Heterologous biosynthesis is a method that involves reproducing a biosynthetic pathway from one organism via the coordinated expression of genes into another host organism. In this experiment, the biosynthetic pathway for erythromycin A is transferred into an e coli host system with the ultimate goal of producing large quantities of this antibiotic. First, the gene cluster responsible for erythromycin formation is isolated from the original bacterial host.
The foreign genetic material is then redesigned in opera format for coordinated expression of the complete enzymatic pathway in e coli. As the e coli strain carrying the heterologous erythromycin pathway replicates in culture, the final erythromycin compound is produced. The antibiotic activity of the compound can then be tested using a bacillus solus functional assay As opposed to production of complex natural products through their original organisms.
Heterologous biosynthesis through e coli offers the opportunity for process speed and engineering potential. This idea began about 10 to 15 years ago, but only recently with various technical breakthroughs and improvements in our technology have we've been able to see real success stories for complex production of natural products using e coli First design and order PCR primers to amplify all of the genes associated with the cluster within the chromosome. For the polymerase chain reaction, use freshly prepared genomic DNA as a template.
If the target genes contain high GC content like the erythromycin A cluster in S eryri, add four microliters of DMSO in a 50 microliter reaction. Confirm the identity of the PCR products by sequencing. Then clone the PCR gene products into an e coli expression system While cin, we used pattern eight as a expression vector for certain genes since the inclusion of terminal leading sequence added protein over expression.
Next, confirm the expression of a functional protein preparation. Using R-T-P-C-R-S-D-S page analysis or convenient phenotypic assays now construct operas based upon genes successfully expressed from individual expression plasmid. If possible, design combinations of compatible cohesive restriction enzymes to sequentially convert genes tos.
During the process of the open constructions, we need to make sure that the restrictions enzyme we used do not cut the individual sequence. If that's the case, we need to considering the gene synthesis Proceed to consolidate the 20 erythromycin A genes into expression plasmids. Considering the factors detailed in the accompanying manuscript.
Now transform e coli strain BAP one with the newly designed biosynthetic plasmids either sequentially or in combination plate the cells on solid LB medium containing a combination of antibiotics to select for plasmid maintenance. This is highly unusual that we use six different antibiotic selections of E Cory transformations, but this is simply because we need six plasmid for the total synthesis of erythromycin A in coline. This section establishes culture conditions for concerted activity of the full erythromycin A pathway.
Gen BP one was designed for the providing of the substrate needed for the cin, a bio synthesis, and to post translationally, modify the IDE mega synthesis. Pick separate colonies of the freshly transformed BAP one and inoculate three 1.5 milliliter cultures in alb media containing the required plasmid selection. Antibiotics, 100 micromolar IPTG two milligrams per milliliter of arabinose to induce gene expression and 20 millimolar sodium propionate.
To support intracellular biosynthetic precursor formation culture, the cells at 22 degrees Celsius and 250 RPM for 24 to 48 hours. Periodically sample the culture for analysis of plasmid stability during growth. Clarify the cultures by centrifugation at 10, 000 RPM.
Remove the supernatant and store the pellet at four degrees Celsius for analysis. To confirm and quantify erythromycin a formation, inject the culture supernatant onto an lc MS system. Also run standard samples of commercially available erythromycin A For cases without the commercially available standards.
The compound will need additional chemical characterizations by a MR and high resolution mass spectrometry. Compare the production between the three preparations and identify the highest producer. Prepare a glycerol stock from the colony source and store at negative 80 degrees Celsius with the highest erythromycin a producing stock.
Start a separate e coli production culture, extract the final supernatant with two volumes of ethyl acetate. Then dry the extract in a speed vac resuspend the pellet in 100 microliters of methanol. Then transfer the extract to a sterile filter disc and air dry separately.
Culture B subtles in LB liquid overnight, add 20 microliters of the B subtles culture to 20 milliliters of liquid lb agar at 45 degrees Celsius and prepare a plate. Now, place the filter disc of erythromycin A on the solid B solus containing agar incubate at 37 degrees Celsius. In this example, the erythromycin gene cluster from S urethra is transferred to e coli after the production of erythromycin A from the e coli heterologous host.
The bioactive product is analyzed in the native S ETH three A chromosome. The erythromycin gene cluster is organized in 55 KB of total DNA following expression, a complex biosynthetic pathway requiring intracellular precursors and poly heide biosynthetic and tailoring enzymes generates erythromycin A for the gene transfer. Individual genes are first isolated through PCR.
Then the genes needed for erythromycin a formation are one, cloned individually into expression vectors to confirm protein production and activity through SDS page and phenotypic assays, and two, consolidated as operas for eventual complete pathway transfer to e coli I certain genes required expression with a five prime leader sequence resulting from the PET 28 expression vector, while others did not. And expression of the individual genes was first analyzed by SDS page. This result shows phenotypic assessment of the ERME gene for erythromycin A resistance erythromycin A within the solid medium is used to assay e coli strains, harboring plasmids with or without the ERME erythromycin a resistance gene.
Clearly cell growth is rescued with ERME expression. The full erythromycin A pathway is then introduced to e coli, including a plasmid for the grow EL chaperone, and a plasmid containing an extra copy of URI K electro transformation is used to introduce the plasmids to e coli. Plasmid stability is then assessed for the final strain.
LCMS results confirm and quantify production of erythromycin. A importantly functionality is confirmed by antibacterial bioassays that produce zones of inhibition in lines of B subtlest bacteria, or limit B subtles culture growth in serial dilutions of erythromycin A.So with this video demonstration of the complex hetero biosis of natural products, you now have the ability to design, implement, and analyze the individual experimental steps required to make a complete heterologous natural product. By applying metabolic engineering to the e coli cell for optimization of various precursors and the introduced pathway, you can maximize production of, say, erythromycin, which is what we talked about today, or any other compound that might be of medicinal value or difficult to produce.
