Comprehensive structural analysis of the terminal Myxalamid reductase domain for the engineered production of primary alcohols Background • The terminal reductase (R) domain from the non- ribosomal peptide synthetase (NRPS) module MxaA in Stigmatella aurantiaca Sga15 catalyzes a non- processive four-electron reduction to produce the myxalamide family of secondary metabolites • Despite widespread use in nature, a lack of structural and mechanistic information concerning reductive release from polyketide synthase (PKS) and NRPS assembly lines principally limits our ability to redesign R domains with altered or improved activity. Approach and Outcomes • The crystal structures of the MxaA reductase in the presence and absence of the cofactor NADPH have been solved. • In addition, computational and structural findings provided a basis for mechanistic investigations and, in the process, delivered a rationally altered variant with improved activity towards highly reduced substrate. Significance • The results presented here provide a comprehensive understanding of these unique termination domains and, in the process, set a strong foundation for future efforts to generate new PKS- or NRPS-based routes to diverse terminal alcohol containing compounds. J.F. Barajas et al. (2015). ”Comprehensive structural analysis of the terminal myxalamid reductase domain for the engineered production of primary alcohols". Chem Biol, DOI: http://dx.doi.org/10.1016/j.chembiol.2015.06.022 PCP PCP NADPH NADPH R R
Transcript
Comprehensive structural analysis of the terminal Myxalamid reductase domain for theengineered production of primary alcohols
Background• The terminal reductase (R) domain from the non-
ribosomal peptide synthetase (NRPS) module MxaAin Stigmatella aurantiaca Sga15 catalyzes a non-processive four-electron reduction to produce themyxalamide family of secondary metabolites
• Despite widespread use in nature, a lack of structuraland mechanistic information concerning reductiverelease from polyketide synthase (PKS) and NRPSassembly lines principally limits our ability to redesignR domains with altered or improved activity.
Approach and Outcomes• The crystal structures of the MxaA reductase in the
presence and absence of the cofactor NADPH havebeen solved.
• In addition, computational and structural findingsprovided a basis for mechanistic investigations and, inthe process, delivered a rationally altered variant withimproved activity towards highly reduced substrate.
Significance• The results presented here provide a comprehensive understanding of these unique termination
domains and, in the process, set a strong foundation for future efforts to generate new PKS- or NRPS-based routes to diverse terminal alcohol containing compounds.
J.F. Barajas et al. (2015). ”Comprehensive structural analysis of the terminal myxalamid reductase domain for the engineered production of primary alcohols". Chem Biol, DOI: http://dx.doi.org/10.1016/j.chembiol.2015.06.022
PCPPCP
NADPH NADPH
RR
Natural products as biofuels and bio-based chemicals: fatty acids and isoprenoids
Background• Natural products are best known for their uses in medicine and
agriculture. To date, more than 20,000 natural products have beenidentified from bacteria, fungi, plants, and marine sources. Althoughthese compounds are not typically considered as candidates for liquidtransportation fuels, a number of fatty acid-derived and isoprenoidnatural products are being developed for use as renewable biofuels andbio-based chemicals.
Significance• This comprehensive review summarizes recent work (2005-2015) on
fatty acid-derived compounds (fatty acid alkyl esters, fatty alcohols,medium- and short-chain methyl ketones, alkanes, -olefins, and long-chain internal alkenes) and isoprenoids, including hemiterpenes (e.g.,isoprene and isopentanol), monoterpenes (e.g., limonene), andsesquiterpenes (e.g., farnesene and bisabolene).
• Accelerated R&D activity in biofuels over the past decade has facilitatedthe discovery of a variety of enzymes that enable biochemicalconversion of fatty acids (and intermediates of fatty acid biosynthesis)to a range of industrially relevant compound classes.
• To date, gram-per-liter titers (in batch culture) and moderate yields havebeen reported for the fatty acid-derived methyl ketones and fatty acidethyl esters and for the isoprenoids isopentanol, bisabolene, andfarnesene. Given the extremely rapid pace of developments insynthetic biology and systems biology, dramatic improvements inproduction are within reach.H. Beller et al. (2015). "Natural products as biofuels and bio‐based chemicals: fatty acids and isoprenoids". Natural Product Reports, doi: 10.1039/c5np00068h .
