Strain Improvement by GR

Strain Improvement Using
Recombinant DNA
Technology
Development Of L-Tryptophan Production(Primary
Metabolite) Strains by Defined Genetic Modification
in Escherichia coli.
(Zhi-Jun-Zhao, Chun Zou, Yi-Xing Zhu, Jun Dai, Sheng Chen, Dab Wu, Jing Wu, Jian Chen)
By -Vaishnavi A. More(203251003)
-Shreyas S. Paregaonkar(203251010)
 INTRODUCTION:
• L-Tryptophan is an essential amino acid for humans and other animals. It is widely used in food, animal
feed, and pharmaceutical industries.
• L- Tryptophan overproducing E.coli was produced by defined genetic modifications like:
Gene/Plasmid Action
1. AroF and TrpED Feedback inhibition of these genes were eliminated by Site Directed
Mutagenesis.
2. psV plasmid Expression of deregulated AroF and TrpED was achieved by a
temperature induced plasmid.
3. TrpR Was deleted to remove transcriptional regulation of Trp repressor.
4. tnaA Deleted to remove L-Tryptophan degradation.
5. pheA and tyrA Deleted to block L-tyrosine and L-phenylalanine biosynthetic pathway
that compete with L-tryptophan biosynthesis.
The biosynthesis of L-Tryptophan in E. coli involves three major steps-
1. The central metabolism pathways towards the condensation of
phosphoenolpyruvate(PEP) and erythrose 4-phosphate (E4P);
2. Secondly, the common aromatic pathway from 3-deoxy-D-arabinoheptulosonate 7-
phosphate (DAHP) to chorismate (CHA);
3. Thirdly, the L-Trp branch pathway leading to L-Trp.
I. Bacterial Strains and Plasmids
1. W3110- Wild type E.coli used as parent strain for PCR product cloned in pACYC177,
gene knock out mutation.
2. psV plasmid.
psV plasmid is formed.
3. pACYC177.
4. pMD18 plasmid. II. Isolation, Manipulation &
5. E. coli JM109. Transformation.
Construction of Low Copy no. vector psV Kits
• constructed by using pACYC177. 1. Plasmid DNA Plasmid Mini Prep Kit(Bio Basic
Inc.).
A 1,345bp DNA fragment containing
2. Chromosomal Genomic DNA Isolation Kit(Bio
1. temperature sensitive lambda repressor cIt3857, E.coli W3110 Basic Inc.).
2. lambda PL and PR promoter. 3. Restriction Used according to manufacturer’s
Enzyme, ligases an manual.
Amplified by PCR using primers- pND_fw_Pst1 and

pND_rv_Pst1; pND_fw_BamH1 and pND_rv_BamH1,
• Plasmid DNA was transferred to competent cells of
Digestion with Pst1 and BamH1, E.coli by Electroporation.
III. Cloning and Expression of aroF and complementary primers aroF_mfw and aroF_mrv.
trpED.
Wild type aroF and trpED. PCR product obtained treated with Dpn-I and
transformed in E.coli.
Amplified by PCR using primers( aroF_fm_BgIII and

aroF_rv_BgIII; trpED_fw_BamHI and Feedback Inhibition Resistant AroF(with Ile deletion
trpED_rv_BamHI). of aroF).
aroF and trpED are ligated into pMD18-T vector. Site directed mutagenesis Using plasmid
pMD18/trpED as template and a pair of
complementary primers trpED_mfw and trpED_mrv.
Plasmid pMD18/aroF and pMD18/trpED transformed
to E.coli JM109.
Feedback Inhibition Resistant TrpED(withSer40Phe
IV. Construction of Feedback mutation of TrpE).
Inhibition Resistant of AroF and
TrpED.
Site directed mutagenesis Using plasmid
pMD18/aroF as template and a pair of
V. Results and Discussion.
1. DAHP Synthase in Common Aromatic Pathway.
The specific activity of TrpE (S40)D in crude extract of E.coli
Wild type and feedback inhibition-resistant aroF were W3110/psV03 was 54.2 U/mg.
cloned in psV vector next to PR promoter resulting in
psV01 and psV02 plasmid.
• To detect the L-Trp production of E.coli W3110
harbouring plasmids, of psV, psV01, psV02, pSV03. Fed
psV01 and psV02 plasmids are transformed in E.coli batch fermentation was performed, the results were-
W3110.
W3110/psV No detectable amount
W3110/psV01 0.05g/l after 30h incubation
The specific activity of DAHP synthase in crude
extract of E.coli W3110/psV01 and E.coli W3110/psV02 0.23g/l
W3110/psV02 were 13.2 U/mg and 12.9 U/mg
respectively W3110/psV03 0.87g/l
2. ANTA Synthase in L-Trp Branch Pathway.
• In addition two competitive amino acids, L-Phe, L-Tyr and
Feedback inhibition resistant trpED was cloned into a precursor of L-Trp, ANTA were accumulated in culture
plasmid psV02 next to the PL promoters resulting in
psV03 plasmid. medium.
psV03 plasmid was transformed in E.coli W3110.

