genetic chemistry: production of non-native compounds in yeast

Available online at www.sciencedirect.com

Genetic chemistry: production of non-native compounds in yeastStanley Goldman

The tools and perspectives that chemists bring to the study of

biological systems have yielded very important discoveries and

opened many new research possibilities over the years

(Hopkins AL: Network pharmacology: the next paradigm in

drug discovery. Nat Chem Biol 2008, 11:682–690; Lehar J,

Stockwell BR, Giaever G, Nislow C: Combination chemical

genetics. Nat Chem Biol 2008, 11:674–681. This work

describes use of genome level data to discover and understand

higher order pleiotropic effects of combinations of drugs).

Chemical biology has an ever-growing toolbox that has been

expanding its reach into many different aspects of the study

and utilization of biological systems (Strombergsson H,

Kleywegt G: A chemogenomic view on protein–ligand

spaces. BMC Bioinformatics 2009, 10(Suppl 6):S13; Bumpus

BB, Evens BS, Thomas PM, Ntai I, Kelleher NI: A proteomics

approach to discovering natural products and their

biosynthetic pathways. Nat Biotechnol 2009, 27:951–956.

This reviews techniques that allow for the identification of

biochemical pathways that produce molecules of interest

under very specific situations; Altamn KH, Buchner J, Kessler

H, Diederich F, Krautler B, Lippard S, Liskamp R, Muller K,

Nolan EM, Samori B, et al.: The state of the art of chemical

biology. Chembiochem 2009, 10:16–29) including the study

and utilization of biological systems in yeast. This review will

describe recent successes in the use of yeast for both

discovery and production of non-native secondary metabolites

focused on pharmaceutically relevant compounds.

Address

Genetic Chemistry Inc. (Evolva Holding Company), 2440 Embarcadero

Way, Palo Alto, CA 94303, United States

Corresponding author: Goldman, Stanley

([email protected])

Current Opinion in Chemical Biology 2010, 14:390–395

This review comes from a themed issue on

Molecular Diversity

Edited by Lisa A. Marcaurelle and Michael A. Foley

Available online 22nd April 2010

1367-5931/$ – see front matter

# 2010 Elsevier Ltd. All rights reserved.

DOI 10.1016/j.cbpa.2010.03.036

IntroductionDefinitions of genetic chemistry differ between research-

ers. For this review genetic chemistry is viewed as the use

of genetic and chemical tools, including purification and

structural determination of small molecules supported by


biochemical, proteomic, transcriptome, and metabolome

analysis for the modification and production of non-native

secondary metabolites. Use of these capabilities range

from the obvious, ever improving, but not simple, ability

to purify natural small molecules and determine their

structures [1] to predict and isolate genes required for the

enzymatic synthesis of newly characterized compounds of

interest [2�] to the newest challenges, the ongoing elu-

cidation of the complex signal and response systems that

control living cells and organisms generally called system

biology [3,4]. Many chemistry-based experimental

approach have yielded exciting and important results

supported by the ever-growing fields of genomics [5�],proteomics [2�,6�], and detailed analysis of global tran-

scription [7�].

Yeast is an ideal organism for use in heterologous expres-

sion efforts because of the fact that it is GRAS (generally

regarded as safe), well characterized physiologically, has

powerful genetic and recombinant tools associated with

it, and is a microorganism. Yeast has already been used

extensively in experimentation and production of many

fine chemicals and drug intermediates [8�,9��]. Com-

pounds such as glycerol [5�] and n-butanol [10], ethanol

from hemicellulose [11], more complex compounds in-

cluding alkaloids [12�], vanillin [13], and drug intermedi-

ates such as taxadiene and artemisinic acid [14,15��] have

all been produced in this microbe. In these programs,

homologous and heterologous biochemical pathways

have been manipulated to achieve industrially relevant

product yields in yeast. Development of expression sys-

tems extend from single gene protein expression based on

plasmids to the production of multigenic artificial

chromosomes [16]. These expanding capabilities have

been essential to a variety of research and industrial

successes extending from the production of enzymes

used in synthesizing commodities to the production of

high quality pharmaceutical intermediates. The com-

bined use of chemistry tools for purification, and struc-

tural determination applied to genetic modification of the

metabolome and expression of heterologous genes has led

to increasing molecular diversity from the flavonoid pro-

ducing pathway [16], as well as the specific expression of

different terpenoids in yeast [14,15��].

