The synergetic effect from the combination of different adsorption resins in batch and semi-continuous cultivations of S. Cerevisiae cell factories to produce acetylated Taxanes precursors of the anticancer drug Taxol.

In situ product recovery is an efficient way to intensify bioprocesses as it can perform adsorption of the desired natural products in the cultivation. However, it is common to use only one adsorbent (liquid or solid) to perform the product recovery. For this study, the use of an in situ product recovery method with three combined commercial resins (HP-20, XAD7HP, and HP-2MG) with different chemical properties was performed. A new yeast strain of Saccharomyces cerevisiae was engineered using CRISPR Cas9 (strain EJ2) to deliver heterologous expression of oxygenated acetylated taxanes that are precursors of the anticancer drug Taxol ® (paclitaxel). Microscale cultivations using a definitive screening design (DSD) were set to get the best resin combinations and concentrations to retrieve high taxane titers. Once the best resin treatment was selected by the DSD, semi-continuous cultivation in high throughput microscale was performed to increase the total taxanes yield up to 783 ± 33 mg/L. The best T5α-yl Acetate yield obtained was up to 95 ± 4 mg/L, the highest titer of this compound ever reported by a heterologous expression. It was also observed that by using a combination of the resins in the cultivation, 8 additional uncharacterized taxanes were found in the gas chromatograms compared to the dodecane overlay method. Lastly, the cell-waste reactive oxygen species concentrations from the yeast were 1.5-fold lower in the resin's treatment compared to the control with no adsorbent aid. The possible future implications of this method could be critical for bioprocess intensification, allowing the transition to a semi-continuous flow bioprocess. Further, this new methodology broadens the use of different organisms for natural product synthesis/discovery benefiting from clear bioprocess intensification advantages.

Bioconversion of cellulose into bisabolene using Ruminococcus flavefaciens and Rhodosporidium toruloides.

In this study, organic acids were demonstrated as a promising carbon source for bisabolene production by the non-conventional yeast, Rhodosporidium toruloides, at microscale with a maximum titre of 1055 ± 7 mg/L. A 125-fold scale-up of the optimal process, enhanced bisabolene titres 2.5-fold to 2606 mg/L. Implementation of a pH controlled organic acid feeding strategy at this scale lead to a further threefold improvement in bisabolene titre to 7758 mg/L, the highest reported microbial titre. Finally, a proof-of-concept sequential bioreactor approach was investigated. Firstly, the cellulolytic bacterium Ruminococcus flavefaciens was employed to ferment cellulose, yielding 4.2 g/L of organic acids. R. toruloides was subsequently cultivated in the resulting supernatant, producing 318 ± 22 mg/L of bisabolene. This highlights the feasibility of a sequential bioprocess for the bioconversion of cellulose, into biojet fuel candidates. Future work will focus on enhancing organic acid yields and the use of real lignocellulosic feedstocks to further enhance bisabolene production.

Rational Design of CRISPR/Cas12a-RPA Based One-Pot COVID-19 Detection with Design of Experiments.

Simple and effective molecular diagnostic methods have gained importance due to the devastating effects of the COVID-19 pandemic. Various isothermal one-pot COVID-19 detection methods have been proposed as favorable alternatives to standard RT-qPCR methods as they do not require sophisticated and/or expensive devices. However, as one-pot reactions are highly complex with a large number of variables, determining the optimum conditions to maximize sensitivity while minimizing diagnostic cost can be cumbersome. Here, statistical design of experiments (DoE) was employed to accelerate the development and optimization of a CRISPR/Cas12a-RPA-based one-pot detection method for the first time. Using a definitive screening design, factors with a significant effect on performance were elucidated and optimized, facilitating the detection of two copies/µL of full-length SARS-CoV-2 (COVID-19) genome using simple instrumentation. The screening revealed that the addition of a reverse transcription buffer and an RNase inhibitor, components generally omitted in one-pot reactions, improved performance significantly, and optimization of reverse transcription had a critical impact on the method's sensitivity. This strategic method was also applied in a second approach involving a DNA sequence of the N gene from the COVID-19 genome. The slight differences in optimal conditions for the methods using RNA and DNA templates highlight the importance of reaction-specific optimization in ensuring robust and efficient diagnostic performance. The proposed detection method is automation-compatible, rendering it suitable for high-throughput testing. This study demonstrated the benefits of DoE for the optimization of complex one-pot molecular diagnostics methods to increase detection sensitivity.

Enhancing Saccharomyces cerevisiae Taxane Biosynthesis and Overcoming Nutritional Stress-Induced Pseudohyphal Growth.

