Dataset Information

Low Carbon Concentration Feeding Improves Medium-Chain-Length Polyhydroxyalkanoate Production in Escherichia coli Strains With Defective ?-Oxidation.

ABSTRACT: Medium-chain-length (MCL) polyhydroxyalkanoates (PHAs) of near homopolymeric composition are unnatural polymers, having almost identical repeating units throughout the polymer chain. These homopolymeric PHAs can be produced by ?-oxidation defective bacterial hosts. Escherichia coli is an attractive workhorse for the production of such genetically engineered PHAs; however, achieving efficient production of the near homopolymers by ?-oxidation defective strains is a major challenge because of a lack of process development studies. In order to address this issue, we investigated the optimization of carbon feeding for efficient production of MCL-PHAs by an E. coli strain with defective ?-oxidation, LSBJ. Engineered bacteria were cultured in shake-flasks with intermittent feeding of a fatty acid substrate [either decanoate (C10) or dodecanoate (C12)] at various concentrations together with a co-carbon source (glucose, glycerol, or xylose) in order to support cell growth. It was found that feeding low concentrations of both fatty acids and co-carbon sources led to an enhanced production of MCL-PHAs. Additionally, the supplementation of yeast extract improved cell growth, resulting in achieving higher titers of MCL-PHA. As a result, poly(3-hydroxydecanoate) [P(3HD)] and poly(3-hydroxydodecanoate) [P(3HDD)] were produced up to 5.44 g/L and 3.50 g/L, respectively, as near homopolymers by employing the developed feeding strategy. To the best of our knowledge, we record the highest titer of P(3HD) ever reported so far.

SUBMITTER: Mohd Fadzil FI

PROVIDER: S-EPMC6287193 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Low Carbon Concentration Feeding Improves Medium-Chain-Length Polyhydroxyalkanoate Production in <i>Escherichia coli</i> Strains With Defective β-Oxidation.

Mohd Fadzil Fakhrul Ikhma FI Mizuno Shoji S Hiroe Ayaka A Nomura Christopher T CT Tsuge Takeharu T

Frontiers in bioengineering and biotechnology 20181130

Medium-chain-length (MCL) polyhydroxyalkanoates (PHAs) of near homopolymeric composition are unnatural polymers, having almost identical repeating units throughout the polymer chain. These homopolymeric PHAs can be produced by β-oxidation defective bacterial hosts. <i>Escherichia coli</i> is an attractive workhorse for the production of such genetically engineered PHAs; however, achieving efficient production of the near homopolymers by β-oxidation defective strains is a major challenge because ...[more]

PMID: 30560122

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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.

Project description:Transcriptomics analysis of biopolymer (medium chain length polyhydroxyalkanoate) producing strain P.putida LS46 cultured with biodiesel derived waste carbon sources: studies of cellular adaptation to the industrial waste streams and metabolic profiling under the polymer producing conditions. We are reporting RNAseq analysis data here as part of our multi-level Omics study of medium chain length polyhydroxyalkanoate (mcl-PHA) producing strain P.putida LS46 culture with biodiesel derived waste glycerol and waste fatty acids. The data presented here will be used in two separate manuscripts. The objectives of this study are a): to evaluate cellular responses of P.putida LS46 under industrial waste stream. b): to study gene expression profile under two selected mcl-PHA producing conditions of P.putida LS46. Comparative multi-level Omics study: for objective a): Exponential P.putida LS46 cell from waste glycerol culture compared against reagent grade pure glycerol culture. For objective b): Two mcl-PHA producing conditions, namely stationary phase waste glycerol culture and exponential phase waste fatty acid culture of P.putida LS46, were compared against exponential phase waste glycerol culture of P.putida LS46. Major results from objective a): The waste glycerol substrate induced expression of a large number of genes putatively involved in heavy metal tolerance, including three gene clusters: a putative cusABC transcript unit and two copies of copAB, which are usually involved in copper resistance and tolerance to other monovalent heavy metals. A local gene relocation was observed in cluster 1 consisting cusABC and copAB relative to the KT2440 type strain according to the phylogenetic and gene neighbourhood analyses on various P. putida strains. P. putida LS46 also contains 11 putative MerR family regulators, which sense various environmental stimuli including heavy metals. MerR-1 is an ortholog of the copper response regulator of other gram-negative bacteria, and was highly up-regulated in waste glycerol cultures. Finally, a number of genes involved in cell responses to high extra-cellular Na+ concentrations, and genes of the fatty acid beta-oxidation pathway were up-regulated in waste glycerol cultures Major results from objective b): Regardless to the type of substrates, up-regulation of two mcl-PHA synthase (PhaC1 and PhaC2), and two phasin proteins (PhaF and PhaI) are the most common genotype under mcl-PHA production conditions. PhaG and possible PhaJ4 connect fatty acid de novo synthesis to mcl-PHA in waste glycerol culture. Interestingly, expression of gene, fabZ, in production of unsaturated fatty acid from fatty acid de novo synthesis was only observed in waste glycerol culture. On the other hand, PhaJ1 and PhaJ4 derived mcl-PHA production via fatty acid beta-oxidation was observed under waste fatty acid culture. These results would help to explain observed different production kinetics and monomer distribution of the polymer. Although under active mcl-PHA production condition, depression on the expression of glpF genes in glycerol transportation system prevent further channelling extra-cellular glycerol into the cell. Waste glycerol culture also triggers trahalose synthesis pathway, a potential competing pathway during mcl-PHA synthesizing. In waste fatty acid culture, the intermediates (acyl-CoA and 3-hydroxyacyl-CoA) of fatty acid beta-oxidation were used for mcl-PHA production and were also likely hydrolysed to their free acid forms via an up-regulated thioesteras coding gene, tesA. Acetyl-CoA cleaved from the pathway was clearly channeled into glyoxylate shut for C2 carbon assimilation over spillage as CO2 through TCA cycle or used in fatty acid biosynthesis pathway.

Dataset Information

Low Carbon Concentration Feeding Improves Medium-Chain-Length Polyhydroxyalkanoate Production in Escherichia coli Strains With Defective ?-Oxidation.

Publications

Low Carbon Concentration Feeding Improves Medium-Chain-Length Polyhydroxyalkanoate Production in <i>Escherichia coli</i> Strains With Defective β-Oxidation.

Similar Datasets

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