454
Metabolic engineering for the microbial production of
1,3-propanediol
Charles E Nakamura and Gregory M Whitedy
Improvements in the biological production of 1,3-propanediol, a
key component of an emerging polymer business, have been
realized. Utilizing genes from natural strains that produce 1,3propanediol from glycerol, metabolic engineering has enabled
the development of a recombinant strain that utilizes the lower
cost feedstock D-glucose. This accomplishment bodes well for
future metabolic engineering efforts and, ultimately, for
increased societal benefit obtained through the production of
chemicals from renewable resources.
to 1,3-propanediol. Specialized enzymatic pathways for
both the oxidation (associated with carbon incorporation
into cell mass) and reduction of glycerol are segregated at
the DNA level and are employed only in the rare event
that glycerol is present but alternate carbon sources (e.g.
D-glucose) are not. To satisfy a conditional requirement of
microbial growth, a complex but robust process for the
anaerobic production of 1,3-propanediol from glycerol has
evolved.
Addresses
DuPont Central Research & Development Experimental Station,
328/245A Wilmington, DE 19880-0328, USA
e-mail: [email protected]
y
Genencor International, Inc., 925 Page Mill Road, Palo Alto,
CA 94304, USA
e-mail: [email protected]
Correspondence: Charles E Nakamura
Over the past few decades, there has been growing
interest in 1,3-propanediol as an industrial chemical. It
has long been known that a polymer prepared from
terephthalic acid and 1,3-propanediol has highly desired
properties for large volume markets (e.g. fiber for use in
apparel and carpet applications). The belief that improved
chemistries, including both traditional petrochemical and
biological routes, could enable the production of 1,3-propanediol with the economy required by these competitive
markets has fueled large efforts in this arena [6,7]. Biological efforts include fermentation optimization of the
natural glycerol-utilizing process and an ambitious metabolic engineering effort, directed towards a more economical process, to build a single microorganism capable of
utilizing the lower cost feedstock D-glucose. As aspects of
the natural fermentation process from glycerol have been
reviewed recently [5], the subject of this review is the
metabolic engineering of the novel D-glucose pathway
and recent developments relating to that effort.
Current Opinion in Biotechnology 2003, 14:454–459
This review comes from a themed issue on
Biochemical engineering
Edited by Jeremy S Edwards and Kenneth J Kauffman
0958-1669/$ – see front matter
ß 2003 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.copbio.2003.08.005
Abbreviations
3-HPA 3-hydroxypropionaldehyde
PEP
phosphoenolpyruvate
PTS
phosphotransferase system
Engineering 1,3-propanediol pathways
The role of coenzyme B12 reactions
Introduction
1,3-Propanediol is produced in nature by the fermentation of glycerol. Since it was first observed in the 19th
century [1], numerous aspects of this niche metabolic
pathway have served as focal points in the areas of
chemistry, enzymology, genetics, microbial physiology
and biochemical engineering [2,3,4,5]. For a large
number of bacteria, including Citrobacter, Clostridium,
Enterobacter, Klebsiella and Lactobacillus species, a consequence of anaerobic growth on glycerol is the generation
of excess reducing equivalents in the form of reduced
nicotinamide adenine dinucleotide (NADH) (Figure 1).
