Pseudomonas putida KT2440 is HV1 certified, not GRAS.

Pseudomonas putida is rapidly becoming a workhorse for industrial production due to its metabolic versatility, genetic accessibility and stress-resistance properties. The P. putida strain KT2440 is often described as Generally Regarded as Safe, or GRAS, indicating the strain is safe to use as food additive. This description is incorrect. P. putida KT2440 is classified by the FDA as HV1 certified, indicating it is safe to use in a P1 or ML1 environment.

After the discovery and full sequencing of the bacterium thus far known as Pseudomonas putida KT2440 (Bagdasarian et al., 1981; Nelson et al., 2002; Timmis, 2002), this strain has rapidly become a workhorse for industrial biotechnology owing to its metabolic versatility, genetic accessibility and stress‐resistance properties (Puchałka et al., 2008; Poblete‐Castro et al., 2012; Nikel and de Lorenzo, 2018). This specific set of characteristics are made more accessible by the recent expansion of its metabolic toolkit, including specific inducible promoters (Cook et al., 2018) and introduction of the CRISPR‐system (Mougiakos et al., 2017; Aparicio et al., 2018; Sun et al., 2018). Its many benefits as a species have launched P. putida KT2440 as a model organism for industrial production of biobased chemicals such as citrulline (Patil et al., 2017), muconate (Johnson et al., 2017), precursors of biobased plastics (Koopman et al., 2010), precursor of biodegradable plastics (Ribera et al., 2001; Poblete‐Castro et al., 2013) and waste biodegradation purposes (Wierckx et al., 2015; Ravi et al., 2017).

Often P. putida KT2440, and sometimes the P. putida species, is described as Generally Regarded as Safe, or GRAS, indicating it is safe to use as a food additive (Nutrition, 2018) (e.g. (Aparicio et al., 2018; Arnfinnsdottir et al., 2016; Belda et al., 2016; Bojanovic, 2016; Calero et al., 2016; Cesarini et al., 2014; Choi et al., 2018; Classen and Pietruszka, 2018; Cook et al., 2018; Cornelissen et al., 2011; Cuenca et al., 2016; Domröse et al., 2015; Dvorak and de Lorenzo, 2018; Escapa et al., 2011; Fröhlich et al., 2014; Gemperlein et al., 2014; Gómez et al., 2016; Gong et al., 2017; de las Heras and de Lorenzo, 2011; Jayakody et al., 2018; Kahlon, 2016; Kim et al., 2019; Klein et al., 2017; Köppen et al., 2014; Kusumawardhani et al., 2018; Lee and Wendisch, 2017; Lieder et al., 2015; Loeschcke and Thies, 2015; Martínez et al., 2014; Mindt et al., 2018; Nikel and de Lorenzo, 2018; Pandi et al., 2017; Pernicova et al., 2019; Poblete‐Castro et al., 2016; Puchałka et al., 2008; Ravi et al., 2017; Rühl et al., 2012; Sun et al., 2018; Taghavi et al., 2011; Troeschel et al., 2012; Wang et al., 2014; Weyens et al., 2009; Wierckx et al., 2015; Wu et al., 2011)).

When a reference is given by the authors for this GRAS claim, the following is used: the US Food and Drug Regulation Administration (FDA) report, specifically the FDA vol. 47, no. 77, Appendix E, page 17197, Certified host–vector systems from 21st April 1982 (see Appendix S1) (National Archives And Records Administration, 1982). However, P. putida KT2440 is herein not certified as GRAS, but as host–vector (HV) system safety level 1: ‘HV1 – Pseudomonas putida strain KT2440 with plasmid vectors pKT262, pKT263 and pKT264’. The HV1 certification indicates that P. putida KT2440 is determined to be safe to work with at a P1 (or ML1) facility, as elaborately described by the FDA on page 17181 of the report (National Archives and Records Administration, 1982). Similarly to E. coli K12, no additional safety measures are required to work with this strain.

The GRAS status determined by the FDA is only awarded to chemicals or specific strains of microorganisms that are proven to be safe for ingestion (Nutrition, C. for F.S. and A., 2018). This is not proven for P. putida KT2440, contrary to food‐related microorganisms such as Lactobacillus helveticus R0052, Lactobacillus casei subsp. paracasei Lpc‐37, Bifidobacterium bifidum R0071 or Streptococcus salivarius K12, which are examples of strains that carry the GRAS status. Intense literature research suggests the GRAS reference occurs due to incorrect transitive referencing. A full GRAS certificate list can be found at the FDA website: https://www.accessdata.fda.gov/scripts/fdcc/?set=GRASNotices.

However, this does not mean that P. putida KT2440 is unsafe to work with. The safety of the strain is proven by the overall absence of virulence factors (Dos Santos et al., 2004; Belda et al., 2016) and the intensive research over the past 40 years by a large community without any fortuitous cases of infection reported. This correct certification does therefore not change the many useful applications of this strain. Furthermore, the widespread use of KT2440, the massive evidence of safe performance and the development of more refined methods for risk assessment than those available in the early 80s ask for a revised classification of the strains at stake under the current FDA and EFSA regulations.

As a note, the phylogenetic classification of P. putida KT2440 within the P. putida group has been questioned for long (Regenhardt et al., 2002), and recent work has provided a new classification (Keshavarz‐Tohid et al., 2019) (Moore et al., in preparation). However, for the purpose of safety certification, the specific bacterium formerly known as P. putida KT2440 remains classified as HV1, regardless of its precise phylogenetic status.

We urge other scientists to properly describe the safety status of all used strains, since we also discovered the same is true for example Corynebacterium glutamicum. Similarly to P. putida KT2440, C. glutamicum is sometimes reported to have GRAS status (e.g. (Baritugo et al., 2018; Kim et al., 2018; Levin, 2002; Neuner et al., 2013)), but when checked using the website stated above, only cultured corn starch fermented by C. glutamicum is GRAS, not the organism itself.

Conflict of interest

None declared.

Author contributions

LFCK conceived the subject. LFCK/RJMV involved in literature search and analysis. LFCK wrote the manuscript. RJMV/VAPMdS supervised the work. VAPMdS arranged the funding.

Appendix S1. FDA report vol. 47, no. 77, Certified host–vector systems from 21st April 1982. Includes Appendix E, page 17197.

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