Construction of a Rhodobacter sphaeroides Strain That Efficiently Produces Hydrogen Gas from Acetate without Poly(β-Hydroxybutyrate) Accumulation: Insight into the Role of PhaR in Acetate Metabolism

Abstract

The purple nonsulfur phototrophic bacterium Rhodobacter sphaeroides produces hydrogen gas (H₂) from acetate. An approach to improve the H₂ production is preventing accumulation of an intracellular energy storage molecule known as poly(β-hydroxybutyrate) (PHB), which competes with H₂ production for reducing power. However, disruption of PHB biosynthesis has been reported to severely impair the acetate assimilation depending on the genetic backgrounds and/or culture conditions. To solve this problem, we analyzed the relationship between PHB accumulation and acetate metabolism in R. sphaeroides. Gene deletion analyses based on the wild-type strain revealed that among the two polyhydroxyalkanoate synthase genes in the genome, phaC1, but not phaC2, is essential for PHB accumulation, and the phaC1 deletion mutant exhibited slow growth with acetate. On the other hand, a strain with the deletion of phaC1 together with phaR, which encodes a transcriptional regulator capable of sensing PHB accumulation, exhibited growth comparable to that of the wild-type strain despite no accumulation of PHB. These results suggest that PHB accumulation is required for normal growth with acetate by altering the expression of genes under the control of phaR. This hypothesis was supported by a transcriptome sequencing (RNA-seq) analysis revealing that phaR is involved in the regulation of the ethylmalonyl coenzyme A pathway for acetate assimilation. Consistent with these findings, deletion of phaC1 in a genetically engineered H₂-producing strain resulted in lower H₂ production from acetate due to growth defects, whereas deletion of phaR together with phaC1 restored growth with acetate and increased H₂ production from acetate without PHB accumulation. IMPORTANCE This study provides a novel approach for increasing the yield of photofermentative H₂ production from acetate by purple nonsulfur phototrophic bacteria. This study further suggests that polyhydroxyalkanoate is not only a storage substance for carbon and energy in bacteria, but may also act as a signaling molecule that mediates bacterial metabolic adaptations to specific environments. This notion will be helpful for understanding the physiology of polyhydroxyalkanoate-producing bacteria, as well as for their metabolic engineering via synthetic biology.

Keywords: PHB; PhaR; Rhodobacter; Rhodobacter sphaeroides; acetate; ethylmalonyl-CoA; hydrogen; hydrogen production; nitrogenase; polyhydroxyalkanoate; polyhydroxybutyrate.

FIG 1

The PHB metabolic pathway and the ethylmalonyl-CoA pathway in R. sphaeroides 2.4.1. (A) The PHB metabolic pathway proposed by Fales et al. (50) and the ethylmalonyl-CoA pathway proposed by Erb et al. (16) are indicated by gray arrows and black arrows, respectively. (B) Regulation of genes involved in PHB metabolism by PhaR in R. sphaeroides FJ1. Expression of phaZ, phaP, and phaR is repressed by PhaR (30). Gene expression of the PhaR-regulon might be altered upon PHB accumulation as observed in Cupriavidus necator (35) and Paracoccus denitrificans (36).

FIG 2

Phenotypes of phaC1 and/or phaC2 deletion mutants during growth with acetate. (A) Photoheterotrophic growth of the wild-type and ΔphaC1 strains with acetate. (B) Dark aerobic growth of the wild-type and ΔphaC1 strains with acetate. (C) Photoheterotrophic growth of the ΔphaC2 and ΔphaC12 strains with acetate. For growth curves, the open symbols indicate phaC1-complemented strains harboring pMGphaC1. Each value represents the mean ± standard deviation of three independent cultures; error bars that are not visible are smaller than symbols. (D) Absorption spectra of the light-harvesting complexes extracted from the wild-type and ΔphaC1 strains grown photoheterotrophically with acetate (OD₆₆₀ ≈ 0.5). (E) PHB accumulation in phaC1 and/or phaC2 deletion mutants in the early stationary phase under photoheterotrophic growth conditions with acetate. Each value represents the mean ± standard deviation of three independent cultures.

FIG 3

Phenotypes of the ΔphaC1 strain during growth with carbon sources other than acetate. (A to C) Photoheterotrophic growth of the wild-type and ΔphaC1 strains with glucose (A), succinate (B), and pyruvate (C). Each value represents the mean ± standard deviation of three independent cultures; error bars that are not visible are smaller than symbols. (D to E) PHB accumulation in the wild-type and ΔphaC1 strains in the early stationary phase under photoheterotrophic growth conditions with glucose (D), succinate (E), and pyruvate (F). Each value represents the mean ± standard deviation of three independent cultures.

FIG 4

Phenotypes of phaR deletion mutants during photoheterotrophic growth with acetate. (A) Photoheterotrophic growth of the ΔphaR strain with acetate. Data for the wild-type strain from Fig. 2A were used for comparison. (B) Photoheterotrophic growth of phaR deletion mutants with the genetic backgrounds of ΔphaC1, ΔphaC2, and ΔphaC12. For growth curves, the open symbols indicate phaR-complemented strains harboring pMGphaR. Each value represents the mean ± standard deviation of three independent cultures; error bars that are not visible are smaller than symbols. (C) PHB accumulation of phaR deletion mutants in the early stationary phase under photoheterotrophic growth conditions with acetate. Data for the wild-type strain from Fig. 2E were used for comparison. Each value represents the mean ± standard deviation of three independent cultures.

FIG 5

Introduction of multicopy ccr restored growth of the ΔphaC1 strain. (A to D) The upstream region of phaP (A), phaZ (B), phaR (C), and the intergenic region between ccr and ecm (D) are shown. The PhaR-binding sequences 5′-CTGCN_3-4GCAG-3′ (30) are boxed. Translational start sites are bolded and underlined. (E) Photoheterotrophic growth of the ΔphaC1 strain harboring pMGccr or pMG180 (empty vector) with acetate. Data for the wild-type strain from Fig. 2A were used for comparison. Each value represents the mean ± standard deviation of three independent cultures; error bars that are not visible are smaller than symbols.

FIG 6

Photofermentative H₂ production by constructed CHP strains with acetate and ammonium chloride in an argon atmosphere. CHP denotes a constitutive hydrogen producer with the genetic background of the ΔhupΔcbbP* strain. (A to D) H₂ production (A), growth (B), acetate consumption (C), and PHB accumulation (D) are shown. Each value indicates the mean ± standard deviation of four independent cultures; error bars that are not visible are smaller than the symbol. (E) Distribution of electrons from consumed acetate. Consumed electrons were calculated from consumed acetate as the oxidation of acetate to CO₂ yields 8 electrons. Electrons used for H₂ production were calculated as the oxidation of H₂ yields 2 electrons. Electrons used for PHB production were calculated as the oxidation of the PHB monomer (C₄H₈O₃) to CO₂ yields 18 electrons. Electrons used for biomass formation were calculated from dry cell weight, which was subtracted with the weight of PHB, using the previously reported elemental composition of R. sphaeroides (CH_1.99O_0.5N_0.19) (51), the oxidation of which to CO₂ and 0.19NH₃ yields 4.5 electrons. Unknown fractions may contain excreted extracellular organic molecules. The dashed line represents the total of H₂ and PHB in the CHP strain, which corresponds to expected H₂ yields by preventing PHB production.