Search

(S)-3-Hydroxybutyrate (S-3HB) can be used as a precursor for the synthesis of biodegradable polymers such as polyhydroxyalkanoate and stereo-specific fine chemicals such as antibiotics, pheromones, and drugs. For the production of S-3HB in yeast, the biosynthetic pathway of S-3HB from acetyl-CoA, consisting of the three enzymes, acetyl-CoA C-acetyltransferase (ACCT), acetoacetyl-CoA reductase (ACR), and 3-hydroxybutyryl-CoA thioesterase (HBT), was introduced into Saccharomyces cerevisiae. An engineered yeast strain overexpressing ERG10, hbd, and tesB genes not only exhibited enzyme activities of AACT, ACR, and HBT, but also produced S-3HB from ethanol. In order to increase the titer of S-3HB, a fed-batch fermentation based on pulse feeding of ethanol as a carbon source was performed, and a final S-3HB titer of 12.0g/L was achieved. This is the first report on the production of 3HB by engineered yeast, utilizing ethanol as the carbon source, suggesting that the industrially preferred S. cerevisiae can be a promising host for producing S-3HB., (Copyright © 2015 Elsevier B.V. All rights reserved.)

To minimize glycerol production during 2,3-BD fermentation by the engineered Saccharomyces cerevisiae, the Lactococcus lactis water-forming NADH oxidase gene (noxE) was expressed at five different levels. The expression of NADH oxidase substantially decreased the intracellular NADH/NAD(+) ratio. The S. cerevisiae BD5_T2nox strain expressing noxE produced 2,3-BD with yield of 0.359 g 2,3-BD/gglucose and glycerol with 0.069gglycerol/gglucose, which are 23.8% higher and 65.3% lower than those of the isogenic strain without noxE. These results demonstrate that the carbon flux could be redirected from glycerol to 2,3-BD through alteration of the NADH/NAD(+) ratio by the expression of NADH oxidase., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Microbial metabolism involves complex, system-level processes implemented via the orchestration of metabolic reactions, gene regulation, and environmental cues. One canonical example of such processes is acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum, during which cells convert carbon sources to organic acids that are later reassimilated to produce solvents as a strategy for cellular survival. The complexity and systems nature of the process have been largely underappreciated, rendering challenges in understanding and optimizing solvent production. Here, we present a system-level computational framework for ABE fermentation that combines metabolic reactions, gene regulation, and environmental cues. We developed the framework by decomposing the entire system into three modules, building each module separately, and then assembling them back into an integrated system. During the model construction, a bottom-up approach was used to link molecular events at the single-cell level into the events at the population level. The integrated model was able to successfully reproduce ABE fermentations of the WT C. acetobutylicum (ATCC 824), as well as its mutants, using data obtained from our own experiments and from literature. Furthermore, the model confers successful predictions of the fermentations with various network perturbations across metabolic, genetic, and environmental aspects. From foundation to applications, the framework advances our understanding of complex clostridial metabolism and physiology and also facilitates the development of systems engineering strategies for the production of advanced biofuels.

The inability of fermenting microorganisms to use mixed carbon components derived from lignocellulosic biomass is a major technical barrier that hinders the development of economically viable cellulosic biofuel production. In this study, we integrated the fermentation pathways of both hexose and pentose sugars and an acetic acid reduction pathway into one Saccharomyces cerevisiae strain for the first time using synthetic biology and metabolic engineering approaches. The engineered strain coutilized cellobiose, xylose, and acetic acid to produce ethanol with a substantially higher yield and productivity than the control strains, and the results showed the unique synergistic effects of pathway coexpression. The mixed substrate coutilization strategy is important for making complete and efficient use of cellulosic carbon and will contribute to the development of consolidated bioprocessing for cellulosic biofuel. The study also presents an innovative metabolic engineering approach whereby multiple substrate consumption pathways can be integrated in a synergistic way for enhanced bioconversion.

Prairie cordgrass (Spartina pectinata L.) has the potential to be a feedstock for bioethanol. It is native to North America, and has extensive genetic diversity. Eleven natural populations of prairie cordgrass harvested in 2011 and 2012 were studied. Compositions of the samples showed significant differences within the same year, and between the two years. Two highest, one medium and two lowest glucan concentration samples from each year were selected to evaluate ethanol yield after dilute acid pretreatment and simultaneous saccharification and co-fermentation using Saccharomycescerevisiae SR8 that can ferment both glucose and xylose. Up to 88% of theoretical ethanol yields were achieved. Our research demonstrates the potential of prairie cordgrass as a dedicated energy crop with ethanol yields of 205.0-275.6 g/kg biomass and 1748-4368 L/ha, depending on feedstock composition and biomass yield. These ethanol yields are comparable with those of switchgrass, corn stover and bagasse., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Fermentation inhibitors present in lignocellulose hydrolysates are inevitable obstacles for achieving economic production of biofuels and biochemicals by industrial microorganisms. Here we show that spermidine (SPD) functions as a chemical elicitor for enhanced tolerance of Saccharomyces cerevisiae against major fermentation inhibitors. In addition, the feasibility of constructing an engineered S. cerevisiae strain capable of tolerating toxic levels of the major inhibitors without exogenous addition of SPD was explored. Specifically, we altered expression levels of the genes in the SPD biosynthetic pathway. Also, OAZ1 coding for ornithine decarboxylase (ODC) antizyme and TPO1 coding for the polyamine transport protein were disrupted to increase intracellular SPD levels through alleviation of feedback inhibition on ODC and prevention of SPD excretion, respectively. Especially, the strain with combination of OAZ1 and TPO1 double disruption and overexpression of SPE3 not only contained spermidine content of 1.1mg SPD/g cell, which was 171% higher than that of the control strain, but also exhibited 60% and 33% shorter lag-phase period than that of the control strain under the medium containing furan derivatives and acetic acid, respectively. While we observed a positive correlation between intracellular SPD contents and tolerance phenotypes among the engineered strains accumulating different amounts of intracellular SPD, too much SPD accumulation is likely to cause metabolic burden. Therefore, genetic perturbations for intracellular SPD levels should be optimized in terms of metabolic burden and SPD contents to construct inhibitor tolerant yeast strains. We also found that the genes involved in purine biosynthesis and cell wall and chromatin stability were related to the enhanced tolerance phenotypes to furfural. The robust strains constructed in this study can be applied for producing chemicals and advanced biofuels from cellulosic hydrolysates., (Copyright © 2015 International Metabolic Engineering Society. All rights reserved.)

The anaerobic spore-forming, gram-positive, solventogenic clostridia are notorious for being difficult to genetically engineer. Based on CRISPR/Cas9 assisted homologous recombination, we demonstrated that clean markerless gene deletion from the chromosome can be easily achieved with a high efficiency through a single-step transformation in Clostridium beijerinckii NCIMB 8052, one of the most prominent strains for acetone, butanol and ethanol (ABE) production. This highly efficient genome engineering system can be further explored for multiplex genome engineering purposes. The protocols and principles developed in this study provided valuable references for genome engineering in other microorganisms lacking developed genetic engineering tools., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Individuals with inactive alleles of the fucosyltransferase 2 gene (FUT2; termed the 'secretor' gene) are common in many populations. Some members of the genus Bifidobacterium, common infant gut commensals, are known to consume 2'-fucosylated glycans found in the breast milk of secretor mothers. We investigated the effects of maternal secretor status on the developing infant microbiota with a special emphasis on bifidobacterial species abundance., Results: On average, bifidobacteria were established earlier and more often in infants fed by secretor mothers than in infants fed by non-secretor mothers. In secretor-fed infants, the relative abundance of the Bifidobacterium longum group was most strongly correlated with high percentages of the order Bifidobacteriales. Conversely, in non-secretor-fed infants, Bifidobacterium breve was positively correlated with Bifidobacteriales, while the B. longum group was negatively correlated. A higher percentage of bifidobacteria isolated from secretor-fed infants consumed 2'-fucosyllactose. Infant feces with high levels of bifidobacteria had lower milk oligosaccharide levels in the feces and higher amounts of lactate. Furthermore, feces containing different bifidobacterial species possessed differing amounts of oligosaccharides, suggesting differential consumption in situ., Conclusions: Infants fed by non-secretor mothers are delayed in the establishment of a bifidobacteria-laden microbiota. This delay may be due to difficulties in the infant acquiring a species of bifidobacteria able to consume the specific milk oligosaccharides delivered by the mother. This work provides mechanistic insight into how milk glycans enrich specific beneficial bacterial populations in infants and reveals clues for enhancing enrichment of bifidobacterial populations in at risk populations - such as premature infants.

