BIOENGINEERING

Metabolic engineering of microbialcompetitive advantage for industrialfermentation processesA. Joe Shaw,1* Felix H. Lam,2 Maureen Hamilton,1 Andrew Consiglio,1 Kyle MacEwen,1

Elena E. Brevnova,1,3† Emily Greenhagen,1‡ W. Greg LaTouf,1 Colin R. South,1§Hans van Dijken,1 Gregory Stephanopoulos1,2

Microbial contamination is an obstacle to widespread production of advanced biofuelsand chemicals. Current practices such as process sterilization or antibiotic dosagecarry excess costs or encourage the development of antibiotic resistance. Weengineered Escherichia coli to assimilate melamine, a xenobiotic compound containingnitrogen. After adaptive laboratory evolution to improve pathway efficiency, the engineeredstrain rapidly outcompeted a control strain when melamine was supplied as the nitrogensource. We additionally engineered the yeasts Saccharomyces cerevisiae and Yarrowialipolytica to assimilate nitrogen from cyanamide and phosphorus from potassiumphosphite, and they outcompeted contaminating strains in several low-cost feedstocks.Supplying essential growth nutrients through xenobiotic or ecologically rare chemicalsprovides microbial competitive advantage with minimal external risks, given thatengineered biocatalysts only have improved fitness within the customized fermentationenvironment.

Microbial bioconversion of plant biomassis a promising route to sustainable liquidfuels and chemicals (1, 2). For compati-bility with society’s petroleum-based infra-structure, researchers have used metabolic

engineering to produce “drop-in”molecules thatmeet existing petrochemical standards (3–5).However, despite many technical advances enabl-ing the production of biofuels and biochemicals,their large-scale production at competitive eco-nomic cost is lagging. One of the major obstaclesfor new bioprocesses is microbial contamination,which negatively affects yield, productivity, andoperability. When scaling up new bioprocesses,contamination can be a major barrier to success-ful operation (6).Conventional biofuel and biochemical fermen-

tations use naturally occurring organisms that arehighly competitive in their specific operating envi-ronment. For example, Saccharomyces cerevisiae,which is used to produce over 100 billion liters ofethanol per year (7, 8), is well suited to exploit theenvironment provided by modern fruit (9), whereit rapidly converts high concentrations of simplesugars to ethanol. Ethanol inhibits many compet-ing microbes and allows the more tolerant S.cerevisiae to dominate the fermentation. Byco-opting this naturally evolved competitive ad-

vantage, bioethanol producers avoid the needfor feedstock refining and sterilization, enablingcost-competitive biofuel production. However,even with this natural competitive advantage,ethanol producers often resort to antibiotic treat-ment to halt the growth of contaminating bacte-

ria (10, 11), particularly Lactobacilli, that toleratehigh ethanol concentrations (12).A consequence of manipulating their central

metabolism to produce advanced biofuels and bio-chemicals is that engineered microorganisms oftenhave reduced growth rates or other competitiveimpairments. We sought to address this challengeby endowing biocatalysts with an engineeredcompetitive advantage. We did this by supply-ing the macronutrients nitrogen and phospho-rus (which are required by all microorganismsfor growth) through low-cost xenobiotic or eco-logically rare compounds and metabolicallyengineering their complementary assimilationpathways (Fig. 1). This combination creates anenvironment where the engineered biocatalystbecomes the dominant microorganism withoutsolely relying on naturally evolved ecological fit-ness. We have termed this strategy “robust op-eration by utilization of substrate technology”(ROBUST).To test the ROBUST strategy, we engineered

Escherichia coli ATCC 10798 with a syntheticsix-step pathway for the conversion of melamine,a xenobiotic compound containing 67% nitrogenby weight, to ammonia and carbon dioxide. Wedemonstrated the utilization of melamine’s sixnitrogen atoms (figs. S1 and S2) through expres-sion of Acidovorax avenae, Pseudomonas sp.,Rhodococcus sp., E. coli, and S. cerevisiae enzymesthat had previously been characterized for thebioremediation of triazides (13–16). When trans-formed with a low-copy plasmid carrying themelamine utilization pathway, E. coli grew withmelamine at a maximum rate of 0.12 ± 0.02 hour−1

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1Novogy, 85 Bolton Street, Cambridge, MA 02140, USA.2Department of Chemical Engineering, MassachusettsInstitute of Technology, Cambridge, MA 02139, USA. 3TotalNew Energies USA, 5858 Horton Street, Emeryville, CA94608, USA.*Corresponding author. Email: jshaw@novogyinc.com †Presentaddress: Evelo Therapeutics, 620 Memorial Drive, Cambridge, MA02139, USA. ‡Present address: Ginkgo Bioworks, 27 DrydockAvenue, Boston, MA 02210, USA. §Present address: Proterro, 66Palmer Avenue, Bronxville, NY 10708, USA.

