iGEM 2024 — Team china-hubu-wuhan

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## Notebook

OAST

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[2023.12.2-2023.12.31](#section1)
[2024.1.1-2024.1.18](#section2)
[2024.1.19-2024.1.31](#section3)
[2024.2.1-2024.2.29](#section4)
[2024.3.1-2024.3.17](#section5)
[2024.3.18-2024.3.31](#section6)
[2024.4.1-2024.4.10](#section7)
[2024.4.11-2024.5.15](#section8)
[2024.5.15-2024.6.8](#section9)
[2024.6.8-2024.7.1](#section10)
[2024.7.2-2024.8.7](#section11)
[2024.8.8-2024.9.20](#section12)
[Back to the top](#top)

## 2023.12.2-2023.12.31

## 2024.1.1-2024.1.18

## 2024.1.19-2024.1.31

## 2024.2.1-2024.2.29

## 2024.3.1-2024.3.17

## 2024.3.18-2024.3.31

## 2024.4.1-2024.4.10

## 2024.4.11-2024.5.15

## 2024.5.15-2024.6.8

## 2024.6.8-2024.7.1

## 2024.7.2-2024.8.7

## 2024.8.8-2024.9.20

{% endblock %}

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## Engineering

OAST

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[Overview](#section1)
[Metabolic engineering of *Zymomonas mobilis* to synthesize 3-HB](#section2)

* [Cycle 1](#subsection1)
* [Cycle 2](#subsection2)
* [Cycle 3](#subsection3)
* [Cycle 4](#subsection4)
* [Cycle 5](#subsection5)
* [Cycle 6](#subsection6)
* [Cycle 7](#subsection7)

[Expression of hypoglycemic peptide and antihypertensive peptide](#section3)

* [Cycle 1](#subsection8)
* [Cycle 2](#subsection9)

[Future study](#section4)

* [References](#subsection10)

[Back to the top](#top)

## Overview

The "One for All——Synbiotic Therapy" project aims to leverage synthetic biology technology for the design and modification of the biosafety strain *Zymomonas mobilis*, with the objective of developing a healthcare solution that integrates preventive care and personalized treatment. By employing techniques such as metabolic engineering, gene editing, and rational protein design, we have successfully engineered highly efficient 3-HB production strains specifically tailored for treating diseases like diabetes and colitis. Moreover, these strains are capable of producing functional products such as fructooligosaccharides and oligosaccharides that can aid in blood pressure reduction or serve as prebiotics. Additionally, we have introduced expression plasmids containing GLP-1 and ACEi into *Zymomonas mobilis* to enable the expression of therapeutic peptides with glucose-lowering and blood pressure-reducing effects. In the future, "One for All - Synbiotic Therapy" will act as a pivotal tool in precisely unlocking various disease treatments, offering patients more convenient, safe, and effective treatment options while achieving comprehensive prevention, control, diagnosis, and treatment across multiple diseases. This endeavor will drive global health innovation in medical development.

Each engineering cycle comprises four interconnected and sequential steps: Design, Build, Test, and Learn. In practice, after conducting thorough research, we prioritized the treatment of colitis and initiated our first trial with theoretical support. As is often the case with initial attempts, challenges emerged. However, through continuous improvement and repetition, we achieved preliminary success in treating colitis which motivated us to proceed with the next iteration of the engineering cycle.
For detailed information on materials and methods including reagents and enzymes used in this study, please refer to the Protocol.

## Metabolic engineering of *Zymomonas mobilis* to synthesize 3-HB

### Cycle 1

#### Design

In order to achieve efficient production of 3-hydroxybutyric acid (3-HB), the team employed genetic modification on ZMNPΔ0038, the initial strain used. During microbial fermentation, substrate and product inhibition often hinders microorganism growth, leading to reduced cell productivity. Given that 3-HB contains hydroxyl and carboxyl groups in close proximity and exhibits some acidity, a tolerance test was conducted on *Zymomonas mobilis* to assess its suitability as a host cell for 3-HB synthesis.

#### Test

The tolerance of *Zymomonas mobilis* to 3-HB was assessed by introducing varying concentrations of 3-HB into RMG2 medium. As the concentration of 3-HB increased from 1 g/L to 40 g/L, the growth rate of strain NP-pEZ15Asp gradually decelerated, leading to a subsequent gradual decline in OD600nm. Based on the experimental findings, it can be inferred that *Z. mobilis* exhibits a maximum tolerance threshold of up to 30 g/L for 3-HB.

#### Learn

This iteration proves the high potential of *Zymomonas mobilis* for efficient production of 3-HB, which supports our next cycle of iteration to further modify it.

### Cycle 2

#### Design

Thioesterases possess the capability to catalyze the hydrolysis of thioester bonds between carbonyl and sulfur atoms in diverse compounds. They are accountable for the hydrolysis of 3-hydroxybutyryl-CoA, leading to the production of 3-HB, which serves as a pivotal enzyme in the biosynthesis pathway of 3-HB. However, due to *Escherichia coli*'s genomic capacity to encode multiple thioesterases with distinct metabolic roles, selecting the most efficient thioesterase is crucial for enhancing 3-HB production. Therefore, team members opted for three different thioesterases from *Escherichia coli* K12 - FadM, YciA, and *tesB* - and engineered them alongside acetyl-CoA acetyltransferase (*phaA*) and acetoacetyl-CoA reductase (Pha B) coding genes using a gene engineering strategy based on RBS-10 sequence (CCATAATCTAGAGAAAGTAAGCAC). Through experimental investigations, we hoped to identify the thioesterase that exhibited the highest yield of 3-HB.

#### Build

The team members constructed a T1AB operon by assembling the *tesB*, *phaA*, and *phaB* genes and integrated them into the pEZ15Asp plasmid. Gene expression was controlled using the *tet*racycline-inducible promoter P*tet*. Subsequently, demethylated *Escherichia coli* trans110 competent cells were transformed with these plasmids to obtain pEZ-PtT1. Similarly, pEZ-PtY and pEZ-PtF plasmids were obtained separately using the same method. These plasmids (pEZ-PtT1, pEZ-PtY, and pEZ-PtF) were then electroporated into ZMNPΔ0038 competent cells to generate strains NP-PtT1, NP-PtY, and NP-PtF capable of producing 3-HB.

#### Test

By employing varying concentrations of tetracycline inducers, the team members conducted comprehensive assessments and analyses on cell growth rate, glucose consumption rate, ethanol production, and 3-HB yield. The strains NP-PtF, NP-PtY, and NP-PtT1 harboring the 3-HB synthesis pathway exhibited consistent growth rates comparable to strain NP-pEZ15Asp when not induced by tetracycline. However, with increasing tetracycline concentration, their growth rates became slower than that of NP-pEZ15Asp. While NP-pEZ15Asp could consistently produce ethanol at 12 hours, both NP-PtF and NP-PtY achieved stable ethanol production at 15 hours. In the case of strain NP-PtT1, it attained stable ethanol production at 12 hours when the tetracycline concentration ranged from 0 to 0.8 µg/mL; however, as the concentration increased to between 0.8 to 1.2 µg/mL, stable ethanol production occurred at 15 hours instead.
Although overall the ethanol production rate of NP-PtF, NP-PtY and NP-PtT1 was slower than that of NP-pEZ15Asp, their final ethanol yields were comparable in magnitude. When the tetracycline concentration reached a level of 1.2 µg/mL, the highest yield of 3-HB was obtained by strains NPP-tF (175 .33 ±3 .06 mg/L), NPP-tY (187 .00 ±2 .00 mg/L), and NPP-tTl (425 .67 ±2 .89 mg/L).