The recent technological advancements in synthetic biology have demonstrated the extensive potential socio-economic benefits at laboratory scale. However, translations of such technologies to industrial scale fermentations remains a major bottleneck. The existence and lack of understanding of the major discrepancies in cultivation conditions between scales often leads to the selection of suboptimal bioprocessing conditions, crippling industrial scale productivity. In this study, strategic design of experiments approaches were coupled with state-of-the-art bioreactor tools to characterize and overcome nutritional stress for the enhanced production of precursors to the blockbuster chemotherapy drug, Taxol, in S. cerevisiae cell factories. The batch-to-batch variation in yeast extract composition was found to trigger nutritional stress at a mini-bioreactor scale, resulting in profound changes in cellular morphology and the inhibition of taxane production. The cells shifted from the typical budding morphology into striking pseudohyphal cells. Doubling initial yeast extract and peptone concentrations (2×YP) delayed filamentous growth, and taxane accumulation improved to 108 mg/L. Through coupling a statistical definitive screening design approach with the state-of-the-art high-throughput micro-bioreactors, the total taxane titers were improved a further two-fold, compared to the 2×YP culture, to 229 mg/L. Filamentous growth was absent in nutrient-limited microscale cultures, underlining the complex and multifactorial nature of yeast stress responses. Validation of the optimal microscale conditions in 1L bioreactors successfully alleviated nutritional stress and improved the titers to 387 mg/L. Production of the key Taxol precursor, T5αAc, was improved two-fold to 22 mg/L compared to previous maxima. The present study highlights the importance of following an interdisciplinary approach combining synthetic biology and bioprocessing technologies for effective process optimization and scale-up.

Definitive screening accelerates Taxol biosynthetic pathway optimization and scale up in Saccharomyces cerevisiae cell factories.

BACKGROUND: Recent technological advancements in synthetic and systems biology have enabled the construction of microbial cell factories expressing diverse heterologous pathways in unprecedentedly short time scales. However, the translation of such laboratory scale breakthroughs to industrial bioprocesses remains a major bottleneck. METHODS AND MAJOR RESULTS: In this study, an accelerated bioprocess development approach was employed to optimize the biosynthetic pathway of the blockbuster chemotherapy drug, Taxol. Statistical design of experiments approaches were coupled with an industrially relevant high-throughput microbioreactor system to optimize production of key Taxol intermediates, Taxadien-5α-ol and Taxadien-5α-yl-acetate, in engineered yeast cell factories. The optimal factor combination was determined via data driven statistical modelling and validated in 1 L bioreactors leading to a 2.1-fold improvement in taxane production compared to a typical defined media. Elucidation and mitigation of nutrient limitation enhanced product titers a further two-fold and titers of the critical Taxol precursors, Taxadien-5α-ol and Taxadien-5α-yl-acetate were improved to 34 and 11 mg L-1 , representing a three-fold improvement compared to the highest literature titers in S. cerevisiae. Comparable titers were obtained when the process was scaled up a further five-fold using 5 L bioreactors. CONCLUSIONS: The results of this study highlight the benefits of a holistic design of experiments guided approach to expedite early stage bioprocess development.

Recent advances in fed-batch microscale bioreactor design.

Advanced fed-batch microbioreactors mitigate scale up risks and more closely mimic industrial cultivation practices. Recently, high throughput microscale feeding strategies have been developed which improve the accessibility of microscale fed-batch cultivation irrespective of experimental budget. This review explores such technologies and their role in accelerating bioprocess development. Diffusion- and enzyme-controlled feeding achieve a continuous supply of substrate while being simple and affordable. More complex feed profiles and greater process control require additional hardware. Automated liquid handling robots may be programmed to predefined feed profiles and have the sensitivity to respond to deviations in process parameters. Microfluidic technologies have been shown to facilitate both continuous and precise feeding. Holistic approaches, which integrate automated high-throughput fed-batch cultivation with strategic design of experiments and model-based optimisation, dramatically enhance process understanding whilst minimising experimental burden. The incorporation of real-time data for online optimisation of feed conditions can further refine screening. Although the technologies discussed in this review hold promise for efficient, low-risk bioprocess development, the expense and complexity of automated cultivation platforms limit their widespread application. Future attention should be directed towards the development of open-source software and reducing the exclusivity of hardware.

Statistical Design of Experiments for Synthetic Biology.

