Keywords

1 Introduction

Over the past 60 years, the development of synthetic polymers has reduced the men’s dependence upon the utilization of plant product-based polymers such as rubber, cotton, wood, etc. But the excessive and ever-growing demands of utilizing synthetic polymers raise the global environmental concerns. Therefore, the scientific community has engaged to discover alternatives of synthetic polymers. As an alternative of synthetic polymers, polyhydroxyalkanoates (PHAs) came into existence. These are the natural products of certain bacterial cells. Therefore, scientists decided to produce biological polymers by using bacterial cells as bioreactors. This was first time exploited in 1980 by Imperial Chemical Industries (Anderson and Dawes 1990). This industry generated a large setup for the bulk production of polyhydroxyalkanoates using polymer-accumulating bacterial strains such as Ralstonia eutropha. But the cost of producing PHAs through this way was very high. So again there were also the requirements of cost-effective alternatives for producing biopolymers. The development of advanced biotechnological tools attracted the concern of global scientists toward the utilization of plant bioreactors for producing renewable biological polymers. Therefore, the scientific community moved toward the utilization of plant bioreactors for generating polyhydroxyalkanoates (PHAs) in a cost-effective and eco-friendly manner. For this purpose, a variety of transgenic plant species including Arabidopsis, tobacco, rapeseed, cotton, alfalfa, flax, sugarcane, Camelina, and oil palm were tested at larger scales and generated new dimensions of producing biopolymers.

Polyhydroxyalkanoates (PHAs) are biopolyesters that are synthesized naturally in a broad range of bacterial cells such as Alcaligenes eutrophus and many other species as an inert carbon and energy reserve accumulated in the cytoplasm up to about 80% of the total dry weight in the form of round-shaped granules with a diameter of 0.2–1.0 μm (Sabbagh and Muhamad 2017). These polymers are made up of about 600–35,000 identical monomer units. Polyhydroxyalkanoates act as water-insoluble storage compounds which are synthesized under environmental stress conditions in the excess of carbon and the limiting quantities of important growth nutrients such as nitrogen, phosphorus, iron, magnesium, potassium, sulfur, zinc, or oxygen (Masood et al. 2014). These biopolymers are depolymerized during the exhausted carbon source conditions. Thus, the degraded products could be used by microbes as an energy and carbon source (Anderson and Dawes 1990).

Polyhydroxyalkanoates (PHAs) are considered similar to the conventional plastics in reference of its properties such as thermoplastic and polypropylene nature (Anjum et al. 2016). Instead of petrochemical plastics, PHAs are natural, nontoxic, biodegradable, and renewable (Sharma et al. 2016). These properties make PHA an attractive alternative of petrochemical plastic. In the near future, it is hopefully projected that the production of synthetic plastic polymers could possibly reached up to about eight hundred ten million tons (810 million tons) by the end of 2050 (Gumel et al. 2013). As it is well known that plastic pollution has been an unbeatable burning issue across the globe, it has been an urgent necessity to find out the eco-friendly alternatives of synthetic plastics. That’s why, the scientific community and industries are engaging to produce synthetic polymers through natural means. Generally microbial bioreactors are utilized in producing PHAs at larger scales, but the whole process of bioplastic polymer production is still highly expensive than the process of producing petrochemical-based synthetic polymers due to the cost of the nutrition for microbial cultures (Baikar et al. 2017). So the scientific community has engaged in optimizing transgenic plants as novel bioreactors for production of PHAs at cheaper costs. The present chapter summarizes the information about PHA, its structure, biosynthesis, and the current status of transgenic plants which were engineered for producing PHAs at cheaper costs.