#### Learn

The experimental results demonstrated the successful production of 3-HB through introduction of the 3-HB pathway into *Escherichia coli* via a plasmid vector. Among these, NP-PtT1 exhibited the highest yield of 3-HB, thus making *tesB* enzyme an ideal candidate for subsequent strain modification. Moreover, considering that the maximum production of 3-HB was achieved at a tetracycline concentration of 1.2 µg/mL, this concentration was selected for further experiments.

### Cycle 3

#### Design

The promoter exerts control over the direction and intensity of transcription, and the selection or design of promoters with varying intensities can impact the expression level of target genes, thereby influencing the production of corresponding enzymes. Strong promoters are typically employed for target gene overexpression to enhance heterologous product titers. The RBS serves as a crucial element in regulating translation intensity and can adjust gene expression strength at the translational level. In an effort to improve 3-HB production, team members endeavored to substitute the tetracycline-inducible P*tet* promoter with a robust P*gap* promoter.

#### Build

In order to construct the pEZ-PgT1 plasmid, reverse amplification of the plasmid was performed upstream of the pEZ-PtT1 promoter, followed by Gibson assembly with the P*gap* promoter. The resulting pEZ-PgT1 plasmid was then introduced into ZMNPΔ0038 competent cells via electroporation to obtain strain NP-PgT1.
Subsequently, the RBS-10 sequence in the pEZ-PtT1 plasmid was replaced with RBS-10K (ATCACAGGGTCTAGAAGGAGGTCGAA) to generate pEZ-PtT1'. Furthermore, concatenation of *tesB*, *phaA*, and *phaB* genes using this sequence resulted in the creation of T1AB' operon. The P*tet* promoter was subsequently PCR amplified and overlapped with T1AB' operon to obtain P*tet*-T1AB' operon. Integration of P*tet*-T1AB' operon into the pEZ15Asp plasmid led to construction of recombinant plasmid pEZ-PtT1'. Finally, electroporation of ZMNPΔ0038 competent cells with the pEZ-PtT1' plasmid resulted in strain NP-PtT1'.

#### Test

The recombinant strain NP-PgT1 obtained will be subjected to fermentation testing, and its growth rate during fermentation is faster than that of NP-PtT1. The final OD600nm nm after reaching the stable phase of fermentation is also higher for NP-PgT1 compared to NP-PtT1. Ethanol production stabilizes at 12 hours for NP-PgT1, while it takes 15 hours for NP-PtT1. Although NP-PgT1 has a growth advantage over NP-PtT1, its 3-HB yield is lower than that of NP-PtT1.
Additionally, the recombinant strain NP-PtT1' with replaced RBS strength was fermented and it was found that its growth rate during fermentation is slower than that of NP- PtT1. Both reach stable ethanol production at 12 hours, but the 3-HB yield of NP- PtT1' is lower than that of NP-PT1.

#### Learn

The replacement of the inducible promoter in the 3-HB strain with a more potent promoter and the adoption of a higher intensity RBS did not yield significant enhancements in 3-HB production; conversely, it resulted in a decline. This prompted team members to extensively review relevant literature and explore potential remedies. They discovered that this could be attributed to an incongruous match between the employed promoters/RBS and gene expression, leading to diminished efficacy, yield, or productivity. Consequently, team members will persist in their endeavors by attempting strain modification using inducible promoters and RBS10 to augment 3-HB production.

### Cycle 4

#### Design

The literature suggests that increasing the copy number of operon required for PHB production pathway can lead to an enhanced yield of PHB, from 5.22±0.23% (DCW) to 16.99±0.88% (DCW). Therefore, team members adopted a similar approach to augmenting the copy number of TAB operon necessary for the 3-HB production pathway in order to optimize the yield of 3-HB.

#### Build

The *tesB*, *phaB*, and *phaA* operons under the control of the P*gap* promoter were integrated into the ZMNPΔ0038 chromosome to construct the recombinant strain NPTΔ1. The pL2R-PgT1 editing plasmid was generated using CRISPR-Cas technology to replace the ZMO1650 locus on the ZMNPΔ0038 genome with the P*gap*-T1AB operons, resulting in strain NPTΔ1. Subsequently, we introduced the pEZ-PtT1 recombinant plasmid to obtain a 3-HB multicopy strain named NPTΔ1-PtT1.

#### Test

During fermentation, the recombinant strain NPTΔ1-PtT1 exhibits a slower growth rate compared to NP-PtT1, resulting in a lower final OD600nm nm in the stable phase. However, despite its slower growth rate, NPTΔ1-PtT1 maintains unaffected ethanol production and shows an increase in 3-HB production compared to NP-PtT1.

#### Learn

The experimental results demonstrate that augmenting the copy number of the TAB operon can effectively enhance 3-HB production. Consequently, team members are encouraged to persist in employing this strategy of increasing the copy number of the TAB operon during subsequent strain modification processes to optimize the final yield of 3-HB.

### Cycle 5

#### Design

The NADPH-dependent acetoacetyl-CoA reductase *phaB* plays a crucial role in the biosynthesis of 3-HB by producing the intermediate 3-hydroxybutyryl-CoA. Previous studies have indicated that *phaB* activity is regulated by the level of NADPH, and increasing NADPH supply has been shown to enhance recombinant *Escherichia coli*'s production of 3-HB. Considering the requirement of NADPH as a cofactor for *phaB* in synthesizing the precursor of 3-HB, it is essential to optimize *Zymomonas mobilis* as a host organism capable of providing higher levels of NADPH. The gene zwf (ZMO0367) on the *Zymomonas mobilis* genome encodes glucose-6-phosphate dehydrogenase, which converts glucose-6-phosphate into 6-phosphogluconolactone while generating NADPH and H+ using NADP+. Therefore, overexpressing zwf may be advantageous for enhancing 3-HB production.

#### Build

The promoter zwf-T1AB was generated by amplifying the zwf and T1AB operon via overlap PCR. Subsequently, the recombinant plasmid pEZ-PtZT1 was constructed utilizing the P*tet* promoter for driving expression, which was then introduced into the NPTΔ1 strain to generate the NPTΔ1-PtZT1 strain.

#### Test

The results of the fermentation experiment indicate that NPTΔ1-PtZT1 exhibited a slower cell growth rate compared to NPTΔ1-PtT1, while demonstrating similar levels of ethanol production. However, there was a further improvement in 3-HB production observed for NPTΔ1-PtZT1.

#### Learn

By overexpressing the endogenous gene zwf, the supply of NADPH can be augmented, thereby facilitating the enhancement of 3-HB production. In subsequent modifications, team members will persist in adopting this strategy and are determined to further amplify the yield of 3-HB.

### Cycle 6

#### Design

The cell wall membrane is associated with various crucial physiological functions, including solute transport, energy metabolism, signal transduction, stress resistance, cell growth, and biochemical production. The absence of PBPs in *Zymomonas mobilis* disrupts the normal synthesis of peptidoglycan, thereby compromising membrane structure and resulting in an elevation of glucosamine concentration in *Z. mobilis* mutants. Consequently, this leads to increased outer membrane permeability that could potentially benefit the extracellular secretion of 3-HB. Both ZMO0959 and ZMO1089 loci in the genome of *Z. mobilis* encode PBPs involved in peptidoglycan biosynthesis on the cell membrane. Therefore, our team members have speculated that knocking out these genes related to peptidoglycan biosynthesis has the potential to enhance 3-HB production.

