Animal-free production of hen egg ovalbumin in engineered Saccharomyces cerevisiae via precision fermentation.

To enable the sustainable production of ovalbumin (OVA) without relying on animal sources, the generally recognized as safe (GRAS) host Saccharomyces cerevisiae was used for the precision fermentation-based production of recombinant OVA. For this purpose, a signal peptide derived from EPX1, the most abundant extracellular protein produced by Pichia pastoris, was identified as a novel signal peptide for the efficient secretion of OVA in S. cerevisiae. To improve OVA secretion and cell growth, three helper proteins (PDI1, KAR2, and HAC1) present in the endoplasmic reticulum were expressed individually or in combination. Notably, the +P1/K2 strain coexpressing PDI1 and KAR2 with OVA produced 2 mg/L of OVA in the medium fraction; this value was 2.6-fold higher than the corresponding value for the control strain without helper proteins. Finally, a glucose-limited fed-batch fermentation process using the +P1/K2 strain yielded 132 mg/L of total OVA with 8 mg/L of extracellular OVA.

Metabolic engineering of Caldicellulosiruptor bescii for hydrogen production.

Hydrogen is an alternative fuel for transportation vehicles because it is clean, sustainable, and highly flammable. However, the production of hydrogen from lignocellulosic biomass by microorganisms presents challenges. This microbial process involves multiple complex steps, including thermal, chemical, and mechanical treatment of biomass to remove hemicellulose and lignin, as well as enzymatic hydrolysis to solubilize the plant cell walls. These steps not only incur costs but also result in the production of toxic hydrolysates, which inhibit microbial growth. A hyper-thermophilic bacterium of Caldicellulosiruptor bescii can produce hydrogen by decomposing and fermenting plant biomass without the need for conventional pretreatment. It is considered as a consolidated bioprocessing (CBP) microorganism. This review summarizes the basic scientific knowledge and hydrogen-producing capacity of C. bescii. Its genetic system and metabolic engineering strategies to improve hydrogen production are also discussed. KEY POINTS: • Hydrogen is an alternative and eco-friendly fuel. • Caldicellulosiruptor bescii produces hydrogen with a high yield in nature. • Metabolic engineering can make C. bescii to improve hydrogen production.

Bioconversion of citrus waste into mucic acid by xylose-fermenting Saccharomyces cerevisiae.

Mucic acid holds promise as a platform chemical for bio-based nylon synthesis; however, its biological production encounters challenges including low yield and productivity. In this study, an efficient and high-yield method for mucic acid production was developed by employing genetically engineered Saccharomyces cerevisiae expressing the NAD+-dependent uronate dehydrogenase (udh) gene. To overcome the NAD+ dependency for the conversion of pectin to mucic acid, xylose was utilized as a co-substrate. Through optimization of the udh expression system, the engineered strain achieved a notable output, producing 20 g/L mucic acid with a highest reported productivity of 0.83 g/L-h and a theoretical yield of 0.18 g/g when processing pectin-containing citrus peel waste. These results suggest promising industrial applications for the biological production of mucic acid. Additionally, there is potential to establish a viable bioprocess by harnessing pectin-rich fruit waste alongside xylose-rich cellulosic biomass as raw materials.

Development of a Novel ATP Bioluminescence Assay Based on Engineered Probiotic Saccharomyces boulardii Expressing Firefly Luciferase.

Quantitative analysis of adenosine triphosphate (ATP) has been widely used as a diagnostic tool in the food and medical industries. Particularly, the pathogenesis of a few diseases including inflammatory bowel disease (IBD) is closely related to high ATP concentrations. A bioluminescent D-luciferin/luciferase system, which includes a luciferase (FLuc) from the firefly Photinus pyralis as a key component, is the most commonly used method for the detection and quantification of ATP. Here, instead of isolating FLuc produced in recombinant Escherichia coli, we aimed to develop a whole-cell biocatalyst system that does not require extraction and purification of FLuc. To this end, the gene coding for FLuc was introduced into the genome of probiotic Saccharomyces boulardii using the CRISPR/Cas9-based genome editing system. The linear relationship (r2 = 0.9561) between ATP levels and bioluminescence generated from the engineered S. boulardii expressing FLuc was observed in vitro. To explore the feasibility of using the engineered S. boulardii expressing FLuc as a whole-cell biosensor to detect inflammation biomarker (i.e., ATP) in the gut, a colitis mouse model was established using dextran sodium sulfate as a colitogenic compound. Our findings demonstrated that the whole-cell biosensor can detect elevated ATP levels during gut inflammation in mice. Therefore, the simple and powerful method developed herein could be applied for non-invasive IBD diagnosis.

