BioEngineering

Metabolic engineering of PHA production in transgenic sugarcane

Major chemical companies worldwide are moving towards partly replacing traditional petrochemical commodity polymers with biopolymers. While of great promotional value, reduced reliance on non-renewable resources is only a minor argument for the change; chemical synthesis accounts for only a small fraction of petrochemical consumption and the energy used for polymer processing amounts to more than the actual chemical content. A more important argument is the possible reduction of xenobiotics in the environment and the reduced load on landfills if biodegradable polymers are used. 

The industry’s interest, however, is mainly driven by the prospect of biotechnology overcoming traditional shortcomings of organic synthesis, notably long product lead time and expensive plant design due to the use of toxic compounds at high pressure, high temperature. In the short term, the bulk biopolymer market will be dominated by semi-natural polymers derived from natural precursors (e.g., lactic acid and 1,3-propandiol) by chemical polymerisation. As biotechnology matures, however, it is likely that these polymers will be replaced with true biopolymers, i.e., polymers fully synthesised in living organisms.

Among the various true biopolymers, polyhydroxyalkanoates (PHAs) are the most promising. PHAs are a class of intracellular energy and carbon storage compounds found in many bacteria (equivalent to glycogen and fats in humans). Using different bacteria and by varying their carbon source, it is possible to produce biomaterials having properties ranging from stiff and brittle plastics to rubbery polymers. PHAs are unbranched polymers predominantly composed of R-(–)-3-hydroxyalkanoic acid monomers ranging from 3 to 14 carbons in length: [-O-CHR-CH₂-CO-]_x with R = [hydrogen or alkyl up to nonyl]. Co-polymers of different length monomers are common and additional variation arise from natural introduction of other monomers (e.g., 4-hydroxybutyrate or 5-hydroxyvalerate), unsaturated bonds and other functional groups.

Poly (3-hydroxybutyrate) (PHB) is the most widespread and thoroughly characterised PHA. It is derived from Acetyl-CoA by the sequential action of three enzymes: 3-ketothiolase, Acetoacetyl-CoA reductase, and PHA synthase. PHB production has been studied extensively in the bacterium Alcaligenes eutrophus, which accumulate as 0.2-0.5 micron PHB inclusions up to 80% by weight, when grown in media with excess carbon (e.g., glucose). Commercially, the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) has been available under the brand name Biopol for almost 20 years, initially produced by ICI and now by Monsanto. PHBV is produced by Alcaligenes eutrophus, when propagated on a mixture of glucose and propionic acid. With a lower melting temperature, PHBV is more amenable for processing than PHB.

Despite high yields and the potential to produce a broad range of polymers through substrate as well as genetic manipulation, PHAs produced by bacterial fermentation is unlikely ever to compete with petrochemical bulk polymers due to high cost. The target price of $1.5/kg is much lower than even the most optimistic projections for fermentation costs of $5/kg. Hence, Biopol has been limited to niche markets, where a biodegradability and a “natural product” label can attract a substantial premium.

Recently, the PHA pathway from Alcaligenes eutrophus has been expressed in transgenic crop plants, a very attractive system for such purpose, with the potential for producing large amounts of chemicals at a low cost. Commercially relevant yields of 14% dry weight has been achieved in the plant Arabidopsis thaliana, a transgenic plant model system commonly used due to the availability of excellent molecular biology tools rather than commercial relevance. In the Australian setting, production of PHAs (and other bulk chemicals) in sugarcane offer a potential for product diversification and reduced reliance on sugar price for Queensland’s rural industry. Hence, Dr Nielsen in collaboration with researchers at BSES has recently commenced a proof of concept pilot study on PHB production in sugarcane.