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Title: A minimum information standard for reproducing bench-scale bacterial cell growth and productivity

Abstract

Reproducing, exchanging, comparing, and building on each other’s work is foundational to technology advances. Advancing biotechnology calls for reliable reuse of engineered organisms. Reliable reuse of engineered organisms requires reproducible growth and productivity. Here, we identify the experimental factors that have the greatest effect on the growth and productivity of our engineered organisms in order to demonstrate reproducibility for biotechnology. Here, we present a draft of a Minimum Information Standard for Engineered Organism Experiments based on this method. We evaluate the effect of 22 factors on Escherichia coli engineered to produce the small molecule lycopene, and 18 factors on E. coli engineered to produce red fluorescent protein. Container geometry and shaking have the greatest effect on product titer and yield. We reproduce our results under two different conditions of reproducibility: conditions of use (different fractional factorial experiments), and time (48 biological replicates performed on 12 different days over four months).

Authors:
 [1];  [2];  [3];  [4]
  1. Joint Initiative for Metrology in Biology, Stanford, CA (United States); National Inst. of Standards and Technology, Stanford, CA (United States); Stanford Univ., Stanford, CA (United States); SLAC National Accelerator Lab., Menlo Park, CA (United States)
  2. Joint Initiative for Metrology in Biology, Stanford, CA (United States); National Inst. of Standards and Technology, Stanford, CA (United States); Stanford Univ., Stanford, CA (United States)
  3. Joint Initiative for Metrology in Biology, Stanford, CA (United States); National Inst. of Standards and Technology, Stanford, CA (United States); Stanford Univ., Stanford, CA (United States); Univ. of Minnesota, Minneapolis, MN (United States)
  4. National Inst. of Standards and Technology (NIST), Gaithersburg, MD (United States)
Publication Date:
Research Org.:
SLAC National Accelerator Lab., Menlo Park, CA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1480469
Grant/Contract Number:  
AC02-76SF00515
Resource Type:
Accepted Manuscript
Journal Name:
Communications Biology
Additional Journal Information:
Journal Volume: 1; Journal Issue: 1; Journal ID: ISSN 2399-3642
Publisher:
Springer Nature
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Salit, Marc, Hecht, Ariel, Munro, Sarah A., and Filliben, James. A minimum information standard for reproducing bench-scale bacterial cell growth and productivity. United States: N. p., 2018. Web. doi:10.1038/s42003-018-0220-6.
Salit, Marc, Hecht, Ariel, Munro, Sarah A., & Filliben, James. A minimum information standard for reproducing bench-scale bacterial cell growth and productivity. United States. doi:10.1038/s42003-018-0220-6.
Salit, Marc, Hecht, Ariel, Munro, Sarah A., and Filliben, James. Sat . "A minimum information standard for reproducing bench-scale bacterial cell growth and productivity". United States. doi:10.1038/s42003-018-0220-6. https://www.osti.gov/servlets/purl/1480469.
@article{osti_1480469,
title = {A minimum information standard for reproducing bench-scale bacterial cell growth and productivity},
author = {Salit, Marc and Hecht, Ariel and Munro, Sarah A. and Filliben, James},
abstractNote = {Reproducing, exchanging, comparing, and building on each other’s work is foundational to technology advances. Advancing biotechnology calls for reliable reuse of engineered organisms. Reliable reuse of engineered organisms requires reproducible growth and productivity. Here, we identify the experimental factors that have the greatest effect on the growth and productivity of our engineered organisms in order to demonstrate reproducibility for biotechnology. Here, we present a draft of a Minimum Information Standard for Engineered Organism Experiments based on this method. We evaluate the effect of 22 factors on Escherichia coli engineered to produce the small molecule lycopene, and 18 factors on E. coli engineered to produce red fluorescent protein. Container geometry and shaking have the greatest effect on product titer and yield. We reproduce our results under two different conditions of reproducibility: conditions of use (different fractional factorial experiments), and time (48 biological replicates performed on 12 different days over four months).},
doi = {10.1038/s42003-018-0220-6},
journal = {Communications Biology},
number = 1,
volume = 1,
place = {United States},
year = {2018},
month = {12}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Figures / Tables:

Table 1 Table 1: 32 experimental factors that have been documented to affect cell growth and productivity

