Naoko Okai

A review of enzymes and microbes for lignocellulosic biorefinery and the possibility of their application to consolidated bioprocessing technology

The biorefinery manufacturing process for producing chemicals and liquid fuels from biomass is a promising approach for securing energy and resources. To establish cost-effective fermentation of lignocellulosic biomass, the consolidation of sacccharification and fermentation processes is a desirable strategy, but requires the development of microorganisms capable of cellulose/hemicellulose hydrolysis and target chemical production. Such an endeavor requires a large number of prerequisites to be realized, including engineering microbial strains with high cellulolytic activity, high product yield, productivities, and titers, ability to use many carbon sources, and resistance to toxic compounds released during the pretreatment of lignocellulosic biomass. Researchers have focused on either engineering naturally cellulolytic microorganisms to improve product-related properties or modifying non-cellulolytic organisms with high product yields to become cellulolytic. This article reviews recent advances in the development of microorganisms for the production of renewable chemicals and advanced biofuels, as well as ethanol, from lignocellulosic materials through consolidated bioprocessing.

High-Throughput Transposon Mutagenesis of Corynebacterium glutamicum and Construction of a Single-Gene Disruptant Mutant Library

ABSTRACT A simple and high-throughput transposon-mediated mutagenesis system employing two different types of transposons in combination with direct genomic DNA amplification and thermal asymmetric interlaced PCR (TAIL-PCR) was developed. Each of the two minitransposons based on IS31831 (ISL3 family) and Tn5 (IS4 family) was integrated into the Corynebacterium glutamicum R genome. By using BLAST and Perl, transposon insertion locations were automatically identified based on the sequences of TAIL-PCR products of mutant cells. Insertion locations of 18,000 mutants were analyzed, and a comprehensive insertion library covering nearly 80% of the 2,990 open reading frames of C. glutamicum R was generated. Eight thousand of the mutants, exhibiting disruption in 2,330 genes, survived on complex medium under normal laboratory conditions, indicating that the genes were not essential for cell survival. Of the 2,330 genes, 30 exhibited high similarity to essential genes of Escherichia coli or Bacillus subtilis. This approach could be useful in furthering genetic understanding of cellular life and facilitating the functional analysis of microorganisms.

Regulation of the expression of phosphoenolpyruvate : carbohydrate phosphotransferase system (PTS) genes in Corynebacterium glutamicum R

The phosphoenolpyruvate : carbohydrate phosphotransferase system (PTS) catalyses the transport of carbohydrates by coupling carbohydrate translocation and phosphorylation. In Corynebacterium glutamicum R, the genes ptsH and ptsI encode general components of the PTS, and genes ptsF, ptsS and ptsG each encode fructose-, sucrose- and glucose-specific components of the PTS, respectively. In this study, we examined the mRNA levels of the pts genes in the presence or absence of PTS sugars. Glucose elevated the expression of ptsG, ptsH and ptsI genes, whereas fructose and sucrose induced the expression of all the pts genes examined, i.e. ptsF, -S, -G, -H and -I. We determined the transcriptional start sites of the pts genes and found that these promoters were activated in the presence of fructose. Disruption of fruR, which is a deoxyribonucleoside repressor (DeoR)-type transcriptional regulator co-transcribed with ptsF, resulted in enhanced induction of the fructose-pts operon, ptsI, and ptsH expression in response to fructose, indicating that FruR attenuates the induction of ptsI, ptsH and fructose-pts by fructose.

Corynebacterium glutamicum glyceraldehyde-3-phosphate dehydrogenase isoforms with opposite, ATP-dependent regulation.

