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: jshaw@novogyinc.com

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

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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.

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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 (NH4)2SO4, 0.1 g/L corn peptone (Amberferm 4500, 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 Na2MoO4·2H2O, 0.2 mg/L CuSO4·5H2O, 40 mg/L H3BO3, 180 mg/L MnSO4·H2O, 75 mg/L MnSO4·H2O, 2 g/L MgSO4·7H2O, 0.8 g/L CaCl2·6H2O, 0.4 g/L NaCl, 1.0 mL/L Antifoam 204 (Sigma Aldrich), 1 mg/L Biotin. Pre-

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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 NH4Cl. Strains NS102 and NS163 were inoculated from 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 (OD600). Using cell density equivalents of 2.7x107 cells·mL-1·OD600

-1 for NS586 and 3.4x107 cells·mL-1·OD600-1

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 OD600 determined, and co-cultures inoculated with a dilution yielding OD600 = 0.05.

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 (NH4)2SO4, 0.1 g/L corn peptone (Amberferm 4500, Sensient), either 4 g/L potassium phosphate

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or 3.53 g/L potassium phosphite (equivalent to 29.4 mM P each), 12 mg/L thiamine, 160 mg/L Na2MoO4·2H2O, 0.2 mg/L CuSO4·5H2O, 40 mg/L H3BO3, 180 mg/L MnSO4·H2O, 75 mg/L MnSO4·H2O, 2 g/L MgSO4·7H2O, 0.8 g/L CaCl2·6H2O, 0.4 g/L NaCl, 1.0 mL/L Antifoam 204 (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

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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).

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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.

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Fig. S2

Growth on melamine for ROBUST pathway engineered E. coli NS163 and control E. coli strain NS102 carrying the pACYC177 reference plasmid. Strains were cultivated in MOPS minimal medium with either NH4Cl or melamine as growth limiting nitrogen source. A standard curve for optical density and NH4Cl concentration was conducted with NS102 at 0, 0.5, 1, and 1.5 mM NH4Cl. NS163 was grown with 0.25 mM melamine, corresponding to 1.5 mM ammonium, or 0 mM melamine, as well as NS102 with 0.25 mM melamine. Optical density readings are reported as mean ± SD, N = 3.

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Fig. S3

Adaptive laboratory evolution for increased melamine growth rate. (A) Plasmid pNC153 was isolated from parental (NS148) and adapted (NS163) strains. A single point mutation in the guaD gene (C → A at nucleotide 7756) created an Arg352Ser amino acid change in the evolved plasmid, termed pNC153*. No other mutations were detected across the entire nucleotide sequence of pNC153* (B) Growth is improved for NS163 (red) compared to NS148 (blue). When pNC153* is re-introduced into wildtype E. coli ATCC 10798 (strains NS734, NS735, NS736, light green to green) growth rate is comparable to NS163. Re-introduction of pNC153 into wildtype E. coli ATCC 10798 (NS739, purple) results in growth comparable to NS148. (C) Maximum growth rate (averaged over 1 hour) and fermentation time at which it occurred.

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Fig. S4

Melamine consumption with strain NS163. Strain NS163 grown in MOPS defined medium with melamine as nitrogen source. The final measured melamine concentration was 0.33 mg/L, below the international standard for maximum acceptable melamine concentration (2.5 mg/kg) set for general food and animal feed products (47).

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Fig. S5

Plasmid maintenance with melamine as nitrogen source. Strain NS163 was grown without antibiotics and with melamine as nitrogen source (melamine consumption is shown in Figure S4), and plasmid maintenance was estimated by comparing total colony forming units to ampicillin resistant colony forming units, since the melamine utilization pathway plasmid also contains the ampR gene. CFU counts are reported as mean ± SD, N = 4.

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Fig. S6

Transferability of melamine pathway to E. coli ATCC 10798, B, MG1655, and Crooks strains. E. coli strains commonly employed by industrial biotechnology were transformed with plasmids containing either the melamine pathway or an empty vector control. Defined medium contained 1 mM melamine as nitrogen source.