Recent progresses in data-driven analysis methods, including network-based approaches, are revolutionizing many classical disciplines. These techniques can also be applied to food and nutrition, which must be studied to design healthy diets. Using nutritional information from over 1,000 raw foods, we systematically evaluated the nutrient composition of each food in regards to satisfying daily nutritional requirements. The nutrient balance of a food was quantified and termed nutritional fitness; this measure was based on the food's frequency of occurrence in nutritionally adequate food combinations. Nutritional fitness offers a way to prioritize recommendable foods within a global network of foods, in which foods are connected based on the similarities of their nutrient compositions. We identified a number of key nutrients, such as choline and α-linolenic acid, whose levels in foods can critically affect the nutritional fitness of the foods. Analogously, pairs of nutrients can have the same effect. In fact, two nutrients can synergistically affect the nutritional fitness, although the individual nutrients alone may not have an impact. This result, involving the tendency among nutrients to exhibit correlations in their abundances across foods, implies a hidden layer of complexity when exploring for foods whose balance of nutrients within pairs holistically helps meet nutritional requirements. Interestingly, foods with high nutritional fitness successfully maintain this nutrient balance. This effect expands our scope to a diverse repertoire of nutrient-nutrient correlations, which are integrated under a common network framework that yields unexpected yet coherent associations between nutrients. Our nutrient-profiling approach combined with a network-based analysis provides a more unbiased, global view of the relationships between foods and nutrients, and can be extended towards nutritional policies, food marketing, and personalized nutrition.

The haloacid dehalogenase (HAD) superfamily is one of the largest enzyme families, consisting mainly of phosphatases. Although intracellular phosphate plays important roles in many cellular activities, the biological functions of HAD enzymes are largely unknown. Pho13 is 1 of 16 putative HAD enzymes in Saccharomyces cerevisiae. Pho13 has not been studied extensively, but previous studies have identified PHO13 to be a deletion target for the generation of industrially attractive phenotypes, namely, efficient xylose fermentation and high tolerance to fermentation inhibitors. In order to understand the molecular mechanisms underlying the improved xylose-fermenting phenotype produced by deletion of PHO13 (pho13Δ), we investigated the response of S. cerevisiae to pho13Δ at the transcriptomic level when cells were grown on glucose or xylose. Transcriptome sequencing analysis revealed that pho13Δ resulted in upregulation of the pentose phosphate (PP) pathway and NADPH-producing enzymes when cells were grown on glucose or xylose. We also found that the transcriptional changes induced by pho13Δ required the transcription factor Stb5, which is activated specifically under NADPH-limiting conditions. Thus, pho13Δ resulted in the upregulation of the PP pathway and NADPH-producing enzymes as a part of an oxidative stress response mediated by activation of Stb5. Because the PP pathway is the primary pathway for xylose, its upregulation by pho13Δ might explain the improved xylose metabolism. These findings will be useful for understanding the biological function of S. cerevisiae Pho13 and the HAD superfamily enzymes and for developing S. cerevisiae strains with industrially attractive phenotypes., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Sustainable biofuel production from renewable biomass will require the efficient and complete use of all abundant sugars in the plant cell wall. Using the cellulolytic fungus Neurospora crassa as a model, we identified a xylodextrin transport and consumption pathway required for its growth on hemicellulose. Reconstitution of this xylodextrin utilization pathway in Saccharomyces cerevisiae revealed that fungal xylose reductases act as xylodextrin reductases, producing xylosyl-xylitol oligomers as metabolic intermediates. These xylosyl-xylitol intermediates are generated by diverse fungi and bacteria, indicating that xylodextrin reduction is widespread in nature. Xylodextrins and xylosyl-xylitol oligomers are then hydrolyzed by two hydrolases to generate intracellular xylose and xylitol. Xylodextrin consumption using a xylodextrin transporter, xylodextrin reductases and tandem intracellular hydrolases in cofermentations with sucrose and glucose greatly expands the capacity of yeast to use plant cell wall-derived sugars and has the potential to increase the efficiency of both first-generation and next-generation biofuel production.

Yeasts are efficient biofuel producers with numerous advantages outcompeting bacterial counterparts. While most synthetic biology tools have been developed and customized for bacteria especially for Escherichia coli, yeast synthetic biological tools have been exploited for improving yeast to produce fuels and chemicals from renewable biomass. Here we review the current status of synthetic biological tools and their applications for biofuel production, focusing on the model strain Saccharomyces cerevisiae We describe assembly techniques that have been developed for constructing genes, pathways, and genomes in yeast. Moreover, we discuss synthetic parts for allowing precise control of gene expression at both transcriptional and translational levels. Applications of these synthetic biological approaches have led to identification of effective gene targets that are responsible for desirable traits, such as cellulosic sugar utilization, advanced biofuel production, and enhanced tolerance against toxic products for biofuel production from renewable biomass. Although an array of synthetic biology tools and devices are available, we observed some gaps existing in tool development to achieve industrial utilization. Looking forward, future tool development should focus on industrial cultivation conditions utilizing industrial strains., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

In order to determine beneficial gene deletions for ethanol production by the yeast Saccharomyces cerevisiae, we performed an in silico gene deletion experiment based on a genome-scale metabolic model. Genes coding for two oxidative phosphorylation reactions (cytochrome c oxidase and ubiquinol cytochrome c reductase) were identified by the model-based simulation as potential deletion targets for enhancing ethanol production and maintaining acceptable overall growth rate in oxygen-limited conditions. Since the two target enzymes are composed of multiple subunits, we conducted a genetic screening study to evaluate the in silico results and compare the effect of deleting various portions of the respiratory enzyme complexes. Over two-thirds of the knockout mutants identified by the in silico study did exhibit experimental behavior in qualitative agreement with model predictions, but the exceptions illustrate the limitation of using a purely stoichiometric model-based approach. Furthermore, there was a substantial quantitative variation in phenotype among the various respiration-deficient mutants that were screened in this study, and three genes encoding respiratory enzyme subunits were identified as the best knockout targets for improving hexose fermentation in microaerobic conditions. Specifically, deletion of either COX9 or QCR9 resulted in higher ethanol production rates than the parental strain by 37% and 27%, respectively, with slight growth disadvantages. Also, deletion of QCR6 led to improved ethanol production rate by 24% with no growth disadvantage. The beneficial effects of these gene deletions were consistently demonstrated in different strain backgrounds and with four common hexoses. The combination of stoichiometric modeling and genetic screening using a systematic knockout collection was useful for narrowing a large set of gene targets and identifying targets of interest., (Copyright © 2014 Elsevier B.V. All rights reserved.)