Corynebacterium glutamicum gapA and gapB encode glyceraldehyde-3-phosphate dehydrogenases (GAPDHs) that differ in molecular weight and activity in the presence of ATP. Comparative genome analysis revealed that GapA, the product of gapA, represented the canonical GAPDH that is highly conserved across the three major life forms. GapB, with an additional 110-residue-long sequence upstream of its GAPDH-specific domain, was homologous only to select microbial putative GAPDHs. Upon gene disruption, the initial growth rates of the wild-type, ΔgapA and ΔgapB strains on glucose (0.77, 0.00 and 0.76 h–1, respectively), lactate (0.20, 0.18 and 0.15 h–1, respectively), pyruvate (0.39, 0.29 and 0.20 h–1, respectively), and acetate (0.06, 0.06 and 0.04 h–1, respectively), implied that GapA was indispensable for growth on glucose, that GapB, but not GapA, affected early growth on acetate, and that GapB had a greater influence on growth under gluconeogenic conditions than GapA. The disruption of either gapA or gapB showed no significant effect on the transcription of any of the other gap cluster genes although it led to reduced triosephosphate isomerase (TPI) activities. Glycolytic GAPDH activity at low in vitro ATP concentrations was solely attributed to the 35.9-kDa GapA. At higher ATP concentrations, the same activity was attributed to the 51.2-kDa GapB. Both enzymes, however, exhibited similar NADP-dependent GAPDH activities at the higher ATP concentrations. In effect therefore, the GAPDH-catalyzed reaction at low ATP concentrations was irreversible, with all the glycolytic activity strictly NAD-dependent and attributed to GapA. At higher ATP concentrations, the reaction was reversible, with glycolytic activity NAD- or NADP-dependent and attributed to GapB, while gluconeogenic activity was attributable to both GapA and GapB.

Cloning, Expression, and Cell Surface Localization of Paenibacillus sp. Strain W-61 Xylanase 5, a Multidomain Xylanase

ABSTRACT We have shown that a xylan-degrading bacterium, W-61, excretes multiple xylanases, including xylanase 5 with a molecular mass of 140 kDa. Here, we emend the previously used classification of the bacterium (i.e., Aeromonas caviae W-61) to Paenibacillus sp. strain W-61 on the basis of the nucleotide sequence of the 16S rRNA gene, and we clone and express the xyn5 gene encoding xylanase 5 (Xyn5) in Escherichia coli and study the subcellular localization of Xyn5. xyn5 encodes 1,326 amino acid residues, including a 27-amino-acid signal sequence. Sequence analysis indicated that Xyn5 comprises two family 22 carbohydrate-binding modules (CBM), a family 10 catalytic domain of glycosyl hydrolases, a family 9 CBM, a domain similar to the lysine-rich region of Clostridium thermocellum SdbA, and three S-layer-homologous (SLH) domains. Recombinant Xyn5 bound to a crystalline cellulose, Avicel PH-101, while an N-terminal 90-kDa fragment of Xyn5, which lacks the C-terminal half of the family 9 CBM, did not bind to Avicel PH-101. Xyn5 was cell bound, and the cell-bound protein was digested by exogenous trypsin to produce immunoreactive and xylanolytic fragments with molecular masses of 80 and 60 kDa. Xyn5 was exclusively distributed in the cell envelope fraction consisting of a peptidoglycan-containing layer and an associated S layer. Thus, Paenibacillus sp. strain W-61 Xyn5 is a cell surface-anchored modular xylanase possessing a functional cellulose-binding module and SLH domains. Possible cooperative action of multiple xylanases produced by strain W-61 is discussed on the basis of the modular structure of Xyn5.

Development of bio-based fine chemical production through synthetic bioengineering

AbstractFine chemicals that are physiologically active, such as pharmaceuticals, cosmetics, nutritional supplements, flavoring agents as well as additives for foods, feed, and fertilizer are produced by enzymatically or through microbial fermentation. The identification of enzymes that catalyze the target reaction makes possible the enzymatic synthesis of the desired fine chemical. The genes encoding these enzymes are then introduced into suitable microbial hosts that are cultured with inexpensive, naturally abundant carbon sources, and other nutrients. Metabolic engineering create efficient microbial cell factories for producing chemicals at higher yields. Molecular genetic techniques are then used to optimize metabolic pathways of genetically and metabolically well-characterized hosts. Synthetic bioengineering represents a novel approach to employ a combination of computer simulation and metabolic analysis to design artificial metabolic pathways suitable for mass production of target chemicals in host strains. In the present review, we summarize recent studies on bio-based fine chemical production and assess the potential of synthetic bioengineering for further improving their productivity.

