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1

Supplementary Information

The phosphoketolase pathway in the central carbon metabolism of

cyanobacteria

Wei Xiong, Tai-Chi Lee, Sarah Rommelfanger, Erica Gjersing, Melissa Cano, Pin-Ching

Maness, Maria Ghirardi, Jianping Yu*

National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, CO 80401,

USA

*Corresponding author:

National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, CO 80401,

USA

Tel.: +1-303-384-6252; Fax: +1-303-384-7836; E-mail: [email protected]

Supplementary Information files include:

S u p p l e m e n t a r y M e t h o d s … … … … … … … … … … … … … … … … … … … … … … … … … … … … 2

Supplementary Da ta 1 ……………………………………………………………………………. . 6

Supplementary Table 1 to 2………………………………………………………………………9

Supplementary Figure 1 to 4……………………………………………………………………… 11

Supplementary References …………………………………………………………………….. . 17

Phosphoketolase pathway contributes to carbon metabolism in cyanobacteria

http://dx.doi.org/10.1038/nplants.2015.187

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SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.187

2

Supplementary Methods

Construction of the ∆slr0453 mutants and complemented strains

The slr0453 open reading frame was disrupted by inserting an antibiotic-resistance gene

cassette, replacing part of the coding sequence. Specifically, regions of the Synechocystis

genome containing the start and the end of the slr0370 open reading frame (bases for

slr0370 is from 3500713 to 3503178; base numbering according to CyanoBase;

http://genome.microbedb.jp/cyanobase) were amplified via PCR with primers (P1:

5’GTGGGTTCTACCCTGGTAGGG3’ and P2: 5’CTAGAGGGGCCAGCGCCAATT3’). The PCR

products were directly inserted into pGEM-T vector (Promega), and then a 1024bp

chloramphenicol resistance gene (CmR)1 amplified by PCR with primers CmR-PF-AgeI and

CmR-PR-HindIII was used to replace the 383bp AgeI-HindIII fragments in slr0453 to create

the deletion plasmid pΔslr0453. Primer CmR-PF-AgeI was 5’

atatatACCGGTTATTTAACGACCCTGCCCTGAAC 3’ and primer CmR-PR-HindIII was 5’

atatatAAGCTTGTATCACGAGGCCCTTTCGTCTTC 3’, where underlines indicate AgeI and

HindIII restriction sites respectively, and lower cases indicate nucleotides added to protect

restriction sites. The plasmid pΔslr0453 was transformed into Synechocystis as previously

reported2. Host Synechocystis strains include the xylAB strain which is a xylose utilizer

expressing E.coli-derived xylose isomerase (XylA) and xylulokinase (XylB), and

∆glgC/xylAB which was further made deficient in glycogen biosynthesis by deleting the

glgC gene (slr1176)3. The detailed information on the construction of these strains are

described in Lee et al. 20154. Full segregation of Δslr0453 was confirmed by PCR with

genomic DNA isolated from parent Synechocystis and deletion strains as templates

(Supplementary Figure 2).

3

To express Slr0453 in the ∆slr0453 strains, the slr0453 open reading frame was cloned

into an expression cassette under the control of the Rubisco large subunit promoter (rbcL)5

(Supplementary Figure 2). The resulting plasmid pXW004 harboring the erythromycin

resistance gene (EmR) was transformed into the ∆slr0453 and xylAB/∆slr0453 mutants. The

expression cassette of slr0453 was integrated at a neutral site between slr1704 and sll1575

to generate the complemented stains. The genotypes of all strains constructed in this study

are summarized in Supplementary Table 1. The integration was verified by PCR using

genomic DNA and primer pairs P2 and P3 (sequence: 5’ CGGCAACAATCATTTCCGGTA3’)

(see Supplementary Figure 2).

Organic acids production assay

Synechocystis strains were cultivated in BG-11 medium until the OD730 reached 1.0. Cells

were collected by centrifugation at 3000 g for 10 minutes. Cell pellets were re-suspended

in either BG-11 medium or BG-11-N medium4, in the presence or absence of xylose/glucose.

