Supplemental materials

Comments: The growth curves were mainly based on non­-labeled L-lactate
and 99% 1-^13^C labeled lactate culture (i.e., 1^st^ position labeled
lactate). The doubling time for growth is about 8 hours. Measurement of
OD~600~ can not be very accurate because the bacteria precipitate metals
during the growth. Instead, protein content was measured to capture the
growth kinetics. We also did the 3-^13^C labeled lactate culture, but
cell can not grow and we have to abandon that experiment (The 3-^13^C
lactate product from Cambridge Isotopic Company is contaminated by
nitrate).

![](media/image1.wmf){width="5.997222222222222in"
height="4.227777777777778in"}

**Figure S-1.** Growth curve of *D. vulgaris* in defined (LS4D)
L-lactate medium.

◊: OD~600~; ■: protein concentration; solid line: Model fitting.

![](media/image2.wmf){width="5.996527777777778in"
height="4.6305555555555555in"}

**Figure S-2.** Lactate and sulfate consumption, acetate production
curves in defined (LS4D) L-lactate medium. ◊: lactate; □: acetate; ▲:
sulfate; solid line: model fitting.

![](media/image3.png){width="6.752083333333333in"
height="4.313194444444444in"}

**Figure S-3.** GC-MS spectrum of glutamate of *Desulfovibrio vulgaris*
lysate.

**Table S-1. Metabolic reactions for lactate metabolism in
*Desulfovibrio vulgaris* Hildenborough.**

  ------------ -------------------------------- -------------------------------
  **Number**   **Reaction (energy balance)\***  **Gene coding for enzymes.**

  1            Lactate ↔ PYR (+NADH)            ***ldh*** DVU0600

  2            PYR ↔ PEP (-ATP)                 ***ppsA*** DVU1833

  3            PEP ↔ PGA (2 teps) (-ATP)        ***eno*** DVU0322 + ***gpmA***
                                                DVU1619/***gpm*** DVU2935

  4            PYR + CO~2~ ↔ OAA (-ATP)         ***pyc*** DVU1834

  5a           PYR↔ ACoA + CO~2~ (NADH or       ***lpdA*** DVU1423
               NADPH)                           

  5b           ACoA + CO~2~↔ PYR(- NADPH)       ***porA*** DVU1569/ ***porB***
                                                DVU1570/ ***por*** DVU3025

  5c           PYR↔ ACoA + FormateCO~2~        DVU2272, DVU2824

  6            Acetate → ACoA(+ATP)             ***acs*** DVU0748

  7            ACoA + OAA ↔ CIT                 Not annotated

  8            PYR + CO~2~ ↔ MAL (-NADH)        ***tme*** DVU0414

  9            CIT ↔ ICT                        ***aco*** DVU1064

  10           ICT ↔ OXO + CO~2~ (NADH)         ***icd*** DVU0477

  11           OXO ↔ SUC + CO~2~(NADH)          ***porA*** DVU1569/ ***oorA***
                                                DVU1945/ ***oorD*** DVU3350

  12           ICT ↔ SUC + Glyoxylate           Not annotated (glyoxlate shunt
                                                may not exist)

  13           FUM ↔ MAL                        ***fumC*** DVU0080/ ***aspA***
                                                DVU1766

  13           SUC ↔ FUM (-FADH~2~)             ***sdhB*** DVU2674/ ***frdC***
                                                DVU3261

  14           MAL ↔ OAA (-NADH)                ***ldh*** DVU0600

  15           ACoA + Glyoxylate ↔ MAL          ***glcB*** DVU0701

  16a          PGA ↔ SER                        Some steps are missing.

  16b          PYR ↔ SER                        ***sda*** DVU2147

  17           SER ↔ GLY + C1                   ***glyA*** DVU1203

  18           PGA ↔ T3P(2 steps)(-ATP-NDAH)    ***pgk*** DVU2529+***gap-1***
                                                DVU0565/ ***gap-2*** DVU2144

  19           T3P + T3P ↔ F6P (2 steps) (-ATP) ***fba*** DVU2143+***glpX***
                                                DVU/ ***fbp*** DVU1841

  20           S7P + T3P ↔ E4P + F6P            DVU1658

  21           C5P + E4P ↔ F6P + T3P            ***tkt*** DVU2530

  22           F6P ↔ G6P                        ***pgi*** DVU3222

  23           G6P ↔ C5P + CO~2~                (4 steps) (NADPH)

               Glucose-6-phosphate              
               dehydrogenase/phosphogluconate   
               dehydrogenase are missing.       

