Production of Bioplastic Compounds

by Genetically Manipulated 7
and Metabolic Engineered
Cyanobacteria

Noriaki Katayama, Hiroko Iijima, and Takashi Osanai

Abstract
Direct conversion of carbon dioxide to valuable compounds is a desirable way to
reduce the environmental burden and switch from fossil to renewable fuels.
Cyanobacteria are photosynthetic bacteria that perform oxygenic photosynthesis
and are able to produce valuable compounds from carbon dioxide in the air.
Synechocystis and Synechococcus species, model unicellular cyanobacteria, can
produce succinate and lactate, which are commodity chemicals used to generate
bioplastics. Several cyanobacteria are also able to produce polyhydroxybutyrate,
a biodegradable polyester that accumulates under nitrogen or phosphorus starva-
tion. Genetic manipulation succeeded in increasing the productivity of succinate,
lactate, and polyhydroxybutyrate from cyanobacteria. We summarize the recent
findings in this review.

Keywords
Cyanobacteria · Lactate · Metabolic engineering · Polyhydroxybutyrate ·
Succinate

7.1 Introduction

An increase in atmospheric carbon dioxide (CO2) is a major global environmental

concern, and advanced technologies to reduce CO2 emissions are needed to stabilize
the climate. Using microorganisms to synthesize biofuels can mitigate climate
change; specifically, microalga can fix CO2 to produce fuel compounds directly

N. Katayama · H. Iijima · T. Osanai (*)

School of Agriculture, Meiji University, Kawasaki, Kanagawa, Japan
e-mail: tosanai@meiji.ac.jp

© Springer Nature Singapore Pte Ltd. 2018 155

W. Zhang, X. Song (eds.), Synthetic Biology of Cyanobacteria,
Advances in Experimental Medicine and Biology 1080,
https://doi.org/10.1007/978-981-13-0854-3_7
156 N. Katayama et al.

[42]. Microalgae include eukaryotic algae and cyanobacteria and comprise a diverse
group of photosynthetic organisms that inhabit various ecological niches [57].
Cyanobacteria, previously called blue-green algae, are a group of bacteria that
includes nitrogen-fixing and non-nitrogen-fixing species that are characterized by
the possession of two photosystems: photosystem I and photosystem
II.  Cyanobacteria are an ideal platform to produce biofuels and bulk chemicals
through efficient, natural CO2 conversion [55]. Model cyanobacteria such as
Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 and PCC 7942
(hereafter referred to as Synechocystis 6803, Synechococcus 7002, and
Synechococcus 7942, respectively) rapidly proliferate; their doubling times are in
the range of 3–6 h under optimal growth conditions [9]. The genome sequences of
Synechocystis 6803 and their sub-strains have been determined [35, 36]. In total, 86
complete and 290 draft genomes of cyanobacteria are available in the genome anno-
tation database CyanoBase (http://genome.microbedb.jp/cyanobase) [19], and the
metabolic pathways of cyanobacteria linked to genomic information can be searched
in the KEGG (Kyoto Encyclopedia of Genes and Genomes) database (http://www.
genome.jp/kegg/) [34]. In this review, we mainly focus on the unicellular, non-­
nitrogen-­fixing cyanobacteria Synechocystis 6803, Synechococcus 7002, and
Synechococcus 7942.
Understanding the primary carbon metabolism of unicellular cyanobacteria is
indispensable because they are the simplest organisms that perform oxygenic pho-
tosynthesis. Many researchers have studied carbon fixation and biosynthesis of gly-
cogen using cyanobacteria, whereas relatively few researchers have focused on
sugar catabolism in cyanobacteria because heterotrophic bacteria such as Escherichia
coli cannot fix CO2, even though sugar catabolic pathways seem to be conserved
among heterotrophic bacteria. Recently, however, much attention has been paid to
cyanobacterial sugar catabolism because of the demand for the generation of vari-
ous compounds from CO2. Fermentation in cyanobacteria is reviewed well by Stal
and Moezelaarat in a publication that appeared 20 years ago [72], and 10 years ago,
there was another review that dealt with cyanobacterial sugar catabolism [56].
Recently, many fine reviews of the biotechnological application and metabolic engi-
neering of cyanobacteria have been published [10, 11, 30, 54, 55, 68]. In this review,
we focus on metabolic engineering for the production of bioplastic compounds such
as succinate, lactate, and polyhydroxybutyrate. Because these metabolites are gen-
erated during pyruvate metabolism and the tricarboxylic acid (TCA) cycle, we first
explain pyruvate metabolism and the TCA cycle in cyanobacteria and then intro-
duce recent studies in metabolic engineering for bioplastic production.

