Abstract
In the face of formidable challenges presented by global public health issues such as diabetes and hypertension, we have introduced an innovative project this year called "One for All - Synbiotic Therapy." Through the application of synthetic biology technology in designing and modifying the biosafety strain Zymomonas mobilis, we hope to develop a cutting-edge healthcare solution that integrates preventive care with personalized treatment. Through a series of metabolic engineering modifications, we have successfully enhanced the production of 3-HB and achieved further improvements in its yield. Concurrently, we introduced expression plasmids pTZ32a containing GLP-1 (glucagon-like peptide-1) and ACEi (angiotensin-converting enzyme inhibitor) into the motile fermenting bacteria to facilitate the expression of therapeutic peptides with bioactive properties. By synergizing with the oligosaccharides generated by the motile fermenting bacteria, our objective is to attain glucose-lowering and blood pressure-reducing effects. Moving forward, "One for All - Synbiotic Therapy" will serve as a pivotal tool to accurately unlock the potential for treating various diseases. This approach simplifies and enhances the treatment and monitoring of diseases while promoting a healthy lifestyle focused on timely intervention and overall well-being.
Parts
| Number | Name | Type |
|---|---|---|
| BBa K5211002 | Pgap | Promoter |
| BBa K5211003 | Ptet | Promoter |
| BBa K5211006 | phaB | Coding Sequence Parts |
| BBa K5211007 | fadM | Coding Sequence Parts |
| BBa K5211008 | yciA | Coding Sequence Parts |
| BBa K1587001 | tesB | Coding Sequence Parts |
| BBa K5211010 | zwf | Coding Sequence Parts |
| BBa K5211011 | ada | Coding Sequence Parts |
| BBa K5211012 | adh2 | Coding Sequence Parts |
| BBa K5211013 | cSAT | Coding Sequence Parts |
| BBa K5211001 | gfp | Coding Sequence Parts |
| BBa K5211014 | GCG | Coding Sequence Parts |
| BBa K5211015 | ACEi | Coding Sequence Parts |
| BBa K5211018 | T1AB | Operon and Pathway |
| BBa K5211019 | T1AB' | Operon and Pathway |
| BBa_K5211023 | Ptet-T1AB' | Operon and Pathway |
| BBa K5211020 | Ptet-T1AB | Operon and Pathway |
Chassis cells
Zymomonas mobilis is a facultatively anaerobic microorganism that utilizes the Entner-Doudoroff (ED) pathway for ethanol production. It exhibits high acid tolerance, has a short fermentation cycle, and demonstrates good biosecurity. This microorganism has been engineered as a microbial cell factory for producing various products, including 2,3-butanediol, isobutanol, and polyhydroxybutyrate (PHB). Recent studies have revealed that Z. mobilis can modulate the gut microbiome and alleviate intestinal diseases, making it a promising probiotic adjunct. Through genetic engineering techniques involving the elimination of endogenous plasmids pZM32, pZM36, pZM33, and pZM39 in the ZMNP strain of Z. mobilis, the complexity of its genome has been reduced while reducing energy consumption. These modifications have resulted in desirable traits such as high transformation efficiency, enhanced tolerance to inhibitors, and improved utilization of secondary mother liquor. Furthermore, a high-throughput CRISPR-based genome editing technology has been developed for Z. mobilis enabling precise design and enhancement of genes and metabolic pathways; thereby accelerating the development of this biofuel-producing strain into an efficient chemical protein and biofuel production cell factory.
Metabolic engineering
In order to efficiently produce 3-hydroxybutyrate (3-HB), our team has utilized genetic engineering techniques to modify the ZMNPΔ0038 strain, which serves as the starting point. We have introduced three enzymes from the exogenous 3-HB synthesis pathway and integrated three essential genes with a tetracycline-induced promoter (Ptet) to form an operon on a plasmid. Subsequently, this plasmid was transferred into ZMNPΔ0038, resulting in a strain capable of producing 3-HB. To enhance the yield of 3-HB, members conducted screenings for efficient thioesterases in the 3-HB synthesis pathway, increased the copy number of 3-HB synthesis genes, overexpressed cofactor supply-related genes, constructed penicillin-binding protein (PBPs) deficient strains, introduced exogenous ethanol utilization pathways (EUP), and optimized culture medium conditions. The final yield was as high as 1525±72.16 mg/L.