2,3-Butanediol (2,3-BD) production from xylose that is abundant in lignocellulosic hydrolyzate would make the production of 2,3-BD more sustainable and economical. Saccharomyces cerevisiae can produce only trace amounts of 2,3-BD, but also cannot ferment xylose. Therefore, it is necessary to introduce both 2,3-BD production and xylose assimilation pathways into S. cerevisiae for producing 2,3-BD from xylose. A pyruvate decarboxylase (Pdc)-deficient mutant (SOS4) was used as a host in order to increase carbon flux toward 2,3-BD instead of ethanol. The XYL1, XYL2, and XYL3 genes coding for xylose assimilating enzymes derived from Scheffersomyces stipitis were introduced into the SOS4 strain to enable xylose utilization. Additionally, the alsS and alsD genes from Bacillus subtilis and endogenous BDH1 gene were overexpressed to increase 2,3-BD production from xylose. As a result, the resulting strain (BD4X) produced 20.7g/L of 2,3-BD from xylose with a yield of 0.27g 2,3-BD/g xylose. The titer of 2,3-BD from xylose increased up to 43.6g/L under a fed-batch fermentation. The BD4X strain produced (R, R)-2,3-BD dominantly (>97% of the total 2,3-BD) with trace amounts of meso-2,3-BD. These results suggest that S. cerevisiae might be a promising host for producing 2,3-BD from lignocellulosic biomass for industrial applications., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Industrial polyploid yeast strains harbor numerous beneficial traits but suffer from a lack of available auxotrophic markers for genetic manipulation. Here we demonstrated a quick and efficient strategy to generate auxotrophic markers in industrial polyploid yeast strains with the RNA-guided Cas9 nuclease. We successfully constructed a quadruple auxotrophic mutant of a popular industrial polyploid yeast strain, Saccharomyces cerevisiae ATCC 4124, with ura3, trp1, leu2, and his3 auxotrophies through RNA-guided Cas9 nuclease. Even though multiple alleles of auxotrophic marker genes had to be disrupted simultaneously, we observed knockouts in up to 60% of the positive colonies after targeted gene disruption. In addition, growth-based spotting assays and fermentation experiments showed that the auxotrophic mutants inherited the beneficial traits of the parental strain, such as tolerance of major fermentation inhibitors and high temperature. Moreover, the auxotrophic mutants could be transformed with plasmids containing selection marker genes. These results indicate that precise gene disruptions based on the RNA-guided Cas9 nuclease now enable metabolic engineering of polyploid S. cerevisiae strains that have been widely used in the wine, beer, and fermentation industries., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Production of renewable fuels and chemicals from cellulosic biomass is a critical step towards energy sustainability and reduced greenhouse gas emissions. Microbial cells have been engineered for producing chemicals from cellulosic sugars. Among these chemicals, 2,3-butanediol (2,3-BDO) is a compound of interest due to its diverse applications. While microbial production of 2,3-BDO with high yields and productivities has been reported, there are concerns associated with utilization of potential pathogenic bacteria and inefficient utilization of cellulosic sugars. To address these problems, we engineered 2,3-BDO production in Saccharomyces cerevisiae, especially from cellobiose, a prevalent sugar in cellulosic hydrolysates. Specifically, we overexpressed alsS and alsD from Bacillus subtilis to convert pyruvate into 2,3-BDO via α-acetolactate and acetoin in an engineered cellobiose fermenting S. cerevisiae. Under oxygen-limited conditions, the resulting strain was able to produce 2,3-BDO. Still, major carbon flux went to ethanol, resulting in substantial amounts of ethanol produced as a byproduct. To enhance pyruvate flux to 2,3-BDO through elimination of the pyruvate decarboxylation reaction, we employed a deletion mutant of both PDC1 and PDC5 for producing 2,3-BDO. When a cellobiose utilization pathway, consisting of a cellobiose transporter and intracellular β-glucosidase, and the 2,3-BDO producing pathway were introduced in a pyruvate decarboxylase deletion mutant, the resulting strain produced 2,3-BDO without ethanol production from cellobiose under oxygen-limited conditions. A titer of 5.29 g/l 2,3-BDO with a productivity of 0.22 g/l h and yield of 0.29 g 2,3-BDO/g cellobiose was attained. These results suggest the possibility of producing 2,3-BDO safely and sustainably from cellulosic hydrolysates.

Saccharomyces cerevisiae can be engineered to ferment cellodextrins produced by cellulases as a product of cellulose hydrolysis. Direct fermentation of cellodextrins instead of glucose is advantageous because glucose inhibits cellulase activity and represses the fermentation of non-glucose sugars present in cellulosic hydrolyzates. To facilitate cellodextrin utilization by S. cerevisiae, a fungal cellodextrin-utilizing pathway from Neurospora crassa consisting of a cellodextrin transporter and a cellodextrin hydrolase has been introduced into S. cerevisiae. Two cellodextrin transporters (CDT-1 and CDT-2) were previously identified in N. crassa, but their kinetic properties and efficiency for cellobiose fermentation have not been studied in detail. In this study, CDT-1 and CDT-2, which are hypothesized to transport cellodextrin with distinct mechanisms, were introduced into S. cerevisiae along with an intracellular β-glucosidase (GH1-1). Cellobiose transport assays with the resulting strains indicated that CDT-1 is a proton symporter while CDT-2 is a simple facilitator. A strain expressing CDT-1 and GH1-1 (DCDT-1G) showed faster cellobiose fermentation than the strain expressing CDT-2 and GH1-1 (DCDT-2G) under various culture conditions with different medium compositions and aeration levels. While CDT-2 is expected to have energetic benefits, the expression levels and kinetic properties of CDT-1 in S. cerevisiae appears to be optimum for cellobiose fermentation. These results suggest CDT-1 is a more effective cellobiose transporter than CDT-2 for engineering S. cerevisiae to ferment cellobiose.