The challengeOver half of the drugs in the present day armamentarium

of the medical profession are based on natural compounds

[17�]. Molecules that have novel modes of action unfor-

tunately have no obvious structural characteristics that

indicate their possible therapeutic value. In addition,

synthesis of some compounds can be beyond the power

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Yeast expressing non-native compounds Goldman 391

of the organic chemist to produce in a cost effective

manner or at all [18]. The problem with ongoing discovery

efforts in the realm of natural products is that they are

technically very difficult [18,19]. Many organisms are too

small, produce too little compound, or only produce the

compound in specific tissues or at a specific time or as a

specific response to some event. This is further encum-

bered by the complexity of the extracts that are initially

acquired and the fact that some activities may be because

of combinatorial or synergistic activities of compounds in

the extract. A single plant extract may have 100 or more

alkaloids [20], more when the extracts are taken from

different plant tissues and from these same tissues during

differentiation, reproduction, stress, and even light and

dark conditions.

The activity of a natural compound is related to the

purpose for which the source organism has synthesized

it. Hence, in many cases the original compound has to be

modified to improve its therapeutic activities and charac-

teristics to make it acceptable as a chemotherapeutic for

humans. This process has historically been completed by

medicinal chemistry efforts and now can also be inves-

tigated by variations in biochemical pathways and com-

binatorial chemistry to produce different and improved

compounds [21�,22,23]. The use of the enzyme lovD with

broad substrate specificity to synthesize simvastatin and

huvastatin instead of lovastatin in Escherichia coli is an

excellent example of using biosynthetic and biocatalytic

capabilities to produce molecules of interest and vari-

ations of natural compounds with improved medicinal

properties [24]. An effort to reproduce this chemistry in

yeast has been reported by Ma et al. [25��]

In the face of all of the challenges in drug discovery using

natural compounds, there are many success stories cover-

ing the range of known pharmaceutical compounds with

activities ranging from antimicrobial and anticancer com-

pounds to analgesics and statins [9��,17�]. Nevertheless,

because of the difficulties in natural product drug dis-

covery this effort has been substantially curtailed by the

big pharmaceutical companies who have turned away

from natural product discovery to efforts focused on using

high-throughput assays technology and synthetically pro-

duced combinatorial chemistry libraries. New efforts in

resolving many of the challenges are coming from the

efforts of chemical biologist who use metabolic engin-

eering, synthetic biology in combination with improved

capabilities in analytical chemistry to discover new com-

pounds as well as produce intermediates or molecules of

therapeutic value [18,21�,22].

SolutionsThe ability to introduce and use foreign genes, gene-

products, and active enzymes has many benefits beyond

the simple production of non-native natural products. To

list just a few: the ability to produce large quantities of


materials such as, amino acids, proteins, secondary metab-

olites, and biopolymers [26]. The benefits range from

gaining access to molecules that would be very rare or

impossible to produce or purify from the naturally occur-

ring sources to the production of medically relevant

materials such as small molecules and drug intermediates

[15��,27]. In particular, the production of complex mol-

ecules using enzymatic systems benefits such efforts as

the production of antibiotics, many of which are semi-

synthetics [28]. There are additional benefits resulting

from the use of living systems to produce molecules of

interest. These include the fact that some molecules can

only be produced in living systems because of overly

complex synthetic routes including single isomer require-

ments or cost of goods limitations. Another consideration

is that production processes using living systems is often

significantly more environmentally friendly since toxic

chemistries and harsh conditions need not be utilized and

thus, expense and environmental impact can be greatly

reduced.

