Isobutanol Production Research Articles

Nutrient composition and safety evaluation of simulated isobutanol distillers dried grains with solubles and associated fermentation metabolites when fed to male Ross 708 broiler chickens (Gallus domesticus).

Saccharomyces cerevisiae genetically engineered to enhance butanol production will be used in a manufacturing process similar to that of fuel ethanol production, including co-production of distillers products for animal feed. A poultry feeding trial was conducted with simulated isobutanol-derived dried distillers grains with solubles (bDDGS), comprising non-fermentable corn solids and heat-inactivated Butamax modified yeast (BMY), to determine potential health effects. Simulated dried distillers grains were produced in 2 variants: bDDGS containing 10% (B10) or 50% (B50) BMY. The BMY concentrations were selected based on a conservative estimate from ethanol-derived distillers grains (eDDGS) approximating 2.5 and 12-fold margins of exposure. The B10 and B50 DDGS were evaluated in a 42-day feeding trial using male Ross 708 broiler chickens fed diets containing eDDGS, B50 DDGS, or B10 DDGS without or with isobutanol, 2,3-butanediol, and isobutyric acid metabolites each at target concentrations of 2 (B10-2), 5 (B10-5), or 10 (B10-10) times the anticipated specification limit in the commercial product. Diets were fed (n = 50 broilers/treatment) in 3 phases: starter phase with 8% DDGS and grower and finisher phases each with 15% DDGS. No statistically significant differences or diet-related effects on mortality, clinical pathology, or organ weights, and no microscopic observations associated with consumption of diets containing B10, B50, or B10 supplemented with metabolites at any targeted exposure level were observed. A lower (P < 0.05) mean absolute bursa of Fabricius weight in the B10-10 group compared to the B10 group was considered to be within the range of biological variability. A non-significant trend toward lower weight, gains, and feed intake, and higher feed:gain ratio was observed in the B10-10 group, and was considered a non-adverse palatability effect of consuming high concentrations of metabolites. These results demonstrate that consumption of phase diets containing simulated DDGS from a novel isobutanol production process was well-tolerated.

Improving isobutanol production with the yeast Saccharomyces cerevisiae by successively blocking competing metabolic pathways as well as ethanol and glycerol formation

BackgroundIsobutanol is a promising candidate as second-generation biofuel and has several advantages compared to bioethanol. Another benefit of isobutanol is that it is already formed as a by-product in fermentations with the yeast Saccharomyces cerevisiae, although only in very small amounts. Isobutanol formation results from valine degradation in the cytosol via the Ehrlich pathway. In contrast, valine is synthesized from pyruvate in mitochondria. This spatial separation into two different cell compartments is one of the limiting factors for higher isobutanol production in yeast. Furthermore, some intermediate metabolites are also substrates for various isobutanol competing pathways, reducing the metabolic flux toward isobutanol production. We hypothesized that a relocation of all enzymes involved in anabolic and catabolic reactions of valine metabolism in only one cell compartment, the cytosol, in combination with blocking non-essential isobutanol competing pathways will increase isobutanol production in yeast.ResultsHere, we overexpressed the three endogenous enzymes acetolactate synthase (Ilv2), acetohydroxyacid reductoisomerase (Ilv5) and dihydroxy-acid dehydratase (Ilv3) of the valine synthesis pathway in the cytosol and blocked the first step of mitochondrial valine synthesis by disrupting endogenous ILV2, leading to a 22-fold increase of isobutanol production up to 0.22 g/L (5.28 mg/g glucose) with aerobic shake flask cultures. Then, we successively deleted essential genes of competing pathways for synthesis of 2,3-butanediol (BDH1 and BDH2), leucine (LEU4 and LEU9), pantothenate (ECM31) and isoleucine (ILV1) resulting in an optimized metabolic flux toward isobutanol and titers of up to 0.56 g/L (13.54 mg/g glucose). Reducing ethanol formation by deletion of the ADH1 gene encoding the major alcohol dehydrogenase did not result in further increased isobutanol production, but in strongly enhanced glycerol formation. Nevertheless, deletion of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2 prevented formation of glycerol and increased isobutanol production up to 1.32 g/L. Finally, additional deletion of aldehyde dehydrogenase gene ALD6 reduced the synthesis of the by-product isobutyrate, thereby further increasing isobutanol production up to 2.09 g/L with a yield of 59.55 mg/g glucose, corresponding to a more than 200-fold increase compared to the wild type.ConclusionsBy overexpressing a cytosolic isobutanol synthesis pathway and by blocking non-essential isobutanol competing pathways, we could achieve isobutanol production with a yield of 59.55 mg/g glucose, which is the highest yield ever obtained with S. cerevisiae in shake flask cultures. Nevertheless, our results indicate a still limiting capacity of the isobutanol synthesis pathway itself.