#### Build

Using the CRISPR-Cas method, we designed editing plasmids pL2R-0959 and pL2R-1089 for targeted gene knockout at positions ZMO0959 and ZMO1089 in the ZMNPΔ0038, resulting in PBP-deficient strains NPΔ0959 and NPΔ1089. Subsequently, recombinant plasmid PtZT1 was introduced into NPΔ0959 and NPΔ1089 separately to generate strains NPΔ0959-PtZT1 and NPΔ1089-PtZT1.

#### Test

After comparing the fermentation processes and outcomes of strains NP-PtZT1, NPΔ0959-PtZT1, and NPΔ1089-PtZT1, it was observed that the recombinant strain NPΔ0959-PtZT1 exhibited the most rapid growth rate, whereas NPΔ1089-PtZT1 displayed the slowest growth rate. However, in terms of 3-HB production, NPΔ1089-PtZT1 demonstrated higher levels compared to both NPΔ0959-PtZT1 and NP-PtZT1.

#### Learn

The experimental results suggest that strains lacking PBP may offer advantages in terms of extracellular secretion of 3-HB. In subsequent modifications, team members will continue to integrate this strategy with other approaches to further enhance the production of 3-HB.

### Cycle 7

#### Design

*Zymomonas mobilis* is an obligate fermentative microorganism capable of ethanol oxidation but unable to utilize it as a carbon source for growth. Hence, employing the EUP pathway to convert the generated ethanol during fermentation into acetyl-CoA represents a strategic approach to augment the availability of acetyl-CoA.

#### Build

Strain NPTΔ2 was generated through deletion of ZMO1089 from the genome of strain NPTΔ1, followed by construction of editing plasmid pL2R-EUP using the CRISPR-Cas method. The EUP operon was then integrated into the ZMO1094 locus in the genome of strain NPTΔ2, resulting in strain NPTΔ3. Plasmid pEZ-PtZT1 was subsequently introduced via electroporation into strain NPTΔ3 to obtain strain NPTΔ3-PtZT1. Furthermore, competent cells were prepared from strain NPTΔ3-PtZT1 and plasmid pEZ39p-PeEUP was electroporated into these cells to generate strain NPTΔ3-PtZT1EUP.

#### Test

The growth rate of NPTΔ1-PtZT1EUP cells exhibits a slower pace compared to that of NPTΔ1-PtT1. By the end of fermentation, the final ethanol production between NPTΔ1-PtZT1EUP and NPTΔ1-PtZT1 remains similar; however, the former demonstrates a significant enhancement in 3-HB production.

#### Learn

The research findings suggest that the EUP pathway serves as a highly efficient route for ethanol conversion into acetyl-CoA, facilitating 3-HB biosynthesis. Consequently, the team members intend to integrate five strategies (Figure 1) to optimize the production of 3-HB.

![Figure 1 The path to increase 3-HB production](https://static.igem.wiki/teams/5211/eng1.jpg)

Figure 1 The path to increase 3-HB production

## Expression of hypoglycemic peptide and antihypertensive peptide

### Cycle 1

#### Design

*Zymomonas mobilis* was utilized as a host cell by the research team, and the expression plasmid pTZ32a was introduced into the strain to facilitate the expression of GLP-1 and ACEi. As a result, therapeutically active peptides were synthesized, leading to hypoglycemic and hypohypertensive effects. By employing the cSAT (Cleavable Self-Aggregating Tag) strategy, extracellular display protein INP guided the expression of 4\*GLP-1 and 5\*LV. Through a series of approaches, GLP-1 was produced and secreted into the extracellular space where it underwent release, transportation via bloodstream, and subsequent binding to β-islet cells' GLP-1 receptor. This activation triggered downstream cAMP signaling pathway resulting in insulin production for achieving hypoglycemic effect. Angiotensin-converting enzyme (ACE) catalyzes angiotensin I conversion into angiotensin II. The angiotensin-converting enzyme inhibitor LV recognizes and inhibits ACE activity causing vasodilation and blood pressure reduction for achieving hypohypertensive effect (Figure 2).

![Figure 2 expression of hypoglycemic peptide and antihypertensive peptide](https://static.igem.wiki/teams/5211/eng2.jpg)

Figure 2 expression of hypoglycemic peptide and antihypertensive peptide

#### Build

By integrating the T7 RNA polymerase gene into the genome of *Zymomonas mobilis*, the team introduced the T7 expression system to enhance target gene expression. To ensure proper protein expression, a fusion protein was formed by attaching a 6×His tag to the therapeutically active peptide and incorporating a cleavable self-assembling cSAT system. Additionally, an extracellular display protein INP was inserted in front of the fusion protein gene sequence and tandem repeat sequences of 4×GLP-1 and 5×LV were designed to construct a polymer, enabling direct secretion of active peptide monomers into the human intestine. The modified expression vector pTZ32a was used for inserting the fusion protein gene which was then expressed using the T7 expression system in *Zymomonas mobilis*.

Furthermore, considering potential degradation during fermentation or exposure to gastric juice, a pH sensor utilizing lactose operon and P170 system from Lactococcus lactis was constructed with rcfB promoter responsive to pH changes. When the pH drops below 5.5, induction of LacI repressor protein occurs leading to its binding to operator O1/O2 and subsequent inhibition of downstream gene expression. Conversely, when environmental pH exceeds 5.5, repression of rcfB promoter takes place resulting in degradation of LACR protein and release from inhibition. For GLP-1, a hypoglycemic peptide is expressed and secreted into extracellular space where it is released and transported via bloodstream for binding to β-islet cell's GLP-1 receptor. This activation triggers downstream cAMP signaling pathway ultimately leading to insulin production. Regarding the hypohypertensive peptide: Angiotensin-converting enzyme (ACE) catalyzes conversion of angiotensin I into angiotensin II. The ACE inhibitor LV recognizes and inhibits ACE activity causing vasodilation and reduction in blood pressure.

![Figure 3 The plasmid map of the bacterial strain for GLP-1 production ](https://static.igem.wiki/teams/5211/eng3.jpg)

Figure 3 The plasmid map of the bacterial strain for GLP-1 production

#### Test

The following SDS-PAGE map was obtained through the induction of strain for protein expression and purification. The results demonstrate successful expression of the corresponding protein.

![Figure 4 Purification of the GLP-1 protein](https://static.igem.wiki/teams/5211/eng4.jpg)

Figure 4 Purification of the GLP-1 protein

#### Learn

The experimental results demonstrate the successful induction of the target protein for expression. However, achieving the project objective of "One for all" remains insufficient at this stage. In the subsequent phase, it is necessary to construct fusion proteins.

### Cycle 2

#### Design

We postulated that simultaneous treatment of diabetes and hypertension using the same strain would yield significant benefits. In consideration of protein expression quantities, we further designed tandem repeat sequences to augment the expression of the therapeutic peptide.