  24           C5P + C5P ↔ S7P +T3P             ***tkt*** DVU2530

  25           Asp + OXO ↔ OAA + Glu            ***aspB*** DVU3223

  26           C5P ↔ His (11 steps)             One of reactions
                                                (phosphoribosyl-ATP
                                                diphosphatase) is missing.

  27           PYR ↔ ALA                        Not annotated

  28           2PEP+E4P ↔ Phe (or Try) +CO~2~   One of reactions
               (9 steps)                        (3-dehydroquinate synthase) is
                                                missing.

  29           Asp ↔ Met                        (4 steps)

  30           C5P ↔ RNA and DNA                (many steps)

               Note: A new Acetyl-CoA           
               degradation route: ACoA ↔ C1     
               pool + \[CO\], which contains    
               some key enzymes.                

  31a          \[CO\] ↔ CO~2~ + \[H\]           ***cooS*** DVU2098

  31b          C1 pool ↔ HCOOH (4 steps)        ***folD*** DVU0323 and
               (ATP+NADH)                       ***metF*** DVU0997

  31c          HCOOH ↔ CO~2~                    ***fdnG-1*** DVU0587/
                                                DVU0577/DVU0578
  ------------ -------------------------------- -------------------------------

\* Energy cost for synthesis of amino acids is listed in Table S-2.

