BIOSYNTHESIS OF FOUR CARBON ALCOHOLS FROM SUSTAINABLE FEEDSTOCK

Ph.DDOCTOR OF PHILOSOPH

Decolourization of remazol brilliant blue R by enzymatic extract and submerged cultures of a newly isolated Pleurotus ostreatus MR3

A local white-rot fungus basidiomycete Pleurotus ostreatus MR3 was isolated from MacRitchie Reservoir Park, Singapore. Among all the ligninolytic activities, laccase was the only enzyme detected in the supernatant when the fungus was grown in liquid culture. This newly isolated white rot fungus was able to completely decolourise remazol brilliant blue R (RBBR) in-vivo on agar plates within five days and in the liquid culture (in the presence of inducers) within three days. The addition of inducers was able to enhance laccase production and therefore enhanced in-vivo RBBR decolourisation. Veratryl alcohol was shown to be the best inducer for laccase production with the maximum laccase activity reaching about 5.99 U/mL. Cu2+ also had a positive effect on laccase production, the laccase activity being enhanced to 5.24 U/mL. In-vitro RBBR decolourisation using the laccase from P. ostreatus MR3 was much comparable to that using the commercial laccase from Trameters versicolor.Keywords: Dyes, remazol brilliant blue R, Pleurotus ostreatus MR3, decolourisation, inducers, laccase activityAfrican Journal of Biotechnology Vol. 12(39), pp. 5778-578

N-Terminal Deletion of Peptide:N-Glycanase Results in Enhanced Deglycosylation Activity

Peptide:N-glycanase catalyzes the detachment of N-linked glycan chains from glycopeptides or glycoproteins by hydrolyzing the β-aspartylglucosaminyl bond. Peptide:N-glycanase in yeast binds to Rad23p through its N-terminus. In this study, the complex formed between Peptide:N-glycanase and Rad23p was found to exhibit enhanced deglycosylation activity, which suggests an important role for this enzyme in the misfolded glycoprotein degradation pathway in vivo. To investigate the role of this enzyme in this pathway, we made stepwise deletions of the N-terminal helices of peptide:N-glycanase. Enzymatic analysis of the deletion mutants showed that deletion of the N-terminal H1 helix (Png1p-ΔH1) enhanced the deglycosylation activity of N-glycanase towards denatured glycoproteins. In addition, this mutant exhibited high deglycosylation activity towards native glycoproteins. Dynamic simulations of the wild type and N-terminal H1 deletion mutant implied that Png1p-ΔH1 is more flexible than wild type Png1p. The efficient deglycosylation of Png1p-ΔH1 towards native and non-native glycoproteins offers a potential biotechnological application

Engineering Scheffersomyces segobiensis for palmitoleic acid‐rich lipid production

Palmitoleic acid (POA; C16:1) is an essential high- value ω- 7- conjugated fatty acid with beneficial bioactivities and potential applications in the nu-traceutical and pharmaceutical industries. Previously, the oleaginous yeast Scheffersomyces segobiensis DSM27193 has been identified as a promis-ing production host as an alternative for POA extraction from plant or animal sources. Here, the POA-producing capacity of this host was further expanded by optimizing the fermentation process and molecular strain engineering. Specifically, a dual fermentation strategy (O-S dynamic regulation strategy) focused on the substrate and dissolved oxygen concentration was designed to eliminate ethanol and pyruvate accumulation during fermentation. Key genes influencing POA production, such as jen, dgat, ole were identified on the transcriptional level and were subsequently over-expressed. Furthermore, the phosphoketolase (Xpk)/phosphotransacetylase (Pta) pathway was intro-duced to improve the yield of the precursor acetyl-CoA from glucose. The resulting cell factory SS-12 produced 7.3 g/L of POA, corresponding to an 11-fold increase compared to the wild type, presenting the highest POA titre reported using oleaginous yeast to date. An economic evaluation based on the raw materials, utilities and facility-dependent costs showed that microbial POA production using S. segobiensis can supersede the current extraction method from plant oil and marine fish. This study reports the construction of a promising cell factory and an effective microbial fermentation strategy for commercial POA production

Genomic comparison of Clostridium species with the potential of utilizing red algal biomass for biobutanol production