The initial OD730 of each culture was adjusted to be identical. Cultures were shaken at 220

rpm either under 50 μEm-2s-1 fluorescent light or in the dark (culture tubes were covered

entirely by aluminum foil) and sampled over time for organic acid measurements. The

culture samples were collected and the medium was separated from cells by filtration

through 0.2 μM-diameter nylon membrane (Acrodisc®) and analyzed with HPLC (Agilent

Technologies1200 series) by injecting 100 μL samples into an HPLC column (Bio-Rad

Aminex HPX-87H), eluting with 5mM sulfuric acid at a flow rate of 0.6 ml/min, and

detecting by either a UV detector at 210 nm for 2-oxoglutarate (retention time: 8.8 min)

and pyruvate (retention time: 9.8 min) or by a Refractive Index Detector for acetate

(retention time: 15.2 min).




2

Supplementary Methods

Construction of the ∆slr0453 mutants and complemented strains

The slr0453 open reading frame was disrupted by inserting an antibiotic-resistance gene

cassette, replacing part of the coding sequence. Specifically, regions of the Synechocystis

genome containing the start and the end of the slr0370 open reading frame (bases for

slr0370 is from 3500713 to 3503178; base numbering according to CyanoBase;

http://genome.microbedb.jp/cyanobase) were amplified via PCR with primers (P1:

5’GTGGGTTCTACCCTGGTAGGG3’ and P2: 5’CTAGAGGGGCCAGCGCCAATT3’). The PCR

products were directly inserted into pGEM-T vector (Promega), and then a 1024bp

chloramphenicol resistance gene (CmR)1 amplified by PCR with primers CmR-PF-AgeI and

CmR-PR-HindIII was used to replace the 383bp AgeI-HindIII fragments in slr0453 to create

the deletion plasmid pΔslr0453. Primer CmR-PF-AgeI was 5’

atatatACCGGTTATTTAACGACCCTGCCCTGAAC 3’ and primer CmR-PR-HindIII was 5’

atatatAAGCTTGTATCACGAGGCCCTTTCGTCTTC 3’, where underlines indicate AgeI and

HindIII restriction sites respectively, and lower cases indicate nucleotides added to protect

restriction sites. The plasmid pΔslr0453 was transformed into Synechocystis as previously

reported2. Host Synechocystis strains include the xylAB strain which is a xylose utilizer

expressing E.coli-derived xylose isomerase (XylA) and xylulokinase (XylB), and

∆glgC/xylAB which was further made deficient in glycogen biosynthesis by deleting the

glgC gene (slr1176)3. The detailed information on the construction of these strains are

described in Lee et al. 20154. Full segregation of Δslr0453 was confirmed by PCR with

genomic DNA isolated from parent Synechocystis and deletion strains as templates

(Supplementary Figure 2).

3

To express Slr0453 in the ∆slr0453 strains, the slr0453 open reading frame was cloned

into an expression cassette under the control of the Rubisco large subunit promoter (rbcL)5

(Supplementary Figure 2). The resulting plasmid pXW004 harboring the erythromycin

resistance gene (EmR) was transformed into the ∆slr0453 and xylAB/∆slr0453 mutants. The

expression cassette of slr0453 was integrated at a neutral site between slr1704 and sll1575

to generate the complemented stains. The genotypes of all strains constructed in this study

are summarized in Supplementary Table 1. The integration was verified by PCR using

genomic DNA and primer pairs P2 and P3 (sequence: 5’ CGGCAACAATCATTTCCGGTA3’)

(see Supplementary Figure 2).

Organic acids production assay

Synechocystis strains were cultivated in BG-11 medium until the OD730 reached 1.0. Cells

were collected by centrifugation at 3000 g for 10 minutes. Cell pellets were re-suspended

in either BG-11 medium or BG-11-N medium4, in the presence or absence of xylose/glucose.