![Figure 5 Schematic Diagram of Tandem Repeat Sequence Construction](https://static.igem.wiki/teams/5211/eng5.jpg)

Figure 5 Schematic Diagram of Tandem Repeat Sequence Construction

#### Build

Subsequently, we will proceed with the construction of a tandemly repeated hypoglycemic peptide sequence (4\*GLP-1) and a tandemly repeated antihypertensive peptide sequence (5\*LV), followed by concatenating each individual therapeutic peptide with an enterokinase sequence. The resulting fusion protein shall be denoted as 4GLP-1-5LV.

![Figure 6 The plasmid map of the bacterial strain for 4GLP-1-5LV production](https://static.igem.wiki/teams/5211/eng6.jpg)

Figure 6 The plasmid map of the bacterial strain for 4GLP-1-5LV production

#### Test

To investigate the potential impact of initial OD600nm and inducer concentration on protein expression levels, we incorporated a gradient experiment into this round of trials. Simultaneously, we introduced plasmids lacking the target sequence into the bacteria to eliminate any influence from proteins expressed by the strain itself. By inducing protein expression in the strain, we obtained the following results:

![Figure 7 The induced expression of 4GLP-1-5LV protein (Number① to ③)](https://static.igem.wiki/teams/5211/eng7.jpg)

Figure 7 The induced expression of 4GLP-1-5LV protein (Number① to ③)

![Figure 8 The induced expression of 4GLP-1-5LV protein (Number④ to ⑥)](https://static.igem.wiki/teams/5211/eng8.jpg)

Figure 8 The induced expression of 4GLP-1-5LV protein (Number④ to ⑥)

![Figure 9 The induced expression of 4GLP-1-5LV protein](https://static.igem.wiki/teams/5211/eng9.jpg)

Figure 9 The induced expression of 4GLP-1-5LV protein

![Figure 10 Group](https://static.igem.wiki/teams/5211/eng10.jpg)

Figure 10 Group

#### Learn

After conducting this experiment, we gained invaluable experiences in gene circuit design and the application of relevant components. Moving forward, we aim to utilize these acquired knowledge and skills to further optimize our models and experimental strategies. This experience has reinforced our determination to advance biological research and develop powerful genetic engineering tools.

### Future study

Despite the experiment being well underway, the team is continuously striving to refine the project. Therefore, in-depth considerations have been made by both members and the principal investigator (PI) regarding the design of a safety mechanism.

After meticulous deliberation, a suicide mechanism has been established based on differentiating between glucose starvation within the human body and that in the natural environment. In response to glucose depletion from its surroundings, induction of T-acrp promoter leads to overexpression of autolysin gene (acmA), ultimately resulting in bacterial autolysis and subsequent demise (Figure 11).

![Figure 11 Safety switch mechanism](https://static.igem.wiki/teams/5211/eng11.jpg)

Figure 11 Safety switch mechanism

Moreover, members employ chitosan and alginate as efficacious biofilms for the encapsulation of probiotics using a layer-by-layer electrostatic self-assembly approach. The encapsulated bacteria exhibit resistance against acidic conditions in the stomach and successfully navigate through various obstacles encountered in the stomach, duodenum, small intestine, and large intestine before ultimately reaching their intended site of action within the intestines.

## References

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2.Lin Z, \*\*g Y, Huang Y, et al. A cleavable self-aggregating tag scheme for the expression and purification of disulfide bonded proteins and peptides. Chemical Engineering Science, 2022, 262: 118052.

3.Singh S M, Eshwari A N S, Garg L C, et al. Isolation, solubilization, refolding, and chromatographic purification of human growth hormone from inclusion bodies of *Escherichia coli* cells: a case study. Therapeutic Proteins: Methods and Protocols, 2005: 163-176.

4.Zeng G, Zheng Y, \*\*ang Y, et al. A novel protein purification scheme based on salt inducible self-assembling peptides. Microbial Cell Factories, 2023, 22(1): 224.

5.Bauer P V, Duca F A, Waise T M Z, et al. Metformin alters upper small intestinal microbiota that impact a glucose-SGLT1-sensing glucoregulatory pathway. Cell metabolism, 2018, 27(1): 101-117. e5.

6.Wu H, Esteve E, Tremaroli V, et al. Metformin alters the gut microbiome of individuals with treatment-naive type 2 diabetes, contributing to the therapeutic effects of the drug. Nature medicine, 2017, 23(7): 850-858.

7.Wu F, Yang L, Hang K, et al. Full-length human GLP-1 receptor structure without orthosteric ligands. Nature communications, 2020, 11(1): 1272.

8.Diz-Chaves Y, Maastor Z, Spuch C, et al. Glucagon-like peptide 1 receptor activation: anti-inflammatory effects in the brain. Neural Regeneration Research, 2024, 19(8): 1671-1677.

9.Müller T D, Finan B, Bloom S R, et al. Glucagon-like peptide 1 (GLP-1). Molecular metabolism, 2019, 30: 72-130.

10.Heijboer A C, Frans A, Lomecky M, et al. Analysis of glucagon-like peptide 1; what to measure? Clinica chimica acta, 2011, 412(13-14): 1191-1194.

11.Huan Y, Jiang Q, Li G, et al. The dual DPP4 inhibitor and GPR119 agonist HBK001 regulates glycemic control and beta cell function ex and in vivo. Scientific reports, 2017, 7(1): 4351.

12.Bothfeld W, Kapov G, Tyo K E J. A glucose-sensing toggle switch for autonomous, high productivity genetic control. ACS synthetic biology, 2017, 6(7): 1296-1304.

13.Zhang Z, Tang R, Bian L, et al. Surface immobilization of human Arginase-1 with an engineered ice nucleation protein display system in *E. Coli*. Plos one, 2016, 11(8): e0160367.

14.Shi L, Liu H, Gao S, et al. Enhanced extracellular production of is PETase in *Escherichia coli* via engineering of the pelB signal peptide. Journal of agricultural and food chemistry, 2021, 69(7): 2245-2252.

15.\*\*ao R, Wang R, Li S, et al. Preliminary Evaluation of Potential Properties of Three Probiotics and Their Combination with Prebiotics on GLP‐1 Secretion and Type 2 Diabetes Alleviation. Journal of Food Quality, 2022, 2022(1): 8586843.

16.Almo M M, Sousa I G, Olinto V G, et al. Therapeutic Effects of *Zymomonas mobilis* on Experimental DSS-Induced Colitis Mouse Model. Microorganisms, 2023, 11(11): 2793.

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18.Gurung M, Li Z, You H, et al. Role of gut microbiota in type 2 diabetes pathophysiology. EBioMedicine, 2020, 51.

19.Sun L, \*\*e C, Wang G, et al. Gut microbiota and intestinal FXR mediate the clinical benefits of metformin. Nature medicine, 2018, 24(12): 1919-1929.

20.Zhu T, Goodarzi M O. Metabolites linking the gut microbiome with risk for type 2 diabetes. Current nutrition reports, 2020, 9: 83-93.

21.Sharma S, Tripathi P. Gut microbiome and type 2 diabetes: where we are and where to go? The Journal of nutritional biochemistry, 2019, 63: 101-108.

22.Fernandes M R, Aggarwal P, Costa R G F, et al. Targeting the gut microbiota for cancer therapy. Nature Reviews Cancer, 2022, 22(12): 703-722.

23.Abenavoli L, Scarpellini E, Colica C, et al. Gut microbiota and obesity: a role for probiotics. Nutrients, 2019, 11(11): 2690.