Abstract Background Sustainable biofuels, which are widely considered as an attractive alternative to fossil fuels, can be generated by utilizing various biomass from the environment. Marine biomass, such as red algal biomass, is regarded as one potential renewable substrate source for biofuels conversion due to its abundance of fermentable sugars (e.g., galactose). Previous studies focused on the enhancement of biofuels production from different Clostridium species; however, there has been limited investigation into their metabolic pathways, especially on the conversion of biofuels from galactose, via whole genomic comparison and evolutionary analysis. Results Two galactose-utilizing Clostridial strains were examined and identified as Clostridium acetobutylicum strain WA and C. beijerinckii strain WB. Via the genomic sequencing of both strains, the comparison of the whole genome together with the relevant protein prediction of 33 other Clostridium species was established to reveal a clear genome profile based upon various genomic features. Among them, five representative strains, including C. beijerinckii NCIMB14988, C. diolis DSM 15410, C. pasteurianum BC1, strain WA and WB, were further discussed to demonstrate the main differences among their respective metabolic pathways, especially in their carbohydrate metabolism. The metabolic pathways involved in the generation of biofuels and other potential products (e.g., riboflavin) were also reconstructed based on the utilization of marine biomass. Finally, a batch fermentation process was performed to verify the fermentative products from strains WA and WB using 60 g/L of galactose, which is the main hydrolysate from algal biomass. It was observed that strain WA and WB could produce up to 16.98 and 12.47 g/L of biobutanol, together with 21,560 and 10,140 mL/L biohydrogen, respectively. Conclusions The determination of the production of various biofuels by both strains WA and WB and their genomic comparisons with other typical Clostridium species on the analysis of various metabolic pathways was presented. Through the identification of their metabolic pathways, which are involved in the conversion of galactose into various potential products, such as biobutanol, the obtained results extend the current insight into the potential capability of utilizing marine red algal biomass and provide a systematic investigation into the relationship between this genus and the generation of sustainable bioenergy

Challenges and Future Perspectives of Promising Biotechnologies for Lignocellulosic Biorefinery

Lignocellulose is a kind of renewable bioresource containing abundant polysaccharides, which can be used for biochemicals and biofuels production. However, the complex structure hinders the final efficiency of lignocellulosic biorefinery. This review comprehensively summarizes the hydrolases and typical microorganisms for lignocellulosic degradation. Moreover, the commonly used bioprocesses for lignocellulosic biorefinery are also discussed, including separated hydrolysis and fermentation, simultaneous saccharification and fermentation and consolidated bioprocessing. Among these methods, construction of microbial co-culturing systems via consolidated bioprocessing is regarded as a potential strategy to efficiently produce biochemicals and biofuels, providing theoretical direction for constructing efficient and stable biorefinery process system in the future

Cofactor Metabolic Engineering of <i>Escherichia coli</i> for Aerobic L-Malate Production with Lower CO₂ Emissions

Escherichia coli has been engineered for L-malate production via aerobic cultivation. However, the maximum yield obtained through this mode is inferior to that of anaerobic fermentation due to massive amounts of CO2 emissions. Here, we aim to address this issue by reducing CO2 emissions of recombinant E. coli during aerobic L-malate production. Our findings indicated that NADH oxidation and ATP-synthesis-related genes were down-regulated with 2 g/L of YE during aerobic cultivations of E. coli E23, as compared to 5 g/L of YE. Then, E23 was engineered via the knockout of nuoA and the introduction of the nonoxidative glycolysis (NOG) pathway, resulting in a reduction of NAD+ and ATP supplies. The results demonstrate that E23 (ΔnuoA, NOG) exhibited decreased CO2 emissions, and it produced 21.3 g/L of L-malate from glucose aerobically with the improved yield of 0.43 g/g. This study suggests that a restricted NAD+ and ATP supply can prompt E. coli to engage in incomplete oxidization of glucose, leading to the accumulation of metabolites instead of utilizing them in cellular respiration

Exploitation of novel wild type solventogenic strains for butanol production

Abstract Butanol has been regarded as an important bulk chemical and advanced biofuel; however, large scaling butanol production by solventogenic Clostridium sp. is still not economically feasible due to the high cost of substrates, low butanol titer and yield caused by the toxicity of butanol and formation of by-products. Renewed interests in biobutanol as biofuel and rapid development in genetic tools have spurred technological advances to strain modifications. Comprehensive reviews regarding these aspects have been reported elsewhere in detail. Meanwhile, more wild type butanol producers with unique properties were also isolated and characterized. However, few reviews addressed these discoveries of novel wild type solventogenic Clostridium sp. strains. Accordingly, this review aims to comprehensively summarize the most recent advances on wild type butanol producers in terms of fermentation patterns, substrate utilization et al. Future perspectives using these native ones as chassis for genetic modification were also discussed

MOESM1 of Genomic comparison of Clostridium species with the potential of utilizing red algal biomass for biobutanol production

Additional file 1: The genomic characteristics of the representative Clostridial strains. Table S1. The genomic characteristics of 35 Clostridium strains (without plasmids). Table S2. The characteristics of 8 plasmids

Current Status, Challenges, and Prospects for the Biological Production of Vanillin

Vanillin has been widely used as a flavoring agent in the food industry and as a precursor in the medicine and polymer industries. However, the use of chemically synthesized vanillin is prohibited in food and some other industries. Additionally, the harsh conditions and toxic substrates in chemically synthesized vanillin lead to some environmental challenges and energy waste. With the rapid development of synthetic biology, the biological production of vanillin from renewable resources through microbial fermentation has gained great attention owing to its high selectivity and environmentally friendly properties. Accordingly, this article will discuss the vanillin biosynthesis technology from the aspects of chassis cell types and substrate types. The key enzymes involved in metabolic pathways are also discussed. Then, we summarize some improvements in the process of vanillin production to increase its production and reduce the toxicity of vanillin in microorganisms, and the possible future directions for vanillin biosynthesis will also be outlined