Analysis of metabolite profiles of Saccharomyces cerevisiae strains suitable for butanol production.

Butanol has advantages over ethanol as a biofuel. Although butanol is naturally produced by some Clostridium species, clostridial fermentation has inherent characteristics that prevent its industrial application. Butanol-producing Saccharomyces cerevisiae strains may be a solution to this problem. The aim of this study was to evaluate the ability of wild-type and industrial Brazilian strains of S. cerevisiae to produce n-butanol using glycine as co-substrate and evaluate the relationship between the production of this alcohol and other metabolites in fermented broth. Of the 48 strains analyzed, 25 were able to produce n-butanol in a glycine-containing medium. Strains exhibited different profiles of n-butanol, isobutanol, ethanol, glycerol and acetic acid production. Some wild-type strains showed substantial n-butanol production capability, for instance UFMG-CM-Y267, which produced about 12.7 mg/L of butanol. Although this concentration is low, it demonstrates that wild-type S. cerevisiae can synthesize butanol, suggesting that selection and genetic modification of this microorganism could yield promising results. The findings presented here may prove useful for future studies aimed at optimizing S. cerevisiae strains for butanol production.

Activation of alternative metabolic pathways diverts carbon flux away from isobutanol formation in an engineered Escherichia coli strain.

Metabolic engineering efforts are guided by identifying gene targets for overexpression and/or deletion. Isobutanol, a biofuel candidate, is biosynthesized using the valine biosynthesis pathway and enzymes of the Ehrlich pathway. Most reported studies for isobutanol production in Escherichia coli employ multicopy plasmids, an approach that suffers from disadvantages such as plasmid instability, increased metabolic burden, and use of antibiotics to maintain selection pressure. Cofactor imbalance is another issue that may limit production of isobutanol, as two enzymes of the pathway utilize NADPH as a cofactor. To address these issues, we constructed E. coli strains with chromosomally-integrated, codon-optimized isobutanol pathway genes (ilvGM, ilvC, kivd, adh) selected on the basis of their cofactor preferences. Genes involved in diverting pyruvate flux toward fermentation byproducts were deleted. Metabolite analyses of the constructed strains revealed extracellular accumulation of significant amounts of isobutyraldehyde, a pathway intermediate, and the overflow metabolites 2,3-butanediol and acetol. These results demonstrate that the genetic modifications carried out led to activation of alternative pathways that diverted carbon flux toward formation of unwanted metabolites. The present study highlights how precursor metabolites can be metabolized through enzymatic routes that have not been considered important in previous studies due to the different strategies employed therein. The insights gained from the present study will allow rational genetic modification of host cells for production of metabolites of interest.

Syntrophic co-culture amplification of production phenotype for high-throughput screening of microbial strain libraries

