Publications by authors named "Christopher V Rao"

85 Publications

Metabolic engineering of non-pathogenic microorganisms for 2,3-butanediol production.

Appl Microbiol Biotechnol 2021 Jul 21. Epub 2021 Jul 21.

Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA.

2,3-Butanediol (2,3-BDO) is a promising commodity chemical with various industrial applications. While petroleum-based chemical processes currently dominate the industrial production of 2,3-BDO, fermentation-based production of 2,3-BDO provides an attractive alternative to chemical-based processes with regards to economic and environmental sustainability. The achievement of high 2,3-BDO titer, yield, and productivity in microbial fermentation is a prerequisite for the production of 2,3-BDO at large scales. Also, enantiopure production of 2,3-BDO production is desirable because 2,3-BDO stereoisomers have unique physicochemical properties. Pursuant to these goals, many metabolic engineering strategies to improve 2,3-BDO production from inexpensive sugars by Klebsiella oxytoca, Bacillus species, and Saccharomyces cerevisiae have been developed. This review summarizes the recent advances in metabolic engineering of non-pathogenic microorganisms to enable efficient and enantiopure production of 2,3-BDO. KEY POINTS: • K. oxytoca, Bacillus species, and S. cerevisiae have been engineered to achieve efficient 2,3-BDO production. • Metabolic engineering of non-pathogenic microorganisms enabled enantiopure production of 2,3-BDO. • Cost-effective 2,3-BDO production can be feasible by using renewable biomass.
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http://dx.doi.org/10.1007/s00253-021-11436-2DOI Listing
July 2021

Investigating the role of the transcriptional regulator Ure2 on the metabolism of Saccharomyces cerevisiae: a multi-omics approach.

Appl Microbiol Biotechnol 2021 Jun 21;105(12):5103-5112. Epub 2021 Jun 21.

DOE Center for Advanced Bioenergy and Bioproducts Innovation, University of Illinois at Urbana-Champaign, Urbana, IL, USA.

Ure2 regulates nitrogen catabolite repression in Saccharomyces cerevisiae. Deletion of URE2 induces a physiological state mimicking the nitrogen starvation and autophagic responses. Previous work has shown that deletion of URE2 increases the fermentation rate of some wine-producing strains of S. cerevisiae. In this work, we investigated the effect of URE2 deletion (ΔURE2) on the metabolism of S. cerevisiae. During growth on glucose, the ΔURE2 mutant grew at a 40% slower rate than the wild type; however, it produced ethanol at a 31% higher rate. To better under the behavior of this mutant, we performed transcriptomics and metabolomics. Analysis of the RNA sequencing results and metabolite levels indicates that the mutant strain exhibited characteristics of both nitrogen starvation and autophagy, including the upregulation of allantoin, urea, and amino acid uptake and utilization pathways and selective autophagic machinery. In addition, pyruvate decarboxylase and alcohol dehydrogenase isoforms were expressed at higher rates than the wild type. The mutant also accumulated less trehalose and glycogen, and produced more lipids. The induction of a nitrogen starvation-like state and increase in lipid production in nitrogen-rich conditions suggest that URE2 may be a promising target for metabolic engineering in S. cerevisiae and other yeasts for the production of lipids and lipid-derived compounds. KEY POINTS: • Deletion of URE2 increases ethanol and lipid production in Saccharomyces cerevisiae. • Deletion of URE2 reduces glycogen and trehalose production. • Metabolic changes mimic nitrogen starvation and autophagic response.
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http://dx.doi.org/10.1007/s00253-021-11394-9DOI Listing
June 2021

HilE is required for synergistic activation of SPI-1 gene expression in Salmonella enterica serovar Typhimurium.

BMC Microbiol 2021 02 16;21(1):49. Epub 2021 Feb 16.

Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 S. Mathews Ave, Urbana, IL, 61801, USA.

Background: Salmonella enterica serovar Typhimurium is an intestinal pathogen capable of infecting a wide range of animals. It initiates infection by invading intestinal epithelial cells using a type III secretion system encoded within Salmonella pathogenicity island 1 (SPI-1). The SPI-1 genes are regulated by multiple interacting transcription factors. The master regulator is HilD. HilE represses SPI-1 gene expression by binding HilD and preventing it from activating its target promoters. Previous work found that acetate and nutrients synergistically induce SPI-1 gene expression. In the present study, we investigated the role of HilE, nominally a repressor of SPI-1 gene expression, in mediating this response to acetate and nutrients.

Results: HilE is necessary for activation of SPI-1 gene expression by acetate and nutrients. In mutants lacking hilE, acetate and nutrients no longer increase SPI-1 gene expression but rather repress it. This puzzling response is not due to the BarA/SirA two component system, which governs the response to acetate. To identify the mechanism, we profiled gene expression using RNAseq in the wild type and a ΔhilE mutant under different growth conditions. Analysis of these data suggested that the Rcs system, which regulates gene expression in response to envelope stress, is involved. Consistent with this hypothesis, acetate and nutrients were able to induce SPI-1 gene expression in mutants lacking hilE and the Rcs system.

Conclusions: While the exact mechanism is unknown, these results demonstrate the HilE, nominally a repressor of SPI-1 gene expression, can also function as an activator under the growth conditions investigated. Collectively, these results provide new insights regarding SPI-1 gene regulation and demonstrate that HilE is more complex than initially envisioned.
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http://dx.doi.org/10.1186/s12866-021-02110-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7887791PMC
February 2021

Renewable biohydrogen production from lignocellulosic biomass using fermentation and integration of systems with other energy generation technologies.

Sci Total Environ 2021 Apr 24;765:144429. Epub 2020 Dec 24.