**Table S-2.** Building blocks needed to produce *D. vulgaris* biomass
\*.

+-------------+--------+------------+----+-----+------+---+----+---+
| Building    | Yield  | Cost of    |    |     |      |   |    |   |
| block       | coeff  | making 1   |    |     |      |   |    |   |
|             | icient | μmol of    |    |     |      |   |    |   |
|             |        | each of    |    |     |      |   |    |   |
|             | (mm    | these      |    |     |      |   |    |   |
|             | ol/mol | building   |    |     |      |   |    |   |
|             | la     | blocks     |    |     |      |   |    |   |
|             | ctate) | (mol/mol)  |    |     |      |   |    |   |
+-------------+--------+------------+----+-----+------+---+----+---+
|             |        | Meta       | A  | N   | N    | 1 | NH | S |
|             |        | bolites^a^ | TP | ADH | ADPH | - | ~4 |   |
|             |        |            |    |     |      | C | ~^ |   |
|             |        |            |    |     |      |   | +^ |   |
+-------------+--------+------------+----+-----+------+---+----+---+
| **Protein   |        |            |    |     |      |   |    |   |
| amino       |        |            |    |     |      |   |    |   |
| acids^\#^** |        |            |    |     |      |   |    |   |
+-------------+--------+------------+----+-----+------+---+----+---+
| Alanine     | 3.22   | 1 pyr      | 0  | 0   | 1    | 0 | 1  | 0 |
+-------------+--------+------------+----+-----+------+---+----+---+
| Arginine    | 1.56   | 1 oxo      | 7  | -1  | 4    | 0 | 4  | 0 |
+-------------+--------+------------+----+-----+------+---+----+---+
| Asparagine  | 1.41   | 1 oaa      | 3  | 0   | 1    | 0 | 2  | 0 |
+-------------+--------+------------+----+-----+------+---+----+---+
| Aspartate   | 1.41   | 1 oaa      | 0  | 0   | 1    | 0 | 1  | 0 |
+-------------+--------+------------+----+-----+------+---+----+---+
| Cysteine    | 0.58   | 1 pga      | 4  | -1  | 5    | 0 | 1  | 1 |
+-------------+--------+------------+----+-----+------+---+----+---+
| Glutamate   | 1.62   | 1 oxo      | 0  | 0   | 1    | 0 | 1  | 0 |
+-------------+--------+------------+----+-----+------+---+----+---+
| Glutamine   | 1.62   | 1 oxo      | 1  | 0   | 1    | 0 | 2  | 0 |
+-------------+--------+------------+----+-----+------+---+----+---+
| Glycine     | 2.74   | 1 pga      | 0  | -1  | 1    | - | 1  | 0 |
|             |        |            |    |     |      | 1 |    |   |
+-------------+--------+------------+----+-----+------+---+----+---+
| Histidine   | 0.55   | 1 C5P      | 6  | -3  | 1    | 1 | 3  | 0 |
+-------------+--------+------------+----+-----+------+---+----+---+
| Isoleucine  | 1.28   | 1oaa, 1pyr | 2  | 0   | 5    | 0 | 1  | 0 |
+-------------+--------+------------+----+-----+------+---+----+---+
| Leucine     | 2.40   | 2 pyr, 1   | 0  | -1  | 2    | 0 | 1  | 0 |
|             |        | acCoA      |    |     |      |   |    |   |
+-------------+--------+------------+----+-----+------+---+----+---+
| Lysine      | 1.65   | 1 oaa, 1   | 2  | 0   | 4    | 0 | 2  | 0 |
|             |        | pyr        |    |     |      |   |    |   |
+-------------+--------+------------+----+-----+------+---+----+---+
| Methionine  | 0.68   | 1 oaa      | 7  | 0   | 8    | 1 | 1  | 1 |
+-------------+--------+------------+----+-----+------+---+----+---+
| Ph          | 1.03   | 1 E4p, 2   | 1  | 0   | 2    | 0 | 1  | 0 |
| enylalanine |        | pep        |    |     |      |   |    |   |
+-------------+--------+------------+----+-----+------+---+----+---+
| Proline     | 1.29   | 1 oxo      | 1  | 0   | 3    | 0 | 1  | 0 |
+-------------+--------+------------+----+-----+------+---+----+---+
| Serine      | 1.29   | 1 pga      | 0  | -1  | 1    | 0 | 1  | 0 |