Gene editing
The I-F CRISPR-Cas gene editing system: The I-F type CRISPR-Cas system identified in Z. mobilis comprises a cluster of interspersed short palindromic repeats and multiple cas genes. The length of the palindromic repeats within the CRISPR cluster is 29 base pairs, while the spacer sequence measures 32 base pairs in length. During the operation of the CRISPR/Cas system, transcription of the spacer sequence occurs within the CRISPR cluster under promoter guidance, resulting in precursor RNA (pre-crRNA) that undergoes subsequent processing to generate guide RNA (gRNA). The mature gRNA then directs Cas protein binding to the target sequence, ultimately facilitating precise cleavage at this site. Following Cas protein-mediated double-strand break (DSB) formation within the genetic sequence, Z. mobilis activates its endogenous repair mechanism to initiate homologous recombination with an identical donor DNA sequence for efficient repair and completion of gene editing.
CRISPR/Cas12a employs a single RNA molecule (crRNA) to direct the Cas12a enzyme towards specific DNA target sites, recognizing them via a distinct PAM (protospacer adjacent motif) sequence located adjacent to the repeat sequences. Subsequently, it induces cleavage of the double-stranded DNA, resulting in staggered DNA double-strand breaks (DSBs).
To overcome the limitations of PAM site selection, we have developed and enhanced a Genome-Wide Iterative and Continuous Editing (GW-ICE) system that is not constrained by sequence. This innovative approach utilizes four compatible shuttle plasmids in conjunction with endogenous I-F type CRISPR-Cas and exogenous CRISPR-Cas12a systems. By harnessing bacterial restriction-modification (R-M), CRISPR/Cas, toxin-antitoxin (T-A) systems, and natural plasmids, we have transformed them into highly efficient gene editing tools. Each gene editing plasmid within this system contains two guide RNAs (gRNAs) capable of simultaneously targeting antibiotic resistance genes on the target plasmid as well as chromosomal genes. Consequently, continuous transformation of editing plasmids into host cells is necessary without relying on counter-selectable markers during genome cycling. As a result, there is no longer a need for an editing plasmid loss step during genome cycling, thereby shortening the genome editing cycle. Furthermore, we have devised a T-A-based strategy for rapid screening and cloning vector construction to enhance the efficiency of editing plasmid loss. Through this methodology, we successfully eliminated four endogenous plasmids from Zymomonas mobilis and generated a plasmid-free mutant strain known as ZMNPΔhypo with improved transformation efficiency after continuous genome editing.
Modeling
The role of 3-HB in the treatment of type 2 diabetes (T2D) is significant. This project employs molecular docking technology to investigate the interaction between 3-HB and HACR2. TesB plays a crucial role in converting short-chain length 3-hydroxybutyrate-CoA into its free fatty acid form, namely 3HB. To enhance the production yield of 3-HB, this project utilizes enzyme modification techniques to modify the structure of TesB. Subsequently, the modified gene is reintroduced into a Zymomonas mobilis, enabling it to produce higher quantities of 3-HB. The objective is to achieve effective regulation of blood glucose through the combined action of GLP-1 and 3HB.
Furthermore, a growth model for the Zymomonas mobilis under intestinal conditions has been developed using growth curves and suicide switch mechanisms, simulating its growth dynamics and impact on GLP-1 expression within the gut environment. Notably, this model considers variations in environmental pH and analyzes growth dynamics before and after activation of the safety switch mechanism. The ultimate aim is to provide a theoretical basis for optimizing GLP-1 protein expression levels and controlling bacterial cell numbers, ensuring safety and efficacy in probiotic treatment strategies.
Human practices
1. A comprehensive curriculum on the principles and applications of synthetic biology was meticulously crafted.
2. Informative articles elucidating the intricacies of diabetes and hypertension from a scientific perspective were authored.
3. A research was conducted in central China to assess the satisfaction of patients with diabetes, colitis and hypertension towards current treatment methods.
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