The fast-developing synthetic biology (SB) has provided many genetic tools to reprogram and engineer cells for improved performance, novel functions, and diverse applications. Such cell engineering resources can play a critical role in the research and development of novel therapeutics. However, there are certain limitations and challenges in applying genetically engineered cells in clinical practice. This literature review updates the recent advances in biomedical applications, including diagnosis, treatment, and drug development, of SB-inspired cell engineering. It describes technologies and relevant examples in a clinical and experimental setup that may significantly impact the biomedicine field. At last, this review concludes the results with future directions to optimize the performances of synthetic gene circuits to regulate the therapeutic activities of cell-based tools in specific diseases. Appropriate interface design makes modules detachable, improving their stability and compatibility in different environments. Whole-genome synthesis, redesign, and reconstruction allow generating of artificial biomimetic/minimal cells, while atomic or module/domain-level protein engineering offers high specificity and controllability over more complex functions. Meanwhile, innovative modification and creative assembly of signaling and communication systems can further optimize cell engineering. In addition, mathematical models for functional module simulation and prediction help streamline circuit design for the development of invaluable research tools.10Modern science is becoming highly multidisciplinary to promote the innovation of new theories and technologies. Synthetic biology (SB) is one of the fastest-growing and most dynamic multidisciplinary fields comprising different projects, approaches, and definitions.12 SB-inspired cell engineering can be commercialized for medical and healthcare. The main goal of medical SB is to genetically modify cells and redesign/synthesize regulatory systems to assist in disease diagnosis and treatment.14 Such cell-based sensors and therapies could achieve more specific detection and treat diseases on a much broader scale.16 Medical SB utilizes rationally designed therapeutic gene circuits, which can be implanted into the human body via vectors to correct the targeted defect. SB-inspired cell engineering with efficient design, specificity, and control can maximize therapeutic effects while minimizing side effects.Engineered cells can have various applications, including the production of industrial or pharmaceutical compounds and increasing agricultural productivity by alleviating pathogens and environmental challenges.26 The ongoing development of engineered cells-derived tools has attracted substantial public attention. Accordingly, this review summarizes the recent preclinical, clinical, and experimental data to discuss the benefits and limitations of SB techniques in cell engineering and biomedical research. Here, we elaborate on various SB-driven cell devices in diagnosis, treatment, and drug development. Also, we address the current and potential future challenges for SB and cell engineering in medical applications.There are several reviews on SB technologies for developing therapeutic strategies or improving clinical effectiveness; most of them covered different aspects or specific approaches of SB in medical applications Fig. .17–26 Th28 Here, we describe the emerging advances of SB-inspired cell engineering approaches in diagnostics that may speed up data acquisition with improved sensitivity, specificity, real-time quantification, and accuracy at a substantially lower cost.Accurate disease characterization and monitoring are critical for therapeutic planning and epidemiological records. However, most traditional diagnostic procedures involving pathological examination of biopsies cannot precisely detect the disease state in real-time and are time-consuming. Thus, there is an urgent need to enhance current medical diagnostics capabilities. SB-inspired cell engineering and programmable biosensors have focused on high-throughput biomolecular/pathogens identification and probing, which can improve clinical diagnosis and disease management protocols Fig. .25,27,2830 Bacterial biosensors can rapidly adapt to test environments to sense biological substances both in vitro and in vivo. Bacterial sensors are preferred for selectivity, sensitivity, automation, miniaturization, and accuracy.31 Engineered bacterial sensors have been successfully used for the analysis of environmental pollutants.33 Many bacterial sensors with specific and sensitive gene circuits for the identification of biological signals can be used to detect widespread clinically relevant biomarkers in vitro, such as in serum and urine.34 In particular, certain symbiotic bacteria that colonize specific tissues and organs are believed to be feasible candidates for cell engineering to serve as in vivo tools for real-time diagnosis.36In the past several decades, bacteria-based biosensors have been extensively used in the food, environment, and medical industries.38 For instance, Zur and ZntR zinc-responsive transcription factors (TFs) modules respond to extracellular concentrations of zinc and produce visible pigments; Escherichia coli cells integrated with this circuit can sense serum zinc levels to identify zinc deficiency.37 Such a bacterial sensor impacts multiple metabolite production pathways through a single TF, reduces diagnostic time, and explains results through visual inspection. Similarly, the glucose-responsive integrase expression cassette (PCpxP–integrase-pA), which expresses the green fluorescent protein (GFP), is an ideal reporter to test glycosuria; hydrogel beads immobilized bacteria containing this module can evaluate urine glucose levels in diabetic patients with high sensitivity and specificity.38Most bacterial-based diagnostic biosensors produce an easily measurable output in the form of a pigment or fluorescent protein upon identification of a target signal.39 QS signaling molecule profiling from specific pathogens are proper biomarkers for bacterial diagnosis; acyl-homoserine lactone (AHL), 2-heptyl-4-quinolone (HHQ), and acyl-homoserine lactones (acyl HSLs) have been used to identify P. aeruginosa infection.42 Importantly, QS signals can be directly monitored by genetically engineered non-pathogenic bacteria.43 Mao et al. reprogrammed the probiotic Lactococcus lactis by introducing a chimeric CqsS-NisK quorum sensing block for the detection of cholera autoinducer 1 (CAI-1) from Vibrio cholerae.44 This system can easily detect V. cholerae in fecal samples and intestinal environments. Another research group generated a probiotic Lactobacillus reuteri strain integrated with an agr quorum sensing (agrQS) biosensor from Staphylococcus aureus, which could identify quorum sensing molecule autoinducer peptide-I (AIP-I) from common pathogenic Staphylococcus sp with high sensitivity in the nanomolar range.45 Engineered probiotic bacteria integrated with a rational antimicrobial module can be novel therapeutic “robots” to track and eliminate pathogenic bacteria.Quorum sensing (QS) systems that determine population behavior are widely distributed among bacteria and have a long evolutionary history.46 The well-defined TCSs have been used for diagnostic signal analysis.47 Holowko et al. integrated three V. cholerae-derived TCS elements CqsS, LuxU, and LuxO with a dCAS9-based GFP-reporter system into E. coli to construct a biosensor with a high sensitivity for QS-ligand cholera CAI-1 indicating the presence and proliferation of V. cholera.48 Previous studies engineered thiosulfate/tetrathionate-responsive E.coli strain by combining TCS sensors ThsSR/TtrSR with pre-installed reporter system or phage lambda cI/Cro-based memory system as potential therapeutics and diagnostics for inflammation50 Recently, a new TCS-based whole-cell biosensor was designed by adopting E. coli NarX-NarL to sense nitrate levels, a biomarker of gut inflammation.51 This highly specific nitrate-responsive genetic circuit allows non-invasive diagnosis of inflammation-related diseases.Two-component system (TCS), including a membrane-bound histidine kinase for signal sensing/transduction and a cognate intracellular response regulator to activate downstream gene expression, is a key mechanism of how bacteria perceive and respond to the environment.52 Another exquisitely engineered micro-bio-electronic device (IMBED) was developed by Mimee et al. They efficiently combined a bacterial-electronic system with SB circuits, electronic sensor, and wireless transmission platform to detect excess heme in blood samples offering rapid in situ diagnosis of gastrointestinal bleeding in pigs.53In addition to in vitro diagnosis, engineered bacteria systems can also be exploited for the diagnosis of complex internal environments. Biomolecular monitoring of the gastrointestinal environment is often limited by access and complexity of the environment. Danino et al. developed a PROP-Z (programmable probiotics with lacZ) platform using a Nissle 1917 (EcN) derived bacterium with a series of expression cassettes. This orally administered probiotic can produce detectable signals in urine reliably indicating hepatic tumors.Microbial biosensors offer inexpensive and feasible disease detection strategies. Similar to engineered bacteria, eukaryotic cell-based biosensors have also been developed for real-time clinical applications.56 One of the advantages of eukaryotic cell biosensors is to incorporate G-protein-coupled receptors (GPCRs) which greatly expands their functional repertoire.59 Diagnostic yeast biosensors have often been used to detect the metabolic rate of oxygen, glucose, lactate, and ethanol through enzyme-catalyzed oxidation-reduction reactions; the generated electrical energy is measured by an implemented electrode which rapidly generates signals with high sensitivity and specificity. A d-lactate-selective biosensor was developed from the debris of the recombinant thermotolerant methylotrophic yeast H. polymorpha. This biosensor overproduces d-lactate: cytochrome c-oxidoreductase (DLDH) and has high selectivity and effective stability for d-lactate due to the deletion of cytochrome c-oxidoreductase  in the C-105 strain genotype (gcr1 catX).60 The same research group further suggested that enriching the flavocytochrome b2 (FC b2) module incorporated H. polymorpha cells by enzyme-bound nanoparticles can improve amperometric current responses.61The number of yeast/fungi-based diagnostic biosensors is growing. Instead of using optical signals such as fluorescence, luminescence, and colorimetry, many yeast-based biosensors utilize electrochemical elements  and growth indicating modules .S. cerevisiae-based olfactory reporter by assembling the endogenous galactose-response system and the gene encoding acetyltransferase I . This system produces enough amount of isoamyl acetate (banana flavor) in response to galactose and is suitable for low-cost non-quantitative/semi-quantitative diagnosis.62 Furthermore, biological paper analytical devices (bioPADs) were created by embedding this bioengineered fluorescent yeast biosensor into a portable paper substrate. This simple but sensitive test strip can detect doxycycline in complex matrices such as in human urine and raw bovine serum.63 It is foreseeable that sustainable developments in fungal genome engineering