Human milk oligosaccharides (HMOs) are one of the major differences between livestock milk and human milk, and the prebiotic functions of HMOs have been verified through in vitro and clinical trials. The most abundant HMOs include 2′-fucysollactose (2′-FL), 3-fucosyllactose (3-FL), lacto-N-neotetraose (LNnT) and lacto-N-tetraose (LNT); their application and synthesis have attracted wide attentions. In recent years, the biotechnological production of 2′-FL, 3-FL, LNnT and LNT have emerged based on techniques such as whole-cell catalysis and fermentation. In particular, the development of metabolic engineering and synthetic biology methods and strategies have facilitated efficient biosynthesis of these HMOs. However, these advantages have not been systematically reviewed yet. In this review, we first discuss the structures and applications of HMOs; secondly, strategies of microbial synthesis of the most abundant 2′-FL, 3-FL, LNnT and LNT are summarized and compared. Finally, challenges and perspectives of efficient microbial production of HMOs as well as strategies for overcoming the challenges are discussed. This review reveals the whole picture of recent development in HMOs microbial synthesis and can further facilitate the understanding of limiting factors, and further propose a few directions to promote the development of efficient production hosts.
Enzymes are considered as functional proteins, also known as biocatalysts, which are required for normal cellular function. The commercial production of enzymes with distinct features always remains the top priority of researchers for industrial biocatalysis. However, the insufficient yield, low stability, low activity, formation of by-products, complex purification process, and many other factors are challenging for the enzyme production industry. Novel advancements in protein engineering, especially the combination of multidisciplinary techniques including post-translational enzyme modification, structured assisted protein tailoring and computational modeling approaches, open a new horizon to more efficient production of biocatalysts. In this review, we focus on the development process of enzyme modification design strategies, such as rational design, semi-rational design and de novo design. Selected examples for each strategy, which are particularly useful for novel enzyme design, are given. We hope this review is helpful for the development of biocatalysts for industrial application.
Enzyme technologies are widely used in the food industry due to their advantages of high efficiency, specificity, and safety. Recently, “future foods” is emerging as a new research hotspot with healthier foods that are more nutritious, delicious, and sustainable; however, these foods still have problems with texture, nutrition, and flavor. Advances in enzyme technology have enabled the development of new tools and approaches to better manipulate food textures and nutritional aspects. In this review, we summarize enzyme technology applications in future food production, focusing on food texture, safety, and flavors. Furthermore, we discuss the prospects of enzyme-based technologies for future food production, including the modification of enzyme activities, the development of suitable food-grade hosts for enzyme production, and the optimization synergistic multi-enzyme systems.
In this study, coffee pulp (Coffea arabica) and green tea (Camellia sinensis) residues were characterized for use as a substrate of solid-state fermentation for cellulases production. The invasion rate was evaluated, as well as cellulases production by strains of Aspergillus niger and Trichoderma asperellum from the western Ghats of India, on coffee pulp, green tea, and a mixture of both substrates (50:50). T. asperellum (AFP) strain was found to have the highest growth rate (0.409 ± 0.021 mm/h) using a mixture of both substrates. The production of cellulases by T. asperellum was unsatisfactory due to the presence of polyphenols in the supports to which A. nigger cellulases are more resistant. The production of cellulases by A. nigger was linked to the pH of the supports, favouring the use of T and TC. It was found that the extracts produced by A. niger (28A strain using a mixture substrate, 28A, and 20A strains using only green tea as a substrate) presented the highest cellulase activities when evaluated using a plate technique producing degradation halos of 2.3 ± 0.1 cm of diameter. Aspergillus 28A strain did not require mineral enrichment media for cellulase production using green tea residues as support of solid-state fermentation.
