Papers by Aindrila Mukhopadhyay
Scientific reports, 2016
Enhancing xylose utilization has been a major focus in Saccharomyces cerevisiae strain-engineerin... more Enhancing xylose utilization has been a major focus in Saccharomyces cerevisiae strain-engineering efforts. The incentive for these studies arises from the need to use all sugars in the typical carbon mixtures that comprise standard renewable plant-biomass-based carbon sources. While major advances have been made in developing utilization pathways, the efficient import of five carbon sugars into the cell remains an important bottleneck in this endeavor. Here we use an engineered S. cerevisiae BY4742 strain, containing an established heterologous xylose utilization pathway, and imposed a laboratory evolution regime with xylose as the sole carbon source. We obtained several evolved strains with improved growth phenotypes and evaluated the best candidate using genome resequencing. We observed remarkably few single nucleotide polymorphisms in the evolved strain, among which we confirmed a single amino acid change in the hexose transporter HXT7 coding sequence to be responsible for the e...
PLoS computational biology, 2015
Current limitations in quantitatively predicting biological behavior hinder our efforts to engine... more Current limitations in quantitatively predicting biological behavior hinder our efforts to engineer biological systems to produce biofuels and other desired chemicals. Here, we present a new method for calculating metabolic fluxes, key targets in metabolic engineering, that incorporates data from 13C labeling experiments and genome-scale models. The data from 13C labeling experiments provide strong flux constraints that eliminate the need to assume an evolutionary optimization principle such as the growth rate optimization assumption used in Flux Balance Analysis (FBA). This effective constraining is achieved by making the simple but biologically relevant assumption that flux flows from core to peripheral metabolism and does not flow back. The new method is significantly more robust than FBA with respect to errors in genome-scale model reconstruction. Furthermore, it can provide a comprehensive picture of metabolite balancing and predictions for unmeasured extracellular fluxes as co...
Applied and Environmental Microbiology, 2015
Limonene, a major component of citrus peel oil, has a number of applications related to microbiol... more Limonene, a major component of citrus peel oil, has a number of applications related to microbiology. The anti-microbial properties of limonene make it a popular disinfectant and food preservative, while its potential as a biofuel component has made it the target of renewable production efforts through microbial metabolic engineering. For both applications, an understanding of microbial sensitivity or tolerance to limonene is crucial, but the mechanism of limonene toxicity remains enigmatic. In this study, we characterized a limonene-tolerant strain of Escherichia coli and found a mutation in ahpC, encoding for alkyl hydroperoxidase, which alleviated limonene toxicity. We show that the acute toxicity previously attributed to limonene is largely due to the common oxidation product limonene-hydroperoxide, which forms spontaneously in aerobic environments. The mutant protein AhpC(L177Q) is able to alleviate this toxicity by reducing the hydroperoxide to a more benign compound. We show that the degree of limonene toxicity is a function of its oxidation level, and that non-oxidized limonene has relatively little toxicity to wild-type E. coli. Our results have implications for both the renewable production of limonene and the applications of limonene as an anti-microbial.
Frontiers in Microbiology, 2015
Biological Soil Crusts (BSCs) are organosedimentary assemblages comprised of microbes and mineral... more Biological Soil Crusts (BSCs) are organosedimentary assemblages comprised of microbes and minerals in topsoil of terrestrial environments. BSCs strongly impact soil quality in dryland ecosystems (e.g., soil structure and nutrient yields) due to pioneer species such as Microcoleus vaginatus; phototrophs that produce filaments that bind the soil together, and support an array of heterotrophic microorganisms. These microorganisms in turn contribute to soil stability and biogeochemistry of BSCs. Non-cyanobacterial populations of BSCs are less well known than cyanobacterial populations. Therefore, we attempted to isolate a broad range of numerically significant and phylogenetically representative BSC aerobic heterotrophs. Combining simple pre-treatments (hydration of BSCs under dark and light) and isolation strategies (media with varying nutrient availability and protection from oxidative stress) we recovered 402 bacterial and one fungal isolate in axenic culture, which comprised 116 phylotypes (at 97% 16S rRNA gene sequence homology), 115 bacterial and one fungal. Each medium enriched a mostly distinct subset of phylotypes, and cultivated phylotypes varied due to the BSC pre-treatment. The fraction of the total phylotype diversity isolated, weighted by relative abundance in the community, was determined by the overlap between isolate sequences and OTUs reconstructed from metagenome or metatranscriptome reads. Together, more than 8% of relative abundance of OTUs in the metagenome was represented by our isolates, a cultivation efficiency much larger than typically expected from most soils. We conclude that simple cultivation procedures combined with specific pre-treatment of samples afford a significant reduction in the culturability gap, enabling physiological and metabolic assays that rely on ecologically relevant axenic cultures.
