Genetic engineering and metabolite profiling for overproduction of polyhydroxybutyrate in cyanobacteria

Abstract

Genetic engineering and metabolite profiling for the overproduction of polyhydroxybutyrate (PHB), which is a carbon material in biodegradable plastics, were examined in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Transconjugants harboring cyanobacterial expression vectors that carried the pha genes for PHB biosynthesis were constructed. The overproduction of PHB by the engineering cells was confirmed through microscopic observations using Nile red, transmission electron microscopy (TEM), or nuclear magnetic resonance (NMR). We successfully recovered PHB from transconjugants prepared from nitrogen-depleted medium without sugar supplementation in which PHB reached approximately 7% (w/w) of the dry cell weight, showing a value of 12-fold higher productivity in the transconjugant than that in the control strain. We also measured the intracellular levels of acetyl-CoA, acetoacetyl-CoA, and 3-hydroxybutyryl-CoA (3HB-CoA), which are intermediate products for PHB. The results obtained indicated that these products were absent or at markedly low levels when cells were subjected to the steady-state growth phase of cultivation under nitrogen depletion for the overproduction of bioplastics. Based on these results, efficient factors were discussed for the overproduction of PHB in recombinant cyanobacteria.

Industrial Production of Poly-β-hydroxybutyrate from CO2: Can Cyanobacteria Meet this Challenge?

“…The accumulation was very high when compared with the 13% PHB of dcw measured for the wild type cultures carried out under the same operating conditions. The PHB content reported for cultures carried out under photoautotrophic growth conditions using just CO 2 as carbon source was lower [78,79] than that measured for cultures with other carbon sources. The overexpression of the native sigE gene (encoding of the minor sigma factors of the organism) provided the integration of the latter in the Synechocystis chromosome, and increased the PHB content from 0.6% to 1.4% [74] when cells were grown in a N-deprived medium.…”

“…The overexpression of the native sigE gene (encoding of the minor sigma factors of the organism) provided the integration of the latter in the Synechocystis chromosome, and increased the PHB content from 0.6% to 1.4% [74] when cells were grown in a N-deprived medium. Hondo et al [78] transformed Synechocystis cells with the vector pAM461c, harboring a PHA biosynthetic operon from Microcystis aeruginosa NIES-843, and reached a PHB content of about 7% in the N-deprived medium [74]. In a recent publication of our group, Carpine et al [74] measured a PHB content larger than 12% (of dcw) by overexpression of phosphoketolase (xfpk), combined with the double deletion of phosphotransacetylase (pta) and acetyl-CoA hydrolase (ach) (under balanced growth conditions, i.e., using BG11 [80] as the growth medium).…”

Identification of Four Polyhydroxyalkanoate Structural Genes in Synechocystis cf. salina PCC6909: In silico Evidences

“…Furthermore, genetically modified transconjugants of Synechocystis sp. PCC6803 produce PHB up to 7% per dry cell weight (12-fold higher than the control), when heterologous PHA genes from Mycrocystis aeruginosa are expressed [28].…”

Interaction of the Nitrogen Regulatory Protein GlnB (PII) with Biotin Carboxyl Carrier Protein (BCCP) Controls Acetyl-CoA Levels in the Cyanobacterium Synechocystis sp. PCC 6803

“…Yet, our understanding of the acetyl-CoA metabolism in cyanobacteria is limited. A few studies, that have addressed the question how acetyl-CoA pools respond during nitrogen deprivation came to controversial results: in some studies, the acetyl-CoA pools increased (Joseph et al, 2014; Anfelt et al, 2015), or were almost unchanged (Schlebusch and Forchhammer, 2010) whereas other reported modest (Osanai et al, 2014) or strong decrease (Hondo et al, 2015) upon nitrogen deprivation. Different growth conditions, extraction procedures, or data normalization could account for the divergence.…”