High-titer production of aromatic amines in metabolically engineered Escherichia coli

Aromatic amines are widely used in the pharmaceutical industry. Here, we reported the establishment of a bacterial platform for synthesizing three types of aromatic amines, namely, tyramine, dopamine, and phenylethylamine. Firstly, we expressed aromatic amino acid decarboxylase from Enterococcus faecium (pheDC) in an Escherichia coli strain with an increased shikimate (SHK) pathway flux toward L-tyrosine or L-phenylalanine synthesis. We found that glycerol served as a better carbon source than glucose, resulting in 940 mg/L tyramine from 4% glycerol. Next, the genes of lactate dehydrogenase (ldhA), formate acetyltransferase (pflB), phosphate acetyltransferase (pta), and alcohol dehydrogenase (adhE) were deleted to mitigate the fermentation byproduct formation. The tyramine level was further increased to 1.965 g/L in shake flasks, corresponding to 2.1 times improvement compared with that of the parental strain. By using a similar strategy, we also managed to produce 703 mg/L dopamine and 555 mg/L phenethylamine. In summary, we have demonstrated that the knockout of ldhA-pflB-pta-adhE is an effective strategy in improving aromatic amine productions, and achieved the highest aromatic amine titers in E. coli under shake flasks reported to date.

Immobilization of Aspergillus oryzae DSM 1863 for l-Malic Acid Production

Whole-cell immobilization by entrapment in natural polymers can be a tool for morphological control and facilitate biomass retention. In this study, the possibility of immobilizing the filamentous fungus Aspergillus oryzae for l-malic acid production was evaluated with the two carbon sources acetate and glucose. A. oryzae conidia were entrapped in alginate, agar, and κ-carrageenan and production was monitored in batch processes in shake flasks and 2.5-L bioreactors. With glucose, the malic acid concentration after 144 h of cultivation using immobilized particles was mostly similar to the control with free biomass. In acetate medium, production with immobilized conidia of A. oryzae in shake flasks was delayed and titers were generally lower compared to cultures with free mycelium. While all immobilization matrices were stable in glucose medium, disintegration of bead material and biomass detachment in acetate medium was observed in later stages of the fermentation. Still, immobilization proved advantageous in bioreactor cultivations with acetate and resulted in increased malic acid titers. This study is the first to evaluate immobilization of A. oryzae for malic acid production and describes the potential but also challenges regarding the application of different matrices in glucose and acetate media.

Establishment of the Carrot-Made LTB-Syn Antigen Cell Line in Shake Flask and Airlift Bioreactor Cultures

Carrot (Daucus carota) cells have been used to effectively manufacture recombinant biopharmaceuticals such as cytokines, vaccines, and antibodies. We generated the carrot cell line Z4, genetically modified to produce the LTB-Syn antigen, which is a fusion protein proposed for immunotherapy against synucleinopathies. In this work, the Z4 cell suspension line was cultivated to produce the LTB-Syn protein in a 250 mL shake flask and 2 L airlift bioreactor cultures grown for 45 and 30 days, respectively. Maximum biomass was obtained on day 15 in both the airlift bioreactor (35.00 ± 0.04 g/L DW) and shake flasks (17.00 ± 0.04 g/L DW). In the bioreactor, the highest LTB-Syn protein yield (1.52 ± 0.03 µg/g FW) was obtained on day 15; while the same occurred on day 18 for shake flasks (0.92 ± 0.02 µg/g FW). LTB-Syn protein levels were analyzed by GM1-ELISA and western blot. PCR analysis confirmed the presence of the transgene in the Z4 line. The obtained data demonstrate that the carrot Z4 cell suspension line grown in airlift bioreactors shows promise for a scale-up cultivation producing an oral LTB-Syn antigen.

Novel Propagation Strategy of Saccharomyces cerevisiae for Enhanced Xylose Metabolism during Fermentation on Softwood Hydrolysate

An economically viable production of second-generation bioethanol by recombinant xylose-fermenting Saccharomyces cerevisiae requires higher xylose fermentation rates and improved glucose–xylose co-consumption. Moreover, xylose-fermenting S. cerevisiae recognises xylose as a non-fermentable rather than a fermentable carbon source, which might partly explain why xylose is not fermented into ethanol as efficiently as glucose. This study proposes propagating S. cerevisiae on non-fermentable carbon sources to enhance xylose metabolism during fermentation. When compared to yeast grown on sucrose, cells propagated on a mix of ethanol and glycerol in shake flasks showed up to 50% higher xylose utilisation rate (in a defined xylose medium) and a double maximum fermentation rate, together with an improved C5/C6 co-consumption (on an industrial softwood hydrolysate). Based on these results, an automated propagation protocol was developed, using a fed-batch approach and the respiratory quotient to guide the ethanol and glycerol-containing feed. This successfully produced 71.29 ± 0.91 g/L yeast with an average productivity of 1.03 ± 0.05 g/L/h. These empirical findings provide the basis for the design of a simple, yet effective yeast production strategy to be used in the second-generation bioethanol industry for increased fermentation efficiency.