+-------------+--------+------------+----+-----+------+---+----+---+
| Threonine   | 1.42   | 1 oaa      | 2  | 0   | 3    | 0 | 1  | 0 |
+-------------+--------+------------+----+-----+------+---+----+---+
| Tryptophan  | 0.26   | 1 C5P, 1   | 5  | -2  | 3    | 0 | 2  | 0 |
|             |        | E4p,       |    |     |      |   |    |   |
|             |        |            |    |     |      |   |    |   |
|             |        | 1 pep      |    |     |      |   |    |   |
+-------------+--------+------------+----+-----+------+---+----+---+
| Tyrosine    | 0.68   | 1 E4p, 2   | 1  | -1  | 2    | 0 | 1  | 0 |
|             |        | pep        |    |     |      |   |    |   |
+-------------+--------+------------+----+-----+------+---+----+---+
| Valine      | 1.98   | 2 pyr      | 0  | 0   | 2    | 0 | 1  | 0 |
+-------------+--------+------------+----+-----+------+---+----+---+
| **RNA       |        |            |    |     |      |   |    |   |
| nu          |        |            |    |     |      |   |    |   |
| cleotides** |        |            |    |     |      |   |    |   |
+-------------+--------+------------+----+-----+------+---+----+---+
| ATP         | 0.98   | 1 C5P, 1   | 11 | -3  | 1    | 1 | 5  | 0 |
|             |        | pga        |    |     |      |   |    |   |
+-------------+--------+------------+----+-----+------+---+----+---+
| GTP         | 1.20   | 1 C5P, 1   | 13 | -3  | 0    | 1 | 5  | 0 |
|             |        | pga        |    |     |      |   |    |   |
+-------------+--------+------------+----+-----+------+---+----+---+
| CTP         | 0.75   | 1 C5P, 1   | 9  | 0   | 1    | 0 | 3  | 0 |
|             |        | oaa        |    |     |      |   |    |   |
+-------------+--------+------------+----+-----+------+---+----+---+
| UTP         | 0.81   | 1 C5P, 1   | 7  | 0   | 1    | 0 | 2  | 0 |
|             |        | oaa        |    |     |      |   |    |   |
+-------------+--------+------------+----+-----+------+---+----+---+
| **DNA       |        |            |    |     |      |   |    |   |
| nu          |        |            |    |     |      |   |    |   |
| cleotides** |        |            |    |     |      |   |    |   |
+-------------+--------+------------+----+-----+------+---+----+---+
| dATP        | 0.15   | 1 C5P, 1   | 11 | -3  | 2    | 1 | 5  | 0 |
|             |        | pga        |    |     |      |   |    |   |
+-------------+--------+------------+----+-----+------+---+----+---+
| dGTP        | 0.15   | 1 C5P, 1   | 13 | -3  | 1    | 1 | 5  | 0 |
|             |        | pga        |    |     |      |   |    |   |
+-------------+--------+------------+----+-----+------+---+----+---+
| dCTP        | 0.15   | 1 C5P, 1   | 9  | 0   | 2    | 0 | 3  | 0 |
|             |        | oaa        |    |     |      |   |    |   |
+-------------+--------+------------+----+-----+------+---+----+---+
| dTTP        | 0.15   | 1 C5P, 1   | 10 | 0   | 3    | 1 | 2  | 0 |
|             |        | oaa        | .5 |     |      |   |    |   |
+-------------+--------+------------+----+-----+------+---+----+---+
| **Average   | 1.6    | 8.2 acCoA  | 7  | 0   | 14   | 0 | 0  | 0 |
| fatty       |        |            | .2 |     |      |   |    |   |
| acid**      |        |            |    |     |      |   |    |   |
+-------------+--------+------------+----+-----+------+---+----+---+
| **Average   | \~7    | 1 G6P      | ND | ND  | ND   | N | ND | N |
| car         |        |            |    |     |      | D |    | D |
| bohydrate** |        |            |    |     |      |   |    |   |
+-------------+--------+------------+----+-----+------+---+----+---+

\* part of data are based on "Stephanopoulos, G. N., A. A. Aristidou,
and J. Nielsen. 1998. Metabolic Engineering Principles and
Methodologies. Academic Press, San Diego."

