Effect of nitrogen availability on the poly-3-d-hydroxybutyrate accumulation by engineered Saccharomyces cerevisiae

Diogo J Portugal-Nunes,Sudhanshu S Pawar,Gunnar Lidén,Marie F Gorwa-Grauslund

doi:10.1186/s13568-017-0335-z

Diogo J Portugal-Nunes, Sudhanshu S Pawar + Show 2 more

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https://doi.org/10.1186/s13568-017-0335-z

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Journal: AMB Express	Publication Date: Feb 7, 2017
Citations: 21	License type: open-access

Affiliation: Lund University

Abstract

Poly-3-d-hydroxybutyrate (or PHB) is a polyester which can be used in the production of biodegradable plastics from renewable resources. It is naturally produced by several bacteria as a response to nutrient starvation in the excess of a carbon source. The yeast Saccharomyces cerevisiae could be an alternative production host as it offers good inhibitor tolerance towards weak acids and phenolic compounds and does not depolymerize the produced PHB. As nitrogen limitation is known to boost the accumulation of PHB in bacteria, the present study aimed at investigating the effect of nitrogen availability on PHB accumulation in two recombinant S. cerevisiae strains harboring different xylose consuming and PHB producing pathways: TMB4443 expressing an NADPH-dependent acetoacetyl-CoA reductase and a wild-type S. stipitis XR with preferential use of NADPH and TMB4425 which expresses an NADH-dependent acetoacetyl-CoA reductase and a mutated XR with a balanced affinity for NADPH/NADH. TMB4443 accumulated most PHB under aerobic conditions and with glucose as sole carbon source, whereas the highest PHB concentrations were obtained with TMB4425 under anaerobic conditions and xylose as carbon source. In both cases, the highest PHB contents were obtained with high availability of nitrogen. The major impact of nitrogen availability was observed in TMB4425, where a 2.7-fold increase in PHB content was obtained. In contrast to what was observed in natural PHB-producing bacteria, nitrogen deficiency did not improve PHB accumulation in S. cerevisiae. Instead the excess available carbon from xylose was shunted into glycogen, indicating a significant gluconeogenic activity on xylose.

Highlights

Poly-3-d-hydroxybutyrate (PHB) was the first discovered microbial produced alkanoate (Khanna and Srivastava 2005)
As initial work indicated that TMB4443 accumulated higher intracellular concentrations of PHB when glucose was used instead of xylose, glucose was chosen as the principle carbon source for TMB4443 in the assessment of effects of nitrogen availability
Upon addition of ammonium sulfate (Glc-nitrogen excess medium (N-high), Fig. 2c), the cells consumed glucose at a rate that was at least twofold higher, as glucose was depleted after 50 h of cultivation compared to approximately 125 h required by the cells in cultures lacking ammonium sulfate (Glc-nitrogen deficient media (N-def), Fig. 2a)

Summary

Introduction

Poly-3-d-hydroxybutyrate (PHB) was the first discovered microbial produced alkanoate (Khanna and Srivastava 2005). PHBs are thermoplastic and biocompatible, which opens the possibility for medical applications (Verlinden et al 2007; Yoneyama et al 2015). These compounds are naturally accumulated as intracellular carbon granules and energy storage material in several bacteria, the so-called ‘natural producers’, such as Cupriavidus necator, Bacillus megaterium and several species belonging to the genus Pseudomonas (Trotsenko and Belova 2000; Verlinden et al 2007). The accumulation of PHB is generally optimal under excess of carbon and limiting levels of nitrogen, phosphorous and/or oxygen (Verlinden et al 2007)

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