Department of Biological Engineering, College of Engineering, Konkuk University, Seoul 05029, Republic of Korea; Institute for Ubiquitous Information Technology and Application, Konkuk University, Seoul 05029, Republic of Korea. Electronic address:

Biohydrogen is a clean and renewable source of energy. It can be produced by using technologies such as thermochemical, electrolysis, photoelectrochemical and biological, etc. Among these technologies, the biological method (dark fermentation) is considered more sustainable and ecofriendly. Dark fermentation involves anaerobic microbes which degrade carbohydrate rich substrate and produce hydrogen. Lignocellulosic biomass is an abundantly available raw material and can be utilized as an economic and renewable substrate for biohydrogen production. Although there are many hurdles, continuous advancements in lignocellulosic biomass pretreatment technology, microbial fermentation (mixed substrate and co-culture fermentation), the involvement of molecular biology techniques, and understanding of various factors (pH, T, addition of nanomaterials) effect on biohydrogen productivity and yield render this technology efficient and capable to meet future energy demands. Further integration of biohydrogen production technology with other products such as bio-alcohol, volatile fatty acids (VFAs), and methane have the potential to improve the efficiency and economics of the overall process. In this article, various methods used for lignocellulosic biomass pretreatment, technologies in trends to produce and improve biohydrogen production, a coproduction of other energy resources, and techno-economic analysis of biohydrogen production from lignocellulosic biomass are reviewed.
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http://dx.doi.org/10.1016/j.scitotenv.2020.144429DOI Listing
April 2021

Engineering xylose metabolism in yeasts to produce biofuels and chemicals.

Curr Opin Biotechnol 2021 02 24;67:15-25. Epub 2020 Nov 24.

Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA; Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA. Electronic address:

Xylose is the second most abundant sugar in lignocellulosic biomass. Efficient and rapid xylose utilization is essential for the economic bioconversion of lignocellulosic biomass into value-added products. Building on previous pathway engineering efforts to enable xylose fermentation in Saccharomyces cerevisiae, recent work has focused on reprogramming regulatory networks to enhance xylose utilization by engineered S. cerevisiae. Also, potential benefits of using xylose for the production of various value-added products have been demonstrated. With increasing needs of lipid-derived bioproducts, activation and enhancement of xylose metabolism in oleaginous yeasts have been attempted. This review highlights recent progress of metabolic engineering to achieve efficient and rapid xylose utilization by S. cerevisiae and oleaginous yeasts, such as Yarrowia lipolytica, Rhodosporidium toruloides, and Lipomyces starkeyi.
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http://dx.doi.org/10.1016/j.copbio.2020.10.012DOI Listing
February 2021

Domesticating a food spoilage yeast into an organic acid-tolerant metabolic engineering host: Lactic acid production by engineered Zygosaccharomyces bailii.

Biotechnol Bioeng 2021 01 8;118(1):372-382. Epub 2020 Oct 8.

Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.

Lactic acid represents an important class of commodity chemicals, which can be produced by microbial cell factories. However, due to the toxicity of lactic acid at lower pH, microbial production requires the usage of neutralizing agents to maintain neutral pH. Zygosaccharomyces bailii, a food spoilage yeast, can grow under the presence of organic acids used as food preservatives. This unique trait of the yeast might be useful for producing lactic acid. With the goal of domesticating the organic acid-tolerant yeast as a metabolic engineering host, seven Z. bailii strains were screened in a minimal medium with 10 g/L of acetic, or 60 g/L of lactic acid at pH 3. The Z. bailii NRRL Y7239 strain was selected as the most robust strain to be engineered for lactic acid production. By applying a PAN-ARS-based CRISPR-Cas9 system consisting of a transfer RNA promoter and NAT selection, we demonstrated the targeted deletion of ADE2 and site-specific integration of Rhizopus oryzae ldhA coding for lactate dehydrogenase into the PDC1 locus. The resulting pdc1::ldhA strain produced 35 g/L of lactic acid without ethanol production. This study demonstrates the feasibility of the CRISPR-Cas9 system in Z. bailii, which can be applied for a fundamental study of the species.
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http://dx.doi.org/10.1002/bit.27576DOI Listing
January 2021

The Unconventional Cytoplasmic Sensing Mechanism for Ethanol Chemotaxis in Bacillus subtilis.

mBio 2020 10 6;11(5). Epub 2020 Oct 6.

Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

Motile bacteria sense chemical gradients using chemoreceptors, which consist of distinct sensing and signaling domains. The general model is that the sensing domain binds the chemical and the signaling domain induces the tactic response. Here, we investigated the unconventional sensing mechanism for ethanol taxis in Ethanol and other short-chain alcohols are attractants for Two chemoreceptors, McpB and HemAT, sense these alcohols. In the case of McpB, the signaling domain directly binds ethanol. We were further able to identify a single amino acid residue, Ala, on the cytoplasmic signaling domain of McpB that, when mutated to serine, reduces taxis to alcohols. Molecular dynamics simulations suggest that the conversion of Ala to serine increases coiled-coil packing within the signaling domain, thereby reducing the ability of ethanol to bind between the helices of the signaling domain. In the case of HemAT, the myoglobin-like sensing domain binds ethanol, likely between the helices encapsulating the heme group. Aside from being sensed by an unconventional mechanism, ethanol also differs from many other chemoattractants because it is not metabolized by and is toxic. We propose that uses ethanol and other short-chain alcohols to locate prey, namely, alcohol-producing microorganisms. Ethanol is a chemoattractant for even though it is not metabolized and inhibits growth. likely uses ethanol to find ethanol-fermenting microorganisms to utilize as prey. Two chemoreceptors sense ethanol: HemAT and McpB. HemAT's myoglobin-like sensing domain directly binds ethanol, but the heme group is not involved. McpB is a transmembrane receptor consisting of an extracellular sensing domain and a cytoplasmic signaling domain. While most attractants bind the extracellular sensing domain, we found that ethanol directly binds between intermonomer helices of the cytoplasmic signaling domain of McpB, using a mechanism akin to those identified in many mammalian ethanol-binding proteins. Our results indicate that the sensory repertoire of chemoreceptors extends beyond the sensing domain and can directly involve the signaling domain.
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http://dx.doi.org/10.1128/mBio.02177-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7542364PMC
October 2020

Two Tandem Mechanisms Control Bimodal Expression of the Flagellar Genes in Salmonella enterica.

