Part:BBa_K4989011

Complete butyrate producing gene cluster

Short Description

In this registry page, we will present our New Improved Part that our team created to be nominated for the gold medal in the iGEM 2023 competition! We designed a whole composite part, with its coding and regulatory sequences, that can potentially produce butyrate, a Short Chain Fatty Acid (SCFA) that could potentially treat depression.

The fundaments of the improvement

To result in the design of our new improved part, we needed to find a corresponding part that requires such changes that would not change the function of the part and the general aim of its initial formation but enhance the overall performance.
Team NRP-UEA-Norwich 2015 designed the BBa_K1618021 composite part that could potentially produce butyrate. We reviewed the part and we realized that it is our ideal candidate to enhance its features and produce a stronger gene cluster of the butyrate production pathway.
Simultaneously, we conducted extensive bibliographic research and we created a draft of the ideal gene cluster and its regulatory elements. In this way, we could compare the ideal draft of the part that we envisioned with the part that was pruned to be improved.

Application in the field of biology

Short-chain fatty acids are vital metabolic byproducts generated through microbial fermentation of dietary fibers in the gut. Gut bacteria, particularly those belonging to the Firmicutes and Bacteroidetes phyla, break down these substrates through various metabolic pathways. There are several fermentation pathways leading to the production of SCFAs, including the glycolytic pathway, the pentose phosphate pathway, and the reductive citric acid cycle. In these pathways, bacteria metabolize sugars and other organic compounds to produce SCFAs as metabolic byproducts.

Acetate Production: Acetate is the most abundant SCFA in the gut. It is primarily generated through the acetyl-CoA pathway, where acetyl-CoA is converted into acetate via various enzymatic reactions.
Propionate Production: Propionate is synthesized mainly via the succinate pathway. Bacteria convert succinate, a metabolic intermediate, into propionate. This pathway is essential for maintaining energy balance and regulating blood glucose levels.
Butyrate Production: Butyrate is generated through several pathways, with the two main pathways being the butyryl-CoA:acetate CoA-transferase pathway and the butyrate kinase pathway. These pathways involve the conversion of acetyl-CoA and butyryl-CoA into butyrate.

From bibliographical research and various experimental data, we concluded that acetate and butyrate are the most profound and effective molecules for the treatment of depression, and generally the overall gut health. Therefore, the design of a probiotic that would include a vector with the gene cluster of the butyrate-producing metabolic pathway, could lead to the production of a psychobiotic that could treat depression. Our selected strain Lactobacillus rhamnosus GG produces only acetate.Lactobacillus rhamnosus GG is a very common probiotic that is Generally Recognised As Safe (GRAS) and our dry lab bioinformatic analysis found that the concentration of the Lactobacillus rhamnosus bacterium is low on depressive people. Thus it made us focus on this probiotic for its utilization as a host of the butyrate-producing pathway.



Pathways for the synthesis of SCFAs. Lange, O., Proczko-Stepaniak, M. & Mika, A. Short-Chain Fatty Acids—A Product of the Microbiome and Its Participation in Two-Way Communication on the Microbiome-Host Mammal Line. Curr Obes Rep 12, 108–126 (2023). https://doi.org/10.1007/s13679-023-00503-6

Configuration of the new part

In this section, we will shortly analyze the re-design process of our part. The extensions of this process and the more detailed handling are analyzed in our team's New Improved Part Wiki section (see at the end of the page).
The re-design project was performed on 3 separate levels:

A. On a sequence level
B. On a regulatory level
C. On a structural level

On a sequence level, we needed to change the whole DNA makeup of the genes and codon-optimize them to be properly expressed in the corresponding hosts. Also, we performed an in-silico optimization of the sequence to not include any restriction sites of the endonucleases that we intended to use.

