Physiochemically and Genetically Engineered Bacteria: Instructive Design Principles and Diverse Applications

Abstract

With the comprehensive understanding of microorganisms and the rapid advances of physiochemical engineering and bioengineering technologies, scientists are advancing rationally-engineered bacteria as emerging drugs for treating various diseases in clinical disease management. Engineered bacteria specifically refer to advanced physiochemical or genetic technologies in combination with cutting edge nanotechnology or physical technologies, which have been validated to play significant roles in lysing tumors, regulating immunity, influencing the metabolic pathways, etc. However, there has no specific reviews that concurrently cover physiochemically- and genetically-engineered bacteria and their derivatives yet, let alone their distinctive design principles and various functions and applications. Herein, the applications of physiochemically and genetically-engineered bacteria, and classify and discuss significant breakthroughs with an emphasis on their specific design principles and engineering methods objective to different specific uses and diseases beyond cancer is described. The combined strategies for developing in vivo biotherapeutic agents based on these physiochemically- and genetically-engineered bacteria or bacterial derivatives, and elucidated how they repress cancer and other diseases is also underlined. Additionally, the challenges faced by clinical translation and the future development directions are discussed. This review is expected to provide an overall impression on physiochemically- and genetically-engineered bacteria and enlighten more researchers.

Keywords: bacterial derivatives; design principles; diverse applications; genetically‐engineered bacteria; physiochemically‐engineered bacteria.

Figure 1

Schematic illustration of design strategies and disease treatment strategies for chemically and genetically engineered bacteria.

Figure 2

Physicochemically‐engineered strategies for designer bacterial interfaces and bacterial derivatives. A) Schematic diagram of preparation of surface sulfhydryl bacteria B) Schematic illustration of chemical reaction‐mediated covalent localization of bacteria. Reproduced with permission.^{[ 65 ]} Copyright 2022, Springer Nature. C) Schematic illustration of electrochemical coating of bacterial surfaces. Reproduced with permission.^{[ 66 ]} Copyright 2023, Springer Nature. D) Schematic diagram of preparation of artificial‐enzyme‐armed probiotics and its simulation of SOD and CAT antioxidant enzymes. Reproduced with permission.^{[ 68 ]} Copyright 2023, Springer Nature. E) Schematic illustration of the LBL preparation process and the mechanism of LBL treatment of ulcerative colitis. Reproduced with permission.^{[ 70 ]} Copyright 2022, John Wiley and Sons. F) Schematic illustration of biointerface mineralization that generates ultraresistant gut microbes. Reproduced with permission.^{[ 73 ]} Copyright 2023, AAAS. G) Schematic diagram of improving cancer chemotherapy by consuming lung microflora with bioinspired nanomedicine. Reproduced with permission.^{[ 82 ]} Copyright 2023, John Wiley and Sons. H) Schematic diagram of OMV from symbiotic bacteria can regulate versatility in intestinal homeostasis through membrane fusion. Reproduced with permission.^{[ 83 ]} Copyright 2023, AAAS.

Figure 3

Physiochemically‐engineered bacteria for specific tissue or organ adhesion and specific tumor microenvironment regulation. A) Schematic diagram of preparation of coated bacteria and its interaction with host. B) Schematic diagram of EcN bioluminescence and BER fluorescence and their corresponding fluorescence intensity. C) Schematic diagram of EcN count in stool samples, average colon length after different treatments and K level of IL‐6 in serum. Reproduced with permission.^{[ 88 ]} Copyright 2023, Elsevier. D) Schematic diagram of adjuvant characteristics for improving immune stimulation by regulating the physical properties of fungal mannan. Reproduced with permission.^{[ 89 ]} Copyright 2022, Cell Press. E) Schematic for illustrating the construction of Sal@PST/DzMN, and its function mechanisms in tumor tissue for enhanced immunotherapy. Reproduced with permission.^{[ 90 ]} Copyright 2023, John Wiley and Sons. F) Schematic illustration of isolation of extremely effective anticancer bacteria from solid tumors. Reproduced with permission.^{[ 91 ]} Copyright 2023, John Wiley and Sons. G) Preparation of Mø@bac and regulation of tumor immunosuppressive microenvironment mediated by Mø@bac. Reproduced with permission.^{[ 28 ]} Copyright 2023, John Wiley and Sons. H) Schematic diagram on how dressed EcN with a hybrid immunoactive surface and induced antiviral and anticancer double immune responses. Reproduced with permission.^{[ 1 ]} Copyright 2023, John Wiley and Sons. I) Schematic illustration of in situ polymerization‐mediated antigen presentation. Reproduced with permission.^{[ 92 ]} Copyright 2023, American Chemical Society. J) Schematic illustration of enhanced antigen presentation of tumors with engineered Bi for chemo‐immunotherapy. Reproduced with permission.^{[ 93 ]} Copyright 2023, American Chemical Society.

