Polyhydroxybutyrate synthesis in Camelina: Towards coproduction of renewable feedstocks for bioplastics and fuels

Abstract

Plant-based co-production of polyhydroxyalkanoates (PHAs) and seed oil has the potential to create a viable domestic source of feedstocks for renewable fuels and plastics. PHAs, a class of biodegradable polyesters, can replace conventional plastics in many applications while providing full degradation in all biologically active environments. Here we report the production of the PHA poly[(R)-3-hydroxybutyrate] (PHB) in the seed cytosol of the emerging bioenergy crop Camelina sativa engineered with a bacterial PHB biosynthetic pathway. Two approaches were used: cytosolic localization of all three enzymes of the PHB pathway in the seed, or localization of the first two enzymes of the pathway in the cytosol and anchoring of the third enzyme required for polymerization to the cytosolic face of the endoplasmic reticulum (ER). The ER-targeted approach was found to provide more stable polymer production with PHB levels up to 10.2% of the mature seed weight achieved in seeds with good viability. These results mark a significant step forward towards engineering lines for commercial use. Plant-based PHA production would enable a direct link between low-cost large-scale agricultural production of biodegradable polymers and seed oil with the global plastics and renewable fuels markets.

Keywords: Camelina sativa; PHA; PHB; biopolymer; polyhydroxyalkanoate; polyhydroxybutyrate.

Figure 1

Strategies for cytosolic PHB production in developing seeds. Cytosolic PHB production using a three‐enzyme pathway of β‐ketothiolase (PhaA), NADPH‐dependent reductase (PhaB), and PHA synthase (PhaC) captures a portion of the acetyl‐CoA that could otherwise be used for fatty acid elongation or synthesis of an array of diverse phytochemicals. Strategies in which PhaC, the enzyme polymerizing substrate R‐3‐hydroxybutyryl‐CoA to polymer, is targeted to the cytosol or anchored to the cytosolic face of the ER membrane are shown.

Figure 2

T₂ seed PHB content and T₂ seedling emergence and survival of select lines obtained from transformations of WT43 with pMBXS394 and pMBXS763. (a) PHB content in T₂ seeds of WT43/pMBXS394. (b) Emergence and survival of T₂ seedlings of WT43/pMBXS394. Lines producing ≥2% PHB in seeds were tested for emergence and survival. (c) PHB content in T₂ seeds of WT43/pMBXS763. 56 lines had ≥2% PHB, only the highest producing 34 lines are shown. (d) Emergence and survival of T₂ seedlings of WT43/pMBXS763. Select T₁ events obtained from pMBXS394 and pMBXS763 transformations were analysed for insert copy number by Southern blot analysis (Figure S4). Where analysed, the insert copy number of the line is shown above the PHB content bar in (a) and (c). Plants without a number were not tested.

Figure 3

Phenotype of C. sativa WT43 control and T₂ seedlings obtained from transformations of WT43 with pMBXS394 and pMBXS763. Seeds were germinated in soil, and seedlings were photographed at the fully expanded cotyledon stage with first true leaves emerging (BBCH stage 10, Martinelli and Galasso, 2011). Seedlings of (a) C. sativa WT43 control, (b) pMBXS394 T₂ line 12‐0415 (4.5% PHB) and (c) pMBXS763 T₂ line 12‐0939 (4.4% PHB) are shown.

Figure 4

Microscopy of seedling cotyledons and imbibed seeds of C. sativa WT43 control and T₂ seedlings obtained from transformations of WT43 with pMBXS394 and pMBXS763. (a–h) Light microscopy images of cross sections of seedling cotyledons at 20× (a, c, e, g) and 40× (b, d, f, h) magnification, respectively. (a, b) C. sativa WT43 control grown alongside and imaged with (c, d) pMBXS394 transformed T₂ line 12‐0415 containing 4.5% PHB in bulk T₂ seed samples. (e, f) C. sativa WT43 control grown alongside and imaged with (g, h) pMBXS763 transformed T₂ line 12‐0933 containing 4.9% PHB in bulk T₂ seeds. Panels f and h are not magnified from panels e and g, respectively, but show a different view of the sample. T₂ seeds were germinated in soil and T₂ seedlings were sampled at the fully expanded cotyledon stage with first true leaves emerging. (i–p) Transmission electron microscopy (TEM) images of longitudinal sections of imbibed seeds. Longitudinal sections for analysis passed through cotyledonary region. (i, j) C. sativa WT43 control grown alongside and imaged with (k, l) pMBXS394 transformed line 12‐0415 containing 4.5% PHB in bulk T₂ seeds. (m, n) C. sativa WT43 control grown alongside and imaged with (o, p) pMBXS763 transformed T₂ line 12‐0933 containing 4.9% PHB in bulk T₂ seed. Seeds were imbibed for 5 h before processing for TEM. CW, cell wall; ER, endoplasmic reticulum; IS, intercellular space; LE, lower epidermis; M, mitochondria; OB, oil bodies; PM, palisade mesophyll; PSV, protein storage vesicles; SM, spongy mesophyll; UE, upper epidermis; VB, vascular bundle. Scale bars are provided at lower left of each image.