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27.Shahzad N, Alzahrani A R, Ibrahim I A A, et al. Therapeutic strategy of biological macromolecules based natural bioactive compounds of diabetes mellitus and future perspectives: A systemic review. Heliyon, 2024.

29.Drucker D J. Advances in oral peptide therapeutics. Nature reviews Drug discovery, 2020, 19(4): 277-289.

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31.Wang Y, Shi Y, Peng X, et al. Biochemotaxis‐Oriented Engineering Bacteria Expressing GLP‐1 Enhance Diabetes Therapy by Regulating the Balance of Immune. Advanced Healthcare Materials, 2024, 13(11): 2303958.

32.Wang Y, Shi Y, Peng X, et al. Biochemotaxis‐Oriented Engineering Bacteria Expressing GLP‐1 Enhance Diabetes Therapy by Regulating the Balance of Immune. Advanced Healthcare Materials, 2024, 13(11): 2303958.

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Li Y, Wang Y, Wang R, et al. Metabolic engineering of *Zymomonas mobilis* for continuous co-production of bioethanol and poly-3-hydroxybutyrate (PHB). Green Chem, 2022, 24(6): 2588-2601.

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![](https://static.igem.wiki/teams/5211/ezgif-com-coalesce-1.gif)

## Experiments

OAST

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[Experimental Design](#section1)
[Genome simplification of *Zymomonas mobilis*](#section2)
[Metabolic engineering of *Zymomonas mobilis* to synthesize 3-HB](#section3)

* [Vector selection](#subsection1)
* [Recombinant strains construction](#subsection2)
* [Results and analysis](#subsection3)

[Expression of hypoglycemic peptides and antihypertensive peptides](#section4)

* [Vector selection](#subsection4)
* [Recombinant strains construction](#subsection5)
* [Results and analysis](#subsection6)

[Safety switch](#section5)
[Back to the top](#top)

## Experimental Design

---

In this project, we employed synthetic biology techniques to modify *Zymomonas mobilis* by introducing specific exogenous genes involved in the 3-hydroxybutyrate (3-HB) synthesis pathway. This enabled the production of 3-HB within the *Zymomonas mobilis*. Subsequently, a series of metabolic engineering modifications were implemented to enhance 3-HB production, including amplifying gene copy numbers, substituting strong promoters, optimizing cofactor supply, incorporating heterologous ethanol utilization pathways, and improving product secretion. Ultimately, these alterations resulted in further enhancements in 3-HB production. Meanwhile, we introduced expression plasmids pTZ32a containing GLP-1 (glucagon-like peptide-1) and ACEi (angiotensin-converting enzyme inhibitor) into motile fermentative bacteria to facilitate the production of therapeutic peptides with bioactive properties. By synergistically harnessing the biosynthesis of fructooligosaccharides and oligosaccharides from *Zymomonas mobilis*, our objective is to achieve dual effects of glycemic control and blood pressure reduction (Figure 1).

In each part, our approach is to initially validate the functionality of each component individually before amalgamating them to evaluate their collective efficacy. Please refer to the protocols section for precise reaction conditions employed in each step of our experimental design.

![](https://static.igem.wiki/teams/5211/exp1.jpg)

Figure 1 Experiment design

To ensure the experimental accuracy, we have implemented the following strategies:

Sequence Verification - Following plasmid construction, members validate its sequence accuracy through Sanger sequencing.

Repeated Experiments - During each experimental session, members conduct multiple replicates or parallel experiments for each experimental group to ensure result consistency.

[Click Here To See Protocols](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D)

[Click Here To See Lab Notebook](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D)

## Genome simplification of *Zymomonas mobilis*

---

The project employs an efficient genome-wide iterative and continuous editing strategy called GW-ICE to eliminate four endogenous plasmids, namely pZM32, pZM36, pZM33, and pZM39 in the strain. This approach harnesses the bacteria's own restriction-modification (R-M), CRISPR/Cas, toxin-antitoxin (T-A) systems, and natural plasmids as effective gene editing tools.

In this system, each gene editing plasmid contains two guide RNAs (gRNAs) that can simultaneously target antibiotic resistance genes of the target plasmid and chromosomal genes. As a result, it only necessitates continuous transformation of editing plasmids into the target host cells without relying on counter-selectable markers. Furthermore, a T-A system-based strategy has been developed in the laboratory for rapid screening and cloning vector construction to enhance efficiency in preventing loss of editing plasmids. By employing this method successfully eliminated four endogenous plasmids in *Zymomonas mobilis* and generated a plasmid-free mutant strain named ZMNPΔhypo which exhibits improved transformation efficiency following continuous genome editing (Figure 2).

The obtained ZMNP strain reduces genomic complexity and energy consumption while possessing desirable traits such as high transformation efficiency and enhanced tolerance to inhibitors.

![](https://static.igem.wiki/teams/5211/exp2.jpg)

Figure 2 Process to obtain the mutant strain ZMNPΔhypo with four native plasmids cured and seven genes encoding hypothetical proteins knocked out after seven continuous rounds of genome editing.

## Metabolic engineering of *Zymomonas mobilis* to synthesize 3-HB

---

### Vector selection

#### Optimization of plasmid selection for 3-HB production strains

1.By selecting or modifying plasmids with varying copy numbers, the copy number of target genes can be manipulated, thereby influencing the expression of target proteins and the production of desired products. To optimize metabolic pathway engineering, such as the 2,3-BDO pathway, we designed and constructed a minimal shuttle vector called pEZ15Asp (pEZ).

2.In order to introduce exogenous EUP pathway, we employed a modified carrier known as pEZ39p.

### Recombinant strains construction

![](https://static.igem.wiki/teams/5211/exp3.jpg)

Figure 3 Schematic diagram of plasmid design for 3-HB production strain

#### 1. Test for 3-HB tolerance of *Zymomonas mobilis*

*Z. mobilis* exhibits a remarkable tolerance to 3-HB concentrations of up to 30 g/L, highlighting its potential for high-yield production of this compound (Figure 4).

![](https://static.igem.wiki/teams/5211/exp4.jpg)

Figure 4 Test for 3-HB tolerance of *Zymomonas mobilis*

#### 2. Introduction of an exogenous 3-HB synthesis pathway into *Zymomonas mobilis*

Initially, we devised a genetic engineering strategy based on the RBS-10 sequence (CCATAATCTAGAGAAAGTAAGCAC) to introduce the coding genes of three exogenous enzymes involved in 3-HB synthesis: thiolase (PhaA), acetoacetyl-CoA reductase (PhaB), and thioesterase (FadM, YciA, TesB). Specifically, we constructed a *T1AB* operon by assembling the *tesB*, *phaA*, and *phaB* genes and integrated it into the pEZ15Asp plasmid. The gene expression was regulated by P*tet* inducible promoter. To enhance the efficiency of plasmid electrotransformation, demethylated Escherichia coli trans110 competent cells were used for transformation followed by PCR verification and cultivation to obtain the desired pEZ-PtT1 plasmids. Similarly, pEZ-PtY and pEZ-PtF plasmids were obtained using identical methods. Subsequently, these three plasmids were individually electroporated into ZMNPΔ0038 competent cells to generate strains NP-PtT1, NP-PtY, and NP-PtF capable of producing 3-HB (Figure 5).