The initial OD730 of each culture was adjusted to be identical. Cultures were shaken at 220

rpm either under 50 μEm-2s-1 fluorescent light or in the dark (culture tubes were covered

entirely by aluminum foil) and sampled over time for organic acid measurements. The

culture samples were collected and the medium was separated from cells by filtration

through 0.2 μM-diameter nylon membrane (Acrodisc®) and analyzed with HPLC (Agilent

Technologies1200 series) by injecting 100 μL samples into an HPLC column (Bio-Rad

Aminex HPX-87H), eluting with 5mM sulfuric acid at a flow rate of 0.6 ml/min, and

detecting by either a UV detector at 210 nm for 2-oxoglutarate (retention time: 8.8 min)

and pyruvate (retention time: 9.8 min) or by a Refractive Index Detector for acetate

(retention time: 15.2 min).




4

1H-NMR analysis for acetate identification

1H-NMR spectra were collected on a Bruker Avance III spectrometer at 14.1T and

600.16MHz. A Bruker 5mm BBO probe was employed with a presaturation water

suppression sequence using an 85dB pulse. A 90° pulse of 15.3us and a recycle delay of 1s

were employed and 512 scans were collected. Automatic phase correction and baseline

correction were applied along with 0.3Hz line broadening. Acetate standard solutions were

prepared at 5, 10, and 20mM and the NMR spectra collected to confirm the chemical shift

assignment of acetate at 1.83ppm. All samples included 10% (by volume) D2O for locking.

13C-labeling and GC-MS analysis

The xylAB and xylAB/∆slr0453 strains were cultivated in BG-11 medium to an OD730 of 1.0.

Cells were then re-inoculated in BG-11 medium supplemented with 100mM sodium

bicarbonate and 10mM unlabeled D-xylose, 1-13C-D-xylose or U-13C-D-xylose, respectively.

Cultures with an initial OD730 of 0.05 were sealed and shaken at 220 rpm under 50 µEm-2 s-1

fluorescent illumination. When OD730 reached 0.8-1, the cultures were sampled. To check

whether the isotope steady state had been reached, cultures were re-sampled at 3 hours

and 6 hours afterwards as well. The isotope steady state allowed constant fractional

labeling of proteinogenic amino acids from biomass harvested at interval time points.

The sample treatment and GC-MS analysis were done as previously reported6, with a few

modifications. Briefly, 5mL of sampled cultures were centrifuged at 10,000 g for 1 minute,

and the cell pellets were digested in 500μL 6M HCl at 105°C for 12 hours. The hydrolysate

was dried under nitrogen gas flow at 65°C, and then dissolved in 50 μL water-free

dimethylformamide. The proteinogenic amino acids were derivatized prior to GC-MS

analysis. The dried hydrolysate, dissolved in N, N-Dimethylformamide was derivatized by

5

N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (TBDMS) with 1% tert-butyl-

dimethylchlorosilane at 85°C for 60 min. 1 μL of the sample in the organic phase was

loaded onto the GC–MS instrument (Agilent GC-6890 gas chromatography equipped with a

Agilent 19091J-413 column (30m×0.32mm×0.25μm) directly connected to a MS-5975C

mass spectrometer). Helium was used as the carrier gas. The oven temperature was

initially held at 50℃ for 2 min; then raised to 150℃ at 5℃/min and held at that value for 2

min; finally it was raised to 320℃ at 7℃/min, and held at that final value for 2 min. Other

settings included splitless and electron impact ionization (EI) at 70 eV. The amino acids

including alanine, aspartate, glutamate, glycine, histidine, isoleucine, leucine, phenylalanine,

proline, serine, threonine, tyrosine and valine were separated and analyzed.

To analyze the isotope labeling pattern of amino acids, a mass isotopomer distribution

vector, MDVα, was assigned according to Nanchen et al 7.

(1)

where m0 is the fractional abundance of molecules with mono-isotopic mass and mi>0 is the

abundance of fragments with heavier masses. The GC-MS data were corrected for the

naturally occurring isotopes of oxygen (O), hydrogen (H) and carbon (C) atoms using a

correction matrix (Eq. 2) as described by Nanchen et al7.

