Engineering The Heterotrophic Carbon Sources Utilization Range of Ralstonia Eutropha H16 For Applications in Biotechnology

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Crit Rev Biotechnol, 2016; 36(6): 978–991 ! 2015 Taylor & Francis. DOI: 10.3109/07388551.2015.1079698
REVIEW ARTICLE
Engineering the heterotrophic carbon sources utilization range of

Ralstonia eutropha H16 for applications in biotechnology
Elena Volodina1, Matthias Raberg1, and Alexander Steinbu ̈chel1,2
1Institut fu ̈r Molekulare Mikrobiologie und Biotechnologie, Westfa ̈lische Wilhelms-Universita ̈t Mu ̈nster, Mu ̈nster,
Germany and 2Environmental Science Department, King Abdulaziz University, Jeddah, Saudi Arabia
Abstract Ralstonia eutropha H16 is an interesting candidate for the biotechnological production of polyesters consisting of
hydroxy- and mercaptoalkanoates, and other compounds. It provides all the necessary characteristics, which are required for a
biotechnological production strain. Due to its metabolic versatility, it can convert a broad range of renewable heterotrophic
resources into diverse valuable compounds. High cell density fermentations of the non- pathogenic R. eutropha can be easily
performed. Furthermore, this bacterium is accessible to engineering of its metabolism by genetic approaches having available a
large repertoire of genetic tools. Since the complete genome sequence of R. eutropha H16 has become available, a variety of
transcriptome, proteome and metabolome studies provided valuable data elucidating its complex metabolism and allowing a
systematic biology approach. However, high production costs for bacterial large-scale production of biomass and
biotechnologically valuable products are still an economic challenge. The application of inexpensive raw materials could
significantly reduce the expenses. Therefore, the conversion of diverse substrates to polyhydroxyalkanoates by R. eutropha was
steadily improved by optimization of cultivation conditions, mutagenesis and metabolic engineering. Industrial by-products and
residual compounds like glycerol, and substrates containing high carbon content per weight like palm, soybean, corn oils as well
as raw sugar-rich materials like molasses, starch and lignocellulose, are the most promising renewable substrates and were
intensively studied.
Keywords Carbohydrates, fatty acids, glycerol, metabolic
engineering, PHAs, PTEs, Ralstonia eutropha, value-added bioproducts
History Received 21 November 2014 Revised 25 May 2015 Accepted 28 May 2015 Published online 27 August 2015
Introduction
Ralstonia eutropha H16 is a Gram-negative, rod-shaped and facultative chemolithoautotrophic β-proteobacterium
isolated from a spring in Germany. Initially described as Hydrogenomonas eutropha (Wilde, 1962), it has been the
subject of frequent taxonomical reclassifications: Alcaligenes eutrophus, R. eutropha, Wautersia eutropha, and
finally Cupriavidus necator (Reinecke & Steinbu ̈chel, 2009). However, the designation Ralstonia eutropha
remained the most established terminus in the scientific community and will be also used throughout this study.
As a ubiquitous inhabitant of soil and freshwater, R. eutropha is well adapted to constantly changing environ-
ments. Metabolic versatility of this microorganism made it to a model organism in microbiology concerning
hydrogen- based chemolithoautotrophy and poly-3-hydroxybutyrate [P(3HB)] synthesis (Reinecke & Steinbu ̈chel,
2009). Although aerobic respiration plays the major role in energy generation, R. eutropha can utilize nitrate and
nitrite as
alternative electron acceptors under anoxic conditions (Aragno & Schlegel, 1992, Pfitzner & Schlegel, 1973).
During heterotrophic growth, diverse carbohydrates, lipids and organic acids serve as carbon and energy source
(Figure 1), whereas in the absence of organic compounds a mixture of H
2
, CO
2
and O
2
enables R. eutropha to grow autotrophically. In dependency on the situation in the
habitat, R. eutropha can easily shift between autotrophy and hetero- trophy (Ka ̈rst & Friedrich, 1984).
Mixotrophical growth is observed when R. eutropha utilizes concomitantly organic and inorganic substrates as
carbon and energy source (Friedrich et al., 1981). The Calvin–Benson–Bassham (CBB) cycle of R. eutropha is
active in the polyhydroxyalkanoate (PHA) synthesis phase even during growth on fructose. On the other side, the
complete tricarboxylic acid cycle (TCC) is also active during autotrophic growth (Schwartz et al., 2009; Shimizu et
al., 2013).
During all nutrition modes, R. eutropha is able to store P(3HB). For this, two molecules of acetyl-CoA are
condensed by a β-ketothiolase (PhaA) to acetoacetyl-CoA. The latter is reduced by the NADPH-dependent
acetoacetyl-CoA reduc-
Address for correspondence: Alexander Steinbu ̈chel, Institut fu ̈r Molekulare Mikrobiologie und Biotechnologie, Westfa
̈lische Wilhelms-Universita ̈t Mu ̈nster, Corrensstraβe 3, D-48149 Mu ̈nster, Germany. E-mail: steinbu@uni-muenster.de
tase (PhaB) to (R)-3-hydroxybutyryl-CoA, which is further polymerized to P(3HB) by the PHA synthase (PhaC)
(Anderson & Dawes, 1990). These three genes are organized in one operon phaCAB, which is constitutively
expressed

independent from the growth conditions (Peplinski et al., 2010). However, intensive synthesis of P(3HB) only starts
if a carbon source is available in excess and if one essential nutritional element like N, O, S, Mg, K or P limits cell
growth. P(3HB) can be stored up to 90% of the cell dry weight (CDW) by R. eutropha H16. Besides
3-hydroxybutyrate (3HB), other short- and medium-chain-length (SCL and
Applications of Ralstonia eutropha H16 in biotechnology 979
DOI: 10.3109/07388551.2015.1079698
Figure 1. Scheme of the central carbon metabolism of R. eutropha illustrating the linkage of KDPG pathway, Calvin-cycle, TCC,
C3/C4-metabolism and FA β-oxidation (Bra ̈mer, 2002; Pohlmann et al., 2006; Schobert NAGPase,
N-acetylglucosamine-6-phosphate deacetylase; GPDA, & Bowien 1984). Abbreviations of enzymes: glucosamine-6-phosphate
deaminase; PGI, PTS
phosphoglucose Nag
, phospho transferase isomerase; system; G6PD, glucose-6-phosphate dehydrogenase; 6PGL, 6-phosphogluconolactonase; EDD,
gluconate-6-phosphate dehydratase; EDA, 2-keto-3-deoxygluconate-6- phosphate aldolase; TPI, triosephosphate isomerase;
FBPA, fructosebisphosphate aldolase; FBPP, fructose bisphosphatase; G3PD, glyceraldehyde-3- phosphate dehydrogenase; PGK,
phosphoglycerate kinase; PGM, phosphoglycerate mutase; ENO, enolase; PK, pyruvate kinase; PPS, phosphoenolpyruvate
synthetase; PRK, phosphoribulokinase; RuBisCo, ribulosebisphosphate carboxylase; PDHC, pyruvate dehydrogenase complex;
ME, malic enzyme; OD, oxaloacetate decarboxylase; PCK, PEP carboxykinase; PC, PEP carboxylase; PEP,
phosphoenolpyruvate; GlyK, glycerol kinase; Gly3P-DH, glycerol-3-phosphate dehydrogenase; FadD, acyl-CoA synthetase;
FadE, acyl-CoA dehydrogenase; FadB, enoyl-CoAhdratase/3- hydroxyacyl-CoA dehydrogenase; FadA, β-ketothiolase.
MCL) hydroxyalkanoates as well as mercaptoalkanoates are polymerized by PhaC
Re
(Table S1; 24, 73). Besides the academic interest, R. eutropha has found applications in
industry for large-scale production of PHAs (Byrom, 1987). PHAs have attracted much interest due to their
biodegradability and origin from renewable resources, as well as to thermoplastic/elastomeric properties similar to

synthetic polymers (Steinbu ̈chel & Valentin, 1995). With more than 150 different constituents, PHAs exhibit a
broad spectrum of properties and functionalities (Chanprateep, 2010). Consequently, various technical and medical
applications have been developed during the last decades (Anderson & Dawes, 1990; Hocking & Marchessault,
1994; Williams, 2005).