![](https://static.igem.wiki/teams/5211/exp5.jpg)

Figure 5 Introduction of an exogenous 3-HB synthesis pathway into *Zymomonas mobilis*

#### 3. Replace strong promoter and high-intensity RBS

The plasmid was reverse-amplified prior to the pEZ-PtT1 promoter, and then assembled with the P*gap* promoter (amplified using ZM4 as the DNA template) through Gibson Assembly to generate the pEZ-PgT1 plasmid. The target strain NP-PgT1 was obtained by electroporating the pEZ-PgT1 plasmid into competent cells of ZMNPΔ0038. By substituting the RBS-10 sequence in the pEZ-PtT1 plasmid with RBS-10K (ATCACAGGGTCTAGAAGGAGGTCGAA), we obtained pEZ-PtT1'. The *T1AB* 'operon was acquired by concatenating *tesB*, *phaA*, and *phaB* genes with an RBS-10K sequence, *Ptet-T1AB'* operon was then obtained through overlap PCR amplification of P*tet* promoter and *T1AB '*operon. The *Ptet-T1AB'* operon was integrated into the pEZ15Asp plasmid to construct a recombinant plasmid named pEZ-PtT1'. Finally, this recombinant plasmid was electrically transferred into competent cells of ZMNPΔ0038 resulting in strains designated as NP-PtT1' (Figure 6).

![](https://static.igem.wiki/teams/5211/exp6.jpg)

Figure 6 Replace strong promoter and high-intensity RBS

#### 4. Amplification of the TAB operon copy number

The pL2R-PgT1 editing plasmid was constructed using the CRISPR-Cas technique, and the *ZMO1650* locus on the genome of ZMNPΔ0038 strain was replaced with the *Ptet-T1AB'* operon to generate NPTΔ1 strain. Subsequently, the recombinant plasmid pEZ-PtT1 was introduced to obtain a 3-HB multi-copy NPTΔ1-PTT1 strain (Figure 7).

![](https://static.igem.wiki/teams/5211/exp7.jpg)

Figure 7 Amplification of the TAB operon copy number

#### 5. Overexpressing cofactor related genes

The overexpression of the *Z. mobilis* endogenous *zwf* gene (*ZMO0367*) has been demonstrated to enhance the biosynthesis of NADPH-dependent metabolites, thereby providing a potential strategy for increasing 3-HB production. In this study, we employed overlap PCR to get the *zwf*-T1AB operon and successfully constructed the recombinant plasmid pEZ-PtZT1 using the P*tet* promoter. Subsequently, this plasmid was transformed into the strain NPTΔ1 to generate strain NPTΔ1-PtZT1 (Figure 8).

![](https://static.igem.wiki/teams/5211/exp8.jpg)

Figure 8 Overexpressing cofactor related genes

#### 6.Construction of penicillin-binding protein (PBPs) deficient strains

The absence of penicillin-binding proteins (PBPs) in *Zymomonas mobilis* can disrupt the normal synthesis of peptidoglycan, thereby affecting membrane structure and resulting in an elevated concentration of glucosamine in *Z. mobilis* mutants. This increased glucosamine concentration leads to enhanced permeability of the outer membrane, facilitating improved exocellular secretion of intracellular substances. Using the CRISPR-Cas technique, plasmids pL2R-0959 and pL2R-1089 were constructed to facilitate gene knockout at positions *ZMO0959* and *ZMO1089* in *Zymomonas mobilis* ZMNPΔ0038. Consequently, defective strains NPΔ0959 and NPΔ1089 were generated. Subsequently, recombinant plasmid PtZT1 was introduced into NPΔ0959 and NPΔ1089 through electroporation to obtain strains NPΔ0959-PtZT1 and NPΔ1089-PtZT1 (Figure 9).

![](https://static.igem.wiki/teams/5211/exp9.jpg)

Figure 9 Construction of penicillin-binding protein (PBPs) deficient strains

#### 7.Introduction of exogenous ethanol utilization pathways

Introducing the exogenous EUP pathway can enhance the acetyl-CoA supply. Therefore, we connected *ada* and *adh2* genes with RBS-10K sequence. Sequentially, the pEZ39p-PeEUP plasmid, which was constructed with an EUP operon driven by the Peno promoter in the shuttle vector pEZ39p, was transferred into NPTΔ1-PtZT1 competent cells to generate the target strain NPTΔ1-PtZT1EUP (Figure 10).

![](https://static.igem.wiki/teams/5211/exp10.jpg)

Figure 10 Introduction of exogenous ethanol utilization pathways

#### 8.Optimization the engineering strain through effective strategy combinations

Several strategies, including manipulation of the *zwf* gene, overexpression of the *zwf* gene, introduction of the ethanol utilization pathway, and knockout of *ZMO1089*, were combined to enhance 3-HB production. Starting with strain NPTΔ1 where *ZMO1089* was knocked out to obtain strain NPTΔ2, CRISPR-Cas technology was employed to construct editing plasmid pL2R-EUP. This plasmid integrated the EUP promoter into the *ZMO1094* locus in strain NPTΔ2, resulting in strain NPTΔ3. Subsequently, plasmid pEZ-PtZT1 was transformed into strain NPTΔ3 to generate NPTΔ3-PtZT1. To further modify this strain, competent cells of NPTΔ3-PtZT1 were introduced with plasmid pEZ39p-PeEUP through electroporation, leading to the creation of strain NPTΔ3-PtZT1EUP (Figure 11).

![](https://static.igem.wiki/teams/5211/exp11.jpg)

Figure 11 Optimization the engineering strain through effective strategy combinations

## Results and analysis

---

#### 1.Construction, testing of heterologous 3-HB production strains and screening of the thioesterase

Under tetracycline induction, the expression of Thioesterase TesB in *Z.mobilis* resulted in the highest yield of 3-HB (425.67±2.89 mg/L). Consequently, Thioesterase TesB was selected for subsequent strain transformation.

#### 2.Replace the inducible promoter of 3-HB strain with a strong promoter and replace the high-intensity RBS

(1)By substituting the strong promoter, the production of 3-HB was reduced to 232.67±2.51 mg/L.

(2)Substituting RBS10K resulted in a decrease in 3-HB production to 405.333±28.989 mg/L (Figure 12).

Therefore, the utilization of inducible promoters and RBS10 was continued for subsequent strain modification.

![](https://static.igem.wiki/teams/5211/exp12.jpg)

Figure 12 the production of ethanol and 3-HB in strong promoter expression strain NP-PgT1、High-strength RBS expression strain NP-PtT1’

#### 3.Construction and evaluation of multi-copy strains for the 3-HB pathway

The production of 3-HB was increased to 533.00±4.36 mg/L by amplifying the gene related to the 3-HB pathway.

#### 4.Construction and testing of cofactor enhanced strains

The overexpression of the cofactor-related gene (*zwf*) resulted in a significant increase in 3-HB production, reaching 685.67±10.69 mg/L.

