Engineering

Evolution of Halomonas TD

Domestication of acetate tolerance

Introduction of formate assimilation pathway

T-switch

Products

P34HB

Amino acid derivatives：Tyrian Purple

Proteins：SOD and phaP

Evolution of Halomonas TD

Domestication of acetate tolerance

First cycle: Adaptive laboratory evolution

Design:
Acetate is the main product obtained through electrochemical catalytic conversion of CO(2).The alkaline and halophilic characteristics of Halomonas TD make it highly tolerant to acetate and give it a significant inherent advantage in utilizing acetate as a carbon source, which meets our requirements perfectly. However, Halomonas TD01 shows lower tolerance and utilization of acetate compared to glucose. When grown in a minimal medium containing up to 75 g/L sodium acetate (NaAc), the cell growth rate of Halomonas TD01 decreases by 56.14%. [1]

Build:
ALE of Halomonas TD1.0 was performed at 37°C using 50MMA agar plates supplemented with NaAC. Initially, single colony was isolated on a 60LB agar plate, and then spread on the 50MMA agar plate, followed by incubation at 37°C for 48 h. Then the single colony with optimal cell growth on the 50MMA agar plates was chose and spread on another new 50MMA agar plate every 48h. The NaAC supplementation was increased in a stepwise manner from 40 g L-1 to 100 g L-1 in a gradient of 20 g L-1 to achieve high-NaAC tolerance.

Test:
We primarily assess the growth of the strain based on its cell morphology observed on the agar plate. A monoclonal strain that appears whiter, fuller, and larger indicates better growth. Due to a marked decline in the strain's growth at a concentration of 100 g/L sodium acetate, we ultimately chose Halomonas TD cultivated on a 50 MMA agar plate containing 80 g/L sodium acetate for whole genome sequencing, resulting in the selection of strain TD80.

Learn:
Based on the strain's growth performance, we ultimately selected TD80 as the most promising candidate for utilizing sodium acetate, with the intention of employing it in fermentation production. Consequently, further exploration is needed to determine the optimal concentration of sodium acetate to use during fermentation.

Second cycle: Optimization of fermentation conditions

Introduction of formate assimilation pathway

·First cycle: Investigating the metabolism and tolerance of TD80 to sodium formate

Design:
Previously, the formate assimilation pathway has typically been studied in Escherichia coli[1][2], Cupriavidus necator[3], Vibrio natriegens[4] and other strains[5]. However, no relevant studies have been conducted on Halomonas TD. Therefore, before constructing plasmids for formate assimilation, we firstly assessed the metabolism and tolerance of TD80 to sodium formate.

Build:
We used sodium formate as the sole carbon source, and set the concentration gradients of 0, 5, 10, 15, 20, 25, 30, 35, and 40 g/L to explore the metabolism of sodium formate by TD80. Simultaneously, we introduced sodium formate with the above concentration gradient while maintaining a carbon source level of 35g/L sodium acetate to evaluate the tolerance of TD80 to sodium formate.

Test:
We inoculated monoclonal colonies of TD80 into a deep well plate system containing 1ml of 50MM medium, supplemented with the carbon sources as described above. After 24 hours of cultivation in a shaker at 37 ℃ and 1000rpm, we measured the OD₆₀₀ of the bacterial solution using spectrophotometer. The remaining concentrations of sodium formate and sodium acetate were then analyzed using a liquid chromatograph.

Learn:
·Due to the significant error associated with the deep well plate, a 150ml shake flask was used as the container for the subsequent experiment.
·Based on the analysis of the data from the sodium formate tolerance experiment, we observed that the decrease trend of OD₆₀₀ was not significant when the concentration of sodium formate was ≤ 35g/L. Therefore, for the fermentation experiment, we tentatively set the concentration of sodium formate at 35g/L.

[1] Yishai O, Bouzon M, Doring V, et al. In vivo assimilation of one-carbon via a synthetic reductive glycine pathway in Escherichia coli[J]. ACS synthetic biology, 2018, 7(9): 2023-2028.
[2] Kim S, Lindner S N, Aslan S, et al. Growth of E. coli on formate and methanol via the reductive glycine pathway[J]. Nature chemical biology, 2020, 16(5): 538-545.
[3] Claassens N J, Bordanaba-Florit G, Cotton C A R, et al. Replacing the Calvin cycle with the reductive glycine pathway in Cupriavidus necator[J]. Metabolic Engineering, 2020, 62: 30-41.
[4] Tian J, Deng W, Zhang Z, et al. Discovery and remodeling of Vibrio natriegens as a microbial platform for efficient formic acid biorefinery[J]. Nature Communications, 2023, 14(1): 7758.
[5] Turlin J, Dronsella B, De Maria A, et al. Integrated rational and evolutionary engineering of genome-reduced Pseudomonas putida strains promotes synthetic formate assimilation[J]. Metabolic Engineering, 2022, 74: 191-205.

