In order to achieve direct and efficient fermentation of optically pure D-lactic acid from raw corn starch, we constructed L-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum and introduced a plasmid encoding Streptococcus bovis 148 ␣-amylase (AmyA). The resulting strain produced only D-lactic acid from glucose and successfully expressed amyA. With the aid of secreting AmyA, direct D-lactic acid fermentation from raw corn starch was accomplished. After 48 h of fermentation, 73.2 g/liter of lactic acid was produced with a high yield (0.85 g per g of consumed sugar) and an optical purity of 99.6%. Moreover, a strain replacing the ldhL1 gene with an amyA-secreting expression cassette was constructed. Using this strain, direct D-lactic acid fermentation from raw corn starch was accomplished in the absence of selective pressure by antibiotics. This is the first report of direct D-lactic acid fermentation from raw starch.
In order to achieve efficient homo L-lactic acid fermentation from xylose, we first carried out addition of xylose assimilation ability to Lactococcus lactis IL 1403 by introducing a plasmid carrying the xylRAB genes from L. lactis IO-1 (pXylRAB). Then modification of xylose assimilation pathway was carried out. L. lactis has two pathways for xylose assimilation called the phosphoketolase pathway (PK pathway) that produces both lactic acid and acetic acid and the pentose phosphate pathway (PP pathway) that produces only lactic acid as a final product. Thus a mutant strain that disrupted its phosphokeolase gene (ptk) was constructed. The Δptk mutant harboring pXylRAB lacked the PK pathway and produced predominantly lactic acid from xylose via the PP pathway, although its fermentation rate slightly decreased. Further introduction of the transketolase gene (tkt) to disrupted ptk locus led restoration of fermentation rate and this was attributed to enhancement of the PP pathway. As a result, ptk::tkt strain harboring pXylRAB produced 50.1 g/l of L-lactic acid from xylose with a high optical purity of 99.6% and a high yield of 1.58 (moles per mole xylose consumed) that is close to theoretical value of 1.67 from xylose.
The glycoside hydrolase family 7 (GH7) member cellobiohydrolase (CBH) is a key enzyme that degrades crystalline cellulose, an important structural component of plant cell walls. As GH7 CBH is a major component in the enzyme mixture used to degrade biomass into fermentable glucose in biorefineries, enhancing its catalytic activity will significantly impact development in this field. GH7 CBH possesses a catalytic tunnel through which cellulose substrates are threaded and hydrolysed. Despite numerous studies dissecting this processive mechanism, the role of amino acid residues in the tunnel remains not fully understood. Herein, we examined the respective contributions of nine amino acid residues in the catalytic tunnel of GH7 CBH from Talaromyces cellulolyticus by substitution with alanine. As a result, N62A and K203A mutants were found to possess significantly higher cellulase activities than wild type. Molecular dynamics simulations showed that the N62 residue interacted strongly with the cellulose substrate, impeding threading, while the N62A mutant allowed cellulose to proceed more smoothly. Furthermore, the W63 residue was observed to facilitate twisting of the cellulose substrate in our simulations. This study helps elucidate cellulose threading and provides insight into biomass hydrolysis.
Sweat sensors that can continuously sample sweat are critical for determining the time-dependent physiological responses occurring in normal daily life. Here, a new device, termed fluidic patch, for collecting human sweat samples at defined time intervals is developed, and the proof-of-concept is demonstrated. The device comprises micropumps and a disposable microfluidic patch attached to the human skin. The fluidic patch continuously collects aliquots of freshly secreted sweat accumulated in the fluidic pathway at accurately defined time windows (typically 5 min). By measuring the weight of the collected samples, the local sweat rate is calculated. The sweat sample collected can be directly subjected to a wide range of chemical analyses. For the proof-of-concept, we compared the sweat rates during passive heating in human trials using the fluidic patch and the conventional ventilated sweat capsule system. Although the sweat rate obtained using the fluidic patch highly correlated with that of the ventilated sweat capsule (R 2 = 0.96, y = 1.4x – 0.05), the fluidic patch overestimated the sweat rate compared with the ventilated capsule system when the sweat rate exceeded 0.5 mg/(cm2·min). The sampled sweat was analyzed for sodium, potassium, chloride, lactate, pyruvate, and cortisol. The device could obtain the time courses of the concentrations of the abovementioned three ions; the concentrations of sodium and chloride increased linearly with the sweat rate during passive heating (R 2 = 0.76 and 0.66, respectively). The device can reliably measure the sweat rate and collect sweat samples for chemical analysis. It can be utilized for real-time physiological investigations toward wider applications.
Background The development of biorefinery systems that use lignocellulosic biomass as a renewable carbon source to produce fuels and chemicals is attracting increasing attention. The process cost of enzymatic saccharification of biomass is a major challenge for commercialization. To decrease this cost, researchers have proposed on-site solid-state fermentation (SSF). This study investigated the feasibility of using Aspergillus oryzae as a host microorganism for SSF recombinant enzyme production with ammonia-treated rice straw as model biomass. Eight A. oryzae strains were tested, all of which are used in the food industry. We evaluated the effects of acetic acid, a fermentation inhibitor. We also developed a platform strain for targeted recombinant enzyme production by gene engineering technologies. Results The SSF validation test showed variation in the visibility of mycelium growth and secreted protein in all eight A. oryzae strains. The strains used to produce shoyu and miso grew better under test conditions. The ammonia-treated rice straw contained noticeable amounts of acetic acid. This acetic acid enhanced the protein production by A. oryzae in a liquid-state fermentation test. The newly developed platform strain successfully secreted three foreign saccharifying enzymes. Conclusions A. oryzae is a promising candidate as a host microorganism for on-site SSF recombinant enzyme production, which bodes well for the future development of a more cost-efficient saccharifying enzyme production system.
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