The methods developed for this kind of research and

production includes whole cell catalysis with substrate

feeding, and then similar efforts with the addition of an

engineered secondary metabolism, with or without sub-

strate feeding. Use of synthetic biology (synthesized gene

sequences) and native gene sequences to produce bioca-

talytic capacity in conjunction with engineered metab-

olisms and finally, the use of heterologous expression

systems to produce molecules from more complex genetic

constructs such as artificial chromosomes [16,29]. It

should be noted that most often successful production

and research efforts use aspects from more than one of the

described methods.

Whole cell biocatalysis using recombinant yeast in com-

bination with specific substrate and precursor feeding

regimes has been highly successful in the production

of active pharmaceutical intermediates (APIs). In a

review published in 2008 Pscheidt and Glieder [8�]describe fine chemical and active pharmaceutical ingre-

dients produced by different strains and species of yeast.

This intrinsic biochemical capacity has produced such

compounds as chiral building blocks using Pichia metha-noli to make ethyl-5-(S)-hydroxyhexanoate and 5-(S)-

hydroxylhexanenitrile, precursors for antagonist inhibit-

ing HMG-CoA reductase cholesterol production and

(1R)-phenylacetylcarbinol, a precursor for antiasthma

and allergy relief drugs.

The next development is the use of recombinant yeasts in

whole cell biocatalysis that have been metabolically

engineered. This has included many different types of

modifications including the addition [15��,16], deletion,

and/or mutation of homologous and heterologous genes

transformed into the yeast genome. The addition of new

genes or genetic modifications to the yeast genome to


392 Molecular Diversity

produce substrates and cofactors include increased pro-

duction of endogenous substrates like farnesyl pyropho-

sphate. In the report from Ro et al. [15��], increase in

farnesyl pyrophosphate production was needed to ensure

an increase of the product output. Not only was the

pathway engineered for higher substrate production

but increased substrate availability was also supported

by down regulation of an alternative sink for the substrate.

Figure 1

(Taken from Naesby et al. [16].) The generic isozymes of the flavonol pathwa

enzymes include phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxyl

isomerase (CHI), flavonone 3-hydroxylases (F3H) and flavonol synthase (FLS


With the addition of two heterologous genes from Artemisiaannua the synthesis of the immediate precursor to artemi-

sinin, an antimalarial drug, artemisinic acid was completed.

The increased production of FPP was accomplished by

direct and indirect upregulation of genes essential to the

synthesis of FPP and the downregulation of erg9, the first

step in sterol synthesis in yeast. Integration into the yeast

genome of a truncated 3-hydroxy-3-methylglutaryl CoA

y that complete the chemical synthesis of flavonols are diagramed. The

ase (C4H), 4 coumarate:CoA ligase (4CL), chalcone synthase (CHS) and

).



reductase significantly added to the level of production

of the required substrate. This truncated gene was

overexpressed in the production of artemisinic acid and

taxadiene, an intermediate in the synthesis of paclitaxel

(taxol), an anticancer drug first isolated from the bark of the

yew tree [14]. The effort to produce a taxol precursor

highlights one of the most critical reasons for pursuing

heterologous natural product production capabilities.

Taxol was originally extracted from the bark of the Pacific

Yew tree in very low yields. The production process today

uses Yew Tree cell lines from which the compound is

isolated. To date, no synthetic route of production makes

sufficient quantities to satisfy medical demand for this

compound. The report by Engels et al. [14], demonstrates

metabolic engineering of yeast to produce increased

amounts of the substrate geranylgeranyl diphosphate in

combination with the heterologous expression of a codon

optimized, heterologous gene taxadiene synthase to

produce the precursor taxadiene.

A similar approach was taken by Herrero et al. [30] who

genetically modified wine yeast to make a monoterpene

with the lis gene of C. breweri encoding S-linalon synthase.