·Second cycle: Build and test pSEVA321-Mmp1-C1M

Iteration 1: Build pSEVA321-Mmp1-C1M

Iteration 2: Fermentation testing of pSEVA321-Mmp1-C1M

Design:
Due to the synthesis of the AM1 genes by the company, pSEVA321-Vib-C1M was prioritized and underwent fermentation validation. we decided to use 35g/L sodium formate as the sole carbon source and make adjustments based on the results. Additionally, initial IPTG induction concentration gradients of 0, 2, 5, 10, and 20mg/L were set to activate the Mmp1 inducible promoter. Based on resistance gene screening, 25 μ g mL-1 chloramphenicol was added to the fermentation broth.

Build:
Please refer to the protocol for details.

Test:
Before fermentation, it was observed that the solution in the shake flask became turbid, raising concerns that this turbidity might affect the OD₆₀₀ measurements. As a result, only the remaining concentration of sodium formate was measured in the end.

Learn:
Through a series of gradient turbidity tests and initial assessments of sodium formate tolerance, we adjusted the concentration of sodium formate during fermentation to 15g/L. Additonally, based on the data obtained from the liquid chromatography, it was determined that it was too early to measure the remaining concentration of sodium formate. Therefore, for the time being, only OD₆₀₀ measurement will be carried out to reflect the metabolism of sodium formate by cells.
After the successful construction of pSEVA321-AM1-C1M, we conducted comparative fermentation using adjusted fermentation conditions. The experimental results showed that the introduction of AM1-C1M increased the growth of TD80 by 67.33% under the culture conditions with sodium formate as the sole carbon source, while the introduction of Vib-C1M increased it by 59.67%
[6] Fu XZ, Tan D, Aibaidula G, Wu Q, Chen JC, Chen GQ. Development of Halomonas TD01 as a host for open production of chemicals. Metab Eng 23, 78-91 (2014).

·Third cycle: Optimize C1M to eliminate the need for inducer

Design:
Given that the Mmp1 promoter requires IPTG for induction, which can be costly, and considering the challenges posed by concentration gradients in large industrial fermenters—where it is difficult to ensure uniform IPTG distribution to all bacteria—we propose converting the inducible promoter into a constitutive promoter.
Previous fermentation data indicated no clear trend in stepwise changes under IPTG concentration gradients. In fact, even at an IPTG concentration of 0, the expression intensity was higher. Therefore, we have decided to replace the Mmp1 inducible promoter with two constitutive promoters, porin194 and porin281, which exhibit weaker expression intensities.
After discussing this with our teacher, we considered that RBS2000 had previously been used as the ribosome binding site for C1M, and it yielded the highest expression intensity among the series of RBSs. Therefore, it would not be meaningful to focus solely on weakening the promoter at this stage. Instead, while adjusting the promoter strength, we decided to change the RBS to the moderately intense B0064 for optimization. Additionally, we divided the C1M gene into two segments for tuning, hoping to achieve varied results.
Based on our own insights and our teacher's advice, we ultimately decided to construct tuning plasmids with RBS B0064 and promoter tuning as the experimental group, while creating control plasmids that only incorporate weak promoter tuning.

Build:
For the construction of tuning plasmids, each group of genes, having their own promoters, was assembled through a two-step reaction process. Firstly, we constructed individual plasmids for each group of genes separately, and then combined the two groups of genes into a complete C1M.
In the first reaction, we utilized a pSEVA321 backbone containing the corresponding porin constitutive promoter, a chloramphenicol resistance gene, and RBS2000, along with a single C1M gene as the target fragment. A set of primers was designed at RBS2000, and due to the only difference in the base groups between B0064 and RBS2000 sequences, the RBS was directly modified during primer design. We then performed Gibson assembly to obtain pSEVA321-porin281/194-B0064-AM1-ftfl, AM1-fch-mtdA, Vib-ftl, and Vib-folD.
In the second reaction, two sets of tuning plasmids corresponding to the complete C1M were spliced together. Considering that the length difference between the assembled two fragments would reduce the success rate, we flexibly adapted and disconnected the pSEVA321 skeleton from the chloramphenicol resistance gene, with the former group of genes carrying the initial half of the skeleton and the latter group of genes carrying the second half of the skeleton. If the transformed strain can grow monoclonal antibodies on LB plates containing chloramphenicol, it indicates that the connection at the skeleton fracture is correct. During sequencing, only the genes at the interface of the two groups of genes need to be tested for mutations.
For the construction of plasmids with only weak promoters, the pSEVA321 backbone with dblTherm terminator, corresponding porphyrin constitutive promoter, and chloramphenicol resistance gene was used. The RBS2000-C1M sequence of the previously constructed pSEVA321-C1M plasmid served as the target fragment for Gibson assembly.
we conducted colony PCR to screen the colonies for the desired construct. Plasmids from positive clones were purified and digested with restriction endonucleases to preliminarily verify the success of cloning. Subsequently, Sanger sequencing (Songong Co., Ltd.(Shang hai, China)) was performed on the cut fragments to exclude the possibility of gene mutations.