MDVα* = Ccorr,COH-1. MDVα (2)




6

where MDVα* is the corrected mass isotopomer distribution vector and Ccorr,COH−1 is the

correction matrix. According to Equation 3, the resulting MDVα* values were then used to

assess the fractional labeling enrichment of serine and alanine whose carbon skeletons are

derived from phosphoglycerate and pyruvate, respectively.

FL = ∑ 𝑖𝑖.𝑚𝑚𝑖𝑖𝑛𝑛𝑖𝑖=0

𝑛𝑛.∑ 𝑚𝑚𝑖𝑖𝑛𝑛𝑖𝑖=0

(3)

where n represents the number of carbon atoms in AKG or pyruvate and i is the mass

isotopomer.

The fractional contribution of phosphoketolase pathway (fa) is calculated based on

Equation 4.

FLPGA1-3 = fa × FLA + fb × FLB + fc × FLC (4)

where FLPGA1-3 is the fractional labeling of 3-P-glycerate with all three carbons during 1-

13C-xylose labeling; FLA , FLB , and FLC are the fractional labeling resulted from the

phosphoketolase pathway, the pentose phosphate pathway and the CBB cycle respectively;

fb and fc are fractional contribution of pentose phosphate pathway and CBB cycle to the

triose pool. The principle of this calculation is illustrated in Supplementary Figure 3b.

Supplementary Data 1

Blast Analysis of Slr0453 against well-characterized phosphoketolase from

Bifidobacterium

7

Query: Phosphoketolase from Bifidobacterium breve (Length = 825) Subject: Slr0453 from Synechocystis sp. PCC 6803 (Length = 821) Score = 760 bits (1962), Expect = 0.0, Method: Compositional matrix adjust. Identities = 380/814 (46%), Positives = 526/814 (64%), Gaps = 27/814 (3%) Query: 1 MTSPVIGTPWKKLNA-----PVSEESLEGVDKYWRVANYLSIGQIYLRSNPLMKAPFTRE 55 +TSP +P+ + + P+ L + +WR ANYL++G IYLR NPL++ P E Sbjct: 18 VTSPFSLSPFGQARSTVTGNPLDPTELNQMHGFWRAANYLAVGMIYLRDNPLLREPLQPE 77 Query: 56 DVKHRLVGHWGTTPGLNFLIGHINRFIADHGQNTVIIMGPGHGGPAGTSQSYLDGTYTET 115 +KHRL+GHWG++PG++FL H+NR I Q+ + ++GPGHG P YL+G+Y+ Sbjct: 78 QIKHRLLGHWGSSPGISFLYTHLNRIIRKFDQDMLYMVGPGHGAPGFLGPCYLEGSYSRF 137 Query: 116 FPKITKDEAGLQKFFRQFSYPGGIPSHFAPETPGSIHEGGELGYALSHAYGAIMDNPSLF 175 F + ++DE G+++FF+QFS+PGGI SH PETPGSIHEGGELGY LSHAYGA DNP+L Sbjct: 138 FAECSEDEDGMKRFFKQFSFPGGIGSHCTPETPGSIHEGGELGYCLSHAYGAAFDNPNLI 197 Query: 176 VPAIVGDGEAETGPLATGWQSNKLVNPRTDGIVLPILHLNGYKIANPTILSRISDEELHE 235 V + GDGE+ETGPLAT W SNK +NP DG VLP+LHLNGYKI NP++LSRIS EEL Sbjct: 198 VVGLAGDGESETGPLATSWHSNKFINPIRDGAVLPVLHLNGYKINNPSVLSRISHEELKA 257 Query: 236 FFHGMGYEPYEFVAGFDDEDHMSIHRRFAELWETIWDEICDIKAAAQTDNVH-RPFYPML 294 F G GY PY FV G D E S+H+ A + EI I+ A++ + RP +PM+ Sbjct: 258 LFEGYGYTPY-FVEGSDPE---SMHQAMAATLDHCVSEIHQIQQEARSTGIAVRPRWPMV 313 Query: 295 IFRTPKGWTCPKYIDGKKTEGSWRAHQVPLASARDTEAHFEVLKNWLESYKPEELFDANG 354 + RTPKGWT P Y+DG K EG WR+HQVP+ + AH + L+ W+ SYKPEELFD G Sbjct: 314 VMRTPKGWTGPDYVDGHKVEGFWRSHQVPMGGMHENPAHLQQLEAWMRSYKPEELFDEQG 373 Query: 355 AVKDDVLAFMPKGELRIGANPNANGGVIRDDLKLPNLEDYEVKEVAEYGHGWGQLEA--T 412 +K A P+G+ R+G+ P ANGG++R LK+P+ Y + G +EA T Sbjct: 374 TLKPGFKAIAPEGDKRLGSTPYANGGLLRRGLKMPDFRQYGIDVDQP-----GTIEAPNT 428 Query: 413 RRLGVYTRDIIKNNPRDFRIFGPDETASNRLQASYEVTNKQWDAGYISDEVD-EHMHVSG 471 LGV+ RD++ NN +FR+FGPDE +SN+L A YEV+ K W A Y+ ++ D + G Sbjct: 429 APLGVFLRDVMANNMTNFRLFGPDENSSNKLHAVYEVSKKFWIAEYLEEDQDGGELSPDG 488 Query: 472 QVVEQLSEHQMEGFLEAYLLTGRHGIWSSYESFVHVIDSMLNQHAKWLEATVREIPWRKP 531 +V+E LSEH +EG+LEAYLLTGRHG +++YESF HVI SM+NQHAKWL+ R + WR Sbjct: 489 RVMEMLSEHTLEGWLEAYLLTGRHGFFATYESFAHVITSMVNQHAKWLD-ICRHLNWRAD 547 Query: 532 IASMNLLVSSHVWRQDHNGFSHQDPGVTSVLLNKCFHNDHVIGIYFATDANMLLAIAEKC 591 I+S+N+L++S VWRQDHNGF+HQDPG V+LNK + V+ IY D N LL++A+ C Sbjct: 548 ISSLNILMTSTVWRQDHNGFTHQDPGFLDVILNK---SPDVVRIYLPPDVNSLLSVADHC 604 Query: 592 YKSTNKINAIIAGKQPAATWLTLDEARAELAKGAAAWDWASTAKNNDEAEVVLAAAGDVP 651 +S N IN I+ KQ + + A KG W+WAS E +VV+AAAGD+P Sbjct: 605 LQSKNYINIIVCDKQAHLQYQDMTSAIRNCTKGVDIWEWASNDAGT-EPDVVMAAAGDIP 663