Hydrogen Production from Barley Straw and Miscanthus by the Hyperthermophilic Bacterium, Cadicellulosirupter bescii.

This work aimed to evaluate the feasibility of biohydrogen production from Barley Straw and Miscanthus. The primary obstacle in plant biomass decomposition is the recalcitrance of the biomass itself. Plant cell walls consist of cellulose, hemicellulose, and lignin, which make the plant robust to decomposition. However, the hyperthermophilic bacterium, Caldicellulosiruptor bescii, can efficiently utilize lignocellulosic feedstocks (Barley Straw and Miscanthus) for energy production, and C. bescii can now be metabolically engineered or isolated to produce more hydrogen and other biochemicals. In the present study, two strains, C. bescii JWCB001 (wild-type) and JWCB018 (ΔpyrFA Δldh ΔcbeI), were tested for their ability to increase hydrogen production from Barley Straw and Miscanthus. The JWCB018 resulted in a redirection of carbon and electron (carried by NADH) flow from lactate production to acetate and hydrogen production. JWCB018 produced ~54% and 63% more acetate and hydrogen from Barley Straw, respectively than its wild-type counterpart, JWCB001. Also, 25% more hydrogen from Miscanthus was obtained by the JWCB018 strain with 33% more acetate relative to JWCB001. It was supported that the engineered C. bescii, such as the JWCB018, can be a parental strain to get more hydrogen and other biochemicals from various biomass.

Relationship between super-leadership and self-directed learning ability in online nursing education: The mediating effects of self-leadership and self-efficacy perceptions.

Background: Self-directed learning ability is a core competence that enables students to enhance their academic achievement, clinical competence, and professional growth in nursing education. Super-leadership-as a strategy to develop the learning process-promotes students' self-leadership, which, in turn, enhances perceived self-efficacy perception and ultimately facilitates self-directed learning ability. Few studies have rigorously determined the influences of super-leadership, self-leadership, and academic self-efficacy perceptions on self-directed learning ability in online nursing education during the COVID-19 pandemic. Objectives: To determine the mediating effects of self-leadership and self-efficacy perceptions in the relationship between perceived super-leadership and self-directed learning ability. Design: This study was conducted using a quantitative mediation analysis design. Participants: One hundred and fifty nursing students were conveniently recruited from two nursing colleges offering the same four-year nursing degree program. Methods: Participants completed a super-leadership scale, a self-leadership scale, an academic self-efficacy scale, and a self-directed learning ability inventory. Data were analyzed with Pearson's correlations, regression analysis, Sobel's test, and Hayes's Process Macro using the bootstrap method. Results: Self-directed learning ability was correlated with perceived super-leadership (r = .47, p < .001), self-leadership (r = 0.69, p < .001), and academic self-efficacy (r = 0.29, p < .001). Super-leadership influenced self-leadership (ß = 0.42, p < .001), academic self-efficacy (ß = 0.22, p = .002), and self-directed learning ability (ß = 0.36, p < .001). Self-leadership and academic self-efficacy mediated the relationship between super-leadership and self-directed learning ability (ß = .42, p < .001; ß = 0.35, p < .001) (z = 1.936, p = .026 by Sobel test). Conclusion: Learning strategies to enhance self-directed learning ability are required for successful super-leadership to boost self-leadership and academic self-efficacy perceptions among nursing students in the online, asynchronous COVID-19 educational environment. Therefore, this study's empirical evidence on integrating super-leadership with self-leadership and academic self-efficacy has practical and future research implications in terms of attaining core academic goals for nursing students.