Test:
Using the same seed culture preparation scheme as in the second cycle, the secondary seed culture medium was inoculated into 20ml of 50MM-F medium at a volume of 5%, which contained 15g/L sodium formate and 25 μ g mL-1 chloramphenicol. The medium was incubated at 37 ℃ and 220rpm in an incubator for 48 hours.
After incubation, dilute the bacterial solution 5 times and measure the OD₆₀₀ using a spectrophotometer. Dilute the bacterial solution 10 times and measure the remaining concentration of sodium formate using liquid chromatography.

Learn:
Analysis of the data revealed that the B0064-porin281 Vib ftl-porin194-Vib folD group had the lowest residual sodium formate concentration, which was 15.3% lower than that of the wild TD80 group. However, regarding OD₆₀₀ measurements, B0064-porin194-AM1 ftfl-porin281-AM1 fch mtdA exhibited the best growth, showing a 66.5% increase compared to wild TD80.
Considering liquid chromatography as a quantitative analysis, the amount of formate assimilated by TD80 was accurately determined. Furthermore, the difference in OD₆₀₀ between the two groups was not significant. Finally, the combination of B0064-porin281-Vib ftl-porin194-Vib folD was selected as the most effective.

·Fourth cycle: dual carbon source fermentation

Design:
Given our hope that Halomonas TD can utilize CO2 electrochemical derivatives instead of formate as their sole carbon source for growth, we initially discovered that the THF system effectively supports Halomonas TD using formate as a single carbon source. Building on this, we began to explore whether the THF system could facilitate dual carbon source fermentation by primarily using acetate, while allowing TD80 to absorb and utilize formate.

Build:
(1) Experimental Group
Recombinant TD80 with pSEVA321-B0064-porin281 Vib ftl-porin194 Vib folD. Set sodium acetate concentration gradients at 0, 2, 4, 6, and 8 g/L, while maintaining a fixed sodium formate concentration of 5 g/L. Monitor whether sodium formate absorption increases with rising sodium acetate concentrations.
(2) Control Group:
1. Wild-type TD80 without any carbon source, using only 50 mM as the cultural medium to eliminate the influence of other factors on TD80 growth.
2. Wild-type TD80 utilizing 5 g/L sodium formate as the sole carbon source, without the introduction of any plasmids.
3. Wild-type TD80 using 8 g/L sodium acetate as the sole carbon source, without the introduction of any plasmids.
4. Wild-type TD80 containing both 5 g/L sodium formate and 8 g/L sodium acetate, without the introduction of any plasmids.

Test:
To ensure the accuracy of the spectrophotometer, we diluted the bacterial solution to OD₆₀₀ between 0.2 and 0.8 for measurement. After diluting the bacterial solution 10 times, measure the remaining concentrations of sodium formate and sodium acetate using liquid chromatography.

Learn:
The experimental results indicated that as the concentration of sodium acetate increased, the absorption and utilization of sodium formate by TD80 also rose. At a sodium acetate concentration of 8 g/L, the group supplemented with 5 g/L sodium formate exhibited a 15% increase in growth compared to the group without added sodium formate. At this stage, following the introduction of the formate assimilation pathway, Halomonas TD can utilize the majority of CO2 electrochemical derivatives (CDE) for growth, resulting in the production of easily recyclable, non-toxic bicarbonate in low concentrations.

