Parts

Overview

Our team worked on strategies to induce or increase the expression of compounds synthesized by cryptic genes, and the compound we chose to use as a model was Cosmomycin D produced by Streptomyces olindensis.

The parts Str(e)pA, Str(e)pB, Str(e)pC, Str(e)pBC, Str(e)pD, Str(e)pE, and Str(e)pBCDE belong to the cosmomycin cluster itself. We obtained them by amplifying the sequences using primers for subsequent cloning to engineer the microorganisms. For the forward primers, the restriction enzyme used was NotI, and for the reverse primers, it was EcoRI. The conjugative plasmid pSET152Ec used in the cloning was provided by Professor Gabriel Padilla from ICB - USP.

The parts Str(e)pF, Str(e)pG, Str(e)pFG, pIJ6021, and CosmoStrep were obtained directly through synthesis by iGEM partner companies. The vector pIJ6021, well-documented in Actinobase, is a multicopy expression vector with resistance genes for kanamycin and thiostrepton. We made a modification to the original vector to render it conjugative.

To help future teams and researchers develop projects, we characterized and disponibilized the new genetic parts we used in the iGEM registry:

Name	Nickname	Type	Description	Length
BBa_K4590001	Str(e)pA	Basic	St(r)epA is SARP (Streptomyces Antibiotic Regulatory Protein) in Streptomyces spp. regulates the expression of the antibiotics. Group Favorite	863 bp
BBa_K4590002	Str(e)pB	Basic	St(r)epB is used in conjunction with St(r)epC. It encodes an ABC Transporter ATP Binding Protein, which is a subunit of an ABC transporter.	990 bp
BBa_K4590003	Str(e)pC	Basic	St(r)epC is used in conjunction with St(r)epB. It encodes an ABC Transporter Membrane Protein, which is a subunit of an ABC transporter.	831 bp
BBa_K4590004	Str(e)pBC	Composite	Contains St(r)epB and St(r)epC together, they encode an ABC transporter, which will be used to export Cosmomycin to the extracellular environment.	1821 bp
BBa_K4590005	Str(e)pD	Basic	St(r)epD has a 99% identity with a gene encoding for a glutathione peroxidase, acting as an antioxidant to counter the damage caused by free radicals in Streptomyces.	483 bp
BBa_K4590006	Str(e)pE	Basic	St(r)epE encodes for a UvrA-like protein, involved in DNA repair in Streptomyces.	2286 bp
BBa_K4590007	Str(e)pBCDE	Composite	It contains St(r)epB, St(r)epC, St(r)epD, and St(r)epC in a single vector.	4811 bp
BBa_K4590008	Str(e)pG	Basic	St(r)epF is a gene from Streptomyces hygroscopicus that encodes a protein similar to a di-tricarboxylate transporter in Rhizobium. It is involved in the incorporation of malonate.	1245 bp
BBa_K4590009	Str(e)pF	Basic	St(r)epG is a gene from Rhizobium leguminosarum that encodes for Malonyl-CoA and is involved in fatty acid synthesis and elongation.	1536 bp
BBa_K4590010	Str(e)pFG	Composite	It contains both St(r)epF and St(r)epG together.	2781 bp
BBa_K4590011	pIJ6021	Plasmid Backbone	pIJ6021 is the plasmid backbone of malonate production	7790 bp
BBa_K4590012	CosmoStrep	Plasmid	The Streptomyces conjugative vector pIJ6021. Its contains both St(r)epF and St(r)epG together.	10571 bp

Bibliography

Arteaga, Roger David Castillo. Mycothiol peroxidase activity as a part of the self-resistance mechanisms against the antitumor antibiotic Cosmomycin D , Microbiology Spectrum, 2022.

Arteaga, Roger David Castillo. Characterization of self-resistance mechanisms to the antitumoral cosmomycin D. Master's Dissertation, Institute of Biomedical Sciences, 2016.

MAHARJAN, Sushila; PARK, Je Won; YOON, Yeo Joon; LEE, Hei Chan; SOHNG, Jae Kyung. Metabolic engineering of Streptomyces venezuelae for malonyl-CoA biosynthesis to enhance heterologous production of polyketides. Biotechnology Letters, [S.L.], v. 32, n. 2, p. 277-282, 17 out. 2009. Springer Science and Business Media LLC. http://dx.doi.org/10.1007/s10529-009-0152-9.

