Production efficiency of the bacterial non-ribosomal peptide indigoidine relies on the respiratory metabolic state in S. cerevisiae

Maren Wehrs,Deepti Tanjore,Aindrila Mukhopadhyay,Yuchen Li,Jan-Philip Prahl,Jay D Keasling,Jadie Moon,Todd Pray

doi:10.1186/s12934-018-1045-1

Abstract

BackgroundBeyond pathway engineering, the metabolic state of the production host is critical in maintaining the efficiency of cellular production. The biotechnologically important yeast Saccharomyces cerevisiae adjusts its energy metabolism based on the availability of oxygen and carbon sources. This transition between respiratory and non-respiratory metabolic state is accompanied by substantial modifications of central carbon metabolism, which impact the efficiency of metabolic pathways and the corresponding final product titers. Non-ribosomal peptide synthetases (NRPS) are an important class of biocatalysts that provide access to a wide array of secondary metabolites. Indigoidine, a blue pigment, is a representative NRP that is valuable by itself as a renewably produced pigment.ResultsSaccharomyces cerevisiae was engineered to express a bacterial NRPS that converts glutamine to indigoidine. We characterize carbon source use and production dynamics, and demonstrate that indigoidine is solely produced during respiratory cell growth. Production of indigoidine is abolished during non-respiratory growth even under aerobic conditions. By promoting respiratory conditions via controlled feeding, we scaled the production to a 2 L bioreactor scale, reaching a maximum titer of 980 mg/L.ConclusionsThis study represents the first use of the Streptomyces lavendulae NRPS (BpsA) in a fungal host and its scale-up. The final product indigoidine is linked to the activity of the TCA cycle and serves as a reporter for the respiratory state of S. cerevisiae. Our approach can be broadly applied to investigate diversion of flux from central carbon metabolism for NRPS and other heterologous pathway engineering, or to follow a population switch between respiratory and non-respiratory modes.

Highlights

Beyond pathway engineering, the metabolic state of the production host is critical in maintaining the efficiency of cellular production
Establishing indigoidine production in Saccharomyces cerevisiae In S. lavendulae, the native pathway to convert l-glutamine into the blue pigment indigoidine consists of the Non-ribosomal peptide synthetases (NRPS) Blue pigment synthetase (BpsA) and a 4′-phosphopantetheinyl transferase (PPTase), needed to activate the apo-NRPS into its holo-form via the addition of a coenzyme A-derived phosphopantetheine moiety (Fig. 1b) [19, 21]
To establish the indigoidine pathway in S. cerevisiae, we genomically integrated the Bacillus subtilis PPtase sfp, previously shown to successfully activate apo-BpsA [22], and the 3.8 kbp NRPS gene bpsA into S. cerevisiae BJ5465, a protease deficient strain reported to functionally express Sfp [23]

Summary

Introduction

The metabolic state of the production host is critical in maintaining the efficiency of cellular production. The biotechnologically important yeast Saccharomyces cerevisiae adjusts its energy metabolism based on the availability of oxygen and carbon sources. This transition between respiratory and nonrespiratory metabolic state is accompanied by substantial modifications of central carbon metabolism, which impact the efficiency of metabolic pathways and the corresponding final product titers. In contrast to many other fungal or bacterial hosts, S. cerevisiae adjusts its energy metabolism based on the nature of available carbon sources via carbon catabolite repression [7]. S. cerevisiae predominantly metabolizes glucose by fermentation leading to the production of ethanol, glycerol and carbon dioxide (Fig. 1a, red arrows) [8, 9]. While the effect of the metabolic state on native pathways and products has been investigated [15,16,17,18], its effect on engineered pathways and biosynthetic products remains understudied

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Conclusion