7.2  yruvate Metabolism and the TCA Cycle

P
in Cyanobacteria

The TCA cycle in cyanobacteria was previously believed to be an incomplete cycle

because 2-oxoglutarate dehydrogenase was lacking [71]. However, in Synechococcus
7002, Zhang and Bryant discovered that 2-oxoglutarate is converted into succinic
7  Production of Bioplastic Compounds by Genetically Manipulated and Metabolic… 157

Phosphoenolpyruvate Polyhydroxybutylate
pyk1, 2 Lactate
(sll0587, sll1275) phaC (slr1829)
pps ddh (slr1556) phaE (slr1830)
ppc (slr0301)
(sll0920) 3-Hydroxybutyryl-CoA
CO2 Pyruvate
phaB
pdhABCD Acetoacetyl-CoA (slr1994)
CO2 (slr1934, sll1721
sll1841, slr1096) phaA pta
me (slr1993) (slr2132)
(slr0721) Acetyl-CoA Acetyl-P ackA
(sll1299)
acs
Oxaloacetate (sll0542) Acetate
gltA
(sll0401)
citH Citrate Glutamine
(sll0891)
acnB Isocitrate icd gltB, gltD, glsF
(slr0665) (slr1289) (sll1502, sll1027,
Malate
sll1499)
2-Oxoglutarate
fumC
(slr0018) Succinyl-CoA
kgd Glutamate
sucC, sucD (sll1981)
(sll1023, sll1557) gad
Fumarate (sll1641)
sdhA, sdhB Succinate Succinic semialdehyde -Aminobutylate
gabD argD
(slr1233, sll0823, sll1625) (slr0370) (slr1022)

Fig. 7.1  Metabolic map of Synechocystis sp. PCC 6803. Metabolic map is drawn based of KEGG
database, and the gene number is derived from CyanoBase. P designates phosphate

semialdehyde by a 2-oxoglutarate decarboxylase (encoded by kgd) and that succinic

semialdehyde is converted into succinate by a succinic semialdehyde dehydroge-
nase (encoded by gabD) [88]. Subsequently, through genetic and metabolic flux
analyses, Synechocystis 6803 was also found to possess 2-oxoglutarate decarboxyl-
ase and succinic semialdehyde dehydrogenase [86, 87].
Xiong et al. also found that a γ-aminobutyric acid (GABA) shunt contributes to
the metabolic flux from 2-oxoglutarate to succinate in Synechocystis 6803 [86].
First, 2-oxoglutarate is converted to glutamate by a glutamate synthase, and then
glutamate is converted to GABA by a glutamate decarboxylase (encoded by gad)
[86]. A 13C-stable isotope analysis revealed that both a 2-oxoglutarate decarboxyl-
ase bypass and GABA shunt can generate succinate and that the GABA shut primar-
ily contributes to flux from 2-oxoglutarate to succinate in Synechocystis 6803 [86]
(Fig. 7.1). In this way, the cyanobacterial TCA cycle is unusual, but it is a closed
cycle, demonstrated by genetic, biochemical, and metabolomic analyses [89].
The principle role of the TCA cycle in cyanobacteria is oxidizing citrate and
generating reductants for respiration. Second, metabolites generated in the TCA
cycle and pyruvate metabolism can serve as precursors for the biosynthesis of
various metabolites. Third, the TCA cycle in cyanobacteria facilitates carbon stor-
age and signal transduction in response to environmental changes. After nitrogen
depletion of Synechocystis 6803 cells, the levels of succinate, fumarate, and malate
increase while those of citrate decrease [63]. Malate and fumarate function as
158 N. Katayama et al.