Microbes can be engineered to synthesize a wide array of bioproducts, yet production phenotype evaluation remains a frequent bottleneck in the design-build-test cycle where strain development requires iterative rounds of library construction and testing. Here, we present Syntrophic Co-culture Amplification of Production phenotype (SnoCAP). Through a metabolic cross-feeding circuit, the production level of a target molecule is translated into highly distinguishable co-culture growth characteristics, which amplifies differences in production into highly distinguishable growth phenotypes. We demonstrate SnoCAP with the screening of Escherichia coli strains for production of two target molecules: 2-ketoisovalerate, a precursor of the drop-in biofuel isobutanol, and L-tryptophan. The dynamic range of the screening can be tuned by employing an inhibitory analog of the target molecule. Screening based on this framework requires compartmentalization of individual producers with the sensor strain. We explore three formats of implementation with increasing throughput capability: confinement in microtiter plates (102-104 assays/experiment), spatial separation on agar plates (104-105 assays/experiment), and encapsulation in microdroplets (105-107 assays/experiment). Using SnoCAP, we identified an efficient isobutanol production strain from a random mutagenesis library, reaching a final titer that is 5-fold higher than that of the parent strain. The framework can also be extended to screening for secondary metabolite production using a push-pull strategy. We expect that SnoCAP can be readily adapted to the screening of various microbial species, to improve production of a wide range of target molecules.

Development of an efficient cytosolic isobutanol production pathway in Saccharomyces cerevisiae by optimizing copy numbers and expression of the pathway genes based on the toxic effect of \u03b1-acetolactate

Isobutanol production in Saccharomyces cerevisiae is limited by subcellular compartmentalization of the pathway enzymes. In this study, we improved isobutanol production in S. cerevisiae by constructing an artificial cytosolic isobutanol biosynthetic pathway consisting of AlsS, α-acetolactate synthase from Bacillus subtilis, and two endogenous mitochondrial enzymes, ketol-acid reductoisomerase (Ilv5) and dihydroxy-acid dehydratase (Ilv3), targeted to the cytosol. B. subtilis AlsS was more active than Ilv2ΔN54, an endogenous α-acetolactate synthase targeted to the cytosol. However, overexpression of alsS led to a growth inhibition, which was alleviated by overexpressing ILV5ΔN48 and ILV3ΔN19, encoding the downstream enzymes targeted to the cytosol. Therefore, accumulation of the intermediate α-acetolactate might be toxic to the cells. Based on these findings, we improved isobutanol production by expressing alsS under the control of a copper-inducible CUP1 promoter, and by increasing translational efficiency of the ILV5ΔN48 and ILV3ΔN19 genes by adding Kozak sequence. Furthermore, strains with multi-copy integration of alsS into the delta-sequences were screened based on growth inhibition upon copper-dependent induction of alsS. Next, the ILV5ΔN48 and ILV3ΔN19 genes were integrated into the rDNA sites of the alsS-integrated strain, and the strains with multi-copy integration were screened based on the growth recovery. After optimizing the induction conditions of alsS, the final engineered strain JHY43D24 produced 263.2 mg/L isobutanol, exhibiting about 3.3-fold increase in production compared to a control strain constitutively expressing ILV2ΔN54, ILV5ΔN48, and ILV3ΔN19 on plasmids.

Establishment of BmoR-based biosensor to screen isobutanol overproducer

BackgroundIsobutanol, a C4 branched-chain higher alcohol, is regarded as an attractive next-generation transport fuel. Metabolic engineering for efficient isobutanol production has been achieved in many studies. BmoR, an alcohol-regulated transcription factor, mediates a σ54-dependent promoter Pbmo of alkane monooxygenase in n-alkane metabolism of Thauera butanivorans and displays high sensitivity to C4–C6 linear alcohols and C3–C5 branched-chain alcohols. In this study, to achieve the high-level production of isobutanol, we established a screening system which relied on the combination of BmoR-based biosensor and isobutanol biosynthetic pathway and then employed it to screen isobutanol overproduction strains from an ARTP mutagenesis library.ResultsFirstly, we constructed and verified a GFP-based BmoR-Pbmo device responding to the isobutanol produced by the host. Then, this screening system was employed to select three mutants which exhibited higher GFP/OD600 values than that of wild type. Significantly, GFP/OD600 of mutant 10 was 190.7 ± 4.8, a 1.4-fold higher value than that of wild type. Correspondingly, the isobutanol titer of that strain was 1597.6 ± 129.6 mg/L, 2.0-fold higher than the wild type. With the overexpression of upstream pathway genes, the isobutanol production from mutant 10 reached 14.0 ± 1.0 g/L after medium optimization in shake flask. The isobutanol titer reached 56.5 ± 1.8 g/L in a fed-batch production experiment.ConclusionsThis work screened out isobutanol overproduction strains from a mutagenesis library by using a screening system which depended on the combination of BmoR-based biosensor and isobutanol biosynthetic pathway. Optimizing fermentation condition and reinforcing upstream pathway could realize the increase of isobutanol production from the overproducer. Lastly, fed-batch fermentation of the mutant enhanced the isobutanol production to 56.5 ± 1.8 g/L.