2 PHA Structure and Biosynthesis

Polyhydroxyalkanoates are generally linear polyesters consisting of several 3-(R)-hydroxy fatty acid monomers (HA) linked together by ester bonds. These ester bonds are produced by the linkage of carboxylic group of one monomer unit to the hydroxyl group of another monomer unit (Sudesh et al. 2000; Lenz and Marchessault 2005). On the basis of the presence of carbon atoms in the monomers, polyhydroxyalkanoates are generally categorized into two major groups. The first group is called short chain length polyhydroxyalkanoates (scl-PHAs), and the second group is medium chain length polyhydroxyalkanoates (mcl-PHAs). Short chain length polyhydroxyalkanoates (scl-PHAs) generally consist of 3–5 carbon atoms, whereas the medium chain length polyhydroxyalkanoates (scl-PHAs) have 6–14 carbons. Under natural conditions, the short chain length polyhydroxyalkanoates (scl-PHAs) are synthesized in Cupriavidus necator, while the medium chain length polyhydroxyalkanoates (scl-PHAs) are accumulated in Pseudomonas species. The examples of short chain length PHAs are P(3HB) [poly-3-hydroxybutyrate], P(4HB) [poly-4-hydroxybutyrate], and P(3HV) [Poly(3-hydroxyvalerate)] or the copolymer P(3HB-co-3HV), whereas the P(3HHx) [poly-3-hydroxyhexanoate], P(3HO) [poly-3-hydroxyoctanoate], and copolymer P(3HHx-co-3HO) are considered as medium chain length PHAs (Kim and Lenz 2001). Each polyhydroxyalkanoate (PHA) polymer generally consists of about 1000–10,000 monomers, but most of them are synthesized by short chain length (SCL) monomer units (Van der walle et al. 2001). The chemical structure of polyhydroxyalkanoates (PHAs) is depicted in Fig. 15.1.

Fig. 15.1
figure 1

The chemical structure of polyhydroxyalkanoates (PHAs). The pendant R groups (shaded boxes) vary in chain length from 1 carbon (C1) to over 14 carbons (C14). Structures shown here are poly-3-hydroxybutyrate (PHB) [R = methyl], poly-3-hydroxyvalerate (PHV) [R = ethyl], and poly-3-hydroxyhexanoate (PHH) or poly-4-hydroxybutyrate (P4HB) [R = propyl]. (Adapted from Suriyamongkol et al. 2007)

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Most of our knowledge about biosynthesis of polyhydroxyalkanoate is mainly based upon the studies on the production of polyhydroxybutyrate (PHB) in cytoplasm of a gram-negative soil bacterium Ralstonia eutrophus or Alcaligenes eutrophus bacteria. This bacterium has the capability of producing polyhydroxyalkanoate in a natural way and could accumulate the polymer up to 85% of its total dry body weight when grown on culture media with excess of glucose. In this way, it acts as an energy source, but the production of this polymer is limited when there is growth-limiting conditions such as lack of macroelements such as nitrogen, phosphorus, and trace elements or the lack of oxygen in culture media (De Koning 1995). Previous studies reported that the polyhydroxybutyrate (PHB) could be depolymerized into acetoacetate and further into acetyl coenzyme A (CoA) by applying growth-limiting conditions (Steinbuchel and Valentin 1995). Therefore, it is clearly demonstrated that the acetyl coenzyme A acts as a precursor of polyhydroxybutyrate biosynthesis in bacterial cell. Polyhydroxybutyrate (PHB) decomposes into 3-hydrobutyrate (3-HB) monomers that can be used by fungi and bacteria as carbon sources. The biosynthesis of PHB was first time described in 1973 in a bacterium Ralstonia eutrophus by Gottingen and Hull (Senior and Dawes 1973). There are three key enzymes, namely, acetoacetyl-CoA reductase, 3-ketothiolase, and PHA synthase which leads to the production of polyhydroxybutyrate by using acetyl-CoA. PHA synthase uses CoA thioester of (R)-hydroxy fatty acids as substrate. The enzyme popularly known as 3-ketothiolase encoded by gene phaA or phbA is mainly responsible for catalyzing the reversible condensation of two molecules of acetyl-CoA into acetoacetyl-CoA molecule. The acetoacetyl-CoA reductase encoded by phaB or phbB gene reduces acetoacetyl-CoA into R-(−)-3-hydroxybutyryl-CoA. After that the R-(−)-3-hydroxybutyryl-CoA finally polymerizes into polyhydroxybutyrate (PHB) by the action of PHA synthase enzyme encoded by a gene called phaC or phbC (Yunus et al. 2008; Kosseva and Rusbandi 2018). The polyhydroxyalkanoates (PHAs) biosynthesis pathway is schematically depicted in Fig. 15.2. Polyhydroxyalkanoates are generally biosynthesized through two possible routes. The first route is based upon β-oxidation pathway intermediates and also on alkanoic acids. In this process, the levorotatory S-3-hydroxyacyl-CoA is converted into R-3-hydroxyacyl-CoA, a dextrorotatory enantiomer by the action of an enzyme epimerase. In the second route, the fatty acid biosynthesis intermediates such as R-3-hydroxyacyl-ACP are used. In this process, the acyl carrier protein (ACP) is replaced by coenzyme A using an important enzyme 3-hydroxyacyl-CoA-ACP transacylase. Both these processes are completed by a gene called phaC [Kosseva and Rusbandi 2018].