T-switch

·First cycle: First try of exploration of useable thermosensitive bio-switch in Halomonas TD

·Second cycle: Second try of exploration of useable thermosensitive bio-switch in Halomonas TD

·Third cycle: CI857 regulatory protein mutation

- Fourth cycle: TCI-42 regulatory protein mutation

·Fifth cycle: promoter replacement of sfGFP and CI857

Products

P34HB

First cycle: Introduction of P34HB synthesis pathway

The existing methods for large-scale production of P34HB primarily rely on microbial fermentation. A key limiting factor in this process is the molar ratio of 4HB. Increasing the 4HB molar ratio can lead to a decrease in the melting temperature and apparent fusion heat of the copolymer, as well as an improvement in the polymer's deformation resistance. Therefore, enhancing the molar ratio of 4HB is crucial for the modification of P34HB.

Iteration 1: Build 341-4hbd sucD ogdA-orfZ

Design:
Prior to embarking on this project, our laboratory had already conducted research on the production of P34HB. It was found that the expression of the 4hbd-sucD-ogdA-orfZ gene cluster could increase the molar ratio of 4HB. Following fermentation using Mmp1 inducible promoter, the porin194 constitutive promoter was considered more suitable based on the concentration gradient induction trend observed with IPTG.
Since two plasmids, pSEVA321 and pSEVA341, are commonly used in the laboratory, the gene cluster has only been previously expressed through the pSEVA321 plasmid. Our intention is to introduce the porin194-4hbd-sucD-ogdA-porin194-orfZ gene cluster into TD80 to synthesize P34HB, utilizing both the pSEVA341 and pSEVA321 plasmids, which allow us to evaluate which plasmid yields better results.

Build:
We obtained pSEVA321-porin194-4hbd-sucD-ogdA-porin194-orfZ directly from the laboratory. For pSEVA341-porin194-4hbd-sucD-ogdA-porin194-orfZ, we utilized the pSEVA341 backbone, which includes spectinomycin resistance gene and kanamycin resistance gene. The 4hbd-sucD-ogd-orfZ gene cluster was sourced from the existing pSEVA321 plasmid.
The primers were synthesized by Songong Co., Ltd (Shanghai,China), accompanied by homologous arms, for assembly using Gibson assembly kit after PCR amplification.
Assembly products were subsequently converted into competent cells of Escherichia coli S17-1.

Test:
We used colony PCR for preliminary screening, followed by Sanger sequencing to rule out the possibility of gene mutations. Please refer to the protocol for more details.

Learn:
By comparing the sequencing results, it was found that there was no mutation in the genes. Therefore, the successfully constructed plasmid can be transferred into TD80 for fermentation validation.

Iteration 2: Fermentation production of P34HB

Design:
Due to the incomplete optimization of formate assimilation, P34HB production initially was carried out in TD80 first. Based on the results of acetate domestication, we have decided to use 35g/L sodium acetate as the sole carbon source.

Build:
Please refer to the protocol for details.

Test:
After 48 hours of cultivation, we measured the dry weight of the strain and used gas chromatography to measure the 4HB molar ratio and PHA production. Please refer to the protocol for details.

Learn:
The experimental results indicated that the 4HB molar ratio achieved by introducing the pSEVA321 was higher than that of the pSEVA341. However, the dry weight decreased somewhat compared to the wild TD80.

Second cycle: Change screening pressure

To stabilize cell dry weight while increasing the 4HB molar ratio, we investigated the effect of the pSEVA321 on cell dry weight. We hypothesized that the addition of chloramphenicol during the fermentation process impacted cell growth. Consequently, we decided to adjust the screening pressure and knock out cysNC, a gene encoding a key enzyme in the sulfate assimilation pathway, in Halomonas TD to block the supply of sulfur sources for methionine synthesis. Simultaneously, we incorporated the cysNC gene into the pSEVA321 backbone to screen for strains that had been successfully transformed with the 321-porin194-4hbd-sucD-ogdA-porin194-orfZ plasmid.

Iteration 1: Constructing knockout plasmids

Design:
We used CRISPR/Cas9 for gene knockout. Due to the challenges of gene editing in Halomonas TD, we employed two methods to block the expression of cysNC. The first method involved knocking out all codons from the start codon (ATG) to the stop codon. The second approach entailed knocking out the core region from its full-length sequence. We designed two sets of gRNAs targeting the specific regions for each knockout method. Both methods were performed simultaneously, and successful knockouts were selected for the subsequent fermentation step.

Build:
We first designed primers to amplify the 1000 bp homologous arms on either side of the core region of the cysNC gene, and the vector backbone. Then, through the Gibson assembly process, we linked the left and right homologous arms with the vector.
After successful sequencing, we designed two pairs of primers to modify the guide RNA in each plasmid. The entire plasmid was divided into two parts at the kanamycin resistance gene to use a medium containing kanamycin for screening bacteria that have successfully taken up the ligated plasmids.

