Supplementary Materials for

34 Pages·2016·1.3 MB·English

www.sciencemag.org/content/353/6299/583/suppl/DC1

Supplementary Materials for

Metabolic engineering of microbial competitive advantage for industrial
fermentation processes
A. Joe Shaw,* Felix H. Lam, Maureen Hamilton, Andrew Consiglio, Kyle MacEwen,
Elena E. Brevnova, Emily Greenhagen, W. Greg LaTouf, Colin R. South, Hans van
Dijken, Gregory Stephanopoulos

*Corresponding author. Email: [email protected]

Published 5 August 2016, Science 353, 583 (2016)
DOI: 10.1126/science.aaf6159

This PDF file includes:

Materials and Methods
Supplementary Text
Figs. S1 to S11
Tables S1 to S6
Full Reference List
Caption for Database S1

Other Supplementary Material for this manuscript includes the following:
(available at www.sciencemag.org/content/353/6299/583/suppl/DC1)

Database S1
Materials and Methods

Strains and routine culturing

The strains used in this study are listed in Table S5. E. coli strains were routinely cultured
in LB medium with 100 µg/mL ampicillin or 50 µg/mL kanamycin when required to maintain
plasmids. All E. coli cultures were grown aerobically at 37°C. S. cerevisiae, Y. lipolytica, and
K. marxianus were routinely cultured aerobically in YPD medium at 30°C, with 300 µg/mL
hygromycin, 200 µg/mL G418, or 100 µg/mL nourseothricin when required for selection.
Cultures were stored at -80°C in 25% w/v glycerol stock vials.

DNA vector and strain construction

DNA vectors were constructed by assembling PCR-amplified and synthetic DNA segments
via homology based yeast gap repair cloning (26). Phusion DNA polymerase and other DNA
manipulation enzymes were from New England Biolabs, Ipswich, MA. Genes used in this study
are listed in Table S6. S. cerevisiae INVSc1 MATa his3D1 leu2 trp1-289 ura3-52 (Invitrogen,
USA) was used for DNA vector assembly with selection for the presence of the URA3 gene with
synthetic defined minus uracil agar medium. Whole DNA was extracted from S. cerevisiae via
phenol-chloroform and ethanol precipitation or mini-prep (Qiagen, USA), and used to transform
E. coli TOP10 electrocompetent cells (Invitrogen) with selection on LB agar media with 100
µg/mL ampicillin for the presence of the ampR gene. E. coli plasmid DNA was extracted by
mini-prep (Qiagen) and used to transform bacteria or yeast to generate strains used in this study.
The DNA sequences of plasmids and DNA vectors used in this study are given in Data Set S1.
E. coli strains were transformed by electrotransformation (27), and lithium acetate protocols
were used for S. cerevisiae (28) and Y. lipolytica (29). Positive transformation was determined
by selective resistance and either restriction band patterning for plasmid DNA transformation or
colony PCR for integrative DNA transformation.

