New insights into the one-carbon metabolic network of Methylobacterium extorquens AM1

Greg Crowther

Dept. of Chemical Engineering

University of WashingtonImage: Dennis Kunkel

Microscopy, Inc.

HCOO-

CH2=H4F

Serine Cycle

CO2

CH2=H4MPT

H4MPT

CH3OH

HCHOH4F

Biomass

The “Central Dogma” of Biology

DNA RNA Proteins

substrates products

microarrays

proteomics,enzyme

activity assays

flux rates

genomics

metabolomics

My primary interest: metabolic fluxes

DNA RNA Proteins

substrates products

flux rates

Clover leaf print showing Methylobacterium strains. Photo by Amy Springer.

Methylotrophic metabolism

• “methyl” = -CH3, “troph” = growth

• methylotrophy = growth on one-carbon compounds such as methanol (CH3OH)

- some methylotrophs can also grow on multicarbon compounds such as succinate (C4H4O4

2-)

Dissimilation

C3

Assimilation

C=OH

HC-OHH

H

H

CO2

Biomass

ATP NAD(P)H

• Carbon cycling

Methane, a greenhouse gas, is consumed by some methylotrophs.

• Bioremediation

Methylotrophs detoxify many nasty compounds (e.g., chloride-containing organics).

• Biocatalysis

Genetic engineering enables synthesis of useful chemicals (e.g., plastics) from CH3OH.

COCO22

CHCH44

Images: Marina Kalyuzhnaya; appa.asso.fr; ouraycolorado.com

Why is methylotrophy important?

Biocatalysis with methylotrophs

• Bacteria are self-replicating multistage catalysts

• Some methylotrophs make biodegradable plastics from methanol, an inexpensive/abundant/renewable feedstock

• Byproducts of metabolism are usually nontoxic

• Long-term goal: redesign methylotrophs for optimal production of useful chemicals from methanol

A B Ck1

k-1

k2

k-2

MEASURE fluxesvia label tracing

MODEL fluxesmathematically

REDIRECT fluxesvia genetic engineering

Methylotrophs are somewhat widespread in domain Bacteria…

Figure: Hugenholtz et al., J Bacteriol 180: 4765, 1998

… but M. extorquens AM1 is the best-studied species

• 100+ genes for one-carbon metabolism are characterized

• genome is sequenced

• we think we know all of the major metabolic pathways

M. extorquens AM1: a model methylotroph

• pink, rod-shaped -proteobacterium

• natural habitat: surface of leaves

demethylation of pectin produces methanol, which is released

through stomata

Meet Methylobacterium extorquens AM1

Clover leaf print showing Methylobacterium strains. Photo by Amy

Springer.

Metabolic fluxes in M. extorquens AM1

HCOO-

CH2=H4F

Serine Cycle

CO2

CH2=H4MPT

H4MPT

CH3OH

HCHOH4F

Biomass

Dissimilation (CO2 production)

HCOO-

CH2=H4F

Serine Cycle

CO2

CH2=H4MPT

H4MPT

CH3OH

HCHOH4F

Biomass

Direct assimilation pathway(Biomass production)

HCOO-

CH2=H4F

Serine Cycle

CO2

CH2=H4MPT

H4MPT

CH3OH

HCHOH4F

Biomass

Long assimilation pathway(Biomass production)