Biocomposite adhesion without added resin:understanding the chemistry of the directconversion of wood into adhesives
Background• Wood is a ubiquitous natural resource that is a
cornerstone material for industrialized society.• Composite materials based on wood require
adhesives, most of which are currently derived fromnon-renewable sources.
• The realization of an effective bio-renewable adhesivefor the production of composite materials is highlydesired.
Approach and Outcomes• Laser modification of wood surfaces created a unique
wood surface that could undergo bonding by hotpressing.
• Compositional analysis of the material at the surfacerevealed it was predominately composed of celluloseand lignin that had been partially depolymerized.
• This produces a very strong bonded interface suitablefor composite materials based on wood.
Significance• As cellulose and lignin are two of the most abundant polymers found in the world, the manipulation
of them into a structural heat setting resin provides a new potential route for the direct utilization of biomass.
J.A. Dolan et al. (2015). "Biocomposite adhesion without added resin: understanding the chemistry of the direct conversion of wood into adhesives". RSC Adv., 5(82), 67267‐67276.
Illustrative model of laser induced changes of a wood surface at the nanoscale level and hot pressed bondline illustrating potential adhesion mechanism.
Tight regulation of plant immune responses by combining promoter and suicide exon elements
Outcomes• Developed a very tight expression system by stacking the suicide exon technology on top of the DEX inducible promoter system • Regenerated viable and healthy plants harboring the resistance gene and the dual regulated effector that can be induced on
demand to initiate the HR phenotype• Demonstrate that this dual regulation strategy is generalizable to control the expression of various toxic proteins or biosynthetic
enzymes
Use of a dual transcription and splicing regulation to eliminate leaky hypersensitive response
Gonzalez, et al. (2015) “Tight regulation of plant immune responses by combining promoter and suicide exon elements.” NAR. DOI: 10.1093/nar/gkv655.
Background• Effector genes regulated by inducible promoters
cause background cell death due to leaky proteinexpression.
• Leaky protein expression from induciblepromoters is a common limitation in transgenicplant generation
Approach• Utilization of a suicide exon to tightly regulate the
expression of effector proteins.• Stacking of the DEX inducible promoter system
on top of the suicide exon technology to furthercontrol the expression of effector proteins
Significance• This dual regulation system is generally applicable to genes that initiate severe or undesired
phenotypes upon leaky expression and provides a straightforward and promising way to generate many previously unattainable transgenic crops.
Phylogenomics databases for facilitatingfunctional genomics in rice
Outcomes• Functional genomics studies of rice genes belonging to a single family can be facilitated by phylogenomic analysis
that integrates diverse types of biological information. • Future prospects to advance analysis of gene function include coupling phylogenomic analyses with computational
predictions of gene function.
BackgroundAmong the 42,653 predicted (non-transposon) genetic elements in the rice genome, 21,998 encode multiple members that make up 3,865 protein families. Thus, each gene within the rice genome has a 51.6% possibility of being functionally redundant (21,998 / 42,653).
ApproachPhylogenomics databases for six large gene families, i.e., those for kinase, glycosyltransferases (GTs), glycoside hydrolases (GHs), transcription factors (TFs), transporters, and cytochrome p450 monooxygenases (P450s), have been constructed.
Significance• In this review key features and applications of these databases has been introduced to
serve as a very useful guide in the post-genomics era of the research.
K.H. Jung et al. (2015). "Phylogenomics databases for facilitating functional genomics in rice". Rice (N Y), 8(1), 60.
Recent innovations in analytical methods for the qualitative and quantitative assessment of ligninBackground• Lignin has frequently been considered a waste‐product from
the deconstruction of plant cell walls, in attempts to isolate polysaccharides that can be hydrolyzed and fermented into fuel or other valuable commodities.
• In order to develop useful applications for lignin, accurate analytical instrumentation and methodologies are required to qualitatively and quantitatively assess.
• This review seeks to provide a comprehensive overview of many of the advancements achieved in evaluating key lignin attributes.
J.S. Lupoi et al., (2015) “Recent innovations in analytical methods for the qualitative and quantitative assessment of lignin”. Renewable and Sustainable Energy Reviews, 49, 871–906.