J Bacteriol 2020 06 9;202(13). Epub 2020 Jun 9.

Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

Flagellar gene expression is bimodal in Under certain growth conditions, some cells express the flagellar genes whereas others do not. This results in mixed populations of motile and nonmotile cells. In the present study, we found that two independent mechanisms control bimodal expression of the flagellar genes. One was previously found to result from a double negative-feedback loop involving the flagellar regulators RflP and FliZ. This feedback loop governs bimodal expression of class 2 genes. In this work, a second mechanism was found to govern bimodal expression of class 3 genes. In particular, class 3 gene expression is still bimodal, even when class 2 gene expression is not. Using a combination of experimental and modeling approaches, we found that class 3 bimodality results from the σ-FlgM developmental checkpoint. Many bacterial use flagella to swim in liquids and swarm over surface. In , over 50 genes are required to assemble flagella. The expression of these genes is tightly regulated. Previous studies have found that flagellar gene expression is bimodal in , which means that only a fraction of cells express flagellar genes and are motile. In the present study, we found that two separate mechanisms induce this bimodal response. One mechanism, which was previously identified, tunes the fraction of motile cells in response to nutrients. The other results from a developmental checkpoint that couples flagellar gene expression to flagellar assembly. Collectively, these results further our understanding of how flagellar gene expression is regulated in .
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http://dx.doi.org/10.1128/JB.00787-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7283601PMC
June 2020

Metabolic engineering of Parageobacillus thermoglucosidasius for the efficient production of (2R, 3R)-butanediol.

Appl Microbiol Biotechnol 2020 May 27;104(10):4303-4311. Epub 2020 Mar 27.

Department of Chemical and Biomolecular Engineering, Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA.

High-temperature fermentation using thermophilic microorganisms may provide cost-effective processes for the industrial production of fuels and chemicals, due to decreased hygiene and cooling costs. In the present study, the genetically trackable thermophile Parageobacillus thermoglucosidasius DSM2542 was engineered to produce (2R, 3R)-butanediol (R-BDO), a valuable chemical with broad industrial applications. The R-BDO biosynthetic pathway was optimized by testing different combinations of pathway enzymes, with acetolactate synthase (AlsS) from Bacillus subtilis and acetolactate decarboxylase (AlsD) from Streptococcus thermophilus yielding the highest production in P. thermoglucosidasius DSM2542. Following fermentation condition optimization, shake flask fermentation at 55 °C resulted in the production of 7.2 g/L R-BDO with ~ 72% theoretical yield. This study details the microbial production of R-BDO at the highest fermentation temperature reported to date and demonstrates that P. thermoglucosidasius DSM2542 is a promising cell factory for the production of fuels and chemicals using high-temperature fermentation.
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http://dx.doi.org/10.1007/s00253-020-10553-8DOI Listing
May 2020

Recent developments in pretreatment technologies on lignocellulosic biomass: Effect of key parameters, technological improvements, and challenges.

Bioresour Technol 2020 Mar 2;300:122724. Epub 2020 Jan 2.

Department of Biological Engineering, College of Engineering, Konkuk University, Seoul 05029, Republic of Korea; Institute for Ubiquitous Information Technology and Application, Konkuk University, Seoul 05029, Republic of Korea. Electronic address:

Lignocellulosic biomass is an inexpensive renewable source that can be used to produce biofuels and bioproducts. The recalcitrance nature of biomass hampers polysaccharide accessibility for enzymes and microbes. Several pretreatment methods have been developed for the conversion of lignocellulosic biomass into value-added products. However, these pretreatment methods also produce a wide range of secondary compounds, which are inhibitory to enzymes and microorganisms. The selection of an effective and efficient pretreatment method discussed in the review and its process optimization can significantly reduce the production of inhibitory compounds and may lead to enhanced production of fermentable sugars and biochemicals. Moreover, evolutionary and genetic engineering approaches are being used for the improvement of microbial tolerance towards inhibitors. Advancements in pretreatment and detoxification technologies may help to increase the productivity of lignocellulose-based biorefinery. In this review, we discuss the recent advancements in lignocellulosic biomass pretreatment technologies and strategies for the removal of inhibitors.
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http://dx.doi.org/10.1016/j.biortech.2019.122724DOI Listing
March 2020

Characterization of the GH16 and GH17 laminarinases from Vibrio breoganii 1C10.

Appl Microbiol Biotechnol 2020 Jan 21;104(1):161-171. Epub 2019 Nov 21.

Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 S. Mathews Ave, Urbana, IL, 61801, USA.

Laminarin is an abundant glucose polymer used as an energy reserve by micro- and macroalgae. Bacteria digest and consume laminarin with laminarinases. Their genomes frequently contain multiple homologs; however, the biological role for this replication remains unclear. We investigated the four laminarinases of glycoside hydrolase families GH16 and GH17 from the marine bacterium Vibrio breoganii 1C10, which can use laminarin as its sole carbon source. All four laminarinases employ an endolytic mechanism and specifically cleave the β-1,3-glycosidic bond. Two primarily produce low-molecular weight laminarin oligomers (DP 3-4) whereas the others primarily produce high-molecular weight oligomers (DP > 8), which suggests that these enzymes sequentially degrade laminarin. The results from this work provide an overview of the laminarinases from a single marine bacterium and also provide insights regarding how multiple laminarinases are used to degrade laminarin.
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http://dx.doi.org/10.1007/s00253-019-10243-0DOI Listing
January 2020

A comprehensive genome-scale model for IFO0880 accounting for functional genomics and phenotypic data.

