Erythromycin production pdf

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Title: biosensors and its applications in fermentation industry ppt Page Link: biosensors and its applications in fermentation industry ppt - Posted By: madhu Created at: Sunday 16th of April AM. I am a professor in Mexico working at the university Chapingo and I am searching areas for nanotechnology development Two additional independently derived transformants, FL and FL, were generated and tested in shake flask fermentations.

FL and FL were also tested in CFM1 medium, though less extensively than FL, and showed a similar reproducible increase in erythromycin production. The strain improvement effect is not seen without including mutR in the duplicated region A strain containing a duplication of the MCM operon region, without co-duplication of mutR, was constructed.

The resulting strain, S. Three independently derived isolates of FL were initially tested in shake flask fermentations and behaved similarly; one strain was extensively NIH-PA Author Manuscript further tested Fig. The results showed no statistically significant difference in the erythromycin production levels between FL and the wild-type strain, showing that inclusion of mutR in the duplicated region is essential for the strain improvement effect observed in FL Duplication of mutR alone does not produce a strain improvement effect To test whether the strain improvement effect in strain FL was solely due to the presence of a duplicate copy of mutR or whether it required co-duplication of the MCM complex genes, a third strain, FL, was constructed and tested in shake flask fermentations.

The chromosomal copy of mutB is inactivated in FL, therefore, integration of an unmodified copy of the MCM region containing mutR would effectively test duplication of only mutR on erythromycin production. Three independently- derived FL strains were initially tested in shake flask fermentations and showed similar Metab Eng.

Page 7 results. Extensive further testing of FL showed no statistically significant differences between itself and the wild-type strain Fig. The kanamycin resistance gene aphI was inserted into ORF1 in the opposite transcriptional orientation using plasmid pFL to create S. The knockout strain displayed normal morphological and pigment characteristics, unlike the mutB knockout strains FL and FL Reeves et al. Furthermore, the ORF1 knockout strains had a wild-type erythromycin production phenotype Fig.

Shake flask fermentations of FL strains showed that erythromycin production was not statistically different between the two strains Figs. Complementation of mutB morphological, pigmentation and growth phenotypes In a previous report, S.

In addition, these mutants were unable to grow on methylmalonic acid as sole carbon source. In this study we showed that the mutant phenotypes associated with mutB knockouts could be complemented through integration of plasmid pFL into the mutB deletion strain FL S.

It can be concluded, therefore, that the genes of the MCM region carried on plasmid pFL, and derivatives of pFL, are expressed upon integration into the chromosome of the S. A red pigment, flaviolin, derived from malonyl-CoA, has been previously reported to be produced by a S. Results from our study showed that both the red variant strain FL Reeves et al. Interestingly, Cortes et al. Our mutation in MCM on the other hand does block pigment production and does have a positive effect on erythromycin production in a carbohydrate-based medium Reeves et al.

One explanation for this result is that carbon needed for red pigment formation could come from the mmCoA pool that feeds into erythromycin, and that blocking pigment formation at the MCM gateway diverts carbon that would normally go into pigment Metab Eng. Page 8 production into the erythromycin pathway.

Blocking pigment production at a step after the MCM reaction and therefore after succinyl-CoA formation, such as at the rppA step, does not have the same benefit simply because once carbon has exited from the mmCoA pool, it may NIH-PA Author Manuscript no longer benefit erythromycin production.

It may be that malonyl-CoA accumulates in rppA mutants but apparently it does not efficiently cycle back to the mmCoA pool in carbohydrate-based medium. Discussion The importance of the mmCoA metabolite node in erythromycin strain improvement was uncovered in a previous study using a reverse engineering approach Reeves et al.

The current study was begun to determine whether we could manipulate the mmCoA metabolite node in an industrially relevant oil-based medium in a way to increase production of erythromycin. The results presented in this report show that increased production of erythromycin is possible through the duplication of the MCM operon which includes mutA, mutB, meaB, and mutR. Previously, mmCoA had been shown to be the limiting factor for erythromycin production in the wild-type strain and that knockouts of the mutB gene, coding for the larger subunit of the MCM, led to significant decreases in erythromycin production in oil-based fermentation NIH-PA Author Manuscript medium Reeves et al.

This model logically suggested that overexpression of the MCM genes should lead to an increase in production of erythromycin in the oil-based medium, and the results in this report are consistent with this prediction.

A model, incorporating the results described in this report, is presented Fig. In oil-based medium, where the MCM acts as a feeder route into the mmCoA metabolite pool, strain FL is postulated to receive additional carbon flow for erythromycin production from the overexpression of MCM complex genes indicated by an enlarged arrow from succinyl-CoA [S] to mmCoA [M].

