Modular and selective biosynthesis of gasoline-range alkanes Supplementary Information

B Titers of C8 and C10 fatty acids observed in culture supernatant upon expression of FatB2 variants... An increased concentration of butyraldehyde was observed in the gas phase using RA

Modular and selective biosynthesis of gasoline-range alkanes: Supplementary Information

Micah J Sheppard1, 2†, Aditya M Kunjapur1, 3†, Kristala L J Prather1, 3*

1 Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

2 Present Address: Provivi, Inc., 1701 Colorado Avenue, Santa Monica, CA 90404, USA

3 Synthetic Biology Engineering Research Center (SynBERC), Massachusetts Institute of Technology, Cambridge, MA 02139, USA

†These authors contributed equally to this work

* Corresponding author:

Department of Chemical Engineering

77 Massachusetts Avenue

Room E17-504G

Cambridge, MA 02139

Phone: 617.253.1950

Fax: 617.258.5042

Email: kljp@mit.edu

SI Figures

Figure S1 Increased soluble expression of FatB2 using truncated variants (A) Anti-His western blot showing expression resulting from four FatB2 variants Increased soluble expression is observed from

FatB2m2 (B) Titers of C8 and C10 fatty acids observed in culture supernatant upon expression of FatB2 variants.

2

Figure S2 GC-FID traces of alkane standards and representative experiments (A) Overlay of GC traces

of an AIRGAS one-point calibration standard mix, n-heptane, n-nonane, and octanal The commercial one-point calibration contained propane, n-butane, and n-pentane, as well as other compounds For

compounds not included in the commercial one-point calibration mix, known volumes were added to a 1.127 L septum capped bottle at 4°C and was then warmed to room temperature After evaporation occurred (< 1 min), 8 ml of gas was sampled from the bottle using a gas-tight syringe and injected into the GC Linear regressions were completed using a (0, 0) intercept (B) Overlay of GC traces from representative experiments

Figure S3 GC-FID traces focused on each alkane synthesized in biological triplicate (A) Production of heptane (B) Production of pentane (C) Production of butane (D) Production of propane

4

Figure S4 Intermediate and byproduct profiles associated with propane synthesis (A) Relative butyraldehyde concentrations in the headspace of cultures containing Modules 2-BC and 4-SA An increased concentration of butyraldehyde was observed in the gas phase using RARE (B) Liquid-phase concentrations of butyraldehyde and butanol in cultures containing Modules 2-BC and 4-SA Increased levels of butyraldehyde and decreased levels of butanol were observed in the liquid phase using RARE

Figure S5 GC-FID traces of isopentane synthesis in biological triplicate and isopentane commercial standard

6

SI Tables

Table S1 Performance and separation metrics for select gasoline alternatives and constituents

Compound

Enthalpy of Combustion (kJ/mol) Ref.

Research Octane Number

Solubility at Room Temperature (mass %) Ref.

Henry's Law Constant k H

(kPa m 3 mol -1 ) Ref.

(Burgess,

(Jin et al.,

(Burgess,

(Morley,

(Gevantman,

(Mackay and Shiu, 1981)

(Burgess,

(Morley,

(Mackay et al., 1993)

Table S2 Strains and modules used in this study.

8

Table S3 Oligonucleotides used in this study.

SI Text:

Codon optimized FATB2 Ch gene sequence:

ATGGTGGCTGCAGCCGCGTCTTCAGCCTTTTTCCCAGTCCCGGCTCCTGGTGCAAGCCCAAAACCGGGTAAATT TGGCAATTGGCCTAGCAGTCTGAGCCCTAGTTTTAAACCAAAATCTATTCCGAACGGTGGCTTCCAAGTTAAAGC CAATGATTCAGCGCATCCAAAAGCTAACGGTTCTGCAGTGTCATTGAAATCCGGCTCTCTGAACACACAAGAAG ATACGTCCTCTTCACCACCGCCTCGCACCTTTCTGCATCAGCTGCCGGATTGGTCACGTCTGTTAACAGCTATCAC CACTGTCTTCGTTAAATCCAAACGCCCGGATATGCACGATCGTAAATCTAAAAGACCTGATATGCTGGTTGATTCC TTTGGTTTAGAATCTACGGTGCAAGATGGCTTAGTCTTTCGCCAGTCATTCAGCATCCGTTCTTATGAAATTGGTA CAGATAGAACGGCAAGCATCGAAACACTGATGAACCATTTGCAAGAAACGAGTCTGAACCACTGTAAATCCACC GGCATCTTGCTGGATGGTTTTGGCAGAACCTTGGAAATGTGCAAACGCGATCTGATTTGGGTTGTGATCAAAATG CAGATTAAAGTCAATCGTTACCCGGCCTGGGGTGATACCGTTGAAATTAACACTAGATTCTCTCGCCTGGGCAAA ATCGGTATGGGCAGAGATTGGTTAATTAGCGATTGTAATACTGGTGAAATCTTGGTGCGCGCGACAAGTGCTTAT GCAATGATGAACCAAAAAACTCGTAGATTATCCAAATTGCCATACGAAGTTCATCAGGAAATTGTCCCTCTGTTT GTTGATTCTCCAGTGATCGAAGATTCAGATTTAAAGGTTCACAAGTTCAAGGTGAAGACGGGTGATTCTATTCAA AAAGGTTTAACCCCAGGCTGGAATGATTTGGATGTCAACCAGCATGTTAGTAACGTGAAGTACATCGGTTGGATT CTGGAATCCATGCCGACAGAAGTTTTAGAAACGCAGGAATTGTGTTCACTGGCTTTAGAATACCGCCGTGAATGC GGTCGTGATAGCGTCTTGGAAAGTGTTACAGCTATGGACCCAAGCAAAGTGGGCGTCCGTAGTCAATATCAGCA CTTATTGAGACTGGAAGATGGTACTGCCATTGTGAATGGCGCGACTGAATGGAGACCTAAAAATGCCGGTGCGA ACGGCGCTATCTCAACCGGTAAAACTAGCAATGGCAACAGTGTTTCCTAA

Codon optimized ADPm gene sequence: (AD_A134FPm has TTT in place of underlined codon)

ATGCCGACCCTGGAAATGCCGGTTGCAGCAGTTCTGGATAGCACCGTTGGTAGCAGCGAAGCACTGCCGGATTT TACCAGCGATCGTTATAAAGATGCATATAGCCGTATTAACGCCATTGTGATTGAAGGTGAACAAGAAGCACACGA TAACTATATTGCAATTGGCACCCTGCTGCCGGATCATGTTGAAGAACTGAAACGTCTGGCAAAAATGGAAATGCG CCATAAAAAAGGTTTTACCGCCTGTGGTAAAAATCTGGGTGTTGAAGCAGATATGGATTTTGCCCGTGAATTTTT TGCACCGCTGCGTGATAATTTTCAGACCGCACTGGGTCAGGGTAAAACCCCGACCTGTCTGCTGATTCAGGCAC TGCTGATTGAAGCATTTGCAATTAGCGCATATCATACCTATATTCCGGTTAGCGATCCGTTTGCACGTAAAATTACC

10

GAAGGTGTTGTGAAAGATGAATACACCCATCTGAATTATGGTGAAGCATGGCTGAAAGCAAATCTGGAAAGCTG TCGTGAGGAACTGCTGGAAGCCAATCGTGAAAATCTGCCGCTGATTCGTCGTATGCTGGATCAGGTTGCCGGTG ATGCAGCCGTGCTGCAGATGGATAAAGAAGATCTGATCGAAGATTTCCTGATCGCCTATCAAGAAAGCCTGACC GAAATTGGTTTTAACACCCGTGAAATTACCCGTATGGCAGCAGCAGCACTGGTTAGCTAA

Codon optimized ADNp gene sequence: (AD_A122FNp has TTT in place of underlined codon)