#### 5.The 3-HB strains lacks penicillin-binding proteins (PBPs) that promote the secretion of 3-HB The production of 3-HB increased to 723.33±9.71 mg/L following the elimination of penicillin-binding protein in the 3-HB strain (Figure 13). ![](https://static.igem.wiki/teams/5211/exp13.jpg) Figure 13 the production of ethanol and 3-HB in PBPs deficient strains NPΔ0959-PtZT1、NPΔ1089-PtZT1 6.Introducing exogenous ethanol utilization to increase the supply of acetyl-coA The implementation of the exogenous ethanol utilization pathway (EUP) resulted in a significant enhancement in 3-HB production, with the yield reaching 1114.33±26.50 mg/L. 7.Optimization of culture medium enhances 3-HB production The yield of 3-HB was enhanced to 1323±22.61 mg/L under a carbon-to-nitrogen ratio of 50/5 (Figure 14). ![](https://static.igem.wiki/teams/5211/exp14.jpg) Figure 14 the production of ethanol and 3-HB in NPTΔ1-PtZT1EUP in different C/N ratio 8.Construction and testing of effective strategy combination strains The highest 3-HB yield achieved in this study was 1525±72.16 mg/L, which resulted from the effective implementation of strategic approaches and optimal medium conditions (Figure 15). ![](https://static.igem.wiki/teams/5211/exp15.jpg) Figure 15 the production of ethanol and 3-HB in strain NPTΔ3-PtZT1EUP(a) in different C/N ratio Expression of hypoglycemic peptides and antihypertensive peptides --- The *Zymomonas mobilis* chassis was employed in our project to genomically integrate the T7 RNA polymerase gene, thereby introducing the T7 expression system and constructing the ZM4-T7 and ZMNP-T7 strains. Vector selectionPlasmid selection of expression strains Integrate the T7 RNA polymerase gene into the genome and insert it into the modified expression plasmid pTZ32a. Replace the f1 sequence on the pET32a vector with zymo\_replication and express it in *Zymomonas mobilis*, including the T7 expression system. Recombinant strains construction Utilizing *Zymomonas mobilis* as the host cells, we integrated the T7RNA polymerase gene into the genome and introduced the T7 expression system. Considering that peptides with hypoglycemic and hypohypertensive effects are susceptible to degradation during fermentation in *Zymomonas mobilis* or upon exposure to gastric juices, we reconstructed a pH sensor by employing the lactose operon and P170 systems from Lactococcus lactis. The rcfB promoter was utilized for acidity sensing, where in under pH < 5.5 conditions, downstream inhibitory protein LacⅠ is expressed, leading to binding with operon O1/O2 and subsequent inhibition of downstream gene expression. To ensure proper protein expression, therapeutic peptides were fused with 6×His tags forming fusion proteins which were incorporated into cleavable self-assembled cSAT system, then it has been inserted into modified expression vector pTZ32a. Using the T7 expression system, the 4×GLP-1/5×LV therapeutic activity peptide was expressed as tandem repeats in *Zymomonas mobilis*. We also incorporated the extracellular display protein INP upstream of the fusion protein gene sequence, and constructed polymers consisting of tandem repeats of 4×GLP-1 and 5×LV to facilitate direct secretion of the active polypeptide in the human gut. These polymers can be degraded into active polypeptide monomers by enterokinase. To encapsulate probiotics, we will employ chitosan and sodium alginate as effective biofilms using a layered electrostatic self-assembly strategy. The resulting coated bacteria are expected to exhibit resistance against acidic conditions in the stomach, successfully navigating through various obstacles from the stomach and duodenum to reach the small and large intestine where they exert their effects. When the environmental pH exceeds 5.5, inhibition of the rcfB promoter occurs, resulting in degradation of the LACR protein and subsequent release of its inhibitory effect. GLP-1 is expressed and secreted into the extracellular space as hypoglycemic peptides, where it is released and binds to GLP-1 receptors on β-islet cells through blood transport, thereby activating downstream cAMP signaling pathway to stimulate insulin production. In the case of antihypertensive peptides: angiotensin-converting enzyme (ACE) catalyzes the conversion of angiotensin I to angiotensin II; ACE inhibitor LV recognizes and inhibits ACE activity, leading to vasodilation and reduction in blood pressure (Figure 16,17). ![](https://static.igem.wiki/teams/5211/exp16.jpg) Figure 16 Plasmid map ![](https://static.igem.wiki/teams/5211/exp17.jpg) Figure 17 Construction of therapeutic strains of hypoglycemic and hypohypertensive peptide Results and analysis --- Analysis of strain of hypoglycemic and hypohypertensive peptides1.The production of GLP-1 peptides by the BL21 (DE3) strain The constructed strain was verified by PCR method (Figure 18). ![](https://static.igem.wiki/teams/5211/exp18.jpg) Figure 18 The constructed strain was verified through Sanger sequencing. ![](https://static.igem.wiki/teams/5211/exp19.jpg) Figure 19 The sequencing results indicate that the constructed strain is correct (Figure 19). Results of protein expression ![](https://static.igem.wiki/teams/5211/exp20.jpg) Figure 20 Due to the excessive disorder in the gel map, where proteins of different sizes were present in almost every interval (Figure 20), we conducted a purification process for it. ![](https://static.igem.wiki/teams/5211/exp21.jpg) Figure 21 By using imidazole solutions with different concentration gradients for elution, no impurities were observed when eluted with a 50mM imidazole concentration, the protein size is approximately 90kDa (Figure 21). 2.The roduction of the 4GLP-1-5LV therapeutic peptides by the Escherichia coli Nissle 1917 (DE3) strain (1)The constructed strain was verified by PCR method (Figure 22). ![](https://static.igem.wiki/teams/5211/exp22.jpg) Figure 22 (2)The constructed strain was verified through Sanger sequencing (Fiure 23,24). ![](https://static.igem.wiki/teams/5211/exp23.jpg) Fiure 23 primer: PT7-SEQ-F ![](https://static.igem.wiki/teams/5211/exp24.jpg) Figure 24 primer: T7-ter The sequencing results indicate that the constructed strain is correct. Results of protein expression ![](https://static.igem.wiki/teams/5211/exp25.jpg) Figure 25 ![](https://static.igem.wiki/teams/5211/exp26.jpg) Figure 26 ![](https://static.igem.wiki/teams/5211/exp27.jpg) Figure 27 The target protein has a size of 24.97 kDa, in addition to the TrxA tag of 11.8 kDa, resulting in a total size of 36.77 kDa. The SDS-PAGE gel shows a protein band approximately at 45 kDa, and there is no significant difference between the control group before and after induction (Figure 25,26,27). 3.The production of 4GLP-1-5LV peptides by the ZMNP strain (1)The constructed strain was verified by PCR method (Figure28). ![](https://static.igem.wiki/teams/5211/exp28.jpg) Figure 28 (2)The constructed strain was verified through Sanger sequencing(Figure 29) ![](https://static.igem.wiki/teams/5211/exp29.jpg) Figure 29 (3)The sequencing results indicate that the constructed strain is correct. Results of protein expression(Figure 30, 31). ![](https://static.igem.wiki/teams/5211/exp300.png) Figuer 30 empty vector ![](https://static.igem.wiki/teams/5211/exp311.png) Figuer 31 pTZ32a/pTZ28a-4GLP-1-5LV 4.The production of 4GLP-1-5LV peptides by the ZM4 strain (1)The constructed strain was verified by PCR method (Figure 32). ![](https://static.igem.wiki/teams/5211/exp30.jpg) Figure 32 (2)The constructed strain was verified through Sanger sequencing (Figure 33). ![](https://static.igem.wiki/teams/5211/exp31.jpg) Figure 33 primer: PT7-SEQ-F/T7-ter (3)Results of protein expression(figure 34, 35) ![](https://static.igem.wiki/teams/5211/exp34.jpg) Figure 34 empty vector ![](https://static.igem.wiki/teams/5211/exp35.jpg) Figure 35 pTZ32a/pTZ28a-4GLP-1-5LV Results of protein expression Safety switch --- The members have identified a mechanism of glucose starvation-induced suicide, which is based on the disparities between the human body and its natural environment. In response to the environmental glucose deficiency, glucose starvation triggers an overexpression of the autolysase gene (*acmA*) through T-αcrp promoter activation, ultimately leading to bacterial autolysis and subsequent demise (Figure 36). ![](https://static.igem.wiki/teams/5211/exp32.jpg) Figure 36 The design of Safety switch