A similar model and concept for strain improvement was demonstrated for the cephamycin C producer but it was arrived at from a much different approach Malmberg and Hu, In cephamycin C strain improvement, a theoretical kinetic analysis was used to predict the rate- limiting factor for cephamycin C biosynthesis to be alpha-aminoadipic acid Malmberg and Hu, This led to the construction of highly improved strains two- to five-fold improvements were obtained through duplication of lat, the gene that codes for lysine e- NIH-PA Author Manuscript aminotransferase.

Overexpression of the LAT gene in cephamycin C biosynthesis, and overexpression of the MCM genes in erythromycin biosynthesis have similar effects on strain improvement presumably because both apparently increase the pool size of the rate-limiting metabolite for biosynthesis of their respective antibiotics.

Together, these two studies present experimental verification of two strain improvement concepts that may have general application: first, that strain improvement can be achieved through increasing carbon flow towards the metabolite node of the limiting factor, and second, that the limiting factor is a metabolite that occupies a boundary position between primary and secondary metabolism. Another remarkably similar strain improvement strategy involving gene duplication by plasmid insertion into a Streptomyces genome has been recently reported by Li et al.

The results from these three highly related studies suggests that a new effective general method for strain improvement could involve the creation and screening of libraries of integrated transformants containing duplicated segments of the chromosome.

Our Metab Eng. Page 9 results suggest that larger fragments would be preferrable, for example, in the range of 10 kb to accommodate larger operons such as the MCM operon. No knowledge of the genes involved would be required in advance, but reverse engineering and follow-up studies would reveal the NIH-PA Author Manuscript genetic basis of the strain improvement effect, in much the same way as gene knockout libraries are now screened and reverse engineered Reeves et al.

The rational strain improvement mechanism presented here is for the S. This is the strain closest in phenotype to most high producing industrial strains. The mechanism works for this strain when it is grown in its most productive fermentation medium, an oil-based medium.

Even though the results presented in this report are for the publicly available wild-type strain, the strain improvement mechanisms could also improve production of higher-producing commercial strains, if mmCoA is also the limiting factor for erythromycin production in those strains.

No information has been reported, to our knowledge, regarding factors that are limiting production of erythromycin in the commercial strains. Nevertheless it would be logical to assume that mmCoA is the limiting factor in commercial strains if they accumulate erythromycin A as the primary end product, and if they show no evidence of a pathway bottleneck, for example, by the accumulation of a pathway intermediate.

This is understandable considering mutR is likely to be transcribed together with the other three genes as part of the MCM operon and also likely to be involved in the regulation of expression of the MCM operon Fig. Another possible explanation for the need to include mutR in the gene duplication comes from inspection of the DNA sequence of mutR and the region downstream of the MCM operon.

A 12 basepair stem-loop secondary structure with only 2 mismatches and a loop of 8 nucleotides is located at the very end of mutR overlapping the TAG stop codon for mutR Fig.

This structure could play a role in transcription termination or transcript stability, and its exclusion from the duplicated region in strain FL could explain why the yield improvement effect was not seen in this strain Pulido and Jimenez, Interestingly, improved erythromycin production was also seen in strain FL fermentations in carbohydrate-based medium. However, the duplication of the MCM operon in strain FL may have had effects on other metabolic pathways besides MCM due to the unknown targets of the mutR regulatory protein.

For example, if mutR duplication also up- regulated carbon flow through the two other feeder pathways in the model fig 5 or down- regulated carbon flow through a drain pathway not diagrammed , then even if the MCM reaction was also up-regulated in strain FL, as predicted, the net metabolic effect would be to increase, not decrease, production of erythromycin in carbohydrate medium. Further study of the mmCoA node to better understand the true strain improvement mechanism in carbohydrate medium could prove helpful for the development of additional rational strain improvement strategies.

At some point in the strain improvement process of the wild type strain the mmCoA metabolite pool may be increased to the point where mmCoA ceases to be the limiting factor affecting Metab Eng. Page 10 erythromycin production. At that point, a new limiting factor may appear. In order to increase production of erythromycin further, efforts would have to be refocused on those metabolic pathways or regulatory circuits that can be manipulated to overcome the scarcity of the new NIH-PA Author Manuscript limiting factor.

In this way, rational strain improvement may in some cases become a step-wise process of increasing the supply of a series of limiting factors. The amount of limiting factor may be increased by making appropriate genetic and physiological manipulations at the proper metabolic or regulatory node for each stage of the strain improvement process.

Future experimental directions will involve optimizing the overexpression of the MCM operon with the hope that further increases in erythromycin yields can still be achieved. Additionally, other genes and pathways connected to the mmCoA metabolite node should be investigated and manipulated to determine their effects on erythromycin production. We also thank Robert Snell for technical support and helpful discussions and Roy Wesley for administrative support and helpful discussions.

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