HCOO-

CH2=H4F

Serine Cycle

CO2

CH2=H4MPT

H4MPT

CH3OH

HCHOH4F

Biomass

Metabolic fluxes in M. extorquens AM1:our view as of 2005

1. The direct assimilation pathway dominates in cells growing on CH3OH.

2. HCHO is the key branch point (Biomass vs. CO2).

3. Formate dehydrogenase (FDH) may not be important?

HCOO-

CH2=H4F

Serine Cycle

CO2

CH2=H4MPT

H4MPT

CH3OH

HCHOH4F

Biomass

Metabolic fluxes in M. extorquens AM1:our view as of 2005

1. The direct assimilation pathway dominates in cells growing on CH3OH.

2. HCHO is the key branch point (Biomass vs. CO2).

3. Formate dehydrogenase (FDH) may not be important?

HCOO-

CH2=H4F

Serine Cycle

CO2

CH2=H4MPT

H4MPT

CH3OH

HCHOH4F

Biomass

Metabolic fluxes in M. extorquens AM1:our view as of 2005

1. The direct assimilation pathway dominates in cells growing on CH3OH.

2. HCHO is the key branch point (Biomass vs. CO2).

3. Formate dehydrogenase (FDH) may not be important?

HCOO-

CH2=H4F

Serine Cycle

CO2

CH2=H4MPT

H4MPT

CH3OH

HCHOH4F

BiomassFDH

The rest of this talk: new insights, 2006-2007

1. The direct assimilation pathway: not important!

HCOO-

CH2=H4F

Serine Cycle

CO2

CH2=H4MPT

H4MPT

CH3OH

HCHOH4F

BiomassFDH

2. Formate dehydrogenase:very important after all!

The direct assimilation pathway

Evidence for this pathway: deuterium assay (Marx et al., PLoS 3:e16, 2005)

GC-MS

extract, derivatize

+2 Serine (D2)+1 Serine (D)

DCOO-

CD2=H4F

CO2

CD2=H4MPT

H4MPT

CD3OD

DCDOH4F

CDH=H4F

90%+ of serine is +2 in CH3OH-grown cells, so the direct pathway appears dominant.

Potential problem with the deuterium assay

If NADPH pool gets contaminated with deuterium, flux through the long pathway will be “counted” as flux through the direct pathway.

GC-MS

extract, derivatize

+2 Serine (D2)+2 Serine (D2)

DCOO-

CD2=H4F

CO2

CD2=H4MPT

H4MPT

CD3OD

DCDOH4F

CD2=H4F NADPD

0

5

10

15

20

25

30

35

40

45

0 5 10 15 20 25 30

Incubation time (s)

+2/

+1

rati

o

If NADPH pool gets contaminated with deuterium, the +2/+1 ratio should increase as incubation time increases (and more deuterium enters the pool).

Conclusion: contamination does occur and might be a problem.

Testing for NADPH contamination

HCOO-

CH2=H4F

Serine Cycle

CO2

CH2=H4MPT

H4MPT

CH3OH

HCHOH4F

Biomass

Other reasons to question the direct pathway

1. If we knock out an enzyme in the long pathway, the cells can’t grow on CH3OH.

Why can’t they just use the direct pathway? HCOO-

CH2=H4F

Serine Cycle

CO2

CH2=H4MPT

H4MPT

CH3OH

HCHOH4F

Biomass

X

Other reasons to question the direct pathway

2. We can’t find an enzyme that catalyzes HCHO + H4F.

• Vorholt et al. (J Bacteriol 2000): cell extracts don’t enhance reaction rate

• My data (J Bacteriol 2005): fae2 and fae3, the genes most likely to encode the enzyme (if it exists), can be knocked out without slowing growth on CH3OH

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 10 20 30

Time (hours)

Cel

l den

sity

(O

D60

0)

wild-type(N=5)

fae2/3(N=5)

HCOO-

CH2=H4F

Serine Cycle

CO2

CH2=H4MPT

H4MPT

CH3OH

HCHOH4F

Biomass

Other reasons to question the direct pathway

3. The nonenzymatic rate constant for HCHO + H4F is much lower than the deuterium and 14C assay data would suggest.

Measure 14C-CO2

Add 14C-CH3OH

Measure 14C-Biomass HCOO-

CH2=H4F

Serine Cycle

CH2=H4MPT

H4MPTHCHOH4F

Estimates of direct pathway flux

Direct pathway flux estimated from 14C-biomass and deuterium assays= (total flux) * (direct flux / total flux) = 0.3 mM/s

14C assay deuterium assay

Is the rate constant for HCHO + H4F high enough to achieve this flux?

HCOO-

CH2=H4F

Serine Cycle

CO2

CH2=H4MPT

H4MPT

CH3OH

HCHOH4F

Biomass

0

0.02

0.04

0.06

0.08

0.1

0.12

6 6.5 6.7 7

pH

v6, 1

/(m

M*s

)

rate = V6*[H4F]*[HCHO]

• V6 < 0.08 mM-1s-1

• [H4F] < 0.15 mM (Vorholt et al. 1998)

• [HCHO] < 1 mM

rate = (0.08 mM-1s-1)*(0.1 mM)*(0.5 mM) = 0.004 mM/s << 0.3 mM/s

Conclusion: the deuterium assay may greatly overestimate the biomass flux coming from the direct pathway.

