Production and secretion of fatty acids

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1 Production and secretion of fatty acids in genetically engineered cyanobacteria presented by Wuthipong Pangjai Xinyao et al. (2010) doi : 10.1073/pnas.1001946107 Roy Curtiss III Xinyao Liu

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LISA STIFFLER, Bio-debatable: Food vs. fuel http://www.seattlepi.com/ Use of resources for biofuel production 2 hot for biofuel production !!

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H 2 O + CO 2 O 2 sugars starch lipid cellulose Algae extraction - labor-intensive - costly 3 biofuel

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Escherichia coli Acyl -ACP Xylose Glycolysis Hemicellulose tesA Fatty acid thioesterase (TE) Fatty acid ‘ tesA need additional carbon 4

Cyanobacteria (blue green algae):

Cyanobacteria (blue green algae) Lake Atitlán , Guatemala sewage photosynthetic membrane - lipids - robust lipid metabolism more genetically manipulatable compared to eukaryotic algae 5

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Membrane lipids biosynthesis in cyanobacteria Calvin cycle 6 membrane TE H 2 O CO 2 Acetyl- CoA (precursor) Malonyl-CoA ass acc Acyl -ACP forward reaction reverse reaction feedback inhibition thioesterase Acetyl- CoA carboxylase a cyl -ACP synthetase acc Acetyl- CoA carboxylase

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to apply a fatty acid secretion strategy in cyanobacteria for biofuel production Objective 7

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(I) gene construction (II) transformation (III) selection (IV) growth and overproduction (V) FFA composition Overview 8

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gene A A A 9 (I) Construction knocked out gene A + overexpressed gene B gene A promoter TER gene B gene A promoter TER gene B A A Homologous recombination system

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expressed mutant E. coli TE gene directed FFAs to medium  10 ‘ tesA Acyl -ACP thioesterase (TE) cell envelope lack of transit peptide  culture medium

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knocked out acyl -ACP synthetase reduced feedback inhibition  11 Acetyl- CoA (precursor) Malonyl-CoA Acyl -ACP acc Lipid ass acyl -ACP synthetase prevent accumulation

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increased the rate controlling enzyme reduced polyester (energy storage) increased lipid precursor  poly-3-hydroxy butyrate rate limiting enzyme 12 Malonyl-CoA Acetyl- CoA (precursor) acc Acetyl- CoA carboxylase overexpress PHB

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expressed plant TE genes weakened s-layer  Umbellularia califomica leaves are aromatic (C12∶0) Cuphea hookeriana (C8∶0, C10:0) TE s-layer 13 Acyl -ACP Lipid

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expressed plant TE gene knocked out cyanophycin synthetase (carbon-storage compound)  Cuphea hookeriana (C8∶0, 10:0) TE 14 Acyl -ACP Lipid Acetyl- CoA (precursor) cyanophycin

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expressed plant TE gene weakened peptidoglycan  TE Cinnamonum camphorum (C14∶0) peptidoglycan 15 Acyl -ACP

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vector Synechocystis sp. PCC 6803 5 h plated onto a selection media grown for 4 days PCR (II) Transformation and (III) selection 16

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(II) Transformation 17  1  1 2  1 2 3  1 2 3 4  1 2 3 4 5 2 transformed new construct into previous transformant 3 4 5 PCC 6803 genome transformant’s generation

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(IV) growth and overproduction cell damage cell density Susan Brown amount of FFAs 18

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WT 23% exponential phase 2 % 24% long lag phase 30 °C in BG-11 medium bubbled with 1% CO 2 -enriched air cell density and cell damage ( transformant #  )  S-layer *  feedback inhibition polyester  E. coli -plant TE  rate controlling enzyme Results 19 0.8%

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secretion  for 4 days secreted FFAs Results lipid droplets 20

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increased FFA-secreting efficiency intracellular FFA amount did not increase overproduction (amount) Results 21

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ACC overproducing strains (rate limiting step) overproduction (amount) 3-fold increase in FFA secretion over their parental strains increasing the substrate amount improved FFA yields Results 22

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deleting S layer from cell envelope overproduction (amount) 3-fold increase in FFA secretion over their parental strains Results 23

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deleting peptidoglycan from cell envelope overproduction (amount) 2- fold increase in FFA secretion over their parental strains removal of the hydrophilic cell wall barrier did facilitate FFA secretion and decrease feedback inhibition Results 24

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overproduction (amount) Results 25  mg/day/L 400 times higher than wild-type 50% of the biomass

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electrospray ionization mass spectrometry (ESI-MS) extracellular FFAs intracellular FFAs (V) chemical composition 26

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Fatty acid weight percentage (%) chemical composition     Results 27  inhibitor  + PHB (polyester)  conEnz + TE plant c8:0, c10:0 C-storage + TE plant c14:0 peptido . + TE plant c8:0, c10:0 c12:0 s-layer

