Metabolic pathway alternation, regulation and control (4)—Fatty acids

Xiaochen Yu01-31-2013

Fatty acids in Microorganism

• Highest energy content of the major nutrients (over 9 kcal/g)

• Crude fat is a mixture of triacylglycerols (TAG), phospholipids, fatty acids, sterols, waxes and pigments.

• TAG: the major storage form of fat in most of microorganisms

Fatty acid catabolism

Fatty acid

Fatty acid

Fatty acyl CoA Fatty acyl-carnitine

Fatty acyl-carnitineFatty acyl CoA

Acetyl-CoA

ACSCAT I

CAT II

β-oxidation

TAC CO2 + H2O

Control the rate of fatty acyl group entry & fatty acid oxidationCytosol

Mitochondria

ACS: acyl-CoA synthetasesCAT I: carnitine acyltransferase ICAT II: carnitine acyltransferase II

β oxidation

• Oxidation of saturated fatty acids with an even # of carbons

R–CH2–CH2–CH2–C–S–CoA

O

R–CH2–CH=C–C–S–CoA

O

H

H

R–CH2–CH–CH2–C–S–CoA

O

OH

H

R–CH2–C–CH2–C–S–CoA

OO

R–CH2–C–S–CoA

O

CH3–C–S–CoA

O

FAD

FADH2

H2O

NAD+ NADH + H+

+

CoA-SH

Palmitoyl-CoA

(C14) Acyl-CoA Acetyl-CoA

Acyl-CoA dehydrogenase

Enoyl-CoA hydratase

3-hydroxyacyl-CoA dehydrogenase

Acyl-CoA acetyltransferase

1st dehydrogenation

Hydration

2nd dehydrogenation

Fatty acid biosynthesis

ONLY exist in oleaginous

microorganism

Fatty acid biosynthesis

Nitrogen limitation

(Papanikolaou and Aggelis, 2011)

AMP

Fatty acid profiles

Docosahexaenoic acid (C22:6 n-3): Crypthecodinium cohnii & Schizochytrium sp.Gamma linoleic acid (C18:3 n-6): Mucor sp.

Metabolic pathway of xylose to fatty acid

Phosphoketolase pathwayPentose phosphate pathway (PPP)

(Tanaka et al., 2002)

Metabolic pathway of glycerol & Acetate

to fatty acid

Acetate

Acetyl-CoA synthetaseATP

ADPAcetyl-CoA

Lipid yield from carbon sources

100 g of glucose (~0.56 moles)~~1.1 moles of acetyl-CoA-------around 0.32 g lipids/g

100 g of xylose (~0.66 moles)-------around 0.34 g lipids/g

Assuming xylose is assimilated for the phosphoketolase pathway (the most efficient) in oleaginous microorganisms.

1. 1.2 moles of acetyl-CoA (phosphoketolase pathway) or2. 1.0 moles of acetyl-CoA (PPP)

100 g of glycerol--------0.30 g lipids/g

(Papanikolaou and Aggelis, 2011)

12

CodeD-Glucose:initCobraToolboxmodel=readCbModel('iNL895');model1=changeRxnBounds(model,'r_51_exchange',10,'u')solution1=optimizeCbModel(model1)

D-Xylosemodel1=changeRxnBounds(model,'r_51_exchange',0,'u')model2=changeRxnBounds(model1,'r_54_exchange',10,'u')

Acetate:model2=changeRxnBounds(model1,'r_30_exchange',10,'u’)

Glycerol:model2=changeRxnBounds(model1,'r_71_exchange',10,'u')

Example 1 Growth rate optimization

• Task Use FBA to predict flux distribution for optimal growth by yeast Yarrowia lipolytica model iNL895 using different carbon sources.

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Uptake rate (10 mmol gDW-1 h-1) Biomass flux (h-1) Aerobic

D-Glucose 3.8879

D-Xylose 3.2862

Acetate 0.7432

Glycerol 2.0828

The maximal theoretical growth rate under different carbon sources

2nd generation

Cellulosic biomassHydro

lysis

Pyrol

ysis

Gasitification

1st generation

Corn starch, Vegetable oils, Animal fats

TranesterificationCra

cking

Where can we get the substrate?

Our choice: Renewable Lipid-based Biofuel Production Strategy

Lignocellulosic biomass

Sugars

Pretreatment & hydrolysis

Fermentation

Characteristics of oleaginous microorganisms applied Similar fatty acid compositions with those of vegetable oils

A wide range of sugars can be utilized

High productivity

Tolerance to some inhibitors

Ash(0-5%)

Cellulose(33-51%)

Hemicellulose(19-36%)

Lignin(21-32%)

Extractive(1-5%)

O

O

OH

OH

CH2OHO

O

OH

OH

CH2OHO

OH

OH

CH2OH

O

OH

OH

CH2OH

OH

OHO OH

OHOH

CH2OH

OH

O OHCH3

OH

OHOH

O OH

OHOH

OH

O OH

OH

OHHOH2C

Sugar

What is cellulosic biomass made of?