Metab Eng Commun 2019 Dec 28;9:e00101. Epub 2019 Aug 28.

Department of Chemical Engineering, The Pennsylvania State University, University Park, 306 Chemical and Biomedical Engineering Building, PA, 16802-4400, USA.

is a red, basidiomycetes yeast that can accumulate a large amount of lipids and produce carotenoids. To better assess this non-model yeast's metabolic capabilities, we reconstructed a genome-scale model of IFO0880's metabolic network (1108) accounting for 2204 reactions, 1985 metabolites and 1108 genes. In this work, we integrated and supplemented the current knowledge with in-house generated biomass composition and experimental measurements pertaining to the organism's metabolic capabilities. Predictions of genotype-phenotype relations were improved through manual curation of gene-protein-reaction rules for 543 reactions leading to correct recapitulations of 84.5% of gene essentiality data (sensitivity of 94.3% and specificity of 53.8%). Organism-specific macromolecular composition and ATP maintenance requirements were experimentally measured for two separate growth conditions: (i) carbon and (ii) nitrogen limitations. Overall, 1108 reproduced 's utilization capabilities for 18 alternate substrates, matched measured wild-type growth yield, and recapitulated the viability of 772 out of 819 deletion mutants. As a demonstration to the model's fidelity in guiding engineering interventions, the OptForce procedure was applied on 1108 for triacylglycerol overproduction. Suggested interventions recapitulated many of the previous successful implementations of genetic modifications and put forth a few new ones.
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http://dx.doi.org/10.1016/j.mec.2019.e00101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6838544PMC
December 2019

The Mechanism of Bidirectional pH Taxis in Bacillus subtilis.

J Bacteriol 2020 01 29;202(4). Epub 2020 Jan 29.

Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

We investigated pH taxis in This bacterium was found to perform bidirectional taxis in response to external pH gradients, enabling it to preferentially migrate to neutral environments. We next investigated the chemoreceptors involved in sensing pH gradients. We identified four chemoreceptors involved in sensing pH: McpA and TlpA for sensing acidic environments and McpB and TlpB for sensing alkaline ones. In addition, TlpA was found to also weakly sense alkaline environments. By analyzing chimeras between McpA and TlpB, the principal acid- and base-sensing chemoreceptors, we identified four critical amino acid residues-Thr, Gln, His, and Glu on McpA and Lys, Glu, Gln, and Asp on TlpB-involved in sensing pH. Swapping these four residues between McpA and TlpB converted the former into a base receptor and the latter into an acid receptor. Based on the results, we propose that disruption of hydrogen bonding between the adjacent residues upon pH changes induces signaling. Collectively, our results further our understanding of chemotaxis in and provide a new model for pH sensing in bacteria. Many bacteria can sense the pH in their environment and then use this information to direct their movement toward more favorable locations. In this study, we investigated the pH sensing mechanism in This bacterium preferentially migrates to neutral environments. It employs four chemoreceptors to sense pH. Two are involved in sensing acidic environments, and two are involved in sensing alkaline ones. To identify the mechanism for pH sensing, we constructed receptor chimeras of acid- and base-sensing chemoreceptors. By analyzing the responses of these chimeric receptors, we were able to identify four critical amino acid residues involved in pH sensing and propose a model for the pH sensing mechanism in .
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http://dx.doi.org/10.1128/JB.00491-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6989800PMC
January 2020

Production of galactitol from galactose by the oleaginous yeast IFO0880.

Biotechnol Biofuels 2019 18;12:250. Epub 2019 Oct 18.

1Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 S. Mathews Ave., Urbana, IL 61801 USA.

Background: Sugar alcohols are commonly used as low-calorie sweeteners and can serve as potential building blocks for bio-based chemicals. Previous work has shown that the oleaginous yeast IFO0880 can natively produce arabitol from xylose at relatively high titers, suggesting that it may be a useful host for sugar alcohol production. In this work, we explored whether can produce additional sugar alcohols.

Results: is able to produce galactitol from galactose. During growth in nitrogen-rich medium, produced 3.2 ± 0.6 g/L, and 8.4 ± 0.8 g/L galactitol from 20 to 40 g/L galactose, respectively. In addition, was able to produce galactitol from galactose at reduced titers during growth in nitrogen-poor medium, which also induces lipid production. These results suggest that can potentially be used for the co-production of lipids and galactitol from galactose. We further characterized the mechanism for galactitol production, including identifying and biochemically characterizing the critical aldose reductase. Intracellular metabolite analysis was also performed to further understand galactose metabolism.

Conclusions: has traditionally been used for the production of lipids and lipid-based chemicals. Our work demonstrates that can also produce galactitol, which can be used to produce polymers with applications in medicine and as a precursor for anti-cancer drugs. Collectively, our results further establish that can produce multiple value-added chemicals from a wide range of sugars.
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http://dx.doi.org/10.1186/s13068-019-1586-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6798376PMC
October 2019

Synergistic action of SPI-1 gene expression in Salmonella enterica serovar typhimurium through transcriptional crosstalk with the flagellar system.

BMC Microbiol 2019 09 5;19(1):211. Epub 2019 Sep 5.

Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 S. Mathews Ave, Urbana, IL, 61801, USA.

Background: Salmonella enterica serovar Typhimurium is a common food-borne pathogen. S. enterica uses a type III secretion system encoded within Salmonella pathogenicity island 1 (SPI-1) to invade intestinal epithelial cells. A complex network of interacting transcription factors regulates SPI-1 gene expression. In addition, SPI-1 gene expression is coupled to flagellar gene expression. Both SPI-1 and flagellar gene expression are bistable, with co-existing populations of cells expressing and not expressing these genes. Previous work demonstrated that nutrients could be used to tune the fraction of cells expressing the flagellar genes. In the present study, we tested whether nutrients could also tune the fraction of cells expressing the SPI-1 genes through transcriptional crosstalk with the flagellar genes.

