Advances in synthetic biology toolboxes paving the way for mechanistic understanding and strain engineering of gut commensal Bacteroides spp. and Clostridium spp.

Tan, Yang; Liang, Jing; Lai, Mingchi; Wan, Sai; Luo, Xiaozhou; Li, Fuli

doi:10.1016/j.biotechadv.2023.108272

Cited by 5 publications

(5 citation statements)

References 186 publications

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“…In order to create plasmid-free clostridia for regulatory approval of COSV, the PyrE loci in the genomes of C. butyricum and C. sporogenes were replaced with high-expression cassettes carrying NY-ESO-1 and CDP-NY-ESO-1, utilizing the previously developed CRISPR-Cas12a system [ 31 ]. The introduction of uracil auxotrophy in the pyrE -deficient clostridia is known to ensure CBT bio-containment [ 16 ]. Consequently, the expression cassettes of miniP c.b _fU- NY-ESO-1 and miniP c.b _fU-CDP- NY-ESO-1 were integrated into the chromosome of C. butyricum , resulting in strains C. butyricum IN pyrE ::ESO and C. butyricum IN pyrE ::CDP-ESO (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Clostridium spp. stands out as a prominent component of the gut microbiota, actively interacting with host organisms and significantly contributing to human health [ 16 ]. The exploration of commensal clostridia for oral vaccination holds great promise in medical applications, presenting a novel avenue beyond the extensively studied E. coli , Salmonella , and lactic acid bacteria in prokaryotic oral vaccination research [ 35 – 37 ].…”

Section: Discussionmentioning

confidence: 99%

“…Advancements in Clostridium synthetic biology tools have played a pivotal role in the development of Clostridium -based bio-therapeutics (CBT) [ 15 , 16 ]. Genetic manipulation of oncological Clostridium sporogenes or probiotic Clostridium butyricum has resulted in the production of bioactive interleukin-2 (IL-2) [ 17 , 18 ], granulocyte macrophage-colony stimulating factor (GM-CSF) [ 19 ], porcine epidermal growth factor (pEGF) [ 20 ], and glucagon-like peptide-1 (GLP-1) [ 21 , 22 ].…”

Section: Introductionmentioning

confidence: 99%

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Multiplex genetic manipulations in Clostridium butyricum and Clostridium sporogenes to secrete recombinant antigen proteins for oral-spore vaccination

Zhang,

Bailey,

Hittmeyer

et al. 2024

Microb Cell Fact

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Background Clostridium spp. has demonstrated therapeutic potential in cancer treatment through intravenous or intratumoral administration. This approach has expanded to include non-pathogenic clostridia for the treatment of various diseases, underscoring the innovative concept of oral-spore vaccination using clostridia. Recent advancements in the field of synthetic biology have significantly enhanced the development of Clostridium-based bio-therapeutics. These advancements are particularly notable in the areas of efficient protein overexpression and secretion, which are crucial for the feasibility of oral vaccination strategies. Here, we present two examples of genetically engineered Clostridium candidates: one as an oral cancer vaccine and the other as an antiviral oral vaccine against SARS-CoV-2. Results Using five validated promoters and a signal peptide derived from Clostridium sporogenes, a series of full-length NY-ESO-1/CTAG1, a promising cancer vaccine candidate, expression vectors were constructed and transformed into C. sporogenes and Clostridium butyricum. Western blotting analysis confirmed efficient expression and secretion of NY-ESO-1 in clostridia, with specific promoters leading to enhanced detection signals. Additionally, the fusion of a reported bacterial adjuvant to NY-ESO-1 for improved immune recognition led to the cloning difficulties in E. coli. The use of an AUU start codon successfully mitigated potential toxicity issues in E. coli, enabling the secretion of recombinant proteins in C. sporogenes and C. butyricum. We further demonstrate the successful replacement of PyrE loci with high-expression cassettes carrying NY-ESO-1 and adjuvant-fused NY-ESO-1, achieving plasmid-free clostridia capable of secreting the antigens. Lastly, the study successfully extends its multiplex genetic manipulations to engineer clostridia for the secretion of SARS-CoV-2-related Spike_S1 antigens. Conclusions This study successfully demonstrated that C. butyricum and C. sporogenes can produce the two recombinant antigen proteins (NY-ESO-1 and SARS-CoV-2-related Spike_S1 antigens) through genetic manipulations, utilizing the AUU start codon. This approach overcomes challenges in cloning difficult proteins in E. coli. These findings underscore the feasibility of harnessing commensal clostridia for antigen protein secretion, emphasizing the applicability of non-canonical translation initiation across diverse species with broad implications for medical or industrial biotechnology.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Multiplex genetic manipulations in Clostridium butyricum and Clostridium sporogenes to secrete recombinant antigen proteins for oral-spore vaccination