Test:
We used colony PCR for preliminary screening, followed by Sanger sequencing to rule out the possibility of gene mutations. Please refer to the protocol for more details.

Learn:
By comparing the sequencing results, it was found that there was no mutation in the gene. Therefore, the successfully constructed plasmid can be used to knock out cysNC or its core region in the TD80 genome.

Iteration 2: Knock out cysNC

Design:
After constructing pSEVA341-gRNA, we used CRISPR/Cas9 to knock out cysNC or its core region in TD80.

Build:
Plasmids were transferred from E. coli S17-1 to H. bluephagenesis TD80 using an optimized conjugation transformation protocol as follows: E. coli S17-1 as the donor cell which harbors the plasmid was cultured in LB medium in the presence of appropriate antibiotic(s). H. bluephagenesis TD80 0r recombinant H. bluephagenesis TD80 were the recipient cells, and were cultured in 60LB. Select monoclonal antibodies and culture them overnight in 5 mL of the appropriate medium. Next, take 20 µL of both the recipient bacterial solution and the donor bacterial solution, and mix them on a 20 LB plate. Incubate the mixture overnight at 37 °C. Finally, resulting bacterial lawn was re-suspended in 60LB medium and spread on a 60LB agar plate with appropriate antibiotics, followed by incubation at 37 °C for 24–48 h to select for transconjugants.
Initially, S17-1 (pSEVA321-Cas9) was conjugated with TD80 and subsequently cultured on a 60AC plate containing ampicillin and chloramphenicol. After achieving monoclonal growth, we obtained TD80 (Cas9) by streaking on the 60AC plate.
Following the acquisition of TD80 (Cas9), it was conjugated with S17-1 (pSEVA341 gRNA) and cultured on a 60CSM plate containing chloramphenicol, spectinomycin, and methionine. Once monoclonal antibodies grow, we can verify the successful knockout of cysNC or its core region.

Test:
After growing monoclonal colonies on a 60CSM plate containing chloramphenicol, spectinomycin, and methionine, we designed a pair of universal primers at 50-100bp around the homologous arm of the genome, along with a specific primer located in the middle of the target knockout gene.
First, we performed colony PCR using the universal primers and sampled the bacteria on a 60LB plate. If the knockout was successful, we expected to see a band around 4500 bp; if it failed, the band would be around 2200 bp. On the following day, we conducted a secondary validation on the successfully knocked-out strains using the specific primers. If the knockout was successful, no bands would be present; if it failed, a band approximately 2000 bp in length would appear.
After successful double validation, in order to facilitate subsequent gene editing and plasmid transfer of the strain, we used a deep well plate to eliminate resistance in the strain at a temperature of 42 °C. The deep well plate consisted of 1 ml of 60LB and 0.2 g/L methionine, and the cultures were incubated for 24 hours.
Starting from the third generation, we verified whether resistance had been removed. Since pSEVA321-Cas9 is easily eliminated, our primary focus was to verify whether pSEVA341-gRNA-cysNC had been removed. We took 5 μL of the bacterial solution and transferred it to a 60ASM plate containing spectinomycin and methionine, followed by incubation at 37 °C for 12 hours. If no colonies grew, this indicated that resistance had been successfully removed.
The next generation was then streaked to select monoclonal colonies from the 60M plate containing methionine for double validation. If successful, the bacteria could be preserved; if not, it would indicate that the bacteria on the sample plate had reverted to the wild type. In such a case, we needed to re-verify whether the bacteria on the sample plate had returned to wild type. If they had, the coated plate should be reconnected; if there was no response, we would begin the de-antibody passage and repeat the above steps until success was achieved.

Learn:
After numerous attempts, t we successfully achieved double validation and the removal of the antibody. The sequencing outcomes revealed that the core region of the cysNC had been successfully knocked out. We can now proceed to the next step of the experiment, which involves constructing cysNC on pSEVA321 as a new selection pressure.

Iteration 3: Build pSEVA321 (C+cysNC) - porin194-4hbd-sucD-ogdA-porin194-orfZ

Design:
After blocking the expression of cysNC in TD80, the strain is unable to synthesize methionine, making it difficult to grow in methionine-deficient media. We aim for only the recombinant TD80 containing the porin194-4hbd-sucD-ogdA-porin194-orfZ gene cluster to thrive in 50 mM medium without the addition of antibiotics. To achieve this, we incorporated the cysNC gene into the pSEVA321 backbone, allowing TD80 transformed with the plasmid to synthesize methionine and grow in 50 mM medium lacking methionine.