Results: Nutrients alone were not found to induce SPI-1 gene expression. However, when the cells were also grown in the presence of acetate, the concentration of nutrients in the growth medium was able to tune the fraction of cells expressing the SPI-1 genes. During growth in nutrient-poor medium, acetate alone was unable to induce SPI-1 gene expression. These results demonstrate that acetate and nutrients synergistically activate SPI-1 gene expression. The response to acetate was governed by the BarA/SirA two-component system and the response to nutrients was governed by transcriptional crosstalk with the flagella system, specifically through the action of the flagellar regulator FliZ.

Conclusions: Acetate and nutrients are capable of synergistically activating SPI-1 gene expression. In addition, these signals were found to tune the fraction of cells expressing the SPI-1 genes. The governing mechanism involves transcriptional crosstalk with the flagellar gene network. Collectively, these results further our understanding of SPI-1 gene regulation and provide the basis for future studies investigating this complex regulatory mechanism.
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http://dx.doi.org/10.1186/s12866-019-1583-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6727558PMC
September 2019

Global Lysine Acetylation in Results from Growth Conditions That Favor Acetate Fermentation.

J Bacteriol 2019 05 9;201(9). Epub 2019 Apr 9.

Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

Lysine acetylation is thought to provide a mechanism for regulating metabolism in diverse bacteria. Indeed, many studies have shown that the majority of enzymes involved in central metabolism are acetylated and that acetylation can alter enzyme activity. However, the details regarding this regulatory mechanism are still unclear, specifically with regard to the signals that induce lysine acetylation. To better understand this global regulatory mechanism, we profiled changes in lysine acetylation during growth of on the hexose glucose or the pentose xylose at both high and low sugar concentrations using label-free mass spectrometry. The goal was to see whether lysine acetylation differed during growth on these two different sugars. No significant differences, however, were observed. Rather, the initial sugar concentration was the principal factor governing changes in lysine acetylation, with higher sugar concentrations causing more acetylation. These results suggest that acetylation does not target specific metabolic pathways but rather simply targets accessible lysines, which may or may not alter enzyme activity. They further suggest that lysine acetylation principally results from conditions that favor accumulation of acetyl phosphate, the principal acetate donor in Bacteria alter their metabolism in response to nutrient availability, growth conditions, and environmental stresses using a number of different mechanisms. One is lysine acetylation, a posttranslational modification known to target many metabolic enzymes. However, little is known about this regulatory mode. We investigated the factors inducing changes in lysine acetylation by comparing growth on glucose and xylose. We found that the specific sugar used for growth did not alter the pattern of acetylation; rather, the amount of sugar did, with more sugar causing more acetylation. These results imply that lysine acetylation is a global regulatory mechanism that is responsive not to the specific carbon source but rather to the accumulation of downstream metabolites.
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http://dx.doi.org/10.1128/JB.00768-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6456854PMC
May 2019

pH selectively regulates citric acid and lipid production in Yarrowia lipolytica W29 during nitrogen-limited growth on glucose.

J Biotechnol 2019 Jan 26;290:10-15. Epub 2018 Nov 26.

Department of Chemical and Biomolecular Engineering, DOE Center for Advanced Bioenergy and Bioproducts Innovation, Carl R. Woese Institute of Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA. Electronic address:

Yarrowia lipolytica has been used to produce both citric acid and lipid-based bioproducts at high titers. In this study, we found that pH differentially affects citric acid and lipid production in Y. lipolytica W29, with citric acid production enhanced at more neutral pH's and lipid production enhanced at more acid pH's. To determine the mechanism governing this pH-dependent switch between citric acid and lipid production, we profiled gene expression at different pH's and found that the relative expression of multiple transporters is increased at neutral pH. These results suggest that this pH-dependent switch is mediated at the level of citric acid transport rather than changes in the expression of the enzymes involved in citric acid and lipid metabolism. In further support of this mechanism, thermodynamic calculations suggest that citric acid secretion is more energetically favorable at neutral pH's, assuming the fully protonated acid is the substrate for secretion. Collectively, these results provide new insights regarding citric acid and lipid production in Y. lipolytica and may offer new strategies for metabolic engineering and process design.
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http://dx.doi.org/10.1016/j.jbiotec.2018.10.012DOI Listing
January 2019

Driving the expression of the Salmonella enterica sv Typhimurium flagellum using flhDC from Escherichia coli results in key regulatory and cellular differences.

Sci Rep 2018 11 12;8(1):16705. Epub 2018 Nov 12.

Centre for Bacterial Cell Biology, Baddiley Clark Building, Newcastle University, Richardson Road, Newcastle upon Tyne, NE2 4AX, UK.

The flagellar systems of Escherichia coli and Salmonella enterica exhibit a significant level of genetic and functional synteny. Both systems are controlled by the flagellar specific master regulator FlhDC. Since the early days of genetic analyses of flagellar systems it has been known that E. coli flhDC can complement a ∆flhDC mutant in S. enterica. The genomic revolution has identified how genetic changes to transcription factors and/or DNA binding sites can impact the phenotypic outcome across related species. We were therefore interested in asking: using modern tools to interrogate flagellar gene expression and assembly, what would the impact be of replacing the flhDC coding sequences in S. enterica for the E. coli genes at the flhDC S. entercia chromosomal locus? We show that even though all strains created are motile, flagellar gene expression is measurably lower when flhDC are present. These changes can be attributed to the impact of FlhDC DNA recognition and the protein-protein interactions required to generate a stable FlhDC complex. Furthermore, our data suggests that in E. coli the internal flagellar FliT regulatory feedback loop has a marked difference with respect to output of the flagellar systems. We argue due diligence is required in making assumptions based on heterologous expression of regulators and that even systems showing significant synteny may not behave in exactly the same manner.
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http://dx.doi.org/10.1038/s41598-018-35005-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6232118PMC
November 2018

Extracellular Acidic pH Inhibits Acetate Consumption by Decreasing Gene Transcription of the Tricarboxylic Acid Cycle and the Glyoxylate Shunt.