Simultaneous fermentation of cellobiose and xylose by an engineered Saccharomyces cerevisiae has been demonstrated in batch fermentation, suggesting the feasibility of continuous co-fermentation of cellulosic sugars. As industrial S. cerevisiae strains have known to possess higher ethanol productivity and robustness compared to laboratory S. cerevisiae strains, xylose and cellobiose metabolic pathways were introduced into a haploid strain derived from an industrial S. cerevisiae. The resulting strain (JX123-BTT) was able to ferment a mixture of cellobiose and xylose simultaneously in batch fermentation with a high ethanol yield (0.38 g/g) and productivity (2.00 g/L · h). Additionally, the JX123-BTT strain co-consumed glucose, cellobiose, and xylose under continuous culture conditions at a dilution rate of 0.05 h(-1) and produced ethanol resulting in 0.38 g/g of ethanol yield and 0.96 g/L · h of productivity. This is the first demonstration of co-fermentation of cellobiose and xylose by an engineered S. cerevisiae under continuous culture conditions., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Functional expression of a cellodextrin transporter and an intracellular β-glucosidase from Neurospora crassa in Saccharomyces cerevisiae enables simultaneous co-fermentation of cellobiose and non-glucose sugars such as xylose. Here we investigate the functional role of aldose 1-epimerase (AEP) in engineered cellobiose utilization. One AEP (Gal10) and two putative AEPs (Yhr210c and Ynr071c sharing 50.6% and 51.0% amino acid identity with Gal10, respectively) were selected. Deletion of GAL10 led to complete loss of both AEP activity and cell growth on cellobiose, while GAL10 complementation restored the AEP activity and cell growth. In addition, deletion of YHR210C or YNR071C resulted in improved cellobiose utilization. These results suggest that the intracellular mutarotation of β-glucose to α-glucose might be a rate controlling step and Gal10 play a crucial role in cellobiose fermentation by engineered S. cerevisiae., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In order to produce 2,3-butanediol (2,3-BD) with a high titer, it is necessary to engineer Saccharomyces cerevisiae by deleting the competing pathway and overexpressing the 2,3-BD biosynthetic pathway. A pyruvate decarboxylase (Pdc)-deficient mutant was constructed and evolved for rapid glucose consumption without ethanol production. Genome re-sequencing of the evolved strain (SOS4) revealed a point mutation (A81P) in MTH1 coding for a transcriptional regulator involved in glucose sensing, unlike the previously reported Pdc-deficient mutant which had internal deletion in MTH1. When alsS and alsD genes from Bacillus subtilis, and endogenous BDH1 gene were overexpressed in SOS4, the resulting strain (BD4) not only produced 2,3-BD efficiently, but also consumed glucose faster than the parental strain. In fed-batch fermentation with optimum aeration, 2,3-BD concentration increased up to 96.2 g/L. These results suggest that S. cerevisiae might be a promising host for producing 2,3-BD for industrial applications., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Simultaneous saccharification and fermentation (SSF) has been considered a promising and economical process for cellulosic ethanol production. Further cost savings could be gained by reducing enzyme loading and engineering host strain for ethanol production. In this study, we demonstrate efficient ethanol production by SSF without supplementation of β-glucosidase using an engineered Saccharomyces cerevisiae strain expressing a cellodextrin transporter and an intracellular β-glucosidase from Neurospora crassa. Ethanol production profiles by the engineered yeast without supplementation of β-glucosidase and by a parental strain with supplementation of β-glucosidase were examined under various fermentation conditions. When initial cell mass concentrations were low, the traditional SSF with supplementation of β-glucosidase showed better ethanol production than SSF with the engineered strain without supplementing β-glucosidase. However, the engineered strain without supplementation of β-glucosidase showed almost the same or even better ethanol productivity than the parental strain with supplementation of β-glucosidase when initial cell mass concentrations were elevated. Our results suggest that efficient ethanol production by SSF could be achieved by engineered yeast capable of fermenting cellobiose without addition of extracellular β-glucosidase, leading to economic production of cellulosic ethanol., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Protein expression patterns of an erythritol-producing yeast, Candida magnoliae, were analyzed to identify differentially expressed proteins in response to glucose perturbation. Specifically, wild type C. magnoliae was grown under high and low glucose conditions and the cells were harvested at both mid-exponential and erythritol production phases for proteomic studies. In order to analyze intracellular protein abundances from the harvested cells quantitatively, total intracellular proteins were extracted and applied to two-dimensional gel electrophoresis for separation and visualization of individual proteins. Among the proteins distributed in the range of pI 4-7 and molecular weight 29-97kDa, five osmo-responsive proteins were drastically changed in response to glucose perturbation. Hsp60 (Heat-shock protein 60), transaldolase and NADH:quinone oxidoreductase were down-regulated under the high glucose condition and Bro1 (BCK1-like Resistance to Osmotic shock) and Eno1 (enolase1) were up-regulated. These proteins are directly or indirectly related with cellular stress response. Importantly, protein expression patterns of Hsp60, Bro1 and Eno1 were strongly correlated with previous studies identifying the proteins perturbed by osmotic stress for other organisms including Saccharomyces cerevisiae., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Efficient regeneration of NADPH is one of the limiting factors that constrain the productivity of biotransformation processes. In order to increase the availability of NADPH for enhanced biotransformation by engineered Escherichia coli, modulation of the pentose phosphate pathway and amplification of the transhydrogenases system have been conventionally attempted as primary solutions. Recently, other approaches for stimulating NADPH regeneration during glycolysis, such as replacement of native glyceradehdye-3-phosphate dehydrogenase (GAPDH) with NADP-dependent GAPDH from Clostridium acetobutylicum and introduction of NADH kinase catalyzing direct phosphorylation of NADH to NADPH from Saccharomyces cerevisiae, were attempted and resulted in remarkable impacts on NADPH-dependent bioprocesses. This review summarizes several metabolic engineering approaches used for improving the NADPH regenerating capacity in engineered E. coli for whole-cell-based bioprocesses and discusses the key features and progress of those attempts.

Saccharomyces cerevisiae can be engineered for xylose fermentation through introduction of wild type or mutant genes (XYL1/XYL1 (R276H), XYL2, and XYL3) coding for xylose metabolic enzymes from Scheffersomyces stipitis. The resulting engineered strains, however, often yielded undesirable phenotypes such as slow xylose assimilation and xylitol accumulation. In this study, we performed the mating of two engineered strains that exhibit suboptimal xylose-fermenting phenotypes in order to develop an improved xylose-fermenting diploid strain. Specifically, we obtained two engineered haploid strains (YSX3 and SX3). The YSX3 strain consumed xylose rapidly and produced a lot of xylitol. On the contrary, the SX3 strain consumed xylose slowly with little xylitol production. After converting the mating type of SX3 from alpha to a, the resulting strain (SX3-2) was mated with YSX3 to construct a heterozygous diploid strain (KSM). The KSM strain assimilated xylose (0.25gxyloseh(-1)gcells(-1)) as fast as YSX3 and accumulated a small amount of xylitol (0.03ggxylose(-1)) as low as SX3, resulting in an improved ethanol yield (0.27ggxylose(-1)). We found that the improvement in xylose fermentation by the KSM strain was not because of heterozygosity or genome duplication but because of the complementation of the two xylose-metabolic pathways. This result suggested that mating of suboptimal haploid strains is a promising strategy to develop engineered yeast strains with improved xylose fermenting capability., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Kluyveromyces marxianus is now considered one of the best choices of option for industrial applications of yeast because the strain is able to grow at high temperature, utilizes various carbon sources, and grows fast. However, the use of K. marxianus as a host for industrial applications is still limited. This limitation is largely due to a lack of knowledge on the characteristics of the promoters since the time and amount of protein expression is strongly dependent on the promoter employed. In this study, four well-known constitutive promoters (P(CYC), P(TEF), P(GPD), and P(ADH)) of Saccharomyces cerevisiae were characterized in K. marxianus in terms of protein expression level and their stochastic behavior. After constructing five URA3-auxotrophic K. marxianus strains and a plasmid vector, four cassettes each comprising one of the promoters--the gene for the green fluorescence protein (GFP)--CYC1 terminator (T(CYC)) were inserted into the vector. GFP expression under the control of each one of the promoters was analyzed by reverse transcription PCR, fluorescence microscopy, and flow cytometer. Using these combined methods, the promoter strength was determined to be in the order of P(GPD) > P(ADH) ∼ P(TEF) >> P(CYC). All promoters except for the P(CYC) exhibited three distinctive populations, including non-expressing cells, weakly expressing cells, and strongly expressing cells. The relative ratios between populations were strongly dependent on the promoter and culture time. Forward scattering was independent of GFP fluorescence intensity, indicating that the different fluorescence intensities were not just due to different cell sizes derived from budding. It also excluded the possibility that the non-expressing cells resulted from plasmid loss because plasmid stability was maintained at almost 100 % over the culture time. The same cassettes, cloned into a single copy plasmid pRS416 and transformed into S. cerevisiae, showed only one population. When the cassettes were integrated into the chromosome, the stochastic behavior was markedly reduced. These combined results imply that the gene expression stochasticity should be overcome in order to use this strain for delicate metabolic engineering, which would require the co-expression of several genes.