Build:
First, a pair of primers was designed to obtain the RBS for cysNC from the genome using PCR. Next, we designed two pairs of primers to divide pSEVA321-porin194-4hbd-sucD-ogdA-porin194-orfZ into two segments, located after the chloramphenicol resistance gene and the 4-hbd gene, respectively. While designing the primers, we included homologous arms to facilitate the connection of these three segments through Gibson assembly, resulting in the formation of a new plasmid: pSEVA321 (C+cysNC) - porin194-4hbd-sucD-ogdA-porin194-orfZ.

Test:
We used colony PCR for preliminary screening, followed by Sanger sequencing to rule out the possibility of gene mutations. Please refer to the protocol for more details.

Iteration 4: test pSEVA321 (C+cysNC) - porin194-4hbd-sucD-ogdA-porin194-orfZ

Design:
After successfully knocking out the key region of the cysNC gene and constructing pSEVA321 (C+cysNC)-porin194-4hbd-sucD-ogdA-porin194-orfZ, we initiated verification to determine whether altering the screening pressure could enhance the 4HB molar ratio.

Build:
We have designed the following experimental groups:

1.TD80（△cysNC-key）&321(C + cysNC) -194-4hbd-sucD-ogd-194-orfZ

This was the key experimental group aimed at determining whether optimal screening pressure was beneficial to the growth of TD80 compared to Group 2.

2.TD80 & 321(C) -194-4hbd-sucD-ogd-194-orfZ

As the control group for Group 1, it was essential to compare whether optimizing the screening pressure had a beneficial effect on the growth of TD80.

1. TD80（△cysNC-key）

To determine whether the knockout of cysNC-key impairs TD80's ability to synthesize methionine.

2.TD80（△cysNC-key）&0.2g/L Met

If Group 3 demonstrated that TD80 (△cysNC-key) could not synthesize methionine, the effect of cysNC supplementation in the form of pSEVA321 on the growth of strains was then observed in this group.

5.TD80

Examine the ability of wild strains to produce P34HB.

Test:
·Measurement of growth curve: 200 microliters of sample were taken at the fermentation time of 2, 4, 6, 8, 10, 12, 16, 20, 24, 28, 32, 40, 48h, diluted to OD₆₀₀ between 0.2~0.8, and measured with a spectrophotometer

·Dry weight measurement: Take 15ml of bacterial solution, centrifuge evenly at 9000rpm for 5 minutes. Discard the supernatant, then add 15ml of water to resuspend the precipitate (2500rpm for 10 minutes). Centrifuge evenly for the second time, discard the supernatant, cover the tube with sealing film and puncture the hole. Place the sample in a -80 °C freezer for 2 days, then transfer it to a freeze dryer for 24 hours before weighing it.

·Content measurement: Take 30-40 mg of the sample into an esterification tube. Add 2 mL of esterification solution and 2 mL of chloroform to each tube. Heat the mixture at 99.9 °C for 4 hours, then cool it down. Add 1 mL of ultrapure water to each tube and mix well. Allow it to stand for 1 hour, then take 1 mL of the lower liquid from the filter head and analyze it using a gas chromatograph.

Learn:
By plotting the growth curves, it is evident that the growth of TD80 (△cysNC-key) is comparable to that of TD80 and TD80 (△cysNC-key) in the presence of 0.2 g/L Met. Consequently, cysNC is not an essential gene in the methionine synthesis pathway and cannot be used as a selective pressure to ensure continuous expression of the gene cluster in TD80.
After gas phase analysis, the molar ratio of P34HB produced by TD80 (△cysNC-key) & 321(C+cysNC)-194-4hbd-sucD-ogd-194-orfZ was significantly lower than that of TD80 & 321(C)-194-4hbd-sucD-ogd-194-orfZ. This suggests that some TD80 cells lost the pSEVA321 plasmid during fermentation and ceased to produce P34HB.
The dry weight results indicated that the growth of recombinant TD80 increased even without the addition of antibiotics. Therefore, identifying a new essential gene may be a viable strategy to enhance the 4HB mol% of P34HB, and this will be a focus of our future efforts.

Amino acid derivatives

Tyrian Purple

·First cycle: Introduce the tyrian purple synthesis pathway in Halomonas TD

Design:
After searching related literatures, we found that there were three key enzymes in the synthesis of Tyrian purple from tryptophan[1]. According to various databases, Halomonas TD doesn't naturally have that three enzymes. Thus, we decided to introduce them into Halomonas TD and test if it could work.

