scholarly journals Efficient Production of Pyruvate Using Metabolically Engineered Lactococcus lactis

2021 ◽  
Vol 8 ◽  
Author(s):  
Fan Suo ◽  
Jianming Liu ◽  
Jun Chen ◽  
Xuanji Li ◽  
Christian Solem ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Carbon Flux ◽  
High Titer ◽  
High Yield ◽  
Efficient Production ◽  
Waste Products ◽  
Commodity Chemicals ◽  
Fed Batch Fermentation ◽  
Microbial Host ◽  
Sole Product

Microbial production of commodity chemicals has gained increasing attention and most of the focus has been on reducing the production cost. Selecting a suitable microorganism, which can grow rapidly on cheap feedstocks, is of key importance when developing an economically feasible bioprocess. We chose Lactococcus lactis, a well-characterized lactic acid bacterium, as our microbial host to produce pyruvate, which is a commodity chemical with various important applications. Here we report the engineering of Lactococcus lactis into becoming an efficient microbial platform for producing pyruvate. The strain obtained, FS1076 (MG1363 Δ3ldh Δpta ΔadhE Δals), was able to produce pyruvate as the sole product. Since all the competitive pathways had been knocked out, we achieved growth-coupled production of pyruvate with high yield. More than 80 percent of the carbon flux was directed toward pyruvate, and a final titer of 54.6 g/L was obtained using a fed-batch fermentation setup. By introducing lactose catabolism into FS1076, we obtained the strain FS1080, which was able to generate pyruvate from lactose. We then demonstrated the potential of FS1080 for valorizing lactose contained in dairy side-streams, by achieving a high titer (40.1 g/L) and high yield (78.6%) of pyruvate using residual whey permeate (RWP) as substrate. The results obtained, show that the L. lactis platform is well-suited for transforming lactose in dairy waste into food-grade pyruvate, and the yields obtained are the highest reported in the literature. These results demonstrate that it is possible to achieve sustainable bioconversion of waste products from the dairy industry (RWP) to valuable products.

scholarly journals Multi-Path Optimization for Efficient Production of 2′-Fucosyllactose in an Engineered Escherichia coli C41 (DE3) Derivative

2020 ◽  
Vol 8 ◽  
Author(s):  
Zhijian Ni ◽  
Zhongkui Li ◽  
Jinyong Wu ◽  
Yuanfei Ge ◽  
Yingxue Liao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Batch Fermentation ◽  
Infant Nutrition ◽  
High Titer ◽  
Production Performance ◽  
Cell Factory ◽  
Efficient Production ◽  
Healthy Development ◽  
Fucosyltransferase Gene ◽  
Fed Batch Fermentation

2′-fucosyllactose (2′-FL), one of the simplest but most abundant oligosaccharides in human milk, has been demonstrated to have many positive benefits for the healthy development of newborns. However, the high-cost production and limited availability restrict its widespread use in infant nutrition and further research on its potential functions. In this study, on the basis of previous achievements, we developed a powerful cell factory by using a lacZ-mutant Escherichia coli C41 (DE3)ΔZ to ulteriorly increase 2′-FL production by feeding inexpensive glycerol. Initially, we co-expressed the genes for GDP-L-fucose biosynthesis and heterologous α-1,2-fucosyltransferase in C41(DE3)ΔZ through different plasmid-based expression combinations, functionally constructing a preferred route for 2′-FL biosynthesis. To further boost the carbon flux from GDP-L-fucose toward 2′-FL synthesis, deletion of chromosomal genes (wcaJ, nudD, and nudK) involved in the degradation of the precursors GDP-L-fucose and GDP-mannose were performed. Notably, the co-introduction of two heterologous positive regulators, RcsA and RcsB, was confirmed to be more conducive to GDP-L-fucose formation and thus 2′-FL production. Further a genomic integration of an individual copy of α-1,2-fucosyltransferase gene, as well as the preliminary optimization of fermentation conditions enabled the resulting engineered strain to achieve a high titer and yield. By collectively taking into account the intracellular lactose utilization, GDP-L-fucose availability, and fucosylation activity for 2′-FL production, ultimately a highest titer of 2′-FL in our optimized conditions reached 6.86 g/L with a yield of 0.92 mol/mol from lactose in the batch fermentation. Moreover, the feasibility of mass production was demonstrated in a 50-L fed-batch fermentation system in which a maximum titer of 66.80 g/L 2′-FL was achieved with a yield of 0.89 mol 2′-FL/mol lactose and a productivity of approximately 0.95 g/L/h 2′-FL. As a proof of concept, our preliminary 2′-FL production demonstrated a superior production performance, which will provide a promising candidate process for further industrial production.