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margin-top: 0.5em; padding-left: 0; line-height:1.8em;"> <li> <span style="color:#7cb342;"> Record Jr, M. Thomas; Courtenay, Elizabeth S.; Cayley, D. Scott</span> </li> <li> Trends in Biochemical Sciences, Vol. 23, Issue 4</li> <li> <span class="text-muted related-url">DOI: <a href="https://doi.org/10.1016/S0968-0004(98)01196-7" class="text-muted" target="_blank" rel="noopener noreferrer">10.1016/S0968-0004(98)01196-7<span class="fa fa-external-link" aria-hidden="true"></span></a></span> </li> </ul> <hr/> </div><div> <h2 class="title" style="margin-bottom:0;" data-apporder=""> <a href="https://doi.org/10.1002/bit.21359" target="_blank" rel="noopener noreferrer" class="name">Scale-up from shake flasks to fermenters in batch and continuous mode withCorynebacterium glutamicum on lactic acid based on oxygen transfer and pH<span class="fa fa-external-link" aria-hidden="true"></span></a> <small class="text-muted" style="text-transform:uppercase; font-size:0.75rem;"><br/> <span class="type">journal</span>, <span class="date" data-date="2007-01-01">January 2007</span></small> </h2> <ul id="references-list" class="small references-list" style="list-style-type:none; margin-top: 0.5em; padding-left: 0; line-height:1.8em;"> <li> <span style="color:#7cb342;"> Seletzky, Juri M.; Noak, Ute; Fricke, Jens</span> </li> <li> Biotechnology and Bioengineering, Vol. 98, Issue 4</li> <li> <span class="text-muted related-url">DOI: <a href="https://doi.org/10.1002/bit.21359" class="text-muted" target="_blank" rel="noopener noreferrer">10.1002/bit.21359<span class="fa fa-external-link" aria-hidden="true"></span></a></span> </li> </ul> <hr/> </div><div> <h2 class="title" style="margin-bottom:0;" data-apporder=""> <a href="https://doi.org/10.1007/BF00258411" target="_blank" rel="noopener noreferrer" class="name">Interaction of cultural conditions and end-product distribution in Bacillus subtilis grown in shake flasks<span class="fa fa-external-link" aria-hidden="true"></span></a> <small class="text-muted" style="text-transform:uppercase; font-size:0.75rem;"><br/> <span class="type">journal</span>, <span class="date" data-date="1989-09-01">September 1989</span></small> </h2> <ul id="references-list" class="small references-list" style="list-style-type:none; margin-top: 0.5em; padding-left: 0; line-height:1.8em;"> <li> <span style="color:#7cb342;"> Delgado, Graciela; Topete, Mayra; Galindo, Enrique</span> </li> <li> Applied Microbiology and Biotechnology, Vol. 31, Issue 3</li> <li> <span class="text-muted related-url">DOI: <a href="https://doi.org/10.1007/BF00258411" class="text-muted" target="_blank" rel="noopener noreferrer">10.1007/BF00258411<span class="fa fa-external-link" aria-hidden="true"></span></a></span> </li> </ul> <hr/> </div></div> <ul class="pagination"></ul> </div> </div> <div class="col-sm-3 order-sm-3"> <ul class="nav nav-stacked"> <li class="active"><a href="" class="reference-type-filter tab-nav" data-tab="biblio-references" data-filter="type" data-pattern="*"><span class="fa fa-angle-right"></span> All References</a></li> <li class="small" style="margin-left:.75em; text-transform:capitalize;"><a href="" class="reference-type-filter tab-nav" data-tab="biblio-references" data-filter="type" data-pattern="journal"><span class="fa fa-angle-right"></span> journal<small class="text-muted"> (67)</small></a></li> </ul> <div style="margin-top:2em;"> <form class="pure-form small text-muted reference-search"> <input class="search form-control pure-input-1" placeholder="Search" style="margin-bottom:10px;" /> <label class="d-block" style="margin-left:1em; 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margin-top:0px;">Similar records in OSTI.GOV collections:</p> <aside> <ul class="item-list" itemscope itemtype="http://schema.org/ItemList" style="padding-left:0; list-style-type: none;"> <li> <div class="article item document" itemprop="itemListElement" itemscope itemtype="http://schema.org/WebPage"><meta itemprop="position" content="0" /><div class="item-info"> <h2 class="title" itemprop="name headline"><a href="/pages/biblio/1379514-cyma-exogenous-flavins-improve-extracellular-electron-transfer-couple-cell-growth-mtr-expressing-escherichia-coli" itemprop="url">CymA and Exogenous Flavins Improve Extracellular Electron Transfer and Couple It to Cell Growth in Mtr-Expressing Escherichia coli</a></h2> <div class="metadata"> <small class="text-muted" style="text-transform:uppercase;display:block;line-height:2.5em;">Journal Article</small><span class="authors"> <span class="author">Jensen, Heather M.</span> ; <span class="author">TerAvest, Michaela A.</span> ; <span class="author">Kokish, Mark G.</span> ; <span class="author">...