Hydroxycitric acid

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• Hydroxycitric acid
• Rapid improvement in production
• by
• Genome shuffling

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Hydroxycitric acid
Citric acid
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Lactone - cyclic ester condensation product of
an alcohol group and a carboxylic acid group in
the same molecule.
Stereoisomers

• isomeric molecules whose atomic connectivity is
  the same but whose atomic arrangement in space is
  different

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(2S,3S) Hydroxycitrate

• Garcinia fruit - G. cambogia, G. indica
• Indian subcontinent and in western Sri Lanka

Per Capsule Sodium 40 mg, Hydroxycitrate 250
mg 500 mg Other Ingredients Vegetable
Cellulose, Vegetable Magnesium Stearate, Silica,
Water
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(2S,3S) Hydroxycitrate effects

• More Carbohydrate - citrate - citrate ATP CoA
  
• acetyl-CoA oxaloacetate.
• ADP Pi
  
• (Fat)
• Malonyl CoA
• carnitine acyl transferase
• (shuttle fatty acid to mitochondria)

More Carbohydrate appetite loss
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(2S, 3R)-HCA

• Hibiscus subdariffa
• pancreatic alpha-amylase complex starches to
  oligosaccharides and
• alpha-glucosidase oligosaccharides-monosaccharid
  es
• Carbohydrate metabolism and
• Blood insulin levels
• attenuated postprandial blood glucose levels
• safe food additive for - diabetes

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(2S, 3R)-HCA
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Production Problems

• Calyxes of Hibiscus subdariffa
• Seasoned flowering plant and depends on climate
• tropical and semitropical areas

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Production Problems
continued..
Stereoselective organic synthesis
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Solution?
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Alternate sources for (2S,3R)-HCA

• Bacillus megaterium G45C (2)
• Streptomyces sp. U121- limited yield (30 mg/l)
• No improved yield by optimizing media
• breeding of industrial microorganisms has
  commonly been performed by classic strain
  improvement, which is robust but, very
  time-consuming also
• Genome shuffling

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Genome shuffling

• Protoplasm fusion
• Two entire protoplast are brought together
• Under right conditions these components fuse to
  form a single hybrid cell which regenerate cell
  wall and proliferates.
• Exchange of genetic information without
  restriction of natural breeding barrier

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Genome shuffling

• Requires a diverse population of mutants
• Protoplast fusion
• Better progenies are selected
• Next round of shuffling

Advantages

• Accelerates directed evolution
• Allows recombination between
• multiple parents at each generation
• Unclear details of regulatory mechanisms

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Comparison of CSI to genome shuffling for
production of tylosin from Streptomyces fradiae.
LETTERS TO NATURE VOL 415 7 FEBRUARY 2002
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• CSI classical strain improvement
• Streptomyces fradiae is used for the commercial
  production of tylosin, a complex polyketide
  antibiotic. Strain SF1 is a stable clone of the
  S. fradiae natural isolate, whereas SF21 produces
  tylosin at a high titre and is derived from SF1
  through 20 cycles of CSI. We aimed to use genome
  shuffling to - high tylosin-producing strains
  from SF1. To generate a diverse population for
  genome shuffling, SF1 was subjected to one round
  of CSI using nitrosoguanidine (NTG) as mutagen

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• Genome shuffling of Streptomyces sp. U121 for
  improved production of hydroxycitric acid
• Hiroyuki Hida Takashi Yamada Yasuhiro Yamada
• Appl Microbiol Biotechnol (2007) 7313871393

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Protocol to obtain the improved mutants

• Spores S. Sp. U121 in tween 80
• 0.01 NTG (nitrosoguanidine) in TrisHCl
• Cultured overnight in garcinia medium
• Spread on garcinia medium - incubated for 6 days
• 200 colonies were randomly selected and HCA
  HPLC
• No strains with improved HCA productivity

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Mutagenesis with EMS (ethyl methane
sulfonate)UVNo improved mutants
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Incubationboiled for 10 min microcentrifugedsup
ernatant filtrated with syringe filter unit and
analyzed by (HPLC)
Measurement of HCA concentration
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Protocol to obtain the improved mutants

continued .