Fig. 1.The ROBUSTstrategy. Macronutrients essential for microbial growth are supplied in the formof xenobiotic or ecologically rare chemicals. Metabolic pathways enabling macronutrient assimilationare engineered in the desired biocatalyst (blue cells), establishing them as the dominant micro-organism over nonutilizing contaminants (brown and red cells) inside the industrial bioreactor en-vironment. NAD+, oxidized nicotinamide adenine dinucleotide; NADH; reduced nicotinamide adeninedinucleotide.

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(n = 3). We next performed adaptive laboratoryevolution spanning about 100 generations indefined medium containing 0.5 mM melamine;we subsequently isolated a colony, designatedstrain NS163, with a threefold increase in growthrate to 0.40 ± 0.04 hour−1 (n = 3).Plasmid isolation from NS163 and reintro-

duction into wild-type E. coli resulted in animmediate growth rate on melamine of 0.4 hour−1

(fig. S3). Sequencing of the parental and reisolatedplasmids identified a single point mutation in theevolved plasmid, resulting in an arginine-to-serinechange at amino acid 352 of the E. coli geneguaD, which encodes ammeline deaminase, thesecond of six steps required for complete mela-mine degradation. That E. coli guaD is a targetof evolutionary selection is perhaps not surprising,given that guaD is a “housekeeping” enzyme withprimary activity for guanine deamination and isthought to catalyze ammeline deamination as aside activity (17).To establish competitive advantage, the mela-

mine utilization pathway ideally operates at arate that makes ammonium available for rapidgrowth, but not so fast that free ammonium isexcreted. With strain NS163, no free ammoniumwas detected during growth onmelamine (fig. S4),although the degradation intermediate cyanuricacid did accumulate to about 13% of the totalmelamine fed, suggesting that further pathwayoptimization is possible. Growth on melaminealso acted to maintain plasmid stability (fig. S5).In coculture experiments in defined medium

with melamine as the nitrogen source, E. colistrain NS163 rapidly outcompeted a control E. colistrain carrying the pACYC177 reference plasmid(Fig. 2A). The utilization pathway additionallyconferred growth on melamine to the industriallyrelevant E. coli B, MG1655, and Crooks strains(fig. S6).

Next, we engineered S. cerevisiae to utilizecyanamide, a nitrile-containing compound, andwe engineered both S. cerevisiae and the oleagi-nous yeast Yarrowia lipolytica to utilize phos-phite, a phosphorus-containing chemical-industryintermediate. For the former, we expressed acyanamide hydratase (18) gene homolog fromAspergillus niger to convert cyanamide to bio-logically accessible urea. After adaptive evolu-tion, this strain grew with cyanamide as thesole nitrogen source. The process was repeatedfor the conversion of phosphite to biologicallyaccessible phosphate by expression of bacterialphosphite dehydrogenase (19) and adaptive evo-lution (supplementary text and fig. S7). Thisresulted in S. cerevisiae strain NS586, whichwas able to simultaneously use cyanamide asa nitrogen source and potassium phosphite asa phosphorus source. We additionally expressedphosphite dehydrogenase in Y. lipolytica wild-type (strain NS324) and lipid-overproducing back-grounds (strain NS392), which enabled phosphitemedium–based growth and lipid production atrates comparable to those in phosphate medium(fig. S8), without the need for adaptive evolution.In competition with a control S. cerevisiae

strain, NS586 demonstrated superior fitness in

cyanamide-containing media (Fig. 2B), despitebaseline growth of the control strain. Likewise,the Y. lipolytica phosphite-assimilating strainNS324 outcompeted a control strain in phosphite-containing media (Fig. 2C).

For the supply of nitrogen and phosphorusthrough synthetic compounds to be econom-ically viable, additional costs must be minimaland more than offset by the use of lower-cost pro-cessing methods and feedstocks. The compounds

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Fig. 2. Competition experiments in glucose medium. Each panel shows colony-forming unit(CFU) counts during cofermentation of ROBUST and control strains inoculated at a 1:1 ratio indefined glucose medium, with either standard or ROBUST chemicals supplying key macronutrients.(A) E. coli strains NS102 (containing the reference plasmid pACYC177) and NS163 (containing themelamine utilization plasmid pNC153), co-inoculated with ammonium chloride or melamine as thenitrogen source. (B) S. cerevisiae reference strain NS891 and cyanamide hydratase–expressingstrain NS586, co-inoculated with urea or cyanamide as the nitrogen source. (C) Y. lipolytica re-ference strain NS535 and phosphite dehydrogenase–expressing strain NS324, co-inoculated withpotassium phosphate or potassium phosphite as the phosphorus source. CFU counts are reportedas means ± SD (n = 4).