Regeneration of oxidized nicotinamide adenine dinucleotide (NADþ) necessitates the formation of a by-product to
serve as an electron sink. In a seemingly unlikely solution
to this redox requirement, glycerol undergoes a difficult
chemical rearrangement to 3-hydroxypropionaldehyde
(3-HPA) followed by an NADH-dependent reduction
Current Opinion in Biotechnology 2003, 14:454–459
Glycerol dehydratase (EC 4.2.1.30) is a coenzyme B12dependent enzyme composed of three polypeptides that
catalyzes the free radical mediated conversion of glycerol
to 3-HPA. As a consequence of the normal catalytic cycle
with glycerol, the coenzyme B12 is occasionally rendered
inactive (B12-inact). The B12-inact remains tightly bound to
the dehydratase and catalysis ceases. An auxiliary
enzyme, glycerol dehydratase reactivase, facilitates the
dissociation of the B12-inact to form glycerol dehydratase
free of cofactor (apoenzyme). The resultant apoenzyme
rebinds coenzyme B12 and glycerol conversion to 3-HPA
resumes. In yet another coenzyme B12 related activity,
coenzyme B12 is regenerated from B12-inact. Clearly, there
are potential issues that confound the use of glycerol
dehydratase as a foundation upon which to build a metabolically engineered strain for 1,3-propanediol production. The ability to dissect natural strains is essential for
engineering improved strains. Fortunately, the multiple
genes that encode these and other functionally related
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Microbial production of 1,3-propanediol Nakamura and Whited 455
Figure 1
OH
OH
Cell mass,
by-products
(e.g. acetate, NADH)
OH
Glycerol dehydratase (dhaB1–3)
H2O
OH
O
H
NADH
1,3-Propanediol oxidoreductase (dhaT)
NAD+
OH
OH
Current Opinion in Biotechnology
1,3-Propanediol production by natural organisms. In the absence of
oxygen, growth occurs by the oxidative consumption of glycerol to
produce cell mass concomitant with the accumulation of by-products
(e.g. acetate) and the generation of excess NADH. The NADH
cannot accumulate and must be converted to NADþ so as to maintain a
steady-state concentration of NADH and NADþ within the cell. Thus,
NADþ is regenerated by the reductive conversion of glycerol to
1,3-propanediol in two steps. The first step is a dehydration of glycerol
to 3-hydroxypropionaldehye, catalyzed by glycerol dehydratase
(encoded by the genes dhaB1–3). The second step is an NADHdependent reduction of the aldehyde to 1,3-propanediol, catalyzed
by 1,3-propanediol oxidoreductase (encoded by the gene dhaT).
activities are physically linked, being tightly clustered in
what in known as the dha regulon. Similarly, diol (1,2propanediol) dehydratase (EC 4.2.1.28), an isoenzyme of
glycerol dehydratase with overlapping substrate specificity, is also associated with a gene cluster (the pdu operon)
that also comprises genes encoding reactivase and B12regenerating activities.
Identification and engineering of new dehydratases
New, naturally occurring bacterial strains, including novel
species, that are able to produce 1,3-propanediol continue
to be identified: for example, lactic acid bacteria, species
of Clostridia and a thermophile (Caloramator viterbensis)
[8–10]. Historically identified by physiological tests
performed on pure cultures, the association between
anaerobic 1,3-propandiol (or 3-HPA) production and conserved regions of DNA has allowed molecular biology
screening techniques to be developed for the identification of these organisms. Ultimately, for the purposes of
metabolic engineering, dissection of the genetic organization [11,12] and biochemical characterization of relevant enzymatic steps [13,14–16] must follow strain
identification. The combined effort provides additional
tools to identify limiting steps in relevant pathways and
increases the knowledge base from which solutions arise
once limiting steps are identified.
www.current-opinion.com
The generation of environmental DNA libraries, by-passing the traditional isolation of pure cultures, allows the
evaluation of specific aspects of microbial diversity. Such
libraries were obtained for dehydratase (mixtures of glycerol and diol dehydratase) genes and screened using
dehydratase-specific DNA probes and activity assays
(growth on glycerol) in a specially constructed Escherichia
coli strain [17]. The objective of this work was the
isolation of dehydratase varients with higher resistance
to inactivation by glycerol and inhibition by 1,3-propanediol. Leading to a particularly interesting result, a
partial diol dehydratase gene set was isolated and the
incomplete portion was complemented with the corresponding DNA from Salmonella enterica. The fusion protein that resulted had greater resistance to inhibition by
1,3-propanediol than control dehydratases. This suggests
a potential role for protein engineering in improving the
performance of particular enzymes that may limit the
overall performance of a pathway. Independently, an
effort to generate mutant libraries of dehydratase genes
that were screened using high-throughput techniques has
provided significantly improved dehydratase enzymes
(X-S Tang, personal communication). Recent crystallographic studies of dehydratase enzymes might also aid
protein engineering efforts [18–20].
The reactivase system
The glycerol dehydratase reactivase reactions from Klebsiella pneumoniae and Citrobacter freundii have been characterized biochemically [14,15]. Like the previously
characterized diol dehydratase reactivase system, the
reaction is ATP-dependent and the reactivase has been
proposed to serve as a molecular chaperone. Recent
crystallographic evidence revealed structural similarity
to known molecular chaperones, supporting this hypothesis [21]. The pduO gene, obtained from the pdu operon of
S. enterica, has been shown to encode an ATP:cob(I)alamin adenosyltransferase that is involved in B12 regeneration [13]. By analogy, the previously unknown function
of genes present in the dha regulon was proposed to be B12
regeneration. In a related advance, a protein system that is
likely to be involved in B12 regeneration, upstream of the
ATP:cob(I)alamin adenosyltransferase reaction, has been
identified [22].
