The commercial production of biopolymer is aimed as bio-degradable alteration to petrochemicals (synthetic) plastics. The homopolymer poly (3-4 hydroxy butyrate) (PHB) shows brittle, whereas copolymer like poly (3-hydroxy butyrate-co-3-hydroxy valerate) (PHBV) are more flexible due to their reduced crystallanity and are well suited for commercial applications. Polyhydroxybutyrate is best characterized polymer synthesized in bacteria Alkaligenes entrophus accumulate upto 70% of the bacterial cell dry weight.
In the molecular farming of plants, production of biodegradable plastics is one of the most key areas of plant biotechnology. Currently, production of biodegradable plastics is restricted to microbial fermentation technology.
Several persistent endeovour has been made to produce bioplastics in bulk in plants. Chemically, bioplastics are class of polymer known as hydroxy alkanoates (PHA) produced by numerous bacterial species as carbon and energy reserve.
Biosynthesis of PHB is relatively a simple three stage pathway utilising acetyl-CoA as starting material. Alcalegene entrophus utilise its three genes for three enzymes involved in the synthesis of PHB (pha A, pha B, pha C genes) (Fig. 17.14). In the production of bioplastic in transgenic plant, initially, only two genes pha B and pha C, encoding acetoacetyl CoA reductase and PHB synthase were transformed into Arabidopsis.
Since acetyl CoA occurs in the cytoplasm of plants transferring phA was found to be unnecessary. All these genes when transferred without targetting sequences, accumulation of biopolymer takes place in the cytoplasm, nucleus, and even vacuole. However, plant growth was found to be retarded with low accumulation of plastics.
In continuation of experiments all the three genes were transferred into Arabidopsis with chloroplast signal peptide. During transgene construct, each gene was fused to a transit peptide signal sequence of rubisco (N-terminal) small sub unit driven under CaMV35S promoter.
Each expression casette was transferred separately into Arabidopsis thaliana by a gene pyramiding technique. All these genes was brought together by a series of crossess between individual transgenic plants. Transgenic Arabidopsis accumulates nearly 14% of plant dry weight.
Production of bioplastic in cotton fibres generates considerable curiosity. It was found that cotton fibres exhibit β-ketothiolase activity, which is committed in the production of PHB. The genes of Alcali entropha pha B and pha were transferred into the seed axial meristem of cotton.
One of the gene pha B was expressed by cotton fibre specific promoter, while pha C was driven by 35S. Small PHB granules were found to accumulate in the cytoplasm of fibre cells. [Fig. 17.14]
A team of researcher from US based monsanto has made considerable effort on large scale production of bioplastic in oil yielding plants. Targetting on oil crops is due to the presence of acetyl CoA reserve and divertion of all these acetyl CoA into the production of biopolymer.
The three genes for PHB synthesis was taken from Ralstonia entropha and fused each gene to seed-specific promoter, which was then transferred to brassica, a major oil yielding plant, for the production of polyhydroxybutyrate. PHB was found to accumulate in oil seed lavcoplast upto 7.5% fresh seed weight.
The crystalline property and brittle nature polyhydroxy butyrate is barrier for its use in commercial way. It is therefore feassible to get polyhydroxy alkaonate co-polymer made from longer monomer. Pseudomonads accumulate medium chain length PHA synthesised from 3. hydroxy acetyl CoA intermediates by β-oxidation of fatty acids.
In an transgenic attempt, the gene pha C, frompseudomonas aeroginosa was transferred to Arabidopsis with a peroxisome targetting from an oil of seed rape isocitrate lyase. The transgenic plant accumulates medium chain length PHA in the peroxisomal and glyoxisomes to a level of 4 mg g-1 dry weight.
In order to overcome the entire problem associated with a brittle poly hydroxy alkanate, a team from the same monsanto company has done successful metabolic engineering in Arabidopsis for the production of co-polymer poly (3 hydroxy butyrate-Co-3 hydroxy (pHBV) valerate). One of the co-polymers PHBV is natural polymer with thermo-plastic properties.
Although PHBV can be produced in bacterial formations using Ralstonia entropha, but the process is not economically viable with polymer production from petrochemicals. Therefore, the production of PHA by genetic engineering of plants is expected to cut down cost to an economic feasibility.
Production of PHB homopolymer in plants has been proved in earlier cases. However, copolymer production (PHBV) in plants is difficult due to the non-availability of metabolic precursor other than acetyl CoA. Thus, monsanto team designed a pathway to produce PHBV in the plastids of Arabidopsis and Brassica plants.
In this pathway acetyl CoA is drawn from plastid metabolism, whereas propionyl CoA is generated from threonine via 2-ketobutyrate. Therefore, this pathway requires transformation of plants with four separate genes such as ilva, bktB, phbB and phbc. All genes were expressed under 35S promoter. The four genes were obtained from the bacteria Ralstonia eutropa and transformed with experimental plants.
In brassica co-transformation of all four genes were expressed from a single vector with seed specific promoter. The four genes for pathway when expressed produced threonine deaminase (ilva), β-ketothiolase (Gene BktB), D-reductase (phbB) and PHB synthase (phbc). The first reaction in the engineering pathway in transformed plants is the convertion of threonine to 2- ketobutyrate by transgene encoded enzyme ilva.
The next step is the formation of propionyl CoA, catalysed by the pyruvate dehydrogenase complex. This is the only plant endogenous enzyme participate in the engineered pathway for the conversion of threonine to polymeric 3- hydroxy valerate. While in Brassica it is capable of converting 2-ketobutyrate to priopionyl CoA.
Once propionyl CoA has been produced, it is along with co-substrate a cetyl CoA are converted to D-3-hydroxy valeryl CoA by the consecutive reactions catalysed by transgenes BktB and phb B. The D-3-hydroxy valeryl CoA and 3-hydroxy butyryl CoA are then converted to PHBV copolymer with the help of another transgene phbc (Fig. 17.15). By designing new pathway, PHBV was produced to a level of 3% in the transgenic plants.
Note:
The gene for enzymes 1, 3, 4 and 4 are derived from microorganism (Ralstonia) and enzyme pyruvate dehydroxygenase is native to the plant.
Production of bioplastic (PHB) in Arabidopsis and Brassica and copolymer (PHBV) by redesigning pathway has been found to have remarkable results. These work were carried out using nuclear transformation. However, nuclear transformation may pose risk of spread of transgenic pollen into the environment. Therefore PHB production in organelle has received greater attention.
A polyster has been produced in the plastids of tobacco. A polyster produced by genes for three bacterial enzymes was transferred to the tobacco plastid genome. The polycistronic phb operon encoding this pathway was cloned into plastome transformation vector.
Transgenic tobacco exhibit retarded in growth, especially in tissue that accumulate PHB, where as in rapidly growing tissue PHB synthesis was reduced. The accumulation of 1.7% PHB in transplastomic tobacco would be too low for profitability and industrial applications of transplastomic PHB plants. These results show successful and first evidence for plastid transformation mediated biopolymer production.