Systems biotechnology – Rational whole‐cell biocatalyst and bioprocess design

Kuhn, Daniel; Blank, Lars M.; Schmid, Andreas; Bühler, Bruno

doi:10.1002/elsc.201000009

Cited by 52 publications

(40 citation statements)

References 122 publications

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“…Such a systems approach requires delicate balancing of factors and strategies and often results in "rather than" instead of "yes or no" answers to given challenges. It agrees with the systems biotechnology concept [256][257][258], but conflicts with the typical chemical engineering approach relying on unit-operations and, yet to some extent, with the synthetic biology type of microbial biocatalyst design, which both rely on modularity [259,260]. In our opinion, a holistic systems approach combining analyses and strategies from different disciplines, as it is also realized in the concept of "functional modules" in chemical engineering [261], is required to successfully develop stable metabolic engineering-derived whole-cell biocatalysts and respective processes (Fig.…”

Section: Implications For Bioprocess Development and Conclusionsupporting

confidence: 85%

Maximizing the stability of metabolic engineering‐derived whole‐cell biocatalysts

Kadisch

Willrodt

Hillen

et al. 2017

Biotechnology Journal

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A systematic and powerful knowledge-based framework exists for improving the activity and stability of chemical catalysts and for empowering the commercialization of respective processes. In contrast, corresponding biotechnological processes are still scarce and characterized by case-by-case development strategies. A systematic understanding of parameters affecting biocatalyst efficiency, that is, biocatalyst activity and stability, is essential for a rational generation of improved biocatalysts. Today, systematic approaches only exist for increasing the activity of whole-cell biocatalysts. They are still largely missing for whole-cell biocatalyst stability. In this review, we structure factors affecting biocatalyst stability and summarize existing, yet not completely exploited strategies to overcome respective limitations. The factors and mechanisms related to biocatalyst destabilization are discussed and demonstrated inter alia based on two case studies. The factors are similar for processes with different objectives regarding target molecule or metabolic pathway complexity and process scale, but are in turn highly interdependent. This review provides a systematic for the stabilization of whole-cell biocatalysts. In combination with our knowledge on strategies to improve biocatalyst activity, this paves the way for the rational design of superior recombinant whole-cell biocatalysts, which can then be employed in economically and ecologically competitive and sustainable bioprocesses.

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Section: Implications For Bioprocess Development and Conclusionsupporting

confidence: 85%

Maximizing the stability of metabolic engineering‐derived whole‐cell biocatalysts

Kadisch

Willrodt

Hillen

et al. 2017

Biotechnology Journal

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“…A critical selection and evaluation of the appropriate whole-cell biocatalyst configuration is key for developing an efficient production process (Kuhn et al, 2010;Willrodt et al, 2015b;Woodley, 2006). As a first step, the maximal specific limonene formation rate was found to be 2.8 times higher during unlimited batch growth as compared to carbon-limited fed-batch cultivation (Willrodt et al, 2014).…”

Section: Productivity Of Resting Cells Is Limited By Microbial Physiomentioning

confidence: 99%

Decoupling production from growth by magnesium sulfate limitation boosts de novo limonene production

Willrodt

Hoschek

Bühler

et al. 2015

Biotech & Bioengineering

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The microbial production of isoprenoids has recently developed into a prime example for successful bottom-up synthetic biology or top-down systems biology strategies. Respective fermentation processes typically rely on growing recombinant microorganisms. However, the fermentative production of isoprenoids has to compete with cellular maintenance and growth for carbon and energy. Non-growing but metabolically active E. coli cells were evaluated in this study as alternative biocatalyst configurations to reduce energy and carbon loss towards biomass formation. The use of non-growing cells in an optimized fermentation medium resulted in more than fivefold increased specific limonene yields on cell dry weight and glucose, as compared to the traditional growing-cell-approach. Initially, the stability of the resting-cell activity was limited. This instability was overcome via the optimization of the minimal fermentation medium enabling high and stable limonene production rates for up to 8 h and a high specific yield of ≥50 mg limonene per gram cell dry weight. Omitting MgSO4 from the fermentation medium was very promising to prohibit growth and allow high productivities. Applying a MgSO4 -limitation also improved limonene formation by growing cells during non-exponential growth involving a reduced biomass yield on glucose and a fourfold increase in specific limonene yields on biomass as compared to non-limited cultures. The control of microbial growth via the medium composition was identified as a key but yet underrated strategy for efficient isoprenoid production. Biotechnol. Bioeng. 2016;113: 1305-1314. © 2015 Wiley Periodicals, Inc.