Schematic representation of lignin including syringyl(S, blue), guaiacyl (G, green), and p-hydroxyphenol (H, red) phenylpropanoid moieties, and lignin–lignin linkages
Common fragments of lignin measured after pyrolysis.
Significance/perspective• High‐throughput, multivariate analysis modeling is an
effective technique to efficiently screen biomass for key biofuel traits.
• Advances in NMR, imaging, mass spectrometry, computational approaches, and vibrational spectroscopy have facilitated the construction of toolbox.
• Techniques developed for probing lignin structure, linkages to carbohydrates, and quantifying specific functionalities and linkages present in lignin and pyMBMS have provided another innovative technique for probing lignin monomer content.
• Further advances in these analytical tools can only build upon the foundation laid by the research described in this review.
Localization of polyhydroxybutyrate in sugarcane using Fourier-transform infrared microspectroscopy and multivariate imagingBackground• The production of bio-based, biodegradable plastics
from or in plants can assist in supplanting those manufactured using fossil fuels.
• Polyhydroxybutyrate (PHB) is a biodegradable polyester that has been evaluated as a possible candidate for relinquishing the use of conventional plastics.
Approach and Outcomes• PHB, possessing similar properties to polyesters
produced from non-renewable sources, has been previously engineered to be expressed in sugarcane.
• This manuscript illustrates the coupling of a Fourier-transform infrared microspectrometer, equipped with a focal plane array (FPA) detector, with multivariate imaging to successfully identify and localize PHB aggregates in planta.
Significance• This study demonstrates the power of IR microspectroscopy to rapidly image plant sections to
provide a snapshot of the chemical composition of the cell. • While PHB was localized in sugarcane, this method is readily transferable to other value-added co-
products expressed in different plant tissues.
Images of polyhydroxybutyrate‐containing sugarcane (a) without and (b) with spectral points selected. The images were collected using a 128 × 128 focal plane array. c Re‐constructed image using the second principal component; (d) Loadings plot for the second principal component used in re‐constructing the image in (c).
J.S. Lupoi et al. (2015). "Localization of polyhydroxybutyrate in sugarcane using Fourier‐transform infrared microspectroscopy and multivariate imaging". Biotechnol Biofuels, 8, 98.
Standards for plant synthetic biology: a common syntax for exchange of DNA parts
Outcomes• Developed standards for Type IIS restriction endonuclease-mediated assembly• Defined a common syntax of 12 fusion sites to enable the facile assembly of eukaryotic transcriptional units
Twelve fusion sites have been defined allowing a multitude of standard parts to be generated to build synthetic genes.
Patron et al. (2015) “Standards for plant synthetic biology: a common syntax for exchange of DNA parts.” New Phytol. DOI: 10.1111/nph.13532 (2015).
Background• Synthetic biology requires the development of
biological parts for the engineering of plants.• In the plant field, synthetic biology tools are
rather limited, none compatible with each others,and are poorly exchangeable between labs
Approach• Standardization of DNA parts will accelerate
plant bioengineering.• An international consortium including inventors,
developers and adopters of Golden Gate cloningmethods agreed to use a Type IIS geneticgrammar for plants that is also extendible to alleukaryotes
Significance• Enables sharing characterized DNA parts between laboratory across the world, speedup
fundamental research discoveries, leverage crop engineering, reduce experimental costs.
Outcomes• The reduced lignin Arabidopsis engineered lines resulted in high levels of monomeric sugar release at lower pretreatment
temperatures as compared to the wild type. • Ionic liquid pretreatment of the engineered Arabidopsis at 70 °C for 5 hours resulted in improved saccharification
efficiency and increased hemicellulose recovery for the pretreated biomass and produced similar total sugar yields as compared to those obtained after pretreatment at 140 °C for 3 hours.
BackgroundIncreasing the accumulation of polysaccharides in biomass and improving biomass digestibility could have significant beneficial impacts on the cost of lignocellulosic biofuel production both by increasing fermentable sugar yield per acre and reducing the severity of pretreatment Approach
ApproachWe engineered Arabidopsis strains withreduced lignin levels that carry mutations of C4H and NST1, which, respectively, restrict lignin biosynthesis to vessels, and enhance polysaccharide levels with little to no apparent negative impact on growth phenotype. These strains were tested using IL pretreatment for reducing severity of the pretreatment.
