Dibutyl phthalate (DBP) is an environmental pollutant that can threaten human health. The strain Arthrobacter sp. ZJUTW, isolated from the sludge of river of Hangzhou city, can efficiently degrade DBP. Its genomic and transcriptomic differences when cultivated with DBP and with glucose revealed specific DBP metabolic pathways in the ZJUTW strain. The degrading gene clusters distribute separately on a circular chromosome and a plasmid pQL1. Genes related to the initial steps of DBP degradation from DBP to phthalic acid (PA), the pehA gene and pht gene cluster, are located on the plasmid pQL1. While pca gene cluster related to the transforming of protocatechuic acid (PCA) to acetyl-CoA, is located on the chromosome. After homologous alignment analysis with the reported gene clusters, we found that there were a series of double copies of homologous genes in pht and pca gene clusters that contribute to the efficient degradation of DBP by ZJUTW. In addition, transcriptomic analysis showed a synergistic effect between pht and pca clusters, which also favor ZJUTW allowing it to efficiently degrade DBP. Combined genomic and transcriptomic analyses affords the complete DBP metabolic pathway in Arthrobacter sp. ZJUTW that is different from that of reported other Arthrobacter strains. After necessary modification based on its metabolic characteristics, Arthrobacter sp. ZJUTW or its mutants might represent promising candidates for use in the bioremediation of DBP pollution.
Glutathione (GSH) plays a central role in the redox balance maintenance in mammalian cells. The study of industrial CHO cell lines have demonstrated a close link between GSH metabolism and clone productivity. However, a deep investigation is still required to understand this correlation and highlights new potential targets for cell engineering. In this study, we have modulated the GSH intracellular content of an industrial cell line under bioprocess conditions in order to further elucidate the role of the GSH synthesis pathway. Two strategies were used : the variation of cystine supply and the direct inhibition of the GSH synthesis using buthionine sulfoximine (BSO). Cysteine supply modulation have revealed a correlation between intracellular GSH and product titer over time. Analysis of metabolites uptake/secretion rates and proteome comparison between BSO-treated cells and non-treated cells has highlighted a slow down of the TCA cycle leading to a secretion of lactate and alanine in the extracellular environment. Moreover, an adaptation of the glutathione related proteome has been observed with a up-regulation of the regulatory subunit of glutamate cysteine ligase and a down-regulation of a specific glutathione transferase subgroup, the Mu family. Surprisingly, the main impact of BSO treatment was observed on a global down-regulation of the cholesterol synthesis pathways. As cholesterol is required for protein secretion, it can be the missing part of the jigsaw to finally elucidate the link between GSH synthesis and productivity.
In order to maximize the productivity of engineered metabolic pathway, in silico model is an established means to provide features of enzyme reaction dynamics. In our previous study, E.coli engineered with acrylate pathway yielded low propionic acid titre. To understand the bottleneck behind this low productivity, a kinetic model was developed that incorporates the enzymatic reactions of the acrylate pathway. The resulting model was capable of simulating the fluxes reported under in vitro studies with good agreement, suggesting repression of propionyl-CoA transferase by carboxylate metabolites as the main limiting factor for propionate production. Furthermore, the predicted ﬂux control coeﬃcients of the pathway enzymes under steady state conditions revealed that the control of ﬂux is shared between propionyl-CoA transferase and lactoyl-CoA dehydratase. Increase in lactate concentration showed gradual decrease in ﬂux control coeﬃcients of propionyl-CoA transferase that in turn confirmed the control exerted by the carboxylate substrate. To interpret these in silico predictions under in vivo system, an organized study was conducted with a Lactic Acid Bacteria (LAB) strain engineered with acrylate pathway. Analysis reported a decreased product formation rate on attainment of inhibitory titre by suspected metabolites and supported the model.