Engineered microbial biofuel production and recovery under supercritical carbon dioxide

Culture contamination, end-product toxicity, and energy efficient product recovery are long-standing bioprocess challenges. To solve these problems, we propose a high-pressure fermentation strategy, coupled with in situ extraction using the abundant and renewable solvent supercritical carbon dioxide (scCO2), which is also known for its broad microbial lethality. Towards this goal, we report the domestication and engineering of a scCO2-tolerant strain of Bacillus megaterium, previously isolated from formation waters from the McElmo Dome CO2 field, to produce branched alcohols that have potential use as biofuels. After establishing induced-expression under scCO2, isobutanol production from 2-ketoisovalerate is observed with greater than 40% yield with co-produced isopentanol. Finally, we present a process model to compare the energy required for our process to other in situ extraction methods, such as gas stripping, finding scCO2 extraction to be potentially competitive, if not superior.

Kinetic Modeling of the Isobutanol Production from Glucose Using Shimwellia blattae (p424IbPSO) Strain: Effect of Initial Substrate Concentration

Isobutanol production from glucose using Shimwellia blattae (p424IbPSO) has been studied. Experiments were carried out under conditions previously determined: medium M92X, in a stirred tank bioreac...

OptSSeq explores enzyme expression and function landscapes to maximize isobutanol production rate

Efficient microbial production of the next-generation biofuel isobutanol (IBA) is limited by metabolic bottlenecks. Overcoming these bottlenecks will be aided by knowing the optimal ratio of enzymes for efficient flux through the IBA biosynthetic pathway. OptSSeq (Optimization by Selection and Sequencing) accomplishes this goal by tracking growth rate-linked selection of optimal expression elements from a combinatorial library. The 5-step pathway to IBA consists of Acetolactate synthase (AlsS), Keto-acid reductoisomerase (KARI), Di-hydroxy acid dehydratase (DHAD), Ketoisovalerate decarboxylase (Kivd) and Alcohol dehydrogenase (Adh). Using OptSSeq, we identified gene expression elements leading to optimal enzyme levels that enabled theoretically maximal productivities per cell biomass in Escherichia coli. We identified KARI as the rate-limiting step, requiring the highest levels of enzymes expression, followed by AlsS and AdhA. DHAD and Kivd required relatively lower levels of expression for optimal IBA production. OptSSeq also enabled the identification of an Adh enzyme variant capable of an improved rate of IBA production. Using models that predict impacts of enzyme synthesis costs on cellular growth rates, we found that optimum levels of pathway enzymes led to maximal IBA production, and that additional limitations lie in the E. coli metabolic network. Our optimized constructs enabled the production of ~3 g IBA per hour per gram dry cell weight and was achieved with 20 % of the total cell protein devoted to IBA-pathway enzymes in the molar ratio 2.5:6.7:2:1:5.2 (AlsS:IlvC:IlvD:Kivd:AdhA). These enzyme levels and ratios optimal for IBA production in E. coli provide a useful starting point for optimizing production of IBA in diverse microbes and fermentation conditions.

Carbon flux distribution in the metabolism of Shimwellia blattae (p424IbPSO) for isobutanol production from glucose as function of oxygen availability