would take yeast biosensors toward precise, accurate, and cost-effective editing.Combining new ideas and technologies based on existing yeast-based biosensors stimulates more innovations. Holly V. Goodson’s research group designed an 64 With the recent technological advances, a few mammalian cell-based diagnostics have been developed. For example, HEK293 cell-based biosensor with a synthetic histamine-responsive signal module  can detect allergies from blood samples of high-risk allergic patients by sensing the extracellular histamine levels and triggering the expression of cAMP-induced SEAP .65 Such engineered mammalian cell systems can be used in high-throughput diagnostic applications such as testing novel drugs for allergic reactions. Many diseases are preceded by a period of asymptomatic pathogenesis and thus require reliable indirect molecular diagnosis methods. Accordingly, Martin Fussenegger’s research group designed a novel HEK293 cell for in vivo diagnostics by employing a calcium-sensing receptor constructed with a synthetic signaling pathway. In response to the increased calcium level in hypercalcemia, this system generates black pigment melanin on the skin via transgenic tyrosinase-mediated enzymatic reaction and thus is ideal for naked-eye detection.66Based on the availability and compatibility of endogenous signal-transduction biomarkers, engineered mammalian cells are more suitable for certain diagnostics applications over bacterial and yeast biosensors.67 Monkey kidney cells (Vero cells) are sensitive to verotoxin Stx (Shiga-like toxins) due to their overproduced Stx receptor GB3 (globotriaosylceramide).68 Bioengineered Vero cells constructed in collagen matrix demonstrated better cell maturation and improved sensitivity to Stx and thus could detect Shiga-like toxin-producing pathogenic bacteria such as Salmonella, Listeria, Citrobacter, Serratia, and Hafnia.69 IgE-mediated vasoactive amines including histamine and serotonin degranulation are characteristics of basophil or mast cells, which make them a good choice for cell-based biosensor development.70 Jiang et al. reported an electrochemical sensor based on rat basophilic leukemia (RBL-2H3) cells exploited on folding paper-based equipment for casein detection.71 Certainly, engineered mammalian cell-based devices for therapeutic and diagnostic applications are a growing area of interest. The diverse cell behaviors in response to different compounds such as antibiotics, antibodies, and chemokines make mammalian cells attractive model for building diagnostic biosensors. Meanwhile, the complexity and packaging of eukaryotic genome have also to be considered when using them for such applications.Except for epithelial cell lines, other mammalian cells including macrophages, neurons cell, mast cells, and B lymphocytes can also be exploited for biosensor applications. Based on the pattern of signal detection and test objective, a specific assay combined with proper cell lines can be used to assess pathogens, toxins, metabolic activity, or even cell proliferation.73 Bioluminescent reporter-based phages have been applied for the diagnosis of pathogens such as Bacillus anthracis,75Vibrio parahaemolyticus,76Salmonella typhimurium,77pathogenic Escherichia coli,79 and Staphylococcus aureus.80 For example, an engineered Yersinia pestis phage was used for the detection of an etiologic agent in bubonic plague.81 A microscopy-based method in association with the fluoromycobacteriophage mCherrybombϕ was used to diagnose M. tuberculosis.82 Recently, Meile et al. engineered a set of nanoluciferase (NLuc)-based phage reporters to identify and differentiate Listeria monocytogenes in potentially contaminated milk, cold food, and other eatable samples.83 These NLuc-based reporter phages enabled rapid and ultrasensitive detection of one CFU of L. monocytogenes in 25 g of artificially contaminated natural food in less than 24 h.Although cell-based biosensor diagnostics can fulfill most needs of clinical applications, the rational design of engineered phages promises some other advantages. Phages are viruses that infect bacteria by adhering to specific host cell surface receptors. A simple model for reporter phage design is that the presence of the correct epitope induces the transfer of the phage genome into bacteria, triggering bioluminescence from luciferase in the target bacteria.84 Niyomdecha et al. used an M13 bacteriophage-modified electrode for the detection of Salmonella spp.: this biosensor binds to target bacteria through the amino acid groups on the filamentous phage.85 Farooq et al. mobilized S. aureus-specific lytic phages in surface-modified bacterial cellulose (BC) matrix conducted by carboxylated multi-walled carbon nanotube (c-MWCNTs). The functionalized electrode could detect S. aureus through impedance measurements.86 Lately, gold electrodes were used as a transducer with chemically modified M13 phages to monitor caspase-3 activity in apoptotic HeLa cells with nanomole range sensitivity.87 Such bacterial cell and phage-based biosensors can effectively sense disease-associated biomarkers, paving the way for next-generation of medical diagnostics.Phages can also be exploited as bioelectrode elements by immobilizing them to an electrode surface or utilizing lytic phages to infect target bacteria to release bacterial cell contents such as adenosine triphosphate (ATP) for electrochemical detection.90 Traditional cancer treatments such as surgical intervention, radiation, and chemotherapeutic drugs have several limitations, including high cytotoxicity and low selectivity.94 SB approaches for cancer treatment are the most vivid and promising trends that enable precise timing, location, and dosing of the drug. Accordingly, engineered mammalian and bacterial cells have been increasingly recognized as promising anticancer devices.97More than 19 million new cases of cancer are diagnosed every year, making it one of the most devastating diseases worldwide.101 Certain bacterial species, including Streptococcus, Caulobacter, Escherichia, Bifidobacterium, and Salmonella, can automatically sense and accumulate in tumor tissues after external administration  to produce cytotoxic toxins or release delivery compounds.102 One of the major advantages of bacterial therapies for cancer is that they can be easily manipulated for the effective delivery of anticancer agents.103 Based on the reported studies, bacterial therapies for cancer majorly involves three types of mechanisms: expression of toxic proteins to directly kill cancer cells, immunotherapeutics that stimulate the immune system to kill cancer cells or the transfer of therapeutic drug delivery vehicles  from enterohemorrhagic E. coli can inhibit cancer cell growth by blocking mitosis.109 The bifunctional Pseudomonas aeruginosa exotoxin T (ExoT) consists of a GTPase-activating protein (GAP) and an ADP-ribosyltransferase encoded from the N- and C-terminus, respectively.111 The GAP protein guides the mitochondrial dysfunction in the host cell through activating host caspases 3 and 9, while the ADP-ribosyltransferase domain upon activating the CT10 regulator of the kinase (Crk) leads to atypical anoikis apoptosis.113 Clostridium perfringens enterotoxin (CPE) can treat colorectal cancers by binding to claudin-3 on the tumor cell surface forming a multi-protein membrane pore complex, altering cellular osmotic equilibrium that leads to cell lysis.115 To improve the specific recognition of bacterial toxins for cancer cells, they can be reprogrammed either to conjugate with tumor surface antigens or ligands that selectively bind to the target cell receptor.116 For instance, P. aeruginosa exotoxin A (PE) was fused to anti-CD25 to form a novel recombinant toxin.117 Native diphtheria toxin (DT) was linked with the B-subunit of Shiga-like toxin (STXB) to improve its affinity for breast cancer cells and showed improved cytotoxicity against Gb3-expressing cancer cells in a dose-dependent manner.118 Besides, bacterial spores can also be exploited as anticancer agents as they can specifically germinate inside the necrotic/hypoxic tissues such as a solid tumor.120Clostridium histolyticum spores showed considerable oncolysis and tumor regression effects in murine models.121 However, the original bacterial spores might not eliminate cancer cells and would require genetic modifications or combinational use with other anticancer strategies such as radiotherapy and chemotherapy.Natural bacterial toxins are emerging as potential anticancer agents to treat established solid tumors.102 Selective expression of cytokines can induce proper immune cells to clear tumors via multiple mechanisms with fewer side effects.122 Interleukin-2 (IL-2) and IL-18 are the most crucial components of cancer-related inflammation and participate in the immune regulation network. They activate the antitumor cytotoxicity of T cells and natural killer cells.124 Engineered Salmonella producing IL-2 and IL-18 was shown to inhibit tumor growth in animal experiments.126 Targeting programmed cell death protein-ligand 1 (PD-L1) and cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) by immune-checkpoint inhibitors is a revolutionary cancer immunotherapy strategy but has side effects such as fatigue, skin rashes, and endocrine disorders.128 Increasing l-arginine levels in tumor tissue can activate the response of immune-checkpoint inhibitors. Engineered E. coli Nissle 1917 strain after accumulation in the tumor tissue converted ammonia to l-arginine recruiting tumor-infiltrating T cells showing a remarkable antitumor effect.129 Gurbatri et al. engineered a probiotic bacteria system to tightly control the release of nanobodies, which targeted PD-L1 and CTLA-4 and activated T cells.130 In another case, engineered Salmonella (△ppGpp) was utilized to secret the development endothelial locus 1 (Del-1) protein promoting the recruitment of M1 macrophage to tumors.131Expression of exogenous cytotoxic proteins may trigger the host immune response raising safety concerns while engineered bacteria to induce specific cytokines can be an alternative anticancer strategy.105 Hu et al. used self-assembled synthetic nanoparticles coated attenuated bacteria to develop a novel oral DNA vaccine to express vascular VEGFR2 and specific antigens.132 This engineered bacteria could activate T cells and induce the production of cytokine for efficacious cancer immunotherapy. In 2017, researchers exploited demi-bacteria (DB) from Bacillus for the efficient accommodation of antigen and cytidine-phosphate-guanosine (CpG) adjuvants, inducing synergistic cellular and humoral immune responses in vivo.133 In combination with traditional chemotherapy and/or phototherapy, SB approaches can significantly promote the anticancer therapeutic outcome. For instance, attenuated Salmonella coated with degradable polydopamine was used for the delivery of photothermal agents to tumors.134 To efficaciously transport the chemotherapeutic drugs to the intestinal environment, Bacillus coagulans spores were modified with deoxycholic acid (DA) and then load them with drugs serving as autonomous nanoparticle (NP) producers.135 In addition, the bacteria-derived outer membrane vesicles (OMVs) have been explored as drug carriers in vaccine developments and cancer therapies.137 Based on oxygenated PDT and immunotherapy, Zhang et al. combined Chlorin e6 (Ce6) with negatively charged catalase (CAT) to form a molecular