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

Abstract

Background: Beyond 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. Results: Saccharomyces 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. In conclusion, this study represents the first use of the Streptomyces lavendulae NRPS (BpsA) in a fungal host and its scale-up. The finalmore » 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.« less

Authors:
 [1];  [2];  [3];  [3];  [2];  [4];  [2]; ORCiD logo [5]
  1. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States. Biological Systems and Engineering Division; Technische Univ. Braunschweig (Germany). Inst. für Genetik; Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States)
  2. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States. Biological Systems and Engineering Division, and Advanced Biofuels and Bioproducts Process Development Unit
  3. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States. Biological Systems and Engineering Division; Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States)
  4. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States. Biological Systems and Engineering Division; Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Univ. of California, Berkeley, CA (United States). Dept. of Plant and Microbial Biology, Dept. of Bioengineering, Dept. of Chemical and Biomolecular Engineering; Technical Univ. of Denmark, Lyngby (Denmark). Novo Nordisk Foundation Center for Biosustainability; Shenzhen Inst. for Advanced Technologies, Shenzhen (China). Synthetic Biochemistry Center, Inst. for Synthetic Biology
  5. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States. Biological Systems and Engineering Division, and Environmental Genomics and Systems Biology Division; Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States)
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1493277
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Accepted Manuscript
Journal Name:
Microbial Cell Factories
Additional Journal Information:
Journal Volume: 17; Journal Issue: 1; Journal ID: ISSN 1475-2859
Publisher:
BioMed Central
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Non-ribosomal peptide synthetase; NRPS, S. cerevisiae; Indigoidine; Metabolic state; TCA cycle activity; Non-ribosomal peptide synthesis; Bioreactor; BpsA; BJ5465

Citation Formats

Wehrs, Maren, Prahl, Jan-Philip, Moon, Jadie, Li, Yuchen, Tanjore, Deepti, Keasling, Jay D., Pray, Todd, and Mukhopadhyay, Aindrila. Production efficiency of the bacterial non-ribosomal peptide indigoidine relies on the respiratory metabolic state in S. cerevisiae. United States: N. p., 2018. Web. doi:10.1186/s12934-018-1045-1.
Wehrs, Maren, Prahl, Jan-Philip, Moon, Jadie, Li, Yuchen, Tanjore, Deepti, Keasling, Jay D., Pray, Todd, & Mukhopadhyay, Aindrila. Production efficiency of the bacterial non-ribosomal peptide indigoidine relies on the respiratory metabolic state in S. cerevisiae. United States. doi:10.1186/s12934-018-1045-1.
Wehrs, Maren, Prahl, Jan-Philip, Moon, Jadie, Li, Yuchen, Tanjore, Deepti, Keasling, Jay D., Pray, Todd, and Mukhopadhyay, Aindrila. Thu . "Production efficiency of the bacterial non-ribosomal peptide indigoidine relies on the respiratory metabolic state in S. cerevisiae". United States. doi:10.1186/s12934-018-1045-1. https://www.osti.gov/servlets/purl/1493277.
@article{osti_1493277,
title = {Production efficiency of the bacterial non-ribosomal peptide indigoidine relies on the respiratory metabolic state in S. cerevisiae},
author = {Wehrs, Maren and Prahl, Jan-Philip and Moon, Jadie and Li, Yuchen and Tanjore, Deepti and Keasling, Jay D. and Pray, Todd and Mukhopadhyay, Aindrila},
abstractNote = {Background: Beyond 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. Results: Saccharomyces 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. In conclusion, this 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.},
doi = {10.1186/s12934-018-1045-1},
journal = {Microbial Cell Factories},
number = 1,
volume = 17,
place = {United States},
year = {2018},
month = {12}
}

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Figures / Tables:

Figure 1 Figure 1: Production of bacterial indigoidine in engineered S. cerevisiae. a S. cerevisiae exhibits two distinct metabolic states which are accompanied with distinct metabolic flux profiles. The width of the arrows represents metabolic flux. Blue arrows represent purely respiratory state, while red arrows represent fully fermentative state. GAP glyceraldehyde 3‑phosphate,more » DHAP dihydroxyacetone phosphate, EtOH ethanol, α-KG α‑ketoglutarate, Glu glutamate, Gln glutamine. Several known pathways for glutamine biosynthesis are shown. The depiction of metabolite intermediates and their cellular localization adapted from Frick et al. Ljungdahl and Daignan‑Fornier, and Chen et al. [10, 48, 49]. b Activation of the apo‑form of the S. lavendulae NRPS, BpsA (blue pigment synthetase A) by the Bacillus subtilis 4′‑phosphopantetheinyl transferase (PPTase; Sfp) via addition of a coenzyme A‑derived moiety to the peptide carrier domain (PCP) into the active holo‑form. The active holo‑BpsA converts two l‑glutamines to one molecule of the blue pigment indigoidine by a catalytic process involving adenylation (a), oxidation (Ox) and thioesterase (TE) domains. c Positive S. cerevisiae transformants exhibit blue pigmentation occurring 3 days after visible colony formation on solid media containing glucose. d Brightfield microscopy of the pigmented colony shows heterogeneity in pigment production, ×63 zoom. The pigment shows punctate subcellular localization, scale bar = 10 µm, increasing non‑linear magnification of boxed areas is depicted by pull‑outs« less

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