As issues surrounding depleting fossil fuels, climate change, and various other environmental impacts are becoming more prevalent, there is a growing interest in technological shifts toward a bio-based economy. Various advanced biotechnological tools have been developed to customize cell factories for the production of a wide range of complex fine chemicals from renewable feedstock. Herein, we report development of a microbial bioprocess for high-level and potentially economical production of 5-aminolevulinic acid (5-ALA), a valuable non-proteinogenic amino acid with multiple applications in medical, agricultural, and food industries, using Escherichia coli as a cell factory. We first implemented the Shemin (i.e., C4) pathway for heterologous 5-ALA biosynthesis in E. coli. To reduce, but not to abolish, the carbon flux toward essential tetrapyrrole/porphyrin biosynthesis, we applied Clustered Regularly Interspersed Short Palindromic Repeats interference (CRISPRi) to repress hemB expression, leading to extracellular 5-ALA accumulation. We then applied metabolic engineering strategies to direct more dissimilated carbon flux toward the key precursor of succinyl-CoA for enhanced 5-ALA biosynthesis. Using these engineered E. coli strains for bioreactor cultivation, we successfully demonstrated high-level 5-ALA biosynthesis solely from glycerol (~30 g l-1) under both microaerobic and aerobic conditions, achieving up to 5.95 g l-1 (36.9% yield) and 6.93 g l-1 (50.9% yield) 5-ALA, respectively. This study represents one of the most effective bio-based production of 5-ALA from a structurally unrelated carbon to date, highlighting the importance of integrated strain engineering and bioprocessing strategies to enhance bio-based production.
Fluorescent in situ hybridization (FISH) has been extensively used in the past decades for the detection and localization of nucleic acid sequences or of the microorganisms themselves within samples. However, a mechanistic approach of the whole FISH process is still missing, and the main limiting steps for the hybridization to occur remain unclear. In here, FISH is approached as a particular case of a diffusion-reaction kinetics, where molecular probes move from the hybridization solution to the target RNA site within the cells. Based on literature models, the characteristic times taken by different molecular probes to diffuse across multiple cellular barriers, and the reaction time associated with the formation of the duplex molecular probe-RNA were estimated. Structural and size differences at the membrane level of bacterial and animal cells were considered. For bacterial cells, the limiting step for diffusion is likely to be the peptidoglycan layer (characteristic time of 2700-4524 s), whereas for animal cells the limiting step should be the diffusion of the probe through the bulk (1.8-5.0 s) followed by the diffusion through the lipid membrane (1 s). The information provided here may serve as a basis to optimize FISH protocols.
Shewanella oneidensis MR-1, a model strain of exoelectrogenic bacteria (EEB), plays a key role in environmental bioremediation and bioelectrochemical systems because of its unique respiration capacity. However, only a narrow range of substrates can be utilized by S. oneidensis MR-1 as carbon sources, resulting in its limited applications. In this work, a rapid, highly efficient and easily manipulated base editing system pCBEso was developed by fusing a Cas9 nickase (Cas9n (D10A)) with the cytidine deaminase rAPOBEC1 in S. oneidensis MR-1. The C-to-T conversion of suitable C within the base editing window could be readily and efficiently achieved by the pCBEso system without requiring double strand break or repair templates. Moreover, double-locus simultaneous editing was successfully accomplished with an efficiency of 87.5. With this tool, the roles of the key genes involving in N-acetyl-glucosamine (GlcNAc) or glucose metabolism in S. oneidensis MR-1 were identified. Furthermore, an engineered strain with expanded carbon source utilization spectra was constructed and exhibited a higher degradation rate for multiple organic pollutants (i.e., azo dyes and organoarsenic compounds) than the wild type when glucose or GlcNAc was used as the sole carbon source. Such a base editing system could be readily applied to other EEB. This work not only enhances the substrate utilization and pollutant degradation capacities of S. oneidensis MR-1, but also accelerates the robust construction of engineered strains for environmental bioremediation.
Tissue constructs of physiologically relevant scale require a vascular system to maintain cell viability. However, in vitro vascularization of engineered tissues is still a major challenge. Successful approaches are based on a feeder layer (FL) to support vascularization. Here, we investigated whether the supporting effect on the self-assembled formation of vascular-like structures by microvascular endothelial cells (mvECs) originates from the FL itself or from its extracellular matrix (ECM). Therefore, we compared the influence of ECM, either derived from adipose-derived stem cells (ASCs) or adipogenic differentiated ASCs, with the classical approaches based on a cellular FL. All cell-derived ECM (cdECM) substrates enable mvEC growth with high viability. Vascular-like structure formation was visualized by immunofluorescence staining of endothelial surface protein CD 31 and can be observed on all cdECM and FL substrates but not on control substrate collagen I. On adipogenic differentiated ECM longer and higher branched structures can be found compared to stem cell cdECM. An increased concentration of pro-angiogenic factors can be found in cdECM substrates and FL approaches compared to controls. Finally, expression of proteins associated with tube formation (E-selectin and thrombomodulin) was confirmed. These results highlight cdECM as promising biomaterial for in vitro vascularization in adipose tissue engineering.
