Redesigning Escherichia coli metabolism for anaerobic production of isobutanol

Fermentation enables the production of reduced metabolites, such as the biofuels ethanol and butanol, from fermentable sugars. This work demonstrates a general approach for designing and constructing a production host that uses a heterologous pathway as an obligately fermentative pathway to produce reduced metabolites, specifically, the biofuel isobutanol. Elementary mode analysis was applied to design an Escherichia coli strain optimized for isobutanol production under strictly anaerobic conditions. The central metabolism of E. coli was decomposed into 38,219 functional, unique, and elementary modes (EMs). The model predictions revealed that during anaerobic growth E. coli cannot produce isobutanol as the sole fermentative product. By deleting 7 chromosomal genes, the total 38,219 EMs were constrained to 12 EMs, 6 of which can produce high yields of isobutanol in a range from 0.29 to 0.41 g isobutanol/g glucose under anaerobic conditions. The remaining 6 EMs rely primarily on the pyruvate dehydrogenase enzyme complex (PDHC) and are typically inhibited under anaerobic conditions. The redesigned E. coli strain was constrained to employ the anaerobic isobutanol pathways through deletion of 7 chromosomal genes, addition of 2 heterologous genes, and overexpression of 5 genes. Here we present the design, construction, and characterization of an isobutanol-producing E. coli strain to illustrate the approach. The model predictions are evaluated in relation to experimental data and strategies proposed to improve anaerobic isobutanol production. We also show that the endogenous alcohol/aldehyde dehydrogenase AdhE is the key enzyme responsible for the production of isobutanol and ethanol under anaerobic conditions. The glycolytic flux can be controlled to regulate the ratio of isobutanol to ethanol production.     

An evolutionary strategy for isobutanol production strain development in Escherichia coli

Higher alcohols such as isobutanol possess several physical characteristics that make them attractive as biofuels such as higher energy densities and infrastructure compatibility. Here we have developed a rapid evolutionary strategy for isolating strains of Escherichia coli that effectively produce isobutanol from glucose utilizing random mutagenesis and a growth selection scheme. By selecting for mutants with the ability to grow in the presence of the valine analog norvaline, we obtained E. coli NV3; a strain with improved 24-h isobutanol production (8.0 g/L) in comparison with a productivity of 5.3 g/L isobutanol obtained with the parental wild type strain. Genomic sequencing of NV3 identified the insertion of a stop codon in the C-terminus of the RNA polymerase σ^s-factor, RpoS. Upon repair of this inhibitory mutation (strain NV3r1), a final isobutanol titer of 21.2 g/L isobutanol was achieved in 99 h with a yield of 0.31 g isobutanol/g glucose or 76% of theoretical maximum. Furthermore, a mutation in ldhA, encoding d-lactate dehydrogenase, was identified in NV3; however, repair of LdhA in NV3r1 had no affect on LdhA activity detected from cell extracts or on isobutanol productivity. Further study of NV3r1 may identify novel genotypes that confer improved isobutanol production.     

Current knowledge on isobutanol production with Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum

Due to steadily rising crude oil prices great efforts have been made to develop designer bugs for the fermentative production of higher alcohols, such as 2-methyl-1-butanol, 3-methyl-1-butanol and 2-Methyl-1-propanol (isobutanol), which all possess quality characteristics comparable to traditional oil based fuels. The common metabolic engineering approach uses the last two steps of the Ehrlich pathway, catalyzed by 2-ketoacid decarboxylase and an alcohol dehydrogenase converting the branched chain 2-ketoacids of L-isoleucine, L-leucine, and L-valine into the respective alcohols. This strategy was successfully used to engineer well suited and industrially employed bacteria, such as Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum for the production of higher alcohols. Among these alcohols, isobutanol is currently the most promising one regarding final titer and yield. This article summarizes the current knowledge and achievements on isobutanol production with E. coli, B. subtilis and C. glutamicum regarding the metabolic engineering approaches and process conditions.     