Inverse metabolic engineering for improving protein content in Saccharomyces cerevisiae.

Production of Saccharomyces cerevisiae-based single cell protein (SCP) has recently received great attention due to the steady increase in the world's population and environmental issues. In this study, an inverse metabolic engineering approach was applied to improve the production of yeast SCP. Specifically, an S. cerevisiae mutant library, generated using UV-random mutagenesis, was screened for three rounds to isolate mutants with improved protein content and/or concentration. The #1021 mutant strain exhibited a respective 31% and 23% higher amino acid content and concentration than the parental S. cerevisiae D452-2 strain. Notably, the content, concentration, and composition of amino acids produced by the PAN2* strain, with a single nucleotide polymorphism in PAN2 coding for a catalytic subunit of the poly(A)-nuclease (PAN) deadenylation complex, were virtually identical to those produced by the #1021 mutant strain. In a glucose-limited fed-batch fermentation, the PAN2* strain produced 19.5 g L-1 amino acids in 89 h, which was 16% higher than that produced by the parental D452-2 strain. This study highlights the benefits of inverse metabolic engineering for enhancing the production titer and yield of target molecules without prior knowledge of rate-limiting steps involved in their biosynthetic pathways.

Simultaneous expression of an endogenous spermidine synthase and a butanol dehydrogenase from Thermoanaerobacter pseudethanolicus in Clostridium thermocellum results in increased resistance to acetic acid and furans, increased ethanol production and an increase in thermotolerance.

BACKGROUND: Sensitivity to inhibitors derived from the pretreatment of plant biomass is a barrier to the consolidated bioprocessing of these complex substrates to fuels and chemicals by microbes. Spermidine is a low molecular weight aliphatic nitrogen compound ubiquitous in microorganisms, plants, and animals and is often associated with tolerance to stress. We recently showed that overexpression of the endogenous spermidine synthase enhanced tolerance of the Gram-positive bacterium, Clostridium thermocellum to the furan derivatives furfural and HMF. RESULTS: Here we show that co-expression with an NADPH-dependent heat-stable butanol dehydrogenase from Thermoanaerobacter pseudethanolicus further enhanced tolerance to furans and acetic acid and most strikingly resulted in an increase in thermotolerance at 65 °C. CONCLUSIONS: Tolerance to fermentation inhibitors will facilitate the use of plant biomass substrates by thermophiles in general and this organism in particular. The ability to grow C. thermocellum at 65 °C has profound implications for metabolic engineering.

Capsule-based colorimetric temperature monitoring system for customizable cold chain management.

The COVID-19 pandemic and the resulting supply chain disruption have rekindled crucial needs for safe storage and transportation of essential items. Despite recent advances, existing temperature monitoring technologies for cold chain management fall short in reliability, cost, and flexibility toward customized cold chain management for various products with different required temperature. In this work, we report a novel capsule-based colorimetric temperature monitoring system with precise and readily tunable temperature ranges. Triple emulsion drop-based microfluidic technique enables rapid production of monodisperse microcapsules with an interstitial phase-change oil (PCO) layer with precise control over its dimension and composition. Liquid-solid phase transition of the PCO layer below its freezing point triggers the release of the encapsulated payload yielding drastic change in color, allowing user-friendly visual monitoring in a highly sensitive manner. Simple tuning of the PCO layer's compositions can further broaden the temperature range in a precisely controlled manner. The proposed simple scheme can readily be formulated to detect both temperature rise in the frozen environment and freeze detection as well as multiple temperature monitoring. Combined, these results support a significant step forward for the development of customizable colorimetric monitoring of a broad range of temperatures with precision.

Efficient production of glutathione in Saccharomyces cerevisiae via a synthetic isozyme system.