Note: small amount of free amino acids (\<0.1mM) including gln, ala,
asp, and cys and phe in cell lyase can also be detected by GC-MS.

\# Amino acids derived from hydrolysis of total protein as measured at
UC Davis Molecular Structural Facility (see material and methods).

**Table S-3.** Atom mapping matrices.

+-------------+-------------+-------------+-------------+-------------+
| PyrPEP     | PEPPGA     | PGA-\>T3P   | CoA        | C1GLY      |
|             |             |             | Acetate     |             |
| 1 0 0       | 1 0 0       | 1 0 0       |             | 0           |
|             |             |             | 1 0         |             |
| 0 1 0       | 0 1 0       | 0 1 0       |             | 1           |
|             |             |             | 0 1         |             |
| 0 0 1       | 0 0 1       | 0 0 1       |             |             |
+-------------+-------------+-------------+-------------+-------------+
| ICTCO2     | ACoA  MAL  | OXOCO2     | OXOGLU     | OXOSUCC    |
|             |             |             |             |             |
| 0 0 0 0 0 1 | 0 0         | 1 0 0 0 0   | 1 0 0 0 0   | 0 1 0 0 0   |
|             |             |             |             |             |
|             | 0 0         |             | 0 1 0 0 0   | 0 0 1 0 0   |
|             |             |             |             |             |
|             | 0 1         |             | 0 0 1 0 0   | 0 0 0 1 0   |
|             |             |             |             |             |
|             | 1 0         |             | 0 0 0 1 0   | 0 0 0 0 1   |
|             |             |             |             |             |
|             |             |             | 0 0 0 0 1   |             |
+-------------+-------------+-------------+-------------+-------------+
| ASPMET     | ACoA  LEU  | PYR LEU    | C1MET      | C1HIS      |
|             |             |             |             |             |
| 1 0 0 0     | 1 0         | 0 0 0       | 0           | 0           |
|             |             |             |             |             |
| 0 1 0 0     | 0 1         | 0 0 0       | 0           | 0           |
|             |             |             |             |             |
| 0 0 1 0     | 0 0         | 0 1 0       | 0           | 0           |
|             |             |             |             |             |
| 0 0 0 1     | 0 0         | 0 1 0       | 0           | 0           |
|             |             |             |             |             |
| 0 0 0 0     | 0 0         | 0 0 1       | 1           | 0           |
|             |             |             |             |             |
|             | 0 0         | 0 0 1       |             | 1           |
+-------------+-------------+-------------+-------------+-------------+
| E4PTYR     | E4PF6P     | E4PPHE     | E4PS7P     | E4PT3P     |
|             |             |             |             |             |
| 0 0 0 0     | 0 0 0 0     | 0 0 0 0     | 0 0 0 0     | 0 1 0 0     |
|             |             |             |             |             |
| 0 0 0 0     | 0 0 0 0     | 0 0 0 0     | 0 0 0 0     | 0 0 1 0     |
|             |             |             |             |             |
| 0 0 0 0     | 1 0 0 0     | 0 0 0 0     | 0 0 0 0     | 0 0 0 1     |
|             |             |             |             |             |
| 0 0 0 0     | 0 1 0 0     | 0 0 0 0     | 1 0 0 0     |             |
|             |             |             |             |             |
| 0 0 0 0     | 0 0 1 0     | 0 0 0 0     | 0 1 0 0     |             |
|             |             |             |             |             |
| 1 0 0 0     | 0 0 0 1     | 1 0 0 0     | 0 0 1 0     |             |
|             |             |             |             |             |
| 0 1 0 0     |             | 0 1 0 0     | 0 0 0 1     |             |
|             |             |             |             |             |
| 0 0 1 0     |             | 0 0 1 0     |             |             |
|             |             |             |             |             |
| 0 0 0 1     |             | 0 0 0 1     |             |             |
+-------------+-------------+-------------+-------------+-------------+
| CO2OAA     | F6PE4P     | F6PG6P     | F6PS7P     | T3PF6P     |
|             |             |             |             |             |
| 0           | 0 0 1 0 0 0 | 1 0 0 0 0 0 | 1 0 0 0 0 0 | 0 0 1       |
|             |             |             |             |             |
| 0           | 0 0 0 1 0 0 | 0 1 0 0 0 0 | 0 1 0 0 0 0 | 0 1 0       |
|             |             |             |             |             |
| 0           | 0 0 0 0 1 0 | 0 0 1 0 0 0 | 0 0 1 0 0 0 | 1 0 0       |
|             |             |             |             |             |
| 1           | 0 0 0 0 0 1 | 0 0 0 1 0 0 | 0 0 0 0 0 0 | 1 0 0       |
|             |             |             |             |             |
|             |             | 0 0 0 0 1 0 | 0 0 0 0 0 0 | 0 1 0       |
|             |             |             |             |             |
|             |             | 0 0 0 0 0 1 | 0 0 0 0 0 0 | 0 0 1       |
|             |             |             |             |             |
|             |             |             | 0 0 0 0 0 0 |             |
+-------------+-------------+-------------+-------------+-------------+
| F6PC5P     | ICTOXO     | F6PG6P     | G6PC5P     | GLXMAL     |
|             |             |             |             |             |
| 1 0 0 0 0 0 | 1 0 0 0 0 0 | 1 0 0 0 0 0 | 0 1 0 0 0 0 | 1 0         |
|             |             |             |             |             |
| 0 1 0 0 0 0 | 0 1 0 0 0 0 | 0 1 0 0 0 0 | 0 0 1 0 0 0 | 0 1         |
|             |             |             |             |             |
| 0 0 0 0 0 0 | 0 0 1 0 0 0 | 0 0 1 0 0 0 | 0 0 0 1 0 0 | 0 0         |
|             |             |             |             |             |
| 0 0 0 0 0 0 | 0 0 0 1 0 0 | 0 0 0 1 0 0 | 0 0 0 0 1 0 | 0 0         |
|             |             |             |             |             |
| 0 0 0 0 0 0 | 0 0 0 0 1 0 | 0 0 0 0 1 0 | 0 0 0 0 0 1 |             |
|             |             |             |             |             |
|             |             | 0 0 0 0 0 1 |             |             |
+-------------+-------------+-------------+-------------+-------------+
| GLYC1      | GLYCO2     | GLYSER     | PYRCO2     | PYRCoA     |
|             |             |             |             |             |
| 0 1         | 1 0         | 1 0         | 1 0 0       | 0 1 0       |
|             |             |             |             |             |
|             |             | 0 1         |             | 0 0 1       |
|             |             |             |             |             |
|             |             | 0 0         |             |             |
+-------------+-------------+-------------+-------------+-------------+
| C1SER      | CO2 MAL    | PYROAA     | ACoAC1     | ACoACO2    |
|             |             |             |             |             |
| 0           | 0           | 1 0 0       | 1.  1       | 2.  0       |
|             |             |             |             |             |
| 0           | 0           | 0 1 0       | (new        | (new        |
|             |             |             | pathway)    | pathway)    |
| 1           | 0           | 0 0 1       |             |             |
|             |             |             |             |             |
|             | 1           | 0 0 0       |             |             |
+-------------+-------------+-------------+-------------+-------------+

