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Photos of the HARV system used to generate SMG (A) and NG (B) conditions, respectively. At the back of HARV there is a gaspermeable membrane for the exchange of O 2 and CO 2 . The tracks of the cells maintained in (C) LSMMG and (D) NG conditions corresponding to (A) and (B), respectively. Cells in the medium would continually suspend under SMG.
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Bioprocesses use cost-effective renewable resources as feedstock to produce various products with less energy consumption under mild conditions. The technical progress in the modification of biocatalysts including enzymes and cells has significantly improved their efficiencies. In this article, the strategies involving molecular, cellular and commu...
Context in source publication
Context 1
... lated microgravity (SMG) that is more applicable and feasible to the study provides a methodology for exploring its applications in bioprocess. The SMG environment can be created by a High Aspect Ratio Vessel (HARV) when the device is rotated hori- zontally with the axis of the vessel perpendicular to the gravi- tational vector, as illustrated in Fig. ...
Citations
... As a result of depleting valuable resources for the production of commodities, global issues such as the production of greenhouse gases and the endangerment of biodiversity are on the rise (Demeneix 2020). However, the advancements of bioprocesses have introduced a new hope to replace existing technologies with those primarily dependent on sustainable resources (Zhang et al. 2015). ...
The successful design of economically viable bioprocesses can help to abate global dependence on petroleum, increase supply chain resilience, and add value to agriculture. Specifically, bioprocessing provides the opportunity to replace petrochemical production methods with biological methods and to develop novel bioproducts. Even though a vast range of chemicals can be biomanufactured, the constraints on economic viability, especially while competing with petrochemicals, are severe. There have been extensive gains in our ability to engineer microbes for improved production metrics and utilization of target carbon sources. The impact of growth medium composition on process cost and organism performance receives less attention in the literature than organism engineering efforts, with media optimization often being performed in proprietary settings. The widespread use of corn steep liquor as a nutrient source demonstrates the viability and importance of “waste” streams in biomanufacturing. There are other promising waste streams that can be used to increase the sustainability of biomanufacturing, such as the use of urea instead of fossil fuel-intensive ammonia and the use of struvite instead of contributing to the depletion of phosphate reserves. In this review, we discuss several process-specific optimizations of micronutrients that increased product titers by twofold or more. This practice of deliberate and thoughtful sourcing and adjustment of nutrients can substantially impact process metrics. Yet the mechanisms are rarely explored, making it difficult to generalize the results to other processes. In this review, we will discuss examples of nutrient sourcing and adjustment as a means of process improvement.
One-Sentence Summary
The potential impact of nutrient adjustments on bioprocess performance, economics, and waste valorization is undervalued and largely undercharacterized.
... In the face of increasing environmental pressures, energy efficiency, waste reduction, safety and health issues have drawn global attention to sustainable development [1], which has led to the potential of biocatalyst applications being explored. As biocatalyst with the advantages of high efficiency, specificity and mild catalytic conditions compared to chemical catalysts, enzymes have been widely used in food [2,3], chemical [4,5], medical [6,7], energy and environmental fields [8][9][10]. ...
... Twenty milligram of Gx-GFP (or Gx-SRGFP) was added into 1 mL of denaturing solutions (50 mM Tris-HCl, 9 M guanidine hydrochloride, 1 mM ethylenediaminetetraacetic acid, 150 mM DL-Dithiothreitol, pH 8.0) and heated in a metal bath at 90 • C for 10 min (3 mg/g), 20 min (5 mg/g) and 30 min (10 mg/g) for rapid inactivation, and the denatured samples were inverted and mixed as described above to determine fluorescence. The relative fluorescence was calculated by referring to formula (1). ...
SpyTag and SpyCatcher can rapidly conjugate covalently to form an irreversible covalent bond. Fusing SpyTag and SpyCatcher to the termini of a protein of interest enables the generation of SpyRing structure, which is often helpful for the stability of protein. In this paper, green fluorescent protein (GFP), L-phenylserine aldolase (LPA) and SRGFP, SRLPA (their SpyRing cyclized form) were covalently bound to glyoxyl-agarose (Gx) beads to prepare immobilized enzymes (Gx-GFP, Gx-LPA, Gx-SRGFP and Gx-SRLPA) with different protein loadings, and the effects of the SpyRing structure on solid-phase refolding were investigated. When the immobilized enzyme was inactivated in high concentrations of N, N-dimethylformamide (DMF) and 9 M guanidine hydrochloride, then reactivated with refolding buffer, the results showed that proteins with the SpyRing structure had a higher renaturation yield than their wild-type counterparts in all three replication cycles. The highest renaturation yields of Gx-SRGFP and Gx-SRLPA were over 90 % and 70 % respectively, which were approximately 15 % and 30 % higher than those of Gx-GFP and Gx-LPA. It suggests that the introduction of the SpyRing structure facilitates the reactivation of immobilized biocatalysts and provides a reference for the regeneration and recycling of industrial biocatalysts.