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Fatty acid weight percentage (%) chemical composition saturated FFA amount increased     Results 28 inhibitor   + PHB (polyester)  conEnz + TE plant c8:0, c10:0 C-storage + TE plant c14:0 peptido . + TE plant c8:0, c10:0 c12:0 s-layer 1.6 23.6

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Fatty acid weight percentage (%) chemical composition Results 29 inhibitor  + PHB (polyester)  conEnz + TE plant c8:0, c10:0 C-storage + TE plant c14:0 peptido . + TE plant c8:0, c10:0 c12:0 s-layer middle chain FFAs are easier to secrete than the longer chain FFAs      middle chain long chain 1.6 17.9 23.6 1.1

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d iscussions - transgenic cells were damaged when CO 2 aeration started at low cell density - always maintain cell densities above 10 7 CFU∕ml at stationary phase, transgenic cell grew better than WT - FFA secretion might be able to relax the overreduced photosynthetic electron transport chain - make the cells healthier in stationary phase 30

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d iscussions - Removal of FFA increased intracellular FFA production because removing the final products from a reaction system into a metabolic sink will push the equilibrium toward products 31

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conclusions cyanobacteria have great potential to produce and export FFAs 32 Overproduction of FFAs - acc and TE overproduction - aas , PHB, S layer and peptidoglycan knock out

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best construct (  ) can produce 24605 liter of biodiesel per 4046 m 2 of culture 20 cm deep at a cell density of 1.5 × 10 8 cells∕ml during a year period conclusions 33

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Acknowledgement Dr. Anchalee Sirikhachornkit 34

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Thank you for your attention 35

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enhance the primary FFA pathway genes attenuate the competing pathway genes improvements of growth conditions to enhance FFA yields (CO 2 concentration, temperature, illumination, pH, and cell density) What’s next ? 36

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39 http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=hmg&part=A2713

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Confirmation of transformation 1,2 primer FadD-F1-seq, FadD-F2-A 3,4 primer S4-seg-100S, S4-seg-100A 5,6 primer S5100S, S5100A 1 2 3 4 5 6 lane 1  wt (parental) lane 2  ∆ ass lane 3  ∆ PHB synthesis (parental) lane 4  ∆ PHB synthesis :: ACC 40

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http://www.cehs.siu.edu 41

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Isolated plant TEs ( thioesterase ) Umbellularia califomica leaves are aromatic lauric acid (C12∶0) Cuphea hookeriana octanoic acid (C8∶0, 10:0) part I engineering (Introduction) Cinnamomum camphorum decanoic acid (C14∶0) 42

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part I engineering (Introduction) suicide vector  levansucrase + sugar  levan sacB 43

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Fatty acid weight percentage (%) chemical composition      Results 44 inhibitor  + PHB (polyester)  conEnz + TE plant c8:0, c10:0 C-storage + TE plant c14:0 peptido . + TE plant c8:0, c10:0 c12:0 s-layer product chain length of plant TEs in 6803 did not totally match their substrate preference in plants or E. coli

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Composition Hemicellulose contains many different sugar monomers. In contrast, cellulose contains only anhydrous glucose. For instance, besides glucose, sugar monomers in hemicellulose can include xylose , mannose , galactose , rhamnose , and arabinose . Hemicelluloses contain most of the D- pentose sugars, and occasionally small amounts of L-sugars as well. Xylose is always the sugar monomer present in the largest amount, but mannuronic acid and galacturonic acid also tend to be present. Structural comparison to cellulose hemicellulose (also a polysaccharide) consists of shorter chains - 500-3,000 sugar units cellulose, 7,000 - 15,000 glucose molecules per polymer seen in cellulose. hemicellulose is a branched polymer, cellulose is unbranched . 45

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Calvin-cycle 46

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part II FFAs analysis (Introduction) SYTOX® Green nucleic acid stain - high-affinity nucleic acid stain - penetrates cells with compromised plasma membranes - not cross the membranes of live cells cell + SYTOX Green microscope / flow cytometry 48

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flow cytometry part II FFAs analysis (Introduction) 49

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electrospray ionization mass spectrometry (ESI-MS) 50

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part I engineering Confirmation of replacement 80 ⁰C/2 min 60 ⁰C 3 times resuspended cell 2 μl water 1 ul PCR 51

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cells∕ml day media cell death 8 × 10 5 10 agar 0.5% 8 × 10 5 3 liquid (1 ml) 0.4% 8 × 10 8 - - 1% 4 × 10 8 1 liquid (200 ml), 1% co 2 14.7% 4 × 10 8 2 liquid (200 ml), 1% co 2 33.7% CO 2 bubbling created significant cell damage part II FFAs analysis low cell density ? growth and cell damage 52

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part I engineering 400 ng suicide vector (target gene) 10 6 SD cells ( sac B / Km® ) 5 h plated onto a sucrose -containing agar plate grown for 3–4 days + media 5–8 days later - kanamycin agar plates - sucrose agar plates candidates for further evaluation sucrose Km transformation 53

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corn sugarcane soybean canola 54 Which plant could use for biofuel production ?

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