PHAs have been industrially produced since the 1970s (Imperial Chemical Industries (ICI); Monsanto:St. Louis,
MO; Metabolix: Cambridge, MA; Zeneca: London, UK, etc.). However, the production costs of bioplastics are still
5–10 times higher than costs for the production of conventional plastics from petrochemicals (Rehm, 2010). The
most critical aspect for industrial production bioproducts is their price competitiveness. Raw materials used for
cultivation have an important impact on total production costs (Chanprateep, 2010). Inexpensive raw sugar-rich
materials, such as molasses, starch and cellulosic resources, promise to reduce the production costs of microbially
synthesized products. Industrial production of plant oils and biodiesel is accom- panied with co-production of
glycerol-, triacylglyceride- and free fatty acid (FA)-rich residuals as by-products (Figure S1). FA-rich feedstocks are
substrates containing high carbon content per weight and can be transformed into biotechnologically relevant
products with a very high yield (Sudesh et al., 2011). The versatility of R. eutropha’s metabolism allows the
utilization of a broad range of carbon sources. Further extension of the carbon substrate range as well as optimiza-
tion of substrate conversion, are key for accelerating competitiveness of the gained products. Besides, the problem
of organic wastes disposal can be partially solved by conversion of residuals to valuable products (Chanprateep,
2010).
Knowledge concerning PHAs and their microbial production is steadily expanding. Novel biopolymers of
3-mercaptoalkanoates and cyanophycin, low-molecular weight compounds and alcohols recently extended the
range of products synthesized by R. eutropha (Grousseau et al., 2014; Li et al., 2012; Lu ̈tke-Eversloh et al., 2001;
Shiraki et al., 2006; Voss & Steinbu ̈chel, 2006). New information encour- aged reviews every few years (Agnew
& Pfleger, 2013; Braunegg et al., 1998; Chanprateep, 2010; Park et al., 2012; Steinbu ̈chel & Valentin, 1995).
Since the genome of R. eutropha H16 has been sequenced (Pohlmann et al., 2006), the energy metabolism of this
bacterium has been reviewed (Cramm, 2009). The genetic potential of this bacterium as a model organism, the
natural and engineered carbon storage pathways for production of PHAs and other bioproducts by R. eutropha were
analyzed (Brigham et al., 2012; Reinecke & Steinbu ̈chel, 2009; Riedel et al., 2014; Steinbu ̈chel & Lu ̈tke-
Eversloh, 2003; Tsuge, 2002).
Recently, a variety of studies about the extension of the carbon source utilization range of R. eutropha and its
optimization became available. To address these items, an understanding of the metabolic network of different
renewable substrates concerning genomic and metabolic back- ground of R. eutropha is essential. This stimulated
to review the recent advances in this field with respect to the application of R. eutropha H16 wild type, mutants and
metabolically engineered strains in biotechnology. The utilization of substrates that are mainly metabolized via the
Entner-Doudoroff
980 E. Volodina et al.
Crit Rev Biotechnol, 2016; 36(6): 978–991
(ED)-pathway or are oxidized in β-oxidation cycle and TCC besides lignin derivatives, amino acids, sulfur
compounds will be discussed and their impact on the synthesis of valuable products will be shortly reviewed.
Utilization of gluconate and carbohydrates
Besides gluconate only two carbohydrates, fructose and N-acetylglucosamine, are utilized as a carbon source by R.
eutropha H16 (Figure 1; Kersters & De Ley, 1984). They are expensive, and their application is therefore
economically not feasible for the production of cheap bulk products. Broadening the substrate range of R. eutropha
will allow using inexpensive and renewable crop-derived feedstocks. Alternatively, second generation products, like
lignocelluloses biomass and other agricultural wastes, provide a good alternative stock of carbon and energy for
microbial conver- sions. Lignocellulose is the most abundant raw material composed of lignin (10–20%) and the
carbohydrate polymers, cellulose (30–50%) and hemicelluloses (20–50%) (Figure S1; Keenan et al., 2006).
Cellulose is a polysaccharide of glucose. Hemicellulose is a heteropolymer consisting of glucose, xylose, mannose,
galactose, rhamnose, and arabinose. Celluloses and hemicelluloses can be microbially fermented or chemically
hydrolyzed into monosaccharides, which can then serve as substrates for biotechnical production of value- added
products (Limayem & Ricke, 2012).
N-Acetylglucosamine, fructose and gluconate
Fructose, N-acetylglucosamine and gluconate are catabolized in the ED-pathway with
2-keto-3-deoxy-6-phosphogluconate aldolase (EDA) as key enzyme, yielding pyruvate (Figure 1).
Phosphofructokinase (Pfk) and 6-phosphogluconate dehydrogenase are absent in R. eutropha H16; therefore, the
Embden– Meyerhof–Parnas (EMP)-pathway and the oxidative pentose phosphate pathways are incomplete.
However, genes for an anabolically operating EMP-pathway (gluconeogenesis) are present in R. eutropha
(Pohlmann et al., 2006). The catabolic EMP-pathway was accomplished in R. eutropha by heterologous expression
of pfk from Escherichia coli (Steinbu ̈chel, 1986). Mutants with a defective ED-pathway serve as a suitable
platform for the establishment of fructose utilization via the EMP-pathway, because the simultaneous function of
EMP- and ED-pathways in the wild type of R. eutropha had a negative effect on growth in presence of fructose.
Alternatively, the ability of EDA-negative mutants to utilize fructose was restored via heterologous expression of
the phosphoketolase gene from Bifidobacterium animalis (Fleige et al., 2011). These experiments showed that
central metabolic pathways can be switched off, while alternative routes can be easily established in R. eutropha.
Pyruvate formed in the ED-pathway is further decarboxylated by the pyruvate dehydrogenase complex (PDHC)
yielding acetyl coenzyme A (CoA). The latter is used in anabolic pathways for the synthesis of essential compounds,
like amino acids and lipids, directed to P(3HB) synthesis and stored as a source of energy and carbon, or directly
oxidized in TCC (Figure 1).
The amino sugar and glucose derivative N-acetylglucosamine is transported by the phosphoenolpyruvate (PEP)-
dependent phosphotransferase transport system (PTSNag)

into cells of R. eutropha (Figure 1). The responsible genes are organized in the nagFECABzwf1 cluster.
N-Acetylglucosamine enters the cell through a porin (NagC) located in the outer membrane (Kaddor & Steinbu
̈chel, 2011). Then, N-acetylglucosamine is phosphorylated by two proteins of the PTSNag, NagF and NagE.
Therefore, PEP provides phosphoryl to the soluble NagF, which comprises three PTS domains,
EINag-HPrNag-EIIANag. Phosphoryl is then trans- ferred from NagF to a cytosolic domain EIIBNag and a
membrane-spanning domain EIICNag of NagE. After phosphorylation of N-acetylglucosamine by NagE the
acetyl- moiety is cleaved off by N-acetylglucosamine-6-phosphate deaminase (NagA), and glucosamine is further
deaminated by glucosamine-6-phosphate deaminase (NagB) yielding fructose-6-phosphate. Fructose uptake is
mediated by a CUT2 family ATP-binding cassette (ABC)-type transporter (frcACB) (Figure 1; Gottschalk, 1964;
Pohlmann et al., 2006). Fructokinase catalyzes the phosphorylation of fructose. Fructose-6-phosphate, which is also
obtained from N- acetylglucosamine, is then converted to glucose-6-phosphate by phosphoglucose isomerase and is
further degraded in ED- pathway with gluconate-6-phosphate as one of the intermediates (Figure 1). If gluconate
serves as the substrate, it enters the ED-pathway as gluconate-6-phosphate. The gluconate transport is mediated by a
gluconate-H+ symporter catalyzed by a gluconate permease (GntP) and a gluconate transporter (GntT) (Kaddor &
Steinbu ̈chel, 2011).
