INSTITUTO TECNOLOGICO Y DE ESTUDIOS SUPERIORES DE MONTERREY CAMPUS ESTADO DE MEXICO
Synthesis of multicomponent polyhydroxyalkanoates from fatty acid sources in Cupriavidus necator H16 and its recombinant strain C. necator H16/pMPJAS03
A thesis submitted in the fulfillment of the requirements for the degree of Doctor of Philosophy in Engineering Sciences
Author: Araceli Flores-Sánchez
Atizapán de Zaragoza, Estado de Mexico, April, 2020
Thesis committee:
1. Dr. Berenice Vergara-Porras (Tecnológico de Monterrey. Atizapán de Zaragoza, Mexico) 2. Dr. María del Rocío López-Cuellar (Universidad Autónoma del Estado de Hidalgo, México) 3. Prof. Dr. Fermín Pérez-Guevara (Centro de Investigación y Estudios Avanzados, México) 4. Prof. Dr. Bruce A. Ramsay (Queen’s University, Kingston, Canada)
5. Prof. Dr. Ulises Figueroa-Lopez (Tecnológico de Monterrey. Atizapán de Zaragoza, Mexico) 6. Dr. Yara C. Almanza-Arjona (Tecnológico de Monterrey. Atizapán de Zaragoza, Mexico)
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Co-Authorship
This thesis is based on 4 manuscripts (1st author) prepared for publication in peer-reviewed journals.
The first part of the experiments (Chapter 3 and Chapter 4) was performed at Tecnológico de Monterrey Campus Estado de Mexico and Centro de Investigación y Estudios Avanzados (CINVESTAV) Biotechnology facilities. The author was supervised by Dr. Berenice Vergara- Porras, Dr. Maria del Rocio Lopez-Cuellar, and Prof. Dr. Fermín Perez-Guevara, who also contributed to the discussion of the results and preparation of the manuscripts.
The second part of the experimental work (Chapter 5 and Chapter 6) was performed at Queens University, ON Canada, at the biopolymer’s laboratory (Chemical Engineering Department). The author was supervised by Prof. Dr. Bruce A. Ramsay and Prof. Dr. Juliana Ramsay, who also contributed to the design of the experiments, discussion of results and preparation of the manuscripts.
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Acknowledgements
The success of completing this thesis would not have been possible without the support of numerous individuals.
I would like to thank Dr. Berenice Vergara-Porras, Dr. Maria del Rocio Lopez-Cuellar and Prof.
Dr. Fermín Perez-Guevara for the initial supervision related to this research, for about 3 years.
Thank you for introducing into Polyhydroxyalkanoates research. The advice and encouragement at the meetings were always appreciated.
I want to express great thanks to Prof. Dr. Bruce A. Ramsay and Prof. Dr. Juliana A. Ramsay for accepting me as a Visiting Research International Student. Thank you for guiding me throughout the investigation for more than a year and for all the fruitful discussions we had in our meetings. I will never forget your kindness and support.
I also would like to thank Joel Alba-Flores for the initial support at the Biotechnology lab (CINVESTAV), especially for teaching me the essential techniques to work with microorganisms.
I thank all the lab members (México and Canada) that I have met throughout this journey and for the great time we shared.
I owe many thanks to Flores-Sánchez family, especially to my parents – Fidelina and Mayo-, and my life partner -Tarik- this thesis would not be complete without your continuous encouragement and patience. Many thanks to my dear friends - Fernando, Walid, Ibrahim, and Mathy- for being part of this journey.
Thanks to the National Council of Science and Technology of Mexico (CONACyT, grant no.
417745), and Natural Science and Engineering Research Council of Canada (NSERC, project no.
388898) for funding this study.
iv Table of Contents
Co-Authorship... ii
Acknowledgements ... iii
List of Figures ... ix
List of Tables ... ixx
Abbreviations ... x
Chapter 1 Introduction ... 2
1.1 Background ... 2
1.2 Chapters and objectives ... 5
1.3 References ... 6
Chapter 2 Literature review ... 8
2.1 Polyhydroxyalkanoates (PHAs) generalities ... 8
2.2 Short-chain-length/ medium-chain-length PHAs ... 12
2.3 PHA biosynthesis in Cupriavidus necator ... 16
2.3.1 PHB operon ... 17
2.3.2 Scl-PHA copolymers biosynthesis ... 18
2.3.3 PHA biosynthesis from fatty acids ... 18
2.4 References ... 22
Chapter 3 Synthesis of Poly-(R-hydroxyalkanoates) by Cupriavidus necator ATCC 17699 using Mexican avocado (Persea americana) Oil as a carbon source ... 26
3.1 Abstract ... 26
3.2 Introduction ... 27
3.3 Materials and methods ... 31
3.3.1 Strain, Medium ... 31
3.3.2 Fermentation Studies... 32
3.3.3 Analytical procedures ... 33
3.3.4 PHA characterization ... 34
3.4 Results ... 35
3.5 Discussion ... 44
3.6 Conclusions ... 48
3.7 References ... 49
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Chapter 4 Biosynthesis of polyhydroxyalkanoates from vegetable oil under the co-expression of fadE and
phaJ genes in Cupriavidus necator ... 53
4.1 Abstract ... 53
4.2 Introduction ... 54
4.3 Materials and Methods ... 57
4.4 Results ... 64
4.5 Discussion ... 73
4.6 Conclusions ... 76
4.7 References ... 77
Chapter 5 High amounts of medium-chain-length polyhydroxyalkanoates subunits can be accumulated in recombinant Cupriavidus necator with wild-type synthase ... 81
5.1 Abstract ... 81
5.2 Introduction ... 82
5.3 Materials and Methods ... 85
5.4 Results ... 90
5.5 Discussion ... 98
5.6 References ... 102
Chapter 6 Fatty acid β-oxidation inhibition in recombinant Cupriavidus necator with wild-type synthase towards the formation of multicomponent polyhydroxyalkanoates ... 107
6.1 Abstract ... 107
6.2 Introduction ... 108
6.3 Materials and methods ... 110
6.4 Results and discussion ... 115
6.5 Conclusions ... 127
6.6 References ... 128
Chapter 7 Conclusions and future work ... 132
7.1 Summary of achievements ... 132
7.2 Opportunities for future work ... 135
vi List of Figures
Figure 1-1 Common PHA structure. Most variants of PHAs have the same backbone and differ only in the monomer side chain, R. ... 3 Figure 2-1 (a) General molecular formula of PHAs. Typically, x=1–8, and n ranges from 100 to 1000 s.
