Engineering Halomonas species TD01 for enhanced polyhydroxyalkanoates synthesis via CRISPRi

Abstract

BackgroundClustered regularly interspaced short palindromic repeats interference (CRISPRi) has provided an efficient approach for targeted gene inhibition. A non-model microorganism Halomonas species TD01 has been developed as a promising industrial producer of polyhydroxyalkanoates (PHA), a family of biodegradable polyesters accumulated by bacteria as a carbon and energy reserve compound. A controllable gene repression system, such as CRISPRi, is needed for Halomonas sp. TD01 to regulate its gene expression levels.ResultsFor the first time CRISPRi was successfully used in Halomonas sp. TD01 to repress expression of ftsZ gene encoding bacterial fission ring formation protein, leading to an elongated cell morphology with typical filamentous shape similar to phenomenon observed with Escherichia coli. CRISPRi was employed to regulate expressions of prpC gene encoding 2-methylcitrate synthase for regulating 3-hydroxyvalerate monomer ratio in PHBV copolymers of 3-hydroxybutyrate (HB) and 3-hydroxyvalerate (HV). Percentages of HV in PHBV copolymers were controllable ranging from less than 1 to 13%. Furthermore, repressions on gltA gene encoding citrate synthase channeled more acetyl-CoA from the tricarboxylic acid (TCA) cycle to poly(3-hydroxybutyrate) (PHB) synthesis. The PHB accumulation by Halomonas sp. TD01 with its gltA gene repressed in various intensities via CRISPRi was increased by approximately 8% compared with the wild type control containing the CRISPRi vector without target.ConclusionsIt has now been confirmed that the CRISPRi system can be applied to Halomonas sp. TD01, a promising industrial strain for production of various PHA and chemicals under open and continuous fermentation process conditions. In details, the CRISPRi system was successfully designed in this study to target genes of ftsZ, prpC and gltA, achieving longer cell sizes, channeling more substrates to PHBV and PHB synthesis, respectively. CRISPRi can be expected to use for more metabolic engineering applications in non-model organisms.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0655-3) contains supplementary material, which is available to authorized users.

Chasing bacterial chassis for metabolic engineering: a perspective review from classical to non‐traditional microorganisms

“…Another case of an alternative, marine chassis microorganism is Halomonas, which can grow in the presence of high salt concentrations under non-sterile conditions (Tan et al, 2011)significantly reducing costs associated with sterilization in the fermentation process. Although the toolbox for genetic manipulation of this bacterial species is somewhat limited, advances have been made to overcome this problem, for example the development of constitutive and inducible promoters for targeted gene expression (Yin et al, 2014;Zhao et al, 2017), construction of knock-out mutants through homologous recombination stimulated by double-strand breaks in the bacterial genome , and the implementation of the CRISPRi technology (Tao et al, 2017). In terms of practical applications, and by combining all these engineering strategies, Halomonas strains have been successfully used for bioproduction of PHB and PHAs copolymers containing 3-hydroxyvalerate , and the osmocompatible solute ectoine .…”

Impact of CRISPR interference on strain development in biotechnology

“…Intensive research has focused on PHB production in E. coli aided by CRISPRi as a gene silencing tool. Enhanced supply of precursors 3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB) toward the production of polyhydroxyalkanoates was achieved by targeting the genes sad, sucC, sucD, sdhA, and [194] Halomonas species TD01 Polyhydroxyalkanoates production gltA Citrate synthase [195] Haloferax volcanii Carotenoid synthesis HVO_2526-HVO_2528 Enzymes involved in carotenoid biosynthesis [196] S. coelicolor as well as S. collinus…”

Next‐Generation Industrial Biotechnology‐Transforming the Current Industrial Biotechnology into Competitive Processes

“…A constitutive chromosomal expression system based on highly expressed porin protein gene porin and an inducible cassette named T7‐like expression system induced by isopropyl‐β‐ d ‐thiogalactoside (IPTG) via expressing RNA polymerases (RNAP) under a lacI‐controlled Ptac promoter have been exploited . As powerful tools for gene engineering in many model organisms, clustered regularly interspaced short palindromic repeats interference (CRISPRi) and CRISPR/Cas9 system have been successfully exploited and adopted in Halomonas spp. Other method such as morphology engineering, effective expression system for increasing oxygen availability, controlling of NADH/NAD + ratio, and promoter engineering for enhancing the accumulation of PHA in Halomonas spp.…”