Genome biology, 2011
Two component regulatory systems are the primary form of signal transduction in bacteria. Althoug... more Two component regulatory systems are the primary form of signal transduction in bacteria. Although genomic binding sites have been determined for several eukaryotic and bacterial transcription factors, comprehensive identification of gene targets of two component response regulators remains challenging due to the lack of knowledge of the signals required for their activation. We focused our study on Desulfovibrio vulgaris Hildenborough, a sulfate reducing bacterium that encodes unusually diverse and largely uncharacterized two component signal transduction systems. We report the first systematic mapping of the genes regulated by all transcriptionally acting response regulators in a single bacterium. Our results enabled functional predictions for several response regulators and include key processes of carbon, nitrogen and energy metabolism, cell motility and biofilm formation, and responses to stresses such as nitrite, low potassium and phosphate starvation. Our study also led to th...
mBio, 2014
Engineering microbial hosts for the production of fungible fuels requires mitigation of limitatio... more Engineering microbial hosts for the production of fungible fuels requires mitigation of limitations posed on the production capacity. One such limitation arises from the inherent toxicity of solvent-like biofuel compounds to production strains, such as Escherichia coli. Here we show the importance of host engineering for the production of short-chain alcohols by studying the overexpression of genes upregulated in response to exogenous isopentenol. Using systems biology data, we selected 40 genes that were upregulated following isopentenol exposure and subsequently overexpressed them in E. coli. Overexpression of several of these candidates improved tolerance to exogenously added isopentenol. Genes conferring isopentenol tolerance phenotypes belonged to diverse functional groups, such as oxidative stress response (soxS, fpr, and nrdH), general stress response (metR, yqhD, and gidB), heat shock-related response (ibpA), and transport (mdlB). To determine if these genes could also impro...
Biotechnology and bioengineering, Jan 2, 2014
Microorganisms can be engineered for the production of chemicals utilized in the polymer industry... more Microorganisms can be engineered for the production of chemicals utilized in the polymer industry. However many such target compounds inhibit microbial growth and might correspondingly limit production levels. Here, we focus on compounds that are precursors to bioplastics, specifically styrene and representative alpha-olefins; 1-hexene, 1-octene, and 1-nonene. We evaluated the role of the Escherichia coli efflux pump, AcrAB-TolC, in enhancing tolerance towards these olefin compounds. AcrAB-TolC is involved in the tolerance towards all four compounds in E. coli. Both styrene and 1-hexene are highly toxic to E. coli. Styrene is a model plastics precursor with an established route for production in E. coli (McKenna and Nielsen, 2011). Though our data indicates that AcrAB-TolC is important for its optimal production, we observed a strong negative selection against the production of styrene in E. coli. Thus we used 1-hexene as a model compound to implement a directed evolution strategy t...
