Zero‐growth bioprocesses: A challenge for microbial production strains and bioprocess engineering

Lange, Julian; Blombach, Bastian

doi:10.1002/elsc.201600108

Cited by 28 publications

(27 citation statements)

References 108 publications

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“…P. putida is regarded as an obligate aerobic bacterium . However, since the implementation of the synthetic isobutanol pathway theoretically enables a closed redox balance, we tested the capabilities of our engineered P. putida strains to produce isobutanol from glucose in a zero‐growth bioprocess under oxygen deprivation conditions . Therefore, we inoculated P. putida WT and Iso2–6 to an OD 600 of 15–20 in closed bottles filled with minimal medium containing 5.4 g/L glucose and characterized substrate consumption and (by‐) product formation (Table ).…”

Section: Resultsmentioning

confidence: 99%

Engineering Pseudomonas putida KT2440 for the production of isobutanol

Nitschel

Ankenbauer

Welsch

et al. 2020

Engineering in Life Sciences

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We engineered P. putida for the production of isobutanol from glucose by preventing product and precursor degradation, inactivation of the soluble transhydrogenase SthA, overexpression of the native ilvC and ilvD genes, and implementation of the feedbackresistant acetolactate synthase AlsS from Bacillus subtilis, ketoacid decarboxylase KivD from Lactococcus lactis, and aldehyde dehydrogenase YqhD from Escherichia coli. The resulting strain P. putida Iso2 produced isobutanol with a substrate specific product yield (Y Iso/S ) of 22 ± 2 mg per gram of glucose under aerobic conditions. Furthermore, we identified the ketoacid decarboxylase from Carnobacterium maltaromaticum to be a suitable alternative for isobutanol production, since replacement of kivD from L. lactis in P. putida Iso2 by the variant from C. maltaromaticum yielded an identical Y Iso/S . Although P. putida is regarded as obligate aerobic, we show that under oxygen deprivation conditions this bacterium does not grow, remains metabolically active, and that engineered producer strains secreted isobutanol also under the non-growing conditions. K E Y W O R D Sisobutanol, ketoacid decarboxylase, metabolic engineering, microaerobic, Pseudomonas putida Abbreviations: 2-KIV, 2-ketoisovalerate; AlsS, acetolactate synthase; BHI, brain-heart infusion; KDC, ketoacid decarboxylase; LB, Lysogeny broth.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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Section: Resultsmentioning

confidence: 99%

Engineering Pseudomonas putida KT2440 for the production of isobutanol

Nitschel

Ankenbauer

Welsch

et al. 2020

Engineering in Life Sciences

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“…Table S1, Blombach et al, 2011). Then, the resulting strain CIsArXy was applied in a zero-growth production processes (Lange et al, 2016), where an aerobic stage was implemented to generate biomass that is used in a subsequent anaerobic, growth-arrested phase to produce isobutanol at high cell densities (cf. Fig.…”

Section: Two-stage Isobutanol Productionmentioning

confidence: 99%

“…Isobutanol production based on the pentoses D-xylose and L-arabinose has so far not been demonstrated and therefore represents a promising example for the valorization of HF within a novel value chain. As a future perspective, a dual-phase process (Lange et al, 2016) is apparent, where an aerobic growth based on acetate within the HF would be directly followed by an anaerobic isobutanol production phase based on the remaining pentoses.…”

Section: Two-stage Isobutanol Productionmentioning

confidence: 99%

Harnessing novel chromosomal integration loci to utilize an organosolv‐derived hemicellulose fraction for isobutanol production with engineered Corynebacterium glutamicum