This plant monoterpene was used as a model for genetic

modification of wine making yeast strains and was found

in subsequent work to not to be detrimental to yeast

survival and functional fermentation under microvinifica-

tion conditions.

Another important successful effort reported by Mutka

and coworkers was the metabolic engineering of yeast to

produce methylmalonyl-CoA as a substrate for sub-

sequent production of complex polyketides [31]. This

effort was accomplished in two different ways with the

use of both propionyl-CoA dependent and propionyl-CoA

independent routes. The presence of methylmalonyl-

CoA substrate allowed for the continued development

of non-native biosynthetic pathways based on modular

polyketides with a clear demonstration of activity by

synthesis of triketide lactone.

The study of the highly iterative polyketide synthase that

is responsible for the synthesis of lovastatin intermediates

in yeast also demonstrates the potential and importance

of heterologous synthetic systems. Xie et al. were able to

demonstrate the production of a number of lovastatin

analogs using lovD, which proved to have broad substrate

specificity toward acyl carriers after the megasynthase

completed synthesis of the precursor nonaketide [24].

Most recently, Ma et al. [25��] was able to express active

megasynthase in yeast while having to engineer the yeast

to express Aspergillus nidulans phospho-pantetheinyl

transferase (npaA). There has been no report to date that

yeast have produced either lovastatin or simvastatin.

Yeast has also been used to produce benzylisquinoline

alkaloids (BIA) as reported by Hawkins and Smolke [12�].


The genetic tools used in the production of BIA include

integration of heterologous P450 enzymes in the yeast

genome, cloning and expression of genes on plasmid

singly or in groups and feeding of substrates that are

not available from the yeast metabolome such as L-dopa-

mine and (R,S)-norlaudanonsoline. Genes that were

expressed on plasmids included norocluarine synthase

that catalyzes the condensation of L-dopamine and 4-

hydroxyphenylacetaldehyde. This product undergoes a

series of methylations and a final hydroxylation to pro-

duce (S)-reticuline, a major branch point for alkaloid

products. Several enzymes from plant flavonoid pathways

have been expressed in yeast as well and are reviewed by

Fowler and Koffas [27]. These include 4-coumaryl:CoA

ligase from P. crispum, chalcone synthase and chalcone

isomerase from P. hybrida, and a P450 from A. thalianawhich has led to the heterologous production of narin-

genin.

The most recent efforts to discover and produce non-

native molecules use another approach, the use of more

complex genetic structures such as artificial chromo-

somes. These efforts promise to greatly increase the

scientist’s capability to discover new molecules of in-

terest. The use of artificial chromosomes includes the

additional possibility of producing and discovering new

molecules that are naturally derived [16,29]. The most

recent expansion of these capabilities uses functionally

modified yeast strains (amino acid auxotrophs) supporting

a randomly assembled biochemical pathway where the

genes of that pathway are incorporated into an artificial

yeast chromosome [16]. This mixing on the level of genes

allows scientists to break the species barrier and offers the

opportunity of production of molecules never before seen

in nature synthesized by enzymatic systems. This method

sources m-RNA from a large variety of species and

biotopes. The mixtures of m-RNA are transcribed into

cDNAs that are integrated into yeast expression cassettes

and then assembled into expression enhanced yeast arti-

ficial chromosomes (eYAC). The randomly assembled

eYAC-mixtures are transformed into baker’s yeast thus

each individual cell expresses enzymes from a different

population of genes. Some of the enzymes will constitute

a metabolic pathway giving rise to novel biochemical

compounds. Naesby et al. [16] demonstrated the func-

tionality of this approach by randomly assembling genes

into artificial yeast chromosomes from the flavonol path-

way. Included are genes from 16 different species repre-

senting plants and fungi, the genes included

phenylalanine ammonia-lyase, cinnamate 4-hydroxylase,

4 coumarate:CoA ligase, chalcone synthase and isomer-

ase, flavonone 3-hydroxylases, and flavonol synthases.

