Crude glycerol, a byproduct of biodiesel production, was utilized as a carbon source to produce microbial lipids by the oleaginous yeast Rhodotorula toruloides in this study. The maximum lipid production and lipid content were 10.56 g/L and 49.52%, respectively, by optimizing fermentation conditions. The obtained biodiesel met the standards of China, the United States, and the European Union. The economic value of biodiesel produced from crude glycerol increased by 48% compared with the sale of crude glycerol. In addition, biodiesel production from crude glycerol could reduce 11,928 tons of carbon dioxide emissions and 55 tons of sulfur dioxide emissions. This study provides a strategy for a closed loop of crude glycerol to biofuel and ensures sustainable and stable development of the biodiesel industries.

Secretory signal peptides (SPs) can increase enhanced green fluorescent protein (eGFP) expression in cytosol. In this study, SPs Iasp (Cry1Ia), Vasp (Vip3A), and their local sequences were used as fusion tags to compare their effects on eGFP expression in Escherichia coli MC4100 and Pichia pastoris GS115. In E coli, the solubility was almost opposite between the proteins encoded by Vegfp and Iegfp. This may be because the overall hydrophobicity of the SPs differed. When the hydrophobic H-region and C-region were removed, the negative effects on eGFP solubility of the N-regions of both SPs (IaN and VN) were significantly reduced without compromise on the expression level. IaN promotes eGFP protein yield 7.1-fold more than Iasp, and using this peptide in tandem (Ia3N) further enhanced fluorescent fusion protein solubility with an efficacy similar to that of a polycationic tag. Furthermore, the GS-IaNeGFP strain produced the highest fluorescent signal intensity when these fusion proteins were expressed in P. pastoris, and the expression was higher than in other strains, including eGFP. In conclusion, we revealed the potential of the N-region of Iasp as a fusion tag in both prokaryotic and eukaryotic cells and further demonstrated the value of the N-regions of abundant SPs.

The COVID-19 pandemic has greatly impacted the global economy and health care systems, illustrating the urgent need for timely and inexpensive responses to pandemic threats in the form of vaccines and antigen tests. Currently, antigen testing is mostly conducted by qualitative flow chromatography or via quantitative ELISA-type assays. The latter mostly utilize materials like protein-adhesive polymers and gold or latex particles. Here we present an alternative ELISA approach using inexpensive, biogenic materials and permitting quick detection based on components produced in the microbial model Ustilago maydis. In this fungus, heterologous proteins like biopharmaceuticals can be exported by fusion to unconventionally secreted chitinase Cts1. As a unique feature, the carrier chitinase binds to chitin allowing its additional use as a purification or immobilization tag. Recent work has demonstrated that nanobodies are suitable target proteins. These proteins represent a very versatile alternative antibody format and can quickly be adapted to detect novel antigens by camelidae immunization or synthetic libraries. In this study, we exemplarily produced different mono- and bivalent SARS-CoV-2 nanobodies directed against the spike protein receptor binding domain (RBD) as Cts1 fusions and screened their antigen binding affinity in vitro and in vivo. Functional nanobody-Cts1 fusions were immobilized on chitin forming an RBD tethering surface. This provides a solid base for future development of inexpensive antigen tests utilizing unconventionally secreted nanobodies as antigen trap and a matching ubiquitous and biogenic surface for immobilization.

Strain robustness during production of recombinant molecules is of major interest to ensure bioprocess profitability. The heterogeneity of populations has been shown in the literature as a source of instability in bioprocesses. Thus, the heterogeneity of the population was studied by evaluating the robustness of the strains (stability of plasmid expression, cultivability, membrane integrity and macroscopic cell behavior) during well-controlled fedbatch cultures. On the context of microbial production of chemical molecules, isopropanol (IPA) has been produced by recombinant strains of Cupriavidus necator. Plasmid stability was monitored by the plate count method to assess the impact of isopropanol production on plasmid stability, depending on implanted plasmid stabilization systems for strain engineering designs. With the reference strain Re2133/pEG7c, an isopropanol titer of 15.1 g·L-1 could be achieved. When the isopropanol concentration has reached about 8 g. L-1, cell permeability increased (up to 25 %) and plasmid stability decreased significantly (up to 1.5 decimal reduction rate) resulting in decreased isopropanol production rates. Bioprocess robustness under isopropanol producing conditions was then investigated with two plasmid construction strategies (1) Post Segregational Killing hok/sok (in Re2133/pEG20) and (2) expression of GroESL chaperon proteins (in Re2133/pEG23). Plasmid stability for strain Re2133/pEG20 (PSK hok/sok) appears to be improved up to 11 g. L-1 of IPA compared to the reference strain (8 g. L-1 IPA). Nevertheless, cell permeability followed the same dynamic as the reference strain with a drastic increase around 8 g. L-1 IPA. On the contrary, the Re2133/pEG23 strain made it possible to minimize the cell permeability (with a constant value at 5 % IP permeability) and to increase the growth capacities in response to increased isopropanol concentrations but plasmid stability was the weakest. The metabolic burden, linked to either the overexpression of GroESL chaperones or the PSK hok/sok system, seems to be deleterious for the overall isopropanol production compared to the reference strain (RE2133/pEG7c) even if we have shown that the overexpression chaperones GroESL improve membrane integrity and PSK system hok/sok improve plasmid stability as long as isopropanol concentration does not exceed 11 g L- 1.

