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BIO 417 L17 2022 (Autosaved)
BIO 417 L17 2022 (Autosaved)
BIO 417 L17 2022 (Autosaved)
L17 2022
1
Biosynthesis of riboflavin….
In Gram-positive bacteria, genes for riboflavin biosynthesis
are organized into an operon
Is there any advantage associated with this type gene
arrangement?
The operon is regulated by an FMN riboswitch
this comprises RNA elements in the 5’ untranslated
region of the rib operon mRNA
this operon consists of an FMN-sensing domain and a
regulatory domain that has the alternating terminator or
‘OFF’ state and the anti-terminator or ‘ON’ state
2
The an FMN riboswitch affords opportunity to switch
riboflavin biosynthesis ‘ON’ or ‘OFF’
Accordingly, and since Gram-positive bacteria can
synthesise riboflavin or acquire it from their
environment, the riboflavin pathway is switched off
when riboflavin is available in their environment
This is essentially, pathway inhibition!
Roseoflavin, a natural analogue of is able to bind to
the FMN riboswitch and shut it down (switch it to
the OFF status)
Hence, roseoflavin is often used to select/screen for
riboflavin over-producers
3
The riboflavin overproducers derived this way
possess a range of mutations including;
single base mutations and deletions in several
regions of the FMN element
InDels (insertions and deletions in the rib
operon)
NB:
The food industry prefers the mutation induction
derived riboflavin over that derived from genetically
engineered organisms
Is there a (strong) case for such a demand or
preference? 4
Riboflavin biosynthesis pathway…
The rib
operon
5
6
Notice the precursors!
Ribulose 5-Phosphate and Guanine Trisphosphate
Ribulose 5-P is derived from the Pentose
Phosphate pathway
GTP is derived from purine metabolism
pathways (de novo synthesis and or salvage
pathway)
Notice the overall significance of the
rib operon
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Industrial production of riboflavin is predominantly based
on microbial fermentation
The choice organisms include Bacillus subtilis and some
flavinogenic fungi, especially A. gossypii
NB: Fungi use oil as substrate while bacteria strictly
use carbohydrate as substrate
When oil is the substrate, many pathways are involved in
the production of riboflavin precursors
Please name the precursors
Please name the critical pathways for producing the
precursors from oil
lipolysis and b-oxidation, TCA, glyoxylate,
gluconeogenesis, pentose phosphate pathway and
purine anabolism 8
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How can we derive the choice/best producer
microrganisms?
screening the available natural diversity
mutation induction followed by screening
genetic engineering followed by assays of;
successful transgene introgression
successful transgene expression
successful transgene function
success of the overall objective of the genetic
transformation undertaking
In industry, the employer often decides on the priority
assay
In academia, prioritisation may be of a very different 10
order!
To date, overexpression of riboflavin synthesis pathway is a
proven method of increasing riboflavin production levels
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Please READ: Mohedano et al. (2019) Frontiers
in Microbiol. 10, 1–13.
doi:10.3389/fmicb.2019.01748 13