Glutathione, a tripeptide consisting of cysteine, glutamic acid, and glycine, has multiple beneficial effects on human health. Previous studies have focused on producing glutathione in Saccharomyces cerevisiae by overexpressing γ-glutamylcysteine synthetase (GSH1) and glutathione synthetase (GSH2), which are the rate-limiting enzymes involved in the glutathione biosynthetic pathway. However, the production yield and titer of glutathione remain low due to the feedback inhibition on GSH1. To overcome this limitation, a synthetic isozyme system consisting of a novel bifunctional enzyme (GshF) from Gram-positive bacteria possessing both GSH1 and GSH2 activities, in addition to GSH1/GSH2, was introduced into S. cerevisiae, as GshF is insensitive to feedback inhibition. Given the HSP60 chaperonin system mismatch between bacteria and S. cerevisiae, co-expression of Group-I HSP60 chaperonins (GroEL and GroES) from Escherichia coli was required for functional expression of GshF. Among various strains constructed in this study, the SKSC222 strain capable of synthesizing glutathione with the synthetic isozyme system produced 240 mg L-1 glutathione with glutathione content and yield of 4.3% and 25.6 mgglutathione /gglucose , respectively. These values were 6.6-, 4.9-, and 4.3-fold higher than the corresponding values of the wild-type strain. In a glucose-limited fed-batch fermentation, the SKSC222 strain produced 2.0 g L-1 glutathione in 67 h. Therefore, this study highlights the benefits of the synthetic isozyme system in enhancing the production titer and yield of value-added chemicals by engineered strains of S. cerevisiae.

Directed Evolution of Soluble α-1,2-Fucosyltransferase Using Kanamycin Resistance Protein as a Phenotypic Reporter for Efficient Production of 2'-Fucosyllactose.

2'-Fucosyllactose (2'-FL), the most abundant fucosylated oligosaccharide in human milk, has multiple beneficial effects on human health. However, its biosynthesis by metabolically engineered Escherichia coli is often hampered owing to the insolubility and instability of α-1,2-fucosyltransferase (the rate-limiting enzyme). In this study, we aimed to enhance 2'-FL production by increasing the expression of soluble α-1,2-fucosyltransferase from Helicobacter pylori (FucT2). Because structural information regarding FucT2 has not been unveiled, we decided to improve the expression of soluble FucT2 in E. coli via directed evolution using a protein solubility biosensor that links protein solubility to antimicrobial resistance. For such a system to be viable, the activity of kanamycin resistance protein (KanR) should be dependent on FucT2 solubility. KanR was fused to the C-terminus of mutant libraries of FucT2, which were generated using a combination of error-prone PCR and DNA shuffling. Notably, one round of the directed evolution process, which consisted of mutant library generation and selection based on kanamycin resistance, resulted in a significant increase in the expression level of soluble FucT2. As a result, a batch fermentation with the ΔL M15 pBCGW strain, expressing the FucT2 mutant (F#1-5) isolated from the first round of the directed evolution process, resulted in the production of 0.31 g/l 2'-FL with a yield of 0.22 g 2'-FL/g lactose, showing 1.72- and 1.51-fold increase in the titer and yield, respectively, compared to those of the control strain. The simple and powerful method developed in this study could be applied to enhance the solubility of other unstable enzymes.

Type D personality, cognitive illness perception, depression, approach coping, and self-management among older adults in long-term care hospitals: Structural equation modeling.

This study aimed to construct a structural equation model to explore the relationship between Type D personality, cognitive illness perception, depression, approach-coping, and self-management. The study was conducted at two long-term care hospitals with 300 or more beds in Korea. Participants were 287 older patients from whom data were collected from February 17 to March 10, 2021, using a structured questionnaire comprising items on the following variables: Type D personality, cognitive illness perception, depression, approach coping, and self-management. Type D personality (ß=-.601, p=.001), cognitive illness perception (ß =.692, p <.001), depression (ß =-.204, p =.011), and approach-coping (ß =.326, p <.001) explained 78.8% of the total variance of self-management, highlighting their impact on how patients accept and manage a disease and their relevance to the self-management of older adults in long-term care hospitals.