3. The development of TrpR knock-out mutant. pathway in E.coli was knocked out and the resulting
strain was named FB-03/psV03..
• trpR gene W3110/psV03 was knocked out by using
Red recombination system and the trpR knock-out • The fermentation resulted in 10.8g/l of L-Trp
strain was named as FB-01/psV03. production.
• Fermentation showed the production of L-Trp of 6. The development of tyrA knock-out mutant.
FB-01/psV03 was increased to 2.8g/l when
compared to W3110/psV03. • tyrA was further knocked out on genome and
resulted in FB-04/psV03.
4. The development of tnaA knock-out mutant.
• When pheA and tyrA were both knocked out, CHA
• The tnaA gene of FB-02/psV03 was further knocked could not be converted in prephenate and as a
out based on trpR background and resulted in the result there was no synthesis of L-Phe and L-Tyr in
strain of FB-02/psV03 the medium.
• Strain FB-02/psV03 produced 7.8g/l L-Trp
compared to the strain of W3110/psV03 and FB-
01/psV03.
5. The development of pheA knock-out mutant.
• To prevent L-Trp carbon flux blockage, L-Phe and L-
Tyr synthesis pathways were blocked.
• phenA which encodes a bifunctional enzyme
catalysing the first two steps of the L-Phe branch
 Rewiring a secondary metabolite pathway towards itaconic acid
production in Aspergillus niger:
(Abeer H. Hossain1,3*, An Li2, Anja Brickwedde2, Lars Wilms1, Martien Caspers2, Karin Overkamp1 and Peter J.
Punt1)
• Itaconic acid (IA) is a C-5 dicarboxylic acid thathas been recognized by the US Department of Energy as one of the top 12
building block chemicals with high potential to be produced using biotechnology.
• Since the 1960s, industrial production is facilitatedby its natural producer Aspergillus terreus, a filamentous fungus.
• Li et al. havepreviously proposed Aspergillus niger as a suitable production host for the industrial production of IA due to the
hosts optimized pathways towards organic acids.
• This gene clusterconsists of the cis-aconitate decarboxylase (cadA),mitochondrial tricarboxylate transporter (mttA) and the
major facilitator superfamily protein (mfsA).
 Biosynthetic Pathway of Itaconic Acid:
 Rewiring the Pathway:
• The cadA gene was introduced in A. niger.
• Expression of cadA in A. niger resulted in IA production at low levels (AB1.13 CAD 4.1).
• This low production was increased by expressing mfsA and mttA in an IA producing A. niger transformant.
• To identify other genes relevant for improving IA production in A. niger a preliminary RNA-Seq analysis was done to
identify genes differentially expressed in relation to IA production.
• One of the identified genes encoded a putative cytosolic citrate synthase, of which overexpression resulted in improved IA
production(citB).
• The overexpression of citB resulted in the conversion of citrate into IA in the cytosol. This further facilitated the
production.
• Therefore, a citB expression vector was designed and introduced in AB1.13 CAD + MFS + MTT#49, being the best IA
producer.
 Results of the Genetic Modification:

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Strain Improvement Using

4. tnaA Deleted to remove L-Tryptophan degradation.

Amplified by PCR using primers- pND_fw_Pst1 and

Amplified by PCR using primers( aroF_fm_BgIII and

psV03 plasmid was transformed in E.coli W3110.

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