carbon sinks in higher plants [13]; thus, these organic acids in the TCA cycle poten-
tially function as a carbon store in unicellular cyanobacteria during periods of nitro-
gen starvation.
During nitrogen starvation, 2-oxoglutarate accumulates and alters the gene
expression of enzymes in nitrogen metabolism pathways [52]. For example,
2-­oxoglutarate directly upregulates transcriptional activity by binding to NtcA, a
global nitrogen transcription factor conserved among cyanobacteria [77]. In addi-
tion, 2-oxoglutarate controls the interactions between NtcA and the small protein
PipX, which functions as a co-activator of NtcA [16, 17]. NtcA and PipX widely
control gene expression related to nitrogen metabolism, carbon metabolism,
translation, transcription, and the cell cycle [18]. Manipulation of ntcA in
Synechocystis 6803 can alter primary carbon metabolism, amino acid levels, and
ethylene production [45, 61]; therefore, NtcA is important for primary metabolism
and metabolic engineering, and the levels of 2-oxoglutarate should be considered
during metabolic engineering.
Biochemical properties of enzymes in the TCA cycle have been studied in uni-
cellular cyanobacteria. NADP-isocitrate dehydrogenase (NADP-IDH) is an enzyme
that produces 2-oxoglutarate from metabolites derived through carbon fixation in
cyanobacteria [51]. NADP-IDH requires NADP+ as an electron acceptor and diva-
lent cations such as Mg2+ or Mn2+ for its enzymatic activity [51] and provides carbon
skeletons for nitrogen assimilation in cyanobacteria [51]. Succinate dehydrogenase
(SDH) provides electrons to the plastoquinone (PQ) pool, and its activity is domi-
nant particularly in dark conditions with cyanide [14]. Thus, SDH plays key roles in
carbon metabolism and redox maintenance in Synechocystis 6803 cells [15].
Phosphoenolpyruvate carboxylase (PEPC) is an important enzyme that catalyzes
irreversible carboxylation of phosphoenolpyruvate with bicarbonate to generate
oxaloacetate and inorganic phosphate in the presence Mg2+ [53]. Synechocystis
6803 PEPC is more tolerant of allosteric inhibition by several metabolites such as
malate, aspartate, and fumarate than other cyanobacterial PEPCs [75]. Takeya et al.
showed that a difference in allosteric regulation of PEPCs is determined by a single
amino acid residue at position 954  in Synechocystis 6803 [75]. In this way, bio-
chemical analyses have provided intriguing insights into the enzymes in the TCA
cycle of unicellular cyanobacteria.

7.3 Succinate and Lactate

Succinate is a metabolite in the TCA cycle that is generated from succinic semial-
dehyde by a succinic semialdehyde dehydrogenase through the oxidative TCA
cycle or from fumarate by a succinate dehydrogenase through the reductive TCA
cycle in cyanobacteria (Fig. 7.1). Succinate is a commodity chemical used as a
surfactant, ion chelator, and additive in agricultural products and food [2]. Succinate
is currently produced petrochemically to satisfy the demand in the chemical market
[47]. Succinate was identified as one of the twelve key building blocks of high-value
bio-­based chemicals and materials, selected by the US Department of Energy [85].
7  Production of Bioplastic Compounds by Genetically Manipulated and Metabolic… 159