Improved production of 2,3-butanediol and isobutanol by engineering electron transport chain in Escherichia coli

The electron transport chain (ETC) is one of the major energy generation pathways in microorganisms under aerobic condition. Higher yield of ATP can be achieved through oxidative phosphorylation with consumption of NADH than with substrate level phosphorylation. However, most value-added metabolites are in an electrochemically reduced state, which requires reducing equivalent NADH as a cofactor. Therefore, optimal production of value-added metabolites should be balanced with ETC in terms of energy production. In this study, we attempted to reduce the activity of ETC to secure availability of NADH. The ETC mutants exhibited poor growth rate and production of fermentative metabolites compared to parental strain. Introduction of heterologous pathways for synthesis of 2,3-butanediol and isobutanol to ETC mutants resulted in increased titres and yields of the metabolites. ETC mutants yielded higher NADH/NAD⁺ ratio but similar ATP content than that by the parental strain. Furthermore, ETC mutants operated fermentative metabolism pathways independent of oxygen supply in large-scale fermenter, resulting in increased yield and titre of 2,3-butanediol. Thus, engineering of ETC is a useful metabolic engineering approach for production of reduced metabolites.     

CRISPR/Cas9-mediated engineering of Escherichia coli for n-butanol production from xylose in defined medium

Journal of Industrial Microbiology & Biotechnology - Butanol production from agricultural residues is the most promising alternative for fossil fuels. To reach the economic viability of...     

产异丁醇关键基因在大肠杆菌中表达的研究

[目的]改造大肠杆菌缬氨酸合成途径,使其能够代谢合成异丁醇.[方法]将乳酸乳球菌(Lactococcus lactis) 1.2829的2-酮异戊酸脱羧酶基因(kivD)和醇脱氢酶基因(adhA)串联克隆到大肠杆菌DH5α宿主中表达.[结果]经过改造的宿主菌发酵24 h后异丁醇产量为0.12 g/L.酶活测定实验发现,kivD和adhA基因在宿主菌中均得到表达,但由于KivD的低表达量导致宿主菌最终的异丁醇合成能力偏低.通过研究温度和pH对KivD和AdhA酶活的影响,最终选定二者的最适温度为30℃,最适pH为6.5. [结论]通过向宿主菌导入外源异丁醇合成基因能够改造其自身代谢途径,从而合成异丁醇.     

Genomic library screens for genes involved in n-butanol tolerance in Escherichia coli

Background

n-Butanol is a promising emerging biofuel, and recent metabolic engineering efforts have demonstrated the use of several microbial hosts for its production. However, most organisms have very low tolerance to n-butanol (up to 2% (v/v)), limiting the economic viability of this biofuel. The rational engineering of more robust n-butanol production hosts relies upon understanding the mechanisms involved in tolerance. However, the existing knowledge of genes involved in n-butanol tolerance is limited. The goal of this study is therefore to identify E. coli genes that are involved in n-butanol tolerance.

Methodology/Principal Findings

Using a genomic library enrichment strategy, we identified approximately 270 genes that were enriched or depleted in n-butanol challenge. The effects of these candidate genes on n-butanol tolerance were experimentally determined using overexpression or deletion libraries. Among the 55 enriched genes tested, 11 were experimentally shown to confer enhanced tolerance to n-butanol when overexpressed compared to the wild-type. Among the 84 depleted genes tested, three conferred increased n-butanol resistance when deleted. The overexpressed genes that conferred the largest increase in n-butanol tolerance were related to iron transport and metabolism, entC and feoA, which increased the n-butanol tolerance by 32.8±4.0% and 49.1±3.3%, respectively. The deleted gene that resulted in the largest increase in resistance to n-butanol was astE, which enhanced n-butanol tolerance by 48.7±6.3%.

Conclusions/Significance

We identified and experimentally verified 14 genes that decreased the inhibitory effect of n-butanol tolerance on E. coli. From the data, we were able to expand the current knowledge on the genes involved in n-butanol tolerance; the results suggest that an increased iron transport and metabolism and decreased acid resistance may enhance n-butanol tolerance. The genes and mechanisms identified in this study will be helpful in the rational engineering of more robust biofuel producers.     