</span> <span class="text-muted pubdata"> - ACS Synthetic Biology</span> </span> </div> <div class="abstract">Introducing extracellular electron transfer pathways into heterologous organisms offers the opportunity to explore fundamental biogeochemical processes and to biologically alter redox states of exogenous metals for various applications. While expression of the MtrCAB electron nanoconduit from Shewanella oneidensis MR-1 permits extracellular electron transfer in Escherichia coli, the low electron flux and absence of growth in these cells limits their practicality for such applications. In this paper, we investigate how the rate of electron transfer to extracellular Fe(III) and cell survival in engineered E. coli are affected by mimicking different features of the S. oneidensis pathway: the number of electron nanoconduits,<a href='#' onclick='$(this).hide().next().show().next().show();return false;' style='margin-left:10px;'>more »</a><span style='display:none;'> the link between the quinol pool and MtrA, and the presence of flavin-dependent electron transfer. While increasing the number of pathways does not significantly improve the extracellular electron transfer rate or cell survival, using the native inner membrane component, CymA, significantly improves the reduction rate of extracellular acceptors and increases cell viability. Strikingly, introducing both CymA and riboflavin to Mtr-expressing E. coli also allowed these cells to couple metal reduction to growth, which is the first time an increase in biomass of an engineered E. coli has been observed under Fe <sub>2</sub>O <sub>3</sub> (s) reducing conditions. Overall and finally, this work provides engineered E. coli strains for modulating extracellular metal reduction and elucidates critical factors for engineering extracellular electron transfer in heterologous organisms.</span><a href='#' onclick='$(this).hide().prev().hide().prev().show();return false;' style='margin-left:10px;display:none;'>« less</a></div><div class="metadata-links small clearfix text-muted" style="margin-top:15px;"> <span class="fa fa-book text-muted" aria-hidden="true"></span> Cited by 9<div class="pure-menu pure-menu-horizontal pull-right" style="width:unset;"> <ul class="pure-menu-list"> <li class="pure-menu-item"><span class="item-info-ftlink">DOI: <a class="misc doi-link " href="https://doi.org/10.1021/acssynbio.5b00279" target="_blank" rel="noopener" title="Link to document DOI" data-ostiid="1379514" data-product-type="Journal Article" data-product-subtype="AM" >10.1021/acssynbio.5b00279</a></span></li> <li class="pure-menu-item"><span class="item-info-ftlink"><a class="misc fulltext-link " href="/pages/servlets/purl/1379514" title="Link to document media" target="_blank" rel="noopener" data-ostiid="1379514" data-product-type="Journal Article" data-product-subtype="AM" >Full Text Available</a></span></li> </ul> </div> </div> </div> <div class="clearfix"></div> </div> </li> <li> <div class="article item document" itemprop="itemListElement" itemscope itemtype="http://schema.org/WebPage"><meta itemprop="position" content="1" /><div class="item-info"> <h2 class="title" itemprop="name headline"><a href="/pages/biblio/1111433-engineering-ralstonia-eutropha-production-isobutanol-ibt-motor-fuel-from-carbon-dioxide-hydrogen-oxygen-project-final-report" itemprop="url">Engineering Ralstonia eutropha for Production of Isobutanol (IBT) Motor Fuel from Carbon Dioxide, Hydrogen, and Oxygen Project Final Report</a></h2> <div class="metadata"> <small class="text-muted" style="text-transform:uppercase;display:block;line-height:2.5em;">Technical Report</small><span class="authors"> <span class="author">Sinskey, Anthony J.</span> ; <span class="author">Worden, Robert Mark</span> ; <span class="author">Brigham, Christopher</span> ; <span class="author">...</span> <span class="text-muted pubdata"></span> </span> </div> <div class="abstract">This research project is a collaboration between the Sinskey laboratory at MIT and the Worden laboratory at Michigan State University. The goal of the project is to produce Isobutanol (IBT), a branched-chain alcohol that can serve as a drop-in transportation fuel, through the engineered microbial biosynthesis of Carbon Dioxide, Hydrogen, and Oxygen using a novel bioreactor. This final technical report presents the findings of both the biological engineering work at MIT that extended the native branched-chain amino acid pathway of the wild type Ralstonia eutropha H16 to perform this biosynthesis, as well as the unique design, modeling, and construction of<a href='#' onclick='$(this).hide().next().show().next().show();return false;' style='margin-left:10px;'>more »</a><span style='display:none;'> a bioreactor for incompatible gasses at Michigan State that enabled the operational testing of the complete system. This 105 page technical report summarizing the three years of research includes 72 figures and 11 tables of