The demand for succinate is expected to increase up to 700,000 tons per year by

2020 [2]. Succinate is a precursor of 1,4-butanediol, tetrahydrofuran, 2-pyrrolidone,
and polybutylene succinate, which are all widely used biodegradable plastics [2,
85]. Bio-based succinates are produced at industrial scale by several companies
using heterotrophic organisms such as E. coli, Saccharomyces cerevisiae, Pichia
kudriavzevii, and Mannheimia succiniciproducens [2, 28].
Succinate production using cyanobacteria has begun recently at laboratory scale.
To our knowledge, Prof. Dismukes’s group has the first to discover that a mutant
lacking ldhA that encodes d-lactate dehydrogenase in Synechococcus 7002 excretes
trace amounts of succinate under anaerobic conditions, whereas the wild-type strain
does not [49]. Synechococcus 7002 cells grown under nitrogen-replete conditions
produce succinate, but cells grown under nitrogen-depleted conditions do not [48].
In the case of Synechocystis 6803, wild-type cells excrete succinate in dark,
anaerobic conditions [64]. Synechocystis 6803 wild-type cells (a glucose-tolerant
strain) produce 15–20 mg/L succinate during 3 days of anaerobic incubation in the
dark [64]. Lactate and acetate are simultaneously excreted from Synechocystis 6803
cells. Acetate is dominant among the excreted metabolites, with titer of approxi-
mately 300 mg/L [64]. Knocking out acetate kinase (encoded by ackA) catalyzes
a reaction from acetylphosphate to acetate, increases succinate and lactate pro-
duction, and decreases acetate production [64], indicating that the suppression of
by-­product generation upregulates succinate production in Synechocystis 6803.
Overexpression of sigE, which encodes RNA polymerase sigma factor SigE, also
enhanced succinate production [64]. Sigma factor is a subunit of an RNA poly-
merase that binds to promoter DNA and initiates transcription. Synechocystis 6803
possesses nine sigma factors (SigA–I). SigE is a positive regulator of sugar catabo-
lism that enhances gene expression of enzymes in glycogen catabolism, oxidative
pentose phosphate (OPP), and glycolysis pathways [58, 60]. Combining genetic
manipulation for the overexpression of sigE and knocking out ackA increased suc-
cinate production additively to ~70 mg/L during a 3-day incubation of Synechocystis
6803 in dark, anaerobic conditions [64].
Succinate production is dependent on growth and incubation conditions.
Succinate and lactate production are upregulated in the presence of potassium
(100–300 mM KCl) in dark, anaerobic conditions [80]. A metabolomic analysis
revealed that potassium enhances glycogen degradation and increases intracellular
levels of organic acids, including succinate, lactate, pyruvate, citrate, 2-oxogluta-
rate, fumarate, and malate after anaerobic incubation in the dark, indicating that
potassium accelerates sugar catabolic reactions and the accumulation of anionic
metabolites in these conditions [80]. Mutant cells overexpressing sigE and lacking
ackA produced ~140 mg/L succinate in the presence of 200 mM KCl during 3 days
of anaerobic incubation in the dark [80]. The addition of sodium bicarbonate
(NaHCO3) during dark, anaerobic incubation also increases succinate production
from Synechocystis 6803 [21]. In the presence of 50–500  mM NaHCO3,
Synechocystis 6803 cells excrete succinate, lactate, acetate, malate, fumarate,
2-oxoglurarate, and citrate [21]. A time-course metabolomic analysis using
13
C-labeling revealed that succinate is synthesized in the reductive TCA cycle in
160 N. Katayama et al.

anaerobic conditions in the dark and that its synthesis was mediated by PEPC,
which catalyzes a reaction from phosphoenolpyruvate to oxaloacetate [21].
Overexpression of the gene encoding PEPC increases succinate production to
192 mg/L in the presence of 100 mM NaHCO3 [21].
Succinate and lactate production requires the reductant NAD(P)H in reactions
from oxaloacetate to malate and pyruvate to lactate, respectively. Synechocystis
6803 cells produce hydrogen (H2) in dark, anaerobic conditions, wasting excess-­
reducing power and recycling NAD(P)H to NAD(P)+ [37]. In non-nitrogen-fixing
cyanobacteria, hydrogen is produced by a hydrogenase that consists of five subunits
of HoxEFHUY [12]. The HoxHY subcomplex exhibits hydrogenase activity to pro-
duce H2, and the HoxEFU subcomplex oxidizes NAD(P)H [12]. In Synechocystis
6803, a mutant with decreased levels of hoxH showed decreased hydrogen produc-
tion and decreased acetate production (to 100 mg/L), whereas succinate and lactate
production increased to approximately 100 mg/L and 300 mg/L, respectively, dur-
ing 3 days of incubation in dark, anaerobic conditions [27]. A hoxH mutant showed
increased levels of SigE proteins and accelerated sugar catabolism, indicating that
excess reductants are consumed through succinate and lactate production in this
mutant [27].
The introduction of external genes into cyanobacteria is also beneficial for
succinate production. Lan and Wei generated an engineered strain of Synechococcus
7942 that overexpressed kgd encoding a 2-oxoglutarate decarboxylase from
Synechococcus 7002 and gabD encoding a succinic semialdehyde dehydrogenase
derived from this gene in E. coli [39]. Succinate production titers from this
engineered strain reached 400 mg/L in 8 days under light, aerobic conditions [39].
This study opened the way for succinate production from cyanobacteria without
maintaining anaerobic conditions [39].
Lactate is widely used as a precursor for biodegradable plastics (polylactic acid,
PLA) and various chemicals. Integration of l-lactate dehydrogenase (LDH) from
Bacillus subtilis into Synechocystis 6803 enabled the cells to stably produce l-­
lactate during batch culture [1, 3, 4]. Co-expression of genes encoding soluble
transhydrogenases additively increased lactate levels to 3.2 mM during 2 weeks
of cultivation [4]. These researchers also discovered that the downregulation of
glycogen biosynthesis and PEPC activity and the upregulation of pyruvate kinase
activity enhanced lactate production from Synechocystis 6803 cells [6, 82].
Joseph et  al. introduced a lactate dehydrogenase from a lactic acid-producing
Lactobacillus species [31]. Co-overexpression of both lactate dehydrogenases and a
lactate transporter from L. plantarum (encoded by lldp) enabled Synechocystis 6803
cells to excrete l-lactate during photoautotrophic conditions [31]. The maximum
levels of secreted lactic acid reached 15.3 g/L (0.17 mM) at 18 days [31].
d-lactate is a chiral chemical used to synthesize PLA and various chemicals,
including cosmetics [41]. The physical properties of PLA are dependent on the opti-
cal purity of lactate; thus, the industry requires the production of optically pure
lactate [24]. Synechocystis 6803 cells can consume d-lactate but not l-lactate as a
carbon source because the genome contains d-lactate (rather than l-lactate) dehy-
drogenase (encoded by ddh, slr1556) [5]. Ddh of Synechocystis 6803 exhibits low
7  Production of Bioplastic Compounds by Genetically Manipulated and Metabolic… 161