Fine-tuning of MXene-nickel oxide-reduced graphene oxide nanocomposite bioelectrode: Sensor for the detection of influenza virus and viral protein.

Influenza viruses can cause epidemics through inter-human transmission, and the social consequences of viral transmission are incalculable. Current diagnostics for virus detection commonly relies on antibodies or nucleic acid as recognition reagent. However, a more advanced and general method for the facile development of new biosensors is increasing in demand. In this study, we report the fabrication of an ultra-sensitive peptide-based nanobiosensor using a nickel oxide (NiO)-reduced graphene oxide (rGO)/MXene nanocomposite to detect active influenza viruses (H1N1 and H5N2) and viral proteins. The sensing mechanism is based on the signal inhibition, the specific interaction between H1N1 (QMGFMTSPKHSV) and H5N1 (GHPHYNNPSLQL) binding peptides anchored on the NiO-rGO/MXene/glassy carbon electrode (GCE) surface and the viral surface protein hemagglutinin (HA) is the critical factor for the decrease in the peak current of the sensor. In this strategy, the NiO-rGO/MXene nanocomposite results in synergistic signal effects, including electrical conductivity, porosity, electroactive surface area, and active site availability when viruses are deposited on the electrode. Based on these observations, the results showed that the developed nanobiosensor was capable of highly sensitive and specific detection of their corresponding influenza viruses and viral proteins with a very low detection limit (3.63 nM of H1N1 and 2.39 nM for H5N1, respectively) and good recovery. The findings demonstrate that the proposed NiO-rGO/MXene-based peptide biosensor can provide insights for developing a wide range of clinical screening tools for detecting affected patients.

Strategies, advances, and challenges associated with the use of graphene-based nanocomposites for electrochemical biosensors.

Graphene is an intriguing two-dimensional honeycomb-like carbon material with a unique basal plane structure, charge carrier mobility, thermal conductivity, wide electrochemical spectrum, and unusual physicochemical properties. Therefore, it has attracted considerable scientific interest in the field of nanoscience and bionanotechnology. The high specific surface area of graphene allows it to support high biomolecule loading for good detection sensitivity. As such, graphene, graphene oxide (GO), and reduced GO are excellent materials for the fabrication of new nanocomposites and electrochemical sensors. Graphene has been widely used as a chemical building block and/or scaffold with various materials to create highly sensitive and selective electrochemical sensing microdevices. Over the past decade, significant advancements have been made by utilizing graphene and graphene-based nanocomposites to design electrochemical sensors with enhanced analytical performance. This review focus on the synthetic strategies, as well as the structure-to-function studies of graphene, electrochemistry, novel multi nanocomposites combining graphene, limit of detection, stability, sensitivity, assay time. Finally, the review describes the challenges, strategies and outlook on the future development of graphene sensors technology that would be usable for the internet of things are also highlighted.

An intradialytic aerobic exercise program ameliorates frailty and improves dialysis adequacy and quality of life among hemodialysis patients: a randomized controlled trial.

BACKGROUND: Hemodialysis patients with chronic kidney disease exhibit impaired exercise tolerance and functional decline. Despite the life-saving benefits of adequate dialysis, those declines translate into frailty and deteriorating quality of life (QoL). This study evaluated the effects of an intradialytic aerobic exercise program on frailty, dialysis adequacy, and QoL among hemodialysis patients. METHODS: Patients at an university hospital-affiliated hemodialysis center were randomly assigned to an exercise group (n = 18) or a control group (n = 21). The 12-week aerobic exercise program comprised 40 to 70 minutes of ergometer cycling 3 times/wk and a single education session. The control group completed only the education session. Outcomes were assessed at the time of enrollment, week 4, week 8, and week 12 using Fried's frailty phenotype measures (gait speed, grip strength, vitality, body mass index, and physical activity), the short physical performance battery (SPPB), Kt/V urea, and the Short Form-36 questionnaire. RESULTS: There were significant interactions between groups and follow-up times in the frailty score (p < 0.001), gait speed (p < 0.001), SPPB (p < 0.001), and mental QoL (p = 0.03). The intention-to-treat and per-protocol analyses revealed that the exercise group exhibited significant improvements in frailty score (p < 0.001), gait speed (p < 0.001), grip strength (p < 0.001), exhaustion (p = 0.02), SPPB (p = 0.01), dialysis adequacy (p = 0.01), and physical QoL (p = 0.003). CONCLUSION: An intradialytic aerobic exercise program could be a safe, feasible, and appropriate additional strategy to routine care among hemodialysis patients for improvements in frailty, dialysis adequacy, and QoL.