We report on the development of a new model of alveolar air-tissue interface consisting of an array of suspended hexagonal monolayers of gelatin nanofibers supported by microframes and a microfluidic device for the patch integration. The suspended monolayers are deformed to a central displacement of 40-80 μm at the air-liquid interface by application of air pressure in the range of 200-1000 Pa. With respect to the diameter of the monolayers that is 500 μm, this displacement corresponds to a linear strain of 2-10% in agreement with the physiological strain range in the lung alveoli. The culture of A549 cells on the monolayers for an incubation time 1-3 days showed viability in the model. We exerted a periodic strain of 5% at a frequency of 0.2 Hz during 1 hour to the cells. We found that the cells were strongly coupled to the nanofibers, but the strain reduced the coupling and induced remodeling of the actin cytoskeleton, which led to a better tissue formation. Our model can serve as a versatile tool in lung investigations such as in inhalation toxicology and therapy.
β-carotene is a natural pigment and health-promoting metabolite, and has been widely used in the nutraceutical, feed and cosmetic industries. Here, we engineered a GRAS yeast Saccharomyces cerevisiae to produce β-carotene from xylose, the second most abundant and inedible sugar component of lignocellulose biomass. Specifically, a β-carotene biosynthetic pathway containing crtYB, crtI and crtE from Xanthophyllomyces dendrorhous were introduced into a xylose-fermenting S. cerevisiae. The resulting strain produced β-carotene from xylose at a titer three-fold higher than from glucose. Interestingly, overexpression of tHMG1, which has been reported as a critical genetic perturbation to enhance metabolic fluxes in the mevalonate (MVA) pathway and β-carotene production in yeast when glucose is used, did not further improve the production of β-carotene from xylose. Through fermentation profiling, metabolites analysis and transcriptional studies, we found the advantages of using xylose as a carbon source instead of glucose for β-carotene production to be a more respiratory feature of xylose consumption, a larger cytosolic acetyl-CoA pool, and up-regulated expression levels of rate-limiting genes in the β-carotene producing pathway, including ACS1 and HMG1. As a result, 772.81 mg/L of β-carotene was obtained in a fed-batch bioreactor culture with xylose feeding. Considering the inevitable production of xylose at large scales when cellulosic biomass-based bioeconomy is implemented, our results suggest xylose utilization is a promising strategy for overproduction of carotenoids and other isoprenoids in engineered S. cerevisiae.
Organs-on-chip are increasingly catching on as a promising and valuable alternative to animal models, in line with the 3Rs initiative, to create 3D tissue microenvironments in which cells behave physiologically and pathologically at unparalleled precision and complexity. Indeed, these platforms offer new opportunities to model human diseases and test the potential therapeutic effect of different drugs as well as their limitations, overtaking the limited predictive accuracy of conventional 2D culture systems. Here, we present a liver-on-a-chip model to investigate the effects of two naturally occurring polyphenols, namely Quercetin and Hydroxytyrosol, on non-alcoholic fatty liver disease (NAFLD) using a method of high-content analysis. NAFLD is currently the most common form of chronic liver disease, whose complex pathogenesis is far from being clear. Besides, no definitive treatment has been established for NAFLD so far. In our experiments, we observed that both polyphenols seem to restrain the progression of the free fatty acid-induced hepatocellular steatosis, showing a cytoprotective effect due to their antioxidant properties. In conclusion, the resulting insights of the present work could guide novel strategies to contrast the onset and progression of NAFLD.
Significant amounts of soluble product aggregates were observed in the low-pH viral inactivation (VI) opertion during an initial scale-up run for an IgG4 monoclonal antibody (mAb IgG4-N1). Being earlier in development, a scale-down model did not exist, nor was it practical to use costly Protein A eluate (PAE) for testing the VI process at scale, thus, a computational fluid dynamics (CFD)-based high molecular weight (HMW) prediction model was developed for troubleshooting and risk mitigation. It was previously reported that the IgG4-N1 molecules upon exposure to low pH tend to change into transient and partially unfolded monomers during VI acidification (i.e., VIA) and form aggregates after neutralization (i.e., VIN) (Jin et al. 2019). Therefore, the CFD model reported here focuses on the VIA step. The model mimics the continuous addition of acid to PAE and tracks acid distribution during VIA. Based on the simulated low-pH zone (≤ pH 3.3) profiles and PAE properties, the integrated low-pH zone (ILPZ) value was obtained to predict HMW level at the VI step. The simulations were performed to examine the operating parameters, such as agitation speed, acid addition rate, and protein concentration of PAE, of the pilot scale (50-200L) runs. The conditions with predictions of no product aggregation risk were recommended to the real scale-up runs, resulted in 100% success rate of the consecutive 12 pilot-scale runs. This work demonstrated that the CFD-based HMW prediction model could be used as a tool to facilitate the scale up of the low-pH VI process directly from bench to pilot/production scale.