Estimates of direct pathway flux

HCOO-

CH2=H4F

Serine Cycle

CO2

CH2=H4MPT

H4MPT

CH3OH

HCHOH4F

Biomass

Testing the direct pathway by studying a long-pathway mutant

Conclusion:

Flux through the direct pathway is insignificant.

If the direct pathway is “real”:

• deuterium assay should detect +2 serine

• 14C assay should detect biomass flux

… NO!

… NO!

HCOO-

CH2=H4F

Serine Cycle

CO2

CH2=H4MPT

H4MPT

CH3OH

HCHOH4F

Biomass

X

Can the long pathway handle the entire CH3OH flux?

Enzyme activities from literature (converted to mM/s):

1. MDH 1.52. Fae 53. MtdA/MtdB 10-284. Mch 115. Fhc 0.2-1.1

Preliminary conclusion:Enzyme activities appear sufficient for handling all CH3OH.

1

2

3

4

5

HCOO-

CH2=H4F

Serine Cycle

CO2

CH2=H4MPT

H4MPT

CH3OH

HCHOH4F

Biomass

This is a question for kinetic modeling (in progress).

Maximum total CH3OH intake = 1.4 mM/s

Conclusion on the direct pathway

Flux through the direct assimilation pathway is insignificant.

Key supporting evidence:

• mutations in long pathway prevent growth on CH3OH

• no detectable enzyme activity

• rate constant for HCHO + H4F is very small

• no +2 serine in long-pathway mutant (deuterium assay)

• no biomass flux in long-pathway mutant (14C assay)

HCOO-

CH2=H4F

Serine Cycle

CO2

CH2=H4MPT

H4MPT

CH3OH

HCHOH4F

Biomass

Formate dehydrogenase (FDH)

Background:

Chistoserdova et al. (J Bacteriol 186: 22, 2004) studied three FDH genes, each of which was shown to be functional in vivo.HCOO-

CH2=H4F

Serine Cycle

CO2

CH2=H4MPT

H4MPT

CH3OH

HCHOH4F

BiomassFDH1-3

Formate dehydrogenase (FDH)

Weird findings for the triple mutant:

• It still grows on CH3OH!

• CO2 production seems unimpaired!

Possible interpretations:

• There is at least one more FDH.

• CO2 is produced “downstream” of the one-carbon network.

HCOO-

CH2=H4F

Serine Cycle

CO2

CH2=H4MPT

H4MPT

CH3OH

HCHOH4F

BiomassFDH1-3X

A fourth formate dehydrogenase!

A gene with sequence homology to other FDHs is upregulated in the triple mutant (E. Skovran’s microarray analysis).

This gene was knocked out along with the other three.

The quadruple mutant cannot grow on CH3OH (L. Chistoserdova), suggesting that there are 4 (and only 4) FDHs.

What do the flux data show?

HCOO-

CH2=H4F

Serine Cycle

CO2

CH2=H4MPT

H4MPT

CH3OH

HCHOH4F

Biomass

0

2

4

6

8

10

12

14

16

wild-type triplemutant

quadruplemutantC

O2

flu

x, n

mo

l/(m

in*m

L*O

D)

My 14C-CO2 flux data

CO2 production is similar in the

wild-type and triple mutant, but is virtually eliminated in the quadruple mutant.

→ Consistent with the hypothesis that there are 4 and only 4 FDHs.

→ Contradicts the hypothesis that there is significant CO2 production “downstream” of the one-carbon network.

HCOO-

CH2=H4F

Serine Cycle

CO2

CH2=H4MPT

H4MPT

CH3OH

HCHOH4F

Biomass

HCOO- consumption data (J. Vorholt)

Capacity to consume HCOO- (as measured by 13C NMR spectroscopy), in nmol/(mg*min)

Wild-type AM1 18.5

Quadruple FDH mutant 4.7

Why does the quadruple mutant still consume some HCOO-, since CO2 production is almost 0?

Does this HCOO- go into biomass via the long pathway?

HCOO-

CH2=H4F

Serine Cycle

CO2

CH2=H4MPT

H4MPT

CH3OH

HCHOH4F

Biomass

Biomass flux is not impaired in either the triple or quadruple mutant.

