CH3OH

Alcohol consumption as a way of life:

the metabolism of methylotrophic bacteria

Greg Crowther(representing the Lidstrom Lab)

CH3OH Today’s outline

What is methylotrophy?

Where are methylotrophs found?

Why are methylotrophs important?

Overview of methylotrophic metabolism

Details of methylotrophic metabolism

A musical summary

CH3OH 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?

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

cs.hiram.edu/~obie

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Why is methylotrophy important?

Carbon cycling

Bioremediation

Biocatalysis

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

CH3OH Bioremediation

Bioremediation = using microorganisms to break down waste or pollutants

Methylotrophs "cometabolize" many toxic compounds (e.g., chloride-containing organics). The toxins aren’t necessarily food (can't support growth) but get converted to less toxic products.

www.alken-murray.com

CH3OH 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

CH3OH Biocatalysis (cont.)

Problem: methylotrophs were not designed to operate well in a bioprocess setting.

Methylotrophs are competitive in natural environments. Optimized parameters:

• low nutrients

• defense systems

• protection from harsh environment

CH3OH 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!

Overview of methylotrophic metabolism

CH3-X

HCHO

CO2

Energy metabolism (dissimilation)

C3

Biomass

Biosynthesis (assimilation)

CH3-X

HCHO

CO2

C3

Biomass

Energy metabolism (dissimilation)

Biosynthesis (assimilation)

“True” methylotrophyAutotrophic

methylotrophy

CH3OH Formaldehyde in cells

• toxic -- reacts with DNA and protein

• produced at a high rate if consumption cut off,

internal [HCHO] = 1 M in minutes

Serine cycle methylotrophs

Acetyl-CoA

Assimilation

Acetoacetyl-CoA

(D)- -Hydroxybutyryl-CoA

Crotonyl-CoA

Butyryl-CoA

NADPH

NADP

Isobutyryl-CoAMethacrylyl-CoA

-hydroxyisobutyryl-CoA

Propionyl-CoA

Succinyl-CoA

Malyl-CoA

Ethylmalonyl-CoA

Methylsuccinyl-CoA

CO2

CO2

CO2

Glyoxylate

Malate

OAA

CO2

NADH

NAD

Formaldehyde

Methanol

Methanol

Methylamine

MethylamineMethylamine dehydrogenase

Formaldehyde

Formate

Methylene H4F

Energy

Metabolism

NAD(P) NAD(P)H

Formaldehyde production

Methylene H4F

FADFADH

FADH FAD

Glycine

Hydroxypyruvate

Glycerate

Serine

PEP2PGA

NADH

NAD

ATP

BIOSYNTHESIS

Formate oxidation

2H

2H

Glyoxylateregenerationcycle

CO2

BIOSYNTHESIS

Formaldehyde oxidation

Methanol dehydrogenase

NADPH

NADP

Serine cycle

CO2

NAD NADH

Hydroxypyruvatereductase

RuMP cycle methylotrophs

Assimilation

Formaldehyde

Methanol

Methanol

Methylamine

MethylamineMethylamine dehydrogenase

Formaldehyde

Formate

Energy

Metabolism

NAD(P) NAD(P)H

Formaldehyde production

BIOSYNTHESIS

Methanol dehydrogenase

CO2

NAD NADH

Hexulose phosphate synthase

Ribulose 5 phosphate

Hexulose 6 phosphate

Glyceraldehyde 3 + phosphate

6 phosphogluconate

CO2 Fructose 6 phosphate

Pyruvate

CH3OH A musical summary

Energy and carbon both Are needed for the growthOf methylotrophs. (Methylotrophs!) But they’ve got the metabolic tools To grow on C1 fuels …

They’re methylotrophs. (Methylotrophs!) And their substrates are all around,So that’s where they are found –On the land and in Puget Sound Are methylotrophs.

CH3OH 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!)

CH3OH A musical summary (cont.)

They take the substrate that’s supplied And make formaldehyde … They’re 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 … They’re methylotrophs!