De Novo Production of Glycyrrhetic Acid 3-O-mono-β-D-glucuronide in Saccharomyces cerevisiae

Glycyrrhetic acid 3-O-mono-β-D-glucuronide (GAMG) is a rare compound in licorice and its short supply limits the wide applications in the pharmaceutical, cosmetic, and food industries. In this study, de novo biosynthesis of GAMG was achieved in engineered Saccharomyces cerevisiae strains based on the CRISPR/Cas9 genome editing technology. The introduction of GAMG biosynthetic pathway resulted in the construction of a GAMG-producing yeast strain for the first time. Through optimizing the biosynthetic pathway, improving the folding and catalysis microenvironment for cytochrome P450 enzymes (CYPs), enhancing the supply of UDP-glucuronic acid (UDP-GlcA), preventing product degradation, and optimizing the fermentation conditions, the production of GAMG was increased from 0.02 μg/L to 92.00 μg/L in shake flasks (4,200-fold), and the conversion rate of glycyrrhetic acid (GA) to GAMG was higher than 56%. The engineered yeast strains provide an alternative approach for the production of glycosylated triterpenoids.

Culturing and Scaling up Stem Cells of Dental Pulp Origin Using Microcarriers

Ectomesenchymal stem cells derived from the dental pulp are of neural crest origin, and as such are promising sources for cell therapy and tissue engineering. For safe upscaling of these cells, microcarrier-based culturing under dynamic conditions is a promising technology. We tested the suitability of two microcarriers, non-porous Cytodex 1 and porous Cytopore 2, for culturing well characterized dental pulp stem cells (DPSCs) using a shake flask system. Human DPSCs were cultured on these microcarriers in 96-well plates, and further expanded in shake flasks for upscaling experiments. Cell viability was measured using the alamarBlue assay, while cell morphology was observed by conventional and two-photon microscopies. Glucose consumption of cells was detected by the glucose oxidase/Clark-electrode method. DPSCs adhered to and grew well on both microcarrier surfaces and were also found in the pores of the Cytopore 2. Cells grown in tissue culture plates (static, non-shaking conditions) yielded 7 × 105 cells/well. In shake flasks, static preincubation promoted cell adhesion to the microcarriers. Under dynamic culture conditions (shaking) 3 × 107 cells were obtained in shake flasks. The DPSCs exhausted their glucose supply from the medium by day seven even with partial batch-feeding. In conclusion, both non-porous and porous microcarriers are suitable for upscaling ectomesenchymal DPSCs under dynamic culture conditions.

Evaluation of the Cultivation of Aspergillus oryzae on Organic Waste-Derived VFA Effluents and Its Potential Application as Alternative Sustainable Nutrient Source for Animal Feed

Considering the projected demand for protein supplementation in animal feed, as well as prioritizing plant-based protein provision for the growing human population, great stress is imposed on conventional protein sources, calling for new sustainable alternatives. In this regard, the production and application of single-cell proteins (SCPs) has proven to be a promising alternative. Therefore, in this study, volatile fatty acids (VFAs) effluents recovered from anaerobically digested FW, CKM, CM, and their combinations were applied for the cultivation of edible filamentous fungi Aspergillus oryzae. The biomass was further evaluated considering its protein, fat and alkali insoluble material contents. The maximum fungal biomass yielded of 0.47 ± 0.00 and 0.37 ± 0.00 g dry biomass/g tVFAsCODeq.consumed, with up to 47% protein and 5% fat content successfully cultivated in shake flasks and bench scale reactors, respectively. In addition to the production of protein-rich biomass, significant reductions in medium COD (25–58%) and ammonium (33–48%) were achieved. The results presented in this research work imply that using waste-derived VFAs for the production of animal feed grade SCP is an innovative approach that can contribute to the economy and sustainability of animal feed production process.

View on a mechanistic model of Chlorella vulgaris in incubated shake flasks

Kinetic growth models are a useful tool for a better understanding of microalgal cultivation and for optimizing cultivation conditions. The evaluation of such models requires experimental data that is laborious to generate in bioreactor settings. The experimental shake flask setting used in this study allows to run 12 experiments at the same time, with 6 individual light intensities and light durations. This way, 54 biomass data sets were generated for the cultivation of the microalgae Chlorella vulgaris. To identify the model parameters, a stepwise parameter estimation procedure was applied. First, light-associated model parameters were estimated using additional measurements of local light intensities at differ heights within medium at different biomass concentrations. Next, substrate related model parameters were estimated, using experiments for which biomass and nitrate data were provided. Afterwards, growth-related model parameters were estimated by application of an extensive cross validation procedure. Graphic abstract

Enhanced Oxytetracycline Production by Streptomyces rimosus in Submerged Co-Cultures with Streptomyces noursei

In the present study, Streptomyces rimosus was confronted with Streptomyces noursei, Penicillium rubens, Aspergillus niger, Chaetomium globosum, or Mucor racemosus in two-species submerged co-cultures in shake flasks with the goal of evaluating the oxytetracycline production and morphological development. The co-culture of S. rimosus with S. noursei exhibited stimulation in oxytetracycline biosynthesis compared with the S. rimosus monoculture, whereas the presence of M. racemosus resulted in a delay in antibiotic production. Different strategies of initiating the “S. rimosus + S. noursei” co-cultures were tested. The improvement in terms of oxytetracycline titers was recorded in the cases where S. noursei was co-inoculated with S. rimosus in the form of spores. As the observed morphological changes were not unique to the co-culture involving S. noursei, there was no evidence that the improvement of oxytetracycline levels could be attributed mainly to morphology-related characteristics.