Fig. 15.2
figure 2

The medium chain length polyhydroxyalkanoates (mcl-PHA) biosynthesis pathways occur in the peroxisomes of transgenic plants. Here, enzyme ACD = acyl-CoA dehydrogenase; ECH = enoyl-CoA hydratase; HCD = l-3-hydroxyacyl-CoA dehydrogenase; and KT = β-ketothiolase. (Reproduced from Dobrogojski et al. 2018)

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3 PHA Production in Transgenic Plants

The production of PHAs by using microorganisms is costly because of various factors such as variety and amount of nutrition supplied for microbes, optimized growth environment, and sterilized conditions (Din et al. 2012; Mozejko-Ciesielska and Kiewisz 2016). Therefore in comparison to microbes, transgenic plants are considered as cheaper eco-friendly alternatives. The biosynthesis of PHAs in transgenic plants mainly depends upon mineral salts, water, light, and carbon dioxide (CO2). The PHA production in transgenic plants is generally based upon the availability of acetyl-CoA, a primary substrate for PHA biosynthesis, because the plant cells do not have the abilities to degrade PHA as the microbes do. Acetyl-CoA is the main metabolite of plant’s catabolic and anabolic processes. The plant cellular compartments such as cytoplasm, mitochondria, peroxisomes, and plastids are rich in acetyl-CoA. Therefore, the scientists targeted these compartments as the major sites for producing and accumulating various PHAs in transgenic plants. The literature showed that the first experimental research attempt for producing PHA was successfully achieved in the cytoplasm of transgenic Arabidopsis thaliana. After this work several another research experiments were also conducted using various plant species. But the deficiency of acetyl-CoA and acetoacetyl-CoA because of their utilization in plant hormone and steroid biosynthesis pathways limited the production of PHAs inside plant cell cytoplasm. Like cytoplasm, mitochondria also have the limitations of the deficiency of acetyl-CoA because of its utilization during cellular respiration. Plastids appear to the best site for PHA biosynthesis in plants because there acetyl-CoA is present in higher concentrations and mainly utilized for the biosynthesis of fatty acids. The plastids are the organelles which work properly despite the structural changes and have the ability to store larger starch granules. But the plastids do not have the stocks of beta ketothiolases. The beta ketothiolases are located in the cell’s cytoplasm. This problem could be overcome by applying specific DNA-encoding plastid-targeted sequences inserting in the vectors. The peroxisomes are also considered as high potential sites for the production of PHAs in transgenic plant cells because of having high reductive strength of NADH and their beta oxidation of fatty acids. Peroxisomes are important cell organelles because of synthesizing medium chain length polyhydroxyalkanoates (mcl-PHAs). Since the last two decades, several scientists are doing research on producing PHAs in transgenic plants. The detailed information regarding the current status of transgenic plants developed for producing polyhydroxyalkanoates are given in Table 15.1.

Table 15.1 The current status of transgenic plants engineered for production of polyhydroxyalkanoates (PHAs)
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4 Conclusion and Future Prospects

The environmental pollution generated through petroleum-based synthetic polymers has become a very big global challenge. The production of synthetic polymers is increasing day by day, and now it has appeared in an unbeatable form of pollutants. The management of the plastic and its products is not an easy task; it takes several hundreds of years to be decomposed. Therefore, it is a need of present time to find out eco-friendly biodegradable alternatives. Polyhydroxyalkanoates (PHAs) have appeared as a smart choice of scientific community as well as industry in the form of plastic alternatives. PHAs are the major class of biodegradable biopolymers which are biosynthesized by microorganisms in a natural way. The production of PHAs by using microorganisms is costly because of various factors such as variety and amount of nutrition supplied for microbes, optimized growth environment, and sterilized conditions. Therefore in comparison to microbes, transgenic plants are considered as cheaper eco-friendly alternatives. The biosynthesis of PHAs in transgenic plants mainly depends upon mineral salts, water, light, and carbon dioxide (CO2). Some cellular compartments such as cytoplasm, mitochondria, peroxisomes, and plastids have been targeted as important sites for producing and accumulating PHAs in transgenic plants. Since the last two decades, several scientists have engaged in research for optimizing transgenic plants as bioreactors for producing PHAs. Several plant species including Arabidopsis thaliana, Camelina, tobacco, sugarcane, maize, rapeseed, flax, cotton, and oil palm have been genetically engineered for producing PHAs. But till date, no one plant species is released for commercial production of biopolymers. The research is on the way, things are optimizing, and we hopefully expect that the transgenic plants would be available in the near future for producing PHAs at commercial scales.