Presented by

Dr. Sanjay Kumar,

Assistant Professor
Department of Microbiology
M. D. University, Rohtak, Haryana

Proteins are growth associated
products so we need to keep
the cells growing for continuous
protein production

Theoretical maximum cell
density for E.coli is 400 g DCW/L

Maximum possible cell density
in real for E.coli is 190 g to 200 g
DCW/L .

No growth possible beyond it
A large Metabolic flux diversion towards Biomass formation results in
low product yield with respect to substrate consumed
Growing cells undergo random Mutations which may cause loss of
productivity
Plasmid instability
Limited operational time
Few drawbacks associated with cell growth
Quiescent cells can provide a solution but there are few
challenges

Not much useful for intracellular products/proteins

No ideal quiescent cells are available

No straightforward approach for making Quiescent cells

Preparation of Genomic Library of E.coli under lac operon in pRSET A plasmid for
random gene silencing
Transformation in BL21 pLysS
Screening of slow growing/Not growing but metabolically active cells by
monitoring growth and glucose uptake rate in submerged culture
Co-expression studies for examining the screened clones to induce growth
retardation and enhance recombinant protein expression (using GFP as model
protein in pBAD33 plasmid expression system under Ara operon.
Primary screening gave 728 potential
clones
Secondary screening gave 70 potential
clones
Screening of slow growing clones on IPTG supplemented LB agar plates

Around 8000 transformants
obtained
17 clones were selected and genes
involved were identified
Methodology for first strategy for preparing Quiescent cells
Best performer carrying gene ribB ((rib3,4 dihydroxy-2butanone-4-phosphate
synthase), gave 7 folds increase over control culture with 347 AU/g DCW
Results


Growth profiles of
induced &
uninduced cultures
of 17 selected
clones
Results
Few transcripts whose blockage lead to growth stoppage
Results
Growth and product profiles of induced & uninduced
cultures of Best performers (ribB & mfd)
Preparation of Genomic Library of E.coli under lac operon in pBAD32 plasmid for
random gene silencing
Transformation in DH5 alpha carrying plasmid pNER31-GFP and grown on agar
plate supplemented with Arabinose and IPTG as inducers
Screening of slow growing colonies showing intense green fluorescence


Around 30,000 transformants
were screened
Finally 4 clones were selected as best
performers and their genes were
identified
Methodology for 2nd strategy
Selected colonies were grown in submerged culture and growth and specific yield
profiles were observed
Best performer identified as kdpF
showed 8.4 folds higher specific yield
than that of control
The results were reproduced using three different media showing that observed
leap in expression was independent of media composition.
Results

Growth profiles
of selected
better
performers
screened by
using 2
nd

strategy
Results
Growth and product profiles of best performer (2
octaprenyl-phenol hydroxylase enzyme) screened using 2
nd

strategy
Conclusion:
A novel strategy for generating a library , consisting of randomly down
regulated metabolic pathways in E. coli was designed by cloning small
genomic DNA fragments in expression vector which transcribed antisense
RNA upon induction.

Genes like ribB (rib3,4 dihydroxy-2butanone-4-phosphate synthase), mfd
(mutation frequency decline protein) kdpF (2 octaprenyl-phenol
hydroxylase enzyme) were found to be useful targets for obtaining
slow/no growth and high expression of recombinant protein.

No direct link at the pathway level between gene function and observed
phenotype could be established. The observed phenotype could be
pertained to complex regulatory response within the cells.

Thus a high throughput screening approach was designed which is a
useful tool for reverse metabolic engineering strategy for the generation
of improved hosts. The approach does not rely on prior knowledge of the
regulated and interconnected nature of cell metabolic network.
Thank you
ribB serves as biosynthetic precursor for xylen ring o riboflavin.


KdpD (Sensory Histidine Kinase) is expressed during osmotic shock otherwise it is
non essential


Show Details
Production of highly potent recombinant siRNAs in Escherichia coli
Linfeng Huang and Judy Lieberman
Nature Protocols 8 p2325 - p2336 31/10/2013doi:10.1038/nprot.2013.149






Efficient and specific gene knockdown by small interfering RNAs produced in
bacteria

Linfeng Huang, Jingmin Jin, Padraig Deighan, Evgeny Kiner, Larry McReynolds, and
Judy Lieberman
Nature Biotechnology 31 (4) 350 - 356 doi:10.1038/nbt.2537
Additional information
Synthetic small interfering RNAs (siRNAs) are an indispensable tool to investigate
gene function in eukaryotic cells and may be used for therapeutic purposes to
knock down genes implicated in disease. Thus far, most synthetic siRNAs have
been produced by chemical synthesis. Here we present a method to produce
highly potent siRNAs in Escherichia coli. This method relies on ectopic expression
of p19, an siRNA-binding protein found in a plant RNA virus. When expressed in E.
coli, p19 stabilizes an 21-nt siRNA-like species produced by bacterial RNase III.
When mammalian cells are transfected by them, siRNAs that were generated in
bacteria expressing p19 and a hairpin RNA encoding 200 or more nucleotides of a
target gene reproducibly knock down target gene expression by 90% without
immunogenicity or off-target effects. Because bacterially produced siRNAs contain
multiple sequences against a target gene, they may be especially useful for
suppressing polymorphic cellular or viral genes.