Sustainable bioproduction of the blue pigment indigoidine: Expanding the range of heterologous products in<i>R. toruloides</i>to include non-ribosomal peptides

Wehrs, Maren; Gladden, John M.; Liu, Yuzhong; Platz, Lukas; Prahl, Jan Philip; Moon, Jadie; Papa, Gabriella; Sundström, Eric; Geiselman, Gina M.; Tanjore, Deepti; Keasling, Jay D.; Pray, Todd; Simmons, Blake A.; Mukhopadhyay, Aindrila

doi:10.1039/c9gc00920e

Cited by 66 publications

(51 citation statements)

References 75 publications

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“…Selecting the best host-final product pair is crucial to developing the ideal production platform, and provides a key consideration in broadening our approach to additional studies. In earlier reports (Supplementary Table 7 ), high indigoidine production was achieved in the oleaginous yeast Rhodosporidium toruloides but remained low in S. cerevisiae , despite similar optimization of cultivation parameters 59 – 61 . This empirical comparison highlights the innate metabolic potential of a given host, and is consistent with our host-constrained calculated maximum theoretical yields for indigoidine (Supplementary Table 2 ).…”

Section: Discussionmentioning

confidence: 79%

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Genome-scale metabolic rewiring improves titers rates and yields of the non-native product indigoidine at scale

et al. 2020

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High titer, rate, yield (TRY), and scalability are challenging metrics to achieve due to trade-offs between carbon use for growth and production. To achieve these metrics, we take the minimal cut set (MCS) approach that predicts metabolic reactions for elimination to couple metabolite production strongly with growth. We compute MCS solution-sets for a non-native product indigoidine, a sustainable pigment, in Pseudomonas putida KT2440, an emerging industrial microbe. From the 63 solution-sets, our omics guided process identifies one experimentally feasible solution requiring 14 simultaneous reaction interventions. We implement a total of 14 genes knockdowns using multiplex-CRISPRi. MCS-based solution shifts production from stationary to exponential phase. We achieve 25.6 g/L, 0.22 g/l/h, and ~50% maximum theoretical yield (0.33 g indigoidine/g glucose). These phenotypes are maintained from batch to fed-batch mode, and across scales (100-ml shake flasks, 250-ml ambr®, and 2-L bioreactors).

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Section: Discussionmentioning

confidence: 79%

“…The purity of extracted indigoidine (Supplementary Fig. 7 ) from both E. coli and P. putida were cross-validated by 1 H-NMR 61 .…”

Section: Methodsmentioning

confidence: 99%

Genome-scale metabolic rewiring improves titers rates and yields of the non-native product indigoidine at scale

et al. 2020

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“…A sustainable alternative to produce these products is through engineering microbial cell factories that can directly use renewable and cost‐effective feedstocks, such as lignocellulose, atmospheric CO 2 , and syngas (Peralta‐Yahya et al, 2012). A vast number of chemicals have been successfully produced in various microbes that feature diverse chemical structures and functional groups (Borodina et al, 2015; Jiang, Qiao, Bentley, Liu, & Zhang, 2017; Lee et al, 2019; Luo, Cho, & Lee, 2019; Wehrs et al, 2019). Recent advances in metabolic engineering and synthetic biology have provided a greatly expanded set of tools necessary to assemble and optimize metabolic pathways for improved titers, productivities and yields (D. Liu, Evans, & Zhang, 2015; Y. Liu & Nielsen, 2019; Salis, Mirsky, & Voigt, 2009).…”

Section: Introductionmentioning

confidence: 99%

Exploiting nonionic surfactants to enhance fatty alcohol production in Rhodosporidium toruloides

Liu

Geiselman

Coradetti

et al. 2020

Biotech & Bioengineering

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Fatty alcohols (FOHs) are important feedstocks in the chemical industry to produce detergents, cosmetics, and lubricants. Microbial production of FOHs has become an attractive alternative to production in plants and animals due to growing energy demands and environmental concerns. However, inhibition of cell growth caused by intracellular FOH accumulation is one major issue that limits FOH titers in microbial hosts. In addition, identification of FOH‐specific exporters remains a challenge and previous studies towards this end are limited. To alleviate the toxicity issue, we exploited nonionic surfactants to promote the export of FOHs in Rhodosporidium toruloides, an oleaginous yeast that is considered an attractive next‐generation host for the production of fatty acid‐derived chemicals. Our results showed FOH export efficiency was dramatically improved and the growth inhibition was alleviated in the presence of small amounts of tergitol and other surfactants. As a result, FOH titers increase by 4.3‐fold at bench scale to 352.6 mg/L. With further process optimization in a 2‐L bioreactor, the titer was further increased to 1.6 g/L. The method we show here can potentially be applied to other microbial hosts and may facilitate the commercialization of microbial FOH production.

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“…11,12 These NRPSs having a 4'-phosphopantetheine (Ppant) modification on the PCP domain (holo form) convert L-glutamine to 5-amino-3H-pyridine-2,6-dione (1), a putative product that is readily oxidized to a 3,3'-bipyridyl natural product, indigoidine (2), which as a bright blue color. [12][13][14][15] Indigoidine synthetase genes have been engineered and introduced into living systems to construct a cross-kingdom reporter system 16 , generate blue rose 17 , and produce 2 as a promising water-insoluble dye 18,19 . Interestingly, indigoidine synthetase genes are widely distributed in bacteria, including Actinobacteria and Proteobacteria ( Figure S1), and frequently colocalized with genes encoding enzymes that glycosylate C-5 of 1, leading to the water-soluble blue pigment indochrome 20,21 , and the C-nucleoside antibiotic, minimycin 13 (Figure 1b).…”

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confidence: 99%

Investigation of Indigoidine Synthetase Reveals a Conserved Active-Site Base Residue of Nonribosomal Peptide Synthetase Oxidases

et al. 2020

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Nonribosomal peptide synthetase (NRPS) oxidase (Ox) domains oxidize protein-bound intermediates to install crucial structural motifs in bioactive natural products. The mechanism of this domain remains elusive. Here, by studying indigoidine synthetase, a single-module NRPS involved in the biosynthesis of indigoidine and several other bacterial secondary metabolites, we demonstrate that its Ox domain utilizes an active-site base residue, tyrosine 665, to deprotonate a protein-bound l-glutaminyl residue. We further validate the generality of this active-site residue among NRPS Ox domains. These findings not only resolve the biosynthetic pathway mediated by indigoidine synthetase but enable mechanistic insight into NRPS Ox domains.

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Sustainable bioproduction of the blue pigment indigoidine: Expanding the range of heterologous products inR. toruloidesto include non-ribosomal peptides

Abstract: Development of R. toruloides as a production host for the sustainable production of the NRP indigoidine.

Cited by 66 publications

References 75 publications

Genome-scale metabolic rewiring improves titers rates and yields of the non-native product indigoidine at scale

Genome-scale metabolic rewiring improves titers rates and yields of the non-native product indigoidine at scale

Exploiting nonionic surfactants to enhance fatty alcohol production in Rhodosporidium toruloides

Investigation of Indigoidine Synthetase Reveals a Conserved Active-Site Base Residue of Nonribosomal Peptide Synthetase Oxidases

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