Expanding the repertoire of counterselection markers for markerless gene deletion in the human gut bacterium Phocaeicola vulgatus

Neff, André; Lück, Rebecca; Hövels, Marcel; Deppenmeier, Uwe

doi:10.1016/j.anaerobe.2023.102742

Cited by 7 publications

(2 citation statements)

References 30 publications

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“…While the tested Bacteroidia lag in yield and product ratios compared to the well-studied propionibacteria, they still hold many opportunities for optimization. The advance in genetic tools for example: with a markerless deletion method already established in P. vulgatus [ 26 ] side product formation of B. graminisolvens can be reduced. Further physiological insights will help to find out, why B. propionicifaciens has a more effective propionate conversion, which can then be applied to species with a broader substrate spectrum.…”

Section: Resultsmentioning

confidence: 99%

Propionate production by Bacteroidia gut bacteria and its dependence on substrate concentrations differs among species

Döring,

Basen

2024

Biotechnol Biofuels

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Background Propionate is a food preservative and platform chemical, but no biological process competes with current petrochemical production routes yet. Although propionate production has been described for gut bacteria of the class Bacteroidia, which also carry great capacity for the degradation of plant polymers, knowledge on propionate yields and productivities across species is scarce. This study aims to compare propionate production from glucose within Bacteroidia and characterize good propionate producers among this group. Results We collected published information on propionate producing Bacteroidia, and selected ten species to be further examined. These species were grown under defined conditions to compare their product formation. While propionate, acetate, succinate, lactate and formate were produced, the product ratios varied greatly among the species. The two species with highest propionate yield, B. propionicifaciens (0.39 gpro/ggluc) and B. graminisolvens (0.25 gpro/ggluc), were further examined. Product formation and growth behavior differed significantly during CO2-limited growth and in resting cells experiments, as only B. graminisolvens depended on external-added NaHCO3, while their genome sequences only revealed few differences in the major catabolic pathways. Carbon mass and electron balances in experiments with resting cells were closed under the assumption that the oxidative pentose pathway was utilized for glucose oxidation next to glycolysis in B. graminisolvens. Finally, during pH-controlled fed-batch cultivation B. propionicifaciens and B. graminisolvens grew up to cell densities (OD600) of 8.1 and 9.8, and produced 119 mM and 33 mM of propionate from 130 and 105 mM glucose, respectively. A significant production of other acids, particularly lactate (25 mM), was observed in B. graminisolvens only. Conclusions We obtained the first broad overview and comparison of propionate production in Bacteroidia strains. A closer look at two species with comparably high propionate yields, showed significant differences in their physiology. Further studies may reveal the molecular basis for high propionate yields in Bacteroidia, paving the road towards their biotechnological application for conversion of biomass-derived sugars to propionate.

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Section: Resultsmentioning

confidence: 99%

Propionate production by Bacteroidia gut bacteria and its dependence on substrate concentrations differs among species

Döring,

Basen

2024

Biotechnol Biofuels

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“…With circa 10 7-14 organisms per milliliter of colonic contents, the gut microbiome is the largest share of bacteria in humans (Vos et al 2022) and plays a crucial part in human health (Wilson et al 2020). The phylum Bacteroidota dominates the human gut (Salyers 1984;Wexler 2007), achieves high yields of organic acids (Macfarlane and Macfarlane 2003;Ríos-Covián et al 2016;Mayhew et al 1975), and can be genetically modified (Lück and Deppenmeier 2022;Neff et al 2023). Moreover, there is evidence that Bacteroidota can produce antibiotic or bioactive compounds (Brinkmann et al 2022;Wexler 2007) and serve as probiotics (Tan et al 2019).…”

Section: Introductionmentioning

confidence: 99%

Process analysis of the anaerobe Phocaeicola vulgatus in a shake flasks and fermenter reveals pH and product inhibition