E. coli melamine assimilation strain construction

A complete melamine assimilation pathway was created in E. coli by assembling individual
steps with evidence of functional activity. Each enzymatic step in the pathway is described
briefly below.
Melamine deaminase: The triA gene from A. citrulli B-12227 (30) was PCR amplified and
cloned in E. coli ATCC 10798 under control of the pNC53 tac promoter (31) and trpT’ rho
dependent terminator (32), resulting in strain NS88. Strain NS88 was evaluated for growth in
MOPS minimal medium with 0.5 mM melamine as sole nitrogen source and found to grow
exponentially in this medium. In comparison, the control strain NS91 harboring the pNC53
plasmid did not grow under the same conditions.
Ammeline deaminase: The native E. coli guanine deaminase (guaD) is reported to
deaminate ammeline (17), and we found E. coli ATCC 10798 grew on 0.5 mM ammeline as sole
nitrogen source in MOPS minimal medium. To assemble the complete melamine assimilation
pathway, the guaD gene was PCR amplified from E. coli ATCC 10798 genomic DNA.
2
Ammelide deaminase: Genes encoding trzC from A. citrulli B-12227 or atzC from pMEL
(33) were cloned in pNC53, transformed into E. coli ATCC 10798, and plated on LB agar
medium with 5 mM ammelide. Ammelide clearing zones after 24 hours of growth were used as
indicators of ammelide deaminase activity. Clearing zones were seen for trzC, but not atzC, E.
coli ATCC 10798 transformants.
Cyanuric acid hydrolase: Genes encoding atzD from Pseudomonas sp. strain ADP (14) and
trzD from Rhodococcus sp. Mel (33) were cloned in pNC53, transformed into E. coli ATCC
10798, and plated on LB agar medium with 10 mM cyanuric acid. Cyanuric acid clearing zones
after 24 hours of growth were used as indicators of ammelide deaminase activity. Strains
expressing either enzyme created cyanuric acid clearing zones, and the atzD from Pseudomonas
sp. strain ADP was chosen for use in the complete melamine assimilation pathway.
Biuret hydrolase and allophanate hydrolyase: A codon optimized gene encoding biuret
hydrolase from Rhodococcus sp. Mel (trzE) (33) and a S. cerevisiae DUR1,2 (34) truncation
gene containing amino acids 1-622 of the allophanate hydrolyase domain were cloned into
pNC53 and transformed into E. coli ATCC 10798. We found these E. coli ATCC 10798
transformants grew on 1 mM biuret as sole nitrogen source in MOPS minimal medium.
To assemble a complete melamine assimilation pathway, the genes identified to function at
separate steps were combined into a single synthetic operon under control of the pNC53 tac
promoter and trpT’ rho dependent terminator. The following inter-gene linkers, with predicted
ribosome binding sites, were used for operon construction: lacZ-lacY (ggaaatccatt), galT-galK
(ggaacgacc), and araB-araA (taaggacacgata). Homology-based yeast gap repair cloning was
used to create the complete operon (plasmid pNC121), and a low-copy version of the operon was
created by PacI/AscI restriction digest of pNC121 and purification of the resulting 7860 bp DNA
fragment and cloning into BsaXI digested pACYC177, resulting in plasmid pNC153 with
replacement of the pACYC177 kanR gene with the melamine assimilation pathway.

S. cerevisiae cyanamide and phosphite utilization strain construction

S. cerevisiae was engineered for utilization of cyanamide by transformation with a
AscI/NotI restriction digested pNC286 vector encoding the A. niger cyanamide hydratase (CAH)
gene homolog under control of the S. cerevisiae TEF1 promoter and S. cerevisiae CYC1
terminator into the Ethanol Red strain, with selection for transformation by hygromycin
resistance. The transformed DNA retained the yeast 2-micron origin of replication. One colony
was isolated, confirmed by colony PCR for the presence of the A. niger CAH gene, and
designated NS379. In order to select for improved growth at higher concentrations of
cyanamide, NS379 was serial transferred in defined Verduyn medium with 5 mM cyanamide as
sole nitrogen source at 30°C in an aerobic drum roller with 14 mL culture tubes. Transfers were
performed by inoculating 4 mL fresh medium with 40 µL of stationary phase cells from the
previous transfer. Six transfers were performed, for an approximate total of 40 generations under
selection. The first two transferred cultures required 48 to 72 hours to reach stationary phase, the
subsequent 4 transfers reached stationary phase after 24 hours. Culture from the final transfer
was struck to isolated colonies on Verduyn agar plates with 5 mM cyanamide as nitrogen source.
Twenty seven isolates were evaluated for growth with 5 mM cyanamide or 5 mM urea as
nitrogen source, and one isolate exhibiting fast growth on both nitrogen sources was stored and
designated NS532.
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To engineer phosphite utilization, pNC360, a vector containing the Pseudomonas stutzeri
ptxD gene under control of the S. cerevisiae TEF1 promoter and S. cerevisiae CYC1 terminator,
was restriction digested with AscI/NotI and transformed into NS532. The transformed DNA
retained the yeast 2-micron origin of replication. Selection for transformation was performed by
plating cells on defined Verduyn agar medium with 1 mM potassium phosphite as sole
phosphorus source. Transformants were re-patched to agar phosphite medium and tested for the
presence of the ptxD gene by colony PCR. One isolate able to grow on phosphite medium and
positive for the ptxD gene by colony PCR was stored and designated NS558. To improve
phosphite growth characteristics, NS558 was serial transferred in defined Verduyn medium with
1 mM potassium phosphite as sole phosphorus source. Transfers were performed by inoculating
4 mL fresh medium with 40 µL of stationary phase cells from the previous transfer. Nine
transfers were performed, for an approximate total of 60 generations under selection. The first
two transferred cultures required 72 hours to reach stationary phase, the subsequent 7 transfers
reached stationary phase after 24 hours. Culture from the final transfer was struck to isolated
colonies on Verduyn agar plates with 1 mM potassium phosphite as phosphorus source. Twenty
isolates were evaluated for growth with 1 mM potassium phosphite or 1 mM potassium
phosphate as phosphorus source, and one isolate exhibiting fast growth on both phosphorus
sources was stored and designated NS586.

