Evaluation of gene expression cassettes and production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with a fine modulated monomer composition by using it in Cupriavidus necator

Abstract

Background Cupriavidus necator has attracted much attention as a platform for the production of polyhydroxyalkanoate (PHA) and other useful materials. Therefore, an appropriate modulation of gene expression is needed for producing the desired materials effectively. However, there is insufficient information on the genetic engineering techniques required for this in C. necator.ResultsWe found that the disruption of a potential ribosome binding site (RBS) in the phaC1 gene in C. necator caused a small decrease in the PhaC1 expression level. We applied this result to finely regulate the expression of other genes. Several gene expression cassettes were constructed by combining three Escherichia coli derived promoters (PlacUV5, Ptrc and Ptrp) to the potential RBS of phaC1 or its disruptant, respectively. Their expression levels were then determined via a lacZ reporter assay in C. necator strains. The promoter strengths were both ranked similarly for the cells that were cultured with fructose or palm kernel oil as a sole carbon source (Ptrc ≥ PlacUV5 > Ptrp), both of which were much stronger than the phaC1 promoter. The disruption of RBS had minute attenuation effect on the expression level of these expression cassettes with E. coli promoters. Furthermore, they were used to finely regulate the (R)-3-hydroxyhexanoate (3HHx) monomer ratio in the production of poly[(R)-3-hydroxybutyrate-co-3-hydroxyhexanoate] (PHBHHx) via R-specific enoyl-CoA hydratases (PhaJs). The 3HHx composition in PHBHHx is crucial because it defines the thermal and mechanical properties of the resulting plastic material. The C. necator mutant strains, whose PhaJ expression was controlled under the gene expression cassettes, could be used to produce PHBHHx with various 3HHx compositions in the same culture conditions.ConclusionsWe constructed and evaluated several gene expression cassettes consisting of promoters and RBSs that finely regulate transcription and translation. These were then applied to finely modulate the monomer composition in the production of PHBHHx by recombinant C. necator.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0583-7) contains supplementary material, which is available to authorized users.

Disruption of poly (3-hydroxyalkanoate) depolymerase gene and overexpression of three poly (3-hydroxybutyrate) biosynthetic genes improve poly (3-hydroxybutyrate) production from nitrogen rich medium by Rhodobacter sphaeroides

“…5). However, the PHB yield achieved in this study is lower than those from other PHB producing microorganisms such as Cupriavidus necator [6, 20, 21]. These microorganisms are often grown aerobically in sugars and oils as carbon sources.…”

“…A lot of studies on PHA production by various kinds of microorganisms have been reported so far [2–6]. Among these microorganisms, purple non-sulfur (PNS) bacteria may be one of the most attractive candidates for PHA production improvements due to their unique characteristics.…”

In-Line Monitoring of Polyhydroxyalkanoate (PHA) Production during High-Cell-Density Plant Oil Cultivations Using Photon Density Wave Spectroscopy

“…Despite alternative substrates and downstream approaches, highly efficient bioprocesses are required for an economic feasible PHA production. Recently, high-cell-density cultivations with R. eutropha on various renewable feedstocks have been published presenting the production of over 100 g·L −1 PHA and space time yields from 1 to 2.5 g PHA ·L −1 ·h −1 [21,24,29,30,31]. However, none of the presented studies describe in-line PAT-based monitoring or control strategies for the enhancement of process results.…”

Functional Genetic Elements for Controlling Gene Expression in Cupriavidus necator H16

“…The heterologous inducible promoters P araBAD ( l -arabinose), P xylS ( m -toluic acid), P lac (lactose), P hpdH (3-hydroxypropionic acid), and P rhaBAD ( l -rhamnose) ( 16 , 27 – 29 ), the synthetic anhydrotetracycline- and cumate-inducible promoters ( 30 , 31 ), and several native promoters ( 27 ) have been shown to be suitable to control and drive gene expression in C. necator . Escherichia coli -derived promoters P lacUV5 , P trc , and P trp have been applied to regulate expression of R-specific enoyl coenzyme A (enoyl-CoA) hydratases, allowing modulation of the proportion of the ( R )-3-hydroxyhexanoate monomer in poly[( R )-3-hydroxybutyrate-co-3-hydroxyhexanoate] ( 32 ). A few constitutive promoters, derived from bacteriophage T5, containing only a core promoter sequence displayed much higher activity in C. necator than the commonly used strong promoter P tac ( 20 ).…”