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in this study are derived from bulk industrialchemical precursors and represent a cost thatis comparable to that of industrial antimicrobialadditives (table S1) and superior to that of sterilefermentation (table S2). We sought next to applyROBUST to minimally refined, low-cost feedstocks.We evaluated sugarcane juice and wheat

straw lignocellulosic hydrolysate, two broadlyavailable industrial feedstocks, for ROBUSTfermentation with S. cerevisiae strain NS586.Sugarcane juice is composed primarily of simplesugars, but also contains some free amino acid–bound nitrogen and 0.1 to 0.6 g of phosphorusper kilogram of dry matter, or 1.7 to 12.5% ofthe phosphorus necessary for complete aerobicconversion of the sugars to yeast biomass (tableS3). We tested batch fermentation of 2% w/vsugarcane juice supplemented with media con-taining 4 mM potassium phosphate or potassiumphosphite. To these media, strain NS586 andKluyveromycesmarxianus CBS 6556, a fast-growing

spoilage yeast (20), were co-inoculated at a 10:1ratio. Despite the presence of a low level ofnaturally occurring phosphate, in the phosphite-supplemented medium, NS586 outcompetedK. marxianus for sugar utilization (Fig. 3A). Withvariability in raw feedstock supplies, ROBUSTmay be more or less effective at different nat-urally occuring phosphate concentrations. Fora given set of operating conditions (e.g., bio-catalyst loading, growth rate, fermentation time),a maximum natural phosphate concentrationmay be established for reliable operation; blend-ing raw sugarcane juice with refined sugar couldbe used to keep phosphate below this level.Wheat straw lignocellulosic hydrolysate is un-

der evaluation for the production of bioethanoland biochemicals, and it requires nitrogen supple-mentation for complete growth of S. cerevisiae(fig. S9).We co-inoculated S. cerevisiaeNS586 andK. marxianus CBS 6556 at a 10:1 initial ratio with5 mM urea or cyanamide in 2% w/v glucan-

equivalent wheat straw hydrolysate. Althoughthe cyanamide condition has a 4- to 5-hour lagrelative to the urea condition, it enables S.cerevisiae NS586 both to grow unhindered byK. marxianus (Fig. 3B) and to achieve a highercellulosic ethanol titer (fig. S10). Other industrialfeedstocks such as sugarcane bagasse, sugar beetmolasses, and corn stover have comparable limitingphosphorusandnitrogencontents (tableS3),makingthem feasible for further ROBUST processing.Corn has the highest annual yield (in metric

tons) of any cereal crop worldwide and producesthe most starch per acre of cultivated crops. Ingeneral, there are two processes for producingfermentable carbohydrate from corn. The first,dry milling, is the lowest-cost route to a fermen-table intermediate (7) but results in a heteroge-neous, semi-solid mash that is challenging tosterilize. The second process, wetmilling, producesa highly refined pure glucose but requires substan-tial capital and operating investment (7) (table S2).We sought to apply ROBUST to dry-milled corn,which could be complicated by the nitrogen andphosphorus present in whole corn kernels. How-ever, by using low-capital dry-mill fractionation(21, 22), 90% of the phosphorus can be readilyseparated from fermentable starch, with copro-duction of protein and oil containing corn germ(Fig. 4A). In simultaneous saccharification andfermentation experiments co-inoculated withY. lipolytica NS392 [engineered for phosphiteutilization and lipid overproduction (23)] andS. cerevisiae Ethanol Red (an industrial ethanolproduction strain used in this study as a conta-minant), the S. cerevisiae strain outcompetedY. lipolytica even at a 10:1 initial disadvantagebecause of rapid ethanol accumulation, whichdecreases Y. lipolytica viability (Fig. 4B). How-ever, when phosphorus was supplied as potas-sium phosphite, Y. lipolytica NS392 continuedto grow and produce lipids, and no ethanol ac-cumulated during fermentation (Fig. 4, C andD). Nearly identical Y. lipolytica performance wasobserved when the yeast antibiotic hygromycinwas added to inhibit S. cerevisiae during phos-phate fermentation (fig. S11). These results demon-strate the capability of ROBUST to fundamentallyshift production toward a desired product in anotherwise identically configured fermentation.Naturally selected microorganisms may emerge

that are capable of assimilating almost anyxenobiotic chemical (table S4), but the abilityto synthesize new nitrogen- and phosphorus-containing compounds and to engineer metabolicassimilation pathways should enable the deploy-ment of future ROBUST compound-pathwayiterations. This dynamic is similar to the armsrace currently underway with multidrug-resistantbacterial strains (24). However, with ROBUST,the competition between industrial biotechnol-ogists and natural evolution can occur withoutthe risk of spreading antibiotic resistance genes(25). Although engineered biocatalysts shouldbe able to consume ROBUST compounds to belowtrace levels, we do anticipate that a full life-cycleanalysis will be required for the use of any newcompound in an industrial bioprocess.