AbstractBACKGROUNDThe carbon flux distribution in Shimwellia blattae (p424IbPSO) cultures when glucose is employed as carbon source is studied in a stirred tank bioreactor changing the oxygen availability. The strain has been constructed to produce isobutanol combining the branched‐chain amino acids pathway and Ehrlich pathway. The information of many similar genetic modified strains for this bioprocess in the literature presents significant discrepancies on the oxygen influence.RESULTSThe study carried out in this work pointed out the presence of other metabolites formed under aerobic conditions, such as isobutyric acid, 2,3‐butanedione, acetoin, 2,3‐butanediol and 3‐oxo‐1‐buten‐2‐yl ether, not previously reported in the literature on the production of isobutanol with S. blattae (p424IbPSO) strain.CONCLUSIONIn this work the routes or the chemical reactions involved in the bioprocess as a function of the availability of oxygen are elucidated. Anaerobic conditions are the best for isobutanol production from glucose employing this strain; around 12 g L−1 of isobutanol are obtained under these conditions. Aerobic conditions are not adequate to produce isobutanol, although the need for oxygen, at least for growth, is also demonstrated. © 2018 Society of Chemical Industry

Enhancement of photosynthetic isobutanol production in engineered cells of Synechocystis PCC 6803

BackgroundCyanobacteria, oxygenic photoautotrophic prokaryotes, can be engineered to produce various valuable chemicals from solar energy and CO2 in direct processes. The concept of photosynthetic production of isobutanol, a promising chemical and drop-in biofuel, has so far been demonstrated for Synechocystis PCC 6803 and Synechococcus elongatus PCC 7942. In Synechocystis PCC 6803, a heterologous expression of α-ketoisovalerate decarboxylase (Kivd) from Lactococcus lactis resulted in an isobutanol and 3-methyl-1-butanol producing strain. Kivd was identified as a bottleneck in the metabolic pathway and its activity was further improved by reducing the size of its substrate-binding pocket with a single replacement of serine-286 to threonine (KivdS286T). However, isobutanol production still remained low.ResultsIn the present study, we report on how cultivation conditions significantly affect the isobutanol production in Synechocystis PCC 6803. A HCl-titrated culture grown under medium light (50 μmol photons m−2 s−1) showed the highest isobutanol production with an in-flask titer of 194 mg l−1 after 10 days and 435 mg l−1 at day 40. This corresponds to a cumulative isobutanol production of 911 mg l−1, with a maximal production rate of 43.6 mg l−1 day−1 observed between days 4 and 6. Additional metabolic bottlenecks in the isobutanol biosynthesis pathway were further addressed. The expression level of KivdS286T was significantly affected when co-expressed with another gene downstream in a single operon and in a convergent oriented operon. Moreover, the expression of the ADH encoded by codon-optimized slr1192 and co-expression of IlvC and IlvD were identified as potential approaches to further enhance isobutanol production in Synechocystis PCC 6803.ConclusionThe present study demonstrates the importance of a suitable cultivation condition to enhance isobutanol production in Synechocystis PCC 6803. Chemostat should be used to further increase both the total titer as well as the rate of production. Furthermore, identified bottleneck, Kivd, should be expressed at the highest level to further enhance isobutanol production.

Resting cells isobutanol production by Shimwellia blattae (p424IbPSO): Influence of growth culture conditions.

Isobutanol is a promising gasoline additive and could even be a potential substitute used directly as combustible. In this work, the production of isobutanol from glucose by Shimwellia blattae (p424IbPSO) in resting cell cultures is studied. This production has two stages, involving a resting cell phase that has not been studied before. The cell growth was carried out under different operating conditions: temperature and medium composition (YE, ammonium, and IPTG concentrations), looking for the highest isobutanol production. Moreover, the cells were collected at three different growth times checking their isobutanol production capacity. The best operating conditions have been determined as: 30°C of temperature, a medium containing 1.5 g L-1 YE and 1.4 g L-1 of ammonium as nitrogen sources, adding 0.5 mM IPTG as inducer. The cells collected at early growth times are significantly more active. The use of S. blattae (p424IbPSO) in resting cells is a good strategy for the production of isobutanol from glucose yielding better results than in batch growth cultures, a yield of 60% attainment of theoretical maximum yield is obtained under optimal conditions. In addition, it has been demonstrated that if the cells are cultured at higher temperatures and with high IPTG concentrations, inclusion bodies are formed in the cytoplasm inhibiting the isobutanol production in the resting cell stage.