J Bacteriol 2019 01 20;201(2). Epub 2018 Dec 20.

Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

produces acetate during aerobic growth on various carbon sources. After consuming the carbon substrate, can further grow on the acetate. This phenomenon is known as the acetate switch, where cells transition from producing acetate to consuming it. In this study, we investigated how pH governs the acetate switch. When was grown on a glucose-supplemented medium initially buffered to pH 7, the cells produced and then consumed the acetate. However, when the initial pH was dropped to 6, the cells still produced acetate but were only able to consume it when little (<10 mM) acetate was produced. When significant acetate was produced in acidic medium, which occurs when the growth medium contains magnesium, amino acids, and sugar, the cells were unable to consume the acetate. To determine the mechanism, we characterized a set of metabolic mutants and found that those defective in the tricarboxylic acid (TCA) cycle or glyoxylate shunt exhibited reduced rates of acetate consumption. We further found that the expression of the genes in these pathways was reduced during growth in acidic medium. The expression of the genes involved in the AckA-Pta pathway, which provides the principal route for both acetate production and consumption, was also inhibited in acidic medium but only after glucose was depleted, which correlates with the acetate consumption phase. On the basis of these results, we conclude that growth in acidic environments inhibits the expression of the acetate catabolism genes, which in turn prevents acetate consumption. Many microorganisms produce fermentation products during aerobic growth on sugars. One of the best-known examples is the production of acetate by during aerobic growth on sugars. In , acetate production is reversible: once the cells consume the available sugar, they can consume the acetate previously produced during aerobic fermentation. We found that pH affects the reversibility of acetate production. When the cells produce significant acetate during growth in acidic environments, they are unable to consume it. Unconsumed acetate may accumulate in the cell and inhibit the expression of pathways required for acetate catabolism. These findings demonstrate how acetate alters cell metabolism; they also may be useful for the design of aerobic fermentation processes.
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http://dx.doi.org/10.1128/JB.00410-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6304666PMC
January 2019

Microbial conversion of xylose into useful bioproducts.

Appl Microbiol Biotechnol 2018 Nov 24;102(21):9015-9036. Epub 2018 Aug 24.

Department of Chemical and Biomolecular Engineering, DOE Center for Advanced Bioenergy and Bioproducts Innovation, University of Illinois at Urbana-Champaign, 600 S. Mathews Ave, Urbana, IL, 61801, USA.

Microorganisms can produce a number of different bioproducts from the sugars in plant biomass. One challenge is devising processes that utilize all of the sugars in lignocellulosic hydrolysates. D-xylose is the second most abundant sugar in these hydrolysates. The microbial conversion of D-xylose to ethanol has been studied extensively; only recently, however, has conversion to bioproducts other than ethanol been explored. Moreover, in the case of yeast, D-xylose may provide a better feedstock for the production of bioproducts other than ethanol, because the relevant pathways are not subject to glucose-dependent repression. In this review, we discuss how different microorganisms are being used to produce novel bioproducts from D-xylose. We also discuss how D-xylose could be potentially used instead of glucose for the production of value-added bioproducts.
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http://dx.doi.org/10.1007/s00253-018-9294-9DOI Listing
November 2018

In Vitro Assay for Measuring Receptor-Kinase Activity in the Bacillus subtilis Chemotaxis Pathway.

Methods Mol Biol 2018 ;1729:95-105

Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA.

The sensing apparatus of the Bacillus subtilis chemotaxis pathway involves a complex consisting of chemoreceptors, the CheA histidine kinase, and the CheV and CheW adaptor proteins. Attractants and repellents alter the rate of CheA autophosphorylation, either by directly binding the receptors or by indirectly interacting with them through intermediate binding proteins. We describe an in vitro assay for measuring receptor-kinase activity in B. subtilis. This assay has been used to investigate the mechanism of signal transduction in B. subtilis chemotaxis and the disparate mechanisms employed by this bacterium for sensory adaptation and gradient sensing.
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http://dx.doi.org/10.1007/978-1-4939-7577-8_10DOI Listing
December 2018

Regulation of metabolism in Escherichia coli during growth on mixtures of the non-glucose sugars: arabinose, lactose, and xylose.

Sci Rep 2018 01 12;8(1):609. Epub 2018 Jan 12.

Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA.

Catabolite repression refers to the process where the metabolism of one sugar represses the genes involved in metabolizing another sugar. While glucose provides the canonical example, many other sugars are also known to induce catabolite repression. However, less is known about the mechanism for catabolite repression by these non-glucose sugars. In this work, we investigated the mechanism of catabolite repression in the bacterium Escherichia coli during growth on lactose, L-arabinose, and D-xylose. The metabolism of these sugars is regulated in a hierarchical manner, where lactose is the preferred sugar, followed by L-arabinose, and then D-xylose. Previously, the preferential utilization of L-arabinose over D-xylose was found to result from transcriptional crosstalk. However, others have proposed that cAMP governs the hierarchical regulation of many non-glucose sugars. We investigated whether lactose-induced repression of L-arabinose and D-xylose gene expression is due to transcriptional crosstalk or cAMP. Our results demonstrate that it is due to cAMP and not transcriptional crosstalk. In addition, we found that repression is reciprocal, where both L-arabinose and D-xylose also repress the lactose gene expression, albeit to a lesser extent and also through a mechanism involving cAMP. Collectively, the results further our understanding of metabolism during growth on multiple sugars.
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http://dx.doi.org/10.1038/s41598-017-18704-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5766520PMC
January 2018

Ancient Regulatory Role of Lysine Acetylation in Central Metabolism.

mBio 2017 11 28;8(6). Epub 2017 Nov 28.

Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA

Lysine acetylation is a common protein post-translational modification in bacteria and eukaryotes. Unlike phosphorylation, whose functional role in signaling has been established, it is unclear what regulatory mechanism acetylation plays and whether it is conserved across evolution. By performing a proteomic analysis of 48 phylogenetically distant bacteria, we discovered conserved acetylation sites on catalytically essential lysine residues that are invariant throughout evolution. Lysine acetylation removes the residue's charge and changes the shape of the pocket required for substrate or cofactor binding. Two-thirds of glycolytic and tricarboxylic acid (TCA) cycle enzymes are acetylated at these critical sites. Our data suggest that acetylation may play a direct role in metabolic regulation by switching off enzyme activity. We propose that protein acetylation is an ancient and widespread mechanism of protein activity regulation. Post-translational modifications can regulate the activity and localization of proteins inside the cell. Similar to phosphorylation, lysine acetylation is present in both eukaryotes and prokaryotes and modifies hundreds to thousands of proteins in cells. However, how lysine acetylation regulates protein function and whether such a mechanism is evolutionarily conserved is still poorly understood. Here, we investigated evolutionary and functional aspects of lysine acetylation by searching for acetylated lysines in a comprehensive proteomic data set from 48 phylogenetically distant bacteria. We found that lysine acetylation occurs in evolutionarily conserved lysine residues in catalytic sites of enzymes involved in central carbon metabolism. Moreover, this modification inhibits enzymatic activity. Our observations suggest that lysine acetylation is an evolutionarily conserved mechanism of controlling central metabolic activity by directly blocking enzyme active sites.
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http://dx.doi.org/10.1128/mBio.01894-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5705920PMC
November 2017

Production of D-arabitol from D-xylose by the oleaginous yeast Rhodosporidium toruloides IFO0880.

Appl Microbiol Biotechnol 2018 Jan 11;102(1):143-151. Epub 2017 Nov 11.

Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 S. Mathews Ave., Urbana, IL, 61801, USA.

The sugar alcohol D-arabitol is one of the Department of Energy's top twelve bio-based building block chemicals. In this study, we found that the oleaginous yeast Rhodosporidium toruloides IFO0880 produces D-arabitol during growth on xylose in nitrogen-rich medium. Efficient xylose utilization was a prerequisite for extracellular D-arabitol production. During growth in complex media, R. toruloides produced 22 ± 2, 32 ± 2, and 49 ± 2 g/L D-arabitol from 70, 105, and 150 g/L xylose, respectively. In addition, we found that R. toruloides could potentially be used for the co-production of lipids and D-arabitol from xylose. These results demonstrate that R. toruloides can be used to produce multiple value-added chemicals from xylose.
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http://dx.doi.org/10.1007/s00253-017-8581-1DOI Listing
January 2018

Exploiting fine-scale genetic and physiological variation of closely related microbes to reveal unknown enzyme functions.

J Biol Chem 2017 08 7;292(31):13056-13067. Epub 2017 Jun 7.

Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801. Electronic address:

Polysaccharide degradation by marine microbes represents one of the largest and most rapid heterotrophic transformations of organic matter in the environment. Microbes employ systems of complementary carbohydrate-specific enzymes to deconstruct algal or plant polysaccharides (glycans) into monosaccharides. Because of the high diversity of glycan substrates, the functions of these enzymes are often difficult to establish. One solution to this problem may lie within naturally occurring microdiversity; varying numbers of enzymes, due to gene loss, duplication, or transfer, among closely related environmental microbes create metabolic differences akin to those generated by knock-out strains engineered in the laboratory used to establish the functions of unknown genes. Inspired by this natural fine-scale microbial diversity, we show here that it can be used to develop hypotheses guiding biochemical experiments for establishing the role of these enzymes in nature. In this work, we investigated alginate degradation among closely related strains of the marine bacterium One strain, 13B01, exhibited high extracellular alginate lyase activity compared with other strains. To identify the enzymes responsible for this high extracellular activity, we compared 13B01 with the previously characterized 12B01, which has low extracellular activity and lacks two alginate lyase genes present in 13B01. Using a combination of genomics, proteomics, biochemical, and functional screening, we identified a polysaccharide lyase family 7 enzyme that is unique to 13B01, secreted, and responsible for the rapid digestion of extracellular alginate. These results demonstrate the value of querying the enzymatic repertoires of closely related microbes to rapidly pinpoint key proteins with beneficial functions.
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http://dx.doi.org/10.1074/jbc.M117.787192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5546043PMC
August 2017

Dynamic Measures of Flagellar Gene Expression.

Methods Mol Biol 2017 ;1593:73-83

Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 S. Mathews Ave, Urbana, IL, 61801, USA.

Many genes are required to assemble flagella. These genes encode not only the structural elements of the flagellum but also a number of regulators that control how the flagellar genes are temporally expressed during the assembly process. These regulators also specify the likelihood that a given cell will express the flagellar genes. In particular, not all cells express the flagellar genes, resulting in mixed populations of motile and non-motile cells. Nutrients provide one signal that specifies the motile fraction. In this chapter, we describe two methods for measuring flagellar gene expression dynamics using fluorescent proteins in Salmonella enterica. Both the methods can be used to investigate the mechanisms governing flagellar gene expression dynamics.
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http://dx.doi.org/10.1007/978-1-4939-6927-2_5DOI Listing
February 2018

Growth rate control of flagellar assembly in Escherichia coli strain RP437.

Sci Rep 2017 01 24;7:41189. Epub 2017 Jan 24.

