Available online 3 March 2023

Author links open overlay panel, , , , ABSTRACT

Strain robustness during production of recombinant molecules is of major interest to ensure bioprocess profitability. The heterogeneity of populations has been shown in the literature as a source of instability in bioprocesses. Thus, the heterogeneity of the population was studied by evaluating the robustness of the strains (stability of plasmid expression, cultivability, membrane integrity and macroscopic cell behavior) during well-controlled fedbatch cultures. On the context of microbial production of chemical molecules, isopropanol (IPA) has been produced by recombinant strains of Cupriavidus necator. Plasmid stability was monitored by the plate count method to assess the impact of isopropanol production on plasmid stability, depending on implanted plasmid stabilization systems for strain engineering designs. With the reference strain Re2133/pEG7c, an isopropanol titer of 15.1 g·L-1 could be achieved. When the isopropanol concentration has reached about 8 g. L-1, cell permeability increased (up to 25%) and plasmid stability decreased significantly (up to 1.5 decimal reduction rate) resulting in decreased isopropanol production rates. Bioprocess robustness under isopropanol producing conditions was then investigated with two plasmid construction strategies (1) Post Segregational Killing hok/sok (in Re2133/pEG20) and (2) expression of GroESL chaperon proteins (in Re2133/pEG23). Plasmid stability for strain Re2133/pEG20 (PSK hok/sok) appears to be improved up to 11 g. L-1 of IPA compared to the reference strain (8 g. L-1 IPA). Nevertheless, cell permeability followed the same dynamic as the reference strain with a drastic increase around 8 g. L-1 IPA. On the contrary, the Re2133/pEG23 strain made it possible to minimize the cell permeability (with a constant value at 5% IP permeability) and to increase the growth capacities in response to increased isopropanol concentrations but plasmid stability was the weakest. The metabolic burden, linked to either the overexpression of GroESL chaperones or the PSK hok/sok system, seems to be deleterious for the overall isopropanol production compared to the reference strain (RE2133/pEG7c) even if we have shown that the overexpression chaperones GroESL improve membrane integrity and PSK system hok/sok improve plasmid stability as long as isopropanol concentration does not exceed 11 g/L.

Availability of data and material

All data generated or analyzed during this study are included in the present work.

Section snippetsINTRODUCTION

Faced with collective ecological awareness, interest in the development of alternative fuels has grown over years. These alternatives to fossil-based fuels are based on microbial production of biofuels and chemical synthons from renewable sources. Isopropanol has been identified as an interesting substitute for biofuel production (Grousseau et al., 2014a) and has already been produced in bioreactors from recombinant plasmid-bearing strains of Cupriavidus necator (Garrigues et al., 2020, Marc et

Strains

C. necator Re2133 (Budde et al., 2011) was used as expression strain. The strain C. necator Re2133 was derived from the wildtype strain C. necator H16 / ATCC1769 whose genes encoding for acetoacetyl-CoA reductases (phaB1B2B3) and for PHA synthase (phaC1) were deleted. C. necator strains were naturally gentamicin resistant (GenR). The strains used for plasmid construction were Escherichia coli S17-1 and Top10.

Plasmids

The plasmids pEG7c, pEG20 and pEG23 were used in this work (Table 1). The design and

RESULTS

In this work, the results concerning the production of isopropanol given by the reference strain Re2133/pEG7c and the best producer Re2133/pEG23 (chaperon GroESL) were compared to the strain Re2133/pEG20, which encoded a PSK system hok/sok. The impact of the PSK system hok/sok was compared to the GroESL over-expressing strategy, on isopropanol production, in fed-batch cultures without a plasmid selection marker.

DISCUSSION

This work aimed to characterize the impact of the plasmid stabilization system PSK hok/sok (pEG20) and of the overexpression of the chaperone protein GroESL (Re2133/pEG23) on the strain robustness during heterologous production of isopropanol by C. necator. The behavior of the strain Re2133/pEG20 was compared to the already characterized strains Re2133/pEG7c (reference strain) and pEG23 (Marc et al., 2017a). In Marc and coworker’s work, the two strains Re2133/pEG7c and pEG23 were characterized

CONCLUSIONS

In conclusion, a strain expressing the PSK hok / sok system (Re2133/pEG20) was compared to a reference strain (Re2133/pEG7c) and a strain expressing GroESL chaperone (Re2133/pEG23), on the basis of their production of isopropanol. Although the strain expressing the chaperones improved tolerance to isopropanol (permeabilization + growth), the reference strain Re2133/pEG7c exhibited the highest titer, yield and isopropanol production rate. Moreover, the expression of the PSK system did not make

Ethical approval

Not applicable, since the work does not involve any study with human participants or animals

AUTHORS' CONTRIBUTIONS STATEMENT

CB, JL, SA, NG and SG conceived and designed research. CB conducted experiments and analyzed data. CB wrote the manuscript and JL, SA, NG and SG reviewed it. All authors read and approved the manuscript.

Conflicts of interest/Competing interests

The authors declare that there are no competing interests.

Consent to participate

Not applicable.

Consent for publication

All authors agreed to publish this work.

Competing interests

The authors declare that there are no competing interests.

Code availability

Not applicable.

Funding

Funding was obtained from Doctoral Research Assignments from the French Ministry of Research.

CRediT authorship contribution statement

Catherine Boy: Investigation, Writing - Original Draft Julie Lesage: Investigation, Writing – Review Sandrine Alfenore: Conceptualization, Supervision, Writing – Review Nathalie Gorret: Conceptualization, Supervision, Writing - Review & Editing Stéphane Guillouet: Conceptualization, Supervision, Writing - Review & Editing

Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Guillouet reports financial support was provided by French Ministry of Research

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