Elimination of biosynthetic pathways for l-valine and l-isoleucine in mitochondria enhances isobutanol production in engineered Saccharomyces cerevisiae

Saccharomyces cerevisiae has a natural ability to produce higher alcohols, making it a promising candidate for production of isobutanol. However, the several pathways competing with isobutanol biosynthesis lead to production of substantial amounts of l-valine and l-isoleucine in mitochondria and isobutyrate, l-leucine, and ethanol in cytosol. To increase flux to isobutanol by removing by-product formation, the genes associated with formation of l-valine (BAT1), l-isoleucine (ILV1), isobutyrate (ALD6), l-leucine (LEU1), and ethanol (ADH1) were disrupted to construct the S. cerevisiae WΔGBIALA1_2vec strain. This strain showed 8.9 and 8.6 folds increases in isobutanol concentration and yield, respectively, relative the corresponding values of the background strain on glucose medium. In a bioreactor fermentation with a gas trapping system, the WΔGBIALA1_2vec strain produced 662 mg/L isobutanol concentration with a yield of 6.71 mgisobutanol/gglucose. With elimination of the competing pathways, the WΔGBIALA1_2vec strain would serve as a platform strain for isobutanol production.

Enhanced isobutanol production from acetate by combinatorial overexpression of acetyl-CoA synthetase and anaplerotic enzymes in engineered Escherichia coli.

Acetic acid is an abundant material that can be used as a carbon source by microorganisms. Despite its abundance, its toxicity and low energy content make it hard to utilize as a sole carbon source for biochemical production. To increase acetate utilization and isobutanol production with engineered Escherichia coli, the feasibility of utilizing acetate and metabolic engineering was investigated. The expression of acs, pckA, and maeB increased isobutanol production by up to 26%, and the addition of TCA cycle intermediates indicated that the intermediates can enhance isobutanol production. For isobutanol production from acetate, acetate uptake rates and the NADPH pool were not limiting factors compared to glucose as a carbon source. This work represents the first approach to produce isobutanol from acetate with pyruvate flux optimization to extend the applicability of acetate. This technique suggests a strategy for biochemical production utilizing acetate as the sole carbon source.

Enhanced isobutanol production from acetate by combinatorial overexpression of acetyl-CoA synthetase and anaplerotic enzymes in engineered Escherichia coli

Enhanced isobutanol production from acetate by combinatorial overexpression of acetyl-CoA synthetase and anaplerotic enzymes in engineered <i>Escherichia coli</i>

Improved production of isobutanol in pervaporation-coupled bioreactor using sugarcane bagasse hydrolysate in engineered Enterobacter aerogenes

A process of isobutanol production from sugarcane bagasse hydrolysates in Enterobacter aerogenes was developed here with a pervaporation-integrated procedure. Isobutanol pathway was overexpressed in a mutant strain with eliminated byproduct-forming enzymes (LdhA, BudA, and PflB). A glucose-and-xylose–coconsuming ptsG mutant was constructed for effective utilization of lignocellulosic biomass. Toxic effects of isobutanol were alleviated by in situ recovery via a pervaporation procedure. Compared to single-batch fermentation, cell growth and isobutanol titer were improved by 60% and 100%, respectively, in the pervaporation-integrated fermentation process. A lab-made cross-linked polydimethylsiloxane membrane was cast on polyvinylidene fluoride and used in the pervaporation process. The membrane-penetrating condensate contained 55–226 g m−2 h−1 isobutanol with 6–25 g L−1 ethanol after separation. This study offers improved fermentative production of isobutanol from lignocellulosic biomass with a pervaporation procedure.

Protein engineering of α-ketoisovalerate decarboxylase for improved isobutanol production in Synechocystis PCC 6803

Protein engineering is a powerful tool to modify e.g. protein stability, activity and substrate selectivity. Heterologous expression of the enzyme α-ketoisovalerate decarboxylase (Kivd) in the unicellular cyanobacterium Synechocystis PCC 6803 results in cells producing isobutanol and 3-methyl-1-butanol, with Kivd identified as a potential bottleneck. In the present study, we used protein engineering of Kivd to improve isobutanol production in Synechocystis PCC 6803. Isobutanol is a flammable compound that can be used as a biofuel due to its high energy density and suitable physical and chemical properties. Single replacement, either Val461 to isoleucine or Ser286 to threonine, increased the Kivd activity significantly, both in vivo and in vitro resulting in increased overall production while isobutanol production was increased more than 3-methyl-1-butanol production. Moreover, among all the engineered strains examined, the strain with the combined modification V461I/S286T showed the highest (2.4 times) improvement of isobutanol-to-3M1B molar ratio, which was due to a decrease of the activity towards 3M1B production. Protein engineering of Kivd resulted in both enhanced total catalytic activity and preferential shift towards isobutanol production in Synechocystis PCC 6803.

