Alcohol consumption as a way of life: the metabolism of methylotrophic bacteria

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Alcohol consumption as a way of lifethe
metabolism of methylotrophic bacteria
Greg Crowther (representing the Lidstrom Lab)
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Todays outline

• What is methylotrophy?
• Where are methylotrophs found?
• Why are methylotrophs important?
• Overview of methylotrophic metabolism
• Details of methylotrophic metabolism
• A musical summary

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What is methylotrophy?
methyl -CH3 troph grow Definition
growth on compounds with no C-C bonds as sole
source of carbon and energy Examples of these
compounds CH4 (methane) CH3Cl (methyl
halides) CH3OH (methanol) CH3NH2 (methylamine)
HCOO- (formate) (CH3)2S (methyl sulfides)
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What is methylotrophy? (cont.)
Methylotrophs cannot grow on CO2 alone. CO2 is a
carbon source but not an energy source. Almost
all methylotrophs are aerobic (need O2).
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Where are methylotrophs found?
cs.hiram.edu/obie
C1 sources in nature methanol produced by
plants methylated amines from degradation of
fish and plant compounds methyl halides and
methyl sulfides produced by marine algae
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Why is methylotrophy important?
Carbon cycling Bioremediation Biocatalysis
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Carbon cycling
Methane (CH4) . . . is an end product of
anaerobic ecosystems is a greenhouse gas
traps 21X as much heat per molecule as CO2 is
consumed by some methylotrophs
www.bbc.co.uk/schools/gcsebitesize
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Bioremediation
Bioremediation
using microorganisms
to break down waste
or pollutants Methylotrophs "cometabolize"
many toxic compounds (e.g., chloride-containing
organics). The toxins arent necessarily food
(can't support growth) but get converted to less
toxic products.
www.alken-murray.com
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Biocatalysis

• Biocatalysis
  conversion of substrates to products using
  isolated enzymes or intact cells
• Can we genetically engineer methylotrophs to be
  environmentally benign chemical factories?
• Methanol as an alternative biofeedstock
• soluble in water
• inexpensive
• pure substrate
• bacteria that use it are well-studied

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Biocatalysis (cont.)
Problem methylotrophs were not designed to
operate well in a bioprocess setting. Methylotrop
hs are competitive in natural environments.
Optimized parameters low nutrients defense
systems protection from harsh environment
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Biocatalysis (cont.)

• Long-term goal redesign methylotrophs with
  industrially valuable parameters optimized.
• redirect metabolism to specific products
• increase metabolic efficiency
• increase process efficiency
• remove unwanted products
• storage products
• extracellular excretion products
• defense systems

This requires a detailed understanding of
methylotrophic metabolism!
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Overview of methylotrophic metabolism
Autotrophic methylotrophy
True methylotrophy
CH3-X
CH3-X
HCHO
HCHO
Energy metabolism (dissimilation)
Biosynthesis (assimilation)
Energy metabolism (dissimilation)
CO2
CO2
C3
Biosynthesis (assimilation)
C3
Biomass
Biomass
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Formaldehyde in cells
toxic -- reacts with DNA and protein
produced at a high rate if consumption cut
off, internal HCHO 1 M in minutes
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Serine cycle methylotrophs
Methanol
Methylamine
Methanol
Methanol dehydrogenase
Formaldehyde production
Methylamine
Formaldehyde
Methylamine dehydrogenase
Energy Metabolism
NAD
NADH
Formaldehyde
Formate
Formate oxidation
Formaldehyde oxidation


Assimilation
Methylene H4F
FADH
FAD
Isobutyryl-CoA
Methacrylyl-CoA
BIOSYNTHESIS
CO
-hydroxyisobutyryl-CoA
b
2
2H
Methylsuccinyl-CoA
CO
2
2H
CO2
Ethylmalonyl-CoA
Propionyl-CoA
PEP
CO2
Glyoxylate regeneration cycle
2PGA
OAA
CO
ATP
2
NADH
Butyryl-CoA
Succinyl-CoA
NAD
Glycerate
Hydroxypyruvate reductase
Malate
Hydroxypyruvate
Crotonyl-CoA
Malyl-CoA
Serine
Glyoxylate
Methylene H4F
Glycine
Acetoacetyl-CoA
BIOSYNTHESIS
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RuMP cycle methylotrophs
Methanol
Methylamine
Methanol
Methanol dehydrogenase
Formaldehyde production
Methylamine
Formaldehyde
Methylamine dehydrogenase
Energy Metabolism
NAD
NADH
Formaldehyde
Formate
Hexulose phosphate synthase



Hexulose 6 phosphate
Ribulose 5 phosphate
Assimilation
Fructose 6 phosphate
Glyceraldehyde 3 phosphate
Pyruvate
6 phosphogluconate
BIOSYNTHESIS
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A musical summary
Energy and carbon both Are needed for the
growth Of methylotrophs. (Methylotrophs!) But
theyve got the metabolic tools To grow on C1
fuels Theyre methylotrophs. (Methylotrophs!)
And their substrates are all around, So thats
where they are found On the land and in Puget
Sound Are methylotrophs.
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A musical summary (cont.)
What can methylotrophs grow on? Repeat after me
Methane. (Methane!) Methyl halides.
(Methyl halides!) Methanol. (Methanol!)
Methylamine. (Methylamine!) Formate.
(Formate!) Formamide. (Formamide!)
Dimethyl sulfide. (Dimethyl sulfide!) And
other stuff. (And other stuff!)
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A musical summary (cont.)
They take the substrate thats supplied And make
formaldehyde Theyre methylotrophs.
(Methylotrophs!) Which then becomes CO2 gas And
also biomass In methylotrophs.
(Methylotrophs!) Yes, they have the ability To
turn C1 molecules to C3 By making serine or RuMP
Theyre methylotrophs!