8

Query: 652 TQEIMAASDKLKEL--GVKFKVVNVADLLSLQSAKENDEALSDEEFADIFTADKPVLFAY 709 T+E +AA+ L++ ++ + V+V DLL LQ E+ LSD +F +FT DKP++F + Sbjct: 664 TKEALAATAMLRQFFPNLRIRFVSVIDLLKLQPESEHPHGLSDRDFDSLFTTDKPIIFNF 723 Query: 710 HSYAHDVRGLIYDRPNHDNFNVHGYEEEGSTTTPYDMVRVNRIDRYELTAEALRMIDADK 769 H+Y + L Y R NH N +V GY+E+G+ TP D+ N+IDR+ L + + + + Sbjct: 724 HAYPWLIHRLTYRRTNHGNLHVRGYKEKGNINTPMDLAIQNQIDRFSLAIDVIDRLPQLR 783 Query: 770 YADK-IDELEK-FRDEAFQFAVDKGYDHPDYTDW 801 A I E+ K + + +A + G D P+ +W Sbjct: 784 VAGAHIKEMLKDMQIDCTNYAYEHGIDMPEIVNW 817

Note: Yellow boxed residues are key sites in Bifidobacterium phosphoketolase for interaction with the sugar-

derived hydroxyl groups. Green boxed residues recognize the distal phosphate group of F6P or Xu5p. All

these residues are conserved in Slr0453.The gray shaded sequences Gly116-to-Pro342, Thr428-to-Ser607,

and Lys618-to-Trp817 are conserved, and correspond to the TPP-binding module, phosphoketolase (XFP)

domain and XFP C-terminal domain, respectively.