Y. lipolytica phosphite utilization strain construction

Wildtype Y. lipolytica was engineered for utilization of phosphite by transformation with a
PmeI restriction digested pNC273 vector containing the Pseudomonas stutzeri ptxD gene under
control of the Y. lipolytica TEF1 promoter and Y. lipolytica CYC1 terminator, with selection for
random (non-homology targeted) chromosomal integration by hygromycin resistance. One
colony was isolated, confirmed by colony PCR for the presence of the ptxD gene, and designated
NS324.
To engineer phosphite utilization in a lipid overproducing strain, the same protocol was
followed as above with a Y. lipolytica strain previously engineered for lipid production via
overexpression of the endogenous Y. lipolytica diacylglycerol acyltransferases DGA1 and DGA2
genes (35) under control of the Y. lipolytica TEF1 promoter and Y. lipolytica CYC1 terminator.
After selection on defined Verduyn agar medium with 1 mM phosphite as sole phosphorus
source, one colony was isolated, confirmed by colony PCR for the presence of the ptxD gene,
and designated NS392.

Cyanamide hydratase activity assay

Cell free extracts for NS379 and wildtype Ethanol Red S. cerevisiae were prepared by
growing cells to an optical density of 1.0 in 50 mL YPD medium, washing twice in an equal
volume of distilled water, and resuspending the wet cell pellet in Y-PER Yeast Protein
Extraction Reagent (Thermo Scientific, Rockford IL) with 250 µL reagent and 100 mg of 0.5
mm acid washed glass beads per 100 mg wet pellet weight. Cells were vortexed vigorously for
20 minutes in 2 mL centrifuge tubes, then centrifuged at 14,000 x g for 10 minutes. Cell free
supernatant was collected and protein concentrations were measured via Bradford assay with
BSA protein standard.

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Cyanamide hydratase activity was measured by monitoring the conversion of NADPH to
NADP+ at 340 nm via a coupled enzymatic reaction for detection of urea and ammonia via
Megazyme test kit K-URAMR (Megazyme, Bray, Ireland). The reaction assay (1 mL)
contained 20 mM cyanamide, 14-24 µg cell free extract, and test kit components. The assay was
measured at room temperature (22-23°C) and pH 8. Baseline conversion of NADPH to NADP+
in the absence of cell free extract was subtracted from cell free extract measurements.
Cyanamide enzymatic activity was reported as μmol NADPH formed per min-1 mg-1 protein.