(b) Some commonly synthesized short-chain-length PHA monomers (scl-PHA) and middle-chain- length PHA monomers (mcl-PHA). 3HB: 3-hydroxybutyrate, 3HV: 3 hydroxyvalerate, 3HHx: 3- hydroxyhexanoate, 3HO: 3-hydroxyoctanoate, 3HD: 3-hydroxydecanoate, 3HDD: 3-
hydroxydodecanoate. ... 10 Figure 2-2 C. necator operon. 1: phaA β-Ketothiolase: 2: phaB: NADPH dependent acetoacetyl-CoA
reductase; 3: phaC PHA synthase. ... 17 Figure 2-3 The oxidation of fatty acids in C. necator provides intermediates to generate PHAs through the
formation of (R)-3-hydroxyacyl-CoA. Supplying enzymes such as Acyl-CoA dehydrogenase (FadE), (R)-specific enoyl-CoA hydratase (PhaJ) provide the PHA intermediates to be polymerize by the PHA synthase (PhaC). ... 20 Figure 3-1 A simplified representation of fatty acid metabolism in Cupriavidus necator. In a non-limiting
growth media –(left side), the tricarboxylic acid (TCA) cycle is active and the main product is biomass itself. Under unbalanced conditions –(right side), caused by a non-carbon nutrient
deficiency, C-source via fatty acid β-oxidation flows towards the poly-R-hydroxyalkanoate (PHA) biosynthesis pathway ... 30 Figure 3-2. Profiles of fructose (▲), ammonium (●), biomass (◊), and poly-R hydroxyalkanoate (PHA)
(□) production during the cultivation of Cupriavidus necator on fructose and avocado oil (20% v/v) in a three-stage fermentation. Stage 1: batch cultivation (a), Stage 2: fed-batch stage (b) additions indicated by black arrows, Stage 3: PHA accumulation (c). Oil addition occurs at 30 h. ... 36 Figure 3-3 Chromatogram obtained from gas chromatography (GC) of A) poly(3-hydroxybutyrate) (PHB)
standard, B) poly(hydroxybutyrate-co-hydroxyvalerate (PHBV) standard with 12 mol% HV content and C) polymer produced from 20% v/v of avocado oil (7 mol% of HV). Benzoic acid was used as internal standard. ... 39 Figure 3-4 Fourier transform infrared spectroscopy (FT-IR) spectra of the poly-R-hydroxyalkanoates
(PHAs) produced by the addition of avocado oil: (a) 0 –Control-; (b) 5, (c) 10, (d) 15, (e) 20 and (f) 25 percentage [v/v]. (A) 600 to 4000 cm-1, (B) 2500 to 4000 cm-1. ... 41 Figure 3-5 Thermograms obtained during the second run of differential scanning calorimetry (DSC) of the poly-R-hydroxyalkanoates (PHAs) produced by the addition of avocado oil at: (a) 0 –Control-; (b) 5, (c) 10, (d) 15, (e) 20 and (f) 25 percentage (v/v). ... 43
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Figure 4-1 Recombinant plasmid pMPJAS03 construction, harboring the fadEEc - phaJ1Pp DNA fragment under araC-PBAD promoter. (Abbreviations: Cm-r: chloramphenicol resistance, km-r: Kanamycin resistance, Amp-r: ampicillin resistance, ori: replication origin, rep: rep protein, lac z: lactose gene) ... 60 Figure 4-2 A) Screening and restriction digestion of plasmid pMPJAS03. Lane M: marker DNA, Lane 1-
8: agarose gel restriction of plasmid DNA from the clones of pMPJAS03 digested with EcoRV, Lane 1and 2: positive clones digested with EcoRV (6606, 2640 bp) digestion. B) Verification of pMPJAS03 orientation. Lane M: marker DNA, Lane 1 and 2: pMPJAS03 digested with HincII + AccII (5055, 2506, and 1685 bp) showed the positive orientation. ... 64 Figure 4-3 Representative kinetic profiles of C. necator H16 and its recombinant -pMPJAS03 strain using
canola oil and 0.1% v/v of arabinose. The cultivations were conducted in two stages: a) cell growth: substrate fructose, b) PHA synthesis: single oil addition at 26 h ... 66 Figure 4-4 Transcriptional level comparison of fadEEc and phaJ1Pp under different arabinose
concentrations. Cell growth phase evaluated at 26 h, and PHA synthesis at 32 and 44 h,
respectively ... 69 Figure 4-5 PHAs produced by C. necator H16/pMPJAS03 with 0.1% v/v of arabinose. Samples analyzed
via gas chromatography (GC-FID), A) octanoic acid, B) avocado oil, C) canola oil. ... 70 Figure 4-6 Thermogravimetric analysis of PHB homopolymer and PHAs produced from C. necator
H16/pMPJAS03 at heating-up rate of 10°C/min. A) TGA and B) DTG curves. ... 72 Figure 5-1 PHAs produced from fatty acid sources rely on the fatty acid degradation pathway (β-