Hypertension is a major risk factor for cardiovascular diseases, with high prevalence in low- and high-income countries. Among the various antihypertensive therapeutic strategies, synthetic Angiotensin I-converting enzyme inhibitors (ACEI) are one of the most used pharmacological agents. However, their use in hypertension therapy has been linked to various side effects. In recent years considerable research has been performed on the use of food-derived ACEI peptides (ACEIp) as antihypertensive agents. Although promising, the industrial production of these ACEIp through conventional methods, such as chemical synthesis and enzymatic hydrolysis of food proteins, has been proven troublesome and expensive. Limitations to the large-scale production of ACEIp for functional foods and supplements can be overcome by producing the precursors of these peptides in heterologous hosts. Bacterial hosts have been the privileged choice, particularly to test the success of the genetic engineering strategies, but new platforms based on plants and microalgae have also been emerging. This work provides an overview of the state of antihypertensive therapy, focusing on ACEI, illustrates the latest advances on ACEIp research, and describes current genetic engineer-based approaches for the heterologous production of ACEIp for antihypertensive therapy.
Technological advancements in the past few decades have made it possible to manufacture nanomaterials at large scale and ENPs are increasingly found in consumer products such as cosmetics, sports products and LED displays. A large amount of these ENPs are in wastewater and potentially impact the performance of wastewater treatment plants (WWTPs). One important function of the WWTP is nitrification, which is carried out by the actions of two groups of bacteria, ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB). Since most ENPs are found to have or are designed to have antimicrobial activities, it is a legitimate concern that ENPs entering WWTPs may have negative impacts on nitrification. In this paper, the effects of ENPs on nitrification is discussed, focusing mainly on autotrophic nitrification by AOBs and NOBs. This review also covers ENPs effects on ANAMMOX. Generally, nitrifiers in pure and mixed culture can be inhibited by a variety of ENPs, but stress response mechanisms may attenuate toxicity. Long-term studies demonstrated that a wide range of NPs can cause severe deterioration of AOBs and/or NOBs when the influent concentration exceeded an inhibition threshold. Proposed mechanisms include the generation of reactive oxygen species, dissolved metals, physical disruption of cell membranes, bacterial engulfment, and intracellular accumulation of ENPs. Future research needs are also discussed.
Homologous recombination over large genomic regions is difficult to achieve due to low efficiencies. Here, we report the successful engineering of a humanized mTert allele, hmTert, in the mouse genome by replacing an 18.1-kb genomic region around the mTert gene with a recombinant fragment of over 45.5-kb, using homologous recombination facilitated by the Crispr/Cas9 technology, in mouse embryonic stem cells (mESCs). In our experiments, with specific sites of DNA double strand breaks (DSBs) by Crispr/Cas9 system, the homologous recombination efficiency was up to 11% and 16% in two mESC lines TC1 and v6.5, respectively. Overall, we obtained a total of 27 mESC clones with heterozygous hmTert/mTert alleles and 3 clones with homozygous hmTert alleles. DSBs induced by Crispr/Cas9 cleavages also caused high rates of genomic DNA deletions and mutations at small guide RNA (sgRNA) target sites. Our results indicated the Crispr/Cas9 system significantly increased the efficiency of homologous recombination-mediated gene editing over a large genomic region in mammal cells, but also inherently caused mutations at unedited target sites. Overall, this strategy provides an efficient and feasible way for manipulating large chromosomal regions.
