Successful Production and Ligninolytic Activity of a Bacterial Laccase, Lac51, Made in Nicotiana benthamiana via Transient Expression

Giant panda could have bamboo as their exclusive diet for about 2 million years because of the contribution of numerous enzymes produced by their gut bacteria, for instance laccases. Laccases are blue multi-copper oxidases that catalyze the oxidation of a broad spectrum of phenolic and aromatic compounds with water as the only byproduct. As a “green enzyme,” laccases have potential in industrial applications, for example, when dealing with degradation of recalcitrant biopolymers, such as lignin. In the current study, a bacterial laccase, Lac51, originating from Pseudomonas putida and identified in the gut microbiome of the giant panda’s gut was transiently expressed in the non-food plant Nicotiana benthamiana and characterized. Our results show that recombinant Lac51 exhibits bacterial laccase properties, with optimal pH and temperature at 7–8 and 40°C, respectively, when using syringaldazine as substrate. Moreover, we demonstrate the functional capability of the plant expressed Lac51 to oxidize lignin using selected lignin monomers that serve as substrates of Lac51. In summary, our study demonstrates the potential of green and non-food plants as a viable enzyme production platform for bacterial laccases. This result enriches our understanding of plant-made enzymes, as, to our knowledge, Lac51 is the first functional recombinant laccase produced in plants.publishedVersio

Genome-wide association identifies nine common variants associated with fasting proinsulin levels and provides new insights into the pathophysiology of type 2 diabetes.

OBJECTIVE: Proinsulin is a precursor of mature insulin and C-peptide. Higher circulating proinsulin levels are associated with impaired β-cell function, raised glucose levels, insulin resistance, and type 2 diabetes (T2D). Studies of the insulin processing pathway could provide new insights about T2D pathophysiology. RESEARCH DESIGN AND METHODS: We have conducted a meta-analysis of genome-wide association tests of ∼2.5 million genotyped or imputed single nucleotide polymorphisms (SNPs) and fasting proinsulin levels in 10,701 nondiabetic adults of European ancestry, with follow-up of 23 loci in up to 16,378 individuals, using additive genetic models adjusted for age, sex, fasting insulin, and study-specific covariates. RESULTS: Nine SNPs at eight loci were associated with proinsulin levels (P < 5 × 10(-8)). Two loci (LARP6 and SGSM2) have not been previously related to metabolic traits, one (MADD) has been associated with fasting glucose, one (PCSK1) has been implicated in obesity, and four (TCF7L2, SLC30A8, VPS13C/C2CD4A/B, and ARAP1, formerly CENTD2) increase T2D risk. The proinsulin-raising allele of ARAP1 was associated with a lower fasting glucose (P = 1.7 × 10(-4)), improved β-cell function (P = 1.1 × 10(-5)), and lower risk of T2D (odds ratio 0.88; P = 7.8 × 10(-6)). Notably, PCSK1 encodes the protein prohormone convertase 1/3, the first enzyme in the insulin processing pathway. A genotype score composed of the nine proinsulin-raising alleles was not associated with coronary disease in two large case-control datasets. CONCLUSIONS: We have identified nine genetic variants associated with fasting proinsulin. Our findings illuminate the biology underlying glucose homeostasis and T2D development in humans and argue against a direct role of proinsulin in coronary artery disease pathogenesis

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Enzymatic conversion of waste paper to mono- and oligosaccharides

Efficient utilization of waste products is a key step in the transition into a sustainable society. Recalcitrant lignocellulosic biomass is a major constituent of industrial and household waste, and its conversion into fuels represents a promising green alternative to fossil sources. Lignocellulosic biomass is a complex composite structure, containing mainly cellulose, hemicelluloses and lignin; this intricate matrix provides tensile strength in the plant cell wall and resistance against enzymatic degradation. There is a high demand for paper products in the world, and consequently, it is a major part of waste products. Cellulose represents the dominant component in paper and its conversion into monosaccharides and oligosaccharides could be a first step in generating value-added products such as fuels. Enzymatic cellulose degradation involves four main types of enzymes: endoglucanases (EGs), cellobiohydrolases (CBHs), lytic polysaccharide monooxygenases (LPMOs) and β-glucosidases (BGs). This thesis focuses on the conversion of waste paper to monosaccharides with a commercial enzyme cocktail (Cellic CTec 2), as well as conversion of oligosaccharides using three purified endoglucanases. Experiments were conducted on shredded office paper (SOP), cut cardboard (CCB) and shredded newspaper (SNP), that were all pretreated by steam explosion. A compositional analysis of SOP, CCB and SNP was performed in order to identify the ratios of the structural components. This analysis also enabled accurate determination of the conversion yields after enzymatic hydrolysis. The cellulose-hemicellulose-lignin-ash-others ratios were (41.4%-8.4%-2.6%-21.2%-26.0%), (58.7%-10.1%-16.3%-11.2%-3.5%), (31.6%-3.5%-39.9%-6.6%-24.1%) for SOP, CCB and SNP, respectively pretreated with steam explosion at 210°C for 14 min. An additive in paper proved a challenge in the conversion using Cellic CTec2 because of its alkaline properties; experiments where the acid loading was varied were therefore performed, in order to achieve the optimal pH for the enzymatic reaction. Following the acid-loading test, a 24-hour hydrolysis was performed, 54%, 46% and 29% of theoretical maximum yields was observed with enzyme loadings of 5 mg/g dry mass (DM) over 24 hours for SOP, CCB and SNP respectively. Furthermore, optimization experiments achieved 89.6%, 55.2% and 56.3% conversions with enzyme loadings of 25mg/g DM and 72-hour incubation. Three endoglucanases were produced using the eukaryotic expression host Pichia pastoris. The purified enzymes were able to produce a range of soluble cellodextrines from both pure cellulose and the waste paper substrates. Yields between 0 to 10% of the theoretical maximum yields were observed. Avicel, which contains highly crystalline cellulose, resulted in yields below 1.3%. Steam exploded SOP, CCB and SNP, on the other hand, resulted in yields of 7.3% in SOP with MaCel45A, 4.1% in CCB with MaCel45A and 13.5% in SNP with TaCel5A In addition to studying the release of soluble products, the effect of the enzymes on insoluble cellulose was tested to better understand how the endoglucanases work. The results demonstrate how the endoglucanases reduce the degree of polymerization by introducing cuts on the insoluble cellulose chains. It thus seem that the endoglucanases hydrolyze the accessible regions of cellulose chain but not to an extent that leads to a high degree of conversion of soluble material.M-BIOTE