oxidation), where the enzymes encoded the fad regulon are responsible for the transport and activation of long-chain fatty acids, and their β-oxidative cleavage into acetyl-CoAs. ... 84 Figure 5-2 Three-stage production of scl/mcl PHA copolymers in C. necator H16/pMPJAS03. a) Initial
cell growth with fructose in batch mode. b) Fed-batch, exponential growth by co-feeding
fructose/canola oil at 9:1 ratio and µ=0.14 h-1. c) PHA synthesis was conducted by constant feeding of canola oil at 7.5 g/h. The values shown are means of two independent samples. ... 92 Figure 5-3 Effect of supplementing DA acid to the substrate at different ratios in the synthesis of mcl
subunits. Changes in 3HD molar content are shown. Results are presented as mean values for two independent samples. ... 94 Figure 5-4 Effect of increasing DA ratio in the substrate on mcl subunits generated during PHA synthesis
(stage 3) ... 95 Figure 5-5 Effect of the increasing of 3HD subunits on the melting temperatures of the polymers ... 97 Figure 6-1 Production of PHA in C. necator H16/pMPJS03 feeding decanoic acid and fructose as co-
substrates. a) Initial cell growth with fructose in batch mode. Continuous cultivation by co-feeding
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fructose and decanoic acid as carbon sources. Dilution rate of 0.14 h-1 and mineral flow of 0.21 L/h, volume 1.5 L. Co-substrates were fed at 5.7 g/L with a molar ratio of 0.69/0.31, fructose and decanoic acid, respectively. b) Non-nutrient limitation, c) Nitrogen limitation cultivation (0.5 g/L NH4+ in the culture). The values shown are means ±standard deviation from three independent samples. ... 116 Figure 6-2 Outflow substrates analysis and total biomass produced at different acrylic acid concentrations, by co-feeding fructose and decanoic acid, with 0.5g/L of NH4. Dilution rate of 0.14 h-1 and mineral flow of 0.21 L/h, volume 1.5 L. Co-substrates were fed at 5.7 g/L with a molar ratio of 0.69/0.31, fructose and decanoic acid, respectively... 120 Figure 6-3 Efficiency of total carbon consumed from decanoic acid at different of acrylic acid
concentrations and its conversion to mcl subunits. Dilution rate of 0.14 h-1 and mineral flow of 0.21 L/h, volume 1.5 L. Co-substrates were fed at 5.7 g/L with a molar ratio of 0.69/0.31, fructose and decanoic acid, respectively. ... 121 Figure 6-4 Effect of co-feeding fructose and mcl fatty acids on cell growth and PHA synthesis in
continuous cultivation by co-feeding fructose and mcl fatty acids. Dilution rate of 0.14 h-1 and mineral flow of 0.21 L/h, volume 1.5 L. Co-substrates were fed at 3.5 g/L with a molar ratio of 0.69/0.31, fructose and mcl fatty acid, respectively. Data shown represent the average of at least 3 steady-state samples and error bars represent the range of the average... 125
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List of Tables
Table 2-1 Thermal properties of some scl-mcl PHA copolymers ... 13 Table 2-2 Mechanical properties of some scl-mcl PHA copolymers... 15 Table 2-3 Scl-mcl PHAs polymerized by the PHA synthase of C. necator H16 ... 21 Table 3-1 Final yields of poly-R-hydroxyalkanoates obtained from a three-stage fermentation of C.
necator ... 388 Table 3-2 Thermal properties and chemical composition of the poly-R-hydroxyalkanoates (PHAs)
produced in C. necator ... 47 Table 3-3 Comparative study for C. necator H16... 47 Table 4-1 Strains, plasmids and genes used to construct the C. necator H16/pMPJAS03 ... 588 Table 4-2 Effect of fadE and phaJ transcriptional levels on cell growth and PHA produced by C. necator H16/pMPJAS03 at different arabinose concentrations ... ¡Error! Marcador no definido.7 Table 4-3 Thermal characterization of the PHAs produced by C. necator H16/pMPJAS03 ... 71 Table 5-1 PHA accumulation and chemical composition of the polymers produced. ¡Error! Marcador no definido.5
Table 5-2 PHA synthesis by the wild-type PHA synthase of C. necator H16 and its recombinant strains from different fatty acid sources ... ¡Error! Marcador no definido.
Table 6-1 Effect of acrylic acid on PHA composition, by co-feeding fructose and decanoic acid. .. ¡Error!
