Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 14 (9), e0222143
eCollection

Analysis of Zobellella Denitrificans ZD1 Draft Genome: Genes and Gene Clusters Responsible for High Polyhydroxybutyrate (PHB) Production From Glycerol Under Saline Conditions and Its CRISPR-Cas System

Affiliations

Analysis of Zobellella Denitrificans ZD1 Draft Genome: Genes and Gene Clusters Responsible for High Polyhydroxybutyrate (PHB) Production From Glycerol Under Saline Conditions and Its CRISPR-Cas System

Yu-Wei Wu et al. PLoS One.

Abstract

Polyhydroxybutyrate (PHB) is biodegradable and renewable and thus considered as a promising alternative to petroleum-based plastics. However, PHB production is costly due to expensive carbon sources for culturing PHB-accumulating microorganisms under sterile conditions. We discovered a hyper PHB-accumulating denitrifying bacterium, Zobellella denitrificans ZD1 (referred as strain ZD1 hereafter) capable of using non-sterile crude glycerol (a waste from biodiesel production) and nitrate to produce high PHB yield under saline conditions. Nevertheless, the underlying genetic mechanisms of PHB production in strain ZD1 have not been elucidated. In this study, we discovered a complete pathway of glycerol conversion to PHB, a novel PHB synthesis gene cluster, a salt-tolerant gene cluster, denitrifying genes, and an assimilatory nitrate reduction gene cluster in the ZD1 genome. Interestingly, the novel PHB synthesis gene cluster was found to be conserved among marine Gammaproteobacteria. Higher levels of PHB accumulation were linked to higher expression levels of the PHB synthesis gene cluster in ZD1 grown with glycerol and nitrate under saline conditions. Additionally, a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas type-I-E antiviral system was found in the ZD1 genome along with a long spacer list, in which most of the spacers belong to either double-stranded DNA viruses or unknown phages. The results of the genome analysis revealed strain ZD1 used the novel PHB gene cluster to produce PHB from non-sterile crude glycerol under saline conditions.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Genome map and phylogenetic tree for Zobellella denitrificans ZD1 strain.
(A) Six rings, from outer to inner, represent (1) assembled scaffolds, (2) genes in the forward strand, (3) genes in the reverse-complement strand, (4) transfer RNA (tRNA) genes, (5) ribosomal RNA (rRNA) genes, and (6) the GC content. (B) Phylogenetic tree constructed from 287 single copy marker genes.
Fig 2
Fig 2
(A) A proposed pathway to convert glycerol to polyhydroxybutyrate (PHB). (B) The PHB synthesis gene cluster identified in the genome of Zobellella denitrificans ZD1. The pathway figure was adapted from Martinez-Gomez et al. [34].
Fig 3
Fig 3. Effects of ammonium (NH4+) and nitrate (NO3-) on the expression of PHB synthesis genes.
(A) phaA (B) phaB (C) phaC and (D) PFP in Zobellella denitrificans ZD1.
Fig 4
Fig 4. Distribution of the bacterial species harboring a polyhydroxybutyrate (PHB) synthesis gene cluster (phaB-phaA-PFP-phaC) similar to that of the Zobellella denitrificans ZD1.
Genus name and the family, order, and class ranks are indicated accordingly. Sp # refers to the number of bacterial species containing the PHB synthesis gene cluster. Only one strain per species was used in the calculation. See "Materials and Methods" for details.
Fig 5
Fig 5. Concatenated protein tree built from four genes of the PHB gene cluster.
The same color indicates the same genus rank. Zobellella denitrificans is indicated by blue color on the right side.
Fig 6
Fig 6. SDS-PAGE analysis of three proteins (EctA, EctB, and EctC) produced in Zobellella denitrificans ZD1 and in E. coli BL21.
1: Mark12 Unstained Standard; 2: supernatant of lysed cells grown with ammonia and 0% NaCl; 3: supernatant of lysed cells grown with ammonia and 3% NaCl; 4: EctA produced by E. coli after 0.2 mM IPTG induction; 5: EctB produced by E. coli after 0.2 mM IPTG induction; 6: EctC produced by E. coli after 0.2 mM IPTG induction; 7: supernatant of lysed cells grown with nitrate and 0% NaCl; and 8: supernatant of lysed cells grown with nitrate and 3% NaCl.
Fig 7
Fig 7. Genes and pathways of denitrification and assimilatory nitrate reduction.
(A) Genes and gene clusters that play roles in denitrification. (B) A pathway to convert NO3- to N2 adapted from previously studies [–40]. (C) A gene cluster for assimilatory nitrate reduction. (D) A pathway for converting nitrate to ammonia.
Fig 8
Fig 8. Identification of clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems from species closely related to Zobellella denitrificans ZD1 strain.
Hexagonal boxes indicate identified CRISPR-Cas systems in these species. Different colors along with the text annotation represent different CRISPR systems.
Fig 9
Fig 9. Phylogenetic trees built from the cas3/cas3HD genes of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems.
Different colors indicate different types of CRISPR genes, and white hexagons in front of the CRISPR-Cas genes contain the numbers of repeats (i.e., the number of spacers) identified from the CRISPR system.
Fig 10
Fig 10. Taxonomic distribution of clustered regularly interspaced short palindromic repeat (CRISPR) spacers.

Similar articles

See all similar articles

References

    1. Jendrossek D, Handrick R. Microbial degradation of polyhydroxyalkanoates. Annu Rev Microbiol. 2002;56:403–32. 10.1146/annurev.micro.56.012302.160838 - DOI - PubMed
    1. van der Walle GA, de Koning GJ, Weusthuis RA, Eggink G. Properties, modifications and applications of biopolyesters. Adv Biochem Eng Biotechnol. 2001;71:263–91. - PubMed
    1. Anderson AJ, Dawes EA. Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev. 1990;54(4):450–72. - PMC - PubMed
    1. Rodriguezvalera F, Lillo JAG. Halobacteria as producers of polyhydroxyalkanoates. FEMS Microbiol Lett. 1992;103(2–4):181–6.
    1. Steinbuchel A, Fuchtenbusch B. Bacterial and other biological systems for polyester production. Trends Biotechnol. 1998;16(10):419–27. - PubMed

Publication types

Supplementary concepts

Grant support

This work was supported by Taipei Medical University grant TMU105-AE1-B19 and Ministry of Science and Technology (TW) grant MOST108-2628-E-038-002-MY3.
Feedback