enzymatic activity, and a ddh-overexpressing strain did not produce d-lactate [5].
Later, Synechocystis 6803 cells were found to produce lactate under dark, anaerobic
conditions [27, 64, 80], although the chirality of the lactate produced has not been
determined. Synechococcus 7002 cells produce d-lactate through autofermentation,
and knockout of nitrate reductase (encoding narB) enhances d-lactate twofold [66].
The introduction of d-lactate dehydrogenase makes unicellular cyanobacteria
produce d-lactate from CO2. Li et al. engineered Synechococcus 7942 to express
d-lactate dehydrogenase from Lactobacillus bulgaricus ATCC11842 to increase its
affinity for NADPH [41]. Introduction of this d-lactate dehydrogenase into
Synechococcus 7942 increased d-lactate production; d-lactate levels reached
798  mg/L after photoautotrophic incubation for 10  days [41]. Aeration at an
increased concentration of CO2 (continuously bubbling the air containing 5% (vol/
vol) CO2) further enhanced d-lactate production from the engineered strain; the titer
reached 1.31 g/L at 10 days [41].
Introduction of a mutated glycerol dehydrogenase (GlyDH*) from a Bacillus
species that produces optically pure d-lactate enhanced d-lactate production from
Synechocystis 6803 [83]. Co-overexpression of GlyDH* and transhydrogenase
(from Pseudomonas aeruginosa) enhanced d-lactate production to 1.14 g/L in pho-
toautotrophic conditions at 24 days of incubation [83]. The addition of acetate to the
medium further upregulated d-lactate production to 2.17 g/L at 24 days [83].
Utilization of anaerobic digestion is an environmentally preferable method for
waste treatment. Anaerobic digestion can provide carbon, nitrogen, and phosphorus
sources for use in microalgal cultivation. The addition of an anaerobic digestion
from the East Lansing Wastewater Treatment Plant (East Lansing, MI, USA) into
the culture medium increased d-lactate production from engineered Synechocystis
6803 cells; the titer reached 1.26 g/L at 20 days [25]. Photomixotrophic conditions,
therefore, may be used to produce d-lactate in cyanobacteria, although the risk of
contamination may be greater than that in photoautotrophic conditions.
Hirokawa et al. showed different ways of producing d-lactate from cyanobacte-
ria [24]. They introduced mgs, which encodes methylglyoxal synthase in E. coli, to
catalyze a reaction from dihydroxyacetone phosphate (DHAP) to methylglyoxal in
Synechococcus 7942, because DHAP is thought to be a metabolite at a high meta-
bolic flux rate in cyanobacteria in photoautotrophic conditions [24]. Methylglyoxal
is converted to s-lactoylglutathione by glyoxalase I (encoded by gloA), and
s-­lactoylglutathione is converted to d-lactate by glyoxalase II (encoded by gloB).
Co-overexpression of mgs, gloA, gloB, and a gene encoding a lactate/H+ symporter
(lldP) from E. coli in Synechococcus 7942 enabled the cells to produce d-lactate at
a high titer (1.23 g/L at 24 days) [24].