Centre for Bacterial Cell Biology, Baddiley Clark Building, Newcastle University, Richardson Road, Newcastle upon Tyne, NE2 4AX, United Kingdom.

The flagellum is a rotary motor that enables bacteria to swim in liquids and swarm over surfaces. Numerous global regulators control flagellar assembly in response to cellular and environmental factors. Previous studies have also shown that flagellar assembly is affected by the growth-rate of the cell. However, a systematic study has not yet been described under controlled growth conditions. Here, we investigated the effect of growth rate on flagellar assembly in Escherichia coli using steady-state chemostat cultures where we could precisely control the cell growth-rate. Our results demonstrate that flagellar abundance correlates with growth rate, where faster growing cells produce more flagella. They also demonstrate that this growth-rate dependent control occurs through the expression of the flagellar master regulator, FlhDC. Collectively, our results demonstrate that motility is intimately coupled to the growth-rate of the cell.
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http://dx.doi.org/10.1038/srep41189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5259725PMC
January 2017

Increasing Growth Yield and Decreasing Acetylation in Escherichia coli by Optimizing the Carbon-to-Magnesium Ratio in Peptide-Based Media.

Appl Environ Microbiol 2017 03 2;83(6). Epub 2017 Mar 2.

Department of Microbiology and Immunology, Stritch School of Medicine, Health Sciences Division, Loyola University Chicago, Maywood, Illinois, USA

Complex media are routinely used to cultivate diverse bacteria. However, this complexity can obscure the factors that govern cell growth. While studying protein acetylation in buffered tryptone broth supplemented with glucose (TB7-glucose), we observed that did not fully consume glucose prior to stationary phase. However, when we supplemented this medium with magnesium, the glucose was completely consumed during exponential growth, with concomitant increases in cell number and biomass but reduced cell size. Similar results were observed with other sugars and other peptide-based media, including lysogeny broth. Magnesium also limited cell growth for and in TB7-glucose. Finally, magnesium supplementation reduced protein acetylation. Based on these results, we conclude that growth in peptide-based media is magnesium limited. We further conclude that magnesium supplementation can be used to tune protein acetylation without genetic manipulation. These results have the potential to reduce potentially deleterious acetylated isoforms of recombinant proteins without negatively affecting cell growth. Bacteria are often grown in complex media. These media are thought to provide the nutrients necessary to grow bacteria to high cell densities. In this work, we found that peptide-based media containing a sugar are magnesium limited for bacterial growth. In particular, magnesium supplementation is necessary for the bacteria to use the sugar for cell growth. Interestingly, in the absence of magnesium supplementation, the bacteria still consume the sugar. However, rather than use it for cell growth, the bacteria instead use the sugar to acetylate lysines on proteins. As lysine acetylation may alter the activity of proteins, this work demonstrates how lysine acetylation can be tuned through magnesium supplementation. These findings may be useful for recombinant protein production, when acetylated isoforms are to be avoided. They also demonstrate how to increase bacterial growth in complex media.
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http://dx.doi.org/10.1128/AEM.03034-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5335519PMC
March 2017

Production of 1-decanol by metabolically engineered Yarrowia lipolytica.

Metab Eng 2016 11 26;38:139-147. Epub 2016 Jul 26.

Department of Chemical and Biomolecular Engineering, Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, United States. Electronic address:

Medium-chain alcohols are used to produce solvents, surfactants, lubricants, waxes, creams, and cosmetics. In this study, we engineered the oleaginous yeast Yarrowia lipolytica to produce 1-decanol from glucose. Expression of a fatty acyl-CoA reductase from Arabidopsis thaliana in strains of Y. lipolytica previously engineered to produce medium-chain fatty acids resulted in the production of 1-decanol. However, the resulting titers were very low (<10mg/mL), most likely due to product catabolism. In addition, these strains produced small quantities of 1-hexadecanol and 1-octadecanol. Deleting the major peroxisome assembly factor Pex10 was found to significantly increase 1-decanol production, resulting in titers exceeding 500mg/L. It also increased 1-hexadecanoland and 1-octadecanol titers, though the resulting increases were less than those for 1-decanol. These results demonstrate that Y. lipolytica can potentially be used for the industrial production of 1-decanol and other fatty alcohols from simple sugars.
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http://dx.doi.org/10.1016/j.ymben.2016.07.011DOI Listing
November 2016

Metabolic engineering of the oleaginous yeast Rhodosporidium toruloides IFO0880 for lipid overproduction during high-density fermentation.

Appl Microbiol Biotechnol 2016 Nov 27;100(21):9393-9405. Epub 2016 Sep 27.

Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 S. Mathews Ave, Urbana, IL, 61810, USA.

Natural lipids can be used to make biodiesel and many other value-added compounds. In this work, we explored a number of different metabolic engineering strategies for increasing lipid production in the oleaginous yeast Rhodosporidium toruloides IFO0880. These included increasing the expression of enzymes involved in different aspects of lipid biosynthesis-malic enzyme (ME), pyruvate carboxylase (PYC1), glycerol-3-P dehydrogenase (GPD), and stearoyl-CoA desaturase (SCD)-and deleting the gene PEX10, required for peroxisome biogenesis. Only malic enzyme and stearoyl-CoA desaturase, when overexpressed, were found to significantly increase lipid production. Only stearoyl-CoA desaturase, when overexpressed, further increased lipid production in a strain previously engineered to overexpress acetyl-CoA carboxylase (ACC1) and diacylglycerol acyltransferase (DGA1). Our best strain produced 27.4 g/L lipid with an average productivity of 0.31 g/L/h during batch growth on glucose and 89.4 g/L lipid with an average productivity of 0.61 g/L/h during fed-batch growth on glucose. These results further establish R. toruloides as a platform organism for the production of lipids and potentially other lipid-derived compounds from sugars.
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http://dx.doi.org/10.1007/s00253-016-7815-yDOI Listing
November 2016