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

Banerjee, Deepanwita; Eng, Thomas; Lau, Andrew K.; Sasaki, Yusuke; Wang, Brenda; Chen, Yan; Prahl, Jan‐Philip; Singan, Vasanth; Herbert, Robin A.; Liu, Yuzhong; Tanjore, Deepti; Kim, Young-Mo; Keasling, Jay D.; Mukhopadhyay, Aindrila

doi:10.1038/s41467-020-19171-4

Cited by 72 publications

(120 citation statements)

References 83 publications

(100 reference statements)

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“…genome scale metabolic model of P. putida, iJN1462 35 and Flux Balance Analysis (FBA), we calculated maximum theoretical yields (MTY) of indigoidine and its precursors for this carbon/substrate pair (Supplementary Table 2). This carbon source to final product MTY pair (pCA/indigoidine) of 0.66 mol/mol is higher than MTY calculated for the glucose/indigoidine pair of 0.54 mol/mol 36 . The predicted flux towards indigoidine in these mutants is summarized in Table 2 and Supplementary Table 3.…”

Section: The Bioconversion Of a Lignin Derived Aromatic To A Heterologous Bioproductcontrasting

confidence: 57%

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High Throughput Fitness Profiling Reveals Loss Of GacS-GacA Regulation Improves Indigoidine Production In Pseudomonas putida

Eng

Banerjee

Lau

et al. 2021

Preprint

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Pseudomonas putida KT2440 is an emerging industrial microbe amenable for use with renewable carbon streams including aromatics such as para-coumarate (pCA). We examined this microbe under common stirred-tank bioreactor parameters with quantitative fitness assays using a pooled transposon library containing nearly all (4,778) non-essential genes. Assessing differential fitness values by monitoring changes in mutant strain abundance over time identified 31 genes with improved fitness in multiple bioreactor-relevant parameters. Twenty-one genes from this subset were reconstructed, including GacA, a signaling protein, TtgB, an ABC transporter, and PP_0063, a lipid A acyltransferase. Twelve deletion strains with roles in varying cellular functions were evaluated for conversion of pCA, to a heterologous bioproduct, indigoidine. Several mutants, such as the ΔgacA strain improved both fitness in a bioreactor and showed an 8-fold improvement in indigoidine production (4.5 g/L, 0.29 g/g pCA, 23% MTY) from pCA as the carbon source.

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Section: The Bioconversion Of a Lignin Derived Aromatic To A Heterologous Bioproductcontrasting

confidence: 57%

“…The purity of indigoidine samples were confirmed using H-NMR as previously described 36 . Indigoidine yields were calculated assuming complete utilization of glucose or p CA based on the amount of fed substrate in minimal media containing no other carbon sources.…”

Section: Methodsmentioning

confidence: 99%

High Throughput Fitness Profiling Reveals Loss Of GacS-GacA Regulation Improves Indigoidine Production In Pseudomonas putida

Eng

Banerjee

Lau

et al. 2021

Preprint

Self Cite

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“…Use of these approaches complemented promising methods to identify coregulated reactions and co-expressed genes [80], and composition of the minimal required substrates in order to produce valuable metabolic products [80,81] (see Table 2.1 in [82] for a list of software packages for enumerating EFMs). Authors in [83] generated hypotheses for strain metabolic engineering guided by the MCS computation of the metabolic network of an industrial microbe (Pseudomonas putida). The experimental implementation of the guided predictions enhances titer, rate and yield of the microbe by coupling its growth to bioproduction, i.e., production starts from late exponential phase.…”

Section: Metabolic Pathway Analysis (Mpa)mentioning

confidence: 99%

Modelling Cell Metabolism: A Review on Constraint-Based Steady-State and Kinetic Approaches