Marcador no definido.18
Table 6-2 Effect co-feeding fructose and different mcl fatty acids at 20 mM of acrylic acid on PHA composition. ... ¡Error! Marcador no definido.23 Table 6-3 Efficiency of diverse mcl fatty acids to produce mcl subunits under the established continuous cultivation conditions. ... ¡Error! Marcador no definido.26
x Abbreviations
PHA Polyhydroxyalkanoates PHB Poly-3-hydroxybutyrate scl-PHA Short-chain-length PHA mcl-PHA Medium-chain-length PHA TAG Triacylglycerol
CoA Coacetyl (A) enzyme 3HA 3-hydroxyalkanoate 3HB 3-hydroxybutyrate 3HV 3-hydroxyvalerate 3HHx 3-hydroxyhexanoate 3HO 3-hydroxyoctanoate
3HD 3-hydroxydecanoate
3HDD 3-hydroxydodecanoate 3HHp 3-hydroxyheptanoate 3HNO 3-hydroxynonanoate phaA β-Ketothiolase
phaB NADPH dependent acetoacetyl-CoA reductase
phaC PHA synthase
FadE Acyl-CoA dehydrogenase
PhaJ (R)-specific enoyl-CoA hydratase
xi araC-PBAD Arabinose promoter
TCA Tricarboxylic cycle DNS Dinitrosalicylic acid Yx/s Biomass yield Yp/s Product yield
GC Gas Chromatography
FTIR Fourier transform infrared DSC Differential scanning calorimetry FID Flame ionized detector
TGA Thermo gravimetric analysis
DTG Derivative thermo gravimetric analysis NMR Nuclear Magnetic Resonance Spectroscopy qRT- PCR Quantitative real time polymerase chain reaction E. coli Escherichia coli
P. putida Pseudomonas putida
pBTB-3 Broad host range vector, origin of replication pBBr1, promoter PBAD, promoter CAT, Cmr, tonB ter
pK18 Broad host range vector, origin of replication Co1E1, Kmr pMPJAS01 Fusion of plasmids pBTB-3 and pK18
pMPJAS02 pBSK gene 1 harboring genes fadEEc and PhaJ1Pp
pMPJAS03 Fusion of plasmids PMPJAS01 and PMPJAS02. fadEEc - PhaJ1Pp downstream of araC-PBAD promoter
Cmr Chloramphenicol resistance
xii Kmr Kanamycin resistance
Tm Melting point
Tg Glass transition temperature ΔHm Enthalpy of fusion
Xc% Crystallinity
CPR CO2 production
DA Decanoic acid
1
2
Chapter 1 Introduction
1.1 Background
Petrochemical plastics have found widespread application in our daily life and currently in wide use being regarded as a major threat of pollution of the environment, including but not limited to contributions to greenhouse gas emissions and accumulation of plastic waste products. Plastics and synthetic polymers do not decompose, so they are stored, burnt or recycled. During combustion, water and carbon dioxide are released into the atmosphere, i.e., an increase in the carbon dioxide concentration in the atmosphere occurs. By recycling polymers, the material quality decreases (Al-Salem et al., 2009). Waste is currently causing serious environmental problems in many countries, for instance, the United States alone generated 31 million tons of plastic waste in 2017 (Geyer et al., 2017). Of this only 8.2%
was recovered for recycling, leaving the majority to accumulate in the landfills or incineration. Synthetic plastics are resistant to degradation, and consequently, their disposal is fueling an international attraction for the development of biodegradable polymers, compatible with our natural ecosystem (Bastioli, 2005). These problems have been the primary motivating factor in the research and development of polyhydroxyalkanoates (PHAs) as a potential substitute for petrochemical-based plastics (Muhammadi et al., 2015).
PHAs are aliphatic polyesters with diverse structures, are the only bioplastics completely synthesized by microorganisms synthesized and intracellularly stored by numerous
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prokaryotes from renewable resources like carbohydrates, alcohols, lipids, or organic acids (Leong et al., 2014; Muhammadi et al., 2015). They have been observed in 1888 by Beijerincka. However, he could not define their role and composition. In 1926 Lemoigne obtained the poly-3-hydroxybutyric acid (P3HB) from Bacillus megaterium Figure 1-1. In 1958 Macrae and Wilkinson proved that PHAs in bacterial cells play the role as the reserved materials of carbon and energy, and they are collected only in an increased carbon to nitrogen ratio (Castilho et al., 2009; Sudesh et al., 2000). Beginning in the 1959s, many companies have been set up to commercialize PHAs as environ-mentally friendly bioplastics, fully independent from petroleum sources. W.R. Grace & Company was the first company that tried to produce P3HB. However, low synthesis efficiency and problems with PHAs purification forced it to close the company. Beginning in 1980s, PHAs were produced under the trade names of Biopol TM, Nodax TM, Biocycle TM, Biomer TM, BioGreen
TM. Nowadays, the PHAs market is very small. Recently, the joint venture Telles, set up by Metabolix and ADM in 2006, aimed at big capacity but hardly sold any PHAs and subsequently collapsed in 2012 (Boyandin et al., 2012; Pakalapati et al., 2018; Rodriguez- Perez et al., 2018). PHAs producers are optimistic and still see potential in this biomaterials claiming that PHAs are new generation of biopolymers and their market needs time to develop.
Figure 1-1 Common PHA structure. Most variants of PHAs have the same backbone and differ only in the monomer side chain, R.
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PHAs with monomers containing up to five carbons are called short-chain-length (scl) PHAs, the most common is polyhydroxybutyrate (PHB). The melt temperature (Tm) of PHB is also high (>170 °C), relative to the region of its thermal decomposition temperature, making PHB much more difficult to manipulate using melt processing equipment used for conventional plastics (Chuah et al., 2013). It was hoped that the incorporation of a co- monomer unit, 3-hydroxyvalerate (3HV), could control the excessively high Tm and crystallinity limitations (Kulkarni et al., 2010). Unfortunately, the desired effect of 3HV incorporation to regulate the crystallinity and Tm was surprisingly limited because of the isodimorphism phenomenon, where 3HV units can be easily included in the crystal lattice of 3-hydroxybutyrate (3HB) units and vice versa without the anticipated disruption of crystallinity (Feng et al., 2004). An alternative structure of PHA copolymers, therefore, had to be designed to overcome this limitation.
The incorporation of medium-chain-length (mcl, C6 to C14) monomers effectively lowers the crystallinity and Tm in a similar manner to the effect of R-olefins in linear low-density polyethylene (Sudesh et al., 2000; Tappel et al., 2014). The Tm can be lowered well below the thermal decomposition temperature of PHAs to make this material much easier to process. The reduced crystallinity provides the ductility and toughness required for many practical applications. The mcl-3HA content regulates the Tm and crystallinity of copolymer almost independently of the mcl size, as long as more than three carbons are present in the side group. On the other hand, the side group chain length of mcl- monomers has a profound effect on the flexibility of the copolymer (Noda et al., 2005).
5 1.2 Chapters and objectives
This thesis focused on developing fermentation strategies for the production of multicomponent PHAs, mainly of the type scl-mcl copolymers with a decreased melting temperature and crystallinity.
The thesis consists of 7 chapters. Chapters 1 and 2 are the introduction and literature review, respectively.