On a regulatory level, we added some very crucial regulatory elements to ensure the expression of the enzymes. We included the P32 promoter with its RBS for the expression to be compatible with our target host Lactobacillus rhamnosus GG. Also, we added at the of the part a terminator sequence specifically designed for Lactobacillus spp..In this way, we ensured our transcriptional termination. Lastly, we included in-between the coding sequences Ribosome Binding Sites, for one transcriptional mRNA to be produced by all the target enzymes of the pathway.

On a structural level, we maintained the order of the genes and we added, upon prompt from our dry lab bioinformatic analysis, the butyryl-CoA:acetyl-CoA transferase that catalyzes the last step of the butyrate production.

Our new improved part has the characteristic of a whole constructed transcriptional cassette. However, we have marginalized all the basic parts (components) for the P32 promoter and the terminator to be removed, always based on the regulatory feature that the cloning vector has.

The obtainment of the sequence and its difficulties

Our initial goal was to synthetically produce the whole sequence. But might be difficult due to the fact that the sequence is large in size and unfortunately, it includes a high rate of complexity. Also, synthesizing the terminator might not be able to happen due to the hairpin that it forms. Twist Bioscience, one of this year's sponsors synthesized our part in gBlocks that we later assembled with the GoldenGate method.

The sequences where obtained from basic parts already recorded in the registry (P32,terminator,RBS), from Coprococcus sp. L2-50 DSM (all the genes up to Eftα) and from Roseburia intestinalis L1-82 (butyryl-CoA:acetyl-CoA transferase).

Biosafety

Our part is safe to be synthesized and utilized on an open bench. Also, the product of the gene does not include any biohazard and does not pose any threat even if by chance there is a leak.

Characterization

The characterization of our part is very important for two main reasons:

1. To compare it with the old part and see if there are major differences in the production of butyrate.

2. To test our new improved part in different hosts, environments, substrates etc.

Unfortunately, due to technical issues with our sequence order, we could not have it before the Wiki Freeze to characterize it. It is expected to arrive just after the Wiki Freeze, thus our team will perform all the designed experiments after the 12th of October. Once we obtain our results that we expect to be very promising, due to the thorough redesign we performed in the old part, we will be able to discuss them with our judges in our Q&A section!

Nonetheless, we managed to test and characterize the old part! The previous team, iGEM NRP-UEA-Norwich 2015, tested the same part and the results came back almost negative for butyrate detection (HPLC). This may be attributed to various reasons that we eliminated with our re-design.

For the characterization of the old sequence and its future use as a control for the characterization of the new part, we cloned the old part's insert into our pNZ8149 food-graded vector, and with this, we transformed E.coli K-12 strains and Lactobacillus rhamnosus GG strains. Then after the bacterial cells were cultured for 24h in MRS and MRS culture medium that included inulin, we measured the production of the SCFAs with HPLC. The results are shown below [Figure 2]. Also, in order for our experiments to be more accurate we measured the production of SCFAs with HPLC from E.coli K-12 strains and Lactobacillus rhamnosus GG strains that they had not been transformed[Figure 1]. Therefore we had one extra control test for the bacteria that have been transformed with the old part.



Figure 1.The results obtained from the HPLC of the bacteria strains that weren’t transformed.



Figure 2. The results were obtained from the HPLC of the bacteria strains that were transformed with the plasmid containing the old part.

What we can understand from the results is that the old part still seems to be completely ineffective in producing butyrate. This is encouraging because our newly designed part could potentially have much better performance.

The characterization that we want to perform to our new improved part includes:

1. The transformation of E.coli K-12, Lactobacillus rhamnosus GG, Lactobacillus plantarum lp-01, and Lactobacillus acidophilus DSM 20079 strains with the pNZ8149 vector, to test the function of the transcriptional cassette and the enzymes in different chassis. Also, we aim to observe the butyrate production of each on of them.

2. The culture of the strains in different polysaccharide substrates (inulin,FOS, GOS etc.) to see if the different substrate enhances the butyrate production and causes higher butyrate yield.