As world energy demand continues to rise and fossil fuel resources are depleted, marine macroalgae (i.e., seaweed) is receiving increasing attention as an attractive renewable source for producing fuels and chemicals. Marine plant biomass has many advantages over terrestrial plant biomass as a feedstock. Recent breakthroughs in converting diverse carbohydrates from seaweed biomass into liquid biofuels (e.g., bioethanol) through metabolic engineering have demonstrated potential for seaweed biomass as a promising, although relatively unexplored, source for biofuels. This review focuses on up-to-date progress in fermentation of sugars from seaweed biomass using either natural or engineered microbial cells, and also provides a comprehensive overview of seaweed properties, cultivation and harvesting methods, and major steps in the bioconversion of seaweed biomass to biofuels., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The focus of this work was to develop a combined acid and alkaline hydrothermal pretreatment of lignocellulose that ensures high recovery of both hexose and pentose. Dilute sulfuric acid and lime pretreatments were employed sequentially. Process performance was optimized in terms of catalyst concentration, retention time, and temperature using response surface methodology. Medium operational conditions in the acid stage and harsh conditions in the alkaline stage were desirable with optimal performance at 0.73 wt% H(2)SO(4), 150 °C, 6.1 min in the first stage, and 0.024 g lime/g biomass, 202 °C, 30 min in the second stage. In comparison to single-stage pretreatments with high recovery of either glucose or xylose, two-stage process showed great promises with >80 % glucose and >70 % xylose recovery. In addition, the method greatly improved ethanol fermentation with yields up to 0.145 g/g Miscanthus, due to significantly reduced formation of inhibitory by-products such as weak acids, furans, and phenols. Supplementing biomimetic acids would further increase glucose yield by up to 15 % and xylose yield by 25 %.

Anaerobic bacteria assimilate cellodextrins from plant biomass by using a phosphorolytic pathway to generate glucose intermediates for growth. The yeast Saccharomyces cerevisiae can also be engineered to ferment cellobiose to ethanol using a cellodextrin transporter and a phosphorolytic pathway. However, strains with an intracellular cellobiose phosphorylase initially fermented cellobiose slowly relative to a strain employing an intracellular β-glucosidase. Fermentations by the phosphorolytic strains were greatly improved by using cellodextrin transporters with elevated rates of cellobiose transport. Furthermore under stress conditions, these phosphorolytic strains had higher biomass and ethanol yields compared to hydrolytic strains. These observations suggest that, although cellobiose phosphorolysis has energetic advantages, phosphorolytic strains are limited by the thermodynamics of cellobiose phosphorolysis (ΔG°=+3.6kJmol(-1)). A thermodynamic "push" from the reaction immediately upstream (transport) is therefore likely to be necessary to achieve high fermentation rates and energetic benefits of phosphorolysis pathways in engineered S. cerevisiae., (Copyright © 2012 Elsevier Inc. All rights reserved.)

As Saccharomyces cerevisiae cannot utilize xylose as a carbon source, expression of XYL1 coding for xylose reductase (XR) from Scheffersomyces (Pichia) stipitis enabled production of xylitol from xylose with a high yield. However, insufficient supply of NAD(P)H for XR and inhibition of xylose uptake by glucose are identified as major constraints for achieving high xylitol productivity. To overcome these problems, we engineered S. cerevisiae capable of converting xylose into xylitol through simultaneous utilization of xylose and cellobiose. An engineered S. cerevisiae (D-10-BT) expressing XR, cellodextrin transporter (cdt-1) and intracellular β-glucosidase (gh1-1) produced xylitol via simultaneous utilization of cellobiose and xylose. The D-10-BT strain exhibited 40% higher volumetric xylitol productivity with co-consumption of cellobiose and xylose compared to sequential utilization of glucose and xylose. Furthermore, the overexpression of S. cerevisiae ALD6, IDP2, or S. stipitis ZWF1 coding for cytosolic NADP(+)-dependent dehydrogenases increased the intracellular NADPH availability of the D-10-BT strain, which resulted in a 37-63% improvement in xylitol productivity when cellobiose and xylose were co-consumed. These results suggest that co-utilization of cellobiose and xylose can lead to improved xylitol production through enhanced xylose uptake and efficient cofactor regeneration., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Engineering of Saccharomyces cerevisiae to produce advanced biofuels such as isobutanol has received much attention because this yeast has a natural capacity to produce higher alcohols. In this study, construction of isobutanol production systems was attempted by overexpression of effective 2-keto acid decarboxylase (KDC) and combinatorial overexpression of valine biosynthetic enzymes in S. cerevisiae D452-2. Among the six putative KDC enzymes from various microorganisms, 2-ketoisovalerate decarboxylase (Kivd) from L. lactis subsp. lactis KACC 13877 was identified as the most suitable KDC for isobutanol production in the yeast. Isobutanol production by the engineered S. cerevisiae was assessed in micro-aerobic batch fermentations using glucose as a sole carbon source. 93 mg/L isobutanol was produced in the Kivd overexpressing strain, which corresponds to a fourfold improvement as compared with the control strain. Isobutanol production was further enhanced to 151 mg/L by additional overexpression of acetolactate synthase (Ilv2p), acetohydroxyacid reductoisomerase (Ilv5p), and dihydroxyacid dehydratase (Ilv3p) in the cytosol.

The traditional ethanologenic yeast Saccharomyces cerevisiae cannot metabolize xylose, which is an abundant sugar in non-crop plants. Engineering this yeast for a practicable fermentation of xylose will therefore improve the economics of bioconversion for the production of fuels and chemicals such as ethanol. One of the most widely employed strategies is to express XYL1, XYL2, and XYL3 genes derived from Scheffersomyces stipitis (formerly Pichia stiptis) in S. cerevisiae. However, the resulting engineered strains have been reported to exhibit large variations in xylitol accumulation and ethanol yields, generating many hypotheses and arguments for elucidating these phenomena. Here we demonstrate that low expression levels of the XYL2 gene, coding for xylitol dehydrogenase (XDH), is a major bottleneck in efficient xylose fermentation. Through an inverse metabolic engineering approach using a genomic library of S. cerevisiae, XYL2 was identified as an overexpression target for improving xylose metabolism. Specifically, we performed serial subculture experiments after transforming a genomic library of wild type S. cerevisiae into an engineered strain harboring integrated copies of XYL1, XYL2 and XYL3. Interestingly, the isolated plasmids from efficient xylose-fermenting transformants contained XYL2. This suggests that the integrated XYL2 migrated into a multi-copy plasmid through homologous recombination. It was also found that additional overexpression of XYL2 under the control of strong constitutive promoters in a xylose-fermenting strain not only reduced xylitol accumulation, but also increased ethanol yields. As the expression levels of XYL2 increased, the ethanol yields gradually improved from 0.1 to 0.3g ethanol/g xylose, while the xylitol yields significantly decreased from 0.4 to 0.1g xylitol/g xylose. These results suggest that strong expression of XYL2 is a necessary condition for developing efficient xylose-fermenting strains., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The lack of microbial strains capable of fermenting all sugars prevalent in plant cell wall hydrolyzates to ethanol is a major challenge. Although naturally existing or engineered microorganisms can ferment mixed sugars (glucose, xylose and galactose) in these hydrolyzates sequentially, the preferential utilization of glucose to non-glucose sugars often results in lower overall yield and productivity of ethanol. Therefore, numerous metabolic engineering approaches have been attempted to construct optimal microorganisms capable of co-fermenting mixed sugars simultaneously. Here, we present recent findings and breakthroughs in engineering yeast for improved ethanol production from mixed sugars. In particular, this review discusses new sugar transporters, various strategies for simultaneous co-fermentation of mixed sugars, and potential applications of co-fermentation for producing fuels and chemicals., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: 2'-Fucosyllactose (2-FL) is a functional oligosaccharide present in human milk which protects against the infection of enteric pathogens. Because 2-FL can be synthesized through the enzymatic fucosylation of lactose with guanosine 5'-diphosphate (GDP)-l-fucose by α-1,2-fucosyltransferase (FucT2), an 2-FL producing Escherichia coli can be constructed through overexpressing genes coding for endogenous GDP- l-fucose biosynthetic enzymes and heterologous fucosyltransferase., Results: The gene for FucT2 from Helicobacter pylori was introduced to the GDP-l-fucose producing recombinant E. coli BL21 star(DE3) strain. However, only small amount of 2-FL was produced in a batch fermentation because the E. coli BL21star(DE3) strain assimilated lactose instead of converting to 2-FL. As an alternative host, the E. coli JM109(DE3) strain which is incapable of assimilating lactose was chosen as a 2-FL producer. Whole cell biosynthesis of 2-FL from lactose was investigated in a series of batch fermentations using various concentrations of lactose. The results of batch fermentations showed that lactose was slowly assimilated by the engineered E. coli JM109(DE3) strain and 2-FL was synthesized without supplementation of another auxiliary sugar for cell growth. A maximum 2-FL concentration of 1.23 g/l was obtained from a batch fermentation with 14.5 g/l lactose. The experimentally obtained yield (g 2-FL/g lactose) corresponded to 20% of the theoretical maximum yield estimated by the elementary flux mode (EFM) analysis., Conclusions: The experimental 2-FL yield in this study corresponded to about 20% of the theoretical maximum yield, which suggests further modifications via metabolic engineering of a host strain or optimization of fermentation processes might be carried out for improving 2-FL yield. Improvement of microbial production of 2-FL from lactose by engineered E. coli would increase the feasibility of utilizing 2-FL as a prebiotic in various foods.