scholarly journals Driving Forces Enable High-Titer Anaerobic 1-Butanol Synthesis in Escherichia coli

2011 ◽  
Vol 77 (9) ◽  
pp. 2905-2915 ◽  
Author(s):  
Claire R. Shen ◽  
Ethan I. Lan ◽  
Yasumasa Dekishima ◽  
Antonino Baez ◽  
Kwang Myung Cho ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Driving Forces ◽  
High Titer ◽  
Catalytic Efficiency ◽  
Anaerobic Growth ◽  
High Yield ◽  
Efficient Production ◽  
Acetyl Coa ◽  
Content Type ◽  
Irreversible Reaction

ABSTRACT1-Butanol, an important chemical feedstock and advanced biofuel, is produced byClostridiumspecies. Various efforts have been made to transfer the clostridial 1-butanol pathway into other microorganisms. However, in contrast to similar compounds, only limited titers of 1-butanol were attained. In this work, we constructed a modified clostridial 1-butanol pathway inEscherichia colito provide an irreversible reaction catalyzed bytrans-enoyl-coenzyme A (CoA) reductase (Ter) and created NADH and acetyl-CoA driving forces to direct the flux. We achieved high-titer (30 g/liter) and high-yield (70 to 88% of the theoretical) production of 1-butanol anaerobically, comparable to or exceeding the levels demonstrated by native producers. Without the NADH and acetyl-CoA driving forces, the Ter reaction alone only achieved about 1/10 the level of production. The engineered host platform also enables the selection of essential enzymes with better catalytic efficiency or expression by anaerobic growth rescue. These results demonstrate the importance of driving forces in the efficient production of nonnative products.

scholarly journals Production of L (+) Lactic Acid by Lactobacillus casei Ke11: Fed Batch Fermentation Strategies

Fermentation ◽  
2021 ◽  
Vol 7 (3) ◽  
pp. 151
Author(s):  
Paola Monteiro de Oliveira ◽  
Larissa Provasi Santos ◽  
Luciana Fontes Coelho ◽  
Paulo Marcelo Avila Neto ◽  
Daiane Cristina Sass ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Batch Fermentation ◽  
Lactobacillus Casei ◽  
Culture Media ◽  
Feeding Strategy ◽  
High Yield ◽  
Feeding Strategies ◽  
Efficient Production ◽  
Fed Batch ◽  
Fed Batch Fermentation

Lactic acid and its derivatives are widely used in pharmaceutical, leather, textile and food industries. However, until now there have been few systematic reports on fed-batch fermentation for efficient production and high concentration of l-lactic acid by lactic acid bacteria. This study describes the obtainment of L (+) lactic acid from sucrose using the Lactobacillus casei Ke11 strain through different feeding strategies using an accessible pH neutralizer such as CaCO3. The exponential feeding strategy can increase lactic acid production and productivity (175.84 g/L and 3.74 g/L/h, respectively) with a 95% yield, avoiding inhibition by high initial substrate concentration and, combined with the selected agent controller, avoids the cellular stress that could be caused by the high osmotic pressure of the culture media. The purification of the acid using charcoal and celite, followed by the use of a cation exchange column proved to be highly efficient, allowing a high yield of lactic acid, high removal of sugars and proteins. The described process shows great potential for the production of lactic acid, as well as the simple, efficient and low-cost purification method. This way, this work is useful to the large-scale fermentation of L. casei Ke11 for production of l-lactic acid.

scholarly journals Deciphering the regulation of the mannitol operon paves the way for efficient production of mannitol in L. lactis

2021 ◽  
Author(s):  
Hang Xiao ◽  
Claus Heiner Bang-Berthelsen ◽  
Peter Ruhdal Jensen ◽  
Christian Solem
Keyword(s):  
Stationary Phase ◽  
Lactococcus Lactis ◽  
Transcriptional Activator ◽  
High Yield ◽  
Efficient Production ◽  
Phosphotransferase System ◽  
Transcriptional Terminator ◽  
Mannitol Production ◽  
Transcriptional Units ◽  
Foreign Genes