scaffold (CAT-Ce6) and transported it using aPDL1 modified (OMV-aPDL1) Salmonella OMVs to inhibit tumor growth.138The deoxygenated inflammatory microenvironment of a tumor is distinct from most other tissues and therefore allows selective accumulation of obligate anaerobes in the tumor for the delivery of bacteria-based vaccine or drug.139 Compared with traditional treatment and engineered bacterial and cell-based therapies have two major advantages: (1) a large number of basic research, clinical trials, and SB can make rapid development in this field, and (2) it offers long-term precise regulation and coordination of immune response in vivo. Current cancer cell therapy includes chimeric antigen receptor (CAR)-T cell, T-cell receptor (TCR) T cell, CAR-natural killer (NK)/NKT cells, tumor-infiltrating lymphocyte (TIL), choline transporter‐like proteins, etc.140 Among them, CAT-T-cell therapy is one of the most innovative gene therapies with the greatest breakthrough in the field.142 At present, five cell therapies have been approved by the Food and Drug Administration (FDA) in July 2021 after clinical trials and all of them are CAR-T-cell therapies (Table 140 Claudin18.2-specific CAR-T cells (CT041) showed good efficacy against digestive tract solid tumors with 48.6% objective remission rate and 73.0% disease control rate exceeding the best result of CAR-T-cell therapy for hematoma treatment (remission rate <30%).143 In the last decade, CAR has been developed to the fourth generation, the present CAR consists of a single-chain variable fragment (scFv), a flexible hinge, a transmembrane domain (CD8a), one or more co-stimulatory domains, and an intracellular CD3ζ domain from the T-cell receptor.139 Here, we discuss the main SB strategies for antitumor CAR engineering, including switchable control, logic gates, and armored CARs, and then discuss the future possible development of cell therapies from a SB perspective  kill switch is one of the most successful designs, which has been used in several clinical trials for CAR T-cell therapies.146 The iCasp9 switch consists of a domain encoding chemically inducible dimerization system derived from FKBP12 (a rapamycin-binding protein) and split caspase-9 protein; the small-molecule AP1903 can induce the dimerization of FKBP12-caspase-9 causing apoptosis. CAR-T cells carrying the iCasp9 switch have shown effective curative effects in several clinical trials.148 There are other non-caspase strategies, such as combining MyD88 and CD40, based on the FKBP12/AP1903 dimerization system, that can overcome the limitation of elimination from the body.149Engineered switches are powerful control systems that minimize the life-threatening toxicities and other unfavorable side effects of CAR-T cells and have two types, named ON and OFF switches.144 All of the antigens present in AND Gate are needed for CAR-T-cell activation. Another common strategy is to separate and fuse the activation signal (CD3ζ) and the co-stimulatory domain to two different antigen-recognizing receptors, such as SynNotch system,150 bispecific tandem CAR (TanCAR)151 and masked CAR (mCAR),152 however, this also has the disadvantage of slow activation kinetics. Following the principle “A and not B”, NOT Gate can restrict off-target attacks of T cells reducing unintended immune attacks; however, this has not been validated in clinical trials so far. Alternatively, the purpose of OR Gate is to overcome antigen heterogeneity and antigen escape, thus multiple-targeting CAR-T therapy is developed by fusing different CAR-T cells or generating specific T cells with different CAR suits. The other two alternative strategies are combining different antigen targeting adapters and generation of bispecific CAR-T cells consisting of two or more different scFvs; however, the concerns of potential cytokine release syndrome, neurotoxicity, and B-cell aplasia remain.144Considering the sophisticated tumor microenvironment and nonspecific antigens on tumor cells, more precise treatments are necessary to balance safety and efficacy. Improved genetic logic circuits, including AND Gate, NOT Gate, and OR Gate, were designed to solve these problems.154 Engineered CAR-T cells can secrete extracellular cytokines , antibodies, and bacterial enzymes increasing the prodrug concentration at the disease site.158C. perfringens neuraminidase (CpNA) can remove sialic acid residues from the target cells. CpNA-restrained CAR-T cells exhibited superior effector function and cytotoxicity in vitro, showing enhanced antitumor efficacy in a Nalm-6 xenograft model of leukemia, xenograft model of glioblastoma, and syngeneic model of melanoma.159 Enabled CAR-T cells with additional natural or genetically modified transmembrane proteins can have more physiological functions such as signal transduction, recognition, and attachment. Kuhn et al. developed CAR-T cells to produce CD40L, a TNF-associated activation protein (TRAP), to recruit and activate immune effectors.160 Moreover, engineered CAR-T cells disturbing the intracellular transduction of inhibitory signaling pathways is another powerful strategy to inhibit the expression of immune-checkpoint molecules, including short-hairpin RNAs or CRISPR-Cas9.162Another strategy for enhancing the antitumor efficacy of the CAR-T cells is to delete or express immunologic factors rather than CAR, named “Armored” CAR-T cells.Although CAR-T therapy offers spectacular therapeutic opportunities against cancer, it has certain limitations such as tumor antigen escape relapse lowering the therapeutic efficacy. To conquer such challenges, novel strategies to optimize the CAR-T cells are needed for increased safety.Certainly, the developments in SB will continue to promote the progress of tumor treatments; designing subtle gene circuits and finding novel biological components and the right targets could be the main endeavors. Meanwhile, controlling biosafety and reducing in vivo toxicity will also be necessary to balance cost and effectiveness.163 Pathogenic infections including those from bacteria, fungi, and viruses are treated by conventional antibiotic therapy, however, many antibiotics have side effects causing intestinal flora disorder, hearing impairment, nephrotoxicity, and allergic reactions. Moreover, the emergence of multidrug-resistant (MDR) bacteria urgently demands new treatment strategies.165 The developing SB could also provide efficient, accurate, and cost-effective tools to treat infectious diseases. Here, we mainly discuss novel strategies against infectious diseases from the SB perspective.Infectious diseases are the major reasons for death and disability worldwide.168 Thus, engineered bacteria can be exploited for novel antimicrobial biotherapeutics. Jana et al. introduced the arabinose-induced Type VI Secretion System (T6SS) into Vibrio natriegens, a safe bacterium, to manipulate the effector against competitors.169 Ting et al. developed programmed inhibitor cells (PICs), a new system expressing surface nanobodies to mediate antigen-specific cell-cell adhesion, exerting T6SS antimicrobial activity directly against selected bacteria.170 Bacteria use chemical language, termed quorum sensing (QS), to communicate in the community and regulate cell density.168 Probiotic E. coli designed to produce AI-2, which is naturally produced by Vibrio cholera, caused significant growth inhibition of V. cholerae and increased the survival rate of infected animals.171 Engineered Δalr ΔdadX E. coli Nissle 1917 strain can produce Dispersin B, Pyocin S5, Lysin E7, and sense autoinducer 3OC12HSL secreted by P. aeruginosa.172 In the presence of 3OC12HSL, this E. coli strain triggers E7 lysozyme-mediated self-lysis releasing Pyocin S5 and Dispersin B that kills P. aeruginosa. In another case, attenuated human lung pathogen Mycoplasma pneumonia was reformed to secrete anti-biofilm microbial enzymes that degraded Staphylococcus aureus biofilm both in in vitro and in vivo.173 Lysostaphin-producing Staphylococcus simulans could treat methicillin-susceptible and methicillin-resistant Staphylococcus aureus infections.174 Although bacteria can be easily tailored to combat microbial pathogens, their use is limited by the on/off switch of effector release, potential toxic effects in human cells, and long-term stability.175 A new research direction has evolved combining Computer-Based Frameworks and SB for the discover of potential antimicrobial peptide (AMP).176Bacteria evolved with a variety of molecular weapons to kill or interfere with competitors, such as bacteriocins, effector proteins, and other chemicals.178 Furthermore, engineered bacteriophage may overcome the limitations of frequent escape of bacterial hosts by modifying cell-wall related receptors, CRISPR-Cas disruption, or restriction-modification systems. E. coli bacteriophage T7 encoding depolymerase Dispersin B (T7DspB) could specifically hydrolyze polymeric b-1,6-N-acetyl-D-glucosamine (PNAG) of bacterial biofilms reducing biofilm formation of E. coli.179 Masuda et al. generated recombinant lnqQ-T7 phage by integrating lnqQ  into the T7 phage genome, which significantly inhibited lacticin Q-susceptible Bacillus coagulans and E. coli.180 Hsu et al. engineered λ phage to express transcriptional repressor of Shiga toxin (Stx), which successfully reduced Shiga toxin production in the E. coli population colonizing the intestinal tract model.181 The phages can also effectively transport molecules  to targeted bacteria. Staphylococcus aureus phage encoding an antimicrobial small acid-soluble spore protein (SASP) was developed to kill S. aureus.182 This engineered phage (SASPject PT1.2) showed 100% activity against the 225 geographically diverse S. aureus isolates. However, bacteriophage treatment will inevitably lead to bacteriophage resistance and serious side effects such as the release of bacterial endotoxins. Also, bacteriophage is highly specific and attacks limited bacteria. In future, SB and genome engineering can expand the scope of phage therapy.182Producing selective endolysins (lysins) to break bacterial cells, bacteriophages are one of the most effective therapeutic approaches against both Gram-positive and Gram-negative bacteria.26 Wei et al. engineered Lactobacillus jensenii, a commensal bacterium that colonizes the cervicovaginal mucosa, to prevent HIV transmission; the engineered strain secretes two-domain CD4 (2D CD4) proteins,183 which associate with HIV type 1 (HIV-1) gp120 and inhibit HIV-1 invasion. Similarly, the probiotic bacterium E. coli Nissle 1917 was designed to secret HIV-gp41-hemolysin peptides, which blocks HIV fusion and subsequent invasion.184 This engineered strain could continuously and stably colonize the colon and cecum and released anti-HIV peptides for several months in mice. Furthermore, a genetically modified Lactobacillus acidophilus ATCC 4356 expressing human CD4 receptors on its surface can adsorb HIV-1 particles.185 The results showed that CD4-carrying bacteria could capture HIV-1 particles and attenuate infection efficiency in vitro. Martins et al. developed a potential microfluidic attenuator containing engineered E. coli to trap Ebola viruses in human blood.186 Along these lines, incorporating engineered probiotics into the vaginal microbiota can inhibit the spread of viruses.SB-based tools can also contribute to the quick diagnosis, therapy, and prevention of viral infections.188 urgently requiring an efficient vaccine. Chen et