Plastic pollution is a severe threat to our environment which necessitates implementation of bioplastics to realize sustainable development for a green world. Polyhydroxyalkanoates (PHA) represent one of the potential candidates for these bioplastics. However, a major challenge faced by PHA is the high production cost which limits its commercial application. Halophiles are considered to be a promising cell factory for PHA synthesis due to its several unique characteristics including high salinity requirement preventing microbial contamination, high intracellular osmotic pressure allowing easy cell lysis for PHA recovery, and capability to utilize wide spectrum of low-cost substrates. Optimization of fermentation parameters has made it plausible to achieve large-scale production at low cost by using halophiles. Further deeper insights into halophiles have revealed the existence of diversified and even novel PHA synthetic pathways within different halophilic species that greatly affects PHA type. Thus, precise metabolic engineering of halophiles with the help of advanced tools and strategies have led to more efficient microbial cell factory for PHA production. This review is an endeavour to summarize the various research achievements in these areas which will help the readers to understand the current developments as well as the future efforts in PHA research.

The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system mediates adaptive immunity against foreign nucleic acids in prokaryotes. However, efficient adaptation of a native CRISPR to purified viruses has only been observed for the type II-A system from a Streptococcus thermophilus industry strain, and rarely reported for laboratory strains. Here, we provide a second native system showing efficient adaptation. Infected by a newly isolated virus HHPV-2, Haloarcula hispanica type I-B CRISPR system acquired spacers discriminatively from viral sequences. Unexpectedly, in addition to Cas1, Cas2 and Cas4, this process also requires Cas3 and at least partial Cascade proteins, which are involved in interference and/or CRISPR RNA maturation. Intriguingly, a preexisting spacer partially matching a viral sequence is also required, and spacer acquisition from upstream and downstream sequences of its target sequence (i.e. priming protospacer) shows different strand bias. These evidences strongly indicate that adaptation in this system strictly requires a priming process. This requirement, if validated also true for other CRISPR systems as implied by our bioinformatic analysis, may help to explain failures to observe efficient adaptation to purified viruses in many laboratory strains, and the discrimination mechanism at the adaptation level that has confused scientists for years.

Small RNAs guard CRISPR-Cas: The microbial adaptive immunity system CRISPR-Cas benefits microbes by warding off genetic invaders, but it also inflicts a fitness cost because of occasional autoimmune reactions, rendering CRISPR loci evolutionarily unstable. Li et al. identified previously unnoticed toxin-antitoxin RNA pairs embedded within diverse CRISPR-Cas loci. The antitoxin RNA mimics a CRISPR RNA and repurposes the CRISPR immunity effector to transcriptionally repress a toxin RNA that would otherwise arrest cell growth by sequestering a rare transfer RNA. These small RNAs thus form a symbiosis with CRISPR, rendering CRISPR addictive to the host despite its fitness cost. These findings reveal how CRISPR-Cas can operate as a selfish genetic element. Science , this issue p. eabe5601

Haloferax 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 Δ 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.

Summary: Polyhydroxyalkanoates (PHA) are a promising and sustainable alternative to the petroleum‐based synthetic plastics. Regulation of PHA synthesis is receiving considerable importance as engineering the regulatory factors might help developing strains with improved PHA‐producing abilities. PHA synthesis is dedicatedly regulated by a number of regulatory networks. They tightly control the PHA content, granule size and their distribution in cells. Most PHA‐accumulating microorganisms have multiple regulatory networks that impart a combined effect on PHA metabolism. Among them, several factors ranging from global to specific regulators, have been identified and characterized till now. This review is an attempt to categorically summarize the diverse regulatory circuits that operate in some important PHA‐producing microorganisms. However, in several organisms, the detailed mechanisms involved in the regulation of PHA synthesis is not well‐explored and hence further research is needed. The information presented in this review might help researcher to identify the prevailing research gaps in PHA regulation.