Chapter 3 to 6 correspond to four specific objectives, described as follows:
Chapter 3: To evaluate the viability of multicomponent PHA biosynthesis from avocado oil in C. necator H16, as an alternative substrate for PHA production.
Chapter 4: To investigate the effect of co-expressing heterologous fadE and phaJ genes in C. necator H16 under the control of the arabinose promoter (araC-PBAD) system and evaluating its impact on scl/mcl PHA production from vegetable oils as carbon source. . Chapter 5: To cultivate in a well-controlled exponential fed-batch followed by constant feeding the recombinant strain C. necator H16/pMPJAS03 in an attempt to increase the incorporation of mcl subunits, derived from canola oil or canola oil/decanoic acid (DA).
Chapter 6: To investigate the full capability of the wild-type synthase in the recombinant strain C. necator H16/pMPJAS03 to polymerize scl-mcl PHAs from different carbon chain-lengths fatty acids in the presence of acrylic acid.
And finally, Chapter 7 gives conclusions and recommends future work.
6 1.3 References
Al-Salem, S., Lettieri, P., Baeyens, J. 2009. Recycling and recovery routes of plastic solid waste (PSW): A review. Waste management, 29(10), 2625-2643.
Bastioli, C. 2005. Handbook of biodegradable polymers. iSmithers Rapra Publishing.
Boyandin, A., Prudnikova, S., Filipenko, M., Khrapov, E., Vasil’ev, A., Volova, T. 2012.
Biodegradation of polyhydroxyalkanoates by soil microbial communities of different structures and detection of PHA degrading microorganisms. Applied biochemistry and microbiology, 48(1), 28-36.
Castilho, L.R., Mitchell, D.A., Freire, D.M. 2009. Production of polyhydroxyalkanoates (PHAs) from waste materials and by-products by submerged and solid-state fermentation.
Bioresource technology, 100(23), 5996-6009.
Chuah, J.-A., Tomizawa, S., Yamada, M., Tsuge, T., Doi, Y., Sudesh, K., Numata, K. 2013.
Characterization of site-specific mutations in a short-chain-length-medium-chain-length polyhydroxyalkanoate synthase: in vivo and in vitro studies on enzymatic activity and substrate specificity. Applied and environmental microbiology, AEM. 00564-13.
Feng, L., Yoshie, N., Asakawa, N., Inoue, Y. 2004. Comonomer‐Unit Compositions, Physical Properties and Biodegradability of Bacterial Copolyhydroxyalkanoates. Macromolecular bioscience, 4(3), 186-198.
Geyer, R., Jambeck, J.R., Law, K.L. 2017. Production, use, and fate of all plastics ever made.
Science advances, 3(7), e1700782.
Kulkarni, S., Kanekar, P., Nilegaonkar, S., Sarnaik, S., Jog, J. 2010. Production and characterization of a biodegradable poly (hydroxybutyrate-co-hydroxyvalerate)(PHB-co- PHV) copolymer by moderately haloalkalitolerant Halomonas campisalis MCM B-1027 isolated from Lonar Lake, India. Bioresource technology, 101(24), 9765-9771.
Leong, Y.K., Show, P.L., Ooi, C.W., Ling, T.C., Lan, J.C.-W. 2014. Current trends in polyhydroxyalkanoates (PHAs) biosynthesis: insights from the recombinant Escherichia coli. Journal of biotechnology, 180, 52-65.
Muhammadi, Shabina, Afzal, M., Hameed, S. 2015. Bacterial polyhydroxyalkanoates-eco-friendly next generation plastic: production, biocompatibility, biodegradation, physical properties and applications. Green Chemistry Letters and Reviews, 8(3-4), 56-77.
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Noda, I., Green, P.R., Satkowski, M.M., Schechtman, L.A. 2005. Preparation and properties of a novel class of polyhydroxyalkanoate copolymers. Biomacromolecules, 6(2), 580-586.
Pakalapati, H., Chang, C.-K., Show, P.L., Arumugasamy, S.K., Lan, J.C.-W. 2018. Development of polyhydroxyalkanoates production from waste feedstocks and applications. Journal of bioscience and bioengineering, 126(3), 282-292.
Rodriguez-Perez, S., Serrano, A., Pantión, A.A., Alonso-Fariñas, B. 2018. Challenges of scaling- up PHA production from waste streams. A review. Journal of environmental management, 205, 215-230.
Sudesh, K., Abe, H., Doi, Y. 2000. Synthesis, structure and properties of polyhydroxyalkanoates:
biological polyesters. Progress in polymer science, 25(10), 1503-1555.
Tappel, R.C., Pan, W., Bergey, N.S., Wang, Q., Patterson, I.L., Ozumba, O.A., Matsumoto, K.i., Taguchi, S., Nomura, C.T. 2014. Engineering Escherichia coli for improved production of short- chain-length-co-medium-chain-length poly [(R)-3-hydroxyalkanoate](SCL-co-MCL PHA) copolymers from renewable nonfatty acid feedstocks. ACS Sustainable Chemistry & Engineering, 2(7), 1879-1887.
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Chapter 2 Literature review
2.1 Polyhydroxyalkanoates (PHAs) generalities
Several microorganisms throughout nature have evolved diverse systems for storing essential nutrients, such as carbon, nitrogen, and phosphorous (Fagerbakke et al., 1996).
This storage frequently entails the accumulation of polymers, which can be depolymerized when the monomers are needed for synthesis of other metabolites or for energy generation (Novak et al., 2015).
In many cases the polymers form insoluble inclusions, which are beneficial because they do not influence reactions involving soluble substrates, and because the polymers do not contribute to the osmotic potential of the cell in which they are stored (Peters et al., 2007;
Rehm, 2003). These storage polymers are synthesized in an opposed manner to DNA, RNA, and proteins, whose synthesis is directed by information encoded in other biopolymers.