The available pneumococcal conjugate vaccines provide protection against only those serotypes that are included in the vaccine, which leads to a selective pressure and serotype replacement in the population. An alternative low-cost, safe and serotype-independent vaccine was developed based on a non-encapsulated pneumococcus strain. This study evaluates process intensification to improve biomass production and shows for the first time the use of perfusion-batch with cell recycling for a bacterial vaccine production. Batch, fed-batch and perfusion-batch were performed at 10 L scale using a complex animal component-free culture medium. Cells were harvested at the highest optical density, concentrated and washed using microfiltration or centrifugation to compare cell separation methods. Higher biomass was achieved using perfusion-batch, which removes lactate while retaining cells. The biomass produced in perfusion-batch would represent at least 4-fold greater number of doses per cultivation than in the previously described batch process. Each strategy yielded similar vaccines in terms of quality as evaluated by Western blot and animal immunization assays, indicating that, so far, perfusion-batch is the best strategy for the intensification of pneumococcal whole cell vaccine production, since it can be integrated to the cell separation process keeping the same vaccine quality.
Many manufacturers of biopharmaceuticals are moving from batch to continuous processing. While this approach offers advantages over batch processing to manufacturers, demonstration of viral clearance for continuous processes is more complex. Regulators expect manufacturers to use an appropriate scale down model, based on solid scientific justification, to verify the viral reduction capacity of the manufacturing process. The output from chromatography columns operated in continuous processes fluctuates in concentration so that the load for the subsequent column is not homogenous. This must be considered when designing viral clearance studies. One way to approach clearance studies is to downscale the continuous process, using multi-column chromatography systems and in-line spiking of virus. The multi-column chromatography systems and experienced operators, however, may not be available at the CRO performing the study. Another approach is to evaluate each step in traditional batch mode, using existing chromatography systems, but to modify the spiking and loading conditions to mimic the variance introduced by the transition between the two connected process steps. Using a standard chromatography system, we have evaluated a flow through anion exchange chromatography step in a monoclonal antibody process using five different methods to introduce the virus to the column. We have shown that regardless of whether the virus spike is introduced in a well-mixed batch mode, introduced as a concentrated pulse of virus with homogeneous mAb or with a concentrated peak of mAb, the clearance of MMV was similar. This study introduces an alternative way to evaluate viral clearance in a continuous process.
Mussel adhesive proteins (MAPs) have great potential as bioglues, in particular in wet conditions. Although in vivo residue-specific incorporation of 3,4-dihydroxyphenylalanine (Dopa) in tyrosine-auxotrophic Escherichia coli cells allows production of bioengineered MAPs (bMAPs), the low production yield hinders the practical application of bMAPs. Such low production yield of Dopa-incorporated bMAPs (Dopa-bMAPs) was known to be caused by low translational activity of a noncanonical amino acid, Dopa, in E. coli cells. Herein, in order to enhance the production yield of Dopa-bMAPs, we investigated the coexpression of Dopa-recognizing tyrosyl-tRNA synthetases (TyrRSs). In order to use the Dopa-specific Methanococcus jannascii TyrRS (MjTyrRS-Dopa), we altered the anti-codon of tyrosyl-tRNA amber suppressor into AUA (MjtRNATyrAUA) to recgonize a tyrosine codon (MjtRNATyrAUA). Co-overexpression of MjTyrRS-Dopa and MjtRNATyrAUA increased the production yield of Dopa-MAP by 57%. Similarly, overexpression of E. coli TyrRS (EcTyrRS) led to a 72% higher production yield of Dopa-incorporated bMAP. Even with coexpression of Dopa-recognizing TyrRSs, Dopa-bMAPs have a high Dopa incorporation yield (over 90%) compared to Dopa-bMAPs prepared without any coexpression of TyrRS.
Clinical use of pancreatic beta islets for regenerative medicine applications requires mass production of functional cells. Current technologies are insufficient for large-scale production in a cost-efficient manner. Here, we evaluate advantages of a porous cellulose scaffold and demonstrate scale-up to a wicking-matrix bioreactor as a platform for culture of human endocrine cells. Scaffold modifications were evaluated in a multi-well platform to find the optimum surface condition for pancreatic cell expansion followed by bioreactor culture to confirm suitability. Preceding scale-up, cell morphology, viability and proliferation of primary pancreatic cells were evaluated. Two optimal surface modifications were chosen and evaluated further for insulin secretion, cell morphology and viable cell density for human induced pluripotent stem cell-derived pancreatic cells at different stages of differentiation. Scale-up was accomplished with uncoated, amine-modified cellulose in a miniature bioreactor, and insulin secretion and cell metabolic profiles were determined for 13 days. We achieved 10-fold cell expansion in the bioreactor along with a significant increase in insulin secretion compared with cultures on tissue-culture plastic. Our findings define a new method for expansion of pancreatic cells on wicking-matrix cellulose platform to advance cell therapy biomanufacturing for diabetes.