Fermentation studies
Traditional glycerol-fed (and glycerol/D-glucose co-fed)
fermentation studies incorporating balance of specific
substrate consumption and by-product formation rates
continue to provide practical information, particularly
when coupled with enzyme assays [23–30]. A particularly
nice example is exemplified by Saint-Amans et al. [27].
Glycerol dehydratase obtained during an examination of
Clostridium butyricum glycerol fermentations was observed
to have striking similarities to a B12-independent, oxygensensitive diol dehydratase previously isolated. Detailed
biochemical evaluation confirmed its B12-independence
Current Opinion in Biotechnology 2003, 14:454–459
456 Biochemical engineering
and full molecular characterization will allow its evaluation in anaerobic processes for 1,3-propanediol production [31].
Production of 1,3-propanediol from
D-glucose
Developing a single organism catalyst
In considering the merit of D-glucose versus glycerol as
feedstock for the production of 1,3-propanediol, it is
immediately apparent that D-glucose is not without its
drawbacks. The oxidation state of D-glucose, glycerol and
1,3-propanediol (on a carbon basis) is D-glucose > glycerol > 1,3-propanediol. An examination of the chemical
transformation of D-glucose to 1,3-propanediol (Equation
1) versus the transformation of glycerol (Equation 2)
illustrates that there is a penalty to be paid in using a
feedstock further removed from product with respect to
redox balance.
1=2 d-Glucose þ 4e þ 4Hþ ! 1;3-Propanediol þ H2 O
(1)
þ
Glycerol þ 2e þ 2H ! 1;3-Propanediol þ H2 O
(2)
A more relevant comparison emerges after imposing
biological pathways, including constraints associated with
sugar metabolism. While the carbon pathway from glycerol remains the same (Equation 3), the route from Dglucose incurs an additional penalty in the form of a highenergy phosphate bond (Pi) which accompanies the
phosphorylation of sugar by ATP or phosphoenolpyruvate
(PEP) (Equation 4).
Glycerol þ NADH þ Hþ
! 1;3-Propanediol þ NADþ þ H2 O
(3)
1=2 d-Glucose þ Pi þ 2NADH þ 2Hþ
! 1;3-Propanediol þ 2NADþ þ Pi þ H2 O
(4)
It is clear that D-glucose is more demanding in terms of
stoichiometric requirements for balance (i.e. the use of Dglucose requires more co-reactants than the use of glycerol). Co-reactants are generated from the substrate
(either D-glucose or glycerol), thus yield (on a carbon basis)
favors the glycerol pathway. However, the great equalizer
is feedstock cost. A comparison considering substrate
cost and yield establishes the merit of a direct D-glucose
route. In addition to yield, the fermentation issues that
dominate the economics of 1,3-propanediol production are
rate and titer. Given assumptions on yield and empirical
values of specific D-glucose consumption rates, encouraging estimates for 1,3-propanediol production rates are
obtained. Additionally, it is reasonable to assume that
the titers currently demonstrated in anaerobic glycerol
to 1,3-propanediol fermentations can be improved.
Parenthetically, the generation of any material from substrate — other than product and required co-reactant(s) —
is deleterious to yield. As a consideration related to the
Current Opinion in Biotechnology 2003, 14:454–459
supply of co-reactants, it is also apparent that, placing
arguments of co-product value and overall process complexity aside, the yield benefits from elimination of acid
by-product, thus implying aerobic metabolism.
The strategy and progress of an effort by DuPont and
Genencor International, Inc. to design and build a single
organism catalyst for the direct conversion of D-glucose to
1,3-propanediol has been revealed in a series of patents
and applications [32–34,35]. Most recently, this work
was also presented at a conference on metabolic engineering (CE Nakamura and P Soucaille, Metabolic Engineering IV: Applied Systems Biology, 6–11 October 2002,
Castelvecchio Pascoli, Italy) (Figure 2). The strain is
based on an E. coli K12 strain, which is eligible for
favorable regulatory status in the United States. The base
strain has only weak capacity to produce glycerol and no
capacity to produce 1,3-propanediol, thus the engineered
strain relies on a predominantly heterologous carbon
pathway that diverts carbon from dihydroxyacetone phosphate (DHAP), a major ‘pipeline’ in central carbon metabolism, to 1,3-propanediol. In direct contrast to processes
that use naturally available organisms, the DuPont/
Genencor process is aerobic. Moreover, significant modifications have been made to the host strain to provide
optimally for the energetic (Pi) and redox demands of
1,3-propanediol production.
