• Microbiology and Biotechnology Letters
• /
• v.24 no.4
• /
• pp.385-392
• /
• 1996

Bacillus stearotheymophilus, a potent xylanolytic bacterium isolated from soil, was tested for the strain's strategies of pentose utilization and the evidence of substrate preferences. The strain metabolized glucose, xylose, ribose, maltose, cellobiose, sucrose, arabinose and xylitol. The efficacy of the sugars as a carbon and energy source in this strain was of the order named above. The organism, however, could not grow on glycerol as a sole growth substrate. During cultivation on a mixture of glucose and xylose or arabinose, the major hydrolytic products of xylan, B. stearothermophilus displayed classical diauxic growth in which glucose was utilized during the first phase. On the other hand, the pentose utilization was prevented immediately upon addition of glucose. Cellobiose was preferred over xylose or arabinose. In contrast, maltose and pentose were co-utilized, and also no preference on between xylose and arabinose. Enzymatic studies indicated that B. stearothermophilus possessed constitutive hexokinase, a key enzyme of the glucose metabolic system. While, the production of $^{D}$-xylose isomerase, $^{D}$-xylulokinase and $^{D}$-arabinose isomerase essential for pentose phosphate pathway were induced by xylose, xylan, and xylitol but repressed by glucose. Taken together, the results suggested that the sequential utilization of B. stearothermophilus would be mediated by catabolite regulatory mechanisms such as catabolite inhibition or inducer exclusion.

• Journal of Microbiology and Biotechnology
• /
• v.29 no.4
• /
• pp.577-586
• /
• 2019

The engineered Aspergillus oryzae has a high NADPH demand for xylose utilization and overproduction of target metabolites. Glucose-6-phosphate dehydrogenase (G6PDH, E.C. 1.1.1.49) is one of two key enzymes in the oxidative part of the pentose phosphate pathway, and is also the main enzyme involved in NADPH regeneration. The open reading frame and cDNA of the putative A. oryzae G6PDH (AoG6PDH) were obtained, followed by heterogeneous expression in Escherichia coli and purification as a his6-tagged protein. The purified protein was characterized to be in possession of G6PDH activity with a molecular mass of 118.0 kDa. The enzyme displayed maximal activity at pH 7.5 and the optimal temperature was $50^{\circ}C$. This enzyme also had a half-life of 33.3 min at $40^{\circ}C$. Kinetics assay showed that AoG6PDH was strictly dependent on $NADP^+$ ($K_m=6.3{\mu}M$, $k_{cat}=1000.0s^{-1}$, $k_{cat}/K_m=158.7s^{-1}{\cdot}{\mu}M^{-1}$) as cofactor. The $K_m$ and $k_{cat}/K_m$ values of glucose-6-phosphate were $109.7s^{-1}{\cdot}{\mu}M^{-1}$ and $9.1s^{-1}{\cdot}{\mu}M^{-1}$ respectively. Initial velocity and product inhibition analyses indicated the catalytic reaction followed a two-substrate, steady-state, ordered BiBi mechanism, where $NADP^+$ was the first substrate bound to the enzyme and NADPH was the second product released from the catalytic complex. The established kinetic model could be applied in further regulation of the pentose phosphate pathway and NADPH regeneration of A. oryzae to improve its xylose utilization and yields of valued metabolites.

• Journal of Microbiology and Biotechnology
• /
• v.29 no.3
• /
• pp.339-346
• /
• 2019

L-Arabinose, a five carbon sugar, has not been considered as an important bioresource because most studies have focused on D-xylose, another type of five-carbon sugar that is prevalent as a monomeric structure of hemicellulose. In fact, L-arabinose is also an important monomer of hemicellulose, but its content is much more significant in pectin (3-22%, g/g pectin), which is considered an alternative biomass due to its low lignin content and mass production as juice-processing waste. This review presents native and engineered microorganisms that can ferment L-arabinose. Saccharomyces cerevisiae is highlighted as the most preferred engineering host for expressing a heterologous arabinose pathway for producing ethanol. Because metabolic engineering efforts have been limited so far, with this review as momentum, more attention to research is needed on the fermentation of L-arabinose as well as the utilization of pectin-rich biomass.

• Microbiology and Biotechnology Letters
• /
• v.1 no.1
• /
• pp.31-36
• /
• 1973

A method for acid-hydrolysis of agricultural wastes and its utilization was investigated. In order to obtain fermentable sugar solution from cellulosic wastes such as cereal straws and hulls, in particular, of rice, barley and wheat, the chemical compositions were analyzed and optimum conditions of hydrolysis determined. The cereal straws contain 42 to 55 % of crude cellulose including hemicellulose. On the hydrolysis with 1% of sulfuric acid at 40 psig, 35.6% of the reducing sugar based on the weight of dry matter was formed from rice straw, (variety Chinheung) in 30 min. More powerful condition of hydrolysis would appear to decompose the sugar formed into other compounds, for instance, furfural. Under atmospheric pressure with 5% of the acid, rice straw was hydrolyzed to 35% of reducing sugar content in 3 hours. Candida utilis could assimilate the sugars in the hydrolyzate up to more than 97%, and a yield of the yeast cells reached 55% to the utilized sugars.

