Expression of foreign pathways often results in suboptimal performance due to unintended factors such as introduction of toxic metabolites, cofactor imbalances or poor expression of pathway ...components. In this study we report a 120% improvement in the production of the isoprenoid-derived sesquiterpene, amorphadiene, produced by an engineered strain of
Escherichia coli developed to express the native seven-gene mevalonate pathway from
Saccharomyces cerevisiae (
Martin et al. 2003). This substantial improvement was made by varying only a single component of the pathway (HMG-CoA reductase) and subsequent host optimization to improve cofactor availability. We characterized and tested five variant HMG-CoA reductases obtained from publicly available genome databases with differing kinetic properties and cofactor requirements. The results of our
in vitro and
in vivo analyses of these enzymes implicate substrate inhibition of mevalonate kinase as an important factor in optimization of the engineered mevalonate pathway. Consequently, the NADH-dependent HMG-CoA reductase from
Delftia acidovorans, which appeared to have the optimal kinetic parameters to balance HMG-CoA levels below the cellular toxicity threshold of
E. coli and those of mevalonate below inhibitory concentrations for mevalonate kinase, was identified as the best producer for amorphadiene (54% improvement over the native pathway enzyme, resulting in 2.5
mM or 520
mg/L of amorphadiene after 48
h). We further enhanced performance of the strain bearing the
D. acidovorans HMG-CoA reductase by increasing the intracellular levels of its preferred cofactor (NADH) using a NAD
+-dependent formate dehydrogenase from
Candida boidinii, along with formate supplementation. This resulted in an overall improvement of the system by 120% resulting in 3.5
mM or 700
mg/L amorphadiene after 48
h of fermentation. This comprehensive study incorporated analysis of several key parameters for metabolic design such as
in vitro and
in vivo kinetic performance of variant enzymes, intracellular levels of protein expression, in-pathway substrate inhibition and cofactor management to enable the observed improvements. These metrics may be applied to a broad range of heterologous pathways for improving the production of biologically derived compounds.
► Variant HMG-CoA reductases to optimize mevalonate pathway engineered in
E. coli. ► Differences in cofactor specificities and kinetic properties. ► Key factors for optimization—HMG-CoA toxicity and mevalonate kinase inhibition. ► NADH-dependent HMG-CoA reductase from
Delftia acidovorans is best performer. ► Further improvement achieved by enhancing intracellular NADH levels.
Microbial engineering strategies that elicit global metabolic perturbations have the capacity to increase organism robustness for targeted metabolite production. In particular, perturbations to ...regulators of cellular systems that impact glycolysis and amino acid production while simultaneously decreasing fermentation by-products such as acetate and CO(2) make ideal targets. Intriguingly, perturbation of the Carbon Storage Regulator (Csr) system has been previously implicated in large changes in central carbon metabolism in E. coli. Therefore, we hypothesized that perturbation of the Csr system through the CsrA-CsrB ribonucleoprotein complex might increase production of biofuels and their intermediates from heterologous pathways.
We engaged the CsrA-CsrB ribonucleoprotein complex of E. coli via overexpression of CsrB. CsrB is a 350-nucleotide non-coding RNA that antagonizes CsrA, an RNA-binding protein that regulates translation of specific mRNA targets. By using shotgun proteomics and targeted metabolomics we established that elevation of CsrB levels leads to alterations in metabolite and protein levels in glycolysis, the TCA cycle and amino acid levels. Consequently, we show that such changes can be suitably applied to improve the production of desired compounds through the native fatty acid and heterologous n-butanol and isoprenoid pathways by up to two-fold. We also observed concomitant decreases in undesirable fermentation by-products such as acetate and CO(2).
We have demonstrated that simple engineering of the RNA-based Csr global regulatory system constitutes a novel approach to obtaining pathway-independent improvements within engineered hosts. Additionally, since Csr is conserved across most prokaryotic species, this approach may also be amenable to a wide variety of production hosts.
DNA from low-biodiversity fracture water collected at 2.8-kilometer depth in a South African gold mine was sequenced and assembled into a single, complete genome. This bacterium, Candidatus ...Desulforudis audaxviator, composes >99.9% of the microorganisms inhabiting the fluid phase of this particular fracture. Its genome indicates a motile, sporulating, sulfate-reducing, chemoautotrophic thermophile that can fix its own nitrogen and carbon by using machinery shared with archaea. Candidatus Desulforudis audaxviator is capable of an independent life-style well suited to long-term isolation from the photosphere deep within Earth's crust and offers an example of a natural ecosystem that appears to have its biological component entirely encoded within a single genome.
Humid tropical forest soils are characterized by low and fluctuating redox, conditions which are thought to inhibit organic matter degradation by microbes. However, evidence suggests that soil ...microbial communities are adapted to the redox conditions in these ecosystems. In this study we tested the hypothesis that soil oxygen (O2) availability as an index of redox conditions structures patterns in litter decomposition and associated microbial community dynamics over space and time in humid tropical forests. We conducted a two year decomposition experiment on a common litter substrate in four sites along an elevational gradient with well described climate and redox dynamics. Microbial community sequencing, potential enzyme activities, and litter chemistry measurements were made on litter and soil to determine the relationship between soil and litter communities and biogeochemistry. Decomposition was slowest in the upper elevation site, which was the wettest and had the lowest average soil O2 availability. However, soil hydrolytic and litter phenol oxidase activities were greatest at this site. Small subunit ribosomal RNA genes were sequenced with universal primers for bacteria, archaea and eukaryotes, yielding 40,850 unique taxa after quality filtering and clustering. Across all sites, microbial succession was observed as increasing litter richness, converging bacterial community profiles, and diverging fungal community profiles. Initial decomposers (1–4 weeks) included many r-selected bacteria, including Alpha-, Beta- and Gammaproteobacteria, Clostridia, Bacteroidetes. We also found evidence of anaerobic fungi such as Cryptococcus, as well as the plant-associated Phialocephala and Phyllachora species, suggesting that anaerobic and plant-associated fungi are prevalent later in decomposition in soils with low and fluctuating redox conditions. Because of the striking similarities between sites in functional potential despite differences in wet tropical soil decomposing communities and litter chemistry, we suggest that future climate-driven disruptions to redox fluctuations could significantly alter the terrestrial carbon (C) cycle in tropical forests.