Several enzymes in the pentose phosphate pathway of Saccharomyces cerevisiae have been identified as relating to the constraint of xylose consumption and conversion to ethanol. However, no strategy has been proposed for simultaneous regulation of all contributing enzymes. If multiple enzymes contribute to constraint, over expression of a native transcription factor controlling the entire constraining pathway may provide optimal pathway wide regulation. Further characterization of this strain on both pure sugars and lignocellulosic hydrolysates would provide an opportunity to identify additional bottlenecks not addressed by the modification of the pentose phosphate pathway expression pattern.
A series of strains were developed expressing STB5 and PGI1 under the control of a novel xylose inducible promoter. Increased transcription of STB5 and its regulatory targets was verified via qRT-PCR. No statistically significant difference was found in terms of xylose consumption or ethanol yield in these strains versus control strains. Xylose consumption through both the fermentative and respiratory pathways appeared to be related to oxygen availability and culture density with high-density (low oxygen) cultures consuming xylose more slowly than low-density cultures. The maximum specific consumption rate for high-density cultures was 0.21 g xylose/gDCW/h versus 0.41 g xylose/gDCW/h in lower density cultures. Statistically similar ethanol yields at high and low density (approximately 0.25 g ethanol/ g xylose) suggest that the maximum rate of fermentation is linked to the rate of respiration in a stoichiometric fashion.
This study did not find evidence supporting the pentose phosphate pathway constraint identified in other works. Instead, NAD + availability mediated by oxygen availability and citric acid cycle flux was suggested to limit fermentation. While increased aeration could provide increased conversion of NAD + to NADH (and a stoichiometric increase in fermentation flux), this increase would not be expected improve ethanol yield beyond 50% of the theoretical maximum. Based on these findings, future work in Saccharomyces cerevisiae development for fermentation of lignocellulosic hydrolysates should focus on balancing NAD + / NADH availability through non-respiratory pathways.
Several enzymes and cofactors have been identified as contributing to the slow utilization of xylose by xylose-fermenting strains of Saccharomyces cerevisiae. However, there has been no consensus on which of these possible bottlenecks are the most important to address. A previous strain characterization study from our lab suggested that insufficient NAD+ limits fermentation and may be the most important bottleneck affecting utilization of xylose for the production of ethanol. The development and validation of a genome scale dynamic flux balance model would help to verify the existence and extent of this and other metabolic bottlenecks and suggest solutions to guide future strain development thereby minimizing bottleneck impact on process economics.
A dynamic flux balance model was developed to identify bottlenecks in several strains of S. cerevisiae, both with wild-type pentose phosphate pathway expression and with the pathway over expressed. ZWF1 was found to be limiting in the oxidative portion of the pentose phosphate pathway under oxygen replete conditions. This pathway is used to regenerate NADPH. Under oxygen limiting conditions, respiration of xylose was limited by the lack of oxygen as a terminal electron acceptor. Ethanol production was also limited under these conditions due to the inability to balance NAD+/NADH. The model suggests the use of the anaplerotic glyoxylate pathway to improve NAD+/NADH balance, increasing ethanol production by 50% while producing succinate as a coproduct at upwards of 20 g/l.
In the production of high value chemicals from biomass, the use of the respiratory metabolism is a waste of feedstock carbon. Bottlenecks previously identified in the oxidative pentose phosphate pathway are currently only relevant under oxygen-replete conditions and cannot impact the partitioning of carbon between the respiratory and fermentative pathways. Focusing future efforts on the non-respiratory balancing of NAD+/NADH, perhaps through the glyoxylate pathway, would improve the economics of ethanol production both directly and through coproduct formation.