9

Supplementary Table

Supplementary Table 1 Doubling times of strains used in this study

Doubling Time1 (h) Genotype/Characterization Reference or

source Wild-type 10.4±0.2 Synechocystis PCC. 6803 Gift from Teruo

Ogawa ∆slr0453 11.6±0.3 slr0453::CmR This study

∆slr0453/slr0453c 16.6±1.0 slr0453::CmR,rbcL-slr0453-EmR This study

xylAB 10.7±0.2 slr0168::psbA-xylAB-SpR 4 xylAB/∆slr0453 11.8±0.4 slr0168::psbA-xylAB-SpR, slr0453::CmR This study

xylAB/∆slr0453/slr0453c 16.9±0.9 slr0168::psbA-xylAB-SpR, slr0453::CmR,rbcL-slr0453-EmR This study

∆glgC/xylAB 12.1±0.2 slr1176::GenR, slr0168::psbA-xylAB-SpR 4 ∆glgC/xylAB/∆slr0453 13.3±0.1 slr1176::GenR, slr0168::psbA-xylAB-SpR,

slr0453::CmR This study

1: The doubling times were measured from triplicate experiments done with cells grown

photoautotrophically under fluorescent light at the intensity of 50 μEm-2s-1 without

antibiotics supplementation to the BG11 medium.




10

Supplementary Table 2 Stoichiometry and theoretical yields for acetate, pyruvate and 2-

oxoglutarate production from phosphoketolase-dependent and -independent pathways

No.1 Product Substrate Phospho-ketolase2 Theoretical yield3 Stoichiometry

a acetate CO2 + ∞ 2 CO2 + 5 ATP + 4 NADPH = 1 Acetate

b acetate CO2 - ∞ 2 CO2 + 6 ATP + 4 NADPH = 1 Acetate

c acetate xylose + 100% 2 Xylose = 5 Acetate + 3 ATP

d acetate xylose - 66.67% 3 Xylose = 5 Acetate + 5 CO2 + 10 ATP + 10 NADPH

e pyruvate CO2 - ∞ 3 CO2 + 7 ATP + 5 NADPH = 1 Pyruvate

f pyruvate xylose - 100.00% 3 Xylose = 5 ATP + 5 NADPH + 5 Pyruvate

g 2-oxoglutarate CO2 + ∞ 5 CO2 + 14 ATP + 8 NADPH = 1 2-

oxoglutarate h 2-

oxoglutarate CO2 - ∞ 5 CO2 + 15 ATP + 8 NADPH = 1 2-oxoglutarate

i 2-oxoglutarate xylose + 100% 1 Xylose = 2 NADPH + 1 2-

oxoglutarate

j 2-oxoglutarate xylose - 83.33% 6 Xylose = 20 NADPH + 5 ATP + 5 2-

oxoglutarate + 5 CO2 1. The No. of each line corresponds to the panel with identical No. in Supplementary Figure

4.

2. The operation of the phosphoketolase pathway can improve acetate and 2-oxoglutarate

but not pyruvate production by enhancing theoretical yield (c, i) or reducing ATP cost (a, g).

3. Theoretical yield = C product / C substrate (organic). The CO2-utilization pathways do not consume

organic carbon, thus are treated here as having theoretical yield of infinity.

11

Supplementary Figures

Supplementary Figure 1 Phosphoketolase pathway and genome information. a. The

phosphoketolase pathway towards acetate production bypasses pyruvate. XI: xylose

isomerase, XK: xylulokinase, PDH: pyruvate dehydrogenase, PAT: phosphate

acetyltransferase, AK: acetate kinase, ACS: acetyl CoA synthetase; XFP: D-xylulose 5-

phosphate/D-Fructose 6-phosphate phosphoketolase. Red arrows denote the

phosphoketolase pathway and dotted arrows denote conversions catalyzed by multi-step

enzymatic reactions. b. Arrangements of putative phosphoketolase genes in cyanobacterial

genomes. Red, purple and blue arrows denote putative genes encoding phosphoketolase,

acetate kinase, and glyceraldehyde 3-phosphate dehydrogenase, respectively. Grey arrows

indicate other predicted open reading frames surrounding phosphoketolase genes.