Keitel,

Miebach,

Rummel

et al. 2024

Ann Microbiol

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Purpose The anaerobic gut bacterium Phocaeicola vulgatus (formerly Bacteroides vulgatus) has a significant role in the human gut microbiome. It can produce bioactive compounds with antimicrobial properties and industrially relevant organic acids like succinate. However, there is a knowledge gap in understanding the metabolism of P. vulgatus, as cultivation of anaerobic gut bacteria is challenging and usually conducted with enriched microbiota cultures. We aim to close this gap by characterizing this anaerobe bacterium in different cultivation conditions and scales. Methods In this work, axenic cultures were studied in a shake flask and 2 L fermenter scale to characterize the influence of initial pH, buffer concentration, osmolality, and product inhibition on growth and organic acid production by P. vulgatus. Both cultivation systems had online gas measurements for total gas and CO2 production. HPLC analysis generated closed carbon balances, accounting for all produced acids. Results Total gas transfer rates and CO2 transfer rates revealed that 65% of produced gas was attributed to H2, while just 35% was connected to CO2 production. A minimum buffer concentration of 50 mM MOPS and an initial pH of 7.3 were identified to mitigate pH inhibition in P. vulgatus cultivations with a defined minimal medium and glucose as substrate. The initial addition of lactate showed an inhibitory effect, starting at a concentration of 1 g L−1. On the contrary, initial acetate addition was beneficial for organic acid production. A comparison of a pH-buffered and a pH-controlled 2 L fermentation demonstrated a switch in acid production toward succinate under pH control. Conclusion The study provides insight into improved cultivation conditions for the gut bacterium P. vulgatus and demonstrates a successful scale-up from the shake flask to the 2 L bioreactor. By applying pH control in the bioreactor, growth was increased, and the organic acid production was switched from lactate to succinate. Even though P. vulgatus could serve as a production organism for interesting bioactive compounds and organic acids, further characterization and improvement are necessary to improve titers.

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Genetic optimization of the human gut bacterium Phocaeicola vulgatus for enhanced succinate production

Gindt,

Lück,

Deppenmeier

2024

Appl Microbiol Biotechnol

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The demand for sustainably produced bulk chemicals is constantly rising. Succinate serves as a fundamental component in various food, chemical, and pharmaceutical products. Succinate can be produced from sustainable raw materials using microbial fermentation and enzyme-based technologies. Bacteroides and Phocaeicola species, widely distributed and prevalent gut commensals, possess enzyme sets for the metabolization of complex plant polysaccharides and synthesize succinate as a fermentative end product. This study employed novel molecular techniques to enhance succinate yields in the natural succinate producer Phocaeicola vulgatus by directing the metabolic carbon flow toward succinate formation. The deletion of the gene encoding the methylmalonyl-CoA mutase (Δmcm, bvu_0309-0310) resulted in a 95% increase in succinate production, as metabolization to propionate was effectively blocked. Furthermore, deletion of genes encoding the lactate dehydrogenase (Δldh, bvu_2499) and the pyruvate:formate lyase (Δpfl, bvu_2880) eliminated the formation of fermentative end products lactate and formate. By overproducing the transketolase (TKT, BVU_2318) in the triple deletion mutant, succinate production increased from 3.9 mmol/g dry weight in the wild type to 10.9 mmol/g dry weight. Overall, succinate yield increased by 180% in the new mutant strain P. vulgatus Δmcm Δldh Δpfl pG106_tkt relative to the parent strain. This approach is a proof of concept, verifying the genetic accessibility of P. vulgatus, and forms the basis for targeted genetic optimization. The increase of efficiency highlights the huge potential of P. vulgatus as a succinate producer with applications in sustainable bioproduction processes. Key points • Deleting methylmalonyl-CoA mutase gene in P. vulgatus doubled succinate production • Triple deletion mutant with transketolase overexpression increased succinate yield by 180% • P. vulgatus shows high potential for sustainable bulk chemical production via genetic optimization

show abstract

Expanding the repertoire of counterselection markers for markerless gene deletion in the human gut bacterium Phocaeicola vulgatus

Cited by 7 publications

References 30 publications

Propionate production by Bacteroidia gut bacteria and its dependence on substrate concentrations differs among species

Propionate production by Bacteroidia gut bacteria and its dependence on substrate concentrations differs among species

Process analysis of the anaerobe Phocaeicola vulgatus in a shake flasks and fermenter reveals pH and product inhibition

Genetic optimization of the human gut bacterium Phocaeicola vulgatus for enhanced succinate production

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