Yasemi¹,

Jolicœur²

2021

Processes

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Studying cell metabolism serves a plethora of objectives such as the enhancement of bioprocess performance, and advancement in the understanding of cell biology, of drug target discovery, and in metabolic therapy. Remarkable successes in these fields emerged from heuristics approaches, for instance, with the introduction of effective strategies for genetic modifications, drug developments and optimization of bioprocess management. However, heuristics approaches have showed significant shortcomings, such as to describe regulation of metabolic pathways and to extrapolate experimental conditions. In the specific case of bioprocess management, such shortcomings limit their capacity to increase product quality, while maintaining desirable productivity and reproducibility levels. For instance, since heuristics approaches are not capable of prediction of the cellular functions under varying experimental conditions, they may lead to sub-optimal processes. Also, such approaches used for bioprocess control often fail in regulating a process under unexpected variations of external conditions. Therefore, methodologies inspired by the systematic mathematical formulation of cell metabolism have been used to address such drawbacks and achieve robust reproducible results. Mathematical modelling approaches are effective for both the characterization of the cell physiology, and the estimation of metabolic pathways utilization, thus allowing to characterize a cell population metabolic behavior. In this article, we present a review on methodology used and promising mathematical modelling approaches, focusing primarily to investigate metabolic events and regulation. Proceeding from a topological representation of the metabolic networks, we first present the metabolic modelling approaches that investigate cell metabolism at steady state, complying to the constraints imposed by mass conservation law and thermodynamics of reactions reversibility. Constraint-based models (CBMs) are reviewed highlighting the set of assumed optimality functions for reaction pathways. We explore models simulating cell growth dynamics, by expanding flux balance models developed at steady state. Then, discussing a change of metabolic modelling paradigm, we describe dynamic kinetic models that are based on the mathematical representation of the mechanistic description of nonlinear enzyme activities. In such approaches metabolic pathway regulations are considered explicitly as a function of the activity of other components of metabolic networks and possibly far from the metabolic steady state. We have also assessed the significance of metabolic model parameterization in kinetic models, summarizing a standard parameter estimation procedure frequently employed in kinetic metabolic modelling literature. Finally, some optimization practices used for the parameter estimation are reviewed.

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“…However, it should be mentioned that indigoidine has also been heterologously produced in bacterial hosts, such as Streptomyces lividans , E. coli and Pseudomonas putida . Especially the industrially relevant P. putida turned out to achieve very high product titers of up to 25.6 g/l [ 182 ].…”

Section: Main Textmentioning

confidence: 99%

Saccharomyces cerevisiae as host for the recombinant production of polyketides and nonribosomal peptides

Tippelt

Nett

2021

Microb Cell Fact

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As a robust, fast growing and genetically tractable organism, the budding yeast Saccharomyces cerevisiae is one of the most widely used hosts in biotechnology. Its applications range from the manufacturing of vaccines and hormones to bulk chemicals and biofuels. In recent years, major efforts have been undertaken to expand this portfolio to include structurally complex natural products, such as polyketides and nonribosomally synthesized peptides. These compounds often have useful pharmacological properties, which make them valuable drugs for the treatment of infectious diseases, cancer, or autoimmune disorders. In nature, polyketides and nonribosomal peptides are generated by consecutive condensation reactions of short chain acyl-CoAs or amino acids, respectively, with the substrates and reaction intermediates being bound to large, multidomain enzymes. For the reconstitution of these multistep catalytic processes, the enzymatic assembly lines need to be functionally expressed and the required substrates must be supplied in reasonable quantities. Furthermore, the production hosts need to be protected from the toxicity of the biosynthetic products. In this review, we will summarize and evaluate the status quo regarding the heterologous production of polyketides and nonribosomal peptides in S. cerevisiae. Based on a comprehensive literature analysis, prerequisites for a successful pathway reconstitution could be deduced, as well as recurring bottlenecks in this microbial host.

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

Cited by 72 publications

References 83 publications

High Throughput Fitness Profiling Reveals Loss Of GacS-GacA Regulation Improves Indigoidine Production In Pseudomonas putida

High Throughput Fitness Profiling Reveals Loss Of GacS-GacA Regulation Improves Indigoidine Production In Pseudomonas putida

Modelling Cell Metabolism: A Review on Constraint-Based Steady-State and Kinetic Approaches

Saccharomyces cerevisiae as host for the recombinant production of polyketides and nonribosomal peptides

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