Factors affecting polyhydroxybutyrate accumulation in mesophyll cells of sugarcane and switchgrass

Abstract

BackgroundPolyhydroxyalkanoates are linear biodegradable polyesters produced by bacteria as a carbon store and used to produce a range of bioplastics. Widespread polyhydroxyalkanoate production in C4 crops would decrease petroleum dependency by producing a renewable supply of biodegradable plastics along with residual biomass that could be converted into biofuels or energy. Increasing yields to commercial levels in biomass crops however remains a challenge. Previously, lower accumulation levels of the short side chain polyhydroxyalkanoate, polyhydroxybutyrate (PHB), were observed in the chloroplasts of mesophyll (M) cells compared to bundle sheath (BS) cells in transgenic maize (Zea mays), sugarcane (Saccharum sp.), and switchgrass (Panicum virgatum L.) leading to a significant decrease in the theoretical yield potential. Here we explore various factors which might affect polymer accumulation in mesophyll cells, including targeting of the PHB pathway enzymes to the mesophyll plastid and their access to substrate.ResultsThe small subunit of Rubisco from pea effectively targeted the PHB biosynthesis enzymes to both M and BS chloroplasts of sugarcane and switchgrass. PHB enzyme activity was retained following targeting to M plastids and was equivalent to that found in the BS plastids. Leaf total fatty acid content was not affected by PHB production. However, when fatty acid synthesis was chemically inhibited, polymer accumulated in M cells.ConclusionsIn this study, we provide evidence that access to substrate and neither poor targeting nor insufficient activity of the PHB biosynthetic enzymes may be the limiting factor for polymer production in mesophyll chloroplasts of C4 plants.

Poly-3-Hydroxybutyrate Functionalization with BioF-Tagged Recombinant Proteins

“…The PHAs constitute, among natural polymers, the largest group of microbial polyesters that displays thermoplastic features (9, 13) (hence the commonly used term "bioplastics"). They have been hailed as potential competitors of oil-derived plastics, not only because of their physical properties but also for their biocompatibility and biodegradability (8,14,15) and the high sustainability of their sources, as they naturally accumulate as a reserve material in several microbial (16) and engineered plant (17) species. The tunable structural features of these polyesters based on their variable compositions make them highly attractive for the development of next-generation biomaterials capable of being functionalized with peptides and proteins.…”

Systems biology and metabolic modelling unveils limitations to polyhydroxybutyrate accumulation in sugarcane leaves; lessons for C4 engineering

“…We further interrogated the model to see whether metabolic impairment can be due to the compartmentalization of PHB synthesis, which mainly occurs in BS chloroplasts (McQualter et al, 2014b;Petrasovits et al, 2012). Figure 5c shows that the relative ATP demand in BS increased at increasing r CD but only when PHB synthesis was compartmentalized to M. When PHB synthesis was all compartmentalized to BS (V DBS /V D = 1) ATP demand in BS decreased for increasing r CD reflecting the ATP advantage of pyruvate decarboxylation in BS.…”

“…Strategies to increase PHB production in plants have relied initially on determining the most suitable subcellular compartment for expression of the PHB pathway (Nawrath et al, 1994;Petrasovits et al, 2007), with sufficient enzyme activity to maximize use of available substrate (Bohmert-Tatarev et al, 2011;Petrasovits et al, 2012;Somleva et al, 2008). Access to substrate in M was found to be problematic in C 4 grasses (McQualter et al, 2014b;Petrasovits et al, 2013) and remedied by the use of an acetoacetyl-CoA synthase in place of a b-ketothiolase (McQualter et al, 2014a). Increasing A by adjusting flux through the RPP cycle (Somleva et al, 2013) significantly corrected adverse phenotypes and increased PHB production in switchgrass plants.…”

The use of an acetoacetyl‐CoA synthase in place of a β‐ketothiolase enhances poly‐3‐hydroxybutyrate production in sugarcane mesophyll cells

“…We previously demonstrated that chemical inhibition of acetyl‐CoA carboxylase (ACCase), the enzyme that commits carbon to FAS, substantially increased PHB accumulation (Petrasovits et al ., ). We also showed that ACCase inhibition leads to a considerable increase in polymer production in M cells (McQualter et al ., ), suggesting that access to substrate limits PHB production in M cells. In an attempt to better utilize the capacity of M cells for PHB synthesis, we have explored a strategy to access the available substrate(s) more effectively using the recently described novel acetoacetyl‐CoA synthase (AACS) encoded by the nphT7 gene of Streptomyces sp.…”