• Asian-Australasian Journal of Animal Sciences
• /
• v.13 no.3
• /
• pp.334-347
• /
• 2000

The present experiment was carried out to study the utilization of glucose in the mammary gland of crossbred Holstein cattle during feeding with different types of roughage. Sixteen first lactating crossbred Holstein cattle which comprised eight animals of two breed types, Holstein Friesian${\times}$Red Sindhi ($50{\times}50=50%$ HF) and Holstein Friesian${\times}$Red Sindhi ($87.5{\times}12.5=87.5%$ HF). They were divided into four groups of 4 animals each of the same breed. The utilization of glucose in the mammary gland was determined by measuring rates of glucose uptake and the incorporation of glucose into milk components in both groups of 50% HF and 87.5% HF animals feeding on either hay or urea treated rice straw. In early lactation, there were no significant differences of the total glucose entry rate and glucose carbon recycling among groups of crossbred animals feeding on either hay or urea treated rice straw. During lactation advance, the total glucose turnover rates and recycling of carbon glucose of crossbred HF animals feeding on urea treated rice straw were markedly higher than those of crossbred HF animals feeding on hay as roughage, whereas there were no significant changes for both groups of crossbred animals feeding on hay. The percentages and values of non-mammary glucose utilization showed an increase during lactation advance in the same group of both 50% HF and 87.5% HF animals. The percentage of glucose uptake for utilization in the synthesis of milk lactose by the mammary gland was approximately 62% for both groups of 87.5% HF and by approximately 55% for both groups of 50% HF animals feeding on either hay or urea treated rice straw. Intracellular glucose 6-phosphate metabolized via the pentose phosphate pathway accounted for the NADPH (reducing equivalent) of fatty acid synthesis in the mammary gland being higher in 87.5% HF animals during mid-lactation. A large proportion of metabolism of glucose via the Embden-Meyerhof pathway in the mammary gland was more apparent in both groups of 50% HF animals than those of 87.5 % HF animals during early and mid-lactation while it markedly increased for both groups of 87.5% HF animals during late lactation. It can be concluded that utilization of glucose in the mammary gland occurs in a different manner for 50% HF and 87.5% HF animals feeding on either hay or urea treated rice straw. The glucose utilization for biosynthetic pathways in the mammary gland of 50% HF animals is maintained in a similar pattern throughout the periods of lactation. A poorer lactation persistency in both groups of 87.5% HF animals occurs during lactation advance, which is related to a decrease in the lactose biosynthetic pathway.

• Journal of Microbiology and Biotechnology
• /
• v.28 no.4
• /
• pp.571-578
• /
• 2018

Biofuel production using lignocellulosic biomass is gaining attention because it can be substituted for fossil fuels without competing with edible resources. However, because Saccharomyces cerevisiae does not have a ${\text\tiny{D}}$-xylose metabolic pathway, oxidoreductase or isomerase pathways must be introduced to utilize ${\text\tiny{D}}$-xylose from lignocellulosic biomass in S. cerevisiae. To elucidate the biochemical properties of xylose isomerase (XI) from Piromyces sp. E2 (PsXI), we determine its crystal structure in complex with substrate mimic glycerol. An amino-acid sequence comparison with other reported XIs and relative activity measurements using five kinds of divalent metal ions confirmed that PsXI belongs to class II XIs. Moreover kinetic analysis of PsXI was also performed using $Mn^{2+}$, the preferred divalent metal ion for PsXI. In addition, the substrate-binding mode of PsXI could be predicted with the substrate mimic glycerol bound to the active site. These studies may provide structural information to enhance ${\text\tiny{D}}$-xylose utilization for biofuel production.

• Genes and Genomics
• /
• v.40 no.11
• /
• pp.1181-1197
• /
• 2018

Tropical plant rubber tree (Hevea brasiliensis) is the sole source of commercial natural rubber and low-temperature stress is the most important limiting factor for its cultivation. To characterize the gene expression profiles of H. brasiliensis under the cold stress and discover the key cold stress-induced genes. Three cDNA libraries, CT (control), LT2 (cold treatment at $4^{\circ}C$ for 2 h) and LT24 (cold treatment at $4^{\circ}C$ for 24 h) were constructed for RNA sequencing (RNA-Seq) and gene expression profiling. Quantitative real time PCR (qRT-PCR) was conducted to validate the RNA-Seq and gene differentially expression results. A total of 1457 and 2328 differentially expressed genes (DEGs) in LT2 and LT24 compared with CT were respectively detected. Most significantly enriched KEGG pathways included flavonoid biosynthesis, phenylpropanoid biosynthesis, plant hormone signal transduction, cutin, suberine and wax biosynthesis, Pentose and glucuronate interconversions, phenylalanine metabolism and starch and sucrose metabolism. A total of 239 transcription factors (TFs) were differentially expressed following 2 h or/and 24 h of cold treatment. Cold-response transcription factor families included ARR-B, B3, BES1, bHLH, C2H, CO-like, Dof, ERF, FAR1, G2-like, GRAS, GRF, HD-ZIP, HSF, LBD, MIKC-MADS, M-type MADS, MYB, MYB-related, NAC, RAV, SRS, TALE, TCP, Trihelix, WOX, WRKY, YABBY and ZF-HD. The genome-wide transcriptional response of rubber tree to the cold treatments were determined and a large number of DEGs were characterized including 239 transcription factors, providing important clues for further elucidation of the mechanisms of cold stress responses in rubber tree.