Although R. eutropha H16 harbors only one specific transport system either for fructose or N-acetylglucosamine,
an additional truncated PTS comprises a variety of PEP-PTS homologous proteins, like PtsN, PtsM, PtsH, and PtsI
(Pohlmann et al., 2006). Recently, it was found that the truncated PTS is indirectly involved in the complex sugar
transport system, regulatory functions of carbon and P(3HB) metabolism (Kaddor & Steinbu ̈chel, 2011; Karstens
et al., 2014). Diminished P(3HB) contents and facilitated P(3HB) mobilization were observed in ptsM, ptsH, and
ptsI single, double, and triple mutants of R. eutropha (Kaddor & Steinbu ̈chel, 2011). The lack of PtsI and PtsH
impairs the transfer of a phosphoryl group to PtsN, as consequence the non- phosphorylated PtsN leads to a lower
P(3HB) content. A deletion of ptsN had an opposite effect and led to an increased P(3HB) content in the cells if they
were grown on gluconate. Further findings demonstrated that protein–protein interactions of non-phosphorylated
PtsN with the key enzyme of a stringent response SpoT1 (GTP diphosphokinase/ppGpp hydrolase) can indirectly
alter the global gene expression in the cell (Karstens et al., 2014). Thus, PtsN, in association with SpoT1, was found
to participate in the regulation of a stringent response in R. eutropha and consequently influences the P(3HB)
content.
Glucose
The wild type of R. eutropha is unable to utilize glucose as a carbon source. However, glucose-6-phosphate is a
common intermediate of the ED-pathway (Figure 1). Crude extracts of fructose-grown cells of strain H16 exhibit
hexokinase activity, which phosphorylates besides fructose, also mannose and glucose (Gottschalk, 1964).
Furthermore, it was demonstrated,
DOI: 10.3109/07388551.2015.1079698
that 14C glucose does not enter the cells. R. eutropha H16’s behavior was designated as cryptic with regard to
glucose, i.e. even cells, which were grown with fructose and equipped with all enzymes necessary for glucose
degradation, were unable to metabolize glucose.
Glucose-utilizing strains of R. eutropha and other bacteria have been intensively studied for biotechnological
production of PHAs. To overcome the incapability of R. eutropha H16 to utilize glucose and to extent the substrate
range, different attempts were undertaken. Historically, the first approaches were based on the isolation of
spontaneous glucose-utilizing mutants (Franz et al., 2012; Ko ̈nig & Schlegel, 1968), later also UV- (Schlegel &
Gottschalk, 1965) and chemically (Kim et al., 1995) induced mutants were obtained. Glucose-utilizing mutants were
provided by Schlegel and co-workers to ICI and one of them (deposited as R. eutropha NCIMB 11599) has been
intensively exploited for the commercial production of PHA (Byrom, 1987; Chanprateep, 2010). The UV-induced
glucose-utilizing mutants were able to transport glucose into the cells and exhibited constitutive
glucose-6-phosphate- dehydrogenase activity (Schlegel & Gottschalk, 1965). Multiple independent spontaneous
glucose-utilizing mutants were also isolated solely by provision of glucose to the media (Franz et al., 2012).
Recently, the glucose transport mechanism in G+1 mutant of R. eutropha H16 was resolved (Raberg et al., 2011,
2012). The glucose-utilizing G+1 mutant was generated in 1965 by Schlegel and Gottschalk by UV-mutagenesis
(Schlegel & Gottschalk, 1965). A mutated and derepressed PTSNag was found to be responsible for the glucose
transport (Raberg et al., 2011, 2012). A point mutation in nagR (a negative regulator of nag genes) of the G+1
mutant introduced a stop codon and caused a truncated repressor protein. Consequently, the derepression of the nag
operon due to inactive NagR led to a constitutive overexpression of the nag operon in mutant G+1. Furthermore, a
missense Ala153Thr mutation in nagE was identified, which positively affected glucose uptake mediated by the
mutated PTSNag in mutant G+1. The R. eutropha H16 AnagRnagEAla153Thr mutant strain showed the same
phenotype as strain G+1 regarding glucose utilization (Raberg et al., 2012). Another independent study also
identified mutations in nagR and nagE in glucose- utilizing R. eutropha NCIMB 11599 (Orita et al., 2012). The
observed single mutation in nagR likewise inactivated the regulator, and an additional mutation Gly265Arg in NagE
also improved glucose conversion. Although these two glucose- utilizing strains harbor different mutations in the
nagE genes, they exhibit the same phenotype. The roles of these amino acids have to be studied. A further important
finding is that the sugar is transported via facilitated diffusion and its transport is uncoupled from phosphorylation
by PTSNag. Intracellular phosphorylation is mediated by glucokinase Glk (Raberg et al., 2012). Another interesting
effect of a constitutively expressed and uncoupled PTSNag transporting sugar by facilitated diffusion is the
avoidance of carbon catabolite repression (Contesse et al., 1969), allowing to use media harboring different carbon
sources that can be utilized in parallel without diauxic growth (Raberg et al., 2012).
An alternative glucose-utilizing strain of R. eutropha was generated by metabolic engineering (Sichwart et al.,
2011).

Heterologous expression of the energy-independent glucose-facilitated diffusion transporter, glf, from Zymomonas
mobilis was used to mediate the glucose transport in the cell. Ralstonia eutropha H16 harboring glf was capable of
growing with glucose as sole carbon source after a lag phase. Co-expression of glf and glk (glucokinase from E.
coli) diminished the lag phase. The heterologous Glk compensated a possible metabolic bottleneck, possibly caused
by insuffi- cient expression/activity of the wild-type glucokinase. Reconstitution of glucose uptake and
phosphorylation in R. eutropha via heterologous expression of glf and glk from Z. mobilis was recently confirmed
(Orita et al., 2012). The strains harboring heterologous genes capable of glucose utilization stored comparable
amounts of P(3HB) like the wild-type R. eutropha grown with gluconate (Sichwart et al., 2011).
Mannose
In contrast to glucose, no spontaneous mannose-utilizing mutants of R. eutropha occurred. Besides the lack of
appropriate transporters for mannose, phosphorylation of mannose by the native hexokinase of R. eutropha was
found to be less efficient than phosphorylation of fructose and glucose (Gottschalk, 1964). Moreover, mannose
inhibits the oxidation of fructose in R. eutropha. An artificial pathway for the utilization of mannose by R. eutropha
H16 was established (Sichwart et al., 2011). For this, mak and pmi genes, encoding mannofructokinase and
phosphomannose isomerase from E. coli, were simultaneously expressed together with glf. Glf mediates not only the
transport of glucose but also of mannose, while Mak phosphorylates mannose and Pmi converts
mannose-6-phosphate to fructose-6-phosphate, which is metabolized in the ED-pathway. Ralstonia eutropha
possesses an own gene encoding Pmi. However, moderate growth of the recombinant strains of R. eutropha,
harboring either glf or glf together with mak, in the presence of mannose as sole carbon source, occurred only after
prior cultivation on fructose (Sichwart et al., 2011). This effect was not observed if the cells were pre-cultivated in
the presence of gluconate. Consequently, fructose seems to serve as inducer of pmi expression in R. eutropha.
However, further investigations and corresponding genetic modifications must be achieved in order to exploit the
native pmi from R. eutropha. Although phosphorylation of mannose is possible by a native hexokinase of R.
eutropha (Gottschalk, 1964), the heterologous Mak improved the growth rate of R. eutropha significantly. P(3HB)
contents of the recombinant strains grown on gluconate or mannose were similar, showing that mannose might be
also utilized for industrial production (Sichwart et al., 2011).
Xylose and arabinose
Xylose and arabinose are sugars which can be derived from hemicelluloses (Figure S2). Ralstonia eutropha H16 is
unable to utilize xylose or arabinose probably because it lacks enzymes mediating both, the uptake and the
catabolism of these two sugars (Pohlmann et al., 2006). Uptake of xylose in E. coli is achieved by an
ABC-transporter. The genome sequence did not reveal any hint for the presence of xylose- specific transporters in R.
eutropha. In E. coli, the catabolism
of xylose is initiated by xylose isomerase (XylA), which converts xylose to xylulose (Jeffries, 1983). Xylulose is
then phosphorylated by xylulokinase (XylB) to xylulose-5-phosphate, which is further converted to
ribulose-5-phosphate by ribulose-phosphate 3-epimerase (RPE, Table S2). In fungi and yeasts, catabolism of xylose
is initiated by an NADP-dependent xylose dehydrogenase, which forms xylonolactone from xylose; then lactonase,
xylonate dehydratase, 2-keto-3-deox- yxylonate dehydratase and aldehyde dehydrogenase convert the lactone via
xylonic acid to 2-oxoglutarate (Jeffries, 1983). Ralstonia eutropha lacks almost all the necessary genes for xylose
uptake and catabolism (Pohlmann et al., 2006). However, application of a xylose transporter (xylE), xylA and xylB
from E. coliin addition to a putativeR. eutropha’s genome encoded RPE was sufficient to establish xylose utilization
in R. eutropha W50 (Liu et al., 2014).