Carbon storage is ubiquitous throughout the eukaryotic and prokaryotic worlds. Mammals produce glycogen and triacylglycerol (TAGs) to store carbon and energy (Kalscheuer et al., 2007). Glycogen is a short term fuel used to buffer the glucose concentration in the blood, while TAGs are used for long term energy storage. Plants store starch and TAGs in their seeds to provide nourishment for growing embryos. Prokaryotes also store carbon,
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and microbial carbon storage molecules have the potential to become resources for the production of many industrial goods. Bacteria have been found to store carbon in the form of glycogen, TAGs, and Polyhydroxyalkanoates (PHAs) (Brigham et al., 2011). PHAs are polyesters synthesized by a wide range of bacteria for carbon and energy storage. PHAs are majorly made up of linear chain of 3-hydroxyacyl CoA (3-HA) monomers. The most common PHA is poly-3-hydroxybutyrate (PHB).
Today PHA monomers are generally characterized as either short chain length (C3 -C5, scl‐PHA) or medium chain length (C6 and greater, mcl‐PHA) (Sudesh et al., 2000). For example, poly(3-hydroxybutyrate) (PHB), poly(3-hydroxyvalerate) (PHV) and their copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) are typical examples of short-chain-length PHAs, whereas poly(3-hydroxyoctanoate) (PHO) and poly(3- hydroxynonanoate) (PHN), which are primarily formed as copolymers with 3- hydroxyhexanoate (HHx), 3-hydroxyheptanoate (HHp) and/or 3-hydroxydecanoate (HD), are typical examples of mcl-PHAs (Chen, 2009). There are now over 150 different types of known basic building blocks for PHA polymers reported.
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Figure 2-1 (a) General molecular formula of PHAs. Typically, x=1–8, and n ranges from 100 to 1000 s. (b) Some commonly synthesized short-chain-length PHA monomers (scl-PHA) and middle-chain-length PHA monomers (mcl-PHA). 3HB: 3-hydroxybutyrate, 3HV: 3 hydroxyvalerate, 3HHx: 3-hydroxyhexanoate, 3HO: 3-hydroxyoctanoate, 3HD: 3- hydroxydecanoate, 3HDD: 3-hydroxydodecanoate. Image taken from (Sudesh et al., 2000)
PHAs have useful properties such as: biodegradability, thermo-plasticity, biocompatibility, non-toxicity, they are considered a replacement for petrochemical polymers. In recent years companies have been interested in the use of PHAs in packaging, biomedical, agricultural applications. It is well known that PHAs were initially used for manufacturing cosmetic containers such as shampoo bottles (Hocking et al., 1994) moisture barriers in sanitary products (Lauzier et al., 1993) or pure chemicals as raw materials for the production of latex paints (Scholz, 2000).
11
Also, they can be used as carriers for long term release of herbicides or insecticides (Galego et al., 2000). Ultrahigh molecular weight of PHAs can be useful to produce ultra-strong fibers for fisheries industry (Bugnicourt et al., 2014). However, in view of their properties PHAs are promising materials especially in a biomedical field. Especially, P(3HB) homopolymer and co-polyester of P(3HB-co-3HV) are the most studied PHAs for medical applications (Chen & Wang, 2013). In recent years they are considered as materials in the fabrication of cardiovascular products (heart valves, stents, vascular grafts), in drug delivery system (tablets, micro carriers for anticancer therapy), in wound management (sutures, nerve cuffs, swabs, strapless), in orthopeady (bone plates, spinal cages) (Chen &
Wang, 2013).
The physical properties of earlier commercial PHAs, like P(3HB-co-3HV) copolymers, are inadequate for many of the applications envisioned for the replacement of commodity plastics. Because of the remarkable stereo-regularity of the perfectly isotactic chain configuration created by the bio-catalyzed polymerization process, PHB homopolymer exhibits an unusually high degree of crystallinity (Sudesh et al., 2000).
The high crystallinity results in a rather hard and brittle material that is not very useful for many applications. The melting temperature (Tm) of PHB is also high, about 180 °C, relative to the region of its thermal decomposition temperature, making PHB much more difficult to manipulate using melt processing equipment used for conventional plastics (Chen, 2009). It was hoped that the incorporation of a co-monomer unit, 3HV, could control the excessively high Tm and crystallinity limitations. Unfortunately, the desired
12
effect of 3HV incorporation to regulate the crystallinity and Tm was surprisingly limited because of the isodimorphism phenomenon, where 3HV units can be easily included in the crystal lattice of 3HB units and vice versa without the anticipated disruption of crystallinity (Pan & Inoue, 2009). An alternative structure of PHA copolymers, therefore, had to be designed to overcome this limitation.
2.2 Short-chain-length/ medium-chain-length PHAs
PHA monomers (3-Hydroxyalkanoates, 3HA) longer than 3HV (mcl monomers that are C6 and longer) have a more pronounced effect on polymer properties. The equilibrium degree of crystallinity and melting temperature Tm of P(3HB‐ co‐ 3HA) decrease linearly as functions of the mol% of mcl in the polymer, and the length of the mcl side chains does not influence how these properties change (Noda et al., 2005).
The glass transition temperature (Tg) of these copolymers also decreases when mcl subunits are added to the polymer, but in this case longer monomers lead to greater decreases in Tg. P (3HB-co-3HA) copolymers are weaker than PHB, but also tougher and more flexible. Generally, the mcl content regulates the Tm and crystallinity of copolymer almost independently of the mcl size, if more than three carbons are present in the side group. On the other hand, the side group chain length of mcl monomer has a profound effect on the flexibility of the copolymer. Some properties of these polymers are summarized in Table 2-1.