al. engineered yeast to stably express the SARS-CoV-2 receptor binding domain (RBD) without impacting protein structure and functionality, which can be a promising candidate vaccine for COVID-19.189 Furthermore, the yeast expressed RBD combined with the 3M-052-alum provoked powerful immune response against SARS-CoV-2 in rhesus monkeys.190 Apart from recombinant proteins, nucleic acid-based (such as messenger RNA) vaccines are potential candidates against SARS-CoV-2. mRNA vaccines can transform host cells into “protein factories”, providing in situ the protein for a balanced immune response for both cellular and humoral immunity. In 2021, clinical trials demonstrated that 2 doses of mRNA vaccine BNT162b2 successfully induced a persistent germinal center B-cell response and robust humoral immunity. This made BNT162b2 to be the first licensed mRNA vaccine against COVID-19 by FDA for people over 16 years of age. Although a considerable number of mRNA vaccines have recently been developed, including PTX-COVID-19-B, SYS6006, and mRNA-LNP, they have several limitations including allergic reactions, uncertain immunity length, and difficult storage.191 Notably, SB tools also can help predict and identify the emerging SARS-CoV-2 variants. Based on reverse genetics, Thao et al. developed a rapid and robust yeast-based synthetic genomics platform for the genetic reconstruction of SARS-CoV-2, which can help generate and functionally characterize evolving viral variants in real-time.192 Scientists have used yeast screening to identify antibody escape mutants of SARS-CoV-2 S RBD to provide new therapeutic strategies against SARS-CoV-2. Importantly, viruses are evolutionarily autonomous; the emergence of new mutations led to the rapid global evolution of influenza viruses.194 Accordingly, the virus evolutionary genetic algorithm should be applied for the development of next-generation SB tools.Recently, COVID-19 (SARS-CoV-2) pandemic became a serious concern for public health safety,197 It is now well-established that human microbiome disturbance can lead to many chronic diseases, such as colorectal cancer (CRC), autism, and diabetes mellitus (DM).201 Microbial population modulation is a new treatment strategy for intestinal and neurodegenerative diseases , which causes gut dysbiosis, is a major cause of morbidity and death worldwide.203 Supplementation with Firmicutes, which convert primary bile acids to secondary bile acids, can significantly lower the growth and virulence of C. difficile by competitively colonizing the infected gut.204 Microbiome product Seres SER-262, which was isolated from healthy donors, could eradicate C. difficile infection. Altered composition, function, and metabolites of the intestinal flora have been associated with the progression of inflammatory bowel disease (IBD).205 Traditional IBD treatment is limited by side effects and complications. Lee et al. developed a hyaluronic acid-bilirubin nanodrug (HABN) which after accumulation restored the epithelial barrier alleviating inflamed colonic epithelia by restoring gut microbiota balance.206 Whelan et al. engineered the probiotic bacterium E.coli Nissle 1917 for the treatment of IBD, which secretes nematode immunomodulator cystine in the intestine.207 This modified probiotic significantly inhibited gut inflammation in murine acute colitis and can potentially treat other similar diseases. A Lactobacillus probiotic was engineered to produce anti-inflammatory molecules, in particular IL-10, in response to murine colitis.208 An engineered E. coli, named EcNL4, improved intestinal microbiota and thereby colitis in mice through the sustainable release of (R)-3-hydroxybutyric acid (3HB).209 An E. coli strain, named SYNB1618, expressing the phenylalanine ammonia-lyase and L-amino acid deaminase genes was engineered to consume phenylalanine in the gut. Overall, the above studies indicated that engineered bacteria can be potentially used to treat rare metabolic diseases.210 Emerging evidence closely associates gut dysbiosis with the development of neurodegenerative diseases through the gut–microbiota–brain axis.212 Wu et al. showed that Enterobacteria infection strongly exacerbated neurodegeneration by promoting the recruitment of immune hemocytes to the brain.213 Being functionally related to the brain immune system, the intestine has been termed the “second brain”. A study showed that oral administration of engineered MG136-pMG36e-GLP-1 strain derived from Lactococcus lactis MG1363 continuously produced Glucagon-like peptide-1 (GLP-1), which dramatically reduced lipopolysaccharide (LPS)-induced memory impairment and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced motor dysfunction, and the abundance of Enterococcus and Proteus pathogens in mice.215 This strain could be a novel intervention in AD and PD.221 Notably, probiotics can compete with pathogenic bacteria for nutrients and binding sites and secrete/produce bacterio-toxins which repress the invasion and adhesion of pathogens.218 Oral bacteriotherapy with SLAB51 probiotic and p62 (SQSTM1)-engineered lactic acid bacteria inhibited the progression of AD, indicating the positive effect of the probiotic intervention in neuronal diseases.219 So far, Lactobacillus and Bifidobacterium strains are the most promising candidates for improving cognitive functioning by reducing the levels of oxidative and inflammatory biomarkers in AD.220Neurodegenerative diseases such as Parkinson’s disease (PD), Alzheimer’s disease (AD), and amyotrophic lateral sclerosis (ALS) have similar features.Lactobacillus gasseri ATCC 33323 to secrete GLP-1 (1–37) to stimulate the conversion of intestinal epithelial cells into insulin-secreting cells in a diabetic rat model. They showed that rats fed daily with engineered bacteria developed insulin-producing cells in the upper intestine, sufficient enough to ameliorate hyperglycemia.214 A recent study showed that an engineered strain MG1363-pMG36e-GLP-1 (M-GLP-1) dramatically decreased body weight and improved glucose intolerance in high-fat diet (HFD) obese mice.215 May-Zhang et al. developed a novel E. coli Nissle 1917 strain expressing N-acyl phosphatidylethanolamines, which are precursors of endogenous lipid satiety factor N-acylethanolamides; the strain significantly reduced the levels of adiposity, hepatic triglycerides, and fatty acid synthesis lowering serum cholesterol levels and extent of necrosis in atherosclerotic mice.217Engineered commensal bacteria also have significant potential in other chronic diseases, i.e., DM and Atherosclerosis (AS). Duan et al. engineered 221 The long-term persistence, potential sensitization, and influence of engineered probiotics on native gut microbiota are yet to be completely comprehended. With the advances in SB tools and massive clinical trial data, this field is anticipated to develop therapies for many chronic diseases in the future.Similar to chemical drugs or cell-based therapies, the safety, tolerability, and efficacy of engineered therapeutic microbes for chronic disease therapies have attracted widespread attention.Microorganisms, plants, marine resources, insects, and even mammals have always been important sources of pharmaceuticals/natural compounds. In nature, such compounds exist in low concentrations and are often difficult to synthesize due to their structural complexity. Advances in SB allow the production of bioactive compounds in non-natural hosts, which has revolutionized the pharmaceutical industry. Using computational and experimental tools, SB can help design chassis cells as novel drug screening platforms or as biofactories for the production of difficult bioactive compounds Fig. .Fig. 6SyE. coli is the best-studied prokaryotic model organism with properties of a well-defined genetic background, rapid reproduction, low cost, and high levels of foreign protein expression. Therefore, it serves as an important component of high-throughput drug screening platforms. For example, multidrug-resistant E. coli strains are utilized to identify novel antimicrobial compounds.222 Male et al. developed a high-throughput gene coding screening platform to screen a SICLOPPS library of cyclic hexapeptides in E. coli that could inhibit the protein interaction between the human CMG2 receptor and the Bacillus anthracis protective antigen (PA).223 Van der Donk and co-workers generated a bicyclic peptide library in E. coli by combining the generation of a lanthipeptide library with a bacterial reverse two-hybrid peptide library (RTHS), and identified interaction inhibitors for the HIV p6 protein and UEV domain of the human TSG101 protein.224E. coli lack the regulation mechanisms of eukaryotic gene expression and therefore is not suitable for screening bioactive compounds against eukaryotic targets.225Saccharomyces cerevisiae is a commonly used eukaryotic model organism for the expression of active eukaryotic proteins undergoing post-translational modifications, including disulfide bond formation and glycosylation. In addition, S. cerevisiae can also tolerate a wide range of pH and high osmotic pressure, which makes it a robust organism in practice.226S. cerevisiae strains, such as a yeast strain expressing human PARP1, have been used to screen inhibitors of enzymes involved in cancer progression.227 A yeast-based chemogenomic platform was used to identify the interaction between the chemotherapeutic agent methotrexate and the protein Dfr1 using haploinsufficiency profiling.228+ T cells for the functional analysis of host factors in HIV infection and pathogenesis.229 Mammalian cells, such as Chinese hamster ovary (CHO) cell lines, are used for primary screening and production of complicated biopharmaceuticals. We will now discuss gene circuits based on mammalian cells. The mammalian cells have a high cost of production and also suffer from the risk of contamination from human-specific pathogens.The complexity of mammalian cells limits their engineering as host cells. However, recent developments in SB are changing the situation. For example, CRISPR-Cas9 technology was applied to create a high-throughput platform of primary human CD4230 The Streptomyces coelicolor Pip was adapted to modulate reporter gene expression  in Chinese Hamster Ovary (CHO) cells in response to streptogramin antibiotics. This screening allowed the identification of new bioavailable and more sensitive antibiotics.231 Another example is the discovery of new anti-tuberculosis drugs. Ethylthionamide repressor (EthR)-based genetic circuit screening was used to identify 2-phenylethyl-butyrate, an anti-tuberculosis drug candidate against multidrug-resistant M. tuberculosis.233Synthetic gene circuits offer a huge opportunity for advanced drug discovery. Antimicrobial resistance (AMR) is emerging as a serious health threat, which can possibly be addressed by synthetic biology. Synthetic mammalian gene circuits can be used for the screening of novel antimicrobial compounds. Streptogramin-inducible (PipON) and streptogramin-repressible (PipOFF) mammalian gene-regulation systems were constructed based on the pristmamycin-responsive interaction between pristinamycin-induced protein (Pip) and its ptr promoter (Pptr) target sequence.Natural products are a valuable source of pharmaceuticals; however, they are difficult to synthesize and exist in low amounts in natural hosts. Rapid advances in SB now allow the production of high-value natural medicines and precursor compounds Table .Table 4A234 Artemisinin and its derivatives also have anticancer,235 anti-inflammation,236 antiviral,237 and anti-SARS-CoV-2 activity.238 Initially, Martin et al. attempted the production of artemisinin precursors in microbial systems; a mevalonate pathway from S. cerevisiae and a synthetic amorpha-4,11-diene synthase (ADS) were heterologously expressed in E. coli, producing amorphadiene  and moss (Physcomitrella patens).243Semi-synthetic artemisinin is a successful case of SB-based synthesis. Artemisinin, a highly effective antimalarial drug, is a sesquiterpene lactone, which is naturally obtained from Artemisia annua (Asteraceae).ene Fig. .239 SyntTaxus brevifolia),244 is widely used to treat breast,245 ovarian,246 pancreatic,247 lung,248 and prostate cancers.249T. brevifolia resources are scarce and have paclitaxel in very low amounts. The chemical synthesis of paclitaxel is complex and costly. Therefore, the industrialized production of paclitaxel by attempted using the tools of SB. Taxadiene, an important intermediate in the paclitaxel biosynthesis pathway, was obtained for the first time by overexpressing DXP synthase, IPP isomerase, GGPP synthase, and taxane synthase genes in E. coli at a titer close to 0.5 mg/L.250 Multiple module metabolic engineering (MMME) in E. coli was used to divide the paclitaxel biosynthetic pathway into an upstream module  for IPP and DMAPP synthesis and a downstream module  for the production of GGPP and taxadiene. The conditions for the optimal balance of the two pathway modules were determined by a systematic multivariate search, resulting in a taxadiene yield of ~1 g/L, which is the highest yield reported so far.251 Since several CYP450s (membrane-bound proteins) are involved in the biosynthesis of paclitaxel, eukaryotic yeasts are more suitable for the heterologous production of paclitaxel and its intermediates. In an S. cerevisiae system, multi-copy chromosomal integration of taxadiene synthase (TXS) harboring fusion solubility tags improved taxadiene titers , a diterpenoid secondary metabolite derived from the stem bark of Chinese yew  in S. cerevisiae.257 By increasing the carbon flux toward GGPP and optimizing the gene module of miltiradiene synthases, the production of miltiradiene was further improved in S. cerevisiae, reaching 3.5 g/L in a 5-L bioreactor.258 Hairy roots are also an attractive system for drug production. Three genes, HMGR, DXS, and GGPPS, involved in the biosynthesis pathway for tanshinone were introduced in Salvia miltiorrhiza hairy root for a high-yield production of tanshinone with higher antioxidant activity.259Tanshinones, including tanshinone I, tanshinone IIA, tanshinone IIB, dihydrotanshnone I, and cryptotanshinone, are bioactive diterpenoid compounds produced from the roots of Salvia miltiorrhiza Bunge (Danshen in Chinese), which have a wide range of pharmacological activities including antibacterial, antioxidant, anti-inflammatory, cardiovascular protective, and antineoplastic properties.260 The complete biosynthesis of the major cannabinoids, cannabigerolic acid, Δ9-tetrahydrocannabinolic acid, cannabidiolic acid, Δ9-tetrahydrocannabivarinic acid, and cannabidivarinic acid, starting from the simple sugar galactose was accomplished in S. cerevisiae.261There have been several efforts for the heterologous production of small-molecule natural drugs based on SB approaches. The full biosynthesis of the opioid compounds thebaine and hydrocodone starting from sugar was engineered in yeast.262 In this section, recent examples of recombinant protein and nucleic acid production will be presented; an overview of typical examples is presented in Table Protein drugs mainly include recombinant protein drugs, monoclonal antibodies, and recombinant protein vaccines. Compared with small-molecule drugs, protein drugs have high activity, strong specificity, low toxicity, clear biological function, and favorable clinical application.E. coli and the disulfide bonds were formed under optimal conditions to obtain bioactive insulin.263 A new and more efficient method for human insulin production was developed using a PCR-based cloning strategy that optimized human insulin expression in E. coli using the pET21b expression vector. It eliminated the use of affinity tags, tedious insulin renaturation, inclusion body recovery steps, and the expensive enzymatic cleavage of C-peptide.264 An insulin secretion expression system in S. cerevisiae has been developed, which expresses a cDNA encoding for a proinsulin-like molecule. ThreonineB30 was deleted for the fusion of proinsulin molecule with the S. cerevisiae α-factor prepro-peptide. This was later replaced by human proinsulin C-peptide with a small C-peptide. The purified proinsulin is subsequently converted into human insulin by tryptic transpeptidation.265 Recombinant human insulin has also been successfully produced in transgenic animals and plants, such as mice,266Arabidopsis thaliana,267 and cucumber.268Recombinant human insulin was the first recombinant protein drug. It was produced by the expression of chemically synthesized cDNAs encoding for the insulin A and B chains separately in E. coli according to three functional modules to facilitate high-titer production of immunoglobulin G (IgG).269 Bacteria, yeast, insect cells, and mammalian cells are used to express recombinant protein vaccines. Trumenba®, produced in E. coli, was developed by Pfizer and uses two variants of the meningococcal factor H-binding protein as antigens.270 Quadrivalent HPV vaccine, Gardasil, is the first commercially available FDA-licensed HPV vaccine; the L1 gene of four human papillomaviruses is expressed in S. cerevisiae and is used with an aluminum adjuvant.271 Most current COVID-19 recombinant protein vaccines are expressed in mammalian cell culture-based expression systems. ZF2001, consisting of tandemly repeated RBD-dimers and aluminum adjuvants, and the RBD-sc-dimers of MERS-CoV and SARS-CoV-2 both were produced at a high yield in an industry-standard CHO cell system.272 The West China Hospital of Sichuan University has developed a recombinant COVID-19 vaccine using baculovirus as a vector; the RBD region of the SARS-CoV-2 spike protein receptor binding domain is expressed in Sf9 insect cells, which showed effective immunogenicity in phase I and phase II clinical trials.273 According to WHO, there were 54 recombinant COVID-19 protein vaccine candidates in the clinical phase as of June 17, 2022.The production of monoclonal antibodies (mAbs) has taken tremendous attention in the pharmaceutical industry. A modular synthetic biology approach is proposed to rationally engineer 274 including recombinant production in bacteria, fungi, and cell-free systems. One way to reduce host toxicity is to produce peptides in E. coli as inclusion bodies. The antimicrobial short peptide (AMP) GKY20 derived from the C-terminus of human thrombin was fused with the C-terminus of Onconase for very high expression as inclusion bodies.275 Fusion proteins also have reduced toxicity for the heterologous expression host. Calmodulin (CaM), a common calcium-sensitive protein in eukaryotes, consists of two independent target binding domains with malleable methionine-rich interaction surfaces that can accommodate many basic and hydrophobic residues.276 CaM can be used as a universal carrier protein to express various types of AMPs in E. coli, such as melittin, fowlicidin-1, tritrpticin, indolicidin, puroindoline A peptide, and human β-defensin 3 (HBD-3). This strategy effectively masks the toxic activity of many AMPs and protects them from degradation during expression and purification.276 Recently, E. coli MSI001, a lethal strain, was isolated from mice; the strain was used for screening bacteria-binding heptapeptides based on an integrative bioinformatics approach, phage display technology, and high-throughput sequencing.277 Yeast is another promising cell factory for the production of peptides. Apidaecins, a series of small and proline-rich antimicrobial peptides act as a defense system against many drug-resistant bacteria. Apidaecin expression in Pichia pastoris was improved after the selection of a mutant strain that limits the loss of the integrated plasmid after induction.278 Additionally, P. pastoris has been used to produce AMPs such as cecropin279 and defensins.280 Cell-free systems provide another solution for the production of natural peptides. Valinomycin is a non-ribosomal peptide with antibacterial, antiviral, and antitumor activities.281 A highly efficient cell-free platform was developed for the rapid, in vitro total biosynthesis of valinomycin with a yield close to 30 mg/L; the cell-free protein synthesis system (CFPS) was coupled with cell-free metabolic engineering system (CFME) by mixing two enzyme-enriched cell lysates to perform the two-stage biosynthesis.282 Other natural peptide products, RiPPs, were also synthesized in cell-free systems. Nisin, a lantipeptide has antibacterial activity and is widely used in the food industry. The cell-free biosynthesis of nisin was achieved by the complete reconstitution of its pathway in a CFPS. The same system can also be used for the genome mining of nisin analogs, screening of mutants, and guiding the overproduction of lanthipeptides in vivo.283 In addition, four different lasso peptides were synthesized using cell-free systems, including known examples such as burhizin, capistruin, and fusilassin and predicted lariat peptides such as ellulassin from Thermobifida halotolerans. This system can rapidly generate and characterize novel lasso peptide variants.284Peptide natural products exhibit diverse biological activities, however, the toxicity, limited bioavailability, and difficult mass production hinder their development as therapeutic agents. Natural peptides can be biologically synthesized with the help of SB,285 INO-4800, a DNA vaccine candidate targeting the SARS-CoV-2 spike antigen,286 is currently in phase 3 trials. BNT162b2 and mRNR-1273 are the approved mRNA vaccines, both of these LNP-formulated nucleoside-modified mRNA vaccines encode PS2 spike protein of SARS-CoV-2.287 There are 16 DNA-based and 37 RNA-based vaccine candidates of SARS-CoV-2 in the clinical phase, together accounting for about 32% of the total vaccine candidates.Nucleic acid vaccines are based on either DNA or mRNA and are amenable to SB design. The target nucleic acid is inserted into human cells, which then produce copies of viral protein. Most of the nucleic acid vaccines against SARS-CoV-2 encode the virus’s spike protein.E. coli (EcNL4) was shown to improve gut microbiome and ameliorate colitis in mice.209 A combination therapy of low-dose anti-CD3 with a clinical-grade self-containing Lactococcus lactis  cured 66% of mice with new-onset diabetes.288 A Salmonella typhimurium strain was engineered to secrete Vibrio vulnificus flagellin B (FlaB) in tumor tissues for cancer immunotherapy.289 Virus-based vaccines, such as polio and measles vaccines, are created utilizing an inactivated or weakened pathogen while maintaining the antigenic properties285 Recently, Beijing Sinovac’s inactivated SARS-CoV-2 vaccine has been approved.With the advancement in SB, bacteria can also be used as active drugs. An engineered 290 For instance, there are currently seven cancer cell therapies through clinical trials that were approved by the FDA by July 2022.291 Despite these successes, SB-inspired cell engineering in medicine still faces