Zhang,

Bailey,

Hittmeyer

et al. 2024

Microb Cell Fact

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“…E. coli can be considered the bacterial version of the C57BL/6 mouse; its genetics are well-defined and easily engineered [189][190][191], and it has become the workhorse for testing the effect of modifications in a given environment. However, the commensal GM consists of far more diversity than just E. coli; thus, researchers are actively seeking a deeper understanding of GM genetics, using, for example, Bacteroides and Clostridium as representative organisms [192,193], as numerous human and mouse studies have demonstrated the important roles of these organisms in health and disease.…”

Section: Understanding Of Bacterial Genes and Functionsmentioning

confidence: 99%

The gut microbiota and diabetes: research, translation, and clinical applications – 2023 Diabetes, Diabetes Care, and Diabetologia Expert Forum

Byndloss,

Devkota,

Duca

et al. 2024

Diabetologia

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This article summarises the state of the science on the role of the gut microbiota (GM) in diabetes from a recent international expert forum organised by Diabetes, Diabetes Care, and Diabetologia, which was held at the European Association for the Study of Diabetes 2023 Annual Meeting in Hamburg, Germany. Forum participants included clinicians and basic scientists who are leading investigators in the field of the intestinal microbiome and metabolism. Their conclusions were as follows: (1) the GM may be involved in the pathophysiology of type 2 diabetes, as microbially produced metabolites associate both positively and negatively with the disease, and mechanistic links of GM functions (e.g. genes for butyrate production) with glucose metabolism have recently emerged through the use of Mendelian randomisation in humans; (2) the highly individualised nature of the GM poses a major research obstacle, and large cohorts and a deep-sequencing metagenomic approach are required for robust assessments of associations and causation; (3) because single time point sampling misses intraindividual GM dynamics, future studies with repeated measures within individuals are needed; and (4) much future research will be required to determine the applicability of this expanding knowledge to diabetes diagnosis and treatment, and novel technologies and improved computational tools will be important to achieve this goal.

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“…The microbiota of the human gut is highly integrated. When the balance is disrupted, various intestinal diseases such as diarrhea and colitis and even extraintestinal conditions like cardiovascular and neurological illnesses may occur (Tan et al, 2023). Probiotics are defined as live microorganisms conferring a health-beneficial effect on the host when administered in appropriate amounts (Sanders, 2003).…”

Section: Introductionmentioning

confidence: 99%

Probiotic properties and the ameliorative effect on DSS‐induced colitis of human milk–derived Lactobacillus gasseri SHMB 0001

Wang,

Dong,

Liu

et al. 2024

Journal of Food Science

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Human milk contains a variety of microorganisms that exert benefit for human health. In the current study, we isolated a novel Lactobacillus gasseri strain named Lactobacillus gasseri (L. gasseri) SHMB 0001 from human milk and aimed to evaluate the probiotic characteristics and protective effects on murine colitis of the strain. The results showed that L. gasseri SHMB 0001 possessed promising potential probiotic characteristics, including good tolerance against artificial gastric and intestinal fluids, adhesion to Caco‐2 cells, susceptibility to antibiotic, no hemolytic activity, and without signs of toxicity or infection in mice. Administration of L. gasseri SHMB 0001 (1 × 108 CFU per gram of mouse weight per day) reduced weight loss, the disease activity index, and colon shortening in mice during murine colitis conditions. Histopathological analysis revealed that L. gasseri SHMB 0001 treatment attenuated epithelial damage and inflammatory infiltration in the colon. L. gasseri SHMB 0001 treatment increased the expression of colonic occludin and claudin‐1 while decreasing the expression of pro‐inflammatory cytokine genes. L. gasseri SHMB 0001 modified the composition and structure of the gut microbiota community and partially recovered the Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways altered by dextran sulfate sodium (DSS). Overall, our results indicated that the human breast milk–derived L. gasseri SHMB 0001 exhibited promising probiotic properties and ameliorative effect on DSS‐induced colitis in mice. L. gasseri SHMB 0001 may be applied as a promising probiotic against intestinal inflammation in the future.Practical ApplicationL. gasseri SHMB 0001 isolated from human breast milk showed good tolerance to gastrointestinal environment, safety, and protective effect against DSS‐induced mice colitis via enforcing gut barrier, downregulating pro‐inflammatory cytokines, and modulating gut microbiota. L. gasseri SHMB 0001 may be a promising probiotic candidate for the treatment of intestinal inflammation.

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Advances in synthetic biology toolboxes paving the way for mechanistic understanding and strain engineering of gut commensal Bacteroides spp. and Clostridium spp.

Cited by 5 publications

References 186 publications

Multiplex genetic manipulations in Clostridium butyricum and Clostridium sporogenes to secrete recombinant antigen proteins for oral-spore vaccination

Multiplex genetic manipulations in Clostridium butyricum and Clostridium sporogenes to secrete recombinant antigen proteins for oral-spore vaccination

The gut microbiota and diabetes: research, translation, and clinical applications – 2023 Diabetes, Diabetes Care, and Diabetologia Expert Forum

Probiotic properties and the ameliorative effect on DSS‐induced colitis of human milk–derived Lactobacillus gasseri SHMB 0001

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