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Luo et al. Microbiome Res Rep 2025;4:10 https://dx.doi.org/10.20517/mrr.2024.57 Page 9 of 25

[87]
sepsis . In 1999, Low et al. made the first attempt to modify the virulence of Salmonella typhimurium by
[88]
removing the msbB gene, which is involved in lipopolysaccharide synthesis . They introduced the
knockout vector pDBMS7, which contains an incomplete fragment of the msbB gene, into Salmonella
typhimurium strain YS501 and used bacterial homologous recombination to replace the wild-type msbB
gene. This resulted in attenuated virulence, which was confirmed in mice and pigs. In a similar approach,
Clairmont et al. deleted the purI gene, an essential enzyme in the purine synthesis pathway, from the
Salmonella genome, creating a purine-deficient strain, VNP20009. This modification limited the bacteria’s
[89]
undesirable bioaccumulation and enhanced its safety profile . The VNP20009 strain demonstrated some
safety in subsequent Phase I clinical studies, although dose-related toxicity was observed, and no significant
[81]
antitumor effects were noted . Another strategy involves targeting Salmonella pathogenicity island 1 (SPI1),
a key virulence factor of Salmonella, whose expression is regulated by ppGpp. Specifically, ppGpp
modulates SPI1 gene expression by activating hilA through the activity-dependent pathway of SpoT .
[90]
Knocking out the relA and spoT genes in Salmonella typhimurium resulted in the ΔppGpp strain, which
proved to be nearly non-toxic in mice, showing potential as a vaccine vector .
[91]
Once the safety concerns are addressed, it will be essential to improve the tumor-targeting and colonization
capabilities of engineered bacteria. Although candidate chassis bacteria have demonstrated significant
colonization abilities in their natural states, the underlying mechanisms are still largely unknown and
challenging to predict. One promising approach to enhance tumor targeting is the incorporation of tumor-
targeting molecules onto the surface of engineered bacteria through synthetic biology. This could
[92]
significantly increase their ability to home in on tumor tissues . For example, the RGD peptide sequence, a
known ligand for αvβ3 integrin, which is overexpressed on various tumor cells and endothelial cells during
tumor angiogenesis, has been used to enhance targeting. By inserting the RGD peptide sequence into the
outer membrane protein A of the attenuated Salmonella strain ΔppGpp, the resulting ΔppGppRGD strain
exhibited strong binding to αvβ3-overexpressing cancer cells and demonstrated efficient targeting of αvβ3-
expressing tumor xenografts in vivo . Similarly, an engineered strain called 2G9-Salmonella was designed
[93]
to specifically infect CD20-positive tumor cells. This was achieved by displaying a single-domain antibody
(VHH) targeting the CD20 antigen on the outer membrane of the SL3261 aroA-deficient Salmonella
strain .
[94]

Drug molecules or protein molecules with antitumor effects carried by engineered bacteria can effectively
kill tumors. For example, the surface of EcN was coupled with the anticancer drug doxorubicin through
pH-sensitive amide bonds, and then ligand-linked with photosensitizer gold nanorods (AuNRs) to form
EcN-Dox-Au microrobots. With the assistance of NIR laser, the AuNRs produced a photothermal effect,
which enhanced the permeability of the tumor cell membrane and promoted the microrobots to deep
tumor. The micro-robots penetrated into the deep tumor tissues. Then, doxorubicin was released under the
acidic condition of the tumor microenvironment to kill the tumor . In another type of scenario, the
[95]
engineered bacteria are designed to express proteins with antitumor effects that act in various ways,
including activating the host immune system, inducing apoptosis, and interfering with tumor cell
metabolism. For example, the Flt3L and OX40L fusion protein expressed by FOLactis promotes DC
maturation and T cell activation, converting cold tumors into hot tumors . In addition, attenuated
[96]
Salmonella typhimurium VNP20009 activates T cells by displaying mPD-1 at the membrane and relieves
tumor cells from immune escape . Lactococcus lactis has been genetically modified to carry the gene
[97]
encoding tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), overcoming its biological half-
[98]
life limitations and demonstrating apoptosis induction in colon cancer cells in vitro . In addition,
engineered Salmonella typhimurium exhibited inhibitory effects on a variety of cancers, including
melanoma, colon and pancreatic cancers, by delivering molecules such as shRNA targeting the inhibin α-