Chemical Engineering Science, 2013
ABSTRACT The role of yhjX, a predicted major facilitator superfamily protein, was examined in con... more ABSTRACT The role of yhjX, a predicted major facilitator superfamily protein, was examined in context of E. coli response to 1,4-butanediol (1,4-BDO). E. coli DH1 and MG1655, two commonly used metabolic engineering hosts, were both sensitive to the presence of 1,4-BDO in the growth medium, but to different extents. The strains also showed differences in the transcriptional response of the yhjX gene that was highly induced in response to 1,4-BDO. yhjX deletion improved growth of the E. coli strains in the control defined medium but did not significantly impact 1,4-BDO sensitivity. Overexpression of yhjX using a plasmid-borne copy and lactose-inducible promoter also did not result in an improvement in 1,4-BDO tolerance. However, the large differential expression of yhjX in response to this diol provided the foundation to develop a biosensor for the detection of 1,4-BDO using a fluorescent gene under the control of the yhjX promoter. A basic PyhjX:GFP biosensor in E. coli DH1 allows the detection of 4–7% 1,4-BDO in the extracellular medium and provides a tool for high throughput engineering for improving 1,4-BDO production strains.
In vivo methods such as ChIP-chip are well-established techniques used to determine global gene t... more In vivo methods such as ChIP-chip are well-established techniques used to determine global gene targets for transcription factors. However, they are of limited use in exploring bacterial two component regulatory systems with uncharacterized activation conditions. Such systems regulate transcription only when activated in the presence of unique signals. Since these signals are often unknown, the in vitro microarray based method described in this video article can be used to determine gene targets and binding sites for response regulators. This DNA-affinity-purified-chip method may be used for any purified regulator in any organism with a sequenced genome. The protocol involves allowing the purified tagged protein to bind to sheared genomic DNA and then affinity purifying the protein-bound DNA, followed by fluorescent labeling of the DNA and hybridization to a custom tiling array. Preceding steps that may be used to optimize the assay for specific regulators are also described. The peaks generated by the array data analysis are used to predict binding site motifs, which are then experimentally validated. The motif predictions can be further used to determine gene targets of orthologous response regulators in closely related species. We demonstrate the applicability of this method by determining the gene targets and binding site motifs and thus predicting the function for a sigma54-dependent response regulator DVU3023 in the environmental bacterium Desulfovibrio vulgaris Hildenborough.
Sulfate-reducing bacteria such as Desulfovibrio vulgaris Hildenborough are often found in environ... more Sulfate-reducing bacteria such as Desulfovibrio vulgaris Hildenborough are often found in environments with limiting growth nutrients. Using lactate as the electron donor and carbon source, and sulfate as the electron acceptor, wild type D. vulgaris shows motility on soft agar plates. We evaluated this phenotype with mutants resulting from insertional inactivation of genes potentially related to motility. Our study revealed that the cheA3 (DVU2072) kinase mutant was impaired in the ability to form motility halos. Insertions in two other cheA loci did not exhibit a loss in this phenotype. The cheA3 mutant was also non-motile in capillary assays. Complementation with a plasmid-borne copy of cheA3 restores wild type phenotypes. The cheA3 mutant displayed a flagellum as observed by electron microscopy, grew normally in liquid medium, and was motile in wet mounts. In the growth conditions used, the D. vulgaris fliA mutant (DVU3229) for FliA, predicted to regulate flagella-related genes including cheA3, was defective both in flagellum formation and in forming the motility halos. In contrast, a deletion of the flp gene (DVU2116) encoding a pilin-related protein was similar to wild type. We conclude that wild type D. vulgaris forms motility halos on solid media that are mediated by flagella-related mechanisms via the CheA3 kinase. The conditions under which the CheA1 (DVU1594) and CheA2 (DVU1960) kinase function remain to be explored.