Build:
There were four genes that we needed to introduce to Halomonas TD:stth,fre,tnaA and fmo.Their specific information could be seen in our part page. For stth and fmo,we obtained them from the genome of E.coli 1655.As for fre and tnaA,we entrusted the company to help synthesise the corresponding gene fragments.Then, we built two plasmids separately carrying these four genes according to the synthesis pathway:pSEVA341-fre-stth and pSEVA321-tnaA-fmo.At last, we transformed this two plasmids into a same strain Halomonas Δ_AFTD,which knocked out pheA and pykF. It was proved that the knock-out of these two genes could improve production of tryptophan, the precursor of tyrian purple[2][3].We obtained the strain from our laboratory.

Test:
We used the constructed strain for 48-hour shake flask fermentation. After the fermentation, the sample was processed and they all seemed depressingly blue, which means there were a large amount of by-product indigo. Because the standard sample of tyrian purple would not arrive for some time, we decided to do some other experiments to see whether the pathway in Halomonas Δ_AFTD could work.

Learn:
Before we could know that whether the strain could produce Tyrian purple, first we should make sure that there were enough 6-Br-Trp provided for follow-up reaction. Thus, before the standard sample of Tyrian purple arrived, we should do another reasearch.

reference:

[1]Lee, J., Kim, J., Song, J. E., Song, W.-S., Kim, E.-J., Kim, Y.-G., Jeong, H.-J., Kim, H. R., Choi, K.-Y., & Kim, B.-G. (2021). Production of Tyrian purple indigoid dye from tryptophan in Escherichia coli. Nature Chemical Biology, 17(1), 104–112.

[2]Guo L, Ding S, Liu Y, Gao C, Hu G, Song W, Liu J, Chen X, Liu L. Enhancing tryptophan production by balancing precursors in Escherichia coli. Biotechnol Bioeng. 2022 Mar;119(3):983-993. doi: 10.1002/bit.28019. Epub 2021 Dec 30. PMID: 34936092.

[3]Xiong B, Zhu Y, Tian D, Jiang S, Fan X, Ma Q, Wu H, Xie X. Flux redistribution of central carbon metabolism for efficient production of l-tryptophan in Escherichia coli. Biotechnol Bioeng. 2021 Mar;118(3):1393-1404. doi: 10.1002/bit.27665. Epub 2021 Jan 13. PMID: 33399214.

·Second cycle: Find out whether the strain could produce enough substrate for follow-up reaction

·Third cycle: Introduce thermosensitive bio-switch into the synthesis of Tyrian purple

Design:
Since in the first cycle the fermentation result seemed depressingly blue, we kept searching information to get the answer. By reading papers we got an important information that Stth and TnaA were isoenzyme, which means they could use tryptophan as substrate to produce different products. When TnaA reacts with the substrate, a large number of indigo by-products are produced, and studies show that tryptophan has a higher preference for TnaA, which means more by-products and fewer target products.Thus, we need to separate this two enzymes to make sure enough substrate for Stth.It was at that time that we came up with the thought of introduce thermosensitive bio-switch into the synthesis of tyrian purple.When looking for relevant literature, we saw a newly published literature this year that also introduced thermalsensitive bio-switch in the production process of tyrian purple to separate the expression of Stth and TnaA enzymes, which finally achieved good results[1].It greatly encouraged us, because it meant that our ideas were feasible.

Build:
When constructing the T-switch, we found that the dynamic regulation performance of the switch was better when switching from low temperature to high temperature. Therefore, when applying to the production of Tyrian purple, we also planned to use the switching from low temperature to high temperature to realise the separate expression of Stth and TnaA. Therefore, we constructed two plasmids, pSEVA341-pr-mmp1-tnaA-fmo and pSEVA321-prm-porin58-fre-porin68-stth, and verified by fermentation.

Test:
We designed to test four strains as below:

No.	Strain	Usage
1	Δ_AFTD+pSEVA341-PRM/pr-tnaA-fmo+pSEVA321-pRM-ci857-mmp1-fre-stth	It was used to check the yield of tyrian purple.
2(Only for deepwell plate fermentation)	Δ_AFTD+pSEVA341-PRM/pr-sfgfp+pSEVA321-pRM-ci857-mmp1-fre-stth	It was used to check the leakage of T-switch.
3	Δ_AFTD+pSEVA341-PRM/pr-tnaA-fmo	As a control of the first strain to assess the yield of Tyrian purple
4	Δ_AFTD+pSEVA321-pRM-ci857-mmp1-fre-stth	It was used to test the yield of 6-Br-Trp at different temperatures.

