Multiple Propionyl Coenzyme A-Supplying Pathways for Production of the Bioplastic Poly(3-Hydroxybutyrate- co -3-Hydroxyvalerate) in Haloferax mediterranei

Abstract

dHaloferax mediterranei is able to accumulate the bioplastic poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with more than 10 mol% 3-hydroxyvalerate (3HV) from unrelated carbon sources. However, the pathways that produce propionyl coenzyme A (propionyl-CoA), an important precursor of 3HV monomer, have not yet been determined. Bioinformatic analysis of H. mediterranei genome indicated that this strain uses multiple pathways for propionyl-CoA biosynthesis, including the citramalate/2-oxobutyrate pathway, the aspartate/2-oxobutyrate pathway, the methylmalonyl-CoA pathway, and a novel 3-hydroxypropionate pathway. Cofeeding of pathway intermediates and inactivating pathway-specific genes supported that these four pathways were indeed involved in the biosynthesis of 3HV monomer. The novel 3-hydroxypropionate pathway that couples CO 2 assimilation with PHBV biosynthesis was further confirmed by analysis of 13 C positional enrichment in 3HV. Notably, 13 C metabolic flux analysis showed that the citramalate/2-oxobutyrate pathway (53.0% flux) and the 3-hydroxypropionate pathway (30.6% flux) were the two main generators of propionyl-CoA from glucose. In addition, genetic perturbation on the transcriptome of the ⌬phaEC mutant (deficient in PHBV accumulation) revealed that a considerable number of genes in the four propionyl-CoA synthetic pathways were significantly downregulated. We determined for the first time four propionyl-CoA-supplying pathways for PHBV production in haloarchaea, particularly including a new 3-hydroxypropionate pathway. These results would provide novel strategies for the production of PHBV with controllable 3HV molar fraction. P olyhydroxyalkanoates (PHAs) are deposited as carbon and energy materials by many bacteria and haloarchaea under unbalanced growth conditions (1, 2). Due to their excellent biodegradability, biocompatibility, and mechanical properties, PHAs have received increased attention as excellent alternatives for petroleum-derived plastics (3). The physical and mechanical properties of PHAs are closely correlated to monomer composition. Among the various types of PHAs, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly-3-hydroxybutyrate (PHB) are the two most extensively studied ones. Due to the incorporation of 3-hydroxyvalerate (3HV) monomer, PHBV becomes more ductile and easier to process and thus possesses a wider range of industrial applications than PHB (4).PHBV is usually produced from acetyl coenzyme A (acetylCoA) and propionyl coenzyme A (propionyl-CoA) via a threestep process catalyzed by ␤-ketothiolase (PhaA/BktB), ␤-ketoacyl-CoA reductase (PhaB), and PHA synthase sequentially (5). Thus far, few bacteria, including Nocardia corallina (6), Rhodococcus spp. (7), a mutant of Ralstonia eutropha (8), several purple nonsulfur bacteria (9), and Bacillus circulans (10) were found to be able to produce propionyl-CoA for PHBV biosynthesis from single unrelated carbon sources. Most bacteria require the addition of propionate in the media to produce PHBV (11). Since propion...

Production of Polyhydroxyalkanoate (PHA) Biopolyesters by Extremophiles?

“…), which contributes considerably to PHBHV´s production cost. This unique metabolic feature is based on particularities of the strain´s PHA-biosynthesis pathway, which accumulates high intracellular pools of the 3HV-precursor propionyl-CoA [46]. In 2006, Don and colleagues fractionated the PHBHV by using a chloroform/acetone mixture and revealed that the polymer consisted of two fractions of different monomeric composition; the predominant fraction (about 93 wt.-% of the total PHA) contained about 10.7 % (mol/ mol) 3HV, whereas the smaller fraction has a higher 3HV content of about 12.3 % [mol/mol] and a drastically lower molecular mass (78.2 kDa vs. 569.5 kDa, respectively), making it soluble even in the classical "PHA-non solvent" acetone.…”

Study on the Production and Re-use of Poly(3-hydroxybutyrate-co-3- hydroxyvalerate) and Extracellular Polysaccharide by the Archaeon Haloferax mediterranei Strain DSM 1411

“…1) from simple carbon sources structurally unrelated to 3-hydroxyvalerate (3HV), such as carbohydrates 9,10,11,36,37 or glycerol 21 ; microbes contemporarily applied for large-scale PHA production convert such substrates to poly(3-[R]-hydroxybutyrate) (PHB) homopolyester. This homopolyester is of modest quality, predominantly due to high crystallinity and high melting point.…”

“…This is an important feature for the large-scale production of easily processible PHBV copolyesters without the need to supply 3HV-related precursors, which are costly, and often negatively impact growth and product formation kinetics. The background of this metabolic particularity was only recently elucidated and understood on the enzymatic and genetic level [37][38][39] . Regarding EPS biosynthesis, the strain is known to excrete an anionic EPS of high molar mass 8,16,40 , resulting in a typical mucous character of colonies grown on solid medium (see also Fig.…”

A Novel DNA-Binding Protein, PhaR, Plays a Central Role in the Regulation of Polyhydroxyalkanoate Accumulation and Granule Formation in the Haloarchaeon Haloferax mediterranei

“…For the P phaRP promoter, the qPCR product, F phaR , was designed to span a region from Ϫ151 to ϩ17 relative to the TSS of phaRP. A promoter region (F glpR ; Ϫ148 to Ϫ12 relative to the translation start site of glpR) of the glpR gene, which is functionally irrelevant to the PHA biosynthesis as shown in the microarray data of the ⌬phaEC mutant (36), was chosen to be a distal/negative-control region. As shown in Fig.…”

“…To achieve economic production of PHA, increasing studies involved in the metabolic pathways of PHA biosynthesis in haloarchaea have been performed (25,36), and close attention has also been paid to the global regulation of PHA metabolism, which was explored through proteomic and transcriptomic approaches (37). In this study, we focused on the elucidation of the role of a specific regulator, PhaR, which was identified previously as a small PGAP (12.0 kDa) of H. mediterranei (20).…”