many challenges, such as the lack of available sensors for essential biomarkers. Current microbial diagnostic and theragnostic approaches mostly rely on the existing sensors while numerous proteins with potential applications remain to be fully elucidated.38 Genome editing and manufacturing is a costly and lengthy process. Also, the therapeutic efficacy and technical difficulties limit their clinical applications.292 In addition, precise control of cellular output in engineered cells is very challenging, including the issues of bacteria diffusing to other tissues or being quickly eliminated by the host immune system. CAR-T-cell therapy is hampered by persistent infusion into patients and susceptibility to tumor antigen escape.293 Thus, additional efforts are required to integrate biological, chemical, and mechanical aspects for next-generation cell engineering.From bacterial-based biosensors and drug development to efficient treatments against pathogens, previously untreatable cancers, and other intractable human pathologies, SB has opened up various possibilities.294 The novel synthetic gene circuits combined with “on/off switches”, suicide gene systems, or CRISPR-Cas9 can create more precise control strategies for diagnostics and cell therapeutics. CRISPR-Cas9 significantly eases genome editing at targeted position(s).162 The iCasp9 kill switch has been licensed for more than 20 clinical trials including CAR-T cell and other type cell therapies and may help overcome limitations of currently approved CAR-T-cell therapies.295 In addition, non-biological factors have also been incorporated in SB approaches, such as temperature, light, rays, and nanomaterials. The integration of chemical biotechnology such as nanomaterial-coated bacteria provides tremendous promise for efficient transport, long-term storage, and spatial-temporal control in host.26 Gujrati et al. engineered bacteria to secrete melanin-containing OMVs with high photothermal conversion efficiency for photoacoustic imaging and photothermal therapy of 4T1 breast tumor.296 Moreover, the tools of computational biology such as machine learning can change the approach of SB by improving the search for biological components, optimizing design, and reforming/creating novel elements.Due to its dynamic multidisciplinary nature comprising different projects, approaches, and definitions, SB is evolving with advancements in biology, pharmacology, materials science, physics, and computational biology.Taken together, recent advances in SB-related cellular diagnostics and therapeutics can significantly transform healthcare paradigms. Currently approved CAR-T therapies and upcoming clinical trials of other engineered cell therapies also inspire the rapid expansion of SB. SB-based biofactories can contribute to the screening and development of new and affordable medicines, including recombinant vaccines. In light of this, SB-inspired cell engineering will enter a new perspective and expand its application for various disease treatments.


Upon exposure to drought, plants undergo complex signal transduction events with concomitant changes in the expression of genes, proteins and metabolites. For example, proteomics studies continue to identify multitudes of drought-responsive proteins with diverse roles in drought adaptation. Among these are protein degradation processes that activate enzymes and signalling peptides, recycle nitrogen sources, and maintain protein turnover and homeostasis under stressful environments. Here, we review the differential expression and functional activities of plant protease and protease inhibitor proteins under drought stress, mainly focusing on comparative studies involving genotypes of contrasting drought phenotypes. We further explore studies of transgenic plants either overexpressing or repressing proteases or their inhibitors under drought conditions and discuss the potential roles of these transgenes in drought response. Overall, the review highlights the integral role of protein degradation during plant survival under water deficits, irrespective of the genotypes’ level of drought resilience. However, drought-sensitive genotypes exhibit higher proteolytic activities, while drought-tolerant genotypes tend to protect proteins from degradation by expressing more protease inhibitors. In addition, transgenic plant biology studies implicate proteases and protease inhibitors in various other physiological functions under drought stress. These include the regulation of stomatal closure, maintenance of relative water content, phytohormonal signalling systems including abscisic acid (ABA) signalling, and the induction of ABA-related stress genes, all of which are essential for maintaining cellular homeostasis under water deficits. Therefore, more validation studies are required to explore the various functions of proteases and their inhibitors under water limitation and their contributions towards drought adaptation. In natuPlants are exposed to water deficit stress when rainfall declines during the growing season and when the rate of transpiration exceeds that of water absorption by roots due to hot and dry conditions . Consequ2 required for photosynthesis and the uptake of nutrients for plant growth (2 absorption and assimilation leads to an electron-rich environment in cells that is conducive for increased accumulation of reactive oxygen species (ROS)  . ABA is t growth . The redes (ROS) , causinges (ROS) , a secones (ROS) . If ROS es (ROS) .via the ABA-dependent pathway of stress response, while others form part of the ABA-independent pathway   , and majhaliana)  have beehaliana)  and genohaliana)  for crophaliana) . In addihaliana) .Undoubtedly, the drought models employed in such studies are quite diverse, and so are the results generated . NonetheHere, we review the differential expression and functional activities of plant protease and protease inhibitor proteins under drought stress, mainly focusing on comparative studies of cereal crops involving genotypes of contrasting drought phenotypes. We further explore studies of transgenic plants that either overexpress or repress proteases or their inhibitors under drought conditions and discuss the potential roles of these transgenes in drought response.2Plant proteases are proteolytic enzymes that hydrolyse peptide bonds in proteins and are found in various plant tissues and organs . Their aProteases are structurally and functionally diverse and are classified based on their catalytic activity, such as aspartic, cysteine, serine and threonine peptidases . Alterna2.1The mechanism of action of proteases, their inhibitors, and different proteolytic pathways in plants have been extensively reviewed elsewhere  and can in vitro recombinant proteins, mutant systems, substrate-trapping assays, and N-terminal degradome techniques coupled with quantitative proteomics and mass spectrometry technologies for substrate identification  protein 33 of the photosystem II (PSII), and heat stressed or photo-damaged D1 reaction centre protein  varieties subjected to water deficits and re-watering. Amongst the observed unique proteome changes was the increased accumulation of a ClpD1 protease in the drought-tolerant IAC1131 rice variety under severe drought conditions and its decreased accumulation upon re-watering. ClpD1 belongs to the caseinolytic protease (Clps) family of proteins (OsClpD1 in rice (proteins  and is e in rice . Clp pro in rice . Wu et aTriticum aestivum) plants subjected to drought stress for 18, 24 and 48 hours  leaves, an aspartate protease and a mitochondrial processing peptidase were up-regulated in the drought-susceptible variety in response to drought stress  plants subjected to drought stress  genotypes subjected to drought stress. The study reported a higher constitutive expression of several drought-responsive genes including protease inhibitors and senescence-related proteases in the drought-tolerant genotype than the sensitive one. Following water deprivation, both genotypes had increased expression of several proteolysis-related genes, but the change was generally much greater in the drought-susceptible variety. The authors suggested that high constitutive expression of drought-stress genes contributes towards the increased drought resilience of the drought-tolerant peanut genotype  plants subjected to limited watering  cultivars subjected to water limitation. The cultivars used, Tiber, Zorin and Starozagorski exhibited various degrees of sensitivity to drought stress, with Tiber being more drought-tolerant. The results showed a general decrease in leaf protein content in all three cultivars, possibly due to increased proteolytic activity. However, the drought-tolerant cultivar exhibited the least protein content decrease and the least amount of proteolytic activity under severe drought stress. Activity assays for a 65 kDa serine protease with a pH optimum of 8.5 showed the greatest increase in the drought-susceptible varieties Starozagorski and Zorin, and a decrease in Tiber, the tolerant cultivar under severe stress. In contrast, the activities of two other serine proteases increased in all three cultivars, possibly illustrating the existence of a large group of proteases with different roles under drought stress in common beans. The study also reported increased activity of an aminopeptidase in the most drought-sensitive Starozagorski and a decrease in the drought-tolerant cultivar. Overall, the findings of the study support the notion of the presence of a wide range of proteolytic enzymes with different substrate specificities, subcellular locations and specific inhibitors in plants. The authors also suggested that regulation of serine proteases in the tolerant variety could guard against premature drought-included leaf senescence in common bean  cultivars in response to drought stress. The cowpea cultivars EPACE-1 and TI83D were reportedly more drought-tolerant than the common bean cultivars Carioca and IPA, with an overall tolerance trend of EPACE-1 > TI83D > IPA > Carioca. The results showed differences in endoproteolytic activity across all four cultivars, being higher in the most drought-susceptible common bean cultivars than in cowpea. For example, under mild-drought conditions, the total endoproteolytic activity increased by 235%, 119%, 95% and 58% for cultivar Carioca, IPA, TI83D and EPACE-1, respectively. The study also investigated the involvement of cysteine, aspartic, serine, and metalloproteases in the drought response of the most drought-sensitive Carioca cultivar using class-specific protease inhibitors. The results suggested limited involvement of metalloproteases in the common bean cultivar under mild stress, but some enzyme activity of cysteine and serine proteases. Furthermore, pepstatin A inhibited about 25% of the total proteolytic activity under mild-drought conditions, thus indicating the presence of drought-responsive aspartic proteases in the bean leaf extract. Subsequent analysis of aspartic protease activity in all four cultivars using a specific peptide substrate revealed that water deficit induced aspartic protease activity in cowpea and bean leaf tissues. However, this enzyme activity increased with increasing levels of sensitivity to drought. The authors suggested that this increase in aspartic protease enzyme activity in the drought-sensitive bean cultivars could be a drought-adaptive response for remobilizing nitrogen to other plant parts