246 nature chemical biology | VOL 10 | APRIL 2014 | wwwnaturecomnaturechemicalbiology

news amp views

METABOLIC ENGINEERING

The sweet smell of biosynthesisMetabolically engineered cells are yielding an expansion of specialty chemicals that rival or supplement traditional petroleum-derived chemicals New results establish a biosynthetic route to volatile esters and larger acetate esters equivalent in size to biodiesel providing an intriguing new direction to create fragrances chemicals or fuels

Brett M Barney

Chemical esters serve a variety of functions throughout biology from performing important

roles as volatile fragrances in flowers and fruits to protecting against water loss and pathogens as cuticular waxes produced by epidermal cells12 Esters can be derived from either petroleum-based feedstocks or extracted directly from biological sources and have a vital commercial role as specialty chemicals2 Esters can also increasingly be synthesized by engineered cells particularly as many desirable alcohol and carboxylic acid building blocks are natural metabolites However metabolic engineers face certain challenges in optimizing these pathways that go beyond just introducing the enzymes Product profiles are determined primarily by the supply of precursors often resulting in suboptimal levels of the desired target esters1 As an ideal design constraint for the engineer the generally applicable enzymes should have only limited specificity for the size or shape of the substrate while still conserving the integrity of the enzymatic reaction catalyzed Enzymes with this broad substrate range have been exploited in biosynthetic routes to produce a wide variety of chemicals from diverse precursors using only a small set of enzymes3ndash5 Rodriguez et al6 now use two such enzymesmdasha keto-acid decarboxylase from lactic acid bacteria3 and alcohol acyltransferases (ATFs) from eukaryotes1mdashin their report of engineering E coli to produce a range of volatile esters

Routes to medium esters such as biodiesel are of keen interest in the field of biosynthetic chemistry Some neutral lipidndashaccumulating bacteria have natural pathways to produce larger esters using the wax ester synthaseacyl-CoAdiacylglycerol acyltransferase (WSDGAT) and fatty acyl reductases (FARs)7ndash9 These products tend to have chain lengths of 28 carbons or more78 the length of which is determined by substrate availability within the cell7

By altering the concentrations of available alternative substrates Kalscheuer et al10 previously developed a route to a microbial-produced biodiesel ester coined lsquomicrodieselrsquo by combining a WSDGAT from Acinetobacter baylyi with enzymes derived from Zymomonas mobilis to overproduce ethanol (Fig 1) In this manner they produced fatty acid ethyl esters that were the combination of ethanol and common indigenous fatty acids (predominantly C18 and C16)

Rodriguez et al6 now demonstrate an alternative biosynthetic approach to reach a unique space within the volatile to medium-chain-length esters Specifically by using an ester-forming ATF in conjunction with a keto-acid decarboxylase to provide adequate levels of desired precursors for the ATF Rodriguez et al6 were able to produce high yields of branched esters such as isobutyl acetate or isoamyl acetate a major component of fragrance in banana oil1 The introduction of additional enzymes allowed Rodriguez et al6 to engineer

medium-chain-length esters that have value in further applications and still have chain lengths or molecular arrangements (such as branching) that allow the esters to exist as liquids at ambient temperatures differentiating them from small volatile esters and longer wax esters1278 Finally inclusion of the Vibrio harveyi LuxCDE enzyme system in conjunction with indigenous enzymes present in Escherichia coli provided long-chain fatty alcohols as substrates to yield a product equivalent in size (but altered in the molecular arrangement) with biodiesel expanding the potential products of the combined enzymes even further Complementing the report from Kalscheuer et al10 Rodriguez et al6 achieved a product with nearly equivalent ester length using essentially inverted substrates combining a long-chain fatty alcohol (C14) with readily available acetate to produce a fatty alcohol acetate ester and providing an intriguing alternative to the pathway used to produce microdiesel (Fig 1) Similarly to the general feature of WSDGAT ATF also has

Figure 1 | Comparison of the final step in two different biosynthetic routes capable of producing a biodiesel equivalent product in E coli The first reaction using an ATF is described by Rodriguez et al6 and the second reaction producing microdiesel was described previously by Kalscheuer et al10

OH

CoAS

O

6

OH

CoAS

O

O

O

6

O

O

6

6

CoA-SH

CoA-SH

ATF

WSDGAT

Fatty alcohol acetate esters

Fatty acid ethyl estersMicrodiesel

npg

copy 2

014

Nat

ure

Am

eric

a In

c A

ll rig

hts

rese

rved

nature chemical biology | VOL 10 | APRIL 2014 | wwwnaturecomnaturechemicalbiology 247

news amp views

a broad substrate range in relation to the size and shape of the fatty alcohols that it will accommodate6810

The work by Rodriguez et al6 further illustrates the potential to expand the range of specialty chemicals that could be biosynthetically produced by combining the roles of several different enzymes each with broad substrate specificity3ndash5 In this manner biosynthetic chemistry can borrow from combinatorial chemistry approaches with whole-cell biocatalysis limited only in its ability to provide the precursor substrates to construct the desired products17 The combined approaches also demonstrate the potential to expand product profiles of these enzymes beyond the realm of their native substrates where Kalscheuer et al10 pushed the limits of the WSDGAT which naturally produces a large wax78 to produce the much smaller fatty acid ethyl ester microdiesel