Elucidating mechanisms of solvent toxicity in ethanologenic Escherichia coli

Ethanol toxicity and its effect on ethanol production by the recombinant ethanologenic Escherichia coli strain KO11 were investigated in batch and continuous fermentation. During batch growth, ethanol produced by KO11 reduced both the specific cell growth rate (µ) and the cell yield (Y_X/S). The extent of inhibition increased with the production of both acetate and lactate. Subsequent accumulation of these metabolites and ethanol resulted in cessation of cell growth, redirection of metabolism to reduce ethanol production, and increased requirements for cell maintenance. These effects were found to depend on both the glycolytic flux and the flux from pyruvate to ethanol. Pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (Adh) activities measured during the batch fermentation suggested that decreased ethanol production resulted from enzyme inhibition rather than down‐regulation of genes in the ethanol‐producing pathway. Ethanol was added in continuous fermentation to provide an ethanol concentration of either 17 or 27 g/L, triggering sustained oscillations in the cell growth rate. Cell concentrations oscillated in‐phase with ethanol and acetate concentrations. The amplitude of oscillations depended on the concentration of ethanol in the fermentor. Through multiple oscillatory cycles, the yield (Y_P/S) and concentration of ethanol decreased, while production of acetate increased. These results suggest that KO11 favorably adapted to improve growth by synthesizing more ATP though acetate production, and recycling NADH by producing more lactate and less ethanol. Implications of these results for strategies to improve ethanol production are described. Biotechnol. Bioeng. 2010;106: 721–730. © 2010 Wiley Periodicals, Inc.     

Salt stress effects on the central and carnitine metabolisms of Escherichia coli

The aim was to understand how interaction of the central carbon and the secondary carnitine metabolisms is affected under salt stress and its effect on the production of L-carnitine by Escherichia coli. The biotransformation of crotonobetaine into L-carnitine by resting cells of E. coli O44 K74 was improved by salt stress, a yield of nearly twofold that for the control being obtained with 0.5 M NaCl. Crotonobetaine and the L-carnitine formed acted as an osmoprotectant during cell growth and biotransformation in the presence of NaCl. The enzyme activities involved in the biotransformation process (crotonobetaine hydration reaction and crotonobetaine reduction reaction), in the synthesis of acetyl-CoA/acetate (pyruvate dehydrogenase, acetyl-CoA synthetase [ACS] and ATP/acetate phosphotransferase) and in the distribution of metabolites for the tricarboxylic acid cycle (isocitrate dehydrogenase [ICDH]) and glyoxylate shunt (isocitrate lyase [ICL]) were followed in batch with resting cells both in the presence and absence of NaCl and in perturbation experiments performed on growing cells in a high density cell recycle membrane reactor. Further, the levels of carnitine, crotonobetaine, gamma-butyrobetaine and ATP and the NADH/NAD(+) ratio were measured in order to know how the metabolic state was modified and coenzyme pools redistributed as a result of NaCl's effect on the energy content of the cell. The results provided the first experimental evidence of the important role played by salt stress during resting and growing cell biotransformation (0.5 M NaCl increased the L-carnitine production in nearly 85%), and the need for high levels of ATP to maintain metabolite transport and biotransformation. Moreover, the main metabolic pathways and carbon flow operating during cell biotransformation was that controlled by the ICDH/ICL ratio, which decreased from 8.0 to 2.5, and the phosphotransferase/ACS ratio, which increased from 2.1 to 5.2, after a NaCl pulse fivefold the steady-state level. Resting E. coli cells were seen to be made up of heterogeneous populations consisting of several types of subpopulation (intact, depolarized, and permeabilized cells) differing in viability and metabolic activity as biotransformation run-time and the NaCl concentration increased. The results are discussed in relation with the general stress response of E. coli, which alters the NADH/NAD(+) ratio, ATP content, and central carbon enzyme activities.     

Micelle-mediated transport disturbance providing extracellular strategy for alleviating n-butanol stress on Escherichia coli

Bioprocess and Biosystems Engineering - One barrier inhibiting further progress in biofuel production is the toxicity of biofuels towards their producers. It is promising to apply gene-based...     

A novel anaerobic needle inoculator for strict obligate anaerobic bacteria

Summary An inoculating needle for picking and transferring colonies of strict anaerobic bacteria was built. A self-refilling syringe was adapted to carry a stainless steel needle within the cannula, and a tee junction allowed continuous flushing with dioxygen-free gas.     