Glycerol dehydrogenase (GldA) from Escherichia coli BW25113, naturally catalyzes the oxidation of glycerol to dihydroxyacetone. It is known that GldA exhibits promiscuity towards short-chain C2-C4 alcohols. However, there are no reports regarding the substrate scope of GldA towards larger substrates. Herein we demonstrate that GldA can accept bulkier C6-C8 alcohols than previously anticipated. Overexpression of the gldA gene in the knockout background, E. coli BW25113 ΔgldA, was strikingly effective converting 2 mM of the compounds: cis-dihydrocatetechol, cis-(1 S,2 R)- 3-methylcyclohexa-3,5-diene-1,2-diol and cis-(1 S,2 R)- 3-ethylcyclohexa-3,5-diene-1,2-diol, into 2.04 ± 0.21 mM of catechol, 0.62 ± 0.11 mM 3-methylcatechol, and 0.16 ± 0.02 mM 3-ethylcatechol, respectively. In-silico studies on the active site of GldA enlightened the decrease in product formation as the steric substrate demand increased. These results are of high interests for E. coli-based cell factories expressing Rieske non-heme iron dioxygenases, producing cis-dihydrocatechols, since such sough-after valuable products can be immediately degraded by GldA, substantially hampering the expected performance of the recombinant platform.

Hyaluronate lyases (HA lyases) have been proved to distribute widely among microorganisms, with large potential in hyaluronan processing. Here, a highly active HA lyase HylC from Citrobacter freundii strain Cf1 is reported. HylC was expressed in Escherichia coli BL21(DE3) under the regulation of T7 promoter, and purified to electrophoretic homogeneity for enzymatic characterization, which suggested its suitable thermo- and pH stability under 45 °C and pH rang of 4-8, and high halotolerancy in 1.5 M NaCl. The enzyme exhibited the optimal activity under 37 °C and pH 5.5, and was activated by Ca2+, K+, Zn2+, Ni2+ and Li+. Analysis of degradation product proved it cleave HA in endolytic manner, releasing unsaturated disaccharides as final product. Then, through optimization of promoter and construction of dual promoter, expression level of HylC improved from 1.10 × 104 U/mL to 2.64 × 104 U/mL on shake-flask level. Finally, through batch fermentation, a highest activity of 2.65×105 U/mL was achieved in a 5-L fermenter. Taken together, this work demonstrates the potential of HylC and its recombinant strain in industrial applications. To our knowledge, the HA lyase production reported in this study was the highest level in literatures to date.

Traditionally produced fish sauce can contain significant amounts of histamine. In some instances, the histamine concentration may be well above the limit recommended by the Codex Alimentarius Commission. The aim of this study was to discover new bacterial strains capable of growing under the stressful environmental conditions of fish sauce fermentation and metabolizing histamine. In this study, 28 bacterial strains were isolated from Vietnamese fish sauce products based on their ability to grow at high salt concentrations (23% NaCl) and tested for their ability to degrade histamine. Strain TT8.5 showed the highest histamine-degradation (45.1 ± 0.2% of initially 5 mM histamine within 7 days) and was identified as Virgibacillus campisalis TT8.5. Its histamine-degrading activity was shown to be localized intracellularly and the enzyme is a putative histamine dehydrogenase. The strain exhibited optimal growth and histamine-degrading activity at 37°C, pH 7%, and 5% NaCl in halophilic archaea (HA) histamine broth. It also showed pronounced histamine-degrading activity in HA histamine broth when cultivated at temperatures of up to 40 °C as well as in the presence of up to 23% NaCl. After treatment with immobilized cells, 17.6-26.9% of the initial histamine in various fish sauce products were reduced within 24 h of incubation, while no significant changes in other parameters of fish sauce quality were observed after this treatment. Our results indicate that V. campisalis TT8.5 is of potential interest to be applied in histamine degradation of traditional fish sauce.

This present study mainly focused on the investigation and optimization of the fermentation performance of oleaginous yeasts on Eucommia ulmoides Oliver hydrolysate (EUOH), which contains abundant and diverse sugars. More importantly, the impacts of the mixed strains fermentation compared with the single strain fermentation were analyzed and evaluated, through systematic investigations of substrate metabolism, cell growth, polysaccharide and lipid production, COD and ammonia-nitrogen removals. It was found that the mixed strains fermentation could effectively promote a more comprehensive and thorough utilization of the various sugars in EUOH, greatly improve COD removal effect, biomass and yeast polysaccharide production, but could not significantly improve the overall lipid content and ammonia nitrogen removal effect. In this study, when the two strains with the highest lipid content (i.e. L. starkeyi and R. toruloides) were mixed-cultured, the maximum lipid yield of 3.82 g/L was achieved, and the yeast polysaccharide yield, COD and ammonia-nitrogen removal rates of the fermentation (LS+RT) were 1.64 g/L, 67.4% and 74.9% respectively. When the strain with the highest polysaccharide content (i.e. R. toruloides) was mixed-cultured with the strains with strong growth activity (i.e. T. cutaneum and T. dermatis), a large amount of yeast polysaccharides could be obtained, which were 2.33 g/L (RT+TC) and 2.38 g/L (RT+TD) respectively. And the lipid yield, COD and ammonia-nitrogen removal rates of the fermentation (RT+TC), (RT+TD) were 3.09 g/L, 77.7%, 81.4% and 2.54 g/L, 74.9%, 80.4%, respectively.