Combined acid catalysis was employed as a pretreatment alternative with combined acid catalysts blending sulfuric acid with two biomimetic acids, trifluoroacetic acid (TFA) and maleic acid (MA), respectively. The influences of acid blending ratio, temperature, and acid dosage on pretreatment performance were investigated. A synergistic effect on hemicellulose decomposition was observed in the combined acid hydrolysis, which greatly increased xylose yield, although TFA/MA would induce more total phenols. Besides, combined TFA pretreatment could efficiently prevent xylose degradation. Fermentation tests of the acid-catalyzed hydrolysates with overliming showed that compared to H(2)SO(4) pretreatment, TFA and MA pretreatments improved overall ethanol yield with an increase by 27-54%. Combined acid catalysis was shown as a feasible pretreatment method for its improved sugar yield, reduced phenols production and catalyst costs., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Efficient fermentation of xylose, which is abundant in hydrolysates of lignocellulosic biomass, is essential for producing cellulosic biofuels economically. While heterologous expression of xylose isomerase in Saccharomyces cerevisiae has been proposed as a strategy to engineer this yeast for xylose fermentation, only a few xylose isomerase genes from fungi and bacteria have been functionally expressed in S. cerevisiae. We cloned two bacterial xylose isomerase genes from anaerobic bacteria (Bacteroides stercoris HJ-15 and Bifidobacterium longum MG1) and introduced them into S. cerevisiae. While the transformant with xylA from B. longum could not assimilate xylose, the transformant with xylA from B. stercoris was able to grow on xylose. This result suggests that the xylose isomerase (BsXI) from B. stercoris is functionally expressed in S. cerevisiae. The engineered S. cerevisiae strain with BsXI consumed xylose and produced ethanol with a good yield (0.31 g/g) under anaerobic conditions. Interestingly, significant amounts of xylitol (0.23 g xylitol/g xylose) were still accumulated during xylose fermentation even though the introduced BsXI might not cause redox imbalance. We investigated the potential inhibitory effects of the accumulated xylitol on xylose fermentation. Although xylitol inhibited in vitro BsXI activity significantly (K(I) = 5.1 ± 1.15 mM), only small decreases (less than 10%) in xylose consumption and ethanol production rates were observed when xylitol was added into the fermentation medium. These results suggest that xylitol accumulation does not inhibit xylose fermentation by engineered S. cerevisiae expressing xylA as severely as it inhibits the xylose isomerase reaction in vitro.

In a phenomenon called persistence, small numbers of bacterial cells survive even after exposure to antibiotics. Recently, bactericidal antibiotics have been demonstrated to kill bacteria by increasing the levels of hydroxyl radicals inside cells. In the present study, we report a direct correlation between intracellular hydroxyl radical formation and bacterial persistence. By conducting flow cytometric analysis in a three-dimensional space, we resolved distinct bacterial populations in terms of intracellular hydroxyl radical levels, morphology and viability. We determined that, upon antibiotic treatment, a small sub-population of Escherichia coli survivors do not overproduce hydroxyl radicals and maintain normal morphology, whereas most bacterial cells were killed by accumulating hydroxyl radicals and displayed filamentous morphology. Our results suggest that bacterial persisters can be formed once they have transient defects in mediating reactions involved in the hydroxyl radical formation pathway. Thus, it is highly probable that persisters do not share a common mechanism but each persister cell respond to antibiotics in different ways, while they all commonly show lowered hydroxyl radical formation and enhanced tolerance to antibiotics., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Although Saccharomyces cerevisiae is capable of fermenting galactose into ethanol, ethanol yield and productivity from galactose are significantly lower than those from glucose. An inverse metabolic engineering approach was undertaken to improve ethanol yield and productivity from galactose in S. cerevisiae. A genome-wide perturbation library was introduced into S. cerevisiae, and then fast galactose-fermenting transformants were screened using three different enrichment methods. The characterization of genetic perturbations in the isolated transformants revealed three target genes whose overexpression elicited enhanced galactose utilization. One confirmatory (SEC53 coding for phosphomannomutase) and two novel targets (SNR84 coding for a small nuclear RNA and a truncated form of TUP1 coding for a general repressor of transcription) were identified as overexpression targets that potentially improve galactose fermentation. Beneficial effects of overexpression of SEC53 may be similar to the mechanisms exerted by overexpression of PGM2 coding for phosphoglucomutase. While the mechanism is largely unknown, overexpression of SNR84, improved both growth and ethanol production from galactose. The most remarkable improvement of galactose fermentation was achieved by overexpression of the truncated TUP1 (tTUP1) gene, resulting in unrivalled galactose fermentation capability, that is 250% higher in both galactose consumption rate and ethanol productivity compared to the control strain. Moreover, the overexpression of tTUP1 significantly shortened lag periods that occurs when substrate is changed from glucose to galactose. Based on these results we proposed a hypothesis that the mutant Tup1 without C-terminal repression domain might bring in earlier and higher expression of GAL genes through partial alleviation of glucose repression. mRNA levels of GAL genes (GAL1, GAL4, and GAL80) indeed increased upon overexpression of tTUP. The results presented in this study illustrate that alteration of global regulatory networks through overexpression of the identified targets (SNR84 and tTUP1) is as effective as overexpression of a rate limiting metabolic gene (PGM2) in the galactose assimilation pathway for efficient galactose fermentation in S. cerevisiae. In addition, these results will be industrially useful in the biofuels area as galactose is one of the abundant sugars in marine plant biomass such as red seaweed as well as cheese whey and molasses., (Copyright © 2010 Wiley Periodicals, Inc.)

The use of plant biomass for biofuel production will require efficient utilization of the sugars in lignocellulose, primarily glucose and xylose. However, strains of Saccharomyces cerevisiae presently used in bioethanol production ferment glucose but not xylose. Yeasts engineered to ferment xylose do so slowly, and cannot utilize xylose until glucose is completely consumed. To overcome these bottlenecks, we engineered yeasts to coferment mixtures of xylose and cellobiose. In these yeast strains, hydrolysis of cellobiose takes place inside yeast cells through the action of an intracellular β-glucosidase following import by a high-affinity cellodextrin transporter. Intracellular hydrolysis of cellobiose minimizes glucose repression of xylose fermentation allowing coconsumption of cellobiose and xylose. The resulting yeast strains, cofermented cellobiose and xylose simultaneously and exhibited improved ethanol yield when compared to fermentation with either cellobiose or xylose as sole carbon sources. We also observed improved yields and productivities from cofermentation experiments performed with simulated cellulosic hydrolyzates, suggesting this is a promising cofermentation strategy for cellulosic biofuel production. The successful integration of cellobiose and xylose fermentation pathways in yeast is a critical step towards enabling economic biofuel production.