The carbon pathway utilizes glycerol 3-phosphate dehydrogenase (DAR1) and glycerol 3-phosphate phosphatase
(GPP2) genes, obtained from Saccharomyces cerevisiae, to
provide glycerol [36,37]. Glycerol dehydratase (dhaB1,
dhaB2, dhaB3) and its reactivating factors (dhaBX, orfX),
obtained from K. pneumoniae, enable the conversion of
glycerol to 3-hydroxypropionaldehyde [32,34]. Surprisingly, a previously uncharacterized oxidoreductase endogenous to E. coli (yqhD) completes the pathway [35].
The preference for YqhD over the more obvious DhaT
(or similar dehydrogenases available from alternate dha
regulons) stems from fed-batch fermentation results in
which strains utilizing YqhD produce 1,3-propanediol
titers of approximately 130 g/L; such high titers are not
obtained in identical strains utilizing DhaT and are
unprecedented in glycerol-fed fermentations using natural 1,3-propanediol-producing organisms. In contrast
to DhaT, YqhD utilizes NADPH rather than NADH
(MH Emptage, personal communication) and it is likely
that the differences in the cofactor reduced/oxidized
ratios contribute to the higher titer.
The D-glucose-utilizing strain incorporates gene deletions that eliminate non-productive reactions. For example, once formed, glycerol is prevented from re-entering
central carbon metabolism by deletion of the genes
encoding glycerol kinase (glpK) and glycerol dehydrogenase (gldA) [33]. It is notable that a triosephosphate
www.current-opinion.com
Microbial production of 1,3-propanediol Nakamura and Whited 457
Figure 2
(a)
(b)
Glucose
Glucose
PEP, ATP
2ATP
ATP-dependent
glucose transport
PEP-dependent
glucose transport
tpi
DHAP
NADH
DAR1
GPP2
GPP2
dhaB1–3
NADH
GAP
NADH
DAR1
Glycerol
tpi
DHAP
GAP
To TCA cycle
and respiration
dhaT
1,3-Propanediol
gap
Glycerol
dhaB1–3
NADPH
To TCA cycle
and respiration
yqhD
1,3-Propanediol
Current Opinion in Biotechnology
Engineering metabolic pathways from D-glucose to 1,3-propanediol: development from (a) an early strain to (b) a late strain. The rationale is described
in the text. Gene names are indicated in blue; intermediate metabolites are indicated in black. Reactions encoded by genes native to the host organism
are indicated by black arrows (deleted native genes by an ‘X’); reactions encoded by genes obtained from donor organisms are indicated by green
arrows. Energy (ATP or PEP) and redox demand (NADH or NADPH) are shown by red arrows. The further metabolism of glyceraldehyde 3-phosphate
(GAP) via the tricarboxylic acid (TCA) cycle and respiration (oxygen consumption) contribute to cell mass, energy, NADH and NADPH generation.
DHAP, dihydroxyacetone phosphate.
isomerase (tpi) deletion was employed at early and intermediate stages of catalyst development. The tpi deletion
forces a 1:1 split of carbon flux at the fructose bisphophate
aldolase step of the Embden-Meyerhof-Parnas pathway:
one branch being forced towards 1,3-propanediol formation and the other towards the tricarboxylic acid cycle.
Yield increased nearly 40% with the incorporation of this
deletion [35]. Although the tpi deletion imposes an
artificial ceiling on yield (42.5% weight yield, 50% carbon
yield; if all carbon passes through fructose bisphosphate
aldolase), its utility in providing a flux control point that is
independent of modulation of enzyme expression was
invaluable in assembling the carbon pathway from DHAP
to 1,3-propanediol, up to the point where the artificial
ceiling on yield was approached.