Alternatively, R. eutropha grows on xylose-containing feedstocks in combination with Lactococcus lactis IO-1 by
two-stage fermentation method (Tanaka et al., 1995). In the first stage, xylose was fermented to lactate and acetate
by L. lactis and in the second stage PHA was synthesized by R. eutropha.
Ralstonia eutropha W50 was enabled to utilize arabinose via heterologous expression of a set of genes for
L
-arabinose uptake and metabolism from E. coli W3110 (Lu et al.,
2013). The uptake of arabinose was catalyzed by the transporter which is encoded by the araFGH operon. In the
cells L
-arabinose was converted to
L
-ribulose by heterologous arabinose isomerase (AraA), then ribulose kinase
(AraB) phosphorylated
L
-ribulose to
L
-ribulose-phosphate, which was finally transformed to
D-
xylulose-phosphate. The putative RPE of R. eutropha might be further involved in the
metabolism of this carbohydrate. It was shown that the expression of the araBAD genes in R. eutropha W50 was
sufficient for utilization of
L
-arabinose; however co- expression of araFGH improved the uptake of
L-arabinose (Lu et al., 2013).
Starch
Starch is one of the most abundant carbohydrates contained in plants (potatoes, wheat, corn, rice, etc.), and
consequently appears in food wastes and starchy wastewaters (Figure S2; 127). The polysaccharide starch consists
of glucose moieties linked by α1–4 glycosidic bonds. Amylases and glucoamylases hydrolyze this polysaccharide
into maltose, maltotriose and glucose. Ralstonia eutropha lacks genes coding for putative amylases or
glucoamylases. Therefore, the utilization of starch by R. eutropha is only possible after prior starch hydrolysis.
Moreover, since glucose is the end product of starch saccharification, a glucose-utilizing strain of R. eutropha must
be available (see above). There are at least two different approaches to transform starch into PHA. One method
applies a two-step fermentation procedure (Yu, 2001). The starchy wastes are converted by the active sludge
containing acidogenic bacteria into volatile FAs (VFAs) such as formic, acetic, propionic and butyric acids. The
VFAs could then be converted into bioplastic by PHA- producing bacteria like R. eutropha during the second
fermentation step. Since propionic acid is available after the

first step, synthesis of P(3HB-co-3-hydroxyvalerate) is possible by this method. The second approach uses
hydrolysis of polysaccharides into sugars by commercially available enzymes (Haas et al., 2008). Thereby, starchy
wastes from a local potato chips factory were successfully converted to P(3HB) by glucose-utilizing R. eutropha
NCIMB 11599 after previous enzymatic hydrolysis. However, the energy- consumption during two-step
fermentation, down-stream processing and saccharification cause additional costs negatively influencing the
competitiveness of PHAs produced by this method. Therefore, the expression of heterologous amylases could be
another promising approach.
Lactose and galactose
Ralstonia eutropha H16 is not able to cleave the disaccharide lactose (Figure S1) or even to utilize galactose.
Consequently, growth of the wild type on these sugars as a sole carbon source is not possible. Cleavage of lactose
was conferred to the glucose-utilizing R. eutropha G+1 by heterologous expression of lacZ (β-galactosidase), lacI
(inducer gene) and lacO (operator) from E. coli (Pries et al., 1990). However, utilization of galactose by R. eutropha
remains unclear. One study showed that R. eutropha H16 was able to phosphorylate glucose and mannose, but not
galactose, arabinose and sorbose (Gottschalk, 1964). Another study demonstrated that the recombinant
glucose-utilizing strain R. eutropha G+1 excreted galactose into the medium, only if the lac genes were applied
(Pries et al., 1990). A concomitant utilization of glucose and galactose was possible only if the gal genes were
heterologously co-expressed with the lac genes. In contrast, recently another study demonstrated that the glu-
cose-utilizing strain of R. eutropha DSM 545 (H1G+3) was able to grow on galactose, lactose or whey permeate as
sole carbon sources harboring only the E. coli lac genes (Povolo et al., 2010). The lac genes were integrated in the
phaZ (PHA- depolymerase)-gene to minimize the mobilization of P(3HB). Since strains G+1 and H1G+3 are
UV-induced mutants, they might differ from the wild type and similar spontaneous mutants regarding the galactose
metabolism. Therefore, it would be interesting to clarify the mechanisms of galactose uptake and utilization.
Utilization of lignin derivatives
Besides hemicellulose lignin is another abundant waste product derived from lignocelluloses (Figure S1). After
chemical, physical or biological pretreatment of lignin, aromatic derivatives as p-coumaric, caffeic, ferulic and
sinapinic acid are released (Tomizawa et al., 2014). These substrates can be further metabolized by some bacteria
(Pseudomonas putida, Sphingomonas paucimobilis) to oxaloacetate and pyruvate with the following intermediates:
vanillic, 4-hydroxybenzoic (4-HBA), 2,5-dihydroxybenzoic/ gentisic (2,5-DHBA),
3,4-dihydroxybenzoic/protocatechuic (3,4-DHBA), and 3,4,5-trihydroxybenzoic/gallic (3,4,5- THBA) acid.
Ralstonia eutropha H16 was able to utilize 4-HBA, 2,5-DHBA and 3,4- DHBA and the cells stored significant
amounts of P(3HB) (Tomizawa et al., 2014). This can serve as a platform for establishment of the entire lignin
degradation pathway in R. eutropha. The introduction of
DOI: 10.3109/07388551.2015.1079698
heterologous genes responsible for the conversion of lignin to 4-HBA, 2,5-DHBA and 3,4-DHBA should
compensate the bottleneck in the lignin degradation pathway of R. eutropha.
Utilization of glycerol
Industrial production of biodiesel and plant oils is still developing; consequently, production of less valuable by-
products is also increasing. Transesterification of oils releases waste rich in glycerol and free FAs (Figure S1; 109).
As a main by-product of biodiesel industry, glycerol is an attractive cheap substrate for enhancing the
competitiveness of PHAs and other biobased products (Posada et al., 2011).
Two glycerol kinases GlyK and two glycerol-3-phosphate dehydrogenases Gly3-DH are located on both
chromosomes of R. eutropha (Table S2; Pohlmann et al., 2006). The genes from the first chromosome were found to
be up-regulated, when R. eutropha H16 was grown on trioleate (Brigham et al., 2010). Glycerol enters the cells
through passive diffusion and is phosphorylated by a kinase (Figure 1). Glycerol-3-phosphate is dehydrogenated to
dihydroxyacetone-phosphate before it is metabolized in the sugar-degrading pathway.
Growth of R. eutropha on glycerol occurs at a low growth rate, which is partially due to high activity of
hydrogenases overproducing reactive oxygen species, such as H
2
O
2
. Cells react with a stress response on oxidative stress grown
on glycerol. The pattern of metabolic response during glycerol utilization does not differ significantly from
autotrophic growth. The activity levels of hydrogenases, ribulose-1,5- bisphosphate-carboxylase/-oxygenase
(RuBisCO) and phosphoribulokinase (PRK) are comparable with autotrophic conditions (Friedrich et al., 1979;
Schwartz et al., 2009).
Several attempts were undertaken to optimize glycerol utilization by R. eutropha strains DSM 545 and IPT 026.
The glycerol and nitrogen contents in growth media were adjusted for P(3HB) synthesis (Campos et al., 2014;
Cavalheiro et al., 2009). Concentrations of waste glycerol higher as 40gLÀ1 were found to inhibit growth, probably
due to impurities in the feedstock (Cavalheiro et al., 2009). Besides P(3HB), synthesis of copolymers
P(3HB-co-3-hydroxyvalerate) and P(3HB-co-4-hydroxybutyrate-co-3-hydroxyvalerate) has been established from
by-products of biodiesel production (Cavalheiro et al., 2012; Garcıa et al., 2013). However, to significantly
accelerate the growth rate it was not sufficient to optimize only the growth conditions. A metabolic network of
intracellular processes during growth on glycerol was analyzed with elementary flux modes and yield space
analysis, and putative links between central metabolism and P(3HB) synthesis were marked (Lopar et al., 2014). A
linkage between Gly3-DH’s cofactor and NAD(H)/NADP(H) trans- hydrogenases was indicated. Facilitation of
crude glycerol uptake and metabolization in R. eutropha might compensate the slow cell growth in order to improve
the marketability of the bioproducts. Enhancing the glycerol uptake and improving the function of GlyK and
Gly3-DH and their interaction with the anabolic EMP-pathway and its metabolites was suggested (Lopar et al.,
2014). Indeed, integration of glpK
Ec
alone or together with aquaglyceroporin (glpF
Ec
) in the R. eutropha H16 genome compensated the slow growth rate and
significantly increased the P(3HB) productivity (Fukui et al., 2014).