Xylose-fermenting Saccharomyces strains are needed for commercialization of ethanol production from lignocellulosic biomass. Engineered Saccharomyces cerevisiae strains expressing XYL1, XYL2 and XYL3 from Pichia stipitis, however, utilize xylose in an oxidative manner, which results in significantly lower ethanol yields from xylose as compared to glucose. As such, we hypothesized that reconfiguration of xylose metabolism from oxidative into fermentative manner might lead to efficient ethanol production from xylose. To this end, we generated a respiration-deficient (RD) mutant in order to enforce engineered S. cerevisiae to utilize xylose only through fermentative metabolic routes. Three different repeated-batch fermentations were performed to characterize characteristics of the respiration-deficient mutant. When fermenting glucose as a sole carbon source, the RD mutant exhibited near theoretical ethanol yields (0.46 g g(-1)) during repeated-batch fermentations by recycling the cells. As the repeated-batch fermentation progressed, the volumetric ethanol productivity increased (from 7.5 to 8.3 g L(-1)h(-1)) because of the increased biomass from previous cultures. On the contrary, the mutant showed decreasing volumetric ethanol productivities during the repeated-batch fermentations using xylose as sole carbon source (from 0.4 to 0.3 g L(-1)h(-1)). The mutant did not grow on xylose and lost fermenting ability gradually, indicating that the RD mutant cannot maintain a good fermenting ability on xylose as a sole carbon source. However, the RD mutant was capable of fermenting a mixture of glucose and xylose with stable yields (0.35 g g(-1)) and productivities (0.52 g L(-1)h(-1)) during the repeated-batch fermentation. In addition, ethanol yields from xylose during the mixed sugar fermentation (0.30 g g(-1)) were higher than ethanol yields from xylose as a sole carbon source (0.21 g g(-1)). These results suggest that a strategy for increasing ethanol yield through respiration-deficiency can be applied for the fermentation of lignocellulosic hydrolyzates containing glucose and xylose., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The economic production of biofuels from renewable biomass using Saccharomyces cerevisiae requires tolerance to high concentrations of sugar and alcohol. Here we applied an inverse metabolic engineering approach to identify endogenous gene targets conferring improved alcohol tolerance in S. cerevisiae. After transformation with a S. cerevisiae genomic library, enrichment of the transformants exhibiting improved tolerance was performed by serial subculture in the presence of iso-butanol (1%). Through sequence analysis of the isolated plasmids from the selected transformants, four endogenous S. cerevisiae genes were identified as overexpression targets eliciting improved tolerance to both iso-butanol and ethanol. Overexpression of INO1, DOG1, HAL1 or a truncated form of MSN2 resulted in remarkably increased tolerance to high concentrations of iso-butanol and ethanol. Overexpression of INO1 elicited the highest ethanol tolerance, resulting in higher titers and volumetric productivities in the fermentation experiments performed with high glucose concentrations. In addition, the INO1-overexpressing strain showed a threefold increase in the specific growth rate as compared to that of the control strain under conditions of high levels of glucose (10%) and ethanol (5%). Although alcohol tolerance in yeast is a complex trait affected by simultaneous interactions of many genes, our results using a genomic library reveal potential target genes for better understanding and possible engineering of metabolic pathways underlying alcohol tolerance phenotypes., (Crown Copyright 2010. Published by Elsevier B.V. All rights reserved.)

Inverse metabolic engineering (IME) is a combinatorial approach for identifying genotypes associated with a particular phenotype of interest. In this study, gene disruptions that increase the biosynthesis of poly-3-hydroxybutyrate (PHB) in the photosynthetic bacterium Synechocystis PCC6803 were identified. A Synechocystis mutant library was constructed by homologous recombination between the Synechocystis genome and a mutagenized genomic plasmid library generated through transposon insertion. Using a fluorescence-activated cell sorting-based high throughput screen, high PHB accumulating mutants from the library grown in different nutrient conditions were isolated and characterized. While several mutants isolated from the screen had increased PHB accumulation, transposon insertions in only two ORFs could be linked to increased PHB production. Disruptions of sll0461, coding for gamma-glutamyl phosphate reductase (proA), and sll0565, a hypothetical protein, resulted in increased accumulation in standard growth media and acetate supplemented media. These genetic perturbations have increased PHB accumulation in Synechocystis and serve as markers for engineering increased polymer production in higher photosynthetic organisms., (2009 American Institute of Chemical Engineers Biotechnol.)

We examined butanol fermentation by Clostridium beijerinckii NCIMB 8052 using various hydrolyzates obtained from rice bran, which is one of the most abundant agricultural by-products in Korea and Japan. In order to increase the amount of fermentable sugars in the hydrolyzates of rice bran, various hydrolysis procedures were applied. Eight different hydrolyzates were prepared using rice bran (RB) and defatted rice bran (DRB) with enzyme or acid treatment or both. Each hydrolyzate was evaluated in terms of total sugar concentration and butanol production after fermentation by C. beijerinckii NCIMB 8052. Acid treatment yielded more sugar than enzyme treatment, and combined treatment with enzyme and acid yielded even more sugars as compared with single treatment with enzyme or acid. As a result, the highest sugar concentration (33 g/l) was observed from the hydrolyzate from DRB (100 g/l) with combined treatment using enzyme and acid. Prior to fermentation of the hydrolyzates, we examined the effect of P2 solution containing yeast extract, buffer, minerals, and vitamins on production of butanol during the fermentation. Fermentation of the hydrolyzates with or without addition of P2 was performed using C. beijerinckii NCIMB 8052 in a 1-l anaerobic bioreactor. Although the RB hydrolyzates were able to support growth and butanol production, addition of P2 solution into the hydrolyzates significantly improved cell growth and butanol production. The highest butanol production (12.24 g/l) was observed from the hydrolyzate of DRB with acid and enzyme treatment after supplementation of P2 solution.

Soluble N-ethylmaleimide sensitive-factor attachment receptor (SNARE) proteins have crucial roles in driving exocytic membrane fusion. Molecular recognition between vesicle-associated (v)-SNARE and target membrane (t)-SNARE leads to the formation of a four-helix bundle, which facilitates the merging of two apposing membranes. Synthetic peptides patterned after the SNARE motifs are predicted to block SNARE complex formation by competing with the parental SNAREs, inhibiting neuronal exocytosis. As an initial attempt to identify the peptide sequences that block SNARE assembly and membrane fusion, we created thirteen 17-residue synthetic peptides derived from the SNARE motifs of v- and t-SNAREs. The effects of these peptides on SNARE-mediated membrane fusion were investigated using an in vitro lipid-mixing assay, in vivo neurotransmitter release and SNARE complex formation assays in PC12 cells. Peptides derived from the N-terminal region of SNARE motifs had significant inhibitory effects on neuroexocytosis, whereas middle- and C-terminal-mimicking peptides did not exhibit much inhibitory function. N-terminal mimicking peptides blocked N-terminal zippering of SNAREs, a rate-limiting step in SNARE-driven membrane fusion. Therefore, the results suggest that the N-terminal regions of SNARE motifs are excellent targets for the development of drugs to block SNARE-mediated membrane fusion and neurotransmitter release.