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“…This adverse effect has been accentuated in OST bacteria because they require additionally high energy and a high NADH level to sustain the solvent tolerance property (Kuhn et al, 2010). In most cases, the problem can be minimized by coupling the system with in situ cofactor regeneration in which a common substrate, such as glucose, is provided for NADH regeneration (Weckbecker et al, 2010).…”

Section: Tode1mentioning

confidence: 99%

Enhanced 3-methylcatechol production by Pseudomonas putida TODE1 in a two-phase biotransformation system

Kongpol¹,

Kato²,

Tajima³

et al. 2014

J. Gen. Appl. Microbiol.

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In this study, genetically engineered Pseudomonas putida TODE1 served as a biocatalyst for the bioproduction of valuable 3-methylcatechol (3MC) from toluene in an aqueous-organic two-phase system. The two-phase system was used as an approach to increase the biocatalyst efficiency. Among the organic solvent tested, n-decanol offered several benefits including having the highest partitioning of 3MC, with a high 3MC yield and low cell toxicity. The effect of media supplementation with carbon/ energy sources (glucose, glycerol, acetate and succinate), divalent metal cations (Mg 2+ , Ca 2+, Mn 2+ and Fe 2+ ), and short-chain alcohols (ethanol, n-propanol and n-butanol) as a cofactor regeneration system on the toluene dioxygenase (TDO) activity, cell viability, and overall 3MC yield were evaluated. Along with the two-step cell preparation protocol, supplementation of the medium with 4 mM glycerol as a carbon/energy source, and 0.4 mM Fe 2+ as a cofactor for TDO significantly enhanced the 3MC production level. When in combination with the use of n-decanol and n-butanol as the organic phase, a maximum overall 3MC concentration of 31.8 mM (166 mM in the organic phase) was obtained in a small-scale production, while it was at 160.5 mM (333.2 mM in the organic phase) in a 2-L scale. To our knowledge, this is the highest 3MC yield obtained from a TDO-based system so far.Key words: an aqueous-organic two-phase fermentation; bioproduction; 3-methylcatechol; Pseudomonas putida TODE1; toluene; toluene dioxygenase Introduction 3-Methylcatechol (3MC) is an important precursor for the production of valuable pharmaceutical compounds such as barbatusol and L-DOPA analogues, as well as synthetic food flavors (Husken et al., 2002;Shirai, 1986). Due to difficulties and relatively low overall production yield from chemical synthesis (Held et al., 1999), 3MC production mainly relies on an economic and efficient biotechnological process, in which toluene is employed as a substrate for a bioconversion to 3MC using bacterial whole-cells as biocatalysts (Faizal et al., 2007;Wery et al., 2000). However, since toluene and 3MC, even at concentrations as low as 1% (v/v), detrimentally affect microbial survival and their catalytic activity, their toxicity, especially to 3MC generated and accumulated in the fermentation system, is the main constraint of the production process (de Smet et al., 1978;Fillet et al., 2012;Husken et al., 2001). Two common approaches to overcoming the toxicity limitation are 1) the use of an organic solvent-tolerant (OST) bacteria, which can thrive in relative high concentrations of organic compounds, as a biocatalyst, and 2) the use of a two-liquid (organicaqueous) phase biotransformation system (referred to as a two-phase system hereafter), where the immiscible organic phase with microbial compatibility acts effectively as a source and a sink for the particular organic substrate and product (Heipieper et al., 2007).3MC is typically produced by a genetically engineered bacterial host from a direct bioconversion of tolue...

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Systems biotechnology – Rational whole‐cell biocatalyst and bioprocess design

Cited by 52 publications

References 122 publications

Maximizing the stability of metabolic engineering‐derived whole‐cell biocatalysts

Maximizing the stability of metabolic engineering‐derived whole‐cell biocatalysts

Decoupling production from growth by magnesium sulfate limitation boosts de novo limonene production

Enhanced 3-methylcatechol production by Pseudomonas putida TODE1 in a two-phase biotransformation system

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