Purified glycerol obtained after acid treatment of crude glycerol solution was used as the carbon source for lipid and citric acid production using Y. lipolytica SKY7. Although purified glycerol was high in phosphorus, it was important to investigate the impact of fortification of trace elements in the medium on cell growth, lipid and citric acid (CA) production. When all the trace elements (including phosphates and sulfates) required for growth and lipid production were added to the purified glycerol medium, high biomass (51.67 g/L) and lipid concentration (19.47 g/L) were observed at 96 h of fed-batch fermentation with low CA concentration of 5.42 g/L. The purified glycerol medium without additional trace elements gave low biomass (27.67 g/L), lipid concentration (9.35 g/L) at 80 h of fed-batch fermentation, but gave high CA concentration (24.51 g/L). When purified glycerol was provided with only sulfates or all elements except KH2PO4, low biomass (32.59 g/L and 38.52 g/L) and citric acid concentration (1 g/L and 2.42 g/L) were obtained at 96 h.
Microalgae have piqued renewed interest as a sustainable biofuel feedstock owing to their high CO2 conversion efficiency. However, the major limitation of microalga-based biofuel production is low productivity. In this study, CO2 in flue gas emitted from the coal-fired power plants was fixed through mass microalgal cultivation using only sunlight as an energy source. To minimize the cost and energy required to supply the flue gas and efficiently utilize the microalgal biomass, a polycarbonate (PC) greenhouse and polymeric photobioreactors were installed near the power plant stack. Four different microalgal strains (Chlamydomonas reinhardtii, Chlorella sorokiniana, Neochloris oleoabundans, and Neochloris oleoabundans #13) were subjected to semi-continuous culturing for 1 month. The maximum biomass productivity was achieved by the N. oleoabundans #13 strain (0.703 g L−1 day−1). Additionally, polymerase chain reaction analysis revealed that the individual microalgal culture was not cross-contaminated with other microalgal cultures in this cultivation system, owing to the structural properties of photobioreactor comprising individual modules. The lipid content and calorific productivity of N. oleoabundans #13 biomass were 45.70% and 3.553 kJ L−1 day−1, respectively, which indicate satisfactory performance of biomass as a direct combustion fuel. The CO2 fixation rate, which was calculated based on the carbon content in the biomass, was 0.309 g CO2 L−1 day−1. Therefore, large amounts of CO2 can be reduced using the large-scale microalgal cultivation system, which enables efficient biological CO2 conversion and maximizes microalgal biomass utilization.
Fish processing towards production of fillet gives rise to wastewater streams that are ultimately directed to biogas production and/or wastewater treatment. However, these wastewater streams are rich in minerals, fat, and proteins that can be converted to protein-rich feed ingredients through submerged cultivation of edible filamentous fungi. In this study, the origin of wastewater stream, initial pH, cultivation time, and extent of washing during sieving, were found to influence the amount of recovered material from the wastewater streams and its protein content, following cultivation with Aspergillus oryzae. Through cultivation of the filamentous fungus in sludge, 330 kg of material per ton of COD were recovered by sieving, corresponding to 121 kg protein per ton of COD, while through its cultivation in salt brine, 210 kg of material were recovered per ton of COD, corresponding to 128 kg protein per ton of COD. Removal ranges of 12–43%, 39–92%, and 32–66% for COD, total solids, and nitrogen, respectively, were obtained after A. oryzae growth and harvesting in the wastewater streams. Therefore, the present study shows the versatility that the integration of fungal cultivation provides to fish processing industries, and should be complemented by economic, environmental, and feeding studies, in order to reveal the most promising valorization strategy.
Byssochlamys fulva AM130, a novel strain of filamentous fungus, could produce ethanol from glucose, xylose, and alkali pretreated rice straw (PRS), while the efficiencies were very low with PRS. Ethanol production of 11.84 g/L was attained by the fungus when grown in glucose, indicating that the limitations while growing on PRS were related to low hydrolytic efficiency. Enzyme profiling of the fungus showed 365 IU/ml of beta-glucosidase and 89 IU/ml of xylanase activity, while endoglucanase and filter paper activity were negligible, which accounts for the low hydrolytic efficiency. The fungus could survive for extended periods under oxygen-limited conditions and produce ethanol. The fungal mycelia could also be used for repeated cycles of anaerobic fermentation, wherein the ethanol yield improved with each consecutive cycle.