Simultaneous production of 2'-fucosyllactose and difucosyllactose by engineered Escherichia coli with high secretion efficiency.

BACKGROUND AND AIM: Difucosyllactose (Di-FL) has strong antimicrobial activity against various pathogens, including group B Streptococcus, identified as the leading cause of neonatal sepsis. In this study, we sought to develop Escherichia coli as a microbial cell factory for efficiently producing Di-FL as well as 2'-fucosyllactose (2'-FL), the most abundant fucosylated oligosaccharide in human milk, by utilizing the salvage guanosine 5'-diphosphate (GDP)-l-fucose biosynthetic pathway. MAIN METHODS AND MAJOR RESULTS: The biosynthetic pathway for producing fucosylated oligosaccharides via the salvage pathway requires two enzymes, l-fucokinase/GDP-l-fucose phosphorylase (FKP) from Bacteroides fragilis and α-1,2-fucosyltransferase (FucT2) from Helicobacter pylori. To decrease the intracellular accumulation of 2'-FL while increasing substrate accessibility to FKP and FucT2, we evaluated whether extracellular secretion of FKP and FucT2 would enhance the production of fucosylated oligosaccharides. Among various engineered strains constructed in this study, the ΔLFAR-YA/FF+P-PLA2 strain expressing phospholipase A2 (PLA2 ) from Streptomyces violaceoruber, whose native signal peptide was replaced with the PelB signal peptide (P-PLA2 ), could secrete both FKP and FucT2 into the culture medium. Notably, it was observed that FKP and FucT2 present in the extracellular fraction could catalyze the synthesis of Di-FL from lactose and fucose. As a result, a batch fermentation with the ΔLFAR-YA/FF+P-PLA2 strain resulted in the production of 1.22 ± 0.01 g L-1 Di-FL and 0.47 ± 0.01 g L-1 2'-FL, whereas the control strain could only produce 0.65 ± 0.01 g L-1 2'-FL. CONCLUSIONS AND IMPLICATIONS: This study highlights the benefits of extracellular secretion of enzymes to improve biotransformation efficiency, as the transport of substrates and/or products across the cell membrane is limited.

Optimization of protein trans-splicing in an inducible plasmid display system for high-throughput screening and selection of soluble proteins.

Directed evolution is widely used to optimize protein folding and solubility in cells. Although the screening and selection of desired mutants is an essential step in directed evolution, it generally requires laborious optimization and/or specialized equipment. With a view toward designing a more practical procedure, we previously developed an inducible plasmid display system, in which the intein (auto-processing) and Oct-1 DNA-binding (DBD) domains were used as the protein trans-splicing domain and DNA-binding module, respectively. Specifically, the N-terminal (CfaN) and C-terminal (CfaC) domains of intein were fused to the C-terminal end of the His-tag and the N-terminal end of Oct-1 DBD to generate His6-CfaN and CfaC-Oct-1, respectively. For such a system to be viable, the efficiency of protein trans-splicing without the protein of interest (POI) should be maximized, such that the probability of occurrence is solely dependent on the solubility of the POI. To this end, we initially prevented the degradation of l-arabinose (the inducer of the PBAD promoter) by employing an Escherichia coli host strain deficient in the metabolism of l-arabinose. Given that a low expression of His6-CfaN, compared with that of CfaC-Oct-1, was found to be conducive to the generation to a soluble product of the protein trans-splicing event, we designed the expression of His6-CfaN and CfaC-Oct-1 to be inducible from the PBAD and PT7 promoters, respectively. The optimized system thus obtained enabled in vitro selection of the plasmid-protein complex with high yield. We believe that the inducible plasmid display system developed in this study would be applicable to high-throughput screening and/or selection of protein variants with enhanced solubility.