Defined medium growth experiments

Chemicals, unless otherwise specified, were purchased from Sigma Aldrich or Fisher
Scientific. Cyanamide (catalog # 181950250) was purchased from Acros Organics. Melamine
(catalog # M2659), cyanuric acid (catalog # 185809), and biuret (catalog # 15270) were
purchased from Sigma Aldrich, St. Louis, MO. Ammeline (catalog # A0676) and ammelide
(catalog # A0645) were purchased from TCI America. Potassium phosphite (Green-T phosphite
30) was purchased from Plant Food Company Inc., Cranbury, NJ.
For growth yield, evolutionary selection, and competition experiments, E. coli was grown in
MOPS medium (36) with 2 g/L glucose and nitrogen sources as indicated in the text and figures.
For evolutionary selection of improved growth on melamine, 0.5 mM melamine was used as
nitrogen source. Cultures were serially transferred by inoculating 5 µL volumes into fresh 5 mL
medium in 14 mL aerobic tubes at 37°C. This was repeated for ten transfers for a total selection
of approximately 100 generations before a single colony was isolated and designated NS163. To
measure growth cells were cultured in 96-well plates with 100 µL media per well, and inoculated
with 2.5 µL of nitrogen limited (3 mM total ammonium equivalent) pre-culture grown overnight
at 37°C. Growth was monitored in a plate reader (Bio-Tek Instruments, Inc., Winooski, VT)
with temperature control at 37°C and 45 seconds of vigorous agitation every 15 minutes. To
measure melamine consumption in strain NS163, a 500 mL shake flask (150 mL liquid volume)
was grown at 37°C and 200 rpm. Ten mL samples were taken and filter sterilized to remove
cells from culture liquid. Melamine, ammeline, ammelide, and cyanuric acid were quantified by
LC-MS-MS following FDA method LIB 4421 (Eurofins Analytical Laboratories Inc., New
Orleans LA). Ammonium ion was quantified by a glutamate dehydrogenase based enzymatic
assay (Megazyme, Bray, Ireland) following the manufacturer’s protocols.
S. cerevisiae was grown in defined medium (Verduyn) with 20 g/L glucose and nitrogen
and phosphorus sources as specified in the text and figures. Growth experiments were monitored
in 96 well plates with 150 µL media per well in a plate reader (Bio-Tek Instruments, Inc.,
Winooski, VT) with temperature control at 30°C and one minute vigorous agitation every 30
minutes. S. cerevisiae fermentations measuring dry cell weight and ethanol production were
performed in initially aerobic, sealed 16 mL Hungate tubes with 4 mL liquid media volume. Dry
cell weight was determined by weighing 3 mL of water washed cell culture. Glucose, ethanol,
and glycerol were quantified by HPLC with an Aminex HPX-87H column.
To measure growth and lipid production, Y. lipolytica was grown in 5 L bioreactors with 60
g/L glucose as carbon source, 1.5 g/L (NH ) SO , 0.1 g/L corn peptone (Amberferm 4500,
4 2 4
Sensient), either 4 g/L potassium phosphate or 3.53 g/L potassium phosphite (equivalent to 29.4
mM P each), 12 mg/L thiamine, 160 mg/L Na MoO ·2H O, 0.2 mg/L CuSO ·5H O, 40 mg/L
2 4 2 4 2
H BO , 180 mg/L MnSO ·H O, 75 mg/L MnSO ·H O, 2 g/L MgSO ·7H O, 0.8 g/L
3 3 4 2 4 2 4 2
CaCl ·6H O, 0.4 g/L NaCl, 1.0 mL/L Antifoam 204 (Sigma Aldrich), 1 mg/L Biotin. Pre-
2 2
5
cultures grown for 24 hours in aerobic shake flasks (200 rpm, 30°C) in defined medium with 20
g/L glucose were inoculated at 2% for Y. lipolytica (NS392). Dry cell weight was determined
by weighing 1 mL of water washed cell culture. Glucose and citric acid were quantified by
HPLC with an Aminex HPX-87H column. Lipids (reported as fatty acid methyl ester (FAME)
equivalents) were quantified via acid-catalyzed in-situ transesterification (23). The sum of 16:0,
16:1, 18:0, 18:1, and 18:2 FAMEs were measured via GC with a flame ionization detector.