Another study demonstrated that the presence of glucose in the medium tion of even at low concentrations reduces
glycerol in R. eutropha DSM 545 (S ˇ
poljaric ́ the consump- et al., 2013). Probably, the high intracellular pool of glycerol- 3-phosphate and
dihydroxyacetone-phosphate serves as a negative feed-back controller of glycerol consumption. These data are also
in accordance with an assumption about inhibition of initial glycerol activation reactions by the products of the
EMP-pathway (Lopar et al., 2014).
Another issue of interest is the molecular weight of P(3HB) derived from glycerol. An unspecific incorporation
of glycerol by PhaC can lead to a polymer chain termination during prolongated glycerol cultivation
(Tanadchangsaeng & Yu, 2012). Several studies reported on the reduction of the molecular weight of polymers
produced from glycerol (Cavalheiro et al., 2009; Tanadchangsaeng & Yu, 2012). Since no significant difference in
molecular weights of PHAs produced from glycerol or fructose were observed in R. eutropha H16 with facilitated
glycerol metabolism (glyK
Ec and glpF
Ec
), the reduction of intracellular glycerol concentration probably reduces the frequency of chain
terminations (Fukui et al., 2014).
Utilization of diverse organic acids
Fatty acids
Since FAs can be transformed into PHAs with a very high yield they are promising substrates for PHA production
and have the potential to reduce production costs. In contrast to carbohydrates, a variety of organic acids are taken
up by R. eutropha H16, so here is no need for substrate range expansions. However, optimization of the conversion
of FAs to products has to be achieved. Organic acids, such as acetic, propionic and succinic acids, are fermentation
products and common intermediates (in CoA-thioester form) of the central metabolism in R. eutropha. Acetic and
succinic acid are oxidized in the TCC, while propionyl-CoA is oxidized in the methylcitrate cycle (MCC).
Acyl-CoA thioesters of butyric and valeric acid (VA) as well as MCL- or long-chain-length FAs first undergo
β-oxidation to yield acetyl-CoA (Figure 1). Since acetyl-CoA can be completely oxidized in TCC, growth on the
above mentioned substrates yielding acetyl- CoA is combined with a loss of carbon as CO
2
and a lack of three-carbon (C3) and four-carbon (C4) units in the
central metabolism. The anaplerotic glyoxylate pathway, which bypasses the decarboxylation steps of the TCC,
mediates the conversion of the acetic residue (C2) to C4-compounds. The glyoxylate pathway comprises cleavage of
isocitrate into succinate and glyoxylate; the latter is further condensed with acetyl-CoA by malate synthase to yield
malate as intermediate (Figure 1). The concomitant product of the glyoxylate bypass, succinate, is metabolized in
the TCC. Another anaplerotic pathway mediates between C3- and C4- compounds during growth on C4-organic
acids like succinate or malate (Schobert & Bowien, 1984). The interconversion of C4- and C3-compounds is
catalyzed by PEP-carboxykinase to yield PEP from oxaloacetate by decarboxylation (Utter & Kolenbrander, 1972).
Alternatively, decarboxylation of malate/oxaloacetate catalyzed by malic enzyme or oxaloacetate decarboxylase
yields pyruvate (Figure 1; Bruland et al.,
2010). Thus, C4-compounds like malate and oxaloacetate are transformed to C3-products like pyruvate and PEP for
gluconeogenesis. Decarboxylation of malate to pyruvate is a NADPH-dependent reaction, and thus the C3/C4
metabolism regenerates the main cofactor of P(3HB) synthesis (Bruland et al., 2010; Yu & Si, 2004).
Acidifying agents commonly occur in the natural environment of R. eutropha, and even moderate
concentrations of them might lower pH and inhibit cell growth. SCL-FAs, like acetic, propionic, butyric and VA, are
often referred to as VFAs. They can freely diffuse through the membrane, acidify the cytoplasm which causes the
toxic effect and consequently slows down metabolite production. Dissociated FAs reduce the proton gradient
through the membrane, increase osmotic pressure and thus interfere with efficient energy metabolism (Wang et al.,
2010). To overcome the toxic effect of FAs, R. eutropha possesses a detoxification mechanism (Lee et al., 2006,
2009). Besides the toxicity, pure-free FAs are relatively expensive. Therefore, the controlled co-feeding at low con-
centrations in the medium is the prevailing method (Steinbu ̈chel & Lu ̈tke-Eversloh, 2003).
Even chain FAs
Even chain FAs provide exclusively 3HB-monomers for PHA synthesis in R. eutropha H16. Among all acids
utilized by R. eutropha, acetic acid exhibits the second highest dissoci- ation constant and consequently causes
severe toxic effects. The detoxification mechanism comprises the rapid metabolization of acetic acid. The tolerance
of R. eutropha towards acetic acid increases with the cell mass concentration and concomitant increase in utilization
rate of acetic acid (Yu & Wang, 2001). Therefore, acetate kinase and/or acetyl-CoA synthase activate(s) the
acetic-residue with CoA. Acetyl-CoA is further directed to P(3HB) synthesis, TCC and other biosynthetic pathways,
such as lipid or amino acid synthesis. Carbon flux analysis showed that the majority of the consumed acetic acid is
divided between P(3HB) synthesis and TCC and only a small part is condensed with glyoxylate (Yu & Si, 2004).
Growth of R. eutropha on acetic acid is associated with the upregulation of such detoxifying enzymes as, for
example, catalases, which are involved in a defensive response of R. eutropha on the toxic effect of organic acids
(Lee et al., 2009). In a mixture of organic acids, acetic acid is consumed after other acids like propionic, lactic or
butyric acid, are exhausted (Yan et al., 2003). Furthermore, two acetic-residues are necessary to produce one
molecule of 3HB. If butyric acid is used as sole carbon source, one molecule of butyric acid provides one molecule
of 3HB or two molecules acetyl-CoA, which are then oxidized in TCC, resulting in better cell growth and P(3HB)
yield. In addition, butyric acid is consumed to a higher rate and less toxic to the cell (Yan et al., 2003, Yang et al.,
2010). Recently, it was shown that butyric acid serves also as precursor for the production of PHAs with C6-units as
described below (Jeon et al., 2014).
γ-Butyrolactone, 4-hydroxybutyric acid (4HB), 4-chlorobutyric acid and 1,4-butanediol are precursors leading
to incorporation of 4HB in PHAs. Ralstonia eutropha synthesizes P(3HB-co-4HB) copolymers with different

4HB-fractions depending on the carbon source (Kunioka et al., 1989). However, although these substrates are taken
up by the cells, partially degraded and incorporated into the copolymer, R. eutropha H16 is not able to grow with
4HB or 1,4-butanediol as sole carbon source (Valentin et al., 1995). γ-Butyrolactone is putatively hydrolyzed,
4-chlorobutyric acid is dechlorinated and 1,4-butanediol is oxidized to 4-hydroxybutyryl-CoA, and the latter is then
incorporated into the copolymer (Kunioka et al., 1989). It is assumed that intracellular degradation of 4HB is carried
out via succinate semialdehyde to succinate (Valentin et al., 1995). P(3HB- co-4HB) exhibits biocompatibility,
decreased crystallinity and consequently better flexible/elastic properties as P(3HB) and is degraded by lipases and
esterases (Jaeger et al., 1995).
Optimization of 4HB uptake in R. eutropha improves conversion of 4HB to acetyl-CoA and its availability for the
central metabolism, while the molar 4HB fraction in the copolymer decreases (Steinbu ̈chel et al., 1994). Synthesis
of P(4HB) homopolyester was possible by P(3HB)-leaky mutants of R. eutropha JMP222, harboring multiple copies
of heterologous PHA biosynthesis genes from R. eutropha H16, if 4HB was used as the carbon source (Steinbu
̈chel et al., 1994).