12

Supplementary Figure 2 Construction of ∆slr0453 mutants. a. Cassettes constructed for

disrupting slr0453. b. Cassettes constructed for slr0453 complementation. c. PCR analysis of

wild-type Synechocystis and mutants using the primer pairs: lane 1-4, P1 and P2, lane 5-7, P2

and P3, and genomic DNA as template: lane 1, wild-type Synechocystis; 2, Δslr0453; 3,

xylAB/Δslr0453; 4, xylAB /ΔglgC/Δslr0453; 5, wild-type; 6, Δslr0453/slr0453c; 7, xylAB

/Δslr0453/slr0453c. The construction procedures and sequences for P1, P2, and P3 are

shown in Supplementary Methods. CmR: Chloramphenicol resistance gene, rbcL: the Rubisco

large subunit promoter, EmR: Erythromycin resistance gene.

13

Supplementary Figure 3 The 13C-xylose tracer experiment quantitatively estimates

the flux of the phophoketolase pathway in the central carbon metabolism. a. Carbon

fate of 1-13C xylose metabolized through phosphoketolase pathway (black arrows), the PPP

(red arrows) and the CBB cycle (green arrows). The solid circles denote 13C-labeled carbon

atoms. b. Calculation of metabolic flux ratios through phosphoketolase pathway. fa

, fb and f

c

are the fractional contribution of the alternative pathways via phosphoketolase, the PPP

and the CBB cycle, respectively. Fractional labeling (FL) is calculated according to Eq. 3 (see

Supplementary Methods). c. Experimental design using U-13C-xylose and 1-13C-xylose to

feed xylAB and xylAB/∆slr0453 strains in the purpose of determining fc

and fa

respectively.




12

Supplementary Figure 2 Construction of ∆slr0453 mutants. a. Cassettes constructed for

disrupting slr0453. b. Cassettes constructed for slr0453 complementation. c. PCR analysis of

wild-type Synechocystis and mutants using the primer pairs: lane 1-4, P1 and P2, lane 5-7, P2

and P3, and genomic DNA as template: lane 1, wild-type Synechocystis; 2, Δslr0453; 3,

xylAB/Δslr0453; 4, xylAB /ΔglgC/Δslr0453; 5, wild-type; 6, Δslr0453/slr0453c; 7, xylAB

/Δslr0453/slr0453c. The construction procedures and sequences for P1, P2, and P3 are

shown in Supplementary Methods. CmR: Chloramphenicol resistance gene, rbcL: the Rubisco

large subunit promoter, EmR: Erythromycin resistance gene.

13

Supplementary Figure 3 The 13C-xylose tracer experiment quantitatively estimates

the flux of the phophoketolase pathway in the central carbon metabolism. a. Carbon

fate of 1-13C xylose metabolized through phosphoketolase pathway (black arrows), the PPP

(red arrows) and the CBB cycle (green arrows). The solid circles denote 13C-labeled carbon

atoms. b. Calculation of metabolic flux ratios through phosphoketolase pathway. fa

, fb and f

c

are the fractional contribution of the alternative pathways via phosphoketolase, the PPP

and the CBB cycle, respectively. Fractional labeling (FL) is calculated according to Eq. 3 (see

Supplementary Methods). c. Experimental design using U-13C-xylose and 1-13C-xylose to

feed xylAB and xylAB/∆slr0453 strains in the purpose of determining fc and f

a respectively.




14

15

Supplementary Figure 4 Phosphoketolase-dependent (Panel a, c, g, h) and independent

(Panel b, d, e, f, h, j) pathways for the production of acetate (Panel a-d), pyruvate (Panel e,

f), and 2-oxoglutarate (Panel g-j) from CO2 (Panel a, b, e, g, h ) or sugars (Panel c, d, f, i, j).

The respective ATP and co-factor costs are summarized in Supplementary Table 2.




16

17

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