**Table S-3 (Continued)**

+-------------+-------------+-------------+-------------+-------------+
| ICTGLX     | ICTSUC     | M           | MALOAA     | PYRMAL     |
|             |             | AL(OAA)CO2 |             |             |
| 1 0 0 0 0 0 | 0 0 0 0 1 0 |             | 1 0 0 0     | 1 0 0       |
|             |             | 0 0 0 1     |             |             |
| 0 1 0 0 0 0 | 0 0 0 1 0 0 |             | 0 1 0 0     | 0 1 0       |
|             |             |             |             |             |
|             | 0 0 1 0 0 0 |             | 0 0 1 0     | 0 0 1       |
|             |             |             |             |             |
|             | 0 0 0 0 0 1 |             | 0 0 0 1     | 0 0 0       |
+-------------+-------------+-------------+-------------+-------------+
| ACoA  ICT  | ACoA  ICT  | OAA-\>ICT   | OAA-\>ICT   | PYRALA     |
|             |             |             | (*E.coli*)  |             |
| (DvH)       | (*Ecoli*)   | (DvH)       |             | 1 0 0       |
|             |             |             | 0 0 0 1     |             |
| 1 0         | 0 0         | 0 0 0 0     |             | 0 1 0       |
|             |             |             | 0 0 1 0     |             |
| 0 1         | 0 0         | 0 0 0 0     |             | 0 0 1       |
|             |             |             | 0 1 0 0     |             |
| 0 0         | 0 0         | 0 1 0 0     |             |             |
|             |             |             | 0 0 0 0     |             |
| 0 0         | 0 1         | 0 0 1 0     |             |             |
|             |             |             | 0 0 0 0     |             |
| 0 0         | 1 0         | 0 0 0 1     |             |             |
|             |             |             | 1 0 0 0     |             |
| 0 0         | 0 0         | 1 0 0 0     |             |             |
+-------------+-------------+-------------+-------------+-------------+
| OAAASP     | OAACO2     | PEPOAA     | OAAPEP     | PEPPYR     |
|             |             |             |             |             |
| 1 0 0 0     | 0 0 0 1     | 1 0 0       | 1 0 0 0     | 1 0 0       |
|             |             |             |             |             |
| 0 1 0 0     |             | 0 1 0       | 0 1 0 0     | 0 1 0       |
|             |             |             |             |             |
| 0 0 1 0     |             | 0 0 1       | 0 0 1 0     | 0 0 1       |
|             |             |             |             |             |
| 0 0 0 1     |             | 0 0 0       |             |             |
+-------------+-------------+-------------+-------------+-------------+
| C5PF6P     | C5PS7P     | G6PCO2     | PEPTYR     | PEPPHE     |
|             |             |             |             |             |
| 1 0 0 0 0   | 1 0 0 0 0   | 1 0 0 0 0 0 | 1 0 0       | 1 0 0       |
|             |             |             |             |             |
| 0 1 0 0 0   | 0 1 0 0 0   |             | 0 1 0       | 0 1 0       |
|             |             |             |             |             |
| 0 0 0 0 0   | 1 0 0 0 0   |             | 0 0 1       | 0 0 1       |
|             |             |             |             |             |
| 0 0 0 0 0   | 0 1 0 0 0   |             | 0 1 0       | 0 1 0       |
|             |             |             |             |             |
| 0 0 0 0 0   | 0 0 1 0 0   |             | 0 0 1       | 0 0 1       |
|             |             |             |             |             |
| 0 0 0 0 0   | 0 0 0 1 0   |             | 0 0 0       | 0 0 0       |
|             |             |             |             |             |
|             | 0 0 0 0 1   |             | 0 0 0       | 0 0 0       |
|             |             |             |             |             |
|             |             |             | 0 0 0       | 0 0 0       |
|             |             |             |             |             |
|             |             |             | 0 0 0       | 0 0 0       |
+-------------+-------------+-------------+-------------+-------------+
| T3PC5P     | PYRLEU     | PYRLYS     | PYRVAL     | C5PHIS     |
|             |             |             |             |             |
| 0 0 0       | 0 0 0       | 0 0 0       | 1 0 0       | 1 0 0 0 0   |
|             |             |             |             |             |
| 0 0 0       | 0 0 0       | 0 0 0       | 0 1 0       | 0 1 0 0 0   |
|             |             |             |             |             |
| 1 0 0       | 0 1 0       | 0 0 0       | 0 2 0       | 0 0 1 0 0   |
|             |             |             |             |             |
| 0 1 0       | 0 2 0       | 0 0 0       | 0 0 2       | 0 0 0 1 0   |
|             |             |             |             |             |
| 0 0 1       | 0 0 2       | 0 1 0       | 0 0 1       | 0 0 0 0 1   |
|             |             |             |             |             |
|             | 0 0 1       | 0 0 1       |             | 0 0 0 0 0   |
+-------------+-------------+-------------+-------------+-------------+
| C5PS7P     | C5PT3P     | S7PE4P     | S7PF6P     | S7P C5P    |
|             |             |             |             |             |
| 1 0 0 0 0   | 0 0 1 0 0   | 0 0 0 1 0 0 | 1 0 0 0 0 0 | 1 0 1 0 0 0 |
|             |             | 0           | 0           | 0           |
| 0 1 0 0 0   | 0 0 0 1 0   |             |             |             |
|             |             | 0 0 0 0 1 0 | 0 1 0 0 0 0 | 0 1 0 1 0 0 |
| 1 0 0 0 0   | 0 0 0 0 1   | 0           | 0           | 0           |
|             |             |             |             |             |
| 0 1 0 0 0   |             | 0 0 0 0 0 1 | 0 0 1 0 0 0 | 0 0 0 0 1 0 |
|             |             | 0           | 0           | 0           |
| 0 0 1 0 0   |             |             |             |             |
|             |             | 0 0 0 0 0 0 | 0 0 0 0 0 0 | 0 0 0 0 0 1 |
| 0 0 0 1 0   |             | 1           | 0           | 0           |
|             |             |             |             |             |
| 0 0 0 0 1   |             |             | 0 0 0 0 0 0 | 0 0 0 0 0 0 |
|             |             |             | 0           | 1           |
|             |             |             |             |             |
|             |             |             | 0 0 0 0 0 0 |             |
|             |             |             | 0           |             |
+-------------+-------------+-------------+-------------+-------------+
| SERC1      | SERGLY     | SUCMALATE  | T3PE4P     | PGASER     |
|             |             |             |             |             |
| 0 0 1       | 1 0 0       | 1 0 0 0     | 0 0 0       | 1 0 0       |
|             |             |             |             |             |
|             | 0 1 0       | 0 1 0 0     | 1 0 0       | 0 1 0       |
|             |             |             |             |             |
|             |             | 0 0 1 0     | 0 1 0       | 0 0 1       |
|             |             |             |             |             |
|             |             | 0 0 0 1     | 0 0 1       |             |
+-------------+-------------+-------------+-------------+-------------+