Engineering Corynebacterium glutamicum for isobutanol production

The production of isobutanol in microorganisms has recently been achieved by harnessing the highly active 2-keto acid pathways. Since these 2-keto acids are precursors of amino acids, we aimed to construct an isobutanol production platform in Corynebacterium glutamicum, a well-known amino-acid-producing microorganism. Analysis of this host’s sensitivity to isobutanol toxicity revealed that C. glutamicum shows an increased tolerance to isobutanol relative to Escherichia coli. Overexpression of alsS of Bacillus subtilis, ilvC and ilvD of C. glutamicum, kivd of Lactococcus lactis, and a native alcohol dehydrogenase, adhA, led to the production of 2.6 g/L isobutanol and 0.4 g/L 3-methyl-1-butanol in 48 h. In addition, other higher chain alcohols such as 1-propanol, 2-methyl-1-butanol, 1-butanol, and 2-phenylethanol were also detected as byproducts. Using longer-term batch cultures, isobutanol titers reached 4.0 g/L after 96 h with wild-type C. glutamicum as a host. Upon the inactivation of several genes to direct more carbon through the isobutanol pathway, we increased production by ∼25% to 4.9 g/L isobutanol in a ∆pyc∆ldh background. These results show promise in engineering C. glutamicum for higher chain alcohol production using the 2-keto acid pathways.     

Relationship between the production of spirosomes and anaerobic glycolysis activity in Escherichia coli B

The effects of culture conditions (aerobic or anaerobic) and glucose in the medium on the production of spirosomes in Escherichia coli B were studied by SDS-PAGE and electron microscopy. The Mr of the spirosome of E. coli B was estimated to be 97,000. Electron microscopy revealed that the amount of spirosomes derived from anaerobic cultures was about eightfold larger than that from aerobic cultures. In SDS-PAGE, the bands of spirosome protein derived from anaerobic cultures were more intense than those derived from aerobic cultures, either in peptone water or in Davis-Mingioli's minimal medium. With increased glucose concentration under aerobic conditions, the intensity of the band of spirosome protein was similar to that observed under anaerobic conditions in basal media. These results suggest that spirosome production by E. coli B is related to its anaerobic glycolysis activity.     

Improved succinate production from galactose-rich feedstocks by engineered Escherichia coli under anaerobic conditions

It is of great economic interest to produce succinate from low-grade carbon sources, which can make it more economically competitive against petrochemical-based succinate. Galactose sugars constitute a significant fraction of the soluble carbohydrate in a meal from agricultural sources which is considered a low value or waste byproduct of oilseed processing. To improve the galactose utilization, the effect of galR and glk on sugars uptake was investigated by deactivation of each gene in three previously engineered host strains. As expected, glk plays an important role in glucose uptake, while, the effect of deactivation of galR is highly dependent on the strength of the downstream module (succinate production module). A new succinate producer FZ661T was constructed by enhancement of the succinate producing module and manipulation of the gal operon. The succinate productivity reached 4.57 g/L/hr when a mixed sugar feedstock was used as a carbon source in shake-flask fermentation, up to 812 mM succinate was accumulated in 80 hr in fed-batch fermentation. When SoyMolaGal hydrolysate was used as a carbon source, 628 mM (74 g/L) succinate was produced within 72 hr. In this study, we demonstrate that FZ661T can produce succinate quickly with relatively high yield, giving it the potential for industrial application.     

Re-engineering Escherichia coli for ethanol production

A lactate producing derivative of Escherichia coli KO11, strain SZ110, was re-engineered for ethanol production by deleting genes encoding all fermentative routes for NADH and randomly inserting a promoterless mini-Tn5 cassette (transpososome) containing the complete Zymomonas mobilis ethanol pathway (pdc, adhA, and adhB) into the chromosome. By selecting for fermentative growth in mineral salts medium containing xylose, a highly productive strain was isolated in which the ethanol cassette had been integrated behind the rrlE promoter, designated strain LY160 (KO11, Δfrd::celY _Ec ΔadhE ΔldhA, ΔackA lacA::casAB _Ko rrlE::(pdc _Zm -adhA _Zm -adhB _Zm -FRT-rrlE) pflB ⁺ ). This strain fermented 9% (w/v) xylose to 4% (w/v) ethanol in 48 h in mineral salts medium, nearly equal to the performance of KO11 with Luria broth.     