Lange

Müller

Blombach

2017

Microbial Biotechnology

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SummaryA successful bioeconomy depends on the manifestation of biorefineries that entirely convert renewable resources to valuable products and energies. Here, the poorly exploited hemicellulose fraction (HF) from beech wood organosolv processing was applied for isobutanol production with Corynebacterium glutamicum. To enable growth of C. glutamicum on HF, we integrated genes required for d‐xylose and l‐arabinose metabolization into two of 16 systematically identified and novel chromosomal integration loci. Under aerobic conditions, this engineered strain CArXy reached growth rates up to 0.34 ± 0.02 h−1 on HF. Based on CArXy, we developed the isobutanol producer strain CIsArXy, which additionally (over)expresses genes of the native l‐valine biosynthetic and the heterologous Ehrlich pathway. CIsArXy produced 7.2 ± 0.2 mM (0.53 ± 0.02 g L−1) isobutanol on HF at a carbon molar yield of 0.31 ± 0.02 C‐mol isobutanol per C‐mol substrate (d‐xylose + l‐arabinose) in an anaerobic zero‐growth production process.

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“…17,18 Hence robustness studies toward oscillating environmental conditions are continuously gaining higher relevance. Many of the current production processes are operated under aerobic conditions, 23 but an increasing number of processes were developed which enable product formation under nonaerated or anaerobic conditions, 24 for example, isobutanol, 25 succinate 26 as well as propionate. 22 Depending on the microbial system and the redox state of substrate and product, microbial production processes can be distinguished.…”

Section: Introductionmentioning

confidence: 99%

“…22 Depending on the microbial system and the redox state of substrate and product, microbial production processes can be distinguished. Many of the current production processes are operated under aerobic conditions, 23 but an increasing number of processes were developed which enable product formation under nonaerated or anaerobic conditions, 24 for example, isobutanol, 25 succinate 26 as well as propionate. 27 The decisive factor for successful production processes under oxygen limitation is a suitable substrate/product combination.…”

Section: Introductionmentioning

confidence: 99%

Comprehensive analysis of metabolic sensitivity of 1,4‐butanediol producing Escherichia coli toward substrate and oxygen availability

Pooth

Gaalen

Trenkamp³

et al. 2019

Biotechnology Progress

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Nowadays, chemical production of 1,4-butanediol is supplemented by biotechnological processes using a genetically modified Escherichia coli strain, which is an industrial showcase of successful application of metabolic engineering. However, large scale bioprocess performance can be affected by presence of physical and chemical gradients in bioreactors which are a consequence of imperfect mixing and limited oxygen transfer. Hence, upscaling comes along with local and time dependent fluctuations of cultivation conditions. This study emphasizes on scale-up related effects of microbial 1,4-butanediol production by comprehensive bioprocess characterization in lab scale. Due to metabolic network constraints 1,4-butanediol formation takes place under oxygen limited microaerobic conditions, which can be hardly realized in large scale bioreactor. The purpose of this study was to assess the extent to which substrate and oxygen availability influence the productivity. It was found, that the substrate specific product yield and the production rate are higher under substrate excess than under substrate limitation. Furthermore, the level of oxygen supply within microaerobic conditions revealed strong effects on product and by-product formation. Under strong oxygen deprivation nearly 30% of the consumed carbon is converted into 1,4-butanediol, whereas an increase in oxygen supply results in 1,4-butanediol reduction of 77%. Strikingly, increasing oxygen availability leads to strong increase of main by-product acetate as well as doubled carbon dioxide formation.The study provides clear evidence that scale-up of microaerobic bioprocesses constitute a substantial challenge. Although oxygen is strictly required for product formation, the data give clear evidence that terms of anaerobic and especially aerobic conditions strongly interfere with 1,4-butanediol production.

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Zero‐growth bioprocesses: A challenge for microbial production strains and bioprocess engineering

Cited by 28 publications

References 108 publications

Engineering Pseudomonas putida KT2440 for the production of isobutanol

Engineering Pseudomonas putida KT2440 for the production of isobutanol

Harnessing novel chromosomal integration loci to utilize an organosolv‐derived hemicellulose fraction for isobutanol production with engineered Corynebacterium glutamicum

Comprehensive analysis of metabolic sensitivity of 1,4‐butanediol producing Escherichia coli toward substrate and oxygen availability

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