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Fig. 3. Improved biocatalyst fitness with industrial feedstocks. Shown are data from aerobicfermentation with ROBUST S. cerevisiae NS586 and contaminant K. marxianus CBS 6556.(A) Sugarcane juice with potassium phosphate or potassium phosphite as the phosphorus source.(B) Wheat straw lignocellulosic hydrolysate with urea or cyanamide as the nitrogen source. CFUcounts are reported as means ± SD (n = 3).

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ACKNOWLEDGMENTS

We thank O. Vidalin and V. Rajgarhia for managerial support,D. Crabtree and J. Afshar for analytical support, H. Blitzblau andV. Tsakraklides for helpful comments, Cereal ProcessTechnologies and the National Corn to Ethanol Research Centerfor materials and insight into corn fractionation processes, BetaRenewables for wheat straw hydrolysate, and Phibro for theyeast Ethanol Red. This work was supported by Novogy andTotal New Energies. F.H.L. and G.S. were supported in part bythe U.S. Department of Energy (grant DE-SC0008744). A.J.S.,C.R.S., H.v.D., E.G., M.H., W.G.L., A.C., K.M., and Novogy havefiled patent applications [WO2014107660 (A3), WO2015031441(A3), and WO2015157431 (A3)] that relate to the use of ROBUSTpathways for fermentation selective advantage. PROESAwheat straw hydrolysate is available from Biochemtex under amaterial transfer agreement. Data are available in thesupplementary materials, and strains are available by request.A.J.S., F.H.L., E.E.B., E.G., W.G.L., C.R.S., H.v.D., and G.S.participated in the design of the experiments and/or theinterpretation of the results. A.J.S., F.H.L., M.H., A.C., K.M.,E.E.B., and E.G. participated in the acquisition and/or analysisof data. A.J.S., F.H.L., and G.S. participated in drafting and/orrevising the manuscript.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/353/6299/583/suppl/DC1Materials and MethodsSupplementary TextFigs. S1 to S11Tables S1 to S6References (26–58)Database S1

20 March 2016; accepted 22 June 201610.1126/science.aaf6159

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Fig. 4. ROBUST-enabled grain-to-lipid fermentation. (A) Dry-mill corn fractionation enables low-costseparation of food- and animal feed–quality germ and fiber from fractionated corn mash. (B) Simul-taneous saccharification and fermentation in fractionated mash co-inoculated with lipid-overproducingY. lipolytica NS392 (engineered for phosphite utilization) and contaminating S. cerevisiae strain EthanolRed at a 10:1 initial ratio, with potassium phosphate supplying phosphorus. (C) Fermentation underidentical conditions, except with potassium phosphite supplying phosphorus. In (B) and (C), CFU countsare reported as means ± SD (n = 4). (D) Lipid accumulation, reported as fatty acid methyl ester (FAME), atthe end of fermentation.

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processesMetabolic engineering of microbial competitive advantage for industrial fermentation

Greg LaTouf, Colin R. South, Hans van Dijken and Gregory StephanopoulosA. Joe Shaw, Felix H. Lam, Maureen Hamilton, Andrew Consiglio, Kyle MacEwen, Elena E. Brevnova, Emily Greenhagen, W.

DOI: 10.1126/science.aaf6159 (6299), 583-586.353Science 

, this issue p. 583; see also p. 542Sciencephosphorus also improved competitive fitness.to outcompete contaminating organisms. Similarly, engineering yeast to use cyanamide for nitrogen or phosphite for

, for example, to consume melamine as a nitrogen source allowed itEscherichia coliEngineering the common biocatalyst engineered bacteria and yeast to use rare compounds as sources of nutrients (see the Perspective by Lennen).et al.

organisms have to be inhibited without using antibiotics because of concerns about spreading antibiotic resistance. Shaw feedstocks, such as sugarcane or dry-milled corn for biofuels or other industrial purposes. The challenge is that foreign

Contaminating microorganisms can be highly detrimental to the large-scale fermentation of complex low-costXenobiotics to the rescue

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