• Resulting colonies
• trans-epoxyaconitic acid (EAA)-
• Antibiotic analog of HCA
• Resistant strains possess
• Excretion of EAA - HCA resistance at higher
  concentration
• Hydration of EAA - more HCA formation

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Synthesis of trans-epoxy aconitic acid.

• prepared from trans-aconitic acid by oxidation
  with H2O2 in the presence of a catalytic
• amount of sodium tungstate in H2O2 at pH 5.0 at
  70 C

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Results

• 200 colonies - rapid growth on EAA medium -
  examined for HCA -

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Genome shuffling

• Improved mutants (60 mg/ml) grown
• Lysozyme
• Observation of protoplast formation
• different growth rates
• Protoplast fusion
• Resuspended in regeneration medium (HCP)
• Diluted and plated incubated for 5-8 days

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Lysozyme -damages bacterial cell walls by
catalyzing hydrolysis of 1,4-beta-linkages
between N-acetylmuramic acid and
N-acetyl-D-glucosamine residues in a
peptidoglycan abundant in tears, saliva,
mucusProtoplast fused by suspension in 10 ml
of SMM containing 30 PEG 4000, 15 dimethyl
sulfoxide (DMSO), and 10 mM CaCl2.
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Lysozyme - damages bacterial cell
walls 1,4-beta-linkages
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200 colonies assayed for HCA production
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Second round Genome shuffling

• limited number of screens (600 screens) and
• non-fused protoplasts also grows

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Third round Genome shuffling

• 200 isolates - HCA productivity
• media non-fused protoplasts not regenerated
• HCP regeneration medium 0.2 EAA or
• acidic medium at pH 5.5
• protoplast fusion - PEG
• mutant protoplasts with EAA resistance were
  formed by fusion and selectively regenerated

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Effect of EAA on selection of mutant strains with
improved HCA productivity
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Improvement of HCA productivity
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Quantification of enhanced EAA resistance in
mutants

• Spores of wild and mutant strains

improved HCA productivity is correlated with EAA
resistance in the mutant strains
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SMM buffer

• 20 mM sodium malate buffer (pH 6.5),
• containing 0.5 M sucrose and
• 20 mM MgCl2,

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• HCP contains (per liter)
• 5.0 g of glucose,
• 3.5 g of K2HPO4,
• 1.5 g of KH2PO4,
• 1.9 g of MgCl2,
• 5.0 g of casamino acid,
• 0.1 g of L-tryptophan, polyvinylpyrrolidone,
• 81.0 g of sodium succinate, and 8.0 g of agar.

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Measurement of HCA production and cell growth in
liquid culture

• Wild-type and mutant strain 361 of Streptomyces
  sp. U121 were each grown in screening medium for
  2 days . The seed culture broths were then each
  transferred to 100 ml of the same medium in
  flask.
• Each strain was cultured by shaking at 220 rpm at
  30C for various numbers of hours (1 to 48 h).
• HCA production was determined by HPLC as
  previously described and cell growth was
  monitored by measurement of wet cell weight.

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Production curve of HCA from wild-type and best
mutant (361) of Streptomyces sp. U121.
EAA resistant strains may have enhanced
activities for excreting EAA, leading to HCA
resistance
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EAA-resistant strains of Streptomyces sp. U121
did nothave activity for the hydrolysis of EAA.
Thus, EAA resistant strains may have enhanced
activities for excreting EAA, leading to HCA
resistance. One clue to the mechanism underlying
the improved performance of mutant strain 361
The result demonstrates that the growth rate of
the 361 strain was higher than that of the wild
type. Hence, enhanced HCA resistance contributed
to enhanced cell growth in the presence of HCA,
and the total amount of improved production of
HCA was achieved in the culture system
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Conclusion

• More than 5 fold higher HCA production
• But not sufficient for commercial production
• More rounds of genome shuffling
• No improved mutants
• Start with
• Larger initial population diversity or
• Original strains with higher HCA yield
• Works are underway in laboratory

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References

• J. Agric. Food Chem. 2002, 50, 10-22
• Biosci. Biotechnol. Biochem., 70 (8), 1972 1974,
  2006
• 3. Biosci. Biotechnol. Biochem., 69 (8),
  15551561, 2005
• A guide to understanding dietary supplements
   By Shawn M.
• 5. Impacts of applied genetics micro-organisms,
  plants, and animals.