Rodriguez et al6 have pushed the limits of ATF which naturally produces a small volatile ester1 to produce the much larger fatty alcohol acetate ester (Fig 1) The discovery of new enzymes or rediscovery of established enzymes in addition to techniques that might expand or improve upon these various enzymes should further pave the way for a much broader range of esters Although efforts that tailor enzymes to produce specific products or expand the range of reactions catalyzed may be important additional goals reengineering cells to supply ample quantities of desired precursors remains a key parameter for consideration by the biosynthetic engineer

Brett M Barney is at the Department of Bioproducts and Biosystems Engineering University of Minnesota St Paul Minnesota USA e-mail bbarneyumnedu

Published online 9 March 2014 doi101038nchembio1480

References1 Beekwilder J et al Plant Physiol 135 1865ndash1878

(2004)2 Jetter R amp Kunst L Plant J 54 670ndash683 (2008) 3 Atsumi S Hanai T amp Liao JC Nature 451 86ndash89

(2008)4 Schirmer A Rude MA Li X Popova E amp

del Cardayre SB Science 329 559ndash562 (2010)5 Steen EJ et al Nature 463 559ndash562 (2010)6 Rodriguez GM Tashiro Y amp Atsumi S Nat Chem Biol

doi101038nchembio1476 (9 March 2014)7 Barney BM Wahlen BD Garner E Wei J amp Seefeldt

LC Appl Environ Microbiol 78 5734ndash5745 (2012)8 Stoumlveken T amp Steinbuumlchel A Angew Chem Int Edn Engl

47 3688ndash3694 (2008)9 Youngquist JT et al Metab Eng 20 177ndash186 (2013)10 Kalscheuer R Stoumllting T amp Steinbuumlchel A Microbiology

152 2529ndash2536 (2006)

Competing financial interestsThe author declares no competing financial interests

npg

copy 2

014

Nat

ure

Am

eric

a In

c A

ll rig

hts

rese

rved

nature chemical biology | VOL 10 | APRIL 2014 | wwwnaturecomnaturechemicalbiology 247

news amp views

a broad substrate range in relation to the size and shape of the fatty alcohols that it will accommodate6810

The work by Rodriguez et al6 further illustrates the potential to expand the range of specialty chemicals that could be biosynthetically produced by combining the roles of several different enzymes each with broad substrate specificity3ndash5 In this manner biosynthetic chemistry can borrow from combinatorial chemistry approaches with whole-cell biocatalysis limited only in its ability to provide the precursor substrates to construct the desired products17 The combined approaches also demonstrate the potential to expand product profiles of these enzymes beyond the realm of their native substrates where Kalscheuer et al10 pushed the limits of the WSDGAT which naturally produces a large wax78 to produce the much smaller fatty acid ethyl ester microdiesel

Rodriguez et al6 have pushed the limits of ATF which naturally produces a small volatile ester1 to produce the much larger fatty alcohol acetate ester (Fig 1) The discovery of new enzymes or rediscovery of established enzymes in addition to techniques that might expand or improve upon these various enzymes should further pave the way for a much broader range of esters Although efforts that tailor enzymes to produce specific products or expand the range of reactions catalyzed may be important additional goals reengineering cells to supply ample quantities of desired precursors remains a key parameter for consideration by the biosynthetic engineer

Brett M Barney is at the Department of Bioproducts and Biosystems Engineering University of Minnesota St Paul Minnesota USA e-mail bbarneyumnedu

Published online 9 March 2014 doi101038nchembio1480

References1 Beekwilder J et al Plant Physiol 135 1865ndash1878

(2004)2 Jetter R amp Kunst L Plant J 54 670ndash683 (2008) 3 Atsumi S Hanai T amp Liao JC Nature 451 86ndash89

(2008)4 Schirmer A Rude MA Li X Popova E amp

del Cardayre SB Science 329 559ndash562 (2010)5 Steen EJ et al Nature 463 559ndash562 (2010)6 Rodriguez GM Tashiro Y amp Atsumi S Nat Chem Biol

doi101038nchembio1476 (9 March 2014)7 Barney BM Wahlen BD Garner E Wei J amp Seefeldt

LC Appl Environ Microbiol 78 5734ndash5745 (2012)8 Stoumlveken T amp Steinbuumlchel A Angew Chem Int Edn Engl

47 3688ndash3694 (2008)9 Youngquist JT et al Metab Eng 20 177ndash186 (2013)10 Kalscheuer R Stoumllting T amp Steinbuumlchel A Microbiology

152 2529ndash2536 (2006)

Competing financial interestsThe author declares no competing financial interests

npg

copy 2

014

Nat

ure

Am

eric

a In

c A

ll rig

hts

rese

rved