Glucose defined medium competition experiments

E. coli competition experiments were performed aerobically at 37°C in 250 mL shake flasks
with 25 mL volume with MOPS medium containing 100 µg/mL ampicillin to maintain plasmids
and either 2 mM melamine or 12 mM NH Cl. Strains NS102 and NS163 were inoculated from
4
overnight cultures grown in LB medium with 100 µg/mL ampicillin. Inocula were washed twice
with nitrogen free MOPS medium and loaded at an initial optical density of 0.1 for each culture.
Colony forming units were counted by dilution plating on agar LB media with either 100 µg/mL
ampicillin (to count total plasmid-harboring E. coli CFU’s) or 100 µg/mL ampicillin and 50
µg/mL kanamycin (to count E. coli CFUs harboring the pACYC177 plasmid). The number of E.
coli CFUs harboring plasmid pNC153 was determined by subtracting the total plasmid harboring
CFUs from pACYC177 harboring CFUs.
For yeast experiments, defined medium (Verduyn) was used as a base formulation with 20
g/L glucose. For competition with S. cerevisiae NS586 and NS891, either 5 mM urea or 5 mM
cyanamide was supplied as nitrogen source. For competition with Y. lipolytica NS324 and
NS535 either 2 mM potassium phosphate or 2 mM potassium phosphite was supplied as
phosphorus source. Colony forming units were measured by plating serial dilutions on YPD
agar plates with antibiotics added for enumeration of specific strains. Plates were incubated at
30°C, and contained 300 µg/mL hygromycin for enumeration of S. cerevisiae NS586 and Y.
lipolytica NS324, with 200 µg/mL G418 for enumeration of S. cerevisiae NS891, and 100
µg/mL nourseothricin for enumeration of Y. lipolytica NS535.

