Development of high cell density Limosilactobacillus reuteri KUB-AC5 for cell factory using oxidative stress reduction approach.

BACKGROUND: Expression systems for lactic acid bacteria have been developed for metabolic engineering applications as well as for food-grade recombinant protein production. But the industrial applications of lactic acid bacteria as cell factories have been limited due to low biomass formation resulted in low efficiency of biomanufacturing process. Limosilactobacillus reuteri KUB-AC5 is a safe probiotic lactic acid bacterium that has been proven as a gut health enhancer, which could be developed as a mucosal delivery vehicle for vaccines or therapeutic proteins, or as expression host for cell factory applications. Similar to many lactic acid bacteria, its oxygen sensitivity is a key factor that limits cell growth and causes low biomass production. The aim of this study is to overcome the oxidative stress in L. reuteri KUB-AC5. Several genes involved in oxidative and anti-oxidative stress were investigated, and strain improvement for higher cell densities despite oxidative stress was performed using genetic engineering. RESULTS: An in-silico study showed that L. reuteri KUB-AC5 genome possesses an incomplete respiratory chain lacking four menaquinone biosynthesis genes as well as a complete biosynthesis pathway for the production of the precursor. The presence of an oxygen consuming enzyme, NADH oxidase (Nox), leads to high ROS formation in aerobic cultivation, resulting in strong growth reduction to approximately 25% compared to anaerobic cultivation. Recombinant strains expressing the ROS scavenging enzymes Mn-catalase and Mn-superoxide dismutase were successfully constructed using the pSIP expression system. The Mn-catalase and Mn-SOD-expressing strains produced activities of 873 U/ml and 1213 U/ml and could minimize the ROS formation in the cell, resulting in fourfold and sevenfold higher biomass formation, respectively. CONCLUSIONS: Expression of Mn-catalase and Mn-SOD in L. reuteri KUB-AC5 successfully reduced oxidative stress and enhanced growth. This finding could be applied for other lactic acid bacteria that are subject to oxidative stress and will be beneficial for applications of lactic acid bacteria for cell factory applications.

Growth modulation effects of CBM2a under the control of AtEXP4 and CaMV35S promoters in Arabidopsis thaliana, Nicotiana tabacum and Eucalyptus camaldulensis.

The expression of cell-wall-targeted Carbohydrate Binding Modules (CBMs) can alter cell wall properties and modulate growth and development in plants such as tobacco and potato. CBM2a identified in xylanase 10A from Cellulomonas fimi is of particular interest for its ability to bind crystalline cellulose. However, its potential for promoting plant growth has not been explored. In this work, we tested the ability of CBM2a to promote growth when expressed using both CaMV35S and a vascular tissue-specific promoter derived from Arabidopsis expansin4 (AtEXP4) in three plant species: Arabidopsis, Nicotiana tabacum and Eucalyptus camaldulensis. In Arabidopsis, the expression of AtEXP4pro:CBM2a showed trends for growth promoting effects including the increase of root and hypocotyl lengths and the enlargements of the vascular xylem area, fiber cells and vessel cells. However, in N. tabacum, the expression of CBM2a under the control of either CaMV35S or AtEXP4 promoter resulted in subtle changes in the plant growth, and the thickness of secondary xylem and vessel and fiber cell sizes were generally reduced in the transgenic lines with AtEXP4pro:CBM2a. In Eucalyptus, while transgenics expressing CaMV35S:CBM2a showed very subtle changes compared to wild type, those transgenics with AtEXP4pro:CBM2a showed increases in plant height, enlargement of xylem areas and xylem fiber and vessel cells. These data provide comparative effects of expressing CBM2a protein in different plant species, and this finding can be applied for plant biomass improvement.

Purification and Immobilization of Superoxide Dismutase Obtained from Saccharomyces cerevisiae TBRC657 on Bacterial Cellulose and Its Protective Effect against Oxidative Damage in Fibroblasts.

Superoxide dismutase (SOD) is an essential enzyme that eliminates harmful reactive oxygen species (ROS) generating inside living cells. Due to its efficacities, SOD is widely applied in many applications. In this study, the purification of SOD produced from Saccharomyces cerevisiae TBRC657 was conducted to obtain the purified SOD that exhibited specific activity of 513.74 U/mg with a purification factor of 10.36-fold. The inhibitory test revealed that the purified SOD was classified as Mn-SOD with an estimated molecular weight of 25 kDa on SDS-PAGE. After investigating the biochemical characterization, the purified SOD exhibited optimal activity under conditions of pH 7.0 and 35 °C, which are suitable for various applications. The stability test showed that the purified SOD rapidly decreased in activity under high temperatures. To overcome this, SOD was successfully immobilized on bacterial cellulose (BC), resulting in enhanced stability under those conditions. The immobilized SOD was investigated for its ability to eliminate ROS in fibroblasts. The results indicated that the immobilized SOD released and retained its function to regulate the ROS level inside the cells. Thus, the immobilized SOD on BC could be a promising candidate for application in many industries that require antioxidant functionality under operating conditions.

Pretreatment of Cellulose from Sugarcane Bagasse with Xylanase for Improving Dyeability with Natural Dyes.

In this study, cellulose was obtained from sugarcane bagasse (SCB) and treated with xylanase to remove residual noncellulosic polymers (hemicellulose and lignin) to improve its dyeability. The cellulose fibers were dyed with natural dye solutions extracted from the heart wood of Ceasalpinia sappan Linn. and Artocarpus heterophyllus Lam. Fourier-transform infrared (FTIR) spectroscopy, Raman analysis, and whiteness index (WI) indicated successful extraction of cellulose by eliminating hemicellulose and lignin. The FTIR analysis of the dyed fibers confirmed successful interaction between natural dyes and cellulose fibers. The absorption (K) and scattering (S) coefficient (K/S) values of the dyed fibers increased in cellulose treated with xylanase before dyeing. Scanning electron microscopy (SEM) analysis showed that the surface of alkaline-bleached fibers (AB-fibers) was smoother than alkaline-bleached xylanase fibers (ABX-fibers), and the presence of dye particles on the surface of dyed fibers was confirmed by energy-dispersive spectrometry (EDS) analysis. The X-ray diffraction (XRD) revealed a higher crystallinity index (CrI), and thermal gravimetric analysis (TGA) also presented higher thermal stability in the dyed fibers with good colorfastness to light. Therefore, xylanase treatment and natural dyes can enhance dyeability and improve the properties of cellulose for various industrial applications.