Lactococcus lactis has great potential for high-yield production of mannitol, which has not yet been fully realized. In this study, we characterize how the mannitol genes in L. lactis are organized and regulated, and use this information to establish efficient mannitol production. Although the organization of the mannitol genes in L. lactis was similar to that in other Gram-positives, mtlF and mtlD , encoding the Enzyme IIA component (EIIA mtl ) of the mannitol phosphotransferase system (PTS), and the mannitol-1-phosphate dehydrogenase, respectively, were separated by a transcriptional terminator, and the mannitol genes were found to be organized in two transcriptional units: an operon comprising mtlA , encoding the Enzyme IIBC component (EIIBC mtl ) of the mannitol PTS, mtlR , encoding a transcriptional activator, and mtlF , and a separately expressed mtlD . The promoters driving expression of the two transcriptional units were somewhat similar, and both contained predicted catabolite responsive elements ( cre ). Presence of carbon catabolite repression was demonstrated, and was shown to be relieved in stationary phase cells. The transcriptional activator MtlR ( mtlR ), in some Gram-positives, is repressed by phosphorylation by EIIA mtl , and when we knocked-out mtlF we indeed observed enhanced expression from the two promotors, which indicated that this mechanism was in place. Finally, by overexpressing the mtlD gene and using stationary phase cells as biocatalysts, we attained 10.1 g/L mannitol with a 55% yield, which is the highest titer ever reported for L. lactis . Summing up, the results of our study should be useful for improving the mannitol producing capacity of this important industrial organism. Importance Lactococcus lactis is the most studied species of the Lactic Acid Bacteria, and it is widely used in various food fermentations. To date, there have been several attempts to persuade L. lactis into producing mannitol, a sugar alcohol with important therapeutic and food applications. Until now, to achieve mannitol production in L. lactis , with significant titer and yield, it has been necessary to introduce and express foreign genes, which precludes the use of such strains in foods, due to their recombinant status. In this study, we systematically characterize how the mannitol genes in L. lactis are regulated, and demonstrate how this impacts on mannitol production capability. We harness this information and manage to establish efficient mannitol production, without introducing foreign genes.

scholarly journals Additive-Enhanced Exfoliation for High-Yield 2D Materials Production

Nanomaterials ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 601
Author(s):  
Dinh-Tuan Nguyen ◽  
Hsiang-An Ting ◽  
Yen-Hsun Su ◽  
Mario Hofmann ◽  
Ya-Ping Hsieh
Keyword(s):  
Large Scale ◽  
2D Materials ◽  
Transition Metal Dichalcogenides ◽  
Spectroscopic Characterization ◽  
High Yield ◽  
Nanometer Scale ◽  
Efficient Production ◽  
Device Performance ◽  
Metal Dichalcogenides ◽  
Scale Deposition

The success of van-der-Waals electronics, which combine large-scale-deposition capabilities with high device performance, relies on the efficient production of suitable 2D materials. Shear exfoliation of 2D materials’ flakes from bulk sources can generate 2D materials with low amounts of defects, but the production yield has been limited below industry requirements. Here, we introduce additive-assisted exfoliation (AAE) as an approach to significantly increase the efficiency of shear exfoliation and produce an exfoliation yield of 30%. By introducing micrometer-sized particles that do not exfoliate, the gap between rotor and stator was dynamically reduced to increase the achievable shear rate. This enhancement was applied to WS2 and MoS2 production, which represent two of the most promising 2D transition-metal dichalcogenides. Spectroscopic characterization and cascade centrifugation reveal a consistent and significant increase in 2D material concentrations across all thickness ranges. Thus, the produced WS2 films exhibit high thickness uniformity in the nanometer-scale and can open up new routes for 2D materials production towards future applications.

scholarly journals A method for the generation of pseudotyped virus particles bearing SARS coronavirus spike protein in high yields

2021 ◽  
Author(s):  
Yoichiro Fujioka ◽  
Sayaka Kashiwagi ◽  
Aiko Yoshida ◽  
Aya O. Satoh ◽  
Mari Fujioka ◽  
...  
Keyword(s):  
Global Economy ◽  
Expression System ◽  
Antiviral Agents ◽  
Production Method ◽  
Spike Protein ◽  
High Yield ◽  
Pseudotyped Virus ◽  
Efficient Production ◽  
Live Virus ◽  
High Yields