D. vulgaris Hildenborough has 72 response regulators. The Desulfovibrio are sulfate reducing bact... more D. vulgaris Hildenborough has 72 response regulators. The Desulfovibrio are sulfate reducing bacteria that are important in the sulfur and carbon cycles in anoxic habitats. Its large number of two componenent systems are probably critical to its ability to sense and respond to its environment. Our goal is to map these RRs to the genes they regulate using a DNA-affinity-purification-chip
Frontiers in microbiology, 2014
We surveyed the eight putative cyclic-di-GMP-modulating response regulators (RRs) in Desulfovibri... more We surveyed the eight putative cyclic-di-GMP-modulating response regulators (RRs) in Desulfovibrio vulgaris Hildenborough that are predicted to function via two-component signaling. Using purified proteins, we examined cyclic-di-GMP (c-di-GMP) production or turnover in vitro of all eight proteins. The two RRs containing only GGDEF domains (DVU2067, DVU0636) demonstrated c-di-GMP production activity in vitro. Of the remaining proteins, three RRs with HD-GYP domains (DVU0722, DVUA0086, and DVU2933) were confirmed to be Mn(2+)-dependent phosphodiesterases (PDEs) in vitro and converted c-di-GMP to its linear form, pGpG. DVU0408, containing both c-di-GMP production (GGDEF) and degradation domains (EAL), showed c-di-GMP turnover activity in vitro also with production of pGpG. No c-di-GMP related activity could be assigned to the RR DVU0330, containing a metal-dependent phosphohydrolase HD-OD domain, or to the HD-GYP domain RR, DVU1181. Studies included examining the impact of overexpresse...
Trends in Biotechnology, 2008
Engineered microorganisms are currently used for the production of food products, pharmaceuticals... more Engineered microorganisms are currently used for the production of food products, pharmaceuticals, ethanol fuel and more. Even so, the enormous potential of this technology has yet to be fully exploited. The need for sustainable sources of transportation fuels has generated a tremendous interest in technologies that enable biofuel production. Decades of work have produced a considerable knowledge-base for the physiology and pathway engineering of microbes, making microbial engineering an ideal strategy for producing biofuel. Although ethanol currently dominates the biofuel market, some of its inherent physical properties make it a less than ideal product. To highlight additional options, we review advances in microbial engineering for the production of other potential fuel molecules, using a variety of biosynthetic pathways.
PLoS Comput Biol, 2014
The study of intracellular metabolic fluxes and inter-species metabolite exchange for microbial c... more The study of intracellular metabolic fluxes and inter-species metabolite exchange for microbial communities is of crucial importance to understand and predict their behaviour. The most authoritative method of measuring intracellular fluxes, 13 C Metabolic Flux Analysis ( 13 C MFA), uses the labeling pattern obtained from metabolites (typically amino acids) during 13 C labeling experiments to derive intracellular fluxes. However, these metabolite labeling patterns cannot easily be obtained for each of the members of the community. Here we propose a new type of 13 C MFA that infers fluxes based on peptide labeling, instead of amino acid labeling. The advantage of this method resides in the fact that the peptide sequence can be used to identify the microbial species it originates from and, simultaneously, the peptide labeling can be used to infer intracellular metabolic fluxes. Peptide identity and labeling patterns can be obtained in a high-throughput manner from modern proteomics techniques. We show that, using this method, it is theoretically possible to recover intracellular metabolic fluxes in the same way as through the standard amino acid based 13 C MFA, and quantify the amount of information lost as a consequence of using peptides instead of amino acids. We show that by using a relatively small number of peptides we can counter this information loss. We computationally tested this method with a well-characterized simple microbial community consisting of two species.
Gene, 2000
A versatile expression vector utilizing a promoter of coliphage T5, PN25 (Gentz and Bujard, 1985.... more A versatile expression vector utilizing a promoter of coliphage T5, PN25 (Gentz and Bujard, 1985. J. Bacteriol. 164, 70–77) and a derivative of the IncW broad-host-range plasmid pJB20 (Beaupré et al., 1997. J. Bacteriol. 179, 78–89) has been developed. This vector successfully expresses virulence proteins of Agrobacterium tumefaciens encoded by virG and a mutant allele of virA, virA (Δ1–284, G665D)
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Papers by Aindrila Mukhopadhyay