•We conducted a 2 year litter decomposition experiment in 4 wet tropical forest sites.•Sites sat along a gradient with well described climate and redox dynamics.•Litter decomposition under fluctuating redox proceeded as quickly as the aerobic site.•Succession on litter emerged as converging bacterial but diverging fungal communities.
We have developed a method that combines the ROSETTA de novo protein folding and refinement protocol with distance constraints derived from homologous structures to build homology models that are ...frequently more accurate than their templates. We test this method by building complete-chain models for a benchmark set of 22 proteins, each with 1 or 2 candidate templates, for a total of 39 test cases. We use structure-based and sequence-based alignments for each of the test cases. All atoms, including hydrogens, are represented explicitly. The resulting models contain approximately the same number of atomic overlaps as experimentally determined crystal structures and maintain good stereochemistry. The most accurate models can be identified by their energies, and in 22 of 39 cases a model that is more accurate than the template over aligned regions is one of the 10 lowest-energy models.
Harnessing the biotechnological potential of the large number of proteins available in sequence databases requires scalable methods for functional characterization. Here we propose a workflow to ...address this challenge by combining phylogenomic guided DNA synthesis with high-throughput mass spectrometry and apply it to the systematic characterization of GH1 β-glucosidases, a family of enzymes necessary for biomass hydrolysis, an important step in the conversion of lignocellulosic feedstocks to fuels and chemicals. We synthesized and expressed 175 GH1s, selected from over 2000 candidate sequences to cover maximum sequence diversity. These enzymes were functionally characterized over a range of temperatures and pHs using nanostructure-initiator mass spectrometry (NIMS), generating over 10,000 data points. When combined with HPLC-based sugar profiling, we observed GH1 enzymes active over a broad temperature range and toward many different β-linked disaccharides. For some GH1s we also observed activity toward laminarin, a more complex oligosaccharide present as a major component of macroalgae. An area of particular interest was the identification of GH1 enzymes compatible with the ionic liquid 1-ethyl-3-methylimidazolium acetate (C2mimOAc), a next-generation biomass pretreatment technology. We thus searched for GH1 enzymes active at 70 °C and 20% (v/v) C2mimOAc over the course of a 24-h saccharification reaction. Using our unbiased approach, we identified multiple enzymes of different phylogentic origin with such activities. Our approach of characterizing sequence diversity through targeted gene synthesis coupled to high-throughput screening technologies is a broadly applicable paradigm for a wide range of biological problems.
We use the Rosetta
de novo structure prediction method to produce three-dimensional structure models for all Pfam-A sequence families with average length under 150 residues and no link to any protein ...of known structure. To estimate the reliability of the predictions, the method was calibrated on 131 proteins of known structure. For approximately 60% of the proteins one of the top five models was correctly predicted for 50 or more residues, and for approximately 35%, the correct SCOP superfamily was identified in a structure-based search of the Protein Data Bank using one of the models. This performance is consistent with results from the fourth critical assessment of structure prediction (CASP4). Correct and incorrect predictions could be partially distinguished using a confidence function based on a combination of simulation convergence, protein length and the similarity of a given structure prediction to known protein structures. While the limited accuracy and reliability of the method precludes definitive conclusions, the Pfam models provide the only tertiary structure information available for the 12% of publicly available sequences represented by these large protein families.
Background: Glycoside hydrolase family 5 (GH5) comprises enzymes with a wide range of activities critical for the deconstruction of lignocellulose.
Results: Concurrent glucan and mannan specificity ...in over 70 members of GH5 can be ascribed to a conserved active site motif.
Conclusion: Single domain multispecific hydrolases are widely prevalent.
Significance: This finding has potential applications in improved enzyme mixture design or microbes engineered for consolidated bioprocessing of lignocellulose.
Enzymes are traditionally viewed as having exquisite substrate specificity; however, recent evidence supports the notion that many enzymes have evolved activities against a range of substrates. The diversity of activities across glycoside hydrolase family 5 (GH5) suggests that this family of enzymes may contain numerous members with activities on multiple substrates. In this study, we combined structure- and sequence-based phylogenetic analysis with biochemical characterization to survey the prevalence of dual specificity for glucan- and mannan-based substrates in the GH5 family. Examination of amino acid profile differences between the subfamilies led to the identification and subsequent experimental confirmation of an active site motif indicative of dual specificity. The motif enabled us to successfully discover several new dually specific members of GH5, and this pattern is present in over 70 other enzymes, strongly suggesting that dual endoglucanase-mannanase activity is widespread in this family. In addition, reinstatement of the conserved motif in a wild type member of GH5 enhanced its catalytic efficiency on glucan and mannan substrates by 175 and 1,600%, respectively. Phylogenetic examination of other GH families further indicates that the prevalence of enzyme multispecificity in GHs may be greater than has been experimentally characterized. Single domain multispecific GHs may be exploited for developing improved enzyme cocktails or facile engineering of microbial hosts for consolidated bioprocessing of lignocellulose.
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