ATGCAGCAGCTGACCGATCAGAGCAAAGAACTGGATTTCAAAAGCGAAACCTATAAAGATGCCTATAGCCGCAT TAACGCCATTGTTATTGAAGGTGAACAAGAAGCCCACGAGAACTATATTACCCTGGCACAACTGCTGCCGGAAA GCCATGATGAACTGATTCGTCTGAGCAAAATGGAAAGCCGTCATAAAAAAGGTTTTGAAGCCTGTGGTCGTAAT CTGGCAGTTACACCGGATCTGCAGTTTGCAAAAGAATTTTTCAGCGGTCTGCATCAGAATTTTCAGACCGCAGC AGCAGAAGGTAAAGTTGTTACCTGCCTGCTGATTCAGAGCCTGATTATTGAATGTTTTGCCATTGCAGCCTACAA CATTTATATCCCGGTTGCAGATGATTTCGCACGCAAAATTACCGAAGGTGTTGTGAAAGAAGAGTATAGCCATCT GAATTTTGGTGAGGTTTGGCTGAAAGAACATTTTGCAGAAAGCAAAGCAGAACTGGAACTGGCAAATCGTCAG AATCTGCCGATTGTTTGGAAAATGCTGAATCAGGTGGAAGGTGATGCACATACCATGGCAATGGAAAAAGATGC ACTGGTGGAAGATTTCATGATTCAGTATGGTGAAGCCCTGAGCAATATTGGTTTTAGCACCCGTGATATTATGCGT CTGAGCGCCTATGGTCTGATTGGTGCATAA

Alkane quantification Gas phase alkane titers were calculated as follows: The gas standard was used

to find the gas phase concentration in ppm As described in methods, this concentration corresponded to

a 2-fold dilution of the original culture head space Multiplication of the gas phase concentration by head-space volume (found by measuring the mass of water used to fill the vial and subtracting the 2 mL culture volume) results in the total mass of alkane in the head-space Division of the total mass of alkane by the culture volume gives the reported titer

6

1 10 mol of product 1 mol of gas 1 L of gas g product 1000 mg of product

1 mol of gas 25.45 L 1000 ml of gas 1 mol product 1 g of product

10 mg of product 25.45 ml of gas 1 ppm of produ

product

product

MW MW









6

3

ct

25.45 ml of gas 1 ppm of product 2 ml of culture 1 L culture

10 mg of product 2.83 L of culture 1 pp

product

product

MW

MW









3

m of product

10 ppm of product standard mg of product FID area of product

FID area of product standard 2.83 L of culture 1 ppm of product

mg of product product titer

L of culture

product MW x

y





Heptane standard concentration calculation The exact volume of 1 liter glass bottle with septum cap

was found by weighing with water The intended gas phase concentration was then calculated as follows:

heptane

heptane 6 bottle

1.1273 L 1 atm 1 mmol

mmol of gas in bottle

0.0821 277.15 K

mol K mmol

10 conc of heptane p mmol

MW



 



12

Supplementary References:

1982 Solubility Data Series, International Union of Pure and Applied Chemistry Pergamon Press, Oxford 1988a Solubility Data Series, International Union of Pure and Applied Chemistry Pergamon Press, Oxford 1988b Solubility Data Series, International Union of Pure and Applied Chemistry Pergamon Press, Oxford.

1996 Soil Screening Guidance, US Environmental Protection Agency In: Agency, U E P., (Ed.) Office of Solid

Waste and Emergency Response, Washington, DC.

Burgess, D R., 2009 Thermochemical Data NIST Chemistry WebBook, NIST Standard Reference Database

Number 69, Eds P.J Linstrom and W.G Mallard National Institute of Standards and Technology, Gaithersburg, MD.

Gevantman, L., 1996 CRC Handbook of Chemistry and Physics CRC Press, Boca Raton, FL.

Hunwartzen, I., 1982 Modification of CFR Test Engine Unit to Determine Octane Numbers of Pure Alcohols and

Gasoline-Alcohol Blends SAE Technical Paper 820002.

Jin, C., Yao, M., Liu, H., Lee, C.-f F., Ji, J., 2011 Progress in the production and application of n-butanol as a

biofuel Renewable and Sustainable Energy Reviews 15, 4080-4106.

Mackay, D., Shiu, W Y., 1981 A Critical Review of Henry's Law Constants for Chemicals of Environmental

Interest J Phys Chem Ref Data 10, 1175-1199.

Mackay, D., Shiu, W Y., Ma, K C., 1993 Illustrated Handbook of Physical-Chemical Properties and

Environmental Fate for Organic Chemicals Lewis Publishers/CRC Press, Boca Raton, FL.

Morley, C., 1987 A Fundamentally Based Correlation Between Alkane Structure and Octane Number

Combustion Science and Technology 55, 115-123.