Expression of heterologous sigma factors enables functional screening of metagenomic and heterologous genomic libraries

Gaida, Stefan M.; Sandoval, Nicholas R.; Nicolaou, Savvas; Chen, Yili; Venkataramanan, Keerthi P.; Papoutsakis, Eleftherios T.

doi:10.1038/ncomms8045

Cited by 60 publications

(45 citation statements)

References 49 publications

(58 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A second possible explanation is that those GH94s potentially present in the library have limited expression, which in many cases is due to the E. coli host RNA polymerase's inability to recognize foreign promoter sequences on the fosmid DNA 42,43 . This limitation can be compensated for by equipping the host strain with additional sigma factors that help it recognize a wider range of promoter sequences, 44 however the host strain used in this study contained only the native E. coli sigma factors. A third possibility is that those GH94s that are expressed are not capable of cleaving DNPGlc.…”

Section: Discussionmentioning

confidence: 99%

Structural and mechanistic analysis of a β-glycoside phosphorylase identified by screening a metagenomic library

Macdonald

Patel

Larmour

et al. 2018

Journal of Biological Chemistry

View full text Add to dashboard Cite

Glycoside phosphorylases have considerable potential as catalysts for the assembly of useful glycans for products ranging from functional foods and prebiotics to novel materials. However, the substrate diversity of currently identified phosphorylases is relatively small, limiting their practical applications. To address this limitation, we developed a high-throughput screening approach using the activated substrate 2,4-dinitrophenyl β-D-glucoside (DNPGlc) and inorganic phosphate for identifying glycoside phosphorylase activity and used it to screen a large insert metagenomic library. The initial screen, based on release of 2,4-dinitrophenol from DNPGlc in the presence of phosphate, identified the gene bglP, encoding a retaining β-glycoside phosphorylase from the CAZy GH3 family. Kinetic and mechanistic analysis of the gene product, BglP, confirmed a double displacement ping-pong mechanism involving a covalent glycosyl-enzyme intermediate. X-ray crystallographic analysis provided insights into the phosphate-binding mode and identified a key glutamine residue in the active site important for substrate recognition. Substituting this glutamine for a serine swapped the substrate specificity from glucoside to Nacetylglucosaminide. In summary, we present a high-throughput screening approach for identifying β-glycoside phosphorylases, which was robust, simple to implement, and useful in identifying active clones within a metagenomics library. GH3 β-glycoside phosphorylase from a metagenomic library 2 Implementation of this screen enabled discovery of a new glycoside phosphorylase class and has paved the way to devising simple ways in which enzyme specificity can be encoded and swapped, which has implications for biotechnological applications.Carbohydrate active enzymes (CAZymes) are the biocatalysts responsible for the assembly, degradation and modification of glycans in biological systems 1 . They are also widely employed enzymes in industry, being used in brewing and food processing, animal feed preparation, industrial pulp and paper applications and increasingly in biofuel and bioproduct development [2][3][4][5] . While the use of CAZymes is cost-effective in glycan degradation, glycan assembly generally requires the use of expensive starting materials, such as nucleotide phosphosugars 6 . The high-cost of these materials makes de novo industrial-scale glycan synthesis difficult and usually non-viable.One class of CAZyme that offers a potential solution to the high costs typically associated with enzymatic glycan synthesis is that of the glycoside phosphorylases (GPs), which are increasingly being recognized and used for the biocatalysis and biotransformation of glycans 7-9 . These enzymes ordinarily carry out phosphorolysis by transferring a glycosyl moiety from the nonreducing end of a di-or polysaccharide substrate onto inorganic phosphate, thereby generating a sugar-1-phosphate 10 ( Figure 1A). GPs distinguish themselves from most CAZymes in that the hydrolytic free energy associated with the glycosidic e...

show abstract

Section: Discussionmentioning

confidence: 99%

Structural and mechanistic analysis of a β-glycoside phosphorylase identified by screening a metagenomic library

Macdonald

Patel

Larmour

et al. 2018

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…As reviewed[2], its membranes contain over 75% vaccenic acid (18:1) in polar lipids, while its cytoplasmic membrane contains hopanoids that apparently stabilize the phospholipid bilayer to resist solvent fluidization. These are but a few of the likely large number of unusual membrane and cell-wall compositions that have evolved in nature to handle chemical toxicity and such capabilities could be explored through modern metagenomic tools and screens[68,69]. Thus, we call for both such naturally inspired and knowledge-driven ab initio design of cell membranes and walls that would exceed the best of the most tolerant organisms known, e.g., species of Pseudomonas , Zymomonas , Lactobacillus , Rhodococcus , Deinococcus , and Nocardioides [2].…”

Section: Recommendations For Future Direction: a Call For A Syntheticmentioning

confidence: 99%

Engineering membrane and cell-wall programs for tolerance to toxic chemicals: Beyond solo genes

Sandoval

Papoutsakis

2016

Current Opinion in Microbiology

View full text Add to dashboard Cite

Metabolite toxicity in microbes, particularly at the membrane, remains a bottleneck in the production of fuels and chemicals. Under chemical stress, native adaptation mechanisms combat hyper-fluidization by modifying the phospholipids in the membrane. Recent work in fluxomics reveals the mechanism of how membrane damage negatively affects energy metabolism while lipidomic and transcriptomic analyses show that strains evolved to be tolerant maintain membrane fluidity under stress through a variety of mechanisms such as incorporation of cyclopropanated fatty acids, trans-unsaturated fatty acids, and upregulation of cell wall biosynthesis genes. Engineered strains with modifications made in the biosynthesis of fatty acids, peptidoglycan, and lipopolysaccharide have shown increased tolerance to exogenous stress as well as increased production of desired metabolites of industrial importance. We review recent advances in elucidation of mechanisms or toxicity and tolerance as well as efforts to engineer the bacterial membrane and cell wall.

show abstract

“…Apart from using alternate hosts, E. coli strains have also been improved to increase metagenomic gene expression. In some studies, heterologous sigma factors, capable of recognizing heterologous promoters, were expressed in E. coli cells enabling higher expression of metagenomic DNA (Stevens, Conway et al 2013, Gaida, Sandoval et al 2015. The insertion of a constitutive promoter at the multiple cloning site (MCS) of the plasmid vector in expression host showed a significantly higher expression of the gene placed downstream to the MCS region (Frebourg and Brison 1988).…”

Section: Choice Of Hostsmentioning

confidence: 99%

Contemporary molecular tools in microbial ecology and their application to advancing biotechnology

Rashid

Stingl

2015

Biotechnology Advances

View full text Add to dashboard Cite

Expression of heterologous sigma factors enables functional screening of metagenomic and heterologous genomic libraries

Cited by 60 publications

References 49 publications

Structural and mechanistic analysis of a β-glycoside phosphorylase identified by screening a metagenomic library

Structural and mechanistic analysis of a β-glycoside phosphorylase identified by screening a metagenomic library

Engineering membrane and cell-wall programs for tolerance to toxic chemicals: Beyond solo genes

Contemporary molecular tools in microbial ecology and their application to advancing biotechnology

Contact Info

Product

Resources

About