Identification of multiple gene targets that exhibit different modes of action toward a desired phenotype is a crucial step in strain improvement. Target identification methods based on traceable genetic perturbations and stoichiometric modeling have been employed before for the mining of putative overexpression and knock-out targets. Most search methods are sequential and, as such, quite limited in the space they can explore. In this study, we investigate a multi-dimensional search approach whereby unknown interactions of gene targets identified by different search methods are assessed by employing orthogonal search strategies. To this end, we combined knock-out and overexpression gene targets, identified through systematic and combinatorial approaches, respectively, in order to improve lycopene production in Escherichia coli. Specifically, we first identified multiple overexpression targets by screening genomic libraries of E. coli in a sequential-iterative manner. Targets so identified confirmed previously amplified genes in the non-mevalonate pathway (dxs and idi) and some regulatory genes (rpoS and appY). Additionally, this method revealed novel gene targets (yjiD, ycgW, yhbL, purDH, and yggT). A two-dimensional search was subsequently undertaken, whereby the selected overexpression targets were combined with the knock-out targets predicted by stoichiometric modeling. All combinations of single (rpoS, appY, yjiD, ycgW, and yhbL), double (yjiD-ycgW) and triple (yjiD-ycgW-yhbL) overexpressions with four gene deletion backgrounds, including single (delta gdhA, or delta aceE), double (delta gdhA delta aceE), and triple (delta gdhA delta aceE delta fdhF) knockouts, were constructed and evaluated for lycopene production. Investigation of the metabolic landscape spanned by these 40 strains identified the best-engineered strain (T5(P)-dxs, T5(P)-idi, rrnB(P)-yjiD-ycgW, delta gdh delta aceE delta fdhF, pACLYC), which accumulated 16,000 ppm (16 mg/g cell) of lycopene within 24 h in a batch shake flask with 5 g/L of glucose in M9 minimal medium.

Xylitol production by Pichia stipitis FPL-YS30, a xyl3-delta1 mutant that metabolizes xylose using an alternative metabolic pathway, was investigated under aerobic and oxygen-limited culture conditions. Under both culture conditions, FPL-YS30 (xyl3-delta1) produced a negligible amount of ethanol and converted xylose mainly into xylitol with comparable yields (0.30 and 0.27 g xylitol/g xylose). However, xylose consumption increased five-fold under aerobic compared to oxygen-limited conditions. This suggests that the efficiency of the alternative route of xylose assimilation is affected by respiration. As a result, the FPL-YS30 strain produced 26 g/l of xylitol, and exhibited a higher volumetric productivity (0.22 g xylitol l(-1) h(-1)) under aerobic conditions.

The identification of genetic targets that are effective in bringing about a desired phenotype change is still an open problem. While random gene knockouts have yielded improved strains in certain cases, it is also important to seek the guidance of cell-wide stoichiometric constraints in identifying promising gene knockout targets. To investigate these issues, we undertook a genome-wide stoichiometric flux balance analysis as an aid in discovering putative genes impacting network properties and cellular phenotype. Specifically, we calculated metabolic fluxes such as to optimize growth and then scanned the genome for single and multiple gene knockouts that yield improved product yield while maintaining acceptable overall growth rate. For the particular case of lycopene biosynthesis in Escherichia coli, we identified such targets that we subsequently tested experimentally by constructing the corresponding single, double and triple gene knockouts. While such strains are suggested (by the stoichiometric calculations) to increase precursor availability, this beneficial effect may be further impacted by kinetic and regulatory effects not captured by the stoichiometric model. For the case of lycopene biosynthesis, the so identified knockout targets yielded a triple knockout construct that exhibited a nearly 40% increase over an engineered, high producing parental strain.

Metabolic pathway engineering is constrained by the thermodynamic and stoichiometric feasibility of enzymatic activities of introduced genes. Engineering of xylose metabolism in Saccharomyces cerevisiae has focused on introducing genes for the initial xylose assimilation steps from Pichia stipitis, a xylose-fermenting yeast, into S. cerevisiae, a yeast traditionally used in ethanol production from hexose. However, recombinant S. cerevisiae created in several laboratories have used xylose oxidatively rather than in the fermentative manner that this yeast metabolizes glucose. To understand the differences between glucose and engineered xylose metabolic networks, we performed a flux balance analysis (FBA) and calculated extreme pathways using a stoichiometric model that describes the biochemistry of yeast cell growth. FBA predicted that the ethanol yield from xylose exhibits a maximum under oxygen-limited conditions, and a fermentation experiment confirmed this finding. Fermentation results were largely consistent with in silico phenotypes based on calculated extreme pathways, which displayed several phases of metabolic phenotype with respect to oxygen availability from anaerobic to aerobic conditions. However, in contrast to the model prediction, xylitol production continued even after the optimum aeration level for ethanol production was attained. These results suggest that oxygen (or some other electron accepting system) is required to resolve the redox imbalance caused by cofactor difference between xylose reductase and xylitol dehydrogenase, and that other factors limit glycolytic flux when xylose is the sole carbon source.

D-Xylulokinase (XK) is essential for the metabolism of D-xylose in yeasts. However, overexpression of genes for XK, such as the Pichia stipitis XYL3 gene and the Saccharomyces cerevisiae XKS gene, can inhibit growth of S. cerevisiae on xylose. We varied the copy number and promoter strength of XYL3 or XKS1 to see how XK activity can affect xylose metabolism in S. cerevisiae. The S. cerevisiae genetic background included single integrated copies of P. stipitis XYL1 and XYL2 driven by the S. cerevisiae TDH1 promoter. Multicopy and single-copy constructs with either XYL3 or XKS1, likewise under control of the TDH1 promoter, or with the native P. stipitis promoter were introduced into the recombinant S. cerevisiae. In vitro enzymatic activity of XK increased with copy number and promoter strength. Overexpression of XYL3 and XKS1 inhibited growth on xylose but did not affect growth on glucose even though XK activities were three times higher in glucose-grown cells. Growth inhibition increased and ethanol yields from xylose decreased with increasing XK activity. Uncontrolled XK expression in recombinant S. cerevisiae is inhibitory in a manner analogous to the substrate-accelerated cell death observed with an S. cerevisiae tps1 mutant during glucose metabolism. To bypass this effect, we transformed cells with a tunable expression vector containing XYL3 under the control of its native promoter into the FPL-YS1020 strain and screened the transformants for growth on, and ethanol production from, xylose. The selected transformant had approximately four copies of XYL3 per haploid genome and had moderate XK activity. It converted xylose into ethanol efficiently.

XYL3, which encodes a D-xylulokinase (EC 2.7.1.17), was isolated from Pichia stipitis CBS 6054 genomic DNA by using primers designed against conserved motifs. Disruption of XYL3 eliminated D-xylulokinase activity, but D-ribulokinase activity was still present. Southern analysis of P. stipitis genomic DNA with XYL3 as a probe confirmed the disruption and did not reveal additional related genes. Disruption of XYL3 stopped ethanol production from xylose, but the resulting mutant still assimilated xylose slowly and formed xylitol and arabinitol. These results indicate that XYL3 is critical for ethanol production from xylose but that P. stipitis has another pathway for xylose assimilation. Expression of XYL3 using its P. stipitis promoter increased Saccharomyces cerevisiae D-xylulose consumption threefold and enabled the transformants to produce ethanol from a mixture of xylose and xylulose, whereas the parental strain only accumulated xylitol. In vitro, D-xylulokinase activity in recombinant S. cerevisiae was sixfold higher with a multicopy than with a single-copy XYL3 plasmid, but ethanol production decreased with increased copy number. These results confirmed the function of XYL3 in S. cerevisiae.