Other variables were the amount of IPTG and tryptophan added.We set two gradients for each of the two variables:IPTG(mg/L)=5, 20 and Trp(g/L)=0.4,0.8.

At 16,24,32h, we took a batch of samples and switched the temperature to 37℃,in case to find a better timing to switch temperature through the yield of 6-Br-Trp.

Strain1, 3 and 4 was for shake flask fermentation to test the production of Tyrian purple.Strain2 was for deepwell plate fermentation to test the leakage of our T-switch in the synthesis of Tyrian purple as a control group.

Learn:

We tested samples taken from group 1 and group 3 as experimental group and control group. The result showed that the experimental group produced 12.31 mg/L and 25.49 mg/L of tyrian purple under the condition of switching to 37°C to continue culture after 36 hours of cultivation at 30°C and keeping in 30°C in the whole process respectively.

The ratio of Tyrian purple to indigo yield expressed under the same switching temperature and Trp,IPTG protocol is as follows.

Growing time at 30℃	Ratio
48h	5.10
32h	2.46

At the same time, we tested the fluorescence intensity(FI) of group 2 to see the expression intensity of pR/pRM-tnaA-fmo in different timing. The result showed that most of the fluorescence leakage were controlled below 20, and a small number of anomalous data are believed to be caused by the poor uniformity of the shaker, which led to the lack of strict temperature control.Fermentation results from deep-well plates showed that the separation of Stth and TnaA expression using our T-switch was effective.

reference:

[1]Feifei Li, Que Chen, Huaxiang Deng, Shumei Ye, Ruidong Chen, Jay D. Keasling, Xiaozhou Luo,One-pot selective biosynthesis of Tyrian purple in Escherichia coli,Metabolic Engineering,Volume 81,2024,Pages 100-109.
[2]Athina Vasileiadou, Ioannis Karapanagiotis, Anastasia Zotou, Determination of Tyrian purple by high performance liquid chromatography with diode array detection,Journal of Chromatography A,Volume 1448,2016,Pages 67-72.
[3]Athina Vasileiadou, Ioannis Karapanagiotis, Anastasia Zotou,
Determination of Tyrian purple by high performance liquid chromatography with diode array detection,Journal of Chromatography A,Volume 1448,2016,Pages 67-72，

Proteins:SOD and phaP

Design:
To enable TD80 to produce more high value-added products, we introduced exogenous protein genes SOD, phaP, amalase, and Meta, and in order to enhance the expression of Meta proteins in TD, we introduced the autopolymerizing peptide PYL gene to enhance the assembly of Meta proteins. The exogenous protein genes were transferred into TD80 by plasmids pSEVA321-SOD, pSEVA321-phaP, pSEVA321-amalase, and pSEVA321-Meta-PYL. Meanwhile, we set up an experimental group and a control group with glucose and sodium acetate as the carbon sources during the fermentation process in order to test whether they could play the TD80 function and the protein expression when sodium acetate was used as the carbon source.

Build:
Plasmids were transferred from E. coli S17-1 to H. bluephagenesis TD80 using an optimized linkage transformation protocol: E. coli S17-1 carrying the plasmids were used as donor cells and cultured in LB medium containing chloramphenicol. h. bluephagenesis TD80 or recombinant H. bluephagenesis TD80 were the H. bluephagenesis TD80 or recombinant H. bluephagenesis TD80 were used as recipient cells and cultured in 60LB medium. Monoclonal antibodies are selected and cultured overnight in 5 mL of appropriate medium. Then, 20 µL of recipient and donor fluids were taken and mixed on 20 LB plates. The mixture was incubated at 37 °C overnight. Finally, the resulting bacterial lawn was resuspended in 60 LB liquid medium and coated on 60 LB plates containing chloramphenicol and ampicillin and incubated at 37 °C for 24-48 hours to select transposons.
Subsequently, TD80 (phaP) and TD80 (SOD) were obtained by picking single clones from the coated plates and streaking them on 60AC plates.

Test:
After adding IPTG for 12 hours of incubation, we measured the dry weight and OD₆₀₀ of the strains and performed protein expression assays by SDS-PAGE.Please refer to the protocol for details.

Learn:
From the fermentation result graph, it can be seen that the exogenous genes phaP and SOD genes can be expressed in TD80, and the amount of protein produced by glucose as carbon source is more than that produced by sodium acetate as carbon source. In addition, the expression of exogenous phaP and SOD genes in TD80 had relatively little effect on the growth of TD80 itself.