Industrial feedstock competition experiments

Sugarcane juice. Concentrated sugar cane juice was purchased from Florida Sugar and
stored under sterile conditions at room temperature until use. Total phosphorus content was
measured by inductively coupled plasma atomic emission spectrometry following the ASTM
D1976 protocol. Total sugar content was measured by HPLC after acid hydrolysis of sucrose.
Sugar cane juice was diluted to 20 g/L total sugars, and fermentations were supplemented with
defined medium base (37) without carbon, nitrogen, or phosphorus, and additionally 2 g/L urea
and either 4 mM potassium phosphate or 4 mM potassium phosphite. Pre-cultures for K.
marxianus and S. cerevisiae strain NS586 were grown in defined medium base with 20 g/L
glucose and 2 mM potassium phosphate (K. marxianus) or 2 mM potassium phosphite (NS586).
Pre-cultures were centrifuged and washed twice with sterile water prior to inoculation at the
levels indicated in the text. Fermentations were performed aerobically at 30°C in 250 mL shake
flasks at 50 mL volume with 200 rpm agitation. Colony forming units were determined in the
same manner as defined medium competition experiments, with YPD plates incubated at 45°C
for enumeration of K. marxianus, and with 300 µg/mL hygromycin for enumeration of S.
cerevisiae NS586.
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Wheat straw lignocellulosic hydrolysate. Starter cultures of yeast strains S. cerevisiae
NS586 and K. marxianus CBS 6556 were grown at 30°C with agitation until saturation in a
defined mineral medium (DM) containing 2% (wt/vol) glucose (38). Cells were pelleted by
centrifugation, washed twice in 3x volumes of water to remove residual sources of nitrogen, and
the optical density of a final 0.5x volume re-suspension determined at 600 nm (OD ). Using
600
cell density equivalents of 2.7x107 cells·mL-1·OD -1 for NS586 and 3.4x107 cells·mL-1·OD -1
600 600
for NS595 (pre-determined by cell counts in a hemacytometer), a stock mixture of the two strains
in a 10:1 (S. cerevisiae:K. marxianus) ratio was prepared, its OD determined, and co-cultures
600
inoculated with a dilution yielding OD = 0.05.
600
Growth media containing lignocellulosic sugars were prepared by formulating DM
without glucose and ammonium sulfate. In place of pure glucose, a biomass hydrolysate derived
from the Proesa™ high temperature and pressure pre-treatment of wheat straw (gift of Beta
Renewables) was added to 38% (vol/vol), an amount that was pre-determined to produce a final
concentration of 2% glucose. In place of ammonium sulfate, either urea or cyanamide, both
freshly prepared, was added at a concentration of 5 mM. All media were adjusted to pH 5.6
using potassium hydroxide and 0.2 µm filter sterilized.
Co-cultures were performed using 25 mL of medium in 250 mL shake flasks maintained
at 30°C with 200 rpm agitation. At each time point, 10 µL of a 1-1 to 40-1 dilution (depending on
growth progress) was analyzed in triplicate by hemacytometer for quantification of total cell
density. To determine the population fraction attributable to NS586 vs. NS595, equal volumes
of an appropriate dilution of co-culture were plated in triplicate on solid YPD medium (39) and
YPD containing 300 µg/mL hygromycin. Following incubation at 30°C for 1–2 d, colonies were
quantified and the ratio of hygromycin resistant colonies to those on (selection free) YPD was
multiplied by the total cell density to determine the portion belonging to NS586 (the remaining
being NS595). Error bars were derived using standard rules of uncertainty propagation of the
standard deviations of the raw measurements. The accuracy of this approach requires that all
cells enumerated in the total cell density measurement are viable; thus, we assessed total cell
vitality in the co-culture by methylene blue staining (1 mg/mL) and verified by microscopy that
>99% of the population was viable at all points throughout the course of growth (data not
shown).
To monitor the consumption of nutrients and production of metabolic byproducts, 0.5 mL
of co-culture was harvested at each time point by centrifugation and the supernatant subjected to
HPLC analysis. Concentrations of glucose, and ethanol were quantified on an Agilent 1200
Series HPLC equipped with an Agilent 1260 Infinity Refractive Index Detector (#G1362A RID)
and Aminex HPX-87H Ion Exclusion Column (Bio-Rad). Analytes were eluted at 0.6 mL/min
using 5 mM sulfuric acid, and column and detector temperatures at 35°C.
Fractionated corn. Dry milled, fractionated corn endosperm starch was produced at the
National Corn to Ethanol Research Center (NCERC) pilot facility with corn fractionation
equipment developed by Cereal Process Technologies (CPT, Overland Park, KS). Endosperm
starch was mashed using standard techniques (40) with addition of 120 μL/L alpha-amylase
(Termamyl SC, Novozymes). Mash was stored frozen with the addition of 30 µg/mL penicillin
G until use. To begin simultaneous saccharification and fermentation (SSF) reactions, 180 μL of
glucoamylase (Saczyme, Novozymes) was added to 1 L mash loaded at 22% w/v solids in
aerobically sparged (0.3 vvm, 1000 rpm) bioreactors at 30°C. pH was controlled at 3.5 with
addition of 10 N NaOH. Medium for fractionated corn fermentations included 1.5 g/L
(NH ) SO , 0.1 g/L corn peptone (Amberferm 4500, Sensient), either 4 g/L potassium phosphate
4 2 4
7
or 3.53 g/L potassium phosphite (equivalent to 29.4 mM P each), 12 mg/L thiamine, 160 mg/L
Na MoO ·2H O, 0.2 mg/L CuSO ·5H O, 40 mg/L H BO , 180 mg/L MnSO ·H O, 75 mg/L
2 4 2 4 2 3 3 4 2
MnSO ·H O, 2 g/L MgSO ·7H O, 0.8 g/L CaCl ·6H O, 0.4 g/L NaCl, 1.0 mL/L Antifoam 204
4 2 4 2 2 2
(Sigma Aldrich), 1 mg/L Biotin. Pre-cultures were grown for 24 hours in aerobic shake flasks
(200 rpm, 30°C) in defined medium with 20 g/L glucose and were inoculated at 2% for Y.
lipolytica (NS392) and at 0.2% v/v for S. cerevisiae Ethanol Red. Glucose and ethanol
concentrations were determined via HPLC with an Aminex HPX-87H Ion Exclusion Column.
Colony forming units were determined in the same manner as defined medium competition
experiments, with dilutions on YPD plates incubated at 30°C. S. cerevisiae Ethanol Red and Y.
lipolytica NS392 were distinguished by colony morphology; additionally Y. lipolytica NS392
CFUs were tracked by plating on YPD plates with 300 µg/mL hygromycin. Lipids (reported as
fatty acid methyl ester (FAME) equivalents) were quantified via acid-catalyzed in-situ
transesterification (23). The sum of 16:0, 16:1, 18:0, 18:1, and 18:2 FAMEs were measured via
GC with a flame ionization detector.

