Effect of Clp protease from Corynebacterium glutamicum on heterologous protein expression

Liu, Xiuxia; Meng, Lingjie; Wang, Xinyue; Yang, Yankun; Bai, Zhonghu

doi:10.1016/j.pep.2021.105928

Cited by 10 publications

(5 citation statements)

References 36 publications

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“…Our results suggest that such a shift in protein degradation specificity might favor cellulose production in K. sucrofermentans . This is in agreement with earlier research on C. glutamicum , which showed that a clpS deletion can increase the expression of a heterologous protein by 65% ( 56 ). In addition to cellulose production, cell growth experiments revealed that the control, evolved, and ΔclpS strains had accelerated growth rates compared to the native strain ( Fig.…”

Section: Resultssupporting

confidence: 93%

Directed evolution of material-producing microorganisms

Laurent,

Jain,

Kan

et al. 2024

Proc. Natl. Acad. Sci. U.S.A.

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Nature is home to a variety of microorganisms that create materials under environmentally friendly conditions. While this offers an attractive approach for sustainable manufacturing, the production of materials by native microorganisms is usually slow and synthetic biology tools to engineer faster microorganisms are only available when prior knowledge of genotype–phenotype links is available. Here, we utilize a high-throughput directed evolution platform to enhance the fitness of whole microorganisms under selection pressure and identify genetic pathways to enhance the material production capabilities of native species. Using Komagataeibacter sucrofermentans as a model cellulose-producing microorganism, we show that our droplet-based microfluidic platform enables the directed evolution of these bacteria toward a small number of cellulose overproducers from an initial pool of 40,000 random mutants. Sequencing of the evolved strains reveals an unexpected link between the cellulose-forming ability of the bacteria and a gene encoding a protease complex responsible for protein turnover in the cell. The ability to enhance the fitness of microorganisms toward a specific phenotype and to unravel genotype–phenotype links makes this high-throughput directed evolution platform a promising tool for the development of new strains for the sustainable manufacturing of materials.

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Section: Resultssupporting

confidence: 93%

Directed evolution of material-producing microorganisms

Laurent,

Jain,

Kan

et al. 2024

Proc. Natl. Acad. Sci. U.S.A.

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“…Although C. glutamicum is considered to have low extracellular protease activity ( Vertès, 2013 ; Freudl, 2018 ), several studies have shown that recombinant protein production by C. glutamicum can be improved by deleting proteases ( Peng et al, 2019 ; Zhang et al, 2019 ; Liu et al, 2022 ). In a recent study, we investigated a possible contribution of extracellular protease activity to reduced GarQ levels during production with C. glutamicum ( Desiderato et al, 2022 ).…”

Section: Discussionmentioning

confidence: 99%

Optimized recombinant production of the bacteriocin garvicin Q by Corynebacterium glutamicum

Desiderato,

Müller,

Schretzmeier

et al. 2024

Front. Microbiol.

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Bacteriocins are antimicrobial peptides applied in food preservation and are interesting candidates as alternatives to conventional antibiotics or as microbiome modulators. Recently, we established Corynebacterium glutamicum as a suitable production host for various bacteriocins including garvicin Q (GarQ). Here, we establish secretion of GarQ by C. glutamicum via the Sec translocon achieving GarQ titers of about 7 mg L–1 in initial fermentations. At neutral pH, the cationic peptide is efficiently adsorbed to the negatively charged envelope of producer bacteria limiting availability of the bacteriocin in culture supernatants. A combination of CaCl2 and Tween 80 efficiently reduces GarQ adsorption to C. glutamicum. Moreover, cultivation in minimal medium supplemented with CaCl2 and Tween 80 improves GarQ production by C. glutamicum to about 15 mg L–1 but Tween 80 resulted in reduced GarQ activity at later timepoints. Using a reporter strain and proteomic analyses, we identified HtrA, a protease associated with secretion stress, as another potential factor limiting GarQ production. Transferring production to HtrA-deficient C. glutamicum K9 improves GarQ titers to close to 40 mg L–1. Applying conditions of low aeration prevented loss in activity at later timepoints and improved GarQ titers to about 100 mg L–1. This is about 50-fold higher than previously shown with a C. glutamicum strain employing the native GarQ transporter GarCD for secretion and in the range of levels observed with the native producer Lactococcus petauri B1726. Additionally, we tested several synthetic variants of GarQ and were able to show that exchange of the methionine in position 5 to a phenylalanine (GarQM5F) results in markedly increased activity against Lactococcus lactis and Listeria monocytogenes. In summary, our findings shed light on several aspects of recombinant GarQ production that may also be of relevance for production with natural producers and other bacteriocins.

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“…C. glutamicum mainly utilizes glucose and fructose to produce nucleotides and amino acids through various metabolic pathways. So far, only three proteases, SprA protease, FtsH protease and Clp protease, were found from C. glutamicum (Liu et al ., 2022). While the activities of the extracellular proteases produced by C. glutamicum remain unclear, the Clp protease of C. glutamicum was recently shown to decrease the production of exogenous proteins.…”

Section: Resultsmentioning

confidence: 99%

The effect of the Corynebacterium glutamicum on the shortening of fermentation time, physicochemical and sensory properties of soy sauce

Gao

Sun‐Waterhouse

Xiang

et al. 2022

Int J of Food Sci Tech

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This study for the first time explores the possibility of using Corynebacterium glutamicum Ⅲ (C. glutamicum) to shorten the soy sauce fermentation period without compromising product quality. C. glutamicum was added to soy sauce that had been fermented with Aspergillus oryzae for 30 days (SS30) to initiate a further 3-day fermentation (yielding SS33). The 3-day fermentation exerted similar impacts on SS30 as the standard fermentation for further 60 days (yielding SS90): that is, increased the contents of total nitrogen, total free amino acids (FAAs), umami FAAs, acids, esters, ketones, furans and sulphurcontaining compounds, whilst decreasing the contents of alcohols and phenols. This explained the similarities in taste, aroma and overall liking between SS33 and SS90. The SS90 was the darkest among the three soy sauces. Compared with SS90, SS33 was less salty and smoky, more umami and kokumi, softer and more mellow, higher overall liking, with more 0.5-1 kDa peptides and fewer peptides <0.5 kDa. Therefore, a 3-day fermentation with C. glutamicum after an initial 30 days of a normal soy sauce fermentation represents a simple and effective way to shorten the soy sauce production time from 90 to 33 days whilst increasing greatly the umami taste and mellow sensation. This approach enhances product competitiveness via lowering manufacturing cost.

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Effect of Clp protease from Corynebacterium glutamicum on heterologous protein expression

Cited by 10 publications

References 36 publications

Directed evolution of material-producing microorganisms

Directed evolution of material-producing microorganisms

Optimized recombinant production of the bacteriocin garvicin Q by Corynebacterium glutamicum

The effect of the Corynebacterium glutamicum on the shortening of fermentation time, physicochemical and sensory properties of soy sauce

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