findings. Ralstonia eutropha (also known as Cupriavidus necator) is a Gram-negative, facultatively chemolithoautotrophic bacteria. It has been the principle organism used for the study of polyhydroxybutyrate (PHB) polymer biosynthesis. The wild-type Ralstonia eutropha H16 produces PHB as an intracellular carbon storage material while under nutrient stress in the presence of excess carbon. Under this stress, it can accumulate approximately 80 % of its cell dry weight (CDW) as this intracellular polymer. With the restoration of the required nutrients, the cells are then able to catabolize this polymer. If extracted from the cell, this PHB polymer can be processed into biodegradable and biocompatible plastics, however for this research, it is the efficient metabolic pathway channeling the captured carbon that is of interest. R. eutropha is further unique in that it contains two carbon-fixation Calvin–Benson–Bassham cycle operons, two oxygen-tolerant hydrogenases, and several formate dehydrogenases. It has also been much studied for its ability in the presence of oxygen, to fix carbon dioxide into complex cellular molecules using the energy from hydrogen. In this research project, engineered strains of R. eutropha redirected the excess carbon from PHB storage into the production of isobutanol and 3-methyl-1-butanol (branched-chain higher alcohols). These branched-chain higher alcohols can be used directly as substitutes for fossil-based fuels and are seen as alternative biofuels to ethanol and biodiesel. Importantly, these alcohols have approximately 98 % of the energy content of gasoline, 17 % higher than the current gasoline additive ethanol, without impacting corn market production for feed or food. Unlike ethanol, these branched-chain alcohols have low vapor pressure, hygroscopicity, and water solubility, which make them readily compatible with the existing pipelines, gasoline pumps, and engines in our transportation infrastructure. While the use of alternative energies from solar, wind, geothermal, and hydroelectric has spread for stationary power applications, these energy sources cannot be effectively or efficiently employed in current or future transportation systems. With the ongoing concerns of fossil fuel availability and price stability over the long term, alternative biofuels like branched-chain higher alcohols hold promise as a suitable transportation fuel in the future. We showed in our research that various mutant strains of R. eutropha with isobutyraldehyde dehydrogenase activity, in combination with the overexpression of plasmid-borne, native branched-chain amino acid biosynthesis pathway genes and the overexpression of heterologous ketoisovalerate decarboxylase gene, would produce isobutanol and 3-methyl-1-butanol when initiated during nitrogen or phosphorus limitation. Early on, we isolated one mutant R. eutropha strain which produced over 180 mg/L branched-chain alcohols in flask culture while being more tolerant of isobutanol toxicity. After the targeted elimination of genes encoding several potential carbon sinks (ilvE, bkdAB, and aceE), the production titer of the improved to 270 mg/L isobutanol and 40 mg/L 3-methyl-1-butanol. Semicontinuous flask cultivation supplied the cells with sufficient nutrients while minimizing the toxicity caused by isobutanol. Under this cultivation, the R. eutropha mutant grew and produced more than 14 g/L branched-chain alcohols over the duration of 50 days. These results demonstrate that R. eutropha carbon flux can be redirected from PHB to branched-chain alcohols and that engineered R. eutropha can be cultivated over prolonged periods of time for product biosynthesis. While this bioengineering work was being done at the Sinskey laboratory at MIT, the researchers at the Worden laboratory at Michigan State were working on the design and construction of the required specialty bioreactor for incompatible gasses (BIG) that would allow the safe feeding of microbes on Carbon Dioxide, Hydrogen, and Oxygen without explosive results. The early design and assembly work in year 1 incorporated a novel microbubble generator to maximize the bioavailability of gasses within the system comprised of small scale hollow fiber reactors. The early success of the microbubble generator eliminated the need to investigate potentially toxic surfactants within the system. For operational control, the system design incorporated a Opto22-based control network. The researchers also selected the specific hollow fiber material suitable for the bioreactor application. A variety of commercially available hollow fiber membranes were compared with regard to their pore sizes, cell affinity, and potential interference to cell viability assays. The selected membrane with its spongy layer was then tested for diffusivity of O2 and CO2. The instrumented