13
Table 2-1 Thermal properties of some scl-mcl PHA copolymers
Monomeric composition of PHAs(mol%)
Tm ΔHm Tg Reference
3HB 3HV 3HHx 3HO 3HD (°C) (J/g) (°C)
92.8 7.1 133.5 66.3 -2.1
(Arikawa &
Matsumoto, 2016)
92.1 7.9 131.1 67.5 -2.2
90.9 9.1 116 18 -1
87.0 13.0 160 55 -9
(Murugan et al., 2016)
81.0 19.0 145 35 0
73.0 27.0 120 18 -1
94.0 1.0 4.0 1.0 155, 173 30 -14 (Valdés et al.,
2018)
100 168 74.4 1.3
(Tappel et al., 2014)
95.0 5.0 mol% (3HHx, 3HO, 3HD) 163.7 37.1 -3
93.0 7.0 mol% (3HHx, 3HO, 3HD) 163.8 36.1 -3.3
92.0 8.0 mol% (3HHx, 3HO, 3HD) 163.9 31.7 -2.9
94.0 6.0 127, 142.0 37.4 -1 (Rajaratanam
et al., 2016)
91.0 9.0 142 -1
(Noda et al., 2005)
91.0 9.0 130 -4
93.0 7.0 138 -12
Tm: Melt temperature, Tg: glass transition temperature, ΔHm: fusion enthalpy temperature
3HB: 3-hydroxybutyrate, 3HV: 3 hydroxyvalerate, 3HHx: 3-hydroxyhexanoate, 3HO: 3-hydroxyoctanoate, 3HD: 3-hydroxydecanoate, 3HDD: 3-hydroxydodecanoate
A small amount of mcl subunits can effectively depress the crystallinity to make PHAs more ductile and tough. It has been reported that, the incorporation of mcl subunits, as low
14
as 5 mol %, can make the mechanical properties of PHAs comparable to those of ductile polyethylene and greatly expand the potential utility of these materials as general purpose plastics. Further incorporation of larger mcl-3HA co-monomer units makes PHAs even more soft and flexible, see Table 2-2.
15
Table 2-2 Mechanical properties of some scl-mcl PHA copolymers
Polymer Tensile
strength
Young's Modulus
Elongation
to break Reference
(MPa) (MPa) (%)
P(3HB) 40 3500 0.4
(Chanprateep &
Kulpreecha, 2006)
P(3HB-co-3HV)
3% 3HV 38 2900 –
9% 3HV 190 1620 37
14% 3HV 150 1500 35
20% 3HV 120 1450 32
25% 3HV 70 1370 30
P(3HB-co-4HB)
3% 4HB 28 – 45
10% 4HB 24 – 242
16% 4HB 26 – 444
44% 4HB 10 – 511
64% 4HB 17 30 591
90% 4HB 65 100 1080
P(3HB-co-3HV-co-
4HB)
10%3HB 40%3HV 50%4HB 9 503 4
11%3HB 34%3HV 55%4HB 10 618 3
11%3HB 23%3HV 66%4HB 9 392 5
12%3HB 12%3HV 76%4HB 4 142 9
10%3HB 6%3HV 84%4HB 9 118 300
4%3HB 3%3HV 93%4HB 14 127 430
P(4HB) 104 149 1000
P(3HB-co-3HV-co-
4HB) 64%3HB 32%3HV 4%4HB 138 18 19 (Ramachandran
et al., 2011)
P(3HB-co-3HHx)
Kaneka corp.
10% 3HHx 21 400
14% 3HHx 23 760
17% 3HHx 20 ˂800
31% 3HHx 6 ˂800
P(3HB-co-3HHx)
(Doi et al., 1995)
10% 3HHx 21 400
15% 3HHx 23 760
17% 3HHx 20 850
P(3HB-co-3HHx)
(Asrar et al., 2002)
2.5% 3HHx 25.7 631 6.7
4.6% 3HHx 22.9 599 6.5
5.4% 3HHx 23.9 493 17.6
7.0% 3HHx 17.3 288 23.6
8.5% 3HHx 15.6 232 34
9.5% 3HHx 8.8 155.3 43
3HB: 3-hydroxybutyrate, 3HV: 3 hydroxyvalerate, 3HHx: 3-hydroxyhexanoate, 3HO: 3-hydroxyoctanoate, 3HD: 3-hydroxydecanoate, 3HDD: 3-hydroxydodecanoate
16 2.3 PHA biosynthesis in Cupriavidus necator
In recent years, hundreds of microorganism possessing the ability to produce different types of PHAs has been investigated. The model organism for studying PHA biosynthesis is Cupriavidus necator H16. C. necator is a Gram‐ negative bacterium of the class Beta- proteobacteria that lives in soil and freshwater environments (Pohlmann et al., 2006). Its importance as a model organism was established when it was discovered that C. necator will accumulate high levels of PHB (~80% of cell dry weight) when grown in media that has plentiful carbon but is limited in some other essential nutrient (Hassan et al., 2013).
PHB synthesis is catalyzed by a PHA synthase, whose in vivo substrates are CoA thioesters (Rehm, 2003). This polymerization is a stereospecific reaction in which only (R)-3- hydroxyacyl-CoA (3HB-CoA) molecules serve as substrates.
The precursor to PHB is 3HB-CoA, which can be synthesized from the central metabolite acetyl-CoA. Wild type C. necator has limited ability to utilize sugars as carbon sources, with fructose and N‐ acetylglucosamine being the only carbohydrates that have been shown to support growth. Fructose is catabolized through the Entner-Doudoroff pathway, and the resulting pyruvate can be converted to acetyl-CoA and used for energy generation in the citric acid cycle, or for production of PHB (Fleige et al., 2011).
17 2.3.1 PHB operon
PHB is synthesized from acetyl-CoA in a three-reaction pathway: two molecules of acetyl- CoA are condensed by an acetyl-CoA acetyl-transferase (PhaA) to form acetoacetyl-CoA, the acetoacetyl-CoA is reduced to form 3HB-CoA by a reductase (PhaB) and the 3HB- CoA is polymerized by the PHA synthase (PhaC1). Historically PhaA has been referred to as a β‐ ketothiolase, which is the enzyme’s reverse reaction. Genes encoding the enzymes that carry out each of these reactions were discovered as an operon (phaC1‐ phaA‐ phaB1) in the C. necator genome (Kichise et al., 1999).