**Table S-4. Measured mass distribution by GC-MS and FT-ICR (in
parenthesis) for ^13^C labeled amino acids from *D. vulgaris*
hydrolysates.**

+-----------+--------+--------+--------+--------+---------------------+
|           | M0     | M1     | M2     | M3     | Comments of         |
|           |        |        |        |        | measurement         |
+-----------+--------+--------+--------+--------+---------------------+
| Glycine   | 0.1    | 0.8    | 0.0    |        | FT-ICR signal for   |
|           | 7±0.02 | 2±0.02 | 1±0.01 |        | M0 is not           |
|           |        |        |        |        | identified          |
|           | \(0\)  | \(1\)  |        |        | (background noise). |
+-----------+--------+--------+--------+--------+---------------------+
| Alanine   | 0.1    | 0.8    | 0.0    |        |                     |
|           | 7±0.02 | 1±0.02 | 2±0.01 |        |                     |
|           | (0.17) | (0.81) |        |        |                     |
|           |        |        | (0.02) |        |                     |
+-----------+--------+--------+--------+--------+---------------------+
| Serine    | 0.1    | 0.8    | 0.02±0 |        | FT-ICR signal for   |
|           | 7±0.02 | 1±0.02 |        |        | M2 is not           |
|           | (0.19) | (0.81) |        |        | identified          |
|           |        |        |        |        | (background noise). |
+-----------+--------+--------+--------+--------+---------------------+
| Proline   | ND     | ND     | ND     |        | GC-MS spectrum      |
|           |        |        |        |        | signal is not       |
|           | (0.23) | (0.73) | (0.04) |        | clear.              |
+-----------+--------+--------+--------+--------+---------------------+
| Valine    | 0.1    | 0.8    | 0.02±0 | 0.01±0 |                     |
|           | 7±0.02 | 0±0.03 |        |        |                     |
|           |        | (0.78) | (0.04) | \(0\)  |                     |
|           | (0.18) |        |        |        |                     |
+-----------+--------+--------+--------+--------+---------------------+
| Threonine | 0.0    | 0.2    | 0.6    |        |                     |
|           | 4±0.03 | 8±0.03 | 7±0.04 |        |                     |
|           | (0.05) | (0.31) | (0.64) |        |                     |
+-----------+--------+--------+--------+--------+---------------------+
| Leucine   | 0.7    | 0.2    | 0.0    |        | FT-ICR can not      |
|           | 5±0.07 | 3±0.04 | 2±0.01 |        | separate leucine    |
|           | (0.59) | (0.40) | (0.01) |        | and isoleucine      |
|           |        |        |        |        | signals. The        |
|           |        |        |        |        | measured value is   |
|           |        |        |        |        | the addition of two |
|           |        |        |        |        | amino acids.        |
+-----------+--------+--------+--------+--------+---------------------+
| I         | 0.4    | 0.5    | 0.0    |        |                     |
| soleucine | 7±0.03 | 1±0.02 | 1±0.01 |        |                     |
|           |        |        |        |        |                     |
|           | (ND)   | (ND)   | (ND)   |        |                     |
+-----------+--------+--------+--------+--------+---------------------+
| Asparate  | 0.0    | 0.2    | 0.6    | 0.0    |                     |
|           | 4±0.02 | 9±0.03 | 6±0.04 | 1±0.01 |                     |
|           | (0.06) |        |        |        |                     |
|           |        | (0.30) | (0.65) | \(0\)  |                     |
+-----------+--------+--------+--------+--------+---------------------+
| Lysine    | 0.0    | 0.3    | 0.6    | 0.01±0 | GC peak is weak.    |
|           | 9±0.02 | 0±0.02 | 0±0.03 |        |                     |
|           | (0.10) | (0.32) | (0.58) |        |                     |
+-----------+--------+--------+--------+--------+---------------------+
| Glutamate | 0.2    | 0.7    | 0.0    |        |                     |
|           | 1±0.03 | 3±0.04 | 6±0.03 |        |                     |
|           |        |        |        |        |                     |
|           | (0.20) | (0.69) | (0.10) |        |                     |
+-----------+--------+--------+--------+--------+---------------------+
| M         | 0.0    | 0.2    | 0.6    | 0.02±0 | Peak tailing in     |
| ethionine | 4±0.03 | 7±0.02 | 5±0.03 |        | GC/MS is observed.  |
|           | (0.07) |        |        |        |                     |
|           |        | (0.27) | (0.66) |        |                     |
+-----------+--------+--------+--------+--------+---------------------+
| Histidine | 0.1    | 0.6    | 0.1    | 0.1    |                     |
|           | 6±0.05 | 2±0.08 | 1±0.04 | 1±0.04 |                     |
|           |        |        |        |        |                     |
|           | (0.19) | (0.62) | (0.11) | (0.08) |                     |
+-----------+--------+--------+--------+--------+---------------------+
| Arginine  | ND     | ND     | ND     | ND     | GC could not have a |
|           |        |        |        |        | clear peak for      |
|           | (0.07) | (0.27) | (0.60) | (0.06) | arginine.           |
+-----------+--------+--------+--------+--------+---------------------+
| Phen      | 0.0    | 0.1    | 0.3    | 0.5    |                     |
| ylalanine | 3±0.02 | 2±0.04 | 5±0.07 | 0±0.08 |                     |
|           |        |        |        |        |                     |
|           | (0.05) | (0.07) | (0.36) | (0.54) |                     |
+-----------+--------+--------+--------+--------+---------------------+
| Tyrosine  | 0.0    | 0.0    | 0.3    | 0.5    | GC peak is weak.    |
|           | 2±0.01 | 8±0.05 | 4±0.06 | 2±0.07 |                     |
|           | (0)    | (0.06) | (0.38) | (0.56) |                     |
+-----------+--------+--------+--------+--------+---------------------+