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## Result

OAST

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Metabolic engineering of *Zymomonas mobilis* to synthesize 3-HB

Expression of hypoglycemic peptide and antihypertensive peptide

## Results and analysis

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### 1.Construction, testing of heterologous 3-HB production strains and screening of the thioesterase

Under tetracycline induction, the expression of Thioesterase TesB in Z.mobilis resulted in the highest yield of 3-HB (425.67±2.89 mg/L). Consequently, Thioesterase TesB was selected for subsequent strain transformation.

### 2.Replace the inducible promoter of 3-HB strain with a strong promoter and replace the high-intensity RBS

(1)By substituting the strong promoter, the production of 3-HB was reduced to 232.67±2.51 mg/L.

(2)Substituting RBS10K resulted in a decrease in 3-HB production to 405.333±28.989 mg/L (Figure 1).

Therefore, the utilization of inducible promoters and RBS10 was continued for subsequent strain modification.

![](https://static.igem.wiki/teams/5211/exp12.jpg)

Figure 1 the production of ethanol and 3-HB in strong promoter expression strain NP-PgT1、High-strength RBS expression strain NP-PtT1’

### 3.Construction and evaluation of multi-copy strains for the 3-HB pathway

The production of 3-HB was increased to 533.00±4.36 mg/L by amplifying the gene related to the 3-HB pathway.

### 4.Construction and testing of cofactor enhanced strains

The overexpression of the cofactor-related gene (zwf) resulted in a significant increase in 3-HB production, reaching 685.67±10.69 mg/L.

### 5.The 3-HB strain lacks penicillin-binding proteins (PBPs) that promote the secretion of 3-HB

The production of 3-HB increased to 723.33±9.71 mg/L following the elimination of penicillin-binding protein in the 3-HB strain (Figure 2).

![](https://static.igem.wiki/teams/5211/exp13.jpg)

Figure2 the production of ethanol and 3-HB in PBPs deficient strains NPΔ0959-PtZT1、NPΔ1089-PtZT1

### 6.Introducing exogenous ethanol utilization to increase the supply of acetyl-coA

The implementation of the exogenous ethanol utilization pathway (EUP) resulted in a significant enhancement in 3-HB production, with the yield reaching 1114.33±26.50 mg/L.

### 7.Optimization of culture medium enhances 3-HB production

The yield of 3-HB was enhanced to 1323±22.61 mg/L under a carbon-to-nitrogen ratio of 50/5 (Figure 3).

![](https://static.igem.wiki/teams/5211/exp14.jpg)

Figure 3 the production of ethanol and 3-HB in NPTΔ1-PtZT1 EUP in different C/N ratio

### 8.Construction and testing of effective strategy combination strains

The highest 3-HB yield achieved in this study was 1525±72.16 mg/L, which resulted from the effective implementation of strategic approaches and optimal medium conditions (Figure 4).

![](https://static.igem.wiki/teams/5211/exp15.jpg)

Figure 4 the production of ethanol and 3-HB in strain NPTΔ3-PtZT1EUP(a) in different C/N ratio

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### Results and analysis

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### Analysis of strain of hypoglycemic and hypohypertensive peptides

#### 1.The production of GLP-1 peptides by the BL21 (DE3) strain

The constructed strain was verified by PCR method (Figure 1).

![](https://static.igem.wiki/teams/5211/exp18.jpg)

Figure 1

The constructed strain was verified through Sanger sequencing.

![](https://static.igem.wiki/teams/5211/exp19.jpg)

Figure 2

The sequencing results indicate that the constructed strain is correct (Figure 2).

Results of protein expression

![](https://static.igem.wiki/teams/5211/exp20.jpg)

Figure 3

Due to the excessive disorder in the gel map, where proteins of different sizes were present in almost every interval (Figure 3), we conducted a purification process for it.

![](https://static.igem.wiki/teams/5211/exp21.jpg)

Figure 4

By using imidazole solutions with different concentration gradients for elution, no impurities were observed when eluted with a 50mM imidazole concentration, the protein size is approximately 90kDa (Figure 4).

#### 2.The roduction of the 4GLP-1-5LV therapeutic peptides by the Escherichia coli Nissle 1917 (DE3) strain

(1)The constructed strain was verified by PCR method (Figure 5).

![](https://static.igem.wiki/teams/5211/exp22.jpg)

Figure 5

(2)The constructed strain was verified through Sanger sequencing (Fiure 6,7).

![](https://static.igem.wiki/teams/5211/exp23.jpg)

Fiure 6 primer: PT7-SEQ-F

![](https://static.igem.wiki/teams/5211/exp24.jpg)

Figure 7 primer: T7-ter

The sequencing results indicate that the constructed strain is correct.

Results of protein expression

![](https://static.igem.wiki/teams/5211/exp25.jpg)

Figure 8

![](https://static.igem.wiki/teams/5211/exp26.jpg)

Figure 9

![](https://static.igem.wiki/teams/5211/exp27.jpg)

Figure 10

The target protein has a size of 24.97 kDa, in addition to the TrxA tag of 11.8 kDa, resulting in a total size of 36.77 kDa. The SDS-PAGE gel shows a protein band approximately at 45 kDa, and there is no significant difference between the control group before and after induction (Figure 8,9,10).

#### 3.The production of 4GLP-1-5LV peptides by the ZMNP strain

(1)The constructed strain was verified by PCR method (Figure 11).

![](https://static.igem.wiki/teams/5211/exp28.jpg)

Figure 11

(2)The constructed strain was verified through Sanger sequencing(Figure 12)

![](https://static.igem.wiki/teams/5211/exp29.jpg)

Figure 12

(3)The sequencing results indicate that the constructed strain is correct.

Results of protein expression(Figure 13, 14).

![](https://static.igem.wiki/teams/5211/exp300.png)

Figuer 13 empty vector

![](https://static.igem.wiki/teams/5211/exp311.png)

Figuer 14 pTZ32a/pTZ28a-4GLP-1-5LV

#### 4.The production of 4GLP-1-5LV peptides by the ZM4 strain

(1)The constructed strain was verified by PCR method (Figure 15).

![](https://static.igem.wiki/teams/5211/exp30.jpg)

Figure 15

(2)The constructed strain was verified through Sanger sequencing (Figure 16).

![](https://static.igem.wiki/teams/5211/exp31.jpg)

Figure 16 primer: PT7-SEQ-F/T7-ter

(3)Results of protein expression(figure 17, 18)

![](https://static.igem.wiki/teams/5211/exp34.jpg)

Figure 17 empty vector

![](https://static.igem.wiki/teams/5211/exp35.jpg)

Figure 18 pTZ32a/pTZ28a-4GLP-1-5LV

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