Figure 2-2 C. necator operon. 1: phaA β-Ketothiolase: 2: phaB: NADPH dependent acetoacetyl-CoA reductase; 3: phaC PHA synthase. Adapted from (Eggers & Steinbüchel, 2013)
18 2.3.2 Scl-PHA copolymers biosynthesis
The copolymer poly (3HB-co-3HV) generated considerable interest when it was discovered that C. necator would accumulate this polymer when odd chain length organic acids were included in the growth medium. Metabolism of these acids leads to the formation of propionyl-CoA, which reacts with acetyl-CoA to form ketovaleryl-CoA.
Reduction of the ketovalerylCoA by PhaB produces (R)-3-hydroxyvaleryl-CoA, allowing HV to be incorporated into the polymer chain (Shang et al., 2004). It was initially believed that PhaA catalyzed the condensation of acetyl-CoA and propionyl-CoA, but later work demonstrated that another β-ketothiolase (BktB) is largely responsible for this reaction in C. necator (Rehm, 2003).
2.3.3 PHA biosynthesis from fatty acids
The strain can grow and synthesize PHAs when is cultivated on mcl- fatty acids or vegetable oil. The fatty acid β‐ oxidation provides precursors for monomer synthesis. In the fatty acid degradation pathway, the acids are first activated by ligation to CoA, Figure 2-3. They are then broken down through a series of reactions, such that every cycle of β- oxidation results in the release of two carbons in the form of acetyl-CoA (Lau et al., 2014;
Magdouli et al., 2015). While some fatty acids in the cell are always turned over and recycled, high flux through this pathway requires that the cells are fed TAGs or fatty acids.
One of the intermediates of β-oxidation is 3-hydroxyacyl-CoA, but it is the (S) form that cannot be polymerized (Sudesh et al., 2000). The existence of a 3-hydroxyacylCoA epimerase has been proposed, but this enzymatic reaction has never been observed.
19
Another intermediate of fatty acid β‐ oxidation is 3-ketoacyl-CoA. The preferred substrate of the C. necator PhaB is acetoacetyl-CoA, but it can also reduce other substrates up to C6 in length (Rehm et al., 2003)
Based on the various types of PHA that were synthesized by C. necator, one common observation is that the incorporated monomers always contain only 3-5 carbon atoms (Madison & Huisman, 1999). This lead to the conclusion that in C. necator, the PHA synthase enzyme which polymerizes the monomers is only active towards scl-PHA (Rehm, 2003). However, the position of the oxidized carbon in the monomer is apparently not a crucial factor, which explains the incorporation of 4- and 5-hydroxyalkanoates (HA) besides the more common 3-HA (Loo et al., 2005).
Very recent findings indicate that the polymerizing enzyme of C. necator may actually have a broader range of substrate specificity. This was realized when the PHA synthase gene of C. necator was expressed in a heterologous environment which can provide for a wider range of HA monomers (Antonio et al., 2000; Dennis et al., 1998). At present, it has been shown that the C. necator PHA synthase enzyme can incorporate small amounts of 3HHx, 3HO and 3HDD units(Rathinasabapathy et al., 2014; Valdés et al., 2018; Volova et al., 2008), Table 2-3. However, the biosynthesis of scl-mcl PHAs in C. necator strains with significant and standardize mcl fraction remains as a challenging task.
20
Figure 2-3 The oxidation of fatty acids in C. necator provides intermediates to generate PHAs through the formation of (R)-3-hydroxyacyl-CoA. Adapted from (Eggers & Steinbüchel, 2013). Supplying enzymes such as Acyl-CoA dehydrogenase (FadE), (R)-specific enoyl-CoA hydratase (PhaJ) provide the PHA intermediates to be polymerize by the PHA synthase (PhaC)
21
Table 2-3 Scl-mcl PHAs polymerized by the PHA synthase of C. necator H16
Strain Modifications Fermentation
mode Substrate Total
biomass PHA 3HB 3HV 3HHx 3HO 3HDD
References
(g/l) % Mol %
C. necator H16
PhaC1, and PhaB overexpression
Shake flask 0.1%
hexanoate 1.96 49 90 10
(Dennis et al., 1998) 0.1%
octanoate 2.25 46 90 10
0.1%
myristate 0.41 51 93 1 6
0.1%
palmitate 0.51 58 93 1 6
0.1% oleate 1.44 57 96 4
E. coli LS1298
PHB operon from C.
necator
Shake flask
Octanoate 15 97.4 2.6
(Antonio et al., 2000)
Decanoate 15 98.6 1.4
Dodecanoate 14 95 1.5 3.5
Dodecanoate + acrylic acid
1.2 100
C. necator H16
Acrylate sodium inhibition
Shake flask (two stage)
Sodium octanoate, sodium acrylate 21.3 mM
2.6 36 97.91 2.09 trace
(Green et al., 2002) Sodium
octanoate, sodium acrylate 29.3 mM
2.5 32 94.32 5.68 trace
C. necator
H16 5l Fermenter (3
stage) Canola oil 92 48 99.81 0.06 0.09 0.04 (Rathinasabapathy et al., 2014) C. necator
H16
Shake flask (two stage)
Canola oil 5.7 67 97.9 0.7 1.4
(Valdés et al., 2018) C. necator
H16
PhaC2P.pu
CA-3 Canola oil 6.2 96 94 1 4 1
PHB-4 PhaC2P.pu
CA-3 Canola oil 3.7 49 96.5 0.8 4 2.7
E. coli: Escherichia coli, P. pu: Pseudomonas putida
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