Attachments

There are two types of regulatory mechanisms in Streptomyces:

1- pyramidal cascades of regulation that usually involve a butyrolactone and its receptor protein triggering the formation of pathway-associated regulatory proteins (SARP - Streptomyces Antibiotic Regulatory Proteins );

2- global regulators that transduce protein phosphorylation signals responding to stress factors.

Membrane transporters mediate either the passive or active transport of solutes ranging from ions to nutrients to signaling molecules and drugs. In passive transports, also known as uniporters or facilitators, conformational transitions between outward and inward-facing states occur spontaneously and randomly, enabling the equalization of substrates across both sides of the membrane. Secondary active transporters concentrate their substrates on one side of a membrane by linking their movements to the symport or antiport of other solutes, usually ions, which themselves move downhill along their electrochemical potentials. They are termed secondary active transporters because they utilize existing gradients of driving solutes as an energy source. Establishing these gradients of driving solutes requires an external energy source. [1]

Pyramidal control

SARP family contains genes that encode activators with a molecular weight about 25 kDa. These activators contain a DNA binding domain near the N-terminus, along with a bacterial transcriptional activation domain. Initiation of expression of the SARP-encoding genes relies on the accumulation of signal molecules formed in response to phosphate, carbon or nitrogen depletion. Regulatory cascades have usually g-butyrolactone synthesizing genes at the top position, and g-butyrolactone receptor proteins that transmit these signals to the SARP-specific regulators. [2]

Malonate incorporation vector

In order to improve cosmomicin D production, our team have been developed a conjugative vector CosmoStrep, and can be digestioned by the following restriction enzymes: EcoRV, PvuII, ApaLI, StuI, DraI, NdeI, HindIII, AcuI, FspAI, BsaBI, XmnI, SbfI, XbaI, AseI, AbsI, AsiSI and MfeI.

The vector contains:

SARP - it comes from Streptomyces olidensis DUAFPE 5622;
cosIJ - it comes from Streptomyces olidensis DUAFPE 5622 and it´s responsible for efflux pumps;
cosIJPU - it comes from Streptomyces olidensis DUAFPE 5622 and it´s responsible for resistence genes;
FkbF - it comes from Streptomyces hygroscopicus var ascomyceticus - di/tricarboxylate transport;
MatA - it comes from Rhizobium leguminosarum - malonyl-coA synthase;
SLIC - site for conjugation;

Figure 3: Plasmid construction for synthesis of malonate

References:

[1]. https://www.annualreviews.org/doi/pdf/10.1146/annurev-biochem-060815-014520

[2].https://www.sciencedirect.com/science/article/abs/pii/S136952741000041X?fr=RR-2&ref=pdf_download&rr=8149ae624f554ecb

New part

Our team has undertaken a groundbreaking project to enhance cosmomicin D production by working with Streptomyces olidensis, a bacterium with great potential for producing valuable secondary metabolites. Streptomyces olidensis is a relatively unexplored organism in the scientific community, making it an exciting candidate for synthetic biology research.

As part of our project, we have characterized and contributed several novel genetic parts to the iGEM Registry of Standard Biological Parts. These parts are designed to serve as valuable tools for future researchers and iGEM teams, making our contribution not only innovative but also highly accessible to the wider scientific community.

Here is a summary of our newly characterized genetic parts:

BBa_K4590001 (St(r)epA): This part encodes a Streptomyces Antibiotic Regulatory Protein (SARP), which plays a crucial role in the regulation of antibiotic production in Streptomyces species.
BBa_K4590002 (St(r)epB): St(r)epB encodes an ABC Transporter ATP Binding Protein, a critical subunit of an ABC transporter.
BBa_K4590003 (St(r)epC): St(r)epC encodes an ABC Transporter Membrane Protein, another vital subunit of an ABC transporter.
BBa_K4590004: This part combines St(r)epB and St(r)epC, creating an ABC transporter that can be used to export cosmomicin to the extracellular environment.
BBa_K4590005 (St(r)epD): St(r)epD encodes a glutathione peroxidase, acting as an antioxidant to counter the damage caused by free radicals in Streptomyces.
BBa_K4590006 (St(r)epE): St(r)epE encodes a UvrA-like protein involved in DNA repair in Streptomyces.
BBa_K4590007: This part combines St(r)epB, St(r)epC, St(r)epD, and St(r)epC in a single vector, offering a comprehensive genetic solution.
BBa_K4590008 (St(r)epF): St(r)epF, a gene from Streptomyces hygroscopicus, encodes a protein similar to a di-tricarboxylate transporter involved in malonate incorporation.
BBa_K4590009 (St(r)epG): St(r)epG, a gene from Rhizobium leguminosarum, encodes Malonyl-CoA synthase, a critical player in fatty acid synthesis and elongation.
BBa_K4590010: This part combines St(r)epF and St(r)epG, offering a dual functionality that can be advantageous for various projects.
BBa_K4590011: The plasmid backbone of malonate production.
BBa_K4590012: The Streptomyces conjugative vector pIJ6021, which incorporates both St(r)epF (BBa_K4590008) and St(r)epG (BBa_K4590009) together.