3. The obtainment of lot's of replicates (at least triplicates) in order to have more precise results.

References

[1]Petra Louis, Sheila I. McCrae, Cédric Charrier, Harry J. Flint, Organization of butyrate synthetic genes in human colonic bacteria: phylogenetic conservation and horizontal gene transfer, FEMS Microbiology Letters, Volume 269, Issue 2, April 2007, Pages 240–247,https://doi.org/10.1111/j.1574-6968.2006.00629.x
[2]Butyrate-Producing Bacterium L2-50 Putative Fe-S Oxidoreductase Gene, Partial Cds; Thiolase, Crotonase, Beta Hydroxybutyryl-CoA Dehydrogenase, Butyryl-CoA Dehydrogenase, Electron Transfer Flavoprotein Beta-Subunit, and Electron Transfer Flavoprotein Alpha-Subunit Genes, Complete Cds; and Putative Multidrug Efflux Pump Gene, Partial Cds.” NCBI Nucleotide, July 2016, https://www.ncbi.nlm.nih.gov/nuccore/DQ987697.1/
[3]Landete, José Maria. “A Review of Food-Grade Vectors in Lactic Acid Bacteria: From the Laboratory to Their Application.” Critical Reviews in Biotechnology, vol. 37, no. 3, Feb. 2016, pp. 296–308, https://doi.org/10.3109/07388551.2016.1144044. Accessed 17 Mar. 2022.
[4]A Review of Food-Grade Vectors in Lactic Acid Bacteria: From the Laboratory to Their Application.” Critical Reviews in Biotechnology, vol. 37, no. 3, Feb. 2016, pp. 296–308, https://doi.org/10.3109/07388551.2016.1144044. T., Takala, and Saris P. “A Food-Grade Cloning Vector for Lactic Acid Bacteria Based on the Nisin Immunity Gene NisI.” Applied Microbiology and Biotechnology, vol. 59, no. 4-5, Jan. 2002, pp. 467–71, https://doi.org/10.1007/s00253-002-1034-4. Accessed 3 Apr. 2021.
[5]Tagliavia, Marcello, and Aldo Nicosia. “Advanced Strategies for Food-Grade Protein Production: A New E. Coli/Lactic Acid Bacteria Shuttle Vector for Improved Cloning and Food-Grade Expression.” Microorganisms, vol. 7, no. 5, Apr. 2019, p. 116, https://doi.org/10.3390/microorganisms7050116.
[6]Cheng, Y., Liu, J., & Ling, Z. (2022). Short-chain fatty acids-producing probiotics: A novel source of psychobiotics. Critical reviews in food science and nutrition, 62(28), 7929-7959.
[7]Dalile, B., Van Oudenhove, L., Vervliet, B., & Verbeke, K. (2019). The role of short-chain fatty acids in microbiota–gut–brain communication. Nature reviews Gastroenterology & hepatology, 16(8), 461-478.
[8]Foster, J. A., & Neufeld, K. M. (2013). Gut–brain axis: how the microbiome influences anxiety and depression. Trends in Neurosciences, 36(5), 305–312. https://doi.org/10.1016/j.tins.2013.01.005
[9]Berezina, Oksana V., et al. “Reconstructing the Clostridial N-Butanol Metabolic Pathway in Lactobacillus Brevis.” Applied Microbiology and Biotechnology, vol. 87, no. 2, Mar. 2010, pp. 635–46, https://doi.org/10.1007/s00253-010-2480-z. Accessed 26 Oct. 2021.
[10]Wang, Liang, et al. “Metabolic Engineering of Escherichia Coli for the Production of Butyric Acid at High Titer and Productivity.” Biotechnology for Biofuels, vol. 12, Mar. 2019, https://doi.org/10.1186/s13068-019-1408-9. Accessed 8 Apr. 2021.

External links

See our Wiki's New Improved Part designing process: https://2023.igem.wiki/thrace/new-improved-part
See our Wiki's Experiments:https://2023.igem.wiki/thrace/experiments
See our Wiki's Part Collection section:https://2023.igem.wiki/thrace/part-collection