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Fig. S7

Adaptive evolution and fermentation properties of S. cerevisiae engineered for cyanamide and phosphite utilization. (A) Initial cyanamide hydratase expressing strain (NS379) and evolved strain (NS532) grown with 5 mM cyanamide as nitrogen source. (B) Initial phosphite dehydrogenase expressing strain (NS558) and evolved strain (NS586) grown with 1 mM potassium phosphite as phosphorus source and 5 mM cyanamide as nitrogen source. (C) Wildtype Ethanol Red strain grown with 5 mM urea and 1 mM potassium phosphate and NS586 grown with 5 mM cyanamide and 1 mM potassium phosphite. (D) Metabolites after 40 hours fermentation with various nitrogen and phosphorus sources. (U = urea, C = cyanamide, PO4 = phosphate, PO3 = phosphite). Initial glucose concentration was 20 g/L. Data are reported as mean ± SD, N = 4.

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Fig. S8

Engineered Y. lipolytica lipid production with phosphate or phosphite. Y. lipolytica strain NS184 engineered for high diacylglycerol acyltransferase activity was grown in defined glucose medium with potassium phosphate, and compared to strain NS392, additionally engineered for phosphite utilization, grown in defined glucose medium with potassium phosphite. Fermentations were performed in 5 L bioreactors at 30°C, and maintained at pH 3.5 and ≥70% dissolved oxygen. Less than 0.25 g/L citric acid was produced in both fermentations. Lipid is reported as fatty acid methyl ester (FAME) equivalents.

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Fig. S9

Evaluation of nitrogen and phosphorus sufficiency in wheat straw hydrolysate. Dry cell weight accumulation of S. cerevisiae Ethanol Red in defined Verduyn medium with 2% glucose, 2% glucose without added nitrogen, 2% glucose without added phosphorus, wheat straw hydrolysate, wheat straw hydrolysate without added nitrogen, and wheat straw hydrolysate without added phosphorus.

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Fig. S10

Glucose consumption and ethanol production during wheat straw hydrolysate competition experiments with urea or cyanamide. Glucose consumption and ethanol production from S. cerevisiae NS586 and K. marxianus CBS 6556 grown in at 10:1 co-culture in wheat straw hydrolysate medium supplemented with 5 mM urea or 5 mM cyanamide.

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Fig. S11

Grain to lipid fermentation with the yeast antibiotic hygromycin. Fractionated corn mash simultaneous saccharification and fermentation (SSF) co-inoculated with lipid overproducing Y. lipolytica NS392 and contaminating S. cerevisiae strain Ethanol Red at a 10:1 initial ratio with potassium phosphate and 300 μg/mL hygromycin.

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Table S1.

Operating cost comparison for ROBUST chemical and antibiotic dosage

Dosage Operating cost

ROBUST chemical/ Antibiotic

$/kg

g chemical / kg yeast

biomass a

mg/L broth

$/m3 (at 10 kg/m3 yeast

biomass)

$/m3 replacing standard N or P b

Melamine $1.00 117.9 - $1.18 $0.25 Ammonium sulfate $0.25 370.5 - $0.93 Potassium phosphite $1.20 50.7 - $0.61 $0.03 Potassium phosphate $1.00 57.5 - $0.58 Cyanamide $1.50 117.9 - $1.77 $1.09 Urea $0.40 168.4 - $0.67 Penicillin G $30 10 $0.30 $0.30 Erythromycin $154 0.5 - 5 $0.08 - $0.77 $0.08 - $0.77 Virginiamycin $100-$200 0.1 - 3 $0.01 - $0.60 $0.01 - $0.60 Oxytetracycline/Neomycin $154 0.5 - 4 $0.08 - $0.62 $0.08 - $0.62 Streptomycin/Penicillin G/Virginiamycin

$154

0.5 - 2.4

$0.08 - $0.37 $0.08 - $0.37

a Modeled at 78.5 grams nitrogen and 13.1 grams phosphorus per kilogram yeast biomass b Melamine replaces ammonium sulfate, potassium phosphite replaces potassium phosphate, cyanamide replaces urea Melamine cost estimate (Alibaba, 2013) Cyanamide cost estimate (Alibaba, 2014) Phosphite cost estimate (Alibaba, 2014) Penicillin G cost estimate (Alibaba, 2014) Erythromycin cost estimate (industry estimate), dosage (Lallemand Biofuels & Distilled Spirits Technical Data Sheet Doc # 20130_Data_Rev.01.03.28.2013) Virginiamycin cost estimate (Alibaba, 2016), dosage (Lallemand Biofuels & Distilled Spirits Technical Data Sheet Doc # 20220_Data_Rev.00.01.01.2013) Oxytetracycline/Neomycincost estimate (industry estimate), dosage (Phibro Ethanol Performance NEOTROL® product guideline data sheet) Streptomycin/Penicillin G/Virginiamycin cost estimate (industry estimate), dosage (Lallemand Biofuels & Distilled Spirits Technical Data Sheet Doc # 20010_Data_Rev.01.03.28.2013)

22

Table S2.