Yeast metabolic engineering for carbon dioxide fixation and its application.

As numerous industrial bioprocesses rely on yeast fermentation, developing CO2-fixing yeast strains can be an attractive option toward sustainable industrial processes and carbon neutrality. Recent studies have shown that the expression of ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) in yeasts, such as Saccharomyces cerevisiae and Kluyveromyces marxianus, enables mixotrophic CO2 fixation and production of biofuels. Also, the expression of a synthetic Calvin-Benson-Bassham (CBB) cycle including RuBisCO in Pichia pastoris enables autotrophic growth on CO2. This review highlights recent advances in metabolic engineering strategies to enable CO2 fixation in yeasts. Also, we discuss the potentials of other natural and synthetic metabolic pathways independent of RuBisCO for developing CO2-fixing yeast strains capable of producing value-added biochemicals.

Functional expression and extracellular secretion of Clostridium thermocellum Cel48S cellulase in Escherichia coli via the signal recognition particle-dependent translocation pathway.

As the only glycoside hydrolase family 48 member in Clostridium thermocellum, the exoglucanase Cel48S plays a crucial role in the extremely high activity of the cellulosome against crystalline cellulose. Although the importance of Cel48S in the hydrolysis of crystalline cellulose has been widely accepted, an efficient production system has not yet been established because Cel48S is usually expressed in Escherichia coli within inactive inclusion bodies. For unstable proteins like Cel48S, translocation across the inner membrane can be more advantageous than cytoplasmic production due to the presence of folding modulators in the periplasm and the absence of cytoplasmic proteases. In this study, we evaluated whether the production of Cel48S in the periplasmic space of E. coli could enhance its functional expression. To do so, we attached the PelB signal peptide, which mediates post-translational secretion, to the N-terminal end of Cel48S (P-Cel48S). The PelB signal peptide allowed catalytically active Cel48S to be successfully produced in the culture medium. In addition, we investigated the role of an alternative co-translational pathway on the extracellular production of Cel48S, finding that co-translational secretion yielded a specific activity of recombinant Cel48S of 135.1 ± 10.0 U/mg cell in the culture medium, which was 2.2 times higher than that associated with P-Cel48S expression. Therefore, we believe that our approach has potential applications for the cost-effective conversion of lignocellulosic biomass and the industrial production of other unstable proteins.

Production of (-)-α-bisabolol in metabolically engineered Saccharomyces cerevisiae.

(-)-α-Bisabolol is a natural monocyclic sesquiterpene alcohol present in German chamomile and has been used as an ingredient of functional foods, cosmetics and pharmaceuticals. In this study, metabolic engineering strategies were attempted to produce (-)-α-bisabolol in Saccharomyces cerevisiae. The codon-optimized MrBBS gene coding for (-)-α-bisabolol synthase from Matricaria recutita was expressed in S. cerevisiae for (-)-α-bisabolol production. The resulting strain (DM) produced 9.5 mg/L of (-)-α-bisabolol in 24 h of batch culture. Additionally, the mevalonate pathway was intensified by introducing a truncated HMG1 gene coding for HMG-CoA reductase and ERG10 encoding acetyl-CoA thiolase. The resulting strain (DtEM) produced a 2.9-fold increased concentration of (-)-α-bisabolol than the DM strain. To increase the acetyl-CoA pool, the ACS1 gene coding for acetyl-CoA synthetase was also overexpressed in the DtEM strain. Finally, the DtEMA strain produced 124 mg/L of (-)-α-bisabolol with 2.7 mg/L-h of productivity in a fed-batch fermentation, which were 13 and 6.8 times higher than the DM strain in batch culture, respectively. Conclusively, these metabolically-engineered approaches might pave the way for the sustainable production of other sesquiterpenes in engineered S. cerevisiae.