Figure 1 shows the general biosynthetic pathway that

was modeled. A number of recombinant yeast containing

eYACs were analyzed by HPLC UV-chromatography

where different yeast showed very different patterns of

secondary metabolite production in the chromatographs.


394 Molecular Diversity

Precursor feeding expreriments were also completed with

the production of compounds such as pinocembrin, galan-

gin, naringenin, and eridictyol based on the specific

substrate feeding experiments.

ConclusionUntil recently the use of these tools in combination:

genome transformation and modification, episomal

expression elements, metabolic engineering for substrate

production, and substrate feeding have represented the

most flexible and successful expression system in yeast.

These capabilities, when combined produce a powerful

tool for the investigation of synthetic mechanisms and

ultimately commercial production of secondary metab-

olites. The addition of the artificial chromosome

assembled with unknown genes from eukaryotic organ-

isms and synthetic genes representing either partial or

completed biochemical pathways greatly increases the

potential diversity of the products and in high-throughput

mode allow the search for truly novel natural compounds

with important pharmaceutical properties.

Conflicts of interestThe author is an employee of Genetic Chemistry Inc., a

holding company of Evolva SA. Work from this company

is mentioned in this review.

AcknowledgementsI would like to thank Gary Liu, Ranjini Chatterjee, and the science teamfrom Evolva SA for reviewing this document.

References and recommended readingPapers of particular interest, published within the period of review,have been highlighted as:

� of special interest�� of outstanding interest

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2.�

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This work describes the use of genome sequence information to identifyproducts from pathways that are rich in producing druggable molecules(polyketide and nonribosomal peptide synthesis hybrids) with medicallyimportant activities.

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Cordier H, Mendes F, Vasconcelos I, Francois J: A metabolic andgenomic study of engineered Saccharomyces cerevisiaestrains for high glycerol production. Metab Eng 2007,9:364-378.

The authors describe using genomic information to tailor the metabolomeof a yeast strain to increase the production of glycerol.

6.�

Bumpus BB, Evens BS, Thomas PM, Ntai I, Kelleher NI: Aproteomics approach to discovering natural products andtheir biosynthetic pathways. Nat Biotechnol 2009, 27:951-956.

This reviews techniques that allow for the identification of biochemicalpathways that produce molecules of interest under very specificsituations.


7.�

Ro D, Ouellet M, Paradise E, Burd H, Eng D, Paddon C, Newman J,Keasling J: Induction of multiple pleiotropic drug resistancegenes in yeast engineered to produce an increased level ofanti-malarial drug precursor artemisinic acid. BMC Biotechnol2008, 8:83-97.

A very interesting analysis of the change in yeast transcriptome indicatingincreased levels of stress because of the expression of heterologousgenes required to synthesize artemisinic acid.

8.�

Pscheidt B, Glieder A: Yeast cell factories for fine chemical andAPI production. Microb Cell Factories 2008, 7:25.

Good recent review of the use of yeast whole cell systems for theproduction of active pharmaceutical ingredients and fine chemicals.

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Excellent review of yeast produced molecules with therapeutic applica-tions.

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12.�

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Heterologous expression of genes that are fundamental to the productionof a large class of alkaloid molecules greatly increasing the diversity ofheterologously produced small compounds.

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15.��

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Excellent demonstration of the use of whole cell biocatalysis, metabolicengineering, and heterologous gene expression to produce a drug inter-mediate in sufficient quantity to include the process in a pharmaceuticalproduct.

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17.�

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21.�

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This paper describes the use of synthetic chemistry tools to modifynatural compounds and expand the diversity of available compounds.

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25.��

Ma S, Li J, Choi J, Zhou H, Lee K, Moorthie V, Xie X, Kealey J, DaSilva N, Vederas J et al.: Complete reconstitution of a highlyiterative polyketide synthase. Science 2009, 326:589-592.

Metabolic engineering and heterologous gene expression effort to under-stand the synthetic components of the megasynthase that makes theprecursor to lovastatin in yeast.

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