system was then fully assembled for experimentally measuring the heterotrophic growth rate of immobilized R. eutropha cells. The requisite procedures for inoculation, measurement, and cleaning were established enabling the system performance to be validated under controlled laboratory conditions. In year 2, the researchers completed the Opto22 based cross-platform control network, and the system’s communications across the Sartorius fermentation system and Bruker gas chromatograph was established via open platform communications (OPC) protocol. Using the revised system, measurements were taken of the R.eutropha cell growth rate and substrate mass transfer rate in the hollow fiber membrane. Several IBT recovery strategies were explored and resin adsorption was determined to be optimal solution for lab scale operations. The adsorption capacity of the resin column was then measured and IBT desorption using methanol has been demonstrated. With the growing body of experimental data in hand, mathematical models were constructed to demonstrate and map the cellular kinetics, mass transfer of heterotrophic and autotrophic substrates in the hollow fiber, and the adsorption process in the resin column. A structured kinetic model was constructed to describe the competition between cell mass generation and IBT production. The reactor was then scaled up from single fiber to a membrane area of 180 cm2 and then further to 1 ft2. In Year 3 of the research, the IBT mass transfer across the membrane was characterized within the system with experiments to empirically measure the IBT diffusion coefficient in the BIG spongy layer. Using the refined mathematical models, the researchers are now able to explain the experimental observations and predict bioreactor performance under a wide range of experimental conditions. The Big system is able to demonstrate continuous controlled operations with the integrated IBT recovery system. Both heterotrophic and autotrophic production have been shown during continuous operation with heterotrophic and autotrophic stages. Performance of BIG system has been measured during continuous run with alternating heterotrophic growth on fructose and autotrophic product formation on H2, CO2, and O2. Volumetric productivities of IBT at 325 mg/(L day) and of 3M1B at 50 mg/(L day) were achieved, which were comparable to that achieved under heterotrophic conditions. Using the mathematical model, researchers are able to predict system performance for scaled-up BIG system. The apparent diffusion coefficient of IBT in the spongy layer of XM-50 hollow fiber membranes has been measured at various lumen liquid flow rates. The experiment is simulated in COMSOL to validate the results. The constructed COMSOL model is able to simulate BIG system performance in both batch and continuous mode. Mathematical simulations of the system performance have been run to identify the most crucial operational conditions, identifying the rate-limiting factors in autotrophic production of IBT, and quantitating the rate of IBT catabolism. Investigations of the productivity of the production system have suggested and the modeling of the system has revealed a particular sensitivity to the catabolism of the produced IBT by the engineered R. eutropha. Experiments have been designed and executed to quantify the IBT catabolism of R. eutropha, which open up possibilities for further system improvements through future, targeted bioengineering of the strain. Finally, the researchers at Michigan State performed an economic analysis of the system, based on the collective results, and their findings are presented in full within the report.</span><a href='#' onclick='$(this).hide().prev().hide().prev().show();return false;' style='margin-left:10px;display:none;'>« less</a></div><div class="metadata-links small clearfix text-muted" style="margin-top:15px;"> <div class="pure-menu pure-menu-horizontal pull-right" style="width:unset;"> <ul class="pure-menu-list"> <li class="pure-menu-item"><span class="item-info-ftlink">DOI: <a class="misc doi-link " href="https://doi.org/10.2172/1111433" target="_blank" rel="noopener" title="Link to document DOI" data-ostiid="1111433" data-product-type="Technical Report" data-product-subtype="" >10.2172/1111433</a></span></li> <li class="pure-menu-item"><span class="item-info-ftlink"><a class="misc fulltext-link " href="/pages/servlets/purl/1111433" title="Link to document media" target="_blank" rel="noopener" data-ostiid="1111433" data-product-type="Technical Report" data-product-subtype="" >Full Text Available</a></span></li> </ul> </div> </div> </div> <div class="clearfix"></div> </div> </li> <li> <div class="article item document" itemprop="itemListElement" itemscope itemtype="http://schema.org/WebPage"><meta itemprop="position" content="2" /><div class="item-info"> <h2 class="title" itemprop="name headline"><a href="/pages/biblio/1479591-machine-learning-reveals-missing-edges-putative-interaction-mechanisms-microbial-ecosystem-networks" itemprop="url">Machine Learning Reveals Missing Edges and Putative Interaction Mechanisms in Microbial Ecosystem Networks</a></h2> <div class="metadata"> <small class="text-muted" style="text-transform:uppercase;display:block;line-height:2.5em;">Journal Article</small><span class="authors"> <span class="author">DiMucci, Demetrius</span> ; <span class="author">Kon, Mark</span> ; <span class="author">Segrè, Daniel</span> ; <span class="author">...