Rational design of a synthetic Entner-Doudoroff pathway for enhancing glucose transformation to isobutanol in Escherichia coli.

Isobutanol as a more desirable biofuel has attracted much attention. In our previous work, an isobutanol-producing strain Escherichia coli LA09 had been obtained by rational redox status improvement under guidance of the genome-scale metabolic model. However, the low transformation from sugar to isobutanol is a limiting factor for isobutanol production by E. coli LA09. In this study, the intracellular metabolic profiles of the isobutanol-producing E. coli LA09 with different initial glucose concentrations were investigated and the metabolic reaction of fructose 6-phosphate to 1, 6-diphosphate fructose in glycolytic pathway was identified as the rate-limiting step of glucose transformation. Thus, redesigned carbon catabolism was implemented by altering flux of sugar metabolism. Here, the heterologous Entner-Doudoroff (ED) pathway from Zymomonas mobilis was constructed, and the adaptation of upper and lower parts of ED pathway was further improved with artificial promoters to alleviate the accumulation of toxic intermediate metabolite 2-keto-3-deoxy-6-phospho-gluconate (KDPG). Finally, the best isobutanol-producing E. coli ED02 with higher glucose transformation and isobutanol production was obtained. In the fermentation of strain E. coli ED02 with 45g/L initial glucose, the isobutanol titer, yield and average producing rate were, respectively, increased by 56.8, 47.4 and 88.1% to 13.67g/L, 0.50 C-mol/C-mol and 0.456g/(L×h) in a shorter time of 30h, compared with that of the starting strain E. coli LA09.

High-level production of valine by expression of the feedback inhibition-insensitive acetohydroxyacid synthase in Saccharomyces cerevisiae

Valine, which is one of the branched-chain amino acids (BCAAs) essential for humans, is widely used in animal feed, dietary supplements and pharmaceuticals. At the commercial level, valine is usually produced by bacterial fermentation from glucose. However, valine biosynthesis can also proceed in the yeast Saccharomyces cerevisiae, which is a useful microorganism in fermentation industry. In S. cerevisiae, valine biosynthesis is regulated by valine itself via the feedback inhibition of acetohydroxyacid synthase (AHAS), which consists of two subunits, the catalytic subunit Ilv2 and the regulatory subunit Ilv6. In this study, to improve the valine productivity of yeast cells, we constructed several variants of Ilv6 by introducing amino acid substitutions based on a protein sequence comparison with the AHAS regulatory subunit of E. coli. Among them, we found that the Asn86Ala, Gly89Asp and Asn104Ala variants resulted in approximately 4-fold higher intracellular valine contents compared with those in cells with the wild-type Ilv6. The computational analysis of Ilv6 predicted that Asn86, Gly89 and Asn104 are located in the vicinity of a valine-binding site, suggesting that amino acid substitutions at these positions induce conformational change of the valine-binding site. To test the effects of these variants on AHAS activity, both recombinant Ilv2 and Ilv6 were purified and reconstituted in vitro. The Ilv6 variants were much less sensitive to feedback inhibition by valine than the wild-type Ilv6. Only a portion of the amino acid changes identified in the E. coli AHAS regulatory subunit IlvH enhanced the valine synthesis, suggesting structural and/or functional differences between the S. cerevisiae and E. coli AHAS regulatory subunits. It should also be noted that these amino acid substitutions did not affect the intracellular pools of the other BCAAs, leucine and isoleucine. The approach described here could be a practical method for the development of industrial yeast strains with high-level production of valine or isobutanol.