However, the 4HB-precursors are toxic for cells at high concentrations and costly, that is why the two-step batch
or controlled co-feeding are the most commonly used proced- ures. The combination of sugar- or FA-containing
cheap substrates as main carbon source with more expensive co- substrates is more beneficial for industrial
production. Soybean, spent palm oil or waste glycerol in combination with 4HB-precursor such as γ-butyrolactone
were successfully applied for production of P(3HB-co-4HB). 4HB fractions of 6–10mol% in P(3HB-co-4HB)
were achieved by co-feeding soybean oil and 0.5–1% (w/v) γ-butyrolactone (Park & Kim, 2011) or even with up to
15mol% with spent palm oil plus 0.5% (w/v) γ-butyrolactone (Rao et al., 2010). Successful variation of
4HB-content in P(3HB-co-4HB) copolymers synthesized by R. eutropha DSM 545 from waste glycerol
supplemented with γ-butyrolactone was demonstrated (Cavalheiro et al., 2012). At higher levels of dissolved
oxygen, more PHA, though with minor 4HB- fraction, was stored and vice versa. The molar composition and in
particular the amount of 4HB in the copolymer can be also manipulated applying alkanoates as co-substrates. This
effect was observed with propionic, butyric, valeric and hexanoic acids; however, with propionic acid the highest
molar content of 4HB was detected (Kimura et al., 1999). The presence of propionic acid inhibited the degradation
of 4HB, and up to 43mol% of 4HB were incorporated in the copolymer (Cavalheiro et al., 2012; Kimura et al.,
1999). Besides, the co-utilization of γ-butyrolactone and propionic acid led to the synthesis of a new
P(3HB-co-4HB-co-3HV) terpolymer. P(4HB) homopolymer could be purified via fractionated isolation of PHA
from R. eutropha grown on mixture of 4HB and propionic acid (Kimura et al., 1999). For further details of
4HB-containing copolymers see below.
After it was shown that PhaC
Re
is also able to utilize MCL- hydroxyalkanoates (Dennis et al., 1998), the
production of copolymers with chain lengths >C5 has been studied intensively in R. eutropha. It is known that
supply of
DOI: 10.3109/07388551.2015.1079698
MCL-precursors is hindered by highly active β-oxidation. Precursors of MCL-PHAs are CoA-thioesters of FAs
that are usually completely degraded to acetyl-CoA. Two operons were found to be responsible for FA degradation
in R. eutropha (Table S2; Brigham et al., 2010). The FA degradation operons seem to be constitutively expressed on
fructose and upregulated in presence of FAs (Shimizu et al., 2013). Consequently, high amounts of 3HB-precursors
are provided by highly active β-oxidation and abundance of acetyl-CoA. Chemical inhibition of β-oxidation by
acrylate resulted, in contrast, in the integration of 3-hydroxyhexanoate (3HHx) and 3-hydroxyoctanoate in
copolymers by R. eutropha (Green et al., 2002). Molecular engineering tech- niques and appropriate carbon sources
optimized P(3HB-co- 3HHx) production in R. eutropha (Budde et al., 2011; Mifune et al., 2010). Therefore, the
efflux of β-oxidation intermediates to PHA synthesis was forced via overexpression of an (R)-enoyl-CoA
hydratase or a 3-hydroxyacyl-ACP:CoA transferase genes. Manipulation of the phaCAB operon resulted in
copolymers with new compositions (Matsumoto et al., 2001; Steinbu ̈chel & Lu ̈tke-Eversloh, 2003). Two
homologues of a (S)-specific 2-enoyl-CoA hydratase/(S)-3-hydroxyacyl-CoA dehydrogenase from the β-oxidation
operons were deleted in order to improve the incorporation of MCL-hydroxyalkanoates (Insomphun et al., 2014).
Alternatively, P(3HB-co- 3HHx) was successfully produced from butyric acid as single carbon source (Jeon et al.,
2014). For this, phaC
Re
was replaced by phaC from Rhodococcus aetherivorans
I24 exhibiting broad substrate specificity, and all three genes encoding for acetoacetyl-CoA reductases were deleted
in R. eutropha. In this mutant, the 3HB-content in the copolymer was reduced. Condensation of an acetic-residue
with butyric acid provided as carbon source yielded 2-ketohexanoyl-CoA, which was thereafter incorporated into the
copolymer. The C2- and C4-CoA thioesters are putatively condensed by one of the multiple β-ketohiolases of R.
eutropha. However, BktB is the most suitable candidate, since it assisted in this reaction in an artificial 1-hexanol
pathway (Dekishima et al., 2011). Further approaches for MCL-PHA biosynthesis based on FA metabolism are
discussed in more detail elsewhere (Riedel et al., 2014).
Odd chain Fas
Usually odd chain FAs are degraded via β-oxidation to acetyl- CoA and propionyl-CoA as end products.
Propionyl-CoA contributes to synthesis of a heteropolymer consisting exclusively of 3HB and 3-hydroxyvaleric
acid (3HV), i.e. P(3HB- co-3HV) (Doi et al., 1988). Different 3HB/3HV ratios influence physical properties of
synthesized copolymers. Generally, the technical applications of P(3HB-co-3HV) are more beneficial than P(3HB)
due to improved flexibility and reduced crystallinity. Melting temperature of these copolymers decreases with
increased 3HV amounts (Wang et al., 2013).
Once propionyl-CoA is formed from propionic acid, it can be directed to the central metabolism or to PHA
metabolism (Lee et al., 2009). The catabolism of propionic acid in MCC yields succinate and pyruvate with the
latter further decarboxylated to acetyl-CoA (Figure 1, 5). Serving as sole

carbon source, propionic acid is mainly converted to acetyl- CoA, resulting in a restricted flux of propionyl-CoA to
the copolyester synthesis. Interestingly, another β-ketothiolase, BktB, is responsible for P(3HB-co-3HV) synthesis.
Whereas BktB is able to condense propionyl-CoA and acetyl-CoA, PhaA is not (Slater et al., 1998). Ralstonia
eutropha lacking bktB grows as the wild type on propionic acid, but is impaired in synthesis of 3HV from
propionyl-CoA (Lindenkamp et al., 2012). Thus, the copolymer from the bktB-lacking mutant contained only up to
3.5 mol% of 3HV.
Propionyl-CoA is condensed by BktB with acetyl-CoA, and reduced by PhaB to 3HV-CoA. If the ratio of
acetyl-CoA/ propionyl-CoA in the cell is high, more 3HB-units are incorporated into the copolymer. In contrast, if
the concentration of propionic acid in the medium is increased, this shifts this ratio towards propionyl-CoA, and
higher 3HV- molar fractions in the copolymer (22–45mol%) (Doi et al., 1987). When acetic and propionic acids are
present in the culture medium, acetyl-CoA is obtained from both substrates (Doi et al., 1987), but it was shown that
propionic acid is more favorably consumed as acetic acid (Yang et al., 2010). Metabolic fluxes show that only small
amounts of propionyl- CoA are converted to 3HV, while MCC consumes the majority of propionyl-CoA (Yu & Si,
2004). Consequently, the acetyl-CoA/propionyl-CoA ratio in the cell is higher only if propionic acid is provided,
and the copolymer contained 2–28mol% of 3HV (Doi et al., 1987). In general, the application of a mixture of acids
allows the modulation of PHA synthesis yielding the desired copolymer composition (Yang et al., 2010).
Provision of butyric (Yu & Si, 2004) or oleic (Marangoni et al., 2000) additionally to propionic acid improves the
total PHA yield. However, only due to an increasing acetyl-CoA/ propionyl-CoA ratio and hence higher 3HB
supply, while total 3HV content is less affected. An interesting effect on carbon distribution in the cell was observed
with the phosphorus limitation strategy during growth of R. eutropha DSM 545 with propionic acid and both
propionic and butyric acids (Grousseau et al., 2014). Unlike nitrogen limitation, phosphorus limitation negatively
affected the specific 3HB production rate and supported 3HV production.
VA also serves as precursor for PHA. Ralstonia eutropha H16 synthesizes the copolymer of P(3HB-co-3HV)
with up to 75 mol% 3HV when grown with VA. If VA is applied as sole carbon source, valeryl-CoA is degraded via
β-oxidation to acetyl-CoA and propionyl-CoA. Simultaneous application of butyric acid and VA reduces 3HV
molar content due to higher 3HB content (Doi et al., 1988). BktB plays also a significant role in metabolism of VA.
Ralstonia eutropha lacking bktB retains the capability to grow on VA and on propionic acid, but incorporates higher
amounts of 3HV (up to 97 mol%) into the copolymer (Lindenkamp et al., 2012).
