; IS, integration site.

Figure 2

The construction of recombinant proteins can be done using three different approaches. Rational design is used to modify a specific amino acid that is known to be related to characteristics that need to be changed. If no information is known about the enzyme’s structure and mechanism, both directed evolution and semi-rational design can be used. For directed evolution, a library is prepared of random mutations, mimicking evaluation. This is followed by a thorough screening and selection process. The semi-rational design combines the knowledge of the enzyme’s structure to know where a change is needed with the methodology of directed evolution to prepare a library of mutations within a specific region.

Figure 3

(A) Overview of the FE production sources mentioned in all submitted dossiers for safety evaluation (within the legal deadline). For each category, the corresponding number of dossiers is given, along with their percentage according to the total number of submitted dossiers. Additionally, for both bacteria and fungi, more details are given on the most used species, along with the total number of dossiers mentioning their usage. (B) The percentage of dossiers mentioning the use of GM strains as the FE production source. For the genetically modified micro-organisms (GMM), more detailed information is given on the percentage of genetically modified (GM) fungal and bacterial strains.

Figure 4

(A) General overview of a developed strategy targeting the unexpected presence of FE-producing micro-organisms, with special attention given to GMM strains. (a) First, by targeting the 16S rRNA sequence and the internal transcribed spacer (ITS) region, the potential presence of bacterial and fungal DNA, respectively< can be detected. Identification of an amplicon is obtained by sequencing followed by blasting against in-house databases. (b) The presence of antimicrobial resistance (AMR) genes is targeted using qPCR amplification. If a positive signal is obtained, the full length of the gene is amplified using a nested PCR and then sequenced. The identification of detected AMR genes is verified by blasting. (c) As an additional analysis, a viability assessment is performed. If colonies are obtained, first the two above methods are repeated to confirm the previously obtained results and to select GMM colonies if AMR genes have been detected. A full characterization can consequently be obtained by whole-genome sequencing (WGS) of the obtained colonies. The genetic modifications can be characterized and the identity of the GMM can be demonstrated. (B) Using the strategy shown in (A), a detected micro-organism can be identified. Here, the example of the GMM-producing protease (RASFF2019.3332) is represented.