Synthesis Gas (Syngas)-Derived Medium-Chain-Length Polyhydroxyalkanoate Synthesis in Engineered Rhodospirillum rubrum

Abstract

The purple nonsulfur alphaproteobacterium Rhodospirillum rubrum S1 was genetically engineered to synthesize a heteropolymer of mainly 3-hydroxydecanoic acid and 3-hydroxyoctanoic acid [P(3HD-co-3HO)] from CO- and CO₂-containing artificial synthesis gas (syngas). For this, genes from Pseudomonas putida KT2440 coding for a 3-hydroxyacyl acyl carrier protein (ACP) thioesterase (phaG), a medium-chain-length (MCL) fatty acid coenzyme A (CoA) ligase (PP_0763), and an MCL polyhydroxyalkanoate (PHA) synthase (phaC1) were cloned and expressed under the control of the CO-inducible promoter P_cooF from R. rubrum S1 in a PHA-negative mutant of R. rubrum P(3HD-co-3HO) was accumulated to up to 7.1% (wt/wt) of the cell dry weight by a recombinant mutant strain utilizing exclusively the provided gaseous feedstock syngas. In addition to an increased synthesis of these medium-chain-length PHAs (PHA_MCL), enhanced gene expression through the P_cooF promoter also led to an increased molar fraction of 3HO in the synthesized copolymer compared with the P_lac promoter, which regulated expression on the original vector. The recombinant strains were able to partially degrade the polymer, and the deletion of phaZ2, which codes for a PHA depolymerase most likely involved in intracellular PHA degradation, did not reduce mobilization of the accumulated polymer significantly. However, an amino acid exchange in the active site of PhaZ2 led to a slight increase in PHA_MCL accumulation. The accumulated polymer was isolated; it exhibited a molecular mass of 124.3 kDa and a melting point of 49.6°C. With the metabolically engineered strains presented in this proof-of-principle study, we demonstrated the synthesis of elastomeric second-generation biopolymers from renewable feedstocks not competing with human nutrition.

Importance: Polyhydroxyalkanoates (PHAs) are natural biodegradable polymers (biopolymers) showing properties similar to those of commonly produced petroleum-based nondegradable polymers. The utilization of cheap substrates for the microbial production of PHAs is crucial to lower production costs. Feedstock not competing with human nutrition is highly favorable. Syngas, a mixture of carbon monoxide, carbon dioxide, and hydrogen, can be obtained by pyrolysis of organic waste and can be utilized for PHA synthesis by several kinds of bacteria. Up to now, the biosynthesis of PHAs from syngas has been limited to short-chain-length PHAs, which results in a stiff and brittle material. In this study, the syngas-utilizing bacterium Rhodospirillum rubrum was genetically modified to synthesize a polymer which consisted of medium-chain-length constituents, resulting in a rubber-like material. This study reports the establishment of a microbial synthesis of these so-called medium-chain-length PHAs from syngas and therefore potentially extends the applications of syngas-derived PHAs.

FIG 1

Synthesis of PHA_MCL from CO and CO₂ in recombinant R. rubrum. Upon entrance of both gases into the cell through the cytoplasmic membrane, carbon monoxide is oxidized with water to carbon dioxide and hydrogen by the carbon monoxide dehydrogenase CODH_{R. rubrum} (1). CO₂ is fixed via ribulose 1,5-bisphosphate carboxylase (RubisCO; Rru_A1998 [2]) through the Calvin cycle, of which the intermediate glyceraldehyde-3-phosphate (G3P) is subsequently converted to the central metabolite acetyl-CoA. Acetyl-CoA is subjected to fatty acid de novo synthesis cycle. The thiolysis of 3-hydroxyacyl-ACP is catalyzed by the 3-hydroxyacyl-ACP thioesterase PhaG_{P. putida} (3). MCL fatty acid CoA ligase PP_0763_{P. putida} (4) catalyzes the activation of free 3-hydroxy fatty acids by the addition of CoA. The resulting 3-hydroxyacyl-CoA precursors are polymerized to poly-3-hydroxyalkanoate (PHA_MCL) by PHA synthase PhaC1_{P. putida} (5). Synthesis of poly(3-hydroxybutyrate) [poly(3HB)] is deactivated by the deletion of phaC1_{R. rubrum} and phaC2_{R. rubrum}, which code for PHA_SCL synthases (6). Additional carboxylases, which might be also involved in assimilating additional CO₂ (16), include a pyruvate synthase (Rru_A2398 [7]), a crotonyl-CoA reductase (Rru_A3063 [8]), a propionyl-CoA carboxylase (Rru_A1943 [9]), and a 2-oxoglutarate synthase (Rru_A2721 [10]). HS-CoA, coenzyme A thiol; HS-ACP, acyl carrier protein thiol.

FIG 2

Cultivation of strains of R. rubrum ΔphaC1 ΔphaC2, harboring pBBR1MCS-2 (empty vector, triangles), pBBR1MCS-2::phaG::phaC1::PP_0763 (circles), or pBBR1MCS-2-P_cooF::phaG::phaC1::PP_0763 (squares). Cells were cultivated in medium modified from Bose et al. (22), with an artificial syngas atmosphere of (by volume) 40% CO, 40% H₂, 10% CO₂, and 10% N₂ (black) or of 40% CO and 60% N₂ (gray) for 5 days. Optical densities of samples, whose standard deviations are represented by error bars, were measured at 680 nm. d, days.

FIG 3

Proteins in crude extracts of cells of R. rubrum ΔphaC1 ΔphaC2 harboring pBBR1MCS-2 (empty vector) (A), pBBR1MCS-2::phaG::phaC1::PP_0763 (B), or pBBR1MCS-2-P_cooF::phaG::phaC1::PP_0763 (C). Cells were harvested after 2 days of cultivation in medium modified from Bose et al. (22) with an artificial syngas atmosphere of (by volume) 40% CO, 40% H₂, 10% CO₂, and 10% N₂. A 40-μg quantity of protein was separated in an 11.5% (wt/vol) SDS-polyacrylamide gel and stained with Coomassie brilliant blue. Molecular masses (in kilodaltons) are indicated on the left.

FIG 4

Polymer accumulation of R. rubrum S1(pBBR1MCS-2) (empty vector) (A), R. rubrum ΔphaC1 ΔphaC2(pBBR1MCS-2) (empty vector) (B), and R. rubrum ΔphaC1 ΔphaC2(pBBR1MCS-2-P_cooF::phaG::phaC1::PP_0763) (C). Cells were stained with Nile red and observed with a fluorescence microscope after 3 days of cultivation in medium modified from Bose et al. (22) with an artificial syngas atmosphere of (by volume) 40% CO, 40% H₂, 10% CO₂, and 10% N₂, applying phase-contrast (left) or fluorescence (right) microscopy.

FIG 5

PHA_MCL synthesis and degradation of R. rubrum ΔphaC1 ΔphaC2 (squares), R. rubrum ΔphaC1 ΔphaC2 ΔphaZ2 (triangles), and R. rubrum ΔphaC1 ΔphaC2 phaZ_C176A (circles) harboring pBBR1MCS-2-P_cooF::phaG::phaC1::PP_0763. Cells were cultivated in medium modified from Bose et al. (22) with an artificial syngas atmosphere of (by volume) 40% CO, 40% H₂, 10% CO₂, and 10% N₂ for 5 days and for a further 2 days in pure N₂. Optical densities of samples, whose standard deviations are represented by error bars, were measured at 680 nm. 3HO (light gray) and 3HD (dark gray) contents were measured by GC analysis of dried cell matter from samples taken after 5 and 7 days of cultivation.