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 tetracycline-inducible promoter Ptet. 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 Ptet promoter with a robust Pgap 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 Pgap 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 Ptet promoter was subsequently PCR amplified and overlapped with T1AB' operon to obtain Ptet-T1AB' operon. Integration of Ptet-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 Pgap 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 Pgap-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 Ptet 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.
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).
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.
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.
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.
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.
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:
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).
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.
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