</span> <span class="text-muted pubdata"> - mSystems</span> </span> </div> <div class="abstract">ABSTRACT Microbes affect each other’s growth in multiple, often elusive, ways. The ensuing interdependencies form complex networks, believed to reflect taxonomic composition as well as community-level functional properties and dynamics. The elucidation of these networks is often pursued by measuring pairwise interactions in coculture experiments. However, the combinatorial complexity precludes an exhaustive experimental analysis of pairwise interactions, even for moderately sized microbial communities. Here, we used a machine learning random forest approach to address this challenge. In particular, we show how partial knowledge of a microbial interaction network, combined with trait-level representations of individual microbial species, can provide accurate inference<a href='#' onclick='$(this).hide().next().show().next().show();return false;' style='margin-left:10px;'>more »</a><span style='display:none;'> of missing edges in the network and putative mechanisms underlying the interactions. We applied our algorithm to three case studies: an experimentally mapped network of interactions between auxotrophic Escherichia coli strains, a community of soil microbes, and a large in silico network of metabolic interdependencies between 100 human gut-associated bacteria. For this last case, 5% of the network was sufficient to predict the remaining 95% with 80% accuracy, and the mechanistic hypotheses produced by the algorithm accurately reflected known metabolic exchanges. Our approach, broadly applicable to any microbial or other ecological network, may drive the discovery of new interactions and new molecular mechanisms, both for therapeutic interventions involving natural communities and for the rational design of synthetic consortia. IMPORTANCE Different organisms in a microbial community may drastically affect each other’s growth phenotypes, significantly affecting the community dynamics, with important implications for human and environmental health. Novel culturing methods and the decreasing costs of sequencing will gradually enable high-throughput measurements of pairwise interactions in systematic coculturing studies. However, a thorough characterization of all interactions that occur within a microbial community is greatly limited both by the combinatorial complexity of possible assortments and by the limited biological insight that interaction measurements typically provide without laborious specific follow-ups. Here, we show how a simple and flexible formal representation of microbial pairs can be used for the classification of interactions via machine learning. The approach we propose predicts with high accuracy the outcome of yet-to-be performed experiments and generates testable hypotheses about the mechanisms of specific interactions.</span><a href='#' onclick='$(this).hide().prev().hide().prev().show();return false;' style='margin-left:10px;display:none;'>« less</a></div><div class="metadata-links small clearfix text-muted" style="margin-top:15px;"> <span class="fa fa-book text-muted" aria-hidden="true"></span> Cited by 2<div class="pure-menu pure-menu-horizontal pull-right" style="width:unset;"> <ul class="pure-menu-list"> <li class="pure-menu-item"><span class="item-info-ftlink">DOI: <a class="misc doi-link " href="https://doi.org/10.1128/msystems.00181-18" target="_blank" rel="noopener" title="Link to document DOI" data-ostiid="1479591" data-product-type="Journal Article" data-product-subtype="PA" >10.1128/msystems.00181-18</a></span></li> </ul> </div> </div> </div> <div class="clearfix"></div> </div> </li> <li> <div class="article item document" itemprop="itemListElement" itemscope itemtype="http://schema.org/WebPage"><meta itemprop="position" content="3" /><div class="item-info"> <h2 class="title" itemprop="name headline"><a href="/pages/biblio/903466-organic-acid-production-filamentous-fungi" itemprop="url">Organic Acid Production by Filamentous Fungi</a></h2> <div class="metadata"> <small class="text-muted" style="text-transform:uppercase;display:block;line-height:2.5em;">Book</small><span class="authors"> <span class="author">Magnuson, Jon K.