under stressful environments  and its biological functions in drought response. The study used wild-type and ASPG1-overexpressing (ASPG1-OE) Arabidopsis plants to investigate tissue-specific gene expression patterns and the roles of ASPG1 in ABA-signalling systems associated with drought. After exposure to a 14-day drought stress treatment followed by re-watering, ASPG1-OE plants exhibited a greater recovery from wilting symptoms and higher survival rates. Gene expression analysis revealed that ASPG1 is drought-inducible in both wild-type and ASPG1-OE plants and is expressed in young seedlings, leaves, stems, flowers, and siliques but not in roots. The gene is also preferentially localized in guard cells where it participates in the ABA-signalling processes, including ROS accumulation triggering stomatal closure. The increased hydrogen peroxide contents are further detoxified by high antioxidant activities of superoxide dismutase and catalase, thus alleviating oxidative stress effects. In addition, increased ABA sensitivity was observed in ASPG1-OE plants under drought stress which could account for increased stomatal closure and a significant reduction in transpiration water loss. The authors suggested that ASPG1 participates in drought response via the ABA-dependent pathway (Drought studies using transgenic plants overexpressing protease and protease inhibitors genes Table 5  pathway .APA1) in drought response using transgenic Arabidopsis plants overexpressing APA1 (OE-APA1), wild-type and apa1 insertional lines under well-watered and mild-water deficit conditions. The study revealed that OE-APA1 lines tolerated drought stress better than the wild-type and apa1 lines. In addition, drought stress did not affect the overall plant phenotype, total chlorophyll content, and principal root length of the OE-APA1 lines relative to the well-watered plants. However, APA1 overexpressing plants exhibited reduced stomatal pore aperture and water loss under mild-drought stress compared to the wild-type and apa1 lines, yet exogenous ABA did not intensify stomatal closure. APA1 was shown to be ABA-responsive, exhibiting a 4-fold increase in expression in well-watered wild-type plants supplemented with exogenous ABA. Furthermore, under well-watered conditions, OE-APA1 plants up-regulated the expression of ABA biosynthetic and signalling genes relative to the wild-type, while the same genes were down-regulation in apa1 insertional lines. As such, the authors suggested that APA1 participates in drought response via the regulation of the ABA-signalling pathway and its location in leaves, vascular tissues, epidermal, and guard cells implicate it in stomatal closure as a water-saving mechanism under conditions of water scarcity (scarcity .Ipomoea batatas) papain-like cysteine protease (SPCP2) in transgenic Arabidopsis plants. Qualitative phenotypic analyses showed that transgenic Arabidopsis plants overexpressing SPCP2 contained a higher number of incompletely developed siliques, and exhibited earlier flowering, reduced fresh weight per seed, and lower germination rates compared to the wild-type controls. It is plausible that the SPCP2 protein degraded silique storage proteins resulting in incomplete development. Furthermore, SPCP2 gene expression was induced during natural leaf senescence, thus suggesting a gene function in senescence. SPCP2 transgenic Arabidopsis plants also showed high tolerance to salt and drought stresses compared to the wild-type, further implicating the gene in osmotic-stress response  are compound inhibitors that inhibit both trypsin and chymotrypsin . Malefo e stress .Oryza sativa chymotrypsin inhibitor-like 1 (OCPI1) gene was transformed into rice plants and characterized at the reproductive stage under drought-stressed field conditions  in transgenic Arabidopsis and apple . The study showed that the MpCYS4 protein is localized in the nucleus, plasma membrane and cytoplasm, consistent with its known cellular locations  senescence-associated gene, ZmSAG39 encoding a cysteine protease in maize plants subjected to either darkness or drought stress. The genotypes used were wild-type, transgenic ZmSAG39 overexpression (OE) lines and ZmSAG39 knockout lines. The results indicated that ZmSAG39 gene expression was induced by darkness and drought stress in the leaves of wild-type plants. The study also investigated seed germination rates in wild-type, ZmSAG39 knockout and ZmSAG39-OE plants under 8% and 12% polyethylene glycol (PEG)-6000 treatment. The results showed that the knockout lines had higher germination rates when compared to the wild and ZmSAG39-OE. Furthermore, the ZmSAG39-OE lines exhibited severe leaf senescence under complete darkness for 5 days, with lower chlorophyll content but higher membrane ion leakage rate and malondialdehyde (MDA) content than wild-type and knockout lines.Plant gene function can also be studied using knockout or knockdown mutants with total or partial loss of gene function . SeveralZmSAG39-OE lines showed greater levels of leaf wilting and senescence than the wild-type. In contrast, the ZmSAG39 knockout lines had an upright and greener phenotype. In addition, the ZmSAG39-OE lines exhibited reduced survival rates and chlorophyll content, higher membrane leakage levels, MDA, ROS, and oxidative stress damage compared to wild-type and knockout lines. Interestingly, the activities of a range of antioxidant enzymes were much greater in the ZmSAG39 knockout lines than in the wild-type and ZmSAG39-OE lines, thus, indicating the enhanced antioxidant capacity of knockout mutants. The expression levels of various stress-responsive genes were also analyzed between the wild-type and transgenic plants. The results showed expression levels of stress-related genes  and chlorophyll synthesis-related genes (chlorophyll a oxygenase) were higher in the drought-stressed knockout lines compared to the wild-type.Similarly, following a 14-day drought-stress treatment imposed by withholding water, the ZmSAG39-OE lines compared to the wild-type. These results indicate the negative regulatory effect of ZmSAG39 on the analysed stress-related and chlorophyll-synthesis-related genes under drought stress. However, under the same conditions of water deficits, ZmSAG39 exhibited a positive regulatory effect on genes associated with senescence and chlorophyll degradation. The authors concluded that ZmSAG39 is associated with leaf senescence, and its increased expression under darkness and drought enhances the sensitivity of maize plants to stress. As such, the knocking out of ZmSAG3 from maize enhanced the resistance of maize plants to darkness and drought tolerance and delayed leaf senescence  and a chlorophyll degradation-related gene (non-yellow colouring 1) were higher in the drought-stressed nescence .HvPap1 and HvPap19, in leaves of barley (Hordeum vulgare) under drought stress. Seven-day-old plants of wild-type, knockdown HvPap1 (KDPap1), and knockdown HvPap19 (KDPap19) were subjected to drought stress by withholding water for 14 days. The wild-type plants were more susceptible to water deprivation, as evidenced by reduced leaf turgor, while both knockdown genotypes remained upright. Furthermore, KDPap1 plants exhibited delayed leaf senescence during their normal growth cycle under well-watered conditions compared to the wild-type. KDPap1 also had a thicker upper epidermis cuticle under normal and drought conditions compared to the wild-type and KDPap19. However, the drought-induced reduction in chlorophyll and carotenoid contents was not significantly different between the three genotypes. Nevertheless, drought-stressed KDPap1 and KDPap19 plants exhibited higher total protein content than wild-type plants, possibly indicating reduced proteolysis in the knockdown mutant lines.KDPap1 and KDPap19 lines, possibly underscoring the critical role of this hormone during drought response. However, ABA levels were much higher in both knockdown lines compared to the wild-type. Conversely, 12-oxo-phytodienoic acid (OPDA), a precursor for jasmonic acid  plants subjected to water deficits using several knockdown lines of different Tr-KPIs. The study utilised two distinct water withholding regimes, a Non-PreStress treatment with direct exposure of plants to water deficits and a PreStress treatment in which plants were initially exposed to drought stress and rehydration, followed by a final drought stress treatment. Both Tr-KPI1 and Tr-KPI5 genes were drought-inducible in three untransformed white clover genotypes with varying degrees of drought tolerance. However, significantly higher gene expression levels were observed in the PreStress treatment, and the genes exhibited different induction levels in leaf and root tissues. The study also showed that proline accumulation and gene expression of 9-cis-epoxycarotenoid dioxygenase (NCED1), a key enzyme in the ABA biosynthesis pathway  synthase 1 (ACS1) and ACC oxidase 1 (ACO1) compared to the wild-type plants. The authors suggested that knockdown lines may experience some level of constitutive stress under well-watered conditions; hence the increased proline accumulation possibly acts as a ROS scavenger. Furthermore, Tr-KPIs may have different tissue-specific expression patterns and target various proteases, resulting in a functionally diverse group of active proteinase inhibitors during plant growth and drought response  pseudo-protease (AtFtsHi3) on the growth and drought tolerance of Arabidopsis plants using knockdown mutants. Pseudo-proteases are catalytically inactive proteolytic enzymes with cell regulatory functions , exhibited a 99% reduction in the transcript expression of the target gene AtFtsHi3 relative to the wild-type. In addition, the mutant seedlings and plants had a pale-green phenotype, were much smaller in rosette size, and had reduced root growth, with lower numbers of lateral roots than the wild-type. The study also generated complementation lines by expressing the FtsHi3 gene in the ftshi3-I (kd) background and designated them ftshi3-I (Comp). Further analysis of chloroplasts ultrastructure between the wild-type, ftshi3-I (Comp), and ftshi3-I (kd) showed that the knockdown mutant lines had distorted chloroplasts with thinner membranes, fewer and distorted thylakoid membranes, and fewer starch granules relative to the other wild-type and Comp lines.In an extensive study, unctions . Gene exftshi3-I (Comp), and ftshi3-I (kd) lines by withholding watering for up to 20 days and observing the drought-related phenotypes. The results showed that ftshi3-I (kd) plants were more tolerant to 12-days of drought treatment, while the wild-type and ftshi3-I (Comp) plants were more drought sensitive with wilting and chlorotic phenotypes. Furthermore, 80% of the ftshi3-I(kd) plants recovered after 20 days of drought stress, followed by 14 days of re-watering, while only 20% of the wild-type and ftshi3-I (Comp) recovered under the same conditions. While all three genotypes had relatively similar leaf ABA levels under control conditions, upon exposure to water limitation, the wild-type and ftshi3-I (Comp) plants had higher ABA content in leaves than the knockdown line. The authors suggested that high endogenous levels of ABA in cotyledons and roots under well-watered conditions could better prime the ftshi3-I(kd) plants for drought than the wild-type. Furthermore, the ftshi3-I(kd) plants had reduced stomatal density but larger stomatal size than the other two