Propionic and VAs are not applicable in high concentrations, because of their toxicity and costs. Utilization of
diverse cheap feedstocks supplemented with propionic or VA are more suitable for P(3HB-co-3HV) synthesis. Up to
90% PHA of CDW with 7 mol% of 3HV was obtained from palm kernel oil with propionic acid as co-substrate or
89% PHA of CDW with 14 mol% of 3HV from olive oil supplemented with VA (Lee et al., 2008). Generally,
higher 3HV fractions from
VA in comparison to propionic acid are explained due to sufficient provision of acetyl-CoA for the central
metabolism from degradation of plant oils. Thus, less VA is cleaved and directed in the central metabolism, but
enhanced integration of 3HV into the copolymer occurs.
Levulinic acid (LA) serves as sole carbon and energy source and as precursor for 3-HV and 4-HV for R.
eutropha. It appears in waste products of wood industry and can be derived from acidic hydrolysis of starch and
lignocellulosic biomass (Assary et al., 2010). LA is the oxidized form of 4HV, and its catabolism starts with
activation to levulinyl- CoA by a membrane-bound acyl-CoA synthetase. The acyl- CoA synthetase activity was
absent in cells grown on acetate, demonstrating an inducible character of LA conversion (Jaremko & Yu, 2011).
Acyl-CoA dehydrogenase and enoyl-CoA hydratase/carnithine racemase were also induced in the presence of LA,
together with glutathione transferase and a chaperon, which might be involved in a detoxifying response (Bra ̈mer,
2002). Acyl-CoA dehydrogenase and enoyl-CoA hydratase/carnithine racemase activity support the assumption that
LA is degraded via β-oxidation. Thus, the expected end products of the catabolism of LA in R. eutropha are
acetyl-CoA and propionyl-CoA. Since the β-oxidation inhibitor acrylate prevented growth of R. eutropha with LA
as sole carbon source, thiolytic cleavage of CoA-derivatives of LA was indirectly approved (Bra ̈mer, 2002).
However, no other intermediates, except acetyl-CoA and propionyl-CoA were observed in in vitro enzyme assays,
supporting the assumption that levulinyl-CoA is directly cleaved into acetyl- CoA and propionyl-CoA (Jaremko &
Yu, 2011).
Since the utilization of LA as sole carbon source yields low values of biomass and PHA content, co-feeding of LA
with glucose or fructose increases the productivity. When R. eutropha was grown under optimized conditions with
glucose and LA, cells reached higher cell densities and stored PHA up to 81% of CDW (Wang et al., 2013). The
3HV monomer composition varied from 25 to 54mol%. It was shown that at low LA-concentrations, it was utilized
at higher rates as glucose and fructose. Concerning PHA synthesis, 3HV-incorporation was less affected by nitrogen
limitation, as it is known for the 3HB-monomer. The highest 3HV fraction in copolymers was observed in the
transition growth phase, when nitrogen was still available in excess and before significant 3HB synthesis started.
Thus, due to the variation of C/N-ratio the desired PHA composition can be achieved (Jaremko & Yu, 2011). During
growth on LA, different monomers, such as 3HV and 4HV, can be incorporated into the copolymer (Valentin &
Steinbu ̈chel, 1995). Furthermore, recombinant R. eutropha was able to synthesize terpolyesters consisting of 3HB,
3HV and minor amounts of 4HV when cultivated with 4HV or 4-valerolactone (Valentin et al., 1992). Application
of 5-chlorovaleric acid alone or in combination with VA as the carbon source resulted in the synthesis of
P(3HB-co-3HV-co-5-hydroxyvalerate) (Doi et al., 1987). Further substrates, such as 5-hydroxyvalerate and
!-pentade- calactone, were successfully used for synthesis of P(3HB-co-3-hydroxypropionate-co-5-hydroxyvalerate)
yielding 5-hydroxyvalerate contents up to 10mol% (Chuah et al., 2013). The PHA-negative mutant R. eutropha
PHB-4 harboring phaC
Re
or mutated copies of phaC
Re
, was cultivated in a

two-step fermentation. In the second step, different concentrations of 5-hydroxyvalerate or !-pentadecalacton were
tested. The obtained lipase-degradable copolymer exhibited reduced crystallinity, melting temperature and tensile
strengths, and increased elongations to break-values in comparison to P(3HB). Alternatively, when other odd chain
compounds like 3-hydroxypropionate (3HP), 1,5-pentanediol or 1,7-heptanediol were used as carbon source,
synthesis of P(3HB-co-3HP) with a 3HP-fraction up to 7mol% was observed. From these, 1,5-pentanediol led to the
highest copolymer content (Nakamura et al., 1991). Copolymers containing 3HP up to 2.1 mol% could be also
produced from structurally unrelated substrates via modified metabolism of R. eutropha (Fukui et al., 2009).
Heterologous malonyl-CoA reductase and the 3HP-CoA synthetase domain of trifunc- tional propionyl-CoA
synthase from CO
2
-fixation pathway of Chloroflexus aurantiacus were introduced in R.
eutropha and catalyzed 3HP-formation from acetyl-CoA via malonyl- CoA, when the cells were grown with fructose
or even chain alkanoates.
Amino acids and modifications on amino acid metabolism
Ralstonia eutropha H16 synthesizes PHAs also from amino acids as sole carbon source although high yield of PHAs
were not obtained, since the dissimilation of amino acids is associated with nitrogen supply (Kimura et al., 2003).
Moreover, single amino acids are costly. Grown on amino acids, R. eutropha accumulates either P(3HB) or
P(3HB-co- 3HV). 3HV is provided from propionyl-CoA obtained during degradation of leucine, isoleucine,
threonine and valine (Figure S2).
L
-Valine was studied in more detail, since it was more effective regarding P(3HB-co-3HV), yielding
the copolymer with 10 mol% of 3HV.
Since propionyl-CoA is involved in the metabolism of some amino acids, an auxotrophic mutant of R. eutropha
H16 with altered anabolism of branched-chain amino synthesized P(3HB-co-3HV) from single unrelated substrates
such as fructose or gluconate. The mutant excreted valine, leucine and isoleucine into the medium, when a nitrogen
source was available in excess, while ammonium limitation yielded P(3HB-co-3HV) in propionic acid-free medium
(Steinbu ̈chel & Pieper, 1992). Such intracellular overproduction of the amino acids or related intermediates
provides propionyl-CoA from renewable resources.
Recombinant R. eutropha PHB-4 harboring phaC from Pseudomonas sp. 61-3 synthesized
P(3HB-co-3-hydroxy-4- methylvalerate) [P(3HB-co-3H4MV)] on fructose medium supplemented with leucine
(Saika et al., 2011). Amino acid co-feeding was chosen as an alternative to supply of costly 4-methylvalerate, since
the leucine backbone is identical with 3H4MV (Figure S2). To increase the 3H4MV fraction without leucine
co-feeding, the end product feedback inhibition was overcome by mutagenesis, and an increased metabolic flux to
leucine biosynthesis was obtained. Consequently, the mutant strain incorporated higher amounts of 3H4MV when
grown on fructose.
L-
Serine,
L-
alanine and
L-
threonine supplemented in addition to β-oxidation-unrelated substrates (acetic acid,
glucose) led to P(3HB-co-4HB) accumulation (Kimura et al., 2008).
DOI: 10.3109/07388551.2015.1079698
amino Supplementation of
L-
acids had a positive effect on CDW with highest values by
L-
threonine and threefold higher P(3HB-co-4HB) yields, as shown in the absence of
amino acids. In presence of
L-
serine higher 4HB-fractions than with
L-
alanine and
L- threonine occurred. This is putatively due to more favorable utilization of
L-
threonine for cell growth and 3HB synthesis.