Supplementary Text
S. cerevisiae and Y. lipolytica strain engineering for cyanamide and phosphite utilization

To construct cyanamide and phosphite utilizing yeast strains, we first expressed a
cyanamide hydratase (CAH) gene homolog from Aspergillus niger in S. cerevisiae, resulting in
strain NS379. A cyanamide hydratase activity of 1.1 ± 0.5 μmol min-1 mg-1 protein was
measured in cell free extracts of NS379 vs. below 0.1 μmol min-1 mg-1 protein in the wildtype
strain. The CAH expressing S. cerevisiae strain grew well at low concentrations of cyanamide
but was inhibited at ≥ 5 mM. We performed serial transfers in defined medium at increasing
concentrations of cyanamide as sole nitrogen source for approximately 100 generations, resulting
in an intermediate isolate NS532 (Figure S7) which exhibited reduced lag time in cyanamide
concentrations up to 10 mM.
Cyanamide slowly degrades to urea in the presence of water (41), and is inefficiently
degraded to urea by the enzyme urease (jack bean urease has a specificity constant of 43 s-1M-1
for cyanamide and 1.2x106 s-1M-1 for urea) (42). For chemical storage and transport, stabilized
formulations of cyanamide can be stored at ambient temperatures for 90 days or longer (43). The
primary degradation product of cyanamide is urea (43), although it has been reported to degrade
at a minor rate to cyanide in the obligate presence of hydrogen peroxide and the enzyme catalase
(44).
NS532 was then transformed with the ptxD gene from Pseudomonas stutzeri. ptxD has
been shown to enable growth on phosphite for plants (45) and the yeasts Schizosaccharomyces
pombe and S. cerevisiae (46), although in the latter, phosphite growth was markedly slower than
phosphate growth. We found that direct engineering of the ptxD gene in S. cerevisiae (strain
NS558) only enabled slow growth on phosphite. To improve growth, we performed phosphite
limited serial batch transfers with strain NS558. After approximately 100 generations of
phosphite limited selection, we isolated several strains with improved growth rates, and one
strain engineered for both cyanamide and phosphite use, designated NS586, was used for further
studies.
NS586 grew on cyanamide and phosphite at rates comparable to that of wildtype S.
cerevisiae Ethanol Red on urea and phosphate (Figure S7). In initially aerobic, air sealed
8
fermentation, the engineered strain achieved comparable dry cell weights on cyanamide,
phosphite, and the combination of both as to the wildtype strain on urea and phosphate. Ethanol
yields were also high, although more glycerol was produced when cyanamide was supplied as
nitrogen source.
To engineer the oleaginous yeast Y. lipolytica, we transformed both a wildtype strain
(NS18) and a lipid overproducing strain expressing additional copies of the Y. lipolytica
diacylglycerol acyltransferase DGA1 and DGA2 genes (NS184) with the P. stutzeri ptxD gene,
resulting in strains NS324 and NS392. We detected no noticeable difference in growth rate or
lipid production rate when these strains were grown on phosphite or phosphate as phosphorus
source. Similarly, the lipid production performance of NS324 grown on phosphite is comparable
to the parental NS184 strain grown on phosphate (Figure S8).

9
Fig. S1.

Steps of the engineered melamine degradation pathway, with enzyme commission (EC)
numbers and gene donor organisms. The first three deamination steps release one ammonia
molecule each, followed by triazine ring breakage, which has been reported to occur via a
carboxybiuret reactive intermediate. The final two steps degrade biuret to ammonia and carbon
dioxide via a codon-optimized biuret hydrolase from Rhodococcus and the allophanate hydrolase
domain of the S. cerevisiae DUR1,2 urea carboxylase. The entire pathway was constructed as a
synthetic gene operon under control of the E. coli tac promoter.

10