Fermentation of glucose & xylose

Glucose and xylose are the major monomeric sugars in the lignocellulosic biomass hydrolysates

A major challenge in efficient utilization of mixed sugars for biofuel production

Many oleaginous yeasts can utilize glucose and xylose but lack the capability of simultaneous fermentation of both sugars, resulting in xylose accumulation

Glucose repression on non-glucose sugar utilization

Microorganisms generally consume the mixed sugars sequentially

Glucose first and then xylose

Allosteric competition for sugar transporters (Kawaguchi et al., 2006)

Solutions

1. Express xylose transporters

2. Introduce cellodextrin transporters

(Kim et al., 2012; Kawaguchi et al., 2006; Zaldivar and Olsson, 2001; Hu et al., 2011; Kastner et al., 1998)

(Ha et al., 2011)

Xylose-specific transporter

(Ha et al., 2011)

Cellodextrin transporters

Sugar transport study

MethodologyDetermine uptake rates of the target sugar by measuring the radio activities of C14 labeled sugar entering into the cells.

Major equipment: Liquid scintillation counter

• Model I, a Michaelis-Menten function: representing a single, saturable carrier

• Model II, a Michaelis-Menten function plus a linear term: representing a single carrier and a non-saturable diffusion process

• Model III, a sum of two Michaelis-Menten functions: representing two saturable carriers in the membrane

Kinetic Model of sugar transport

Example 2 Kinetics of xylose transport

S (mM) V(umol/min/g DCW)2.00 2.024.00 3.646.00 3.878.00 5.5910.00 4.5012.00 6.7414.00 5.5516.00 8.4320.00 6.6150.00 7.87

150.00 10.49200.00 10.74500.00 14.25

Experiment

Measure the uptake rates in C. curvatus at different concentrations of xylose

Task Determine the model of xylose transport in Cryptococcus curvatus

Data analysis: Non-linear regression

Data analysis: Non-linear regression

Data analysis: Non-linear regression

Results

Xylose (mM)0 100 200 300 400 500 600

V (nm

ol/min/m

g DC

W)

0

2

4

6

8

10

12

14

16

Model IModel IIModel III

Parameter Model I Model II Model III

V1 12.2 9.0 9.0

V2 - - -488254.5

Km1 12.6 6.3 6.3

Km2 - --

45118858.3

k - 0.0108 -

Variance 1.4 0.6337 0.6971

Other application of Michaelis-Menten kinetics

Enzyme behavior ----- competitive, non-competitive and uncompetitive inhibition

Hill reaction---- the laboratory equivalent of the light reaction of photosynthesis

The reaction center of photosystem II can be saturated with photons of red light just like the active site of an enzyme can be saturated with substrate molecules.

(Savage, 2011)

Final biofuel products

Comparison of chemical properties of transportation

fuels

Structural features influencing the physical and fuel properties of a fatty ester molecule

• Carbon chain length• degree of saturation• branching of the chain

(Li et al., 2010)

Energy yields of various fuels

FuelMass energy density

(MJ kg-1)Volumetric energy density

(MJ L-1)Max biochemical yield

(g g-1)a

Stoichiometric yield (g g-1)

Energy yield from glucose (%) b

Gasoline 42.7 32.0 - - -

Jet fuel 43.8 34.8 - - -

Diesel 45.5 38.7 - - -

Ethanol 29.7 20.8 0.511 0.5111 97.6

Fatty acids (C12-C22)

37-41 33-35 0.35-0.39 0.34-0.37 89-90

(Li et al., 2010)

20082009

20102011

20122013

20142015

20162017

20182019

20202021

20220

5

10

15

20

25

30

35

40Other

Cellulosic biofuels

Biomass-based diesel

Conventional biofuels

Year

Bil

lion

s of

Gal

lon

sU.S. Renewable Fuel Standard (RFS)

Consumption Mandate

36 billions

16 billions

How can we achieve the goal?

Progress in biofuel research, development and commercialization

Solazyme, Inc.

Algae-based biodiesel fuel now available at pump 11.14.2012 abc news

Reference• Liping Xiao, 2010. Evaluation of Extraction Methods for Recovery of Fatty Acids from Marine Products.

Retrieved from http://www.cursos.ualg.pt/emqal/documents/thesis/Liping_Xiao.pdf

• Tanaka, K. et al, 2002. Two different pathways for D-xylose metabolism and the effect of xylose concentration on the yield coefficient of L-lactate in mixed-acid fermentation by the lactic acid bacterium Lactococcus lactis IO-1. Appl Microbiol Biotechnol 60:160–167

• Papanikolaou, S. and Aggelis, G. 2011. Lipids of oleaginous yeasts. Part I: Biochemistry of single cell oil production. Eur. J. Lipid. Sci. Technol. 113: 1031-1051

• Kawaguchi, H., et al., Engineering of a Xylose Metabolic Pathway in Corynebacterium glutamicum. Applied and Environmental Microbiology, 2006. 72(5): p. 3418-3428.

• Kim, S.R., et al., Simultaneous co-fermentation of mixed sugars: a promising strategy for producing cellulosic ethanol. Trends in Biotechnology, 2012. 30(5): p. 274-282.

• Zaldivar, J., J. Nielsen, and L. Olsson, Fuel ethanol production from lignocellulose: a challenge for metabolic engineering and process integration. Applied Microbiology and Biotechnology, 2001. 56(1): p. 17-34.

• Hu, C., et al., Simultaneous utilization of glucose and xylose for lipid production by Trichosporon cutaneum. Biotechnology for Biofuels, 2011. 4: p. 25.

• Kastner, J.R., W.J. Jones, and R.S. Roberts, Simultaneous utilization of glucose and D-xylose by Candida shehatae in a chemostat. Journal of Industrial Microbiology & Biotechnology, 1998. 20(6): p. 339-343.

• Li, H., et al., Biofuels: Biomolecular Engineering Fundamentals and Advances. Annu. Rev. Chem. Biomol. Eng. 2010. 1:19–36