</span> ; <span class="author">Lasure, Linda L.</span> <span class="text-muted pubdata"></span> </span> </div> <div class="abstract">Many of the commercial production processes for organic acids are excellent examples of fungal biotechnology. However, unlike penicillin, the organic acids have had a less visible impact on human well-being. Indeed, organic acid fermentations are often not even identified as fungal bioprocesses, having been overshadowed by the successful deployment of the β-lactam processes. Yet, in terms of productivity, fungal organic acid processes may be the best examples of all. For example, commercial processes using Aspergillus niger in aerated stirred-tank-reactors can convert glucose to citric acid with greater than 80% efficiency and at final concentrations in hundreds of grams per liter.<a href='#' onclick='$(this).hide().next().show().next().show();return false;' style='margin-left:10px;'>more »</a><span style='display:none;'> Surprisingly, this phenomenal productivity has been the object of relatively few research programs. Perhaps a greater understanding of this extraordinary capacity of filamentous fungi to produce organic acids in high concentrations will allow greater exploitation of these organisms via application of new knowledge in this era of genomics-based biotechnology. In this chapter, we will explore the biochemistry and modern genetic aspects of the current and potential commercial processes for making organic acids. The organisms involved, with a few exceptions, are filamentous fungi, and this review is limited to that group. Although yeasts including Saccharomyces cerevisiae, species of Rhodotorula, Pichia, and Hansenula are important organisms in fungal biotechnology, they have not been significant for commercial organic acid production, with one exception. The yeast, Yarrowia lipolytica, and related yeast species, may be in use commercially to produce citric acid (Lopez-Garcia, 2002). Furthermore, in the near future engineered yeasts may provide new commercial processes to make lactic acid (Porro, Bianchi, Ranzi, Frontali, Vai, Winkler, & Alberghina, 2002). This chapter is divided into two parts. The first contains a review of the commercial aspects of current and potential large-scale processes for fungal organic acid production. The second presents a detailed review of current knowledge of the biochemistry and genetic regulation of organic acid biosynthesis. The organic acids considered are limited to polyfunctional acids containing one or more carboxyl groups, hydroxyl groups, or both, that are closely tied to central metabolic pathways. A major objective of the review is to link the biochemistry of organic acid production to the available genomic data.</span><a href='#' onclick='$(this).hide().prev().hide().prev().show();return false;' style='margin-left:10px;display:none;'>« less</a></div><div class="metadata-links small clearfix text-muted" style="margin-top:15px;"> <div class="pure-menu pure-menu-horizontal pull-right" style="width:unset;"> </div> </div> </div> <div class="clearfix"></div> </div> </li> <li> <div class="article item document" itemprop="itemListElement" itemscope itemtype="http://schema.org/WebPage"><meta itemprop="position" content="4" /><div class="item-info"> <h2 class="title" itemprop="name headline"><a href="/pages/biblio/1548398-iterative-genome-editing-escherichia-coli-hydroxypropionic-acid-production" itemprop="url">Iterative genome editing of <em>Escherichia coli</em> for 3-hydroxypropionic acid production</a></h2> <div class="metadata"> <small class="text-muted" style="text-transform:uppercase;display:block;line-height:2.5em;">Journal Article</small><span class="authors"> <span class="author">Liu, Rongming</span> ; <span class="author">Liang, Liya</span> ; <span class="author">Choudhury, Alaksh</span> ; <span class="author">...</span> <span class="text-muted pubdata"> - Metabolic Engineering</span> </span> </div> <div class="abstract">Synthetic biology requires strategies for the targeted, efficient, and combinatorial engineering of biological sub-systems at the molecular level. Here, we report the use of the iterative CRISPR EnAbled Trackable genome Engineering (iCREATE) method for the rapid construction of combinatorially modified genomes. We coupled this genome engineering strategy with high-throughput phenotypic screening and selections to recursively engineer multiple traits in <em>Escherichia coli</em> for improved production of the platform chemical 3- hydroxypropionic acid (3HP). Particularly, we engineered i) central carbon metabolism, ii) 3HP synthesis, and (iii) 3HP tolerance through design, construction and testing of ~162,000 mutations across 115 genes spanning global regulators,<a href='#' onclick='$(this).hide().next().show().next().show();return false;' style='margin-left:10px;'>more »</a><span style='display:none;'> transcription factors, and enzymes involved in 3HP synthesis and tolerance. The iCREATE process required ~1 month to perform 13 rounds of combinatorial genome modifications with targeted gene knockouts, expression modification by ribosomal binding site (RBS) engineering, and genome-level site-saturation mutagenesis. Specific mutants conferring increased 3HP titer, yield, and productivity were identified and then combined to produce 3HP at a yield and concentration ~60-fold higher than the wild-type strain.