Scaling production of GFP1-10 detector protein in E. coli for secretion screening by split GFP assay

Background The split GFP assay is a well-known technology for activity-independent screening of target proteins. A superfolder GFP is split into two non-fluorescent parts, GFP11 which is fused to the target protein and GFP1-10. In the presence of both, GFP1-10 and the GFP11-tag are self-assembled and a functional chromophore is formed. However, it relies on the availability and quality of GFP1-10 detector protein to develop fluorescence by assembly with the GFP11-tag connected to the target protein. GFP1-10 detector protein is often produced in small scale shake flask cultivation and purified from inclusion bodies. Results The production of GFP1-10 in inclusion bodies and purification was comprehensively studied based on Escherichia coli as host. Cultivation in complex and defined medium as well as different feed strategies were tested in laboratory-scale bioreactor cultivation and a standardized process was developed providing high quantity of GFP1-10 detector protein with suitable quality. Split GFP assay was standardized to obtain robust and reliable assay results from cutinase secretion strains of Corynebacterium glutamicum with Bacillus subtilis Sec signal peptides NprE and Pel. Influencing factors from environmental conditions, such as pH and temperature were thoroughly investigated. Conclusions GFP1-10 detector protein production could be successfully scaled from shake flask to laboratory scale bioreactor. A single run yielded sufficient material for up to 385 96-well plate screening runs. The application study with cutinase secretory strains showed very high correlation between measured cutinase activity to split GFP fluorescence signal proofing applicability for larger screening studies.

Production of Indole Auxins by Enterobacter sp. Strain P-36 under Submerged Conditions

Bioactive compounds produced by plant growth-promoting bacteria through a fermentation process can be valuable for developing innovative second-generation plant biostimulants. The purpose of this study is to investigate the biotechnological potential of Enterobacter on the production of auxin—a hormone with multiple roles in plant growth and development. The experiments were carried in Erlenmeyer flasks and a 2-L fermenter under batch operating mode. The auxin production by Enterobacter sp. strain P-36 can be doubled by replacing casein with vegetable peptone in the culture medium. Cultivation of strain P36 in the benchtop fermenter indicates that by increasing the inoculum size 2-fold, it is possible to reduce the fermentation time from 72 (shake flask cultivation) to 24 h (bioreactor cultivation) and increase the auxin volumetric productivity from 6.4 to 17.2 mg [IAAequ]/L/h. Finally, an efficient storage procedure to preserve the bacterial auxin was developed. It is noteworthy that by sterilizing the clarified fermentation broth by filtration and storing the filtrated samples at +4 °C, the level of auxin remains unchanged for at least three months.

Novel molecular biological tools for the efficient expression of fungal lytic polysaccharide monooxygenases in Pichia pastoris

Background Lytic polysaccharide monooxygenases (LPMOs) are attracting large attention due their ability to degrade recalcitrant polysaccharides in biomass conversion and to perform powerful redox chemistry. Results We have established a universal Pichia pastoris platform for the expression of fungal LPMOs using state-of-the-art recombination cloning and modern molecular biological tools to achieve high yields from shake-flask cultivation and simple tag-less single-step purification. Yields are very favorable with up to 42 mg per liter medium for four different LPMOs spanning three different families. Moreover, we report for the first time of a yeast-originating signal peptide from the dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit 1 (OST1) form S. cerevisiae efficiently secreting and successfully processes the N-terminus of LPMOs yielding in fully functional enzymes. Conclusion The work demonstrates that the industrially most relevant expression host P. pastoris can be used to express fungal LPMOs from different families in high yields and inherent purity. The presented protocols are standardized and require little equipment with an additional advantage with short cultivation periods.