genotypes. Overall, this study showed that ftshi3-I(kd) plants had improved drought tolerance compared to wild-type and ftshi3-I (Comp) plants  plants .2.3In summary, plants encounter drought-induced osmotic and oxidative stresses, which disrupt cell structure and function . In turnResults from comparative proteomics and enzyme activity assays 6. For eKDPap1, an HvPap1 knockdown mutant line, could have been due to pleiotropic effects associated with the knocked-down cysteine protease gene. In addition, knockdown mutants exhibit some residual mRNA expression or protein accumulation of the knocked-down gene to varying extents , relative to the wild-type, other studies reported much lower reduction rates of the target genes (Furthermore, gene function studies using loss-of-function technologies such as knockout/down mutants also provide additional experimental evidence on the broader roles of plant proteases and their inhibitors in drought response ( extents , hence t extents . For exaet genes .Tr-KPIs in their study. However, the RNA interference procedure also affected the expression of other untargeted members of the Tr-KPI gene family. Consequently, evaluating resultant plant mutant phenotypes after exposure to drought stress becomes challenging. Therefore, despite the steady progress in gene function investigations using transgenic mutant plants, more studies with multiple transgenic lines are required to ascertain the bona fide physiological effects and roles of target protease and protease inhibitor genes.Furthermore, although the knockdown procedure would have specifically intended to reduce the expression level of a target gene, in some cases, untargeted genes are also affected . For exaAs discussed earlier, phytohormones may interact during plant growth and development, as well as in response to drought stress, ultimately contributing towards the overall plant phenotype . In someFor example, gibberellins regulate gene expression through DELLA proteins, which are repressors of plant growth and development . Under nOther studies have reported that exogenous application of ABA increases chymotrypsin inhibitory activity in barley leaves, while jasmonic acid treatment enhances trypsin inhibitor activity . These o3Protein degradation and its regulatory processes in plants are crucial for maintaining cellular homeostasis under water deficits; otherwise, damaged proteins would accumulate and disrupt cell structure and metabolism. However, the drought-induced differential expression of proteases and protease inhibitor proteins in contrasting genotypes are also unravelling the complex networking events during drought responses in plants. For example, increased proteolysis and reduced total protein in drought-sensitive genotypes could indicate massive protein damage events and less robust protective machinery against drought and its secondary effects, which result in sub-optimal metabolic and growth processes. In contrast, the more drought-tolerant genotypes exhibit reduced levels of proteolysis due to fewer proteases and more protease inhibitors. Together with enhanced protective machinery and improved targeted stress-induced gene transcriptional and translational activities, these factors collectively contribute towards greater performance and survival rates under water-limitation conditions.Nevertheless, the types and functions of proteases and protease inhibitors are quite diverse, and these proteins could be involved in many more physiological roles, such as stomatal control and ABA, jasmonic acid, and ethylene signalling. Furthermore, with this wide structural and functional diversity, their differential expression patterns between drought-tolerant and susceptible genotypes possibly make proteases and protease inhibitors important groups of proteins with potential use in crop improvement strategies. For instance, could the reduced level of protein degradation in drought-tolerant genotypes be used as a marker for drought tolerance and vice versa? Nevertheless, more functional validation studies are required to unravel additional roles of protein degradation events in plants under drought stress as we continue to find ways of generating crops with enhanced resilience to hot and drier climates. Furthermore, meta-analyses of protease and protease inhibitor gene and/or protein expression datasets between contrasting plant species and genotypes are required to unravel the expansive role of protein degradation in plant drought response.Conceptualisation, RN. Writing-manuscript preparation, SJM, RN. Writing-review and editing, RN. Funding acquisition, supervision, RN. Both authors contributed to the article and approved the submitted version.


A substantial body of clinical research involving individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evaluated the association between in-hospital biomarkers and severe SARS-CoV-2 outcomes, including intubation and death. However, most existing studies considered each of multiple biomarkers independently and focused analysis on baseline or peak values.We propose a two-stage analytic strategy combining functional principal component analysis (FPCA) and sparse-group LASSO (SGL) to characterize associations between biomarkers and 30-day mortality rates. Unlike prior reports, our proposed approach leverages: 1) time-varying biomarker trajectories, 2) multiple biomarkers simultaneously, and 3) the pathophysiological grouping of these biomarkers. We apply this method to a retrospective cohort of 12, 941 patients hospitalized at Massachusetts General Hospital or Brigham and Women’s Hospital and conduct simulation studies to assess performance.Renal, inflammatory, and cardio-thrombotic biomarkers were associated with 30-day mortality rates among hospitalized SARS-CoV-2 patients. Sex-stratified analysis revealed that hematogolical biomarkers were associated with higher mortality in men while this association was not identified in women. In simulation studies, our proposed method maintained high true positive rates and outperformed alternative approaches using baseline or peak values only with respect to false positive rates.The proposed two-stage approach is a robust strategy for identifying biomarkers that associate with disease severity among SARS-CoV-2-infected individuals. By leveraging information on multiple, grouped biomarkers’ longitudinal trajectories, our method offers an important first step in unraveling disease etiology and defining meaningful risk strata.The online version contains supplementary material available at 10.1186/s12874-023-02076-3. Since the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in December 2019, more than 670 million confirmed cases and 6.8 million associated deaths have been reported worldwide, with a large proportion of these deaths preceded by hospitalization [Among SARS-CoV-2 infected individuals, multiple biomarkers are typically measured repeatedly over the duration of hospitalization. An extensive literature has identified correlations between biomarker levels and severe outcomes, including intubation, admission to intensive care units, and death among hospitalized SARS-CoV-2 infected individuals . HoweverMethods for joint modeling of multiple longitudinal biomarkers and time-to-event outcomes have also been described –18. HoweData derived from a retrospective cohort of 12,941 patients infected with SARS-CoV-2 based on hospital record ICD-10 codes  and positive PCR tests between March 1, 2020 and November 30, 2021 were used for analysis  and the Massachusetts Registry of Vital Records and Statistics were obtained, and in the case of an inconsistency between the two sources, death dates from the Registry were adopted. Exposures are repeated laboratory measurements of mentclass2pt{minimi has a set of coefficients for these eigenfunctions called FPC scores, denoted as i, M) to adopt, resulting in A two-stage analytic approach was considered. First, FPCA was performed separately on each of the nt}Xk(t) . Throughker data . To implker data . Based oomarkers .J demographic characteristics Second, using the SGL package in R, we performed Cox SGL regression with the k over all biomarkers in that pathophysiological group and M FPC scores; and SGL estimates the sparsity :\documenThis two-stage strategy, FPCA decomposition of the biomarker trajectoris followed by SGL, allowed us to identify biomarkers associated with 30-day mortality while accounting for within-group correlations between biomarkers as well as the time varying biomarker trajectories. Both stages of analysis were stratified by sex.We selected the overall regularization parameter We set the weight for the group LASSO and LASSO penalty terms to K biomarkers in place of the FPC scores. Here we log transformed the baseline and peak measurements to ensure they were approximately normally distributed, and again imputed missing values with available biomarker measures using the MICE package in R [For comparison, we considered application of SGL using only the baseline or peak measurements of each of the age in R . AnalyseTo characterize the performance of our proposed two-stage approach, we conducted simulation studies including 200 repetitions with sample sizes of ondition , 28  among SARS-CoV-2 patients and discovered that AKI was related to more severe outcomes including death, respiratory failure, and disseminated intravascular coagulation , 10, 29.A limited number of studies have specifically investigated the effect of sex on the associations between biomarker levels and disease severity –14. We hMethodologically, our proposed FPCA-SGL approach is an easy-to-implement and computationally efficient analytic strategy that is able to simultaneously consider multiple biomarkers as well as their longitudinal trajectories in evaluating associations with severe SARS-CoV-2 outcomes. It is a versatile alternative to existing methods concerning multiple longitudinal measurements and a survival outcome and could be applied in other areas. Using simulation studies, we demonstrated that FPCA-SGL retained high TPR and outperformed alternative approaches using baseline or peak values with respect to FPR. In particular, we observed a substantial “survival bias” (inflated FPR) in our simulations when using peak measurements, because they are endogenous covariates, meaning their values and future paths are directly affected by the survival outcome of interest . For exaWe presented a two-stage analytic approach that combined FPCA and SGL to study the associations between hospitalized SARS-CoV-2 patients’ multiple biomarker trajectories with their 30-day mortality rates. We demonstrated that this method had high TPR and outperformed simpler comparator approaches using biomarkers’ baseline or peak measurements with respect to FPR. Using data from a retrospective cohort of 12,941 patients, we showed that renal biomarkers , inflammatory biomarkers , cardio-thrombotic biomarkers (d-dimer) were associated with 30-day mortality rates among hospitalized SARS-CoV-2 patients. Our sex-stratified analysis also revealed that hematological biomarkers  were associated with higher mortality only among males. This study recognized the prognostic value of biomarkers as well as the underlying potential sex difference. These results provide insights into assessment of SARS-CoV-2 disease severity and effective risk stratification.Additional file 1. Supplementary Figures and Tables.Additional file 2. Supplementary Methods.