The design and optimization of biological systems is an inherently complex undertaking that requires careful balancing of myriad synergistic and antagonistic variables. However, despite this complexity, much synthetic biology research is predicated on One Factor at A Time (OFAT) experimentation; the genetic and environmental variables affecting the activity of a system of interest are sequentially altered while all other variables are held constant. Beyond being time and resource intensive, OFAT experimentation crucially ignores the effect of interactions between factors. Given the ubiquity of interacting genetic and environmental factors in biology this failure to account for interaction effects in OFAT experimentation can result in the development of suboptimal systems. To address these limitations, an increasing number of studies have turned to Design of Experiments (DoE), a suite of methods that enable efficient, systematic exploration and exploitation of complex design spaces. This review provides an overview of DoE for synthetic biologists. Key concepts and commonly used experimental designs are introduced, and we discuss the advantages of DoE as compared to OFAT experimentation. We dissect the applicability of DoE in the context of synthetic biology and review studies which have successfully employed these methods, illustrating the potential of statistical experimental design to guide the design, characterization, and optimization of biological protocols, pathways, and processes.

Optimizing the biosynthesis of oxygenated and acetylated Taxol precursors in Saccharomyces cerevisiae using advanced bioprocessing strategies.

Taxadien-5α-hydroxylase and taxadien-5α-ol O-acetyltransferase catalyze the oxidation of taxadiene to taxadien-5α-ol and subsequent acetylation to taxadien-5α-yl-acetate in the biosynthesis of the blockbuster anticancer drug, paclitaxel (Taxol®). Despite decades of research, the promiscuous and multispecific CYP725A4 enzyme remains a major bottleneck in microbial biosynthetic pathway development. In this study, an interdisciplinary approach was applied for the construction and optimization of the early pathway in Saccharomyces cerevisiae, across a range of bioreactor scales. High-throughput microscale optimization enhanced total oxygenated taxane titer to 39.0 ± 5.7 mg/L and total taxane product titers were comparable at micro and minibioreactor scale at 95.4 ± 18.0 and 98.9 mg/L, respectively. The introduction of pH control successfully mitigated a reduction of oxygenated taxane production, enhancing the potential taxadien-5α-ol isomer titer to 19.2 mg/L, comparable with the 23.8 ± 3.7 mg/L achieved at microscale. A combination of bioprocess optimization and increased gas chromatography-mass spectrometry resolution at 1 L bioreactor scale facilitated taxadien-5α-yl-acetate detection with a final titer of 3.7 mg/L. Total oxygenated taxane titers were improved 2.7-fold at this scale to 78 mg/L, the highest reported titer in yeast. Critical parameters affecting the productivity of the engineered strain were identified across a range of scales, providing a foundation for the development of robust integrated bioprocess control systems.

Multiplex Genome Engineering Methods for Yeast Cell Factory Development.

As biotechnological applications of synthetic biology tools including multiplex genome engineering are expanding rapidly, the construction of strategically designed yeast cell factories becomes increasingly possible. This is largely due to recent advancements in genome editing methods like CRISPR/Cas tech and high-throughput omics tools. The model organism, baker's yeast (Saccharomyces cerevisiae) is an important synthetic biology chassis for high-value metabolite production. Multiplex genome engineering approaches can expedite the construction and fine tuning of effective heterologous pathways in yeast cell factories. Numerous multiplex genome editing techniques have emerged to capitalize on this recently. This review focuses on recent advancements in such tools, such as delta integration and rDNA cluster integration coupled with CRISPR-Cas tools to greatly enhance multi-integration efficiency. Examples of pre-placed gate systems which are an innovative alternative approach for multi-copy gene integration were also reviewed. In addition to multiple integration studies, multiplexing of alternative genome editing methods are also discussed. Finally, multiplex genome editing studies involving non-conventional yeasts and the importance of automation for efficient cell factory design and construction are considered. Coupling the CRISPR/Cas system with traditional yeast multiplex genome integration or donor DNA delivery methods expedites strain development through increased efficiency and accuracy. Novel approaches such as pre-placing synthetic sequences in the genome along with improved bioinformatics tools and automation technologies have the potential to further streamline the strain development process. In addition, the techniques discussed to engineer S. cerevisiae, can be adapted for use in other industrially important yeast species for cell factory development.

Sustainable Production of Microbial Isoprenoid Derived Advanced Biojet Fuels Using Different Generation Feedstocks: A Review.