Cost estimates for sanitary and sterile fermentation Capital and variable costs per m3 fermentation volume

Sanitary + antibiotic

Sanitary + ROBUST

Steam sterile

756,000 L capital costs ($) Sanitary 304 stainless steel tank & piping $1,982,400 $1,982,400 Sterile 316 stainless steel tank, piping, steam generation

$5,947,200

Batch cycle time (hr) 50.33 50.33 57.1 Batches per year 157 157 139

m3 processed per year (756,000 L fermenter) 118965 118965 104860 20 yr depreciation with 7% discount (9.5% of capital expense) ($/m3)

$1.58 $1.58 $5.39

Variable operating costs ($/m3)

Steam $2.38 Caustic for CIP $0.01 $0.01 $0.01 Antibiotic $0.25 ROBUST nutrient $0.25

Feedstock cost ($/m3)

Corn mash $51.00 $51.00

Glucose (DE 95 wet milled) $87.00 Capital & variable operating costs ($/m3) $1.84 $1.84 $7.78

Total cost ($/m3) $52.84 $52.84 $94.78

Fermenter and equipment cost estimates

Vendor quotations from a National Renewable Energy Laboratory 2011 Technical Report (48) for 7,560 L (2,000 gal) and 756,000 L (200,000 gal) fermenters were used as a basis for equipment calculations. These specifications called for sanitary 304 grade stainless steel design with clean-in-place (CIP) treatment and atmospheric pressure operation. They were not designed with the pressure rating or high electropolish surface finish (Ra ≤ 0.3 μm) (49) required for full sterile operation via steam sterilization. The uninstalled, equipment costs for these fermenters were (adjusted from 2009 to 2016 dollars by a 112% consumer price index adjustment) $197,120 for 7,560 L and $660,800 for 756,000 L.

Starting from these values, two scenarios were developed for 304 stainless steel (sanitary, non-sterile) fermenters and for high electropolish, pressure rated 316 stainless steel (sterile) fermenters using a material cost factor (50) of 3.0 for conversion of 304 stainless steel to high polish, pressure rated 316 stainless steel with steam generation equipment. Additionally, a Lang installation factor of 3 times the fermenter cost was used to estimate piping, valves, and related equipment of the same material quality (51). This resulted in the following total capital costs, per fermenter:

23

Capital cost estimates 7,560 L Sanitary 304 stainless steel $167,552 ($35.02/L capacity) 7,560 L Sterile 316 stainless steel $1,005,312 ($105.07/L capacity) 756,000 L Sanitary 304 stainless steel $561,680 ($1.31/L capacity) 756,000 L Sterile 316 stainless steel $3,370,080 ($7.87/L capacity)

To convert capital costs to a variable cost per m3 fermentation broth for the 756,000 L

volumes, the following factors were assumed. A fermentation cycle time of 50 hours was employed, including the time to fill and drain the fermentation vessel. Additionally, a 20 min clean-in-place time was included (49) to batch cycle time. For sterilization, a batch-style steam sterilization method was modeled (52). Sterilization was assumed to proceed at 121°C, with a 20-fold decimal reduction in Bacillus stearothermophilus spores (decimal reduction time of 2.26 min at 121°C) for sterility (53). This results in a sterilization time of 45 minutes, plus the time required to heat the vessel from 20°C to 121°C and cool to 35°C. The heating and cooling times were each estimated at 180 minutes (52), for a total of 7.1 hours of sterilization and cleaning between batches. The fermenters were assumed to operate 24 hours per day for 330 days per year, for a 20 year plant life with a 7% discount rate for an annual depreciation cost of 9.5% of capital costs. Variable cost estimates Antibiotic and ROBUST chemical costs were approximated from Table S1. Caustic rinse cost ($0.01/m3 fermenter volume) was estimated from an industry value. Steam cost for direct injection batch sterilization was estimated by calculating a total heat requirement using the equation Q = m• cp •dT, where Q = quantity of heat (kJ), m = mass of liquid to be heated (kg), cp = specific heat of water (4.2 kJ kg-1 °C-1), and dT = temperature change (°C). From this, a total heat requirement of 3.2x108 kJ was calculated to bring a 756,000 L fermenter volume from 20°C to 121°C. A steam pressure of 500 kPa was used following a heuristic to supply steam at approximately 300 kPa above the desired operating pressure (220 kPa for 121°C). At this pressure, steam has an evaporation energy of 2107 kJ/kg, and a total of 1.5x105 kg steam was assumed necessary to raise temperature and pressure. To generate steam, a natural gas (at $4/MMBtu) fed boiler was modeled at 85.7% efficiency, with a requirement of 2592 Btu/kg steam generated (54). This results in a steam cost at $0.012/kg, and a usage of 198 kg steam/ m3 fermenter volume, for a cost of $2.38/m3 fermenter volume. Cooling water to return the fermenter temperature to 35°C was not included in this estimation. Feedstock cost estimates

