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Fructose-Based Production of Short-Chain-Length and Medium-Chain-Length Polyhydroxyalkanoate Copolymer by Arctic Pseudomonas sp. B14-6.

ABSTRACT: Arctic bacteria employ various mechanisms to survive harsh conditions, one of which is to accumulate carbon and energy inside the cell in the form of polyhydroxyalkanoate (PHA). Whole-genome sequencing of a new Arctic soil bacterium Pseudomonas sp. B14-6 revealed two PHA-production-related gene clusters containing four PHA synthase genes (phaC). Pseudomonas sp. B14-6 produced poly(6% 3-hydroxybutyrate-co-94% 3-hydroxyalkanoate) from various carbon sources, containing short-chain-length PHA (scl-PHA) and medium-chain-length PHA (mcl-PHA) composed of various monomers analyzed by GC-MS, such as 3-hydroxybutyrate, 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, 3-hydroxydodecenoic acid, 3-hydroxydodecanoic acid, and 3-hydroxytetradecanoic acid. By optimizing the PHA production media, we achieved 34.6% PHA content using 5% fructose, and 23.7% PHA content using 5% fructose syrup. Differential scanning calorimetry of the scl-co-mcl PHA determined a glass transition temperature (T_g) of 15.3 °C, melting temperature of 112.8 °C, crystallization temperature of 86.8 °C, and 3.82% crystallinity. In addition, gel permeation chromatography revealed a number average molecular weight of 3.6 × 10⁴, weight average molecular weight of 9.1 × 10⁴, and polydispersity index value of 2.5. Overall, the novel Pseudomonas sp. B14-6 produced a polymer with high medium-chain-length content, low T_g, and low crystallinity, indicating its potential use in medical applications.

SUBMITTER: Choi TR

PROVIDER: S-EPMC8123457 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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 PcooF 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 (PHAMCL), enhanced gene expression through the PcooF promoter also led to an increased molar fraction of 3HO in the synthesized copolymer compared with the Plac 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 PHAMCL 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.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.

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Fructose-Based Production of Short-Chain-Length and Medium-Chain-Length Polyhydroxyalkanoate Copolymer by Arctic Pseudomonas sp. B14-6.

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