Mercaptoalkanoic acids
Cultivated in presence of sulfur-compounds, such as 3-mercaptopropionic (3MP), 3,30-thiodipropionic (TDP),
3,30-dithiopropionic (DTDP), 3-mercaptobutyric (3MB) or 3-mercaptovaleric (3MV) acid, R. eutropha incorporates
3MP, 3MB or 3MV into the corresponding copolymers with 3HB (Lu ̈tke-Eversloh et al., 2001; Lu ̈tke-Eversloh
& Steinbu ̈chel, 2003). However, R. eutropha cannot utilize these substrates as the sole carbon source. The
enzymes mediating uptake of 3MP, TDP and DTDP or cleavage of TDP and DTDP are important for improving the
availability of 3MP for polythioester (PTE) synthesis. In contrast, the activity of enzymes metabolizing 3MP to
propionyl-CoA are less favorable, since they compete with PTE synthesis for 3MP. PTEs were first identified in
2001 (Lu ̈tke-Eversloh et al., 2001). The copolymers of 3HB and 3-mercaptoalkanoates exhibit biotechnologically
attractive features differing from PHAs in their melting point or glass transition temperature. Moreover, in contrast
to PHAs, PTEs are not biodegradable (Elbanna et al., 2004).
The metabolism of 3MP, TDP and DTDP in R. eutropha is still not completely clear. Genome-wide
transcriptome analysis of R. eutropha H16 revealed genes which were upregulated and might participate in the
utilization of TDP or DTDP (Peplinski et al., 2010). Consequently, several deletion mutants were generated to
identify genes required for the metabolism of these substrates (Doberstein et al., 2014). Single and double deletion
mutants lacking ABC-type transporter(s) exhibited slower growth with DTDP (Table S2). Interestingly, only a
deletion of the cluster H16_A0357- A0359 led to significantly lower 3MP-fractions in the copolymer and only
during growth with DTDP. In contrast, double deletion AH16_A0357-A0359AH16_A3658-A3660 resulted in
higher 3MP-fractions. Applying DTDP as precursor, less 3MP was also incorporated if bug genes encoding
putative Bordetella-type uptake proteins were deleted. DTDP is probably metabolized by a disulfide reductase to
3MP, while TDP is converted by a sulfide hydrolase to 3MP with concomitant 3HP release. Disulfide interchange
proteins DsbA, DsbD, FrnE, rhodonase-related sulfurtransferase and also hydrolase S-adenosylhomocysteinase,
might be involved in the cleavage of DTDP since the expression of the corresponding genes was upregulated in
presence of sulfur- compounds (Peplinski et al., 2010). However, only deletion of dsbD led to a significant reduction
of 3MP-molar fraction in the copolymer (Doberstein et al., 2014).
3MP is further activated to 3MP-CoA and polymerized by PhaC. PhaB3 seems also to be important for
P(3HB-co-3MP) synthesis, since its expression was two- to five-fold higher than during growth on gluconate
(Peplinski et al., 2010). On the other hand, the expression of phaB3 was reduced upon the

onset of significant P(3HB) synthesis (Budde et al., 2010). Genes of FA metabolism were also upregulated during
growth with DTDP or TDP, suggesting the metabolization of 3MP and 3-hydroxypropionate to propionyl-CoA
(Table S2; Peplinski et al., 2010). Other genes, encoding cysteine dioxygenase (CdoA), 2-methylcitrate synthase
(PrpC1) and propionate CoA transferase (Pct), of R. eutropha were not differentially expressed in the microarray
analysis. However, these enzymes have also been studied in terms of PTE synthesis. CdoA converts 3MP to
3-sulfinopropionate (Bruland et al., 2009). 3-Sulfinopropionate can be converted to propionyl-CoA and oxidized in
the MCC. The activity of the key MCC-enzyme, PrpC1, was increased in the late stationary phase in presence of
3MP-precursors (Peplinski et al., 2010). The lack of pct in the corresponding negative mutant of R. eutropha did not
affect 3MP fraction in the copolymer (Lindenkamp et al., 2013). The gene might be silent but the enzyme was able
to activate 3MP with CoA, which suggests that overexpression of pct might increase the 3MP fraction in the
copolymer.
Since synthesis of P(3MP) homopolythioesters was shown only for E. coli, but not for R. eutropha, suppression
of 3HB-CoA synthesis and deletion of β-ketothiolases was investigated (Lindenkamp et al., 2010). Deletion of 9 of
14 β-ketothiolases in fact resulted in the increased molar 3MP- fraction in the copolymer due to lowered
3HB-contents.
Thus, it was shown that the 3MP-fraction in the copolymer synthesized by R. eutropha can be varied. The most
important drawbacks like low yield copolymer synthesis and costliness of the precursors need to be addressed.
Further improvements by metabolic engineering and establishment of copolymer synthesis from inorganic sulfur as
PTE-precursor would be advantageous (Wu ̈bbeler & Steinbu ̈chel, 2014).
Utilization of C1-compounds
Among biotechnologically relevant C1-substrates such as CO, CO
2
, methanol and formate, only CO
2
and formate are utilized by R. eutropha as the sole carbon source
(Friedrich et al., 1979). CO
2
and formate differ from other substrates discussed in this study, since they are metabolized in a
similar manner via the CBB cycle. In the presence of CO
2
as the sole carbon and H
2
as energy source, R. eutropha assimilates CO
2
by the key enzyme RuBisCO (Figure 1), while three distinct
oxygen-tolerant [NiFe]-hydrogenases deliver energy via H
2
oxidation (Lenz et al., 2010). One hydrogenase is membrane-bound
and channels electrons into the electron transport chain. The second hydrogenase is soluble and generates reducing
equiva- lents (NADH), which are especially needed in the absence of organic compounds for CO
2
-fixation. The third regulatory hydrogenase controls gene expression of the
membrane-bound and soluble hydrogenases in response to the availability of H
2 (Burgdorf et al., 2005; Kleihues et al., 2000).
If formate serves as a substrate, hydrogenases and enzymes of CBB cycle are highly active (Friedrich et al.,
1979). The activities of two formate dehydrogenases are also increased. These enzymes oxidize formate to CO
2
, which is then fixed in CBB cycle. As formic acid is very toxic, it is not
applicable at high concentrations, as aldehyde it may inhibit enzymes of
the central metabolism. In general, it was shown that the cells reduce the amino acid biosynthesis (Shikimate
pathway), pyrimidine and purine formation, while the P(3HB) synthesis enzymes demonstrate higher activity in
presence of formate (Lee et al., 2006). Due to its toxicity, low energy potential and low PHA/biomass yield, formate
is not suitable as sole carbon source. However, like other VFAs it is a common ingredient of carbohydrate
hydrolysates. Moreover, since formic acid can be electrochemically generated from CO
2
and water, it can be further microbially converted to diverse products.
Electricity generated from regenerative energies sources can be stored in form of electromicrobial fuels.
Recombinant strains of R. eutropha were used electromicrobial conversion of CO
2
/formate to higher alcohols (Jeon et al., 2013; Li et al., 2012). These auto- and mixotrophical approaches
comprise, on one hand, the possibility to store energy and on the other, consume CO
2
, which is also beneficial for the environment.
Conclusions
In summary, the metabolic versatility of R. eutropha has made this bacterium to a model production strain for PHA
metabolism in the past. The understanding of its metabolism based on the availability of the genome sequence and a
range of transcriptomic, proteomic and metabolomic data, gene- deletion vectors and expression systems recently
broadened the spectrum of applications of R. eutropha as a production platform. Thus, the potential for high-yield
production of diverse tailor-made biopolymers by R. eutropha H16 has been demonstrated. The application of
metabolically engineered strains of R. eutropha revealed a potential of this bacterium as a platform for large-scale
biotechnological production of various chemicals and offers an alternative for chemical synthesis. Various ways to
extend and optimize the conversion of inexpensive renewable feedstocks have been established. The autotrophical
production of bio-based products is another important separate topic and was only briefly mentioned in this review.
However, production of valuable products, derived from the P(3HB) or central metabolism, applying CO
2
as carbon source has been intensively studied. In parallel to chemical synthesis and the application of other
production strains, synthesis of additional chemicals like organic acids by R. eutropha seems to be promising and
should be established in future to replenish the range of synthesized bioproducts. The chemical production of
organic acids (acetic, succinic or pyruvic acid) may be replaced by sustainable microbial production. The versatile
metabolism of R. eutropha can be successfully modulated based on research experience of more than 50 years. Thus,
simple, renewable and inexpensive feedstocks can be used by R. eutropha. The pathway’s bottlenecks can be
identified and reconstructed, undesired metabolic fluxes and by-products can be reduced or eliminated based on the
advances in genome sequencing, functional genomics, transcriptomics, proteomics and metabolomics.
Declaration of interest
The authors report no declarations of interest. The authors alone are responsible for the content and writing of this
article.

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Supplementary material available online Supplementary Figures S1, S2 and Tables S1, S2

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