Genetic engineering of Synechocystis sp. PCC6803 for poly-β-hydroxybutyrate overproduction

Roberta Carpine,Wei Du,Giuseppe Olivieri,Antonino Pollio,Klaas J Hellingwerf,Antonio Marzocchella,Filipe Branco Dos Santos

doi:10.1016/j.algal.2017.05.013

Roberta Carpine, Wei Du + Show 5 more

Open Access

https://doi.org/10.1016/j.algal.2017.05.013

Copy DOI

Abstract

The biosynthesis of poly-β-hydroxybutyrate (PHB) directly from carbon dioxide is a sustainable alternative for non-renewable, petroleum-based polymer production. Synechocystis sp. PCC6803 can naturally accumulate PHB using CO2 as the sole carbon source, particularly when major nutrients such as nitrogen become limiting. Many previous studies have tried to genetically engineer PHB overproduction; mostly by increasing the expression of enzymes directly involved in its biosynthesis pathway. Here, we have instead concentrated on engineering the central carbon metabolism of Synechocystis such that (i) the PHB synthesis pathway becomes deregulated, and/or (ii) the levels of its substrate, acetyl-CoA, were increased. Seven different mutants were constructed harboring, separately or in combination, three different genetic modifications to Synechocystis' metabolic network. These were the deletions of phosphotransacetylase (Pta) and acetyl-CoA hydrolase (Ach), and the expression of a heterologous phosphoketolase (XfpK) from Bifidobacterium breve. The wild type Synechocystis and the derivative strains were compared in terms of biomass and the PHB production capability during photoautotrophic growth. This was performed in a photobioreactor exposed to a diel light/dark rhythm and using standard BG11 as the growth medium. We found that the strain that combined all three genetic modifications, i.e. xfpk overexpression in a double pta and ach deletion background, showed the highest levels of PHB production from all the strains tested here. Encouragingly, the production levels obtained: 232mgL−1, ~12% (w/w) of the dry biomass weight, and a productivity of 7.3mgL−1d−1; are to the best of our knowledge, the highest ever reported for PHB production directly from CO2.

Highlights

The growing world population and the accompanying increased demand of plastic materials drive our need for more sustainable production of biodegradable polymers [1]
The biosynthesis of poly-β-hydroxybutyrate (PHB) directly from carbon dioxide is a sustainable alternative for non-renewable, petroleum-based polymer production
Seven different mutants were constructed harboring, separately or in combination, three different genetic modifications to Synechocystis' metabolic network. These were the deletions of phosphotransacetylase (Pta) and acetyl-CoA hydrolase (Ach), and the expression of a heterologous phosphoketolase (XfpK) from Bifidobacterium breve

Summary

Introduction

The growing world population and the accompanying increased demand of plastic materials drive our need for more sustainable production of biodegradable polymers [1]. Costs of raw materials for PHB production by microbial fermentation are still very high, making it of paramount importance to find other sustainable production routes. This has brought plants into focus as an alternative low cost photosynthetic production system [4,5]. Plant-based expression systems compete directly with subsistence crops for agricultural acreage, and raise further ethical concerns, as the dissemination of transgenic plants is difficult to control. The latter has led to strict regulatory restrictions of transgenic plants in many countries [6]. Cyanobacteria, share all the advantages of photoautotrophic microorganisms when compared to chemotrophs, having the potential to use (sun)light energy to directly convert CO2 into a product of interest such as PHB, but circumvent the drawbacks of competing with the agro-food market for resources [7,8,9]

Objectives

Methods

Results

Conclusion