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

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.

From Organic Wastes to Bioplastics: Feasibility of Nonsterile Poly(3-hydroxybutyrate) Production by Zobellella denitrificans ZD1

“…The results were consistent with our previous findings. 30 Interestingly, the band corresponding to EctB band was only observed in cells in the stationary phase (lane 4) and not in cells in the exponential growth phase, suggesting this was a linkage of the enhanced expression of ectABC between the improved cellular growth and PHB accumulation under high NaCl conditions (3%) ( Table 1 ). However, future studies to determine ectoine concentrations and confirmation of N-terminal protein sequencing of each of the corresponding bands on the SDS-PAGE gel are needed to positively confirm the above observations.…”

“… 52 The genes involved in the biosynthesis of ectoine are ectA , ectB , and ectC , 53 , 54 which have been previously identified in the ZD1 genome. 30 Accordingly, our experiments were designed to determine whether ZD1 would express these genes to produce ectoine as an osmolyte under high salinity conditions. Our results suggest that ectoine might be the possible osmolyte in ZD1 grown in saline media, as the presence of these bands corresponds to the three predicted enzymes (EctA, B, and C) according to SDS-PAGE analysis ( Figure S4 ).…”

“…The synergetic effects of nitrate and salts on enhanced cellular growth and PHB production in ZD1 were observed in this study, but the underlying reasons and mechanisms remain unclear. Our previous study on genome analysis of ZD1 30 has revealed many unique genes, such as the presence of a phasin-family protein ( PFP ) gene, which is known to play a significant role in the formation of a PHB granule. In the study, much higher gene expression levels of PHB synthesis genes ( pha A–C ) and PFP were observed in nitrate-grown ZD1 with 3% NaCl than those in ammonia-grown culture with 3% NaCl, suggesting a linkage between the nitrogen source type and PHB synthesis in ZD1.…”

“…In the study, much higher gene expression levels of PHB synthesis genes ( pha A–C ) and PFP were observed in nitrate-grown ZD1 with 3% NaCl than those in ammonia-grown culture with 3% NaCl, suggesting a linkage between the nitrogen source type and PHB synthesis in ZD1. Given that genes associated with complete denitrification, a complete glycerol-to-PHB pathway, and ectoine synthesis enzymes have been identified in the genome of ZD1, 30 further studies are required to substantiate the interactions among these genes in relation to PHB accumulation observed under different nitrogen sources and saline conditions.…”

“…SDS-PAGE analysis was used to examine the expression of ectoine synthesis genes ( ectA , ectB , and ectC ) by strain ZD1, as previously described. 30 Briefly, each of the ectoine synthesis genes was constructed into a plasmid that was then inserted into Escherichia coli for overexpression. A detailed description of the construct of the plasmids for the expression of ectoine synthesis genes in E. coli and SDS-PAGE analysis is provided in Tables S1 and S2 in the Supporting Information.…”