Figure 4

Design strategies for physiochemically‐engineered bacteria‐combined physical therapy. A) Schematic illustration of the bacterial biohybrid microrobots, conjugated with NLs and mNPs. B) Schematic of the NL synthesis. C) Cumulative drug release profile under different conditions. Reproduced with permission.^{[ 94 ]} Copyright 2022, AAAS. D) Schematic illustration of the preparation of LA&LDH and their in‐situ activation by the TME for tumor‐targeted NIR‐II photodynamic cancer therapy. Reproduced with permission.^{[ 96 ]} Copyright 2023, John Wiley and Sons. E) Schematic illustration of preparing tumor‐resident living immunotherapeutics by decorating bacteria with triple immune nanoactivators. F) Schematic illustration of decorated bacteria‐mediated reversal of the tumor immunosuppressive microenvironment. Reproduced with permission.^{[ 97 ]} Copyright 2022, John Wiley and Sons.

Figure 5

Genetic circuit design for oncolysis and improving bacterial colonization and promoting immune regulations. A) Schematic diagram of sequential strain inhibition of strains. B) Genetic diagram of the quorum‐sensing SLC and TA module. Reproduced with permission.^{[ 105 ]} Copyright 2019, AAAS. C) Schematic diagram of intrinsically disordered region required in the B. thetaiotaomicron Rho protein. D) Phase separation of transcription termination factor Rho in commensal bacterium B. thetaiotaomicron governs gene expression and promotes bacterial fitness. Reproduced with permission.^{[ 110 ]} Copyright 2023, AAAS. E) Schematic demonstrating the ProCAR platform. Reproduced with permission.^{[ 115 ]} Copyright 2023, AAAS. F) Diagram of anti‐SasA CAR and CASP11 short hairpin RNA structure in plasmid DNA. G) Schematic illustration of the preparation of pDNA‐laden peptide nanoparticle. H) Schematic illustration of the pPNP coating on an implant (Ti‐pPNP). I) Schematic illustration of the locoregional generation of S. aureus–specific super CAR‐MΦs at the bone‐implant interface for preventing periprosthetic joint infection. Reproduced with permission.^{[ 116 ]} Copyright 2023, AAAS. J) Schematic diagram of genetically engineered bacteria‐derived‐OMV‐based oral tumor vaccine. Reproduced with permission.^{[ 119 ]} Copyright 2022, Springer Nature. K) Schematic diagram of in situ production and release of nano‐vaccine for tumor immunotherapy by the engineered oral bacterial hydrogel. Reproduced with permission.^{[ 71 ]} Copyright 2023, Elsevier. L) Schematic showing the mechanism by which engineered bacteria controllably release constitutively produced PD‐L1 and CTLA‐4 blocking nanobodies intratumorally. M) Lysis circuit diagram in which plux drove the transcription of luxl and ϕX174E genes under a single promoter. Reproduced with permission.^{[ 120 ]} Copyright 2020, Springer Nature. N) Representative anti‐CD11b‐antibody‐stained sections from the lungs, liver, spleen and bone marrow of mice treated with ANP‐C. Reproduced with permission.^{[ 121 ]} Copyright 2023, AAAS.

Figure 6

Schematic illustration of bacterial activation of immune system. A) Nanometer‐sized bacterial OMV enrichment in an anoxic tumor environment. B) Engineered bacteria carrying therapeutic cargo targeting immune cells.