SignificanceSimilar sugar recovery at the lower temperature pretreatment supports the hypothesis that reducinglignin can reduce the necessary severity of pretreatment needed, and increased polysaccharide
deposition can increase glucose recovery on a mass basis.
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C. Scullin et al. (2015) "Restricting lignin and enhancing sugar deposition in secondary cell walls enhances monomeric sugar release after low temperature ionic liquid pretreatment". Biotechnol Biofuels, 8, 95.
Outcomes• Protocols can be translated for liquid-handling robots, microfluidic devices, automated microscopes, and people.• DNA assembly and mutatagenesis protocols were successfully performed on a robot, a microfluidic device, and by hand.
L. Sun et al. (2015). "Non‐invasive imaging of cellulose microfibril orientation within plant cell walls by polarized Raman microspectroscopy". Biotechnol Bioeng. doi, 10.1002/bit.25690
Background• Reliable screening methods
are needed to assess the consequence of genetic mutation on the cell wall properties, ideally with predictive value on the mechanical stability of the feedstock plant.
Approach• Develop an optical approach
(including automated analysis) that combines the orientation sensitivity of polarized light with the macromolecular specificity of Raman Microspectroscopy
Significance• Enables quick assessment of mutation on the order of plant cell wall microfibril
organization for a predictive understanding of mechanical plant properties.
MUCI10 produces galactoglucomannan that maintains pectin and cellulose architecture in Arabidopsis seed mucilage
Background• Mannans are abundant hemicelluloses in plants• The biosynthesis is not fully understood.
Approach and Outcomes• muci10 is a mutants with altered seed mucilage• muci10 is mutated in a glycosyltransferase in the GT34 family.• Arabidopsis seed mucilage was shown to contain abundant
galactoglucomannan. This is different from seedgalactomannans described in other species and from mannan invegetative tissue.
• MUCI10 is an alpha-1,6-galactosyltransferase that addsgalactose to the glucomannan backbone.
• Branched mannans are important for cellulose deposition.
Significance• Most glycosyltransferases still have unknown function. This study
increases our basic understanding of cell wall biosynthesis andprovides new tools for cell wall engineering. Understandinggalactoglucomannan biosynthesis is important since it is themost abundant hemicellulose in gymnosperm wood. Knowing theenzymes required for glucomannan biosynthesis enablesengineering of bioenergy crops plants with increasedhexose/pentose ratio, which would be advantageous forfermentation into fuels and bioproducts.
Voiniciuc et al. (2015). "MUCI10 Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage”. Plant Physiol, doi 10.1104/pp.15.00851.
muci10 has decreased seed mucilage. The csla2mutant which cannot synthesize the glucomannanbackbone is shown for comparison
MUCI10 adds alpha‐1,6‐linked galactoseresidues to the glucomannanbackbone
Interlaboratory study to evaluate the robustness of capillary electrophoresis with mass spectrometry for peptide mapping
Background• Due to the different separation mechanism, peptides
missed in reversed-phase LC–MS can be detected by CE–MS, rendering this technology an attractive complementary or alternative tool for peptide mapping.
• In an effort to illustrate the robustness and portability of CE–MS for peptide mapping, an international team was formed, which includes 13 independent laboratories from companies and universities in the United States, Canada, Australia, and Europe.
Approach and Outcomes• Trypsin digested BSA was chosen as a model system. • All laboratories analyzed the sample on two
consecutive days. • Migration time and peak intensity of a representative
set of ten target peptides were evaluated and a statistical analysis was performed based on the principles of the ISO 5725-2 guideline
Significance• Repeatability levels of 9–12% RSD obtained by these laboratories agree with results reported by
former studies employing CE–MS- or LC–MS-based methods.• This study emphasizes the importance of tightly controlled system suitability tests for a successful
method transfer across multiple sites.
C. Wenz et al. (2015). "Interlaboratory study to evaluate the robustness of capillary electrophoresis with mass spectrometry for peptide mapping". J Sep Sci. doi, 10.1002/jssc.201500551
Repeatability andreproducibility (given as % RSD) of tm(laboratories 1–11) (A) and %A (laboratories 1–9) (B) were plotted against their general meanfor all target peptides.
Linear regression trendlines are indicated as dashed lines. Correlation coefficient (R2) values are given asinsets.