Coenzyme Q10 Biosynthesis Established in the Non-Ubiquinone Containing Corynebacterium glutamicum by Metabolic Engineering

Coenzyme Q10 (CoQ10) serves as an electron carrier in aerobic respiration and has become an interesting target for biotechnological production due to its antioxidative effect and benefits in supplementation to patients with various diseases. For the microbial production, so far only bacteria have been used that naturally synthesize CoQ10 or a related CoQ species. Since the whole pathway involves many enzymatic steps and has not been fully elucidated yet, the set of genes required for transfer of CoQ10 synthesis to a bacterium not naturally synthesizing CoQ species remained unknown. Here, we established CoQ10 biosynthesis in the non-ubiquinone-containing Gram-positive Corynebacterium glutamicum by metabolic engineering. CoQ10 biosynthesis involves prenylation and, thus, requires farnesyl diphosphate as precursor. A carotenoid-deficient strain was engineered to synthesize an increased supply of the precursor molecule farnesyl diphosphate. Increased farnesyl diphosphate supply was demonstrated indirectly by increased conversion to amorpha-4,11-diene. To provide the first CoQ10 precursor decaprenyl diphosphate (DPP) from farnesyl diphosphate, DPP synthase gene ddsA from Paracoccus denitrificans was expressed. Improved supply of the second CoQ10 precursor, para-hydroxybenzoate (pHBA), resulted from metabolic engineering of the shikimate pathway. Prenylation of pHBA with DPP and subsequent decarboxylation, hydroxylation, and methylation reactions to yield CoQ10 was achieved by expression of ubi genes from Escherichia coli. CoQ10 biosynthesis was demonstrated in shake-flask cultivation and verified by liquid chromatography mass spectrometry analysis. To the best of our knowledge, this is the first report of CoQ10 production in a non-ubiquinone-containing bacterium.

Multiplex Design of the Metabolic Network for Production of L-Homoserine in Escherichia coli

ABSTRACT l-Homoserine, which is one of the few amino acids that is not produced on a large scale by microbial fermentation, plays a significant role in the synthesis of a series of valuable chemicals. In this study, systematic metabolic engineering was applied to target Escherichia coli W3110 for the production of l-homoserine. Initially, a basic l-homoserine producer was engineered through the strategies of overexpressing thrA (encoding homoserine dehydrogenase), removing the degradative and competitive pathways by knocking out metA (encoding homoserine O-succinyltransferase) and thrB (encoding homoserine kinase), reinforcing the transport system, and redirecting the carbon flux by deleting iclR (encoding the isocitrate lyase regulator). The resulting strain constructed by these strategies yielded 3.21 g/liter of l-homoserine in batch cultures. Moreover, based on CRISPR-Cas9/dCas9 (nuclease-dead Cas9)-mediated gene repression for 50 genes, the iterative genetic modifications of biosynthesis pathways improved the l-homoserine yield in a stepwise manner. The rational integration of glucose uptake and recovery of l-glutamate increased l-homoserine production to 7.25 g/liter in shake flask cultivation. Furthermore, the intracellular metabolic analysis further provided targets for strain modification by introducing the anaplerotic route afforded by pyruvate carboxylase to oxaloacetate formation, which resulted in accumulating 8.54 g/liter l-homoserine (0.33 g/g glucose, 62.4% of the maximum theoretical yield) in shake flask cultivation. Finally, a rationally designed strain gave 37.57 g/liter l-homoserine under fed-batch fermentation, with a yield of 0.31 g/g glucose. IMPORTANCE In this study, the bottlenecks that sequentially limit l-homoserine biosynthesis were identified and resolved, based on rational and efficient metabolic-engineering strategies, coupled with CRISPR interference (CRISPRi)-based systematic analysis. The metabolomics data largely expanded our understanding of metabolic effects and revealed relevant targets for further modification to achieve better performance. The systematic analysis strategy, as well as metabolomics analysis, can be used to rationally design cell factories for the production of highly valuable chemicals.

