Sialic Acid Production by Metabolically Engineered Escherichia coli

Benjamin R. Lundgren and Christopher N. Boddy*

Syracuse University, Syracuse, NY 13244

Sialic acid is a key molecule in cell adhesion and cell signaling

OR3O

OR5OR4

R2HNOH

R1O

CO2HR1 = AcR2 = Ac or -CO-CH2OHR3 = AcR4 = Ac, CH3 or SO3

R5 = S-lactyl or PO32

1 R1 =R3 =R4 =R5 = H, R2 = Ac

N-acetylneuraminic acid, Neu5Ac, undergoes extensive modifications to generate the diversity

of the sialic acid family

Sialic acid encompasses a large family of cell-surface carbohydrates

Biological Function• Cell Adhesion• Cell Signaling• Glycoprotein Stability• Bacterial Virulence• Tumor Metastasis

Medical Importance• Influenza Inhibitors• Marker for Disorders• Biologics• Diagnostics

Limited supply of sialic acid analogs has hindered advancement in basic research, diagnostic development and therapeutic production

Current resources for sialic acid are scarce, costly, and non-scalable

Natural Resources

Chemical Synthesis

Enzymatic Synthesis

Microbial Fermentation?

low yield& purity

costly & not readily scalable

OHO

OH OH

AcHNOH

HO

CO2H

challenging

Fermentation as an alternative route in sialic acid production

• Low cost• Scalable• Can be crystallized from aqueous solutions at

concentrations > 150 g/L

• Bacterial sialic acid metabolism is well characterized

Harness the chemistry of biological pathways in bacteria to produce sialic acid

OHO

OHOH

AcHNOH

HO

CO2HOHO

HONH2

OH

OH

GeneExpression

Plasmid

Host DNALacks genes for

sialic acid degradation

E. coli CellEncodes sialic acid biosynthesis

Feedstock Sialic Acid

SialicAcid

SialicAcid ManNAc + Pyruvate

ManNAc-6-P

GlcNAc-6-P GlcN-6-P

Fructose-6-P

NanA

NanK

NanENagA

NagB

NanT

OUT IN

Removal of sialic acid catabolism in E. coli is crucial for sialic acid production

ATP

ADP

Deletion of transporter and aldolase eliminates sialic acid degradation

TransporterAldolase

NeuC and NeuB catalyze the de novo biosynthesis of intracellular sialic acid in

N. meningitidis group B

UDP-GlcNAc ManNAc SialicAcid

CMP-Sialic Acid[Sialic Acid]n

NeuC NeuB

NeuANeuS

PEP Pi CTPPPi

ExportPolysialic Acid Capsule

Insertion of NeuB and NeuC allows in vivo synthesis of sialic acid

H2O UDP

Glucosamine synthase increases the synthesis of key metabolite for sialic acid biosynthesis

Fru-6-P GlcN-6-P GlcN-1-P

GlcNAc-1-PUDP-GlcNAc

GlmS

Gln Glu

GlmM

Ac-CoA

CoAGlmU

GlmU

UTPPPi

NeuBC pathway

Sialic Acid

Increases production of sialic acid from low-cost feedstocks

Sialic acid production by genetically & metabolically engineered E. coli

Various carbon sources lead to sialic acid production

Sialic acid production from glucose is the cheapest

Multiple feedings of glucose leads to a sialic acid titer of 1.7 grams per liter

Production costs: <$1 per gram of sialic acid

Sialic Acid Analogs: Tools for discovery in sialic acid research

Serve as biological probes, components of drugs and diagnostics

OHO

OH OH

NHOH

HO

CO2H

O

N3

N-azido sialic acid

OHO

OH OH

NHOH

HO

CO2H

O

H3C n

N-acyl sialic acid

imaging of cells in vivomodulate cell-cell interactions

Analogs can be produced using chemically modified feedstocks

OHOHO

NHOH

OH

RO

OHO

OH OH

NHOH

HO

CO2H

R

O

N-acyl glucosamine N-acyl sialic acid

GlmS NeuC NeuB

Feeding of N-acyl glucosamines to engineered E. coli results in production of N-acyl sialic acids

Analogs can be produced in vivo by expressing tailoring enzymes

OHOHO

OHOH

OH

OHO

OH OH

NHOH

HO

CO2H

O

OHO

OH OH

NHOH

HO

CO2H

O

HO

GlmS NeuC NeuB

Hydroxylase

Glucose Sialic Acid

Produce gram quantities of important analogs

N-glycolyl sialic acid

Sialic acid production by microbial fermentation

• Is efficient, rapid, and cost effective

• Higher yields from dense-cell cultures • Generate large amounts of analogs

• Shows the feasibility to produce complex, unavailable small molecules

Acknowledgements

• Christopher N. Boddy• The Blattner lab at University of WI-Madison• Timothy J. Durfee • Kinya Hotta • The Borer and Doyle labs at Syracuse University• Syracuse University and the Structural Biology,

Biochemistry and Biophysics (SB3) graduate program.