0

0.2

0.4

0.6

0.8

1

1.2

wild-type triplemutant

quadruplemutant

Bio

mas

s fl

ux,

n

mo

l/(m

in*m

L*O

D)

My 14C-Biomass flux data

→ HCOO- consumed by the quadruple mutant goes into biomass via the long pathway.

(This happens in the wild-type and triple mutant as well.)

HCOO-

CH2=H4F

Serine Cycle

CO2

CH2=H4MPT

H4MPT

CH3OH

HCHOH4F

Biomass

Conclusions on formate dehydrogenase

We have now identified all of the major FDHs (4 of them).

CH3OH can enter the serine cycle via the long pathway, but cannot easily be converted to CO2 at that point. Thus cells lacking FDH1-4 cannot grow on CH3OH alone.

HCOO-

CH2=H4F

Serine Cycle

CO2

CH2=H4MPT

H4MPT

CH3OH

HCHOH4F

BiomassFDH1-4

Metabolic fluxes in M. extorquens AM1:our view as of 2007

1. The enzyme-mediated long pathway is the cell’s only significant route for assimilating CH3OH.

2. Therefore HCOO-, not HCHO, is the key branch point.

3. Formate dehydrogenase (FDH) is important after all!

HCOO-

CH2=H4F

Serine Cycle

CO2

CH2=H4MPT

H4MPT

CH3OH

HCHOH4F

Biomass

Metabolic fluxes in M. extorquens AM1:our view as of 2007

1. The enzyme-mediated long pathway is the cell’s only significant route for assimilating CH3OH.

2. Therefore HCOO-, not HCHO, is the key branch point.

3. Formate dehydrogenase (FDH) is important after all!

HCOO-

CH2=H4F

Serine Cycle

CO2

CH2=H4MPT

H4MPT

CH3OH

HCHOH4F

Biomass

Metabolic fluxes in M. extorquens AM1:our view as of 2007

1. The enzyme-mediated long pathway is the cell’s only significant route for assimilating CH3OH.

2. Therefore HCOO-, not HCHO, is the key branch point.

3. Formate dehydrogenase (FDH) is important after all!

HCOO-

CH2=H4F

Serine Cycle

CO2

CH2=H4MPT

H4MPT

CH3OH

HCHOH4F

BiomassFDH1-4

Ongoing and future work

C1 metabolic network

• Since HCOO- is the key branch point, study the regulation of FDHs and FtfL.

HCOO-

CH2=H4F

Serine Cycle

CO2

CH2=H4MPT

H4MPT

CH3OH

HCHOH4F

BiomassFDH1-4

FtfL

Ongoing and future work

HCOO-

CH2=H4F

Serine Cycle

CO2

CH2=H4MPT

H4MPT

CH3OH

HCHOH4F

Biomass

Interaction of C1 and multicarbon networks

• Two versions of the glyoxylate regeneration cycle have been proposed (Korotkova et al. 2002; Albers et al. 2006). Which occurs in AM1?

• How (in terms of enzyme regulation/activity, metabolite levels, and fluxes) do cells transition from succinate (C4H4O4

2-) use to CH3OH use?

Undergrads can do this stuff!

Accessible techniques

• enzyme and metabolite assays

• cloning, transformation, PCR

• growth assays

• mathematical modeling

Preliminary success

• Dan Yates

• Jason Lum

Summary of metabolism research

A B Ck1

k-1

k2

k-2

MEASURE fluxesvia label tracing

MODEL fluxesmathematically

REDIRECT fluxesvia genetic engineering

Long-term goal:

Redesign methylotrophs for optimal production of useful chemicals from methanol.

Lidstrom lab members:

Mila Chistoserdova, Ph.D.

Marina Kalyuzhnaya, Ph.D.

Mary Lidstrom, Ph.D.

Jonathan Miller, M.S.

Betsy Skovran, Ph.D.

Tim Strovas, Ph.D.

Acknowledgments

Former lab members:Kelly FitzGerald, Ph.D. (UW Tech Transfer)Xiaofeng Guo, Ph.D. (Brigham & Women’s)Chris Marx, Ph.D. (Harvard)Steve Van Dien, Ph.D. (Genomatica)Julia Vorholt, Ph.D. (ETH Zurich)

Additional collaborator:George Kosály, Ph.D. (UW Mech Eng)

Funding: Kirschstein NRSA (NIH)

Acknowledgments

The End

Image: hancockpub.lib.in.us