Aerobic and anaerobic alcohol dehydrogenases in Escherichia coli

Abstract Mutants unable to use ethanol for carbon and energy were counterselected from an ethanolutilizing mutant of Escherichia coli K12 derepressed for alcohol dehydrogenase (ADH). Mutants of one class were devoid of ADH activity under anaerobic conditions but exhibited aerobic activities comparable to those of wild-type E. coli. Mutants of a second class exhibited ADH activity levels intermediate between those of the wild-type and derepressed parent. Immunological studies showed that mutants of the former class synthesized far less ADH protein than did the derepressed parent while mutants of the latter class synthesized about the same amount. The ADH mutations in both classes were located within the previously described adh region which contains the structural gene for the activity that is derepressed in the parent. An Eth⁻ adh-lac fusion mutant with an insertion in the structural gene was also isolated and characterized. It exhibited no ADH activity under anaerobic conditions and wild-type levels under aerobic conditions. These data are consistent with the existence in E. coli of distinct aerobic and anaerobic ADH enzymes and a derepression of the anaerobic but not the aerobic enzyme in the ethanol utilizing strain.     

A statistical method for enhancing the production of succinic acid from Escherichia coli under anaerobic conditions

The most influential parameters for succinic acid production obtained through one at a time method were sucrose, tryptone, magnesium carbonate, inoculum size and incubation period. These resulted in the production of 7.0 g L(-1) of succinic acid in 60 h from Escherichia coli W3110 under anaerobic conditions. Based on these results, a statistical method, face centered central composite design (FCCCD) falling under response surface method (RSM) was employed for further enhancing the succinic acid production and to monitor the interactive effect of these parameters, which resulted in a twofold increase in yield (14.3 g L(-1) in 48 h). The analysis of variance (ANOVA) showed the adequacy of the model and the verification experiments confirmed its validity. On subsequent scale-up in a 10-L bioreactor using conditions optimized through RSM, 24.2 g L(-1) of succinic acid was obtained in 30 h. This clearly indicated that the model stood valid even on large-scale. Thus, the statistical optimization strategy led to a 3.5-fold increase in the yield of succinic acid. This is the first report on the use of FCCCD to improve succinic acid production from E. coli.     

Corynebacterium glutamicum tailored for efficient isobutanol production

We recently engineered Corynebacterium glutamicum for aerobic production of 2-ketoisovalerate by inactivation of the pyruvate dehydrogenase complex, pyruvate:quinone oxidoreductase, transaminase B, and additional overexpression of the ilvBNCD genes, encoding acetohydroxyacid synthase, acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. Based on this strain, we engineered C. glutamicum for the production of isobutanol from glucose under oxygen deprivation conditions by inactivation of l-lactate and malate dehydrogenases, implementation of ketoacid decarboxylase from Lactococcus lactis, alcohol dehydrogenase 2 (ADH2) from Saccharomyces cerevisiae, and expression of the pntAB transhydrogenase genes from Escherichia coli. The resulting strain produced isobutanol with a substrate-specific yield (Y_P/S) of 0.60 ± 0.02 mol per mol of glucose. Interestingly, a chromosomally encoded alcohol dehydrogenase rather than the plasmid-encoded ADH2 from S. cerevisiae was involved in isobutanol formation with C. glutamicum, and overexpression of the corresponding adhA gene increased the Y_P/S to 0.77 ± 0.01 mol of isobutanol per mol of glucose. Inactivation of the malic enzyme significantly reduced the Y_P/S, indicating that the metabolic cycle consisting of pyruvate and/or phosphoenolpyruvate carboxylase, malate dehydrogenase, and malic enzyme is responsible for the conversion of NADH+H⁺ to NADPH+H⁺. In fed-batch fermentations with an aerobic growth phase and an oxygen-depleted production phase, the most promising strain, C. glutamicum ΔaceE Δpqo ΔilvE ΔldhA Δmdh(pJC4ilvBNCD-pntAB)(pBB1kivd-adhA), produced about 175 mM isobutanol, with a volumetric productivity of 4.4 mM h⁻¹, and showed an overall Y_P/S of about 0.48 mol per mol of glucose in the production phase.