</span><a href='#' onclick='$(this).hide().prev().hide().prev().show();return false;' style='margin-left:10px;display:none;'>« less</a></div><div class="metadata-links small clearfix text-muted" style="margin-top:15px;"> <span class="fa fa-book text-muted" aria-hidden="true"></span> Cited by 4<div class="pure-menu pure-menu-horizontal pull-right" style="width:unset;"> <ul class="pure-menu-list"> <li class="pure-menu-item"><span class="item-info-ftlink">DOI: <a class="misc doi-link " href="https://doi.org/10.1016/j.ymben.2018.04.007" target="_blank" rel="noopener" title="Link to document DOI" data-ostiid="1548398" data-product-type="Journal Article" data-product-subtype="AM" >10.1016/j.ymben.2018.04.007</a></span></li> <li class="pure-menu-item"><span class="item-info-ftlink"><a class="misc fulltext-link " href="/pages/servlets/purl/1548398" title="Link to document media" target="_blank" rel="noopener" data-ostiid="1548398" data-product-type="Journal Article" data-product-subtype="AM" >Full Text Available</a></span></li> </ul> </div> </div> </div> <div class="clearfix"></div> </div> </li> </ul> </aside> </div> </section> </div> <div class="col-sm-3 order-sm-3"> <ul class="nav nav-stacked"> <li class="active"><a class="tab-nav disabled" data-tab="related" style="color: #636c72 !important; opacity: 1;"><span class="fa fa-angle-right"></span> Similar Records</a></li> </ul> </div> </div> </section> </div></div> </div> </div> </section> <footer class="" style="background-color:#f9f9f9; /* padding-top: 0.5rem; */"> <div class="footer-minor"> <div class="container"> <hr class="footer-separator" /> <div class="text-center" style="margin-top:1.25rem;"> <div class="pure-menu pure-menu-horizontal"> <ul class="pure-menu-list" id="footer-org-menu"> <li class="pure-menu-item"> <a href="https://energy.gov" target="_blank" rel="noopener noreferrer"> <img src="data:image/gif;base64,R0lGODlhAQABAIAAAP///wAAACH5BAEAAAAALAAAAAABAAEAAAICRAEAOw==" class="sprite sprite-footer-us-doe-min" alt="U.S. Department of Energy" /> </a> </li> <li class="pure-menu-item"> <a href="https://www.energy.gov/science/office-science" target="_blank" rel="noopener noreferrer"> <img src="data:image/gif;base64,R0lGODlhAQABAIAAAP///wAAACH5BAEAAAAALAAAAAABAAEAAAICRAEAOw==" class="sprite sprite-footer-office-of-science-min" alt="Office of Science" /> </a> </li> <li class="pure-menu-item"> <a href="/"> <img src="data:image/gif;base64,R0lGODlhAQABAIAAAP///wAAACH5BAEAAAAALAAAAAABAAEAAAICRAEAOw==" class="sprite sprite-footer-osti-min" alt="Office of Scientific and Technical Information" /> </a> </li> </ul> </div> </div> <div class="text-center small" style="margin-top:0.5em;margin-bottom:2.0rem;"> <div class="pure-menu pure-menu-horizontal"> <ul class="pure-menu-list"> <li class="pure-menu-item"><a href="/disclaim" class="pure-menu-link"><span class="fa fa-institution"></span> Website Policies <span class="hidden-xs">/ Important Links</span></a></li> <li class="pure-menu-item"><a href="/pages/contact" class="pure-menu-link"><span class="fa fa-comments-o"></span> Contact Us</a></li> <li class="d-block d-md-none"></li> <li class="pure-menu-item"><a href="https://www.facebook.com/ostigov" target="_blank" rel="noopener noreferrer" class="pure-menu-link social"><span class="fa fa-facebook" style=""></span></a></li> <li class="pure-menu-item"><a href="https://twitter.com/OSTIgov" target="_blank" rel="noopener noreferrer" class="pure-menu-link social"><span class="fa fa-twitter" style=""></span></a></li> <li class="pure-menu-item"><a href="https://www.youtube.com/user/ostigov" target="_blank" rel="noopener noreferrer" class="pure-menu-link social"><span class="fa fa-youtube-play" style=""></span></a></li> </ul> </div> </div> </div> </div> </footer> <link href="/pages/css/pages.fonts.191122.1355.css" rel="stylesheet"> <script src="/pages/js/pages.191122.1355.js"></script><noscript></noscript> <script defer src="/pages/js/pages.biblio.191122.1355.js"></script><noscript></noscript> <script defer src="/pages/js/lity.js"></script><noscript></noscript><script async type="text/javascript" src="/pages/js/Universal-Federated-Analytics-Min.js?agency=DOE" id="_fed_an_ua_tag"></script><noscript></noscript></body> <!-- DOE PAGES v.191122.1355 --> </html>