As the fastest mode of transport, the aircraft is a major driver for globalization and economic growth. The development of alternative advanced liquid fuels is critical to sustainable development within the sector. Such fuels should be compatible with existing infrastructure and derived from second generation feedstocks to avoid competition with food markets. With properties similar to petroleum based fuels, isoprenoid derived compounds such as limonene, bisabolane, farnesane, and pinene dimers are of increasing interest as "drop-in" replacement jet fuels. In this review potential isoprenoid derived jet fuels and progress toward their microbial production was discussed in detail. Although substantial advancements have been achieved, the use of first generation feedstocks remains ubiquitous. Lignocellulosic biomass is the most abundant raw material available for biofuel production, however, technological constraints associated with its pretreatment and saccharification hinder its economic feasibility for low-value commodity production. Non-conventional microbes with novel characteristics including cellulolytic bacteria and fungi capable of highly efficient lignocellulose degradation and xylose fermenting oleaginous yeast with enhanced lignin-associated inhibitor tolerance were investigated as alternatives to traditional model hosts. Finally, innovative bioprocessing methods including consolidated bioprocessing and sequential bioreactor approaches, with potential to capitalize on such unique natural capabilities were considered.

Enhanced production of taxadiene in Saccharomyces cerevisiae.

BACKGROUND: Cost-effective production of the highly effective anti-cancer drug, paclitaxel (Taxol®), remains limited despite growing global demands. Low yields of the critical taxadiene precursor remains a key bottleneck in microbial production. In this study, the key challenge of poor taxadiene synthase (TASY) solubility in S. cerevisiae was revealed, and the strains were strategically engineered to relieve this bottleneck. RESULTS: Multi-copy chromosomal integration of TASY harbouring a selection of fusion solubility tags improved taxadiene titres 22-fold, up to 57 ± 3 mg/L at 30 °C at microscale, compared to expressing a single episomal copy of TASY. The scalability of the process was highlighted through achieving similar titres during scale up to 25 mL and 250 mL in shake flask and bioreactor cultivations, respectively at 20 and 30 °C. Maximum taxadiene titres of 129 ± 15 mg/L and 127 mg/L were achieved through shake flask and bioreactor cultivations, respectively, of the optimal strain at a reduced temperature of 20 °C. CONCLUSIONS: The results of this study highlight the benefit of employing a combination of molecular biology and bioprocess tools during synthetic pathway development, with which TASY activity was successfully improved by 6.5-fold compared to the highest literature titre in S. cerevisiae cell factories.

Seroprevalence of measles, mumps and rubella among children in American Samoa, 2011, and progress towards West Pacific Region goals of elimination.

INTRODUCTION: In line with the global goals for measles elimination, countries in the West Pacific Region (WPR) have set a goal to eliminate measles by 2012. Due to its contagiousness, high population immunity is needed for achieving and documenting measles elimination. We assessed population immunity to measles, mumps and rubella among first grade children in American Samoa (AS) through a seroprevalance study. METHODS: Using commercial indirect enzyme-linked immunosorbant IgG assays (Wampole Laboratories, Cranbury, NJ) we determined IgG antibodies against the measles, mumps, and rubella (MMR) viruses in sera collected from first grade students in AS in April-May 2011. Vaccination status was retrieved from the immunization cards. Factors associated with seropositivity of measles, mumps, and rubella were analyzed separately. RESULT: Among 509 first grade students, measles, mumps, and rubella seroprevalence were 92%, 90%, and 93%, respectively. The proportions of first grade students with documented one or two doses of MMR vaccine were 93% and 84%, respectively. The vaccination status of 6% of the first graders was unknown and 1% was unvaccinated. Receiving two-doses of MMR vaccines was associated with high measles and mumps seropositivity (p<0.01). CONCLUSION: The high measles seroprevalence among children shows the progress by American Samoa towards measles elimination. Achieving and maintaining high two-dose MMR vaccine coverage in all age groups will aid in attaining the measles elimination status and prevent transmission of measles from potential imported measles cases from other countries.

Of atoms, oaks, and cannibals; or, more things that talk.

While literary works are often treated as museum pieces, an alternative Romantic/ Pragmatic aesthetic emphasizes instead the rootedness of all texts in lived experience. This suggests that both literary and scientific texts may be approached as performances that weave together discursive and material elements, giving language to matter, both making, and becoming, "things that talk." Three authors are contrasted: Emerson uses natural objects as metaphors to complete his thought; Thoreau uses natural objects as mediators who enroll him to speak for them in the name of a wider ecology; Humboldt attempts to enroll nonhumans, namely cannibals, into the global civil community by asking them to speak for themselves. The resulting quandary unsettles the Cartesian boundary between human and nonhuman, subject and object; as scholars divided by this boundary, we must multiply our own relations, the better to understand the ties that bind us into the common project of building the Cosmos.

Evaluation of the immune response to a 2-dose measles vaccination schedule administered at 6 and 9 months of age to HIV-infected and HIV-uninfected children in Malawi.

