Abstract:The aim of this study was to establish isobutanol production on chemically defined medium in Escherichia coli. By individually expressing each gene of the pathway, we constructed a plasmid library for isobutanol production. Strain screening on chemically defined medium showed successful production in the robust E. coli W strain, and expression vector IB 4 was selected as the most promising construct due to its high isobutanol yields and efficient substrate uptake. The investigation of different aeration strate… Show more
“…However, costeffective production of pyruvate-derived metabolites such as 2-KIV requires additional flux rerouting (Gu et al, 2017;Noda et al, 2019). Nowadays, redirection of carbon flux towards pyruvate has been unlocked in a large variety of microorganisms (Buchholz et al, 2013;Gu et al, 2017;Novak et al, 2020), although most studies have focused on disabling pyruvate consumption along competing pathways (Noda et al, 2019;Novak et al, 2020). For instance, detailed analysis of studies dealing with overproduction of 2-KIV from glucose (Supplementary Figure S4; Supplementary Table S6) revealed Frontiers in Bioengineering and Biotechnology frontiersin.org three main knockout strategies.…”
Section: Resultsmentioning
confidence: 99%
“…In recent years, much research has been conducted into the use of whey as a fermentation medium to produce lipids, organic acids and alcohols ( Cao et al, 2020 ; Mano et al, 2020 ; Novak et al, 2020 ). The physicochemical composition of whey is behind this growing interest, as it typically contains around 88% lactose, 4% protein, 1.4% (w/w) lipids and trace minerals in powder product ( Carranza-Saavedra, Sánchez Henao and Zapata Montoya, 2021 ).…”
Replacing traditional substrates in industrial bioprocesses to advance the sustainable production of chemicals is an urgent need in the context of the circular economy. However, since the limited degradability of non-conventional carbon sources often returns lower yields, effective exploitation of such substrates requires a multi-layer optimization which includes not only the provision of a suitable feedstock but the use of highly robust and metabolically versatile microbial biocatalysts. We tackled this challenge by means of systems metabolic engineering and validated Escherichia coli W as a promising cell factory for the production of the key building block chemical 2-ketoisovalerate (2-KIV) using whey as carbon source, a widely available and low-cost agro-industrial waste. First, we assessed the growth performance of Escherichia coli W on mono and disaccharides and demonstrated that using whey as carbon source enhances it significantly. Second, we searched the available literature and used metabolic modeling approaches to scrutinize the metabolic space of E. coli and explore its potential for overproduction of 2-KIV identifying as basic strategies the block of pyruvate depletion and the modulation of NAD/NADP ratio. We then used our model predictions to construct a suitable microbial chassis capable of overproducing 2-KIV with minimal genetic perturbations, i.e., deleting the pyruvate dehydrogenase and malate dehydrogenase. Finally, we used modular cloning to construct a synthetic 2-KIV pathway that was not sensitive to negative feedback, which effectively resulted in a rerouting of pyruvate towards 2-KIV. The resulting strain shows titers of up to 3.22 ± 0.07 g/L of 2-KIV and 1.40 ± 0.04 g/L of L-valine in 24 h using whey in batch cultures. Additionally, we obtained yields of up to 0.81 g 2-KIV/g substrate. The optimal microbial chassis we present here has minimal genetic modifications and is free of nutritional autotrophies to deliver high 2-KIV production rates using whey as a non-conventional substrate.
“…However, costeffective production of pyruvate-derived metabolites such as 2-KIV requires additional flux rerouting (Gu et al, 2017;Noda et al, 2019). Nowadays, redirection of carbon flux towards pyruvate has been unlocked in a large variety of microorganisms (Buchholz et al, 2013;Gu et al, 2017;Novak et al, 2020), although most studies have focused on disabling pyruvate consumption along competing pathways (Noda et al, 2019;Novak et al, 2020). For instance, detailed analysis of studies dealing with overproduction of 2-KIV from glucose (Supplementary Figure S4; Supplementary Table S6) revealed Frontiers in Bioengineering and Biotechnology frontiersin.org three main knockout strategies.…”
Section: Resultsmentioning
confidence: 99%
“…In recent years, much research has been conducted into the use of whey as a fermentation medium to produce lipids, organic acids and alcohols ( Cao et al, 2020 ; Mano et al, 2020 ; Novak et al, 2020 ). The physicochemical composition of whey is behind this growing interest, as it typically contains around 88% lactose, 4% protein, 1.4% (w/w) lipids and trace minerals in powder product ( Carranza-Saavedra, Sánchez Henao and Zapata Montoya, 2021 ).…”
Replacing traditional substrates in industrial bioprocesses to advance the sustainable production of chemicals is an urgent need in the context of the circular economy. However, since the limited degradability of non-conventional carbon sources often returns lower yields, effective exploitation of such substrates requires a multi-layer optimization which includes not only the provision of a suitable feedstock but the use of highly robust and metabolically versatile microbial biocatalysts. We tackled this challenge by means of systems metabolic engineering and validated Escherichia coli W as a promising cell factory for the production of the key building block chemical 2-ketoisovalerate (2-KIV) using whey as carbon source, a widely available and low-cost agro-industrial waste. First, we assessed the growth performance of Escherichia coli W on mono and disaccharides and demonstrated that using whey as carbon source enhances it significantly. Second, we searched the available literature and used metabolic modeling approaches to scrutinize the metabolic space of E. coli and explore its potential for overproduction of 2-KIV identifying as basic strategies the block of pyruvate depletion and the modulation of NAD/NADP ratio. We then used our model predictions to construct a suitable microbial chassis capable of overproducing 2-KIV with minimal genetic perturbations, i.e., deleting the pyruvate dehydrogenase and malate dehydrogenase. Finally, we used modular cloning to construct a synthetic 2-KIV pathway that was not sensitive to negative feedback, which effectively resulted in a rerouting of pyruvate towards 2-KIV. The resulting strain shows titers of up to 3.22 ± 0.07 g/L of 2-KIV and 1.40 ± 0.04 g/L of L-valine in 24 h using whey in batch cultures. Additionally, we obtained yields of up to 0.81 g 2-KIV/g substrate. The optimal microbial chassis we present here has minimal genetic modifications and is free of nutritional autotrophies to deliver high 2-KIV production rates using whey as a non-conventional substrate.