7.4 Polyhydroxybutyrates (PHBs)

Polyhydroxybutyrates (homopolymers of [R]-3-hydroxybutyrate, PHBs) are poly-

esters grouped into polyhydroxyalkanoates (PHAs), which are biodegradable plas-
tics (Fig.  7.2a)  [81]. A life cycle assessment suggested that using PHBs is more
162 N. Katayama et al.

Fig. 7.2 (a) Chemical structure of polyhydroxybutyrate. (b) Gene loci of phaAB and phaEC in a
Synechocystis sp. PCC 6803 genome

environmentally acceptable than using petroleum-derived plastics [81]. PHBs accu-

mulate in many bacterial cells as carbon and energy sources in nitrogen- or phos-
phorus-starved conditions. Synechocystis 6803 cells accumulate PHBs during
nitrogen or phosphorus starvation, with the production of PHBs activated by
acetylphosphate [46]. Nostoc species, Spirulina platensis, and Calothrix scytonemi-
cola accumulate PHBs [70, 33, 65], although Synechococcus 7942 and 7002 do not.
Synechocystis 6803 possesses a two-component PHA synthase composed of
PhaC (slr1830) and PhaE (slr1829), which catalyze the reaction from
3-­hydroxybutyryl-CoA to a PHB (Fig.  7.1) [23]. In addition, 3-hydroxybutyryl-­
CoA is synthesized from acetyl-CoA by two enzymes: β-ketothiolase (PhaA,
slr1993) and acetoacetyl-CoA reductase (PhaB, slr1994) (Fig.  7.1) [78].
Cyanobacterial PHA synthases are classified as type III synthases [20], and phaAB
and phaEC together comprise an operon (Fig. 7.2b).
Metabolic engineering to increase the amount of PHBs produced by Synechocystis
6803 has been attempted for 15 years. Introduction of a gene encoding PHA syn-
thase from Ralstonia eutropha (Cupriavidus necator) into Synechocystis 6803 cells
increased PHA synthase activity, but did not increase the PHB amount [73]. Tyo
et  al. discovered that disruption of a gene encoding gamma-glutamyl phosphate
reductase (proA, sll0461) or sll0565 (encoding a hypothetical protein) increased
PHB levels from a transposon library of Synechocystis 6803 mutants [79]. Knockout
of sll0783, whose expression is highly activated during nitrogen starvation, abol-
ished PHB accumulation during nitrogen starvation, although the function of the
product of sll0783 is unknown [69]. The levels of phaABCE transcripts are not
7  Production of Bioplastic Compounds by Genetically Manipulated and Metabolic… 163

altered by knockout of sll0783, indicating the posttranslational regulation of PHB