Characterization of the mannitol catabolic operon of Corynebacterium glutamicum

Corynebacterium glutamicum encodes a mannitol catabolic operon, which comprises three genes: the DeoR-type repressor coding gene mtlR (sucR), an MFS transporter gene (mtlT), and a mannitol 2-dehydrogenase gene (mtlD). The mtlR gene is located upstream of the mtlTD genes in the opposite orientation. In spite of this, wild-type C. glutamicum lacks the ability to utilize mannitol. This wild-type phenotype results from the genetic regulation of the genes coding for mannitol transport and catalytic proteins mediated by the autoregulated MtlR protein since mtlR mutants grow on mannitol as the sole carbon source. MtlR binds to sites near the mtlR (two sites) and mtlTD promoters (one site downstream of the promoter), with the consensus sequence 5′-TCTAACA-3′ being required for its binding. The newly discovered operon comprises the three basic functional elements required for mannitol utilization: regulation, transport, and metabolism to fructose, further processed to the common intermediate of glycolysis fructose-6-phosphate. When relieved from MtlR repression, C. glutamicum, which lacks a functional fructokinase, excretes the fructose derived from mannitol and imports it by the fructose-specific PTS. In order to use mannitol from seaweed biomass hydrolysates as a carbon source for the production of useful commodity chemicals and materials, an overexpression system using the tac promoter was developed. For congruence with the operon, we propose to rename sucR as the mtlR gene.

3-Amino-4-hydroxybenzoic acid production from sweet sorghum juice by recombinant Corynebacterium glutamicum

The production of the bioplastic precursor 3-amino-4-hydroxybenzoic acid (3,4-AHBA) from sweet sorghum juice, which contains amino acids and the fermentable sugars sucrose, glucose and fructose, was assessed to address the limitations of producing bio-based chemicals from renewable feedstocks. Recombinant Corynebacterium glutamicum strain KT01 expressing griH and griI derived from Streptomyces griseus produced 3,4-AHBA from the sweet sorghum juice of cultivar SIL-05 at a final concentration (1.0 g l(-1)) that was 5-fold higher than that from pure sucrose. Fractionation of sweet sorghum juice by nanofiltration (NF) membrane separation (molecular weight cut-off 150) revealed that the NF-concentrated fraction, which contained the highest concentrations of amino acids, increased 3,4-AHBA production, whereas the NF-filtrated fraction inhibited 3,4-AHBA biosynthesis. Amino acid supplementation experiments revealed that leucine specifically enhanced 3,4-AHBA production by strain KT01. Taken together, these results suggest that sweet sorghum juice is a potentially suitable feedstock for 3,4-AHBA production by recombinant C. glutamicum.

Development of a glutathione production process from proteinaceous biomass resources using protease-displaying Saccharomyces cerevisiae.

Glutathione is a valuable tri-peptide that is widely used in the pharmaceutical, food, and cosmetic industries. Glutathione is produced industrially by fermentation using Saccharomyces cerevisiae, and supplementation of fermentation with several amino acids can increase intracellular GSH content. More recently, however, focus has been given to protein as a resource for biofuel and fine chemical production. We demonstrate that expression of a protease on the cell surface of S. cerevisiae enables the direct use of keratin and soy protein as a source of amino acids and that these substrates enhanced intracellular GSH content. Furthermore, fermentation using soy protein also enhanced cell concentration. GSH fermentation from keratin and to a greater extent from soy protein using protease-displaying yeast yielded greater GSH productivity compared to GSH fermentation with amino acid supplementation. This protease-displaying yeast is potentially applicable to a variety of processes for the bio-production of value-added chemicals from proteinaceous biomass resources.

Thermal stability and starch degradation profile of α-amylase from Streptomyces avermitilis.

Amylases from Streptomyces are useful in the production of maltooligosaccharides, but they have weak thermal stability at temperatures higher than 40 °C. In this study, α-amylase (SAV5981 gene of Streptomyces avermitilis) was expressed from Streptomyces lividans 1326 and purified by ammonium sulfate fractionation followed by anionic chromatography (Q-HP sepharose). The properties of the purified SAV5981 amylase were determined by the starch-iodine method. The effect of metal ions on amylase activity was investigated. The optimal temperature shifted from 25 to 50 °C with the addition of the Ca2+ ion. The thermal stability of SAV5981 was also dramatically enhanced by the addition of 10 mM CaCl2. Improvement of the thermal stability of SAV5981 was examined by CD spectra in the presence and the absence of the Ca2+ ion. Thin-layer chromatography (TLC) analysis and HPLC analysis of starch degradation revealed that SAV5981 mainly produced maltose and maltotriose, not glucose. The maltoorigosaccharide-producing amylase examined in this study has the potential in the industrial application of oligosaccharide production.