“…Later, the iBuOH pathway was further enhanced by increasing the activity and optimizing the expression level of the involved enzymes [ 18 – 20 ]. Comprehensive summaries of the performance parameters reached with different E. coli strains (and other organisms), substrates, media additives, and culture conditions can be found in [ 21 , 22 ]. The E. coli strain RL3000Δferm-pIBA4 [ 19 ] exhibits one of the best performances in regard to iBuOH production so far, but reached, under anaerobic conditions in defined minimal medium, only very low biomass concentrations not relevant for industrial application.…”
Section: Introductionmentioning
confidence: 99%
“…The E. coli strain RL3000Δferm-pIBA4 [ 19 ] exhibits one of the best performances in regard to iBuOH production so far, but reached, under anaerobic conditions in defined minimal medium, only very low biomass concentrations not relevant for industrial application. For cultivations in a bioreactor with higher biomass concentrations, yeast extract was again added to the medium [ 19 ], making a fair comparison of performance parameters with other strains or process conditions challenging [ 21 , 23 ]. Fig.…”
Background
The microbial production of isobutanol holds promise to become a sustainable alternative to fossil-based synthesis routes for this important chemical. Escherichia coli has been considered as one production host, however, due to redox imbalance, growth-coupled anaerobic production of isobutanol from glucose in E. coli is only possible if complex media additives or small amounts of oxygen are provided. These strategies have a negative impact on product yield, productivity, reproducibility, and production costs.
Results
In this study, we propose a strategy based on acetate as co-substrate for resolving the redox imbalance. We constructed the E. coli background strain SB001 (ΔldhA ΔfrdA ΔpflB) with blocked pathways from glucose to alternative fermentation products but with an enabled pathway for acetate uptake and subsequent conversion to ethanol via acetyl-CoA. This strain, if equipped with the isobutanol production plasmid pIBA4, showed robust exponential growth (µ = 0.05 h−1) under anaerobic conditions in minimal glucose medium supplemented with small amounts of acetate. In small-scale batch cultivations, the strain reached a glucose uptake rate of 4.8 mmol gDW−1 h−1, a titer of 74 mM and 89% of the theoretical maximal isobutanol/glucose yield, while secreting only small amounts of ethanol synthesized from acetate. Furthermore, we show that the strain keeps a high metabolic activity also in a pulsed fed-batch bioreactor cultivation, even if cell growth is impaired by the accumulation of isobutanol in the medium.
Conclusions
This study showcases the beneficial utilization of acetate as a co-substrate and redox sink to facilitate growth-coupled production of isobutanol under anaerobic conditions. This approach holds potential for other applications with different production hosts and/or substrate–product combinations.
“…Most butanol and isobutanol are currently synthesized from petrochemicals, however, Clostridium acetobutylicum will naturally produce 1-butanol and Saccharomyces cerevisiae, bacteria and cyanobacteria have been engineered to produce these alcohols [4][5][6][7][8]. Isobutanol production by E. coli and S. cerevisiae has used glucose as feedstock but microbial strains have been engineered to achieve isobutanol synthesis from xylose, cellulose, methanol, cheese whey and acetate [9][10][11][12][13].…”
Isobutanol is an important and valuable platform chemical and an appealing biofuel that is compatible with contemporary combustion engines and existing fuel distribution infrastructure. The present study aimed to compare the potential of triticale, wheat and barley starch as feedstock for isobutanol production using an engineered strain of Saccharomyces cerevisiae. A simultaneous saccharification and fermentation (SSF) approach showed that all three starches were viable feedstock for co-production of isobutanol and ethanol and could produce titres similar to that produced using purified sugar as feedstock. A fed-batch process using triticale starch yielded 0.006 g isobutanol and 0.28 g ethanol/g starch. Additionally, it is demonstrated that Fusarium graminearum infected grain starch contaminated with mycotoxin can be used as an effective feedstock for isobutanol and ethanol co-production. These findings demonstrate the potential for triticale as a purpose grown energy crop and show that mycotoxin-contaminated grain starch can be used as feedstock for isobutanol biosynthesis, thus adding value to a grain that would otherwise be of limited use.
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