Note: ^13^C-Labeled biomass was sampled in the mid-log phase in the LS4D
medium. Standard deviation is for mass distributions of GC-MS data is
based on two biological replicates. Each biological replicate contained
2 technical replicates. The FT-ICR MS data is based on single
measurements.

**Table S-5.** CAD (MS^2^) spectrum of \[^13^C-Aps+H\]^+^ generated by
ESI.

+---------------------------+------------+----------+----------+-----+
| **Ion structures**        | **Neutral  | **       | **The    | *   |
|                           | Loss**     | Measured | oretical | *Er |
| **(abundance of           |            | m/z**    | m/z**    | ror |
| ^13^C/^12^C)**            |            |          |          | (   |
|                           |            |          |          | ppm |
|                           |            |          |          | )** |
+---------------------------+------------+----------+----------+-----+
| ![](media/image4.wmf)     |            | 13       | 13       | 1.3 |
|                           |            | 5.047964 | 5.048139 |     |
| \[^13^C-Asp + H\]^+^      |            |          |          |     |
+---------------------------+------------+----------+----------+-----+
| ![](media/image5.wmf)     | H~2~O      | 11       | 11       | 1.2 |
|                           |            | 7.037434 | 7.037574 |     |
+---------------------------+------------+----------+----------+-----+
| ![](media/image6.wmf)     | H~2~O + CO | 8        | 8        | 0.8 |
|                           |            | 9.042586 | 9.042660 |     |
| (Ratio = 1.4)             | H~2~O +    |          |          | 0.9 |
|                           | ^13^CO     | 8        | 8        |     |
|                           |            | 8.039228 | 8.039305 |     |
+---------------------------+------------+----------+----------+-----+
| ![](media/image7.wmf)     | CH~2~CO    | 7        | 7        | 0.3 |
|                           |            | 5.026984 | 5.027010 |     |
| (Ratio = 0.4)             | C          |          |          | 0.3 |
|                           | H~2~^13^CO | 7        | 7        |     |
|                           |            | 4.023636 | 4.023655 |     |
+---------------------------+------------+----------+----------+-----+

Note: the fragmentation data indicate that two carboxyl groups of
aspartic acid should be labeled.