... The design of the microbial cell factories involves several steps, including: identification of a de novo biosynthetic pathway for the desired product; selection of a microbial chassis (host); enzyme and metabolic engineering to allow the formation of the new products in the microbial cell factory host (Pscheidt and Glieder, 2008;Fisher et al., 2014;Zhang et al., 2015). The strategies for the production of the desired compound(s) in microorganisms involve: the application of heterologous genes to assemble a new biosynthetic pathway in the host; improvement of the pathway flux by, for example, augmenting substrate availability, down-regulating competing pathways or increasing the expression of key enzymes; the improvement of certain enzymes of the pathway by protein engineering (Pearsall et al., 2015). ...
Microorganisms have been exposed to a myriad of substrates and environmental conditions throughout evolution resulting in countless metabolites and enzymatic activities. Although mankind have been using these properties for centuries, we have only recently learned to control their production, to develop new biocatalysts with high stability and productivity and to improve their yields under new operational conditions. However, microbial cells still provide the best known environment for enzymes, preventing conformational changes in the protein structure in non-conventional medium and under harsh reaction conditions, while being able to efficiently regenerate necessary cofactors and to carry out cascades of reactions. Besides, a still unknown microbe is probably already producing a compound that will cure cancer, Alzeihmer's disease or kill the most resistant pathogen. In this review, the latest developments in screening desirable activities and improving production yields are discussed.
A SpyRing cyclized cephalosporin C acylase (SRCCA) was obtained by fusing SpyTag and SpyCatcher to the N- and C- termini of cephalosporin C acylase (CCA), respectively. The results suggested that the introduction of the SpyRing (head-to-tail cyclization via SpyTag and SpyCatcher) did not affect the active center of the SRCCA (the specific activities of CCA and SRCCA are 15.71 U/mg and 13.11 U/mg, respectively). Also, the thermostability, organic solvents tolerance, and denaturant tolerance of the free enzyme SRCCA were improved. Since glyoxyl agarose carrier favors the covalent immobilization of enzymes through its surface regions having the highest lysine residues density, SRCCA permitted its multipoint and oriented immobilization because SpyRing is very rich in Lys residues, while CCA is quite poor in Lys residues and immobilization is via less enzyme support-bonds. When the enzyme loading amount was 10 mg/g carrier, the expressed activity of SRCCA was 22 % higher than that of CCA. The stability of the immobilized SRCCA was also significantly improved; the half-life of the immobilized SRCCA at 50 °C was 125 min, which was about 5 times the half-life of the immobilized CCA.
Pyrroloquinoline quinone (PQQ) is a cofactor of various membrane-bound dehydrogenases. The amount of endogenous PQQ is generally regarded as a bottleneck to achieving higher catalytic efficiency of PQQ-dependent dehydrogenases. Proteins that biosynthesize PQQ in Gluconobacter oxydans WSH-003 are encoded by the pqqABCDE gene cluster and the tldD gene. In this study, PQQ overproduction in G. oxydans was attempted by overexpressing PQQ biosynthetic genes using the promoter of pqqA and the elongation factor TU (tufB). Overexpression of each single gene could enhance PQQ biosynthesis except for the tldD gene. Overexpression of pqqA, pqqB, pqqC, pqqD and pqqE with the pqqA promoter enhanced the extracellular PQQ concentration by 38.5%, 68.4%, 19.9%, 30.3% and 8.2%, respectively, whereas production was enhanced by 59.4%, 85.7%, 30.9%, 42.2% and 19.3%, respectively, using the tufB promoter. In addition, the results show that PQQ biosynthesis could be enhanced by overexpressing some of the individual genes in the gene cluster in G. oxydans and the PQQ levels were positively correlated with the efficiency of conversion of d-sorbitol to l-sorbose. The results demonstrated that cofactor engineering of PQQ in G. oxydans is beneficial for enhancing the production of quinoprotein-related products.
The greenness of chemical processes turns around two main axes: the selectivity of catalytic transformations, and the separation of pure products. The transfer of the exquisite catalytic efficiency shown by enzymes in nature to chemical processes is an important challenge. By using appropriate reaction systems, the combination of biopolymers with supercritical carbon dioxide (scCO2) and ionic liquids (ILs) resulted in synergetic and outstanding platforms for developing (multi)catalytic green chemical processes, even under flow conditions. The stabilization of biocatalysts, together with the design of straightforward approaches for separation of pure products including the full recovery and reuse of enzymes/ILs systems, are essential elements for developing clean chemical processes. By understanding structure-function relationships of biopolymers in ILs, as well as for ILs themselves (e.g. sponge-like ionic liquids, SLILs; supported ionic liquids-like phases, SILLPs, etc.), several integral green chemical processes of (bio)catalytic transformation and pure product separation are pointed out (e.g. the biocatalytic production of biodiesel in SLILs, etc.). Other developments based in DNA/ILs systems, as pathfinder works for further technological applications in the near future, are also considered.