Combinatorial modular pathway engineering for guanosine 5′-diphosphate-L-fucose production in recombinant Escherichia coli

Background: Guanosine 5′-diphosphate (GDP)-L-fucose is a vital nucleotide sugar involved in the synthesis of fucosylated oligosaccharides, such as fucosylated human milk oligosaccharides, which play important roles in physiological and pathological processes.Results: In this study, a combinatorial modular pathway engineering strategy was implemented to efficiently increase the intracellular titers of GDP-L-fucose in engineered Escherichia coli. The de novo GDP-L-fucose synthesis pathway was partitioned into two modules and fine-tuned in both transcriptional and translational levels, which remarkably improved the GDP-L-fucose production. In addition, the gene encoding the UDP-glucose lipid carrier transferase (WcaJ) was inactivated to eliminate the competing metabolite pathway from GDP-L-fucose to colanic acid. Furthermore, cofactors regeneration was underpinned to promote biocatalysis. Taken together, the final engineered strain EWL37, which could achieve the titers of 18.33 mg/L in shake-flask cultivation, was able to produce 106.21 mg/L (4.28 mg/g DCW) GDP-L-fucose through fed-batch cultivation.Conclusion: To date, this is the first utilization of a modular pathway optimization approach to increase the production potential of the de novo synthesized GDP-L-fucose. In general, this study manifests that via combinatorial modular pathway engineering, the GDP-L-fucose biosynthesis can be significantly improved.

Expression of a Recombinant Endo-β-1,4-Xylanase in Pichia pastoris and its Application in Degradation of Corn Stalk

The optimization of xylanase expression by recombinant Pichia pastoris were carried out in this study. Several factors were evaluated and the conclusion were as follow: the optimal conditions were in shake flask cultivation with the rotate speed 180 r/min using BMGY medium with initial pH 7.0, initial OD600 1.0, 0.1% histidine, 0.05% tween80. 1.0% methanol was added into the culture every 24 h. The xylanase activity was up to 1527 U/mL at the optimal conditions after 15 days. The optimum pH and temperature were pH5.0 and 50°C. The recombinant xylanase was stable over a pH range of 2.0-8.0. The optimal conditions of degradation were as below: after 20 h, with the pH 5.0 and temperature 45°C, 2.0% of substrate concentration, 100 U/mL of enzyme dosage and 0.05% of tween80 concentration,the degradation of xylan was the best, which indicating great potential in the bioconversion of lignocellulosic waste to xylooligosaccharide.

Screening of Actinomycetes with High Producing Xylanase

Xylanase has a wide range of potential biotechnological applications in pulp and bleaching processes, textile industries, food and bread making, fruit juice clarification and so on. In order to explore and utilize xylanases using actinomycetes, collected dozens of soil samples beneath decaying wood or leaf debris of different parts of China, 102 xylanase producing actinomycetes were isolated by plate screening with home-made corncob xylan as the sole carbon source. All strains degraded xylan and produced evident xylan hydrolyzed circles. 37 actinomycetes were selected by shake flask cultivation, and among them 7 stains with high producing xylanase were submerged fermentation again. Xylanase activities of 7 strains except L1904 (158U/ml) were all beyond 200U/ml and stable. L2001 as the most promising strain, xylanolytic activity of which was 815U/ml. The project set up foundation for the further study in the future.

Examination of Humicola lutea Immobilized in Sol-Gel Matrices: Effective Source of α-Galactosidase

α-Galactosidase production by the fungus Humicola lutea 120-5 immobilized in a hybrid sol-gel matrix, consisting of tetraethylorthosilicate (TEOS) as a precursor and a mixture of polyethyleneglycol (PEG) and polyvinylalcohol (PVA), was investigated under semicontinuous shake flask cultivation and compared to the enzyme secretion by free cells. The influence of the carrier weight on the α-galactosidase biosynthesis in repeated batch experiments was followed. Best results were obtained with 2 g of the sol-gel particles per culture flask using 144-h runs. The growth behaviour of the immobilized mycelium during both the growth and productive phases was observed by scanning electron microscopy. The presence of abundant mycelial growth of intact hyphae correlated with a 2-fold higher enzyme activity compared to free cells. The obtained biocatalyst retained a high level of enzyme titer exceeding the activity of free cells during four cycles of operation (24 days). This result is confirmed by the micrographs showing the retained viability of the growing vegetative cells due to the protective role of the carrier.