For this calculation, corn was used for the primary carbohydrate-rich feedstock. Non-sterile operation with antibiotic dosage can be operated with hammer milled, cooked corn as part of the low-cost “dry-grind” process. Whole corn contains (in addition to starch), protein, oil, and fiber. For this basic calculation, it was assumed that the sale of co-products (distiller’s dried grain and solubles (DDGS), corn oil, high protein corn germ, and fiber) off-sets the purchase and processing costs of these co-materials at zero profit (or loss) for the manufacturer. In practice, the sale of co-products is a valuable revenue stream for dry-grind ethanol production (55, 7). For non-sterile ROBUST operation, a dry-grind fractionation is required to separate phosphate-rich corn germ from endosperm prior to mash cooking. Although this fractionation step requires some additional capital, it is assumed that the higher value co-product (corn germ vs DDGS) off-

24

sets this charge (21). For sterile operation, corn mash suspended solids are incompatible with steam sterilization as insoluble material insulates microorganisms and prevents uniform steam exposure. Thus, refined glucose from a corn wet-mill process is required as feedstock. Although corn co-products are also produced in this process, more unit operations and capital are required to extract refined glucose. A published cost model was used to estimate the production cost of purified starch via the wet-mill process (56), plus an additional $0.02/kg starch for cost of amylase enzyme to convert starch to glucose (DE 95 glucose). These estimates resulted in a cost of $170/metric ton for dry-milled corn, and $290/metric ton for DE 95 glucose (with corn purchase price of $170/metric ton). To convert to a feedstock cost per m3 fermenter volume, a loading of 300 g/L carbohydrate was assumed in each case.

25

Table S3.

Nitrogen and phosphorus concentrations in industrially relevant feedstocks, and concentration required for yeast biomass generation. For estimation of the amount of the yeast biomass that can be produced from feedstock carbohydrates, an aerobic yield of 0.5 g yeast biomass/ g carbohydrate was used.

g/kg dry matter a

Nitrogen Phosphorus Carbohydrate b Sugarcane juice 1.3 0.6 731 Sugarcane juice c 3.5 0.1 891 Sugarcane bagasse 2.9 ± 0.5 0.6 ± 0.8 744 ± 61 Beet molasses 22.9 ± 2.4 0.3 ± 0.2 632 ± 36 Wheat straw 6.7 ± 1.1 0.7 ± 0.2 703 ± 42 Corn stover (fresh) 11 ± 0.2 2.0 ± 0.6 607 ± 87 Corn stover (dried) 5.9 ± 0.6 0.7 ± 0.3 740 ± 28 Baker's yeast d 78.5 ± 0.8 13.1 ± 0.5 -- a Values reported here are from the Institut National de la Recherche Agronomique Animal feed resources information system (57) unless otherwise noted. b Simple sugars, cellulose, and hemicellulose c Values obtained from material used in this study d Value obtained from (58)

26

Table S4.

Native xenobiotic and rare chemical utilizers reported in the literature Organism Xenobiotic/rare chemical Reference

Myrothecium verrucaria cyanamide U. H. Maier-Greiner et al., Isolation and properties of a nitrile hydratase from the soil fungus Myrothecium verrucaria that is highly

specific for the fertilizer cyanamide and cloning of its gene. Proc. Natl. Acad. Sci U.S.A. 88, 4260–4264 (1991).

Micrococcus sp. melamine (complete) W. S. El-Sayed, A. F. El-Baz, A. M. Othman, Biodegradation of melamine formaldehyde by Micrococcus sp. strain MF-1 isolated

from aminoplastic wastewater effluent. Inter. Biodeter. Biodegr. 57, 75–81 (2006).

Melaminivora alkalimesophila melamine (complete) H. Wang et al., Melaminivora alkalimesophila gen. nov., sp. nov., a melamine-degrading betaproteobacterium isolated from a

melamine-producing factory. Int. J. Syst. Evol. Microbiol.. 64, 1938–1944 (2014).