biosynthesis by Sll0783 [69].
Genetic manipulation of transcriptional regulators increases PHB levels in
Synechocystis during nitrogen starvation. sigE overexpression increases the levels
of glycogen phosphorylases (encoded by glgPs), isoamylases (encoded by glgXs),
glucose-6-phosphate dehydrogenase (encoded by zwf), and 6-phosphogluconate
dehydrogenase (encoded by gnd) [59]. Protein levels of PhaA, PhaB, PhaC, and
PhaE increase with sigE overexpression in either nitrogen-replete or nitrogen-­
depleted conditions [59]. PHB levels in the wild-type and sigE-overexpressing
strains were 5.7 mg/L and 14.0 mg/L after cultivation for 9 days [59].
Rre37 is an OmpR-type transcriptional regulator whose expression increases
during nitrogen starvation and in the presence of glucose [8, 74]. Rre37 upregulates
the expression of genes encoding enzymes that function in glycogen catabolism and
glycolysis [8, 44, 74] and promotes sugar catabolism [32, 62]. Further, rre37 over-
expression in Synechocystis 6803 increased the levels of PhaA, PhaB, PhaC, and
PhaE in either nitrogen-replete or nitrogen-depleted conditions [62]. PHB levels in
the wild-type and rre37-overexpressing strains were 6.2  mg/L and 11.5  mg/L,
respectively, after cultivation for 9 days [62], and co-overexpression of rre37 and
sigE additively increased PHB levels to 17.2 mg/L [62].
Hauf et al. identified the small ORF ssl2501, which encodes a protein, phasin
PhaP in Synechocystis 6803, that coats the surfaces of PHB granules [22]. PhaP
fused with green fluorescent protein localized in PHB granules, and deletion of
phaP decreased the number and increased the size of PHB granules [22].
Overexpression of the Synechocystis phaABEC gene cluster in Synechocystis
6803 cells increased PHB levels [26]. A plasmid containing the phaABEC gene
cluster fused with the promoter of psbAII (encoding a Photosystem II D1 protein)
was introduced into Synechocystis 6803 cells, and PHB levels reached 7% (w/w) of
the dried cells, which were 12 times higher than levels in the wild-type cells [26].
They also showed that intracellular levels of acetyl-CoA, acetoacetyl-CoA, and
3-hydroxybutyryl-CoA increased with overproduction of the Synechocystis 6803
phaABEC gene cluster, indicating that pathway-level regulation is important to
increasing PHB levels [26]. Another group enhanced PHB levels by overexpressing
the native phaAB gene operon, with levels reaching 26% (w/w) of the dried cells
after cultivation for 9 days in nitrogen-starved conditions [38].
Selection of cyanobacterial species and improvement of cultivation methods
enhance PHB levels. Monshupanee et al. revealed that Chlorogloea fritschii TISTR
8527, which is a unicellular cyanobacterium that is easily harvested by auto-­
sedimentation, accumulates PHB at high levels [50]. Two-stage cultivation, first in
photoautotrophic conditions and second in heterotrophic conditions with acetate as
a carbon source, caused the cells to produce 513 mg/L PHB after 22 days of cultiva-
tion [50]. The maximum conversion efficiency from acetate to PHB reached 51%,
which is comparable to the level of theoretical efficiency [50].
Recently, Oscillatoria okeni TISTR 8549 was found to produce poly(3-­
hydroxybutyrate-­co-3-hydroxyvalerate) (PHBV) [76]. O. okeni cells grown in the
medium deprived of a nitrogen source and supplemented with acetate accumulated
164 N. Katayama et al.

PHBV to 42% (w/w) of the dried cells [76]. Thus, their study opens the possibility
that cyanobacteria can be engineered to produce various PHAs, in addition to PHB.

7.5 Conclusion

In this review, we summarized studies on metabolic engineering of unicellular cya-

nobacteria, particularly to produce bioplastic compounds such as succinate, lactate,
and PHBs. Cyanobacteria can directly convert CO2 to these valuable compounds,
and this increased productivity is indispensable for reducing the consumption of
fossil fuels and emission of greenhouse gases. At present, the titers of succinate,
lactate, and PHBs produced from cyanobacteria are lower than from heterotrophic
bacteria or yeasts. For example, titers of succinate produced by the yeast Pichia
kudriavzevii and E. coli reached 48.2 g/L and 96.0 g/L, respectively [2]. Production
in these organisms, however, required sugar derivatives, which may compete with
available food. Further genetic and metabolic engineering of cyanobacteria is
required to increase the productivity of bioplastic compounds and lower the cost of
bio-production. Improving methods to cultivate cyanobacteria, increasing the
growth rate, reducing the cost, utilizing wastewater, and suppressing contamination
are also critical to the industrialization of cyanobacteria. New biotechnologies to
engineer cyanobacteria thus command the attention of not only scientists but also
the general public.

7.6 Future Trends

The number of the published papers on bioplastic production using cyanobacteria

and microalgae has been increasing and will continue to increase. Several fine
reviews on organic acid production have been published by several groups ([1, 43,
84, 90, 29]. PHA production will be also expanding, and there are several recent
reviews on improving the quantity and quality of PHAs produced [7, 40, 67]. The
efficiency of bioplastics production from microalga can be improved by further
studies in the fields of metabolic engineering, molecular biology, and biochemistry,
offering the potential that CO2 emissions due to human actions can be limited.

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