Figure 7

Genetic circuit design for evading host defense and transporting therapeutic payloads, and strategies for fabricating genetically‐engineered microbial chassis and bacteriophage. A) Programmable CAP system for control over bacterial encapsulation and in vivo delivery profiles. Reproduced with permission.^{[ 122 ]} Copyright 2022, Springer Nature. B) Schematic diagram of designing a bacterial vector by genetic engineering. Reproduced with permission.^{[ 126 ]} Copyright 2021, Springer Nature. C) Kaplan–Meier curve (top panel) and therapeutic response (bottom panel) of ANP‐C. D) Representative anti‐CD11b‐antibody‐stained sections from the lungs, liver, spleen and bone marrow of mice treated with ANP‐C. Reproduced with permission.^{[ 127 ]} Copyright 2018, Springer Nature. E) Experimental strategy of engineered native bacteria. Reproduced with permission.^{[ 128 ]} Copyright 2022, Cell Press. F) A hybrid‐glycolysis yeast that disrupts the Embden‐Meyerhof‐Parnas glycolysis pathway and introduced components of the phosphoketolase pathway. G) Schematic diagram of fluxes and gene expression for the electron transport chain and ATP synthase. Reproduced with permission.^{[ 130 ]} Copyright 2023, Cell Press. H) Schematic diagram of phage consortium targeting inhibition of human IBD‐related intestinal microflora symbionts in the treatment of intestinal inflammation. Reproduced with permission.^{[ 135 ]} Copyright 2022, Cell Press. I) Mechanism model diagram of Tad1. Reproduced with permission.^{[ 136 ]} Copyright 2022, Springer Nature. J) Bacteriophages antagonize cGAS‐like bacterial immunity by sequestering immune signaling molecules and acquiring capsid gene mutations. Reproduced with permission.^{[ 137 ]} Copyright 2023, Cell Press.

Figure 8

Modularized multiple‐gene circuit designs for combined treatment actions. A) Schematic illustration of genetic circuit design for programming bacterial lifestyles. B) Representative confocal microscopy image of frozen tumor sections taken from mice in group D3 and M3. Reproduced with permission.^{[ 49 ]} Copyright 2023, Science China Press. C) Schematic diagram of ultrasound‐responsive bacteria in controlling IFN‐γ expression by focused ultrasound and their mechanisms for cancer immunotherapy. Reproduced with permission.^{[ 140 ]} Copyright 2022, Springer Nature. D) Schematic design of ABC sugar transporter. Reproduced with permission.^{[ 141 ]} Copyright 2023, Springer Nature. E) Schematic diagram of engineered bacteria for detecting tumor DNA. F) Schematic diagram of intrinsically disordered region required in the B. thetaiotaomicron Rho protein. Reproduced with permission.^{[ 142 ]} Copyright 2023, AAAS. G) Schematic diagram of sequence homology of GvpA/B. Reproduced with permission.^{[ 144 ]} Copyright 2018, Springer Nature. H) Diagram of the construct from a with Axe‐Txe47 added, creating pBAD‐bARGSer‐AxeTxe, to enable plasmid maintenance in the absence of antibiotics. I) Diagram of the in vivo protocol for assessing in situ bARGSer expression in tumors. Reproduced with permission.^{[ 145 ]} Copyright 2023, Springer Nature.

Figure 9

Schematic illustration of physiochemically‐ or/and genetically‐engineered bacteria combined with physical therapy. Genetically and physiochemically‐combined engineered bacteria can be combined with physical therapies such as PDT, PTT, SDT and RT to enhance tumor targeting and permeability, and produce cytotoxic reactive oxygen species, thus destroying tumor cells.

Figure 10

Genetic circuit design for executing biosensing. A) An illustration of AMF‐Bac, comprising five modules. active navigation, signal decoding, signal feedback, signal process, and signal output. B) An illustration of the assembly process and working principle of AMF‐Bac. C) Schematic illustration of the type I IFN pathway and adaptive immunity activated by AMF‐Bac. Reproduced with permission.^{[ 154 ]} Copyright 2025, Springer Nature.