Nocardioides sp. melamine to cyanuric acid K. Takagi, K. Fujii, K. Yamazaki, N. Harada, A. Iwasaki, Biodegradation of melamine and its hydroxy derivatives by a bacterial

consortium containing a novel Nocardioides species. Appl. Microbiol. Biotechnol. 94, 1647–1656 (2012).

Microbacterium esteramaticum melamine (complete) N. Shiomi, M. Ako. Biodegradation of melamine and cyanuric acid by a newly-isolated Microbacterium strain. Adv. Microbio. 2,

303-309 (2012).

Raoultella terrigena melamine to cyanuric acid X. Zheng et al., Melamine-induced renal toxicity Is mediated by the gut microbiota. Sci. Transl. Med. 5, 172ra22 (2013).

Arthrobacter spp. melamine (complete) T. Hatakeyama et al., Mineralization of melamine and cyanuric acid as sole nitrogen source by newly isolated Arthrobacter spp.

using a soil-charcoal perfusion method. World J. Microbiol. Biotechnol. 31, 785–793 (2015). Acidovorax avenae subsp. Citrulli

melamine (complete) R. W. Eaton, J. S. Karns, Cloning and analysis of s-triazine catabolic genes from Pseudomonas sp. strain NRRLB-12227. J. Bact.

173, 1215–1222 (1991).

Rhodococcus sp. melamine (complete) A. G. Dodge, L. P. Wackett, M. J. Sadowsky, Plasmid localization and organization of melamine degradation genes in Rhodococcus

sp. strain Mel. Appl. Environ. Microbiol. 78, 1397–1403 (2012).

Rhodococcus corallinus melamine (complete) A. M. Cook, R. Huetter, Deethylsimazine: bacterial dechlorination, deamination, and complete degradation. J. Agric. Food Chem.

32, 581–585 (1984).

Pseudomonas stutzeri phosphite A. M. Costas, A. K. White, W. W. Metcalf, Purification and characterization of a novel phosphorus-oxidizing enzyme from

Pseudomonas stutzeri WM88. J. Biol. Chem. 276, 17429–17436 (2001).

Escherichia coli phosphite W. W. Metcalf, B. L. Wanner, Involvement of the Escherichia coli phn (psiD) gene cluster in assimilation of phosphorus in the

form of phosphonates, phosphite, Pi esters, and Pi. J. Bacteriol. 173, 587–600 (1991).

Proclorococcus marinus phosphite A. Martínez, M. S. Osburne, A. K. Sharma, E. F. DeLong, S. W. Chisholm, Phosphite utilization by the marine picocyanobacterium

Prochlorococcus MIT9301. Environ. Microbiol. 14, 1363–1377 (2012).

Alcaligenes faecalis phosphite M. M. Wilson, W. W. Metcalf, Genetic diversity and horizontal transfer of genes involved in oxidation of reduced phosphorus

compounds by Alcaligenes faecalis WM2072. Appl. Environ. Microbiol. 71, 290–296 (2005).

Desulfotignum phosphitoxidans phosphite B. Schink, V. Thiemann, H. Laue, M. W. Friedrich, Desulfotignum phosphitoxidans sp. nov., a new marine sulfate reducer that

oxidizes phosphite to phosphate. Arch. Microbiol. 177, 381–391 (2002).

Bacillus sp. phosphite T. L. Foster, L. Winans, S. J. Helms, Anaerobic utilization of phosphite and hypophosphite by Bacillus sp. Appl. Environ.

Microbiol. 35, 937–944 (1978).

Klebsiella aerogenes phosphite K. Imazu et al., Enhanced Utilization of Phosphonate and Phosphite by Klebsiella aerogenes. Appl. Environ. Microbiol. 64, 3754–

3758 (1998).

Pseudomonas aeruginosa phosphite L. E. Casida, Microbial oxidation and utilization of orthophosphite during growth. J Bacteriol. 80, 237–241 (1960).

Pseudomonas fluorescens phosphite L. E. Casida, Microbial oxidation and utilization of orthophosphite during growth. J Bacteriol. 80, 237–241 (1960).

Rhizobium trifolii phosphite L. E. Casida, Microbial oxidation and utilization of orthophosphite during growth. J Bacteriol. 80, 237–241 (1960).

Agrobacterium radiobacter phosphite L. E. Casida, Microbial oxidation and utilization of orthophosphite during growth. J Bacteriol. 80, 237–241 (1960).