Our contributions are especially relevant for the "Best New Part" special prize category, as we have not only expanded the iGEM Registry with a diverse array of genetic parts but also improved the understanding of Streptomyces regulation and secondary metabolite production. Streptomyces is known for its complex regulatory mechanisms, and our parts enable researchers to explore the pyramidal cascades and global regulators involved in Streptomyces antibiotic production.

In addition, we have developed a novel "Malonate incorporation vector" to advance cosmomicin D production. This vector is a powerful tool for genetic engineering applications, underscoring our team's commitment to innovation and impact in the field of synthetic biology.

By offering these meticulously characterized and readily accessible genetic parts, we are providing a valuable resource for future iGEM teams and researchers to address critical challenges in the field. Our work aligns perfectly with the spirit of the "Best New Part" special prize, as it represents a significant contribution to the iGEM community and the broader scientific community.

Our dedication to pushing the boundaries of synthetic biology and our commitment to collaboration, open-source sharing, and responsible research make us strong contenders for the "Best New Part" special prize in the iGEM competition.

St(r)epA (BBa_K4590001) - Our Gold and Best New Part

Among the array of genetic parts we have characterized and contributed, St(r)epA (BBa_K4590001) stands out as the Gold Part and, without a doubt, our Best New Part. St(r)epA, or Streptomyces Antibiotic Regulatory Protein, plays a pivotal role in regulating antibiotic production in Streptomyces species, making it a key genetic component with several noteworthy attributes that distinguish it as the crowning jewel of our project.

1. Central to Antibiotic Regulation: St(r)epA is the cornerstone of antibiotic regulation in Streptomyces, governing the expression of antibiotics. This regulatory function is critical in Streptomyces biology, as it allows us to modulate and fine-tune antibiotic production. Our team's comprehensive characterization of St(r)epA is not only novel but also holds the potential to transform antibiotic research and production.

2. Essential for Pathway Optimization: The precise control of antibiotic production is vital for enhancing the efficiency of antibiotic biosynthesis processes. By providing St(r)epA as a well-characterized and accessible genetic part, we empower researchers to optimize and engineer antibiotic biosynthesis pathways with precision and reliability. This is especially important in the context of increasing the production of valuable compounds like cosmomicin D.

3. Pyramidal Control: St(r)epA is part of the pyramidal cascades of regulation in Streptomyces, involving butyrolactone signaling and pathway-specific regulatory proteins. This unique regulatory mechanism ensures that antibiotics are produced in response to specific environmental cues. By offering St(r)epA, we open the door to a deeper understanding of this intricate regulatory network, paving the way for more targeted and efficient antibiotic production.

4. Wider Implications for Synthetic Biology: Beyond its immediate applications in antibiotic production, St(r)epA has broader implications for the field of synthetic biology. The ability to control gene expression with precision is a fundamental requirement in synthetic biology projects, and St(r)epA provides a model for understanding such regulatory mechanisms.

5. Collaboration and Open-Source Sharing: Our commitment to collaboration and open-source sharing aligns perfectly with the iGEM spirit. By making St(r)epA accessible to the iGEM community and the scientific world, we facilitate collective progress in the field and enable other researchers to harness its potential.

In summary, St(r)epA (BBa_K4590001) is not only a genetic part but a gateway to unlocking the intricate world of Streptomyces antibiotic regulation. Its significance in antibiotic production, pathway optimization, and synthetic biology research cannot be overstated. As our Gold Part and the Best New Part, St(r)epA embodies the spirit of innovation, collaboration, and scientific advancement that defines the iGEM competition. It represents a beacon of discovery, a source of knowledge, and a catalyst for future breakthroughs in the fascinating world of synthetic biology.

Our team is excited to share the transformative potential of St(r)epA with the iGEM community and is committed to its responsible and open use. We proudly present St(r)epA as the epitome of our iGEM project, embodying the spirit of excellence and innovation that characterizes the "Best New Part" special prize category.