BACKGROUND: The World Health Organization recommends that infants at high risk for developing measles before 9 months of age, including human immunodeficiency virus (HIV)-infected infants, receive measles vaccination (MV) at 6 and 9 months of age. METHODS: Children born to HIV-infected mothers received MV at 6 and 9 months, and children of HIV-uninfected mothers were randomized to receive MV at 6 and 9 months, MV at 9 months, or routine MV without follow-up. Blood samples were obtained before and 3 months after each MV. Data were collected on adverse events for 21 days after each MV, at all clinic visits, on any hospitalization, and for subjects who died. HIV-infection status was determined by antibody assays and polymerase chain reaction; the presence of measles IgG was determined by EIA. RESULTS: Twenty-two hundred mother-infant pairs were enrolled. After the first and second doses of measles vaccine, respectively, the percentages of children who were measles seropositive were 59% (36 of 61) and 64% (29 of 45) among HIV-infected children, 68% (152 of 223) and 94% (189 of 202) among HIV-exposed but uninfected children, and 62% (288 of 467) and 92% (385 of 417) among HIV-unexposed children. Of 521 HIV-unexposed children vaccinated only at 9 months, 398 (76%) were measles seropositive at 12 months. No serious vaccine-related adverse events were identified. CONCLUSIONS: An early, 2-dose MV schedule was immunogenic, but a higher proportion of HIV-infected children remained susceptible to measles, compared with HIV-uninfected children (whether HIV exposed or HIV unexposed).

Lack of evidence of measles virus shedding in people with inapparent measles virus infections.

Serological evidence of measles virus infection has been detected among people exposed to measles who do not exhibit classical clinical symptoms. Throat swabs, lymphocytes, and serum and urine samples were collected from contacts of individuals with confirmed measles 12-16 days after exposure, during measles outbreaks occurring in 1998. Follow-up serum samples were drawn 2 weeks later. Samples were tested for measles IgM antibody by enzyme immunoassays and plaque reduction neutralization testing. Virus isolation and reverse transcriptase-polymerase chain reaction testing was attempted for all samples. None of the 133 contacts developed classical measles disease; 11 (8%) had serological evidence of infection. Duration of exposure of >or=3 h was the only significant risk factor for developing serological response (24% vs. 4% among contacts exposed for 1-2 h; relative risk, 6.0; 95% confidence interval, 1.9-19.2). None of the 133 contacts had virological evidence of infection by culture or polymerase chain reaction. We found no evidence that persons with inapparent measles virus infections shed measles virus.

Immunologic studies of specific mucosal and systemic immune responses in Mexican school children after booster aerosol or subcutaneous immunization with measles vaccine.

The purpose of the present study was to compare serum and mucosal immune responses following either aerosol (Aer) or subcutaneous (SQ) measles immunization of Mexican school children. A cohort of 49 children from 6 to 7 years of age received either Aer ( n = 22) or SQ ( n = 27) Edmonston-Zagreb (EZ) measles vaccine. Serum and nasal secretions were collected prior to (Pre), 1 and 3 months (mos) intervals and analyzed for immunoglobulin (Ig) concentrations and measles specific Ig isotype-associated antibody by enzyme immunoassay (EIA). Serum and nasal IgG and IgA antibody responses were stimulated following immunization with live, attenuated EZ measles vaccine administered either by SQ or Aer routes but these responses were significantly greater by the Aer compared to the SQ route. These studies also suggest that the level of antibody in these secretions may serve as an important marker of immunity to measles and lend further support for aerosol immunization as an effective alternative vaccine delivery strategy for measles eradication.

Progress toward measles elimination in Romania after a mass vaccination campaign and implementation of enhanced measles surveillance.

In response to an outbreak of >33,000 measles cases in 1996-1998 and to prevent an outbreak predicted for 2002, Romania conducted a nationwide measles-rubella vaccination campaign in October 1998. Some 2.1 million children aged 7-18 years were vaccinated. Data from national surveillance and seroprevalence studies conducted in three districts were used to assess the campaign and status of measles control. Surveillance data showed a dramatic drop in measles despite enhanced surveillance starting in October 1999. From October 1999 to December 2001, 400 suspected measles cases were reported, down from about 5000 cases annually in non-outbreak years. Only 29 (8%) of 386 cases with specimens were laboratory confirmed; 14 were clinically confirmed. Seroprevalence estimates showed high measles antibody levels before (92.9%) and after (94.4%) the campaign. The low number of laboratory-confirmed cases and high population immunity suggest that interruption of indigenous measles virus transmission is a real possibility for Romania.