Erwinia amylovora phosphite L. E. Casida, Microbial oxidation and utilization of orthophosphite during growth. J Bacteriol. 80, 237–241 (1960).

27

Table S5.

Strains used in this study Strain Organism Description Reference/Source

INVSc1 Saccharomyces cerevisiae MATa his3D1 leu2 trp1-289 ura3-52 Invitrogen

TOP10 Escherichia coli F– mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara leu) 7697 galU galK rpsL (StrR) endA1 nupG

Invitrogen

ATCC 10798

Escherichia coli Wildtype (designation K-12) NRRL B-3707

B Escherichia coli wildtype ATCC 11303

Crooks Escherichia coli wildtype ATCC 8739

MG1655 Escherichia coli wildtype ATCC 47076

NRRL B-12227

Acidovorax citrulli wildtype CECT 4423

NS88 Escherichia coli ATCC 10798

pNC85 this study

NS91 Escherichia coli ATCC 10798

pNC53 this study

NS102 Escherichia coli ATCC 10798

pACYC177 this study

NS110 Escherichia coli ATCC 10798

pNC121 this study

NS120 Escherichia coli MG1655 pNC53 this study

NS121 Escherichia coli MG1655 pNC121 this study

NS122 Escherichia coli B pNC121 this study

NS123 Escherichia coli Crooks pNC53 this study

NS124 Escherichia coli Crooks pNC121 this study

NS148 Escherichia coli ATCC 10798

pNC153 this study

NS163 Escherichia coli ATCC 10798

pNC153* this study

Ethanol Red

Saccharomyces cerevisiae Industrial wildtype strain Phibro

CBS 6556 Kluyveromyces marxianus wildtype ATCC 26548

NS379 Saccharomyces cerevisiae CAH (pNC286 integration) this study

NS532 Saccharomyces cerevisiae CAH (pNC286 integration) serial transfer cyanamide medium this study

NS558 Saccharomyces cerevisiae CAH (pNC286 integration) serial transfer cyanamide medium, ptxD (pNC360 integration)

this study

NS586 Saccharomyces cerevisiae CAH (pNC286 integration) serial transfer cyanamide medium, ptxD (pNC360 integration) serial transfer phosphite medium

this study

NS18 Yarrowia lipolytica wildtype NRRL YB-392

NS184 Yarrowia lipolytica YlDGA1 (pNC104 integration) YlDGA2 (pNC160 integration) HygR NatR this study

NS324 Yarrowia lipolytica ptxD (pNC273 integration) HygR this study

NS392 Yarrowia lipolytica ptxD (pNC273 integration) YlDGA1 (pNC104 integration) YlDGA2 (pNC160 integration) HygR NatR

this study

NS535 Yarrowia lipolytica NatR this study

NS891 Saccharomyces cerevisiae G418R this study

28

Table S6.

Genes used in this study Gene Enzyme Source Organism Notes

bla β-lactamase Escherichia coli Ampicillin resistance cloning marker used for DNA propagation

URA3 Orotidine 5'-phosphate decarboxylase

Saccharomyces cerevisiae Cloning marker used for DNA propagation

hph hygromycin phosphotransferase Streptomyces hygroscopicus Selection marker used for strain construction

nat nourseothricin acetyl transferase

Streptomyces noursei Selection marker used for strain construction

DGA1 Diacylglycerol O-acyltransferase Yarrowia lipolytica lipid overproducer enzyme

DGA2 Diacylglycerol O-acyltransferase Yarrowia lipolytica lipid overproducer enzyme

triA melamine deaminase Acidovorax avenae subsp. citrulli melamine utilization pathway

guaD guanine (ammeline) deaminase Escherichia coli melamine utilization pathway

trzC ammelide deaminase Acidovorax avenae subsp. citrulli melamine utilization pathway

atzD cyanuric acid hydrolase Pseudomonas sp. strain ADP melamine utilization pathway

trzE Biuret hydrolase Rhodococcus sp. Mel

codon optimized, melamine utilization pathway

DUR1,2 urea amidolyase Saccharomyces cerevisiae melamine utilization pathway, Allophanate hydrolase domain only

CAH cyanamide hydratase Aspergillus niger cyanamide utilization pathway

ptxD phosphite dehydrogenase Pseudomonas stutzeri phosphite utilization pathway

29

Database S1 (separate file)

Database S1 contains DNA sequences of plasmids and transformation vectors used in this study.

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