Yunjon Han

A novel multifunctional cellulolytic enzyme screened from metagenomic resources representing ruminal bacteria

Metagenomic resources representing ruminal bacteria were screened for novel exocellulases using a robotic, high-throughput screening system, the novel CelEx-BR12 gene was identified and the predicted CelEx-BR12 protein was characterized. The CelEx-BR12 gene had an open reading frame (ORF) of 1140 base pairs that encoded a 380-amino-acid-protein with a predicted molecular mass of 41.8 kDa. The amino acid sequence was 83% identical to that of a family 5 glycosyl hydrolase from Prevotella ruminicola 23. Codon-optimized CelEx-BR12 was overexpressed in Escherichia coli and purified using Ni-NTA affinity chromatography. The Michaelis-Menten constant (Km value) and maximal reaction velocity (Vmax values) for exocellulase activity were 12.92 μM and 1.55 × 10(-)(4 )μmol min(-1), respectively, and the enzyme was optimally active at pH 5.0 and 37°C. Multifunctional activities were observed against fluorogenic and natural glycosides, such as 4-methylumbelliferyl-β-d-cellobioside (0.3 U mg(-1)), CMC (105.9 U mg(-1)), birch wood xylan (132.3 U mg(-1)), oat spelt xylan (67.9 U mg(-1)), and 2-hydroxyethyl-cellulose (26.3 U mg(-1)). Based on these findings, we believe that CelEx-BR12 is an efficient multifunctional enzyme as endocellulase/exocellulase/xylanase activities that may prove useful for biotechnological applications.

Strategy for screening metagenomic resources for exocellulase activity using a robotic, high-throughput screening system

Exocellulases play a key role in cleaving the accessible ends of cellulose molecules to release soluble glucose and cellobiose. To date, there have been no screens for exocellulase owing to assay protocol limitations, the high cost of substrates, and low activity of exocellulases compared with endocellulases. This study is the first to demonstrate direct screening for exocellulase activity using a robotic, high-throughput screening (HTS) system. Cell growth in 96-well plates was measured by monitoring optical density over 11-14h at 37°C with agitation. Fluorescence of methylumbelliferyl groups released from 4-methylumbelliferyl-β-D-cellobioside was determined using a VICTOR3 microplate reader. This new HTS system enabled activity verification of more than 10(4) clones per day. As a result, we obtained four exocellulases clones (CelEx-SF301, CelEx-SF309, CelEx-BR12 and CelEx-BR15) from 29,006 metagenomic fosmid clones that had previously been prepared from sweet potato field soil microbes and rumen fluid. This powerful approach could be effectively applied to screen various metagenomic resources for new enzymes.

Itaconic acid production from glycerol using Escherichia coli harboring a random synonymous codon-substituted 5'-coding region variant of the cadA gene.

Aspergillus terreus cadA, encoding cis‐aconitate decarboxylase, is an essential gene for itaconic acid (IA) biosynthesis, but it is primarily expressed as insoluble aggregates in most industrial hosts. This has been a hurdle for the development of recombinant strategies for IA production. Here, we created a library of synonymous codon variants (scv) of the cadA gene containing synonymous codons in the first 10 codons (except ATG) and screened it in Escherichia coli. Among positive clones, E. coli scvCadA_No8 showed more than 95% of expressed CadA in the soluble fraction, and in production runs, produced threefold more IA than wild‐type E. coli in Luria‐Bertani broth supplemented with 0.5% glucose. In M9 minimal media containing 0.85 g/L citrate and 1% glycerol, E. coli scvCadA_No8 produced 985.6 ± 33.4 mg/L IA during a 72‐h culture after induction with isopropyl β‐D‐1‐thiogalactopyranoside. In a 2‐L fed‐batch fermentation consisting of two stages (growth and nitrogen limitation conditions), we obtained 7.2 g/L IA by using E. coli by introducing only the scv_cadA gene and optimizing culture conditions for IA production. These results could be combined with metabolic engineering and generate an E. coli strain as an industrial IA producer. Biotechnol. Bioeng. 2016;113: 1504–1510.

A Rapid and Simple Method for Preparing an Insoluble Substrate for Screening of Microbial Xylanase

Several types of enzymes, including cellulases and xylanases, are required to degrade hemicelluloses and cellulose, which are major components of lignocellulosic biomass. Such degradative processes can be used to produce various useful industrial biomaterials. Screening methods for detecting polysaccharide-degrading microorganisms include the use of dye-labeled substrates in growth medium and culture plate staining techniques. However, the preparation of screening plates, which typically involves chemical cross-linking to synthesize a dye-labeled substrate, is a complicated and time-consuming process. Moreover, such commercial substrates are very expensive, costing tenfold more than the natural xylan. Staining methods are also problematic because they may damage relevant microorganisms and are associated with contamination of colonies of desirable organisms with adjacent unwanted bacteria. In the present study, we describe a sonication method for the simple and rapid preparation of an insoluble substrate that can be used to screen for xylanase-expressing bacteria in microbial populations. Using this new method, we have successfully isolated a novel xylanase gene from a xylolytic microorganism termed Xyl02-KBRB and Xyl14-KBRB in the bovine rumen.