Indigo dye is an organic compound with a distinctive blue color. Most of the indigo currently used in industry is produced via chemical synthesis, which generates a large amount of wastewater. Therefore, several studies have recently been conducted to find ways to produce indigo eco-friendly using microorganisms. Here, we produced indigo using recombinant Escherichia coli with both an indigo-producing plasmid and a cyclopropane fatty acid (CFA)-regulating plasmid. The CFA-regulating plasmid contains the cfa gene, and its expression increases the CFA composition of the phospholipid fatty acids of the cell membrane. Overexpression of cfa showed cytotoxicity resistance of indole, an intermediate product formed during the indigo production process. This had a positive effect on indigo production and cfa originated from Pseudomonas sp. B 14-6 was used. Optimal conditions for indigo production were determined by adjusting the expression strain, culture temperature, shaking speed, and isopropyl β-D-1-thiogalactopyranoside concentration. Treatment with Tween 80 at a particular concentration to increase the permeability of the cell membrane had a positive effect on indigo production. The strain with the CFA plasmid produced 4.1 mM of indigo after 24 h of culture and produced 1.5-fold higher indigo than the control strain without the CFA plasmid that produced 2.7 mM.

It is well-known that enzymes are molecules particularly susceptible to pH and temperature variations. Immobilization techniques may overcome this weakness besides improving the reusability of the biocatalysts. Given the strong push toward a circular economy, the use of natural lignocellulosic wastes as supports for enzyme immobilization has been increasingly attractive in recent years. This fact is mainly due to their high availability, low costs, and the possibility of reducing the environmental impact that can occur when they are improperly stored. In addition, they have physical and chemical characteristics suitable for enzyme immobilization (large surface area, high rigidity, porosity, reactive functional groups, etc.). This review aims to guide readers and provide them with the tools necessary to select the most suitable methodology for lipase immobilization on lignocellulosic wastes. The importance and the characteristics of an increasingly interesting enzyme, such as lipase, and the advantages and disadvantages of the different immobilization methods will be discussed. The various kinds of lignocellulosic wastes and the processing required to make them suitable as carriers will be also reported.

There is a demand for increasing the current manufacturing capacities for recombinant protein-based drugs. Novel expression systems such as plants are being explored as faster, more flexible, and possibly cheaper platforms. Many of these therapeutic proteins are glycosylated and require terminal sialylation to attain full biological activity. In planta protein sialylation has been achieved by the introduction of an entire mammalian biosynthetic pathway in Nicotiana benthamiana, comprising the coordinated expression of the genes for (i) biosynthesis, (ii) activation, (iii) transport, and (iv) transfer of Neu5Ac to terminal galactose. Here we address technical issues that can compromise the efficacy of protein sialylation and how they can be overcome. We used the same reporter protein to compared three strategies to transiently deliver the sialylation pathway-genes evaluating efficacy, heterogeneity and batch-to-batch consistency. In addition, we assess the ability of the single-step method to sialylated additional recombinant proteins with different complexity and number of glycosylation sites. Finally, we show that efficient protein sialylation can be up-scaled for large-scale production of sialylated proteins in plants.

V160 is a viral vaccine candidate against human cytomegalovirus (HCMV) that is manufactured using Adult Retinal Pigment Epithelial cells (ARPE-19) grown on Cytodex-1 microcarriers. The microcarriers are generally hydrated, washed, and autoclaved prior to use, which can be limiting at large production scales. To minimize microcarrier preparation and sterilization, the use of gamma irradiated Cytodex-1 was investigated. Similar ARPE-19 cell growth was observed on heat-sterilized and gamma irradiated Cytodex-1; however, significantly reduced virus production was observed in cultures exposed to gamma irradiated Cytodex-1. Additional experiments suggest that infection inhibition is not exclusive to ARPE-19 but is most directly linked to HCMV V160, as evidenced by similar inhibition of V160 with Vero cells and no inhibition of Measles virus with either cell type. These observations suggest a putative impact on HCMV infection from the presence of extractable(s)/leachable(s) in the gamma irradiated microcarriers. Thorough aseptic rinsing of gamma irradiated Cytodex-1 prior to use can mitigate this impact and enable comparable process performance to heat-sterilized Cytodex-1. Though not fully a "ready-to-use" product for the HCMV V160 production process, utilization of Cytodex-1 microcarriers was possible without requiring heat sterilization, suggesting a potential path forward for large scale production of V160.