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has threatened human health and the global economy. Development of additional vaccines and therapeutics is urgently required, but such development with live virus must be conducted with biosafety level 3 confinement. Pseudotyped viruses have been widely adopted for studies of virus entry and pharmaceutical development to overcome this restriction. Here we describe a modified protocol to generate vesicular stomatitis virus (VSV) pseudotyped with SARS-CoV or SARS-CoV-2 Spike protein in high yield. We found that pseudovirions produced with the conventional transient expression system lacked coronavirus Spike protein at their surface as a result of inhibition of parental VSV infection by overexpression of this protein. Establishment of stable cell lines with an optimal expression level of coronavirus Spike protein allowed the efficient production of progeny pseudoviruses decorated with Spike protein. This improved VSV pseudovirus production method should facilitate studies of coronavirus entry and development of antiviral agents.

scholarly journals Efficient production of vindoline from tabersonine by metabolically engineered Saccharomyces cerevisiae

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Tengfei Liu ◽  
Ying Huang ◽  
Lihong Jiang ◽  
Chang Dong ◽  
Yuanwei Gou ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
De Novo ◽  
Indole Alkaloid ◽  
Functional Expression ◽  
High Yield ◽  
Efficient Production ◽  
Cytochrome P450 Enzymes ◽  
Copy Numbers ◽  
Yield Production ◽  
Engineered Saccharomyces Cerevisiae

AbstractVindoline is a plant derived monoterpene indole alkaloid (MIA) with potential therapeutic applications and more importantly serves as the precursor to vinblastine and vincristine. To obtain a yeast strain for high yield production of vindoline from tabersonine, multiple metabolic engineering strategies were employed via the CRISPR/Cas9 mediated multiplex genome integration technology in the present study. Through increasing and tuning the copy numbers of the pathway genes, pairing cytochrome P450 enzymes (CYPs) with appropriate cytochrome P450 reductases (CPRs), engineering the microenvironment for functional expression of CYPs, enhancing cofactor supply, and optimizing fermentation conditions, the production of vindoline was increased to a final titer as high as ∼16.5 mg/L, which is more than 3,800,000-fold higher than the parent strain and the highest tabersonine to vindoline conversion yield ever reported. This work represents a key step of the engineering efforts to establish de novo biosynthetic pathways for vindoline, vinblastine, and vincristine.

scholarly journals Requirements for Efficient Production and Transduction of Human Immunodeficiency Virus Type 1-Based Vectors

1999 ◽  
Vol 73 (3) ◽  
pp. 1828-1834 ◽  
Author(s):  
Mehdi Gasmi ◽  
Jacqueline Glynn ◽  
Ming-Jie Jin ◽  
Douglas J. Jolly ◽  
Jiing-Kuan Yee ◽  
...  
Keyword(s):  
High Titer ◽  
Regulatory Element ◽  
Target Cells ◽  
Efficient Production ◽  
Replication Competent Virus ◽  
Constitutive Transport Element ◽  
Hiv 1

ABSTRACT A number of human immunodeficiency type 1 (HIV-1)-based vectors have recently been shown to transduce nondividing cells in vivo as well as in vitro. However, if these vectors are to be considered for eventual clinical use, a major consideration is to reduce the probability of unintended generation of replication-competent virus. This can be achieved by eliminating viral genetic elements involved in the generation of replication-competent virus without impairing vector production. We have designed a system to transiently produce HIV-1-based vectors by using expression plasmids encoding Gag, Pol, and Tat of HIV-1 under the control of the cytomegalovirus immediate-early promoter. Our data show that the best vector yield is achieved in the presence of the Rev/Rev-responsive element (RRE) system. However, the constitutive transport element of Mason-Pfizer monkey virus can substitute for RRE and Rev at least to some extent, whereas the posttranscriptional regulatory element of human hepatitis B virus appeared to be inefficient. In addition, we show that high-titer virus preparations can be obtained in the presence of sodium butyrate, which activates the expression of both the packaging construct and the vector genome. Finally, our results suggest that efficient infectivity of vectors defective in the accessory proteins Vif, Vpr, Vpu, and Nef depends on the nature of the target cells.

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