The two most fundamental changes imposed on the
natural reactions of the E. coli-based strain are the elimination of D-glucose transport by the phosphotransferase
system (PTS) and downregulation of glyceraldehyde 3phosphate dehydrogenase (gap) (CE Nakamura, GM
Whited unpublished results). The former modification
addresses the limitation that D-glucose phosphoryation in
E. coli is largely PEP-dependent. If PEP is generated
solely via the enolase reaction of the Embden-MeyerhofParnas pathway, this constraint imposes a second artificial
ceiling of 42.5% weight yield in 1,3-propanediol production. Although alternate routes for the generation of PEP
exist, glucose phosphorylation is energetically more efficient if the PEP-dependent system is replaced with ATPwww.current-opinion.com
dependent phosphorylation. As a result, the PTS system
is replaced with a synthetic system comprising galactose
permease (galP) and glucokinase (glk), both genes are
endogenous to E. coli. The gap modification is motivated
by the requirement to remove the artificial ceiling
imposed by the tpi deletion (discussed above). Downregulation of gap, concomitant with reinstatement of tpi,
provides an improved flux control point.
Several additional reactions, outside of the direct carbon
pathway to 1,3-propanediol, were examined to maximize
biocatalyst efficiency. The examination involved modulation of rationally targeted enzyme expression levels and
as a result of these studies several further changes were
incorporated into the host strain. Although disclosure of
the details must await the publication of patent applications, the end result is a metabolically engineered organism that provides 1,3-propanediol at a rate of 3.5 g/L/h, a
titer of 135 g/L, and a weight yield of 51% in D-glucose
fed-batch 10 L fermentations. By contrast, typical maximum values for rate, titer and weight yield of 1,3-propanediol from the anaerobic fermentation of glycerol are
3.0 g/L/h, 78 g/L and 55%, respectively [5].
Conclusions
Biology, when compared to traditional chemistry, offers
alternate catalysts for the production of chemicals, often
through synthetic pathways and using starting materials
not available through traditional chemistry. Such is the
case for the production of 1,3-propanediol. The direct
Current Opinion in Biotechnology 2003, 14:454–459
458 Biochemical engineering
fermentation of glycerol allows the use of a renewable
feedstock in contrast to heterogeneous catalysis routes
that are dependent on petrochemical feedstocks, ethylene oxide or acrolein. Today, the value of 1,3-propanediol
lies predominantly in its use in polymers prepared from
1,3-propanediol and terephthalic acid, demand of which is
estimated to be one to two billion pounds per year in 10
years. Providing low-cost 1,3-propanediol will be the key
to competitiveness in this new market. It is noteable that
a leader in the chemical industry has elected to pursue a
biological route [38]; it is all the more noteable that a
genetic engineering effort, arguably unprecedented in
scope, resulted in the catalyst that is the centerpiece of
that process.
Toraya T: Radical catalysis of B12 enzymes: structure,
mechanism, inactivation, and reactivation of diol and glycerol
dehydratases. Cell Mol Life Sci 2000, 57:106-127.
Reviews the biochemistry and genetic organization of diol and glycerol
dehydratases with respect to catalysis, inactivation and reactivation by
their respective protein reactivating factors.
In fermentation, metabolic engineering expands the
range of chemical process solutions and enables improved
cost effectiveness of options based on natural biological
routes. The story of 1,3-propanediol represents a benchmark in metabolic engineering. Expanding the glycerolbased natural process to a more efficient process based
on lower cost carbon feedstock involved several steps:
changing an anaerobic process to an aerobic one; replacing
the feedstock uptake (transport) mechanism of the host
organism; intergeneric transfer of complex metabolic
pathways; and both the design and implementation of
an optimum solution to the balance of carbon, redox and
energy with respect to microbial growth and product
formation. The expectation of success presupposed that
sufficient biochemical, genetic and physiological knowledge was available for rational design and that the tools
necessary to design and implement genetic improvements were available. What was essential, but unavailable, was anticipated to be made available by concerted
effort. The success of the project bodes well for future
metabolic engineering efforts and, ultimately, for increased societal benefit obtained through the production
of chemicals from renewable resources.
Acknowledgements
We thank the many Genencor and DuPont individuals whose contributions
are cited in this paper. We gratefully acknowledge the significant
contributions of the broader scientific and engineering community that
served as the foundation upon which this work was based. Finally, we thank
Mark J Nelson and Scott E Nichols for helpful discussions and reading of this
manuscript.
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