Biochemical characterization of two cellobiose 2-epimerases and application for efficient production of lactulose and epilactose

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Biochemical characterization of two cellobiose 2-epimerases and application for efficient production of lactulose and epilactose

publishedVersio

Discovery of a Thermostable GH10 Xylanase with Broad Substrate Specificity from the Arctic Mid-Ocean Ridge Vent System

acceptedVersio

HIST 520 American Urban History

Course syllabus for HIST 520 American Urban History Course description: Presents an historical examination of the process of modernization and its effect on the American city and people. Emphasizes migration and mobility, family roles, machine politics, and various reform movements

In-depth characterization of Trichoderma reesei cellobiohydrolase TrCel7A produced in Nicotiana benthamiana reveals limitations of cellulase production in plants by host-specific post-translational modifications

Sustainable production of biofuels from lignocellulose feedstocks depends on cheap enzymes for degradation of such biomass. Plants offer a safe and cost‐effective production platform for biopharmaceuticals, vaccines and industrial enzymes boosting biomass conversion to biofuels. Production of intact and functional protein is a prerequisite for large‐scale protein production, and extensive host‐specific post‐translational modifications (PTMs) often affect the catalytic properties and stability of recombinant enzymes. Here we investigated the impact of plant PTMs on enzyme performance and stability of the major cellobiohydrolase TrCel7A from Trichoderma reesei, an industrially relevant enzyme. TrCel7A was produced in Nicotiana benthamiana using a vacuum‐based transient expression technology, and this recombinant enzyme (TrCel7Arec) was compared with the native fungal enzyme (TrCel7Anat) in terms of PTMs and catalytic activity on commercial and industrial substrates. We show that the N‐terminal glutamate of TrCel7Arec was correctly processed by N. benthamiana to a pyroglutamate, critical for protein structure, while the linker region of TrCel7Arec was vulnerable to proteolytic digestion during protein production due to the absence of O‐mannosylation in the plant host as compared with the native protein. In general, the purified full‐length TrCel7Arec had 25% lower catalytic activity than TrCel7Anat and impaired substrate‐binding properties, which can be attributed to larger N‐glycans and lack of O‐glycans in TrCel7Arec. All in all, our study reveals that the glycosylation machinery of N. benthamiana needs tailoring to optimize the production of efficient cellulases

In-depth characterization of Trichoderma reesei cellobiohydrolase TrCel7A produced in Nicotiana benthamiana reveals limitations of cellulase production in plants by host-specific post-translational modifications

Sustainable production of biofuels from lignocellulose feedstocks depends on cheap enzymes for degradation of such biomass. Plants offer a safe and cost‐effective production platform for biopharmaceuticals, vaccines and industrial enzymes boosting biomass conversion to biofuels. Production of intact and functional protein is a prerequisite for large‐scale protein production, and extensive host‐specific post‐translational modifications (PTMs) often affect the catalytic properties and stability of recombinant enzymes. Here we investigated the impact of plant PTMs on enzyme performance and stability of the major cellobiohydrolase TrCel7A from Trichoderma reesei, an industrially relevant enzyme. TrCel7A was produced in Nicotiana benthamiana using a vacuum‐based transient expression technology, and this recombinant enzyme (TrCel7Arec) was compared with the native fungal enzyme (TrCel7Anat) in terms of PTMs and catalytic activity on commercial and industrial substrates. We show that the N‐terminal glutamate of TrCel7Arec was correctly processed by N. benthamiana to a pyroglutamate, critical for protein structure, while the linker region of TrCel7Arec was vulnerable to proteolytic digestion during protein production due to the absence of O‐mannosylation in the plant host as compared with the native protein. In general, the purified full‐length TrCel7Arec had 25% lower catalytic activity than TrCel7Anat and impaired substrate‐binding properties, which can be attributed to larger N‐glycans and lack of O‐glycans in TrCel7Arec. All in all, our study reveals that the glycosylation machinery of N. benthamiana needs tailoring to optimize the production of efficient cellulases