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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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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 class="small references-list" style="list-style-type:none; margin-top: 0.5em; padding-left: 0; line-height:1.8em;"> <li> <span style="color:#5C7B2D;"> 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 class="small references-list" style="list-style-type:none; margin-top: 0.5em; padding-left: 0; line-height:1.8em;"> <li> <span style="color:#5C7B2D;"> 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> <div class="pagination-container small"> <a class="pure-button prev page" href="#" rel="prev"><span class="fa fa-angle-left"></span></a><ul class="pagination d-inline-block" style="padding-left:.2em;"></ul><a class="pure-button next page" href="#" rel="next"><span class="fa fa-angle-right"></span></a> </div> </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; 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margin-top:0px;">Similar Records in DOE PAGES and 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="2" /><div class="item-info"> <h2 class="title" itemprop="name headline"><a href="/pages/biblio/1213053-improving-microbial-biogasoline-production-escherichia-coli-using-tolerance-engineering" itemprop="url">Improving microbial biogasoline production in <i>Escherichia coli</i> using tolerance engineering</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">Foo, Jee Loon</span> ; <span class="author">Jensen, Heather M.</span> ; <span class="author">Dahl, Robert H.</span> ; <span class="author">...</span> <span class="text-muted pubdata"> - mBio (Online)</span> </span> </div> <div class="abstract">Engineering microbial hosts for the production of fungible fuels requires mitigation of limitations posed on the production capacity. One such limitation arises from the inherent toxicity of solvent-like biofuel compounds to production strains, such as <i>Escherichia coli</i>. Here we show the importance of host engineering for the production of short-chain alcohols by studying the overexpression of genes upregulated in response to exogenous isopentenol. Using systems biology data, we selected 40 genes that were upregulated following isopentenol exposure and subsequently overexpressed them in <i>E. coli</i>. Overexpression of several of these candidates improved tolerance to exogenously added isopentenol. Genes conferring isopentenol tolerance<a href='#' onclick='$(this).hide().next().show().next().show();return false;' style='margin-left:10px;'>more »</a><span style='display:none;'> phenotypes belonged to diverse functional groups, such as oxidative stress response (<i>soxS</i>, <i>fpr</i>, and <i>nrdH</i>), general stress response (<i>metR</i>, <i>yqhD</i>, and <i>gidB</i>), heat shock-related response (<i>ibpA</i>), and transport (<i>mdlB</i>). To determine if these genes could also improve isopentenol production, we coexpressed the tolerance-enhancing genes individually with an isopentenol production pathway. Our data show that expression of 6 of the 8 candidates improved the production of isopentenol in <i>E. coli</i>, with the methionine biosynthesis regulator MetR improving the titer for isopentenol production by 55%. Additionally, expression of MdlB, an ABC transporter, facilitated a 12% improvement in isopentenol production. To our knowledge, MdlB is the first example of a transporter that can be used to improve production of a short-chain alcohol and provides a valuable new avenue for host engineering in biogasoline production.The use of microbial host platforms for the production of bulk commodities, such as chemicals and fuels, is now a focus of many biotechnology efforts. Many of these compounds are inherently toxic to the host microbe, which in turn places a limit on production despite efforts to optimize the bioconversion pathways. In order to achieve economically viable production levels, it is also necessary to engineer production strains with improved tolerance to these compounds. We demonstrate that microbial tolerance engineering using transcriptomics data can also identify targets that improve production. Our results include an exporter and a methionine biosynthesis regulator that improve isopentenol production, providing a starting point to further engineer the host for biogasoline production.</span><a href='#' onclick='$(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 24<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/mBio.01932-14" target="_blank" rel="noopener" title="Link to document DOI" data-ostiid="1213053" data-product-type="Journal Article" data-product-subtype="AM" >10.1128/mBio.01932-14</a></span></li> <li class="pure-menu-item"><span class="item-info-ftlink"><a class="misc fulltext-link " href="/pages/servlets/purl/1213053" title="Link to document media" target="_blank" rel="noopener" data-ostiid="1213053" 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="3" /><div class="item-info"> <h2 class="title" itemprop="name headline"><a href="/pages/biblio/1604827-vivo-thermodynamic-analysis-glycolysis-clostridium-thermocellum-thermoanaerobacterium-saccharolyticum-using-tracers" itemprop="url"><em>In Vivo</em> Thermodynamic Analysis of Glycolysis in <em>Clostridium thermocellum</em> and <em>Thermoanaerobacterium saccharolyticum</em> Using <sup>13</sup> C and <sup>2</sup> H Tracers</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">Jacobson, Tyler B.</span> ; <span class="author">Korosh, Travis K.</span> ; <span class="author">Stevenson, David M.</span> ; <span class="author">...</span> <span class="text-muted pubdata"> - mSystems</span> </span> </div> <div class="abstract">Clostridium thermocellumandThermoanaerobacterium saccharolyticumare thermophilic anaerobic bacteria with complementary metabolic capabilities that utilize distinct glycolytic pathways for the conversion of cellulosic sugars to biofuels. We integrated quantitative metabolomics with <sup>2</sup>H and<sup>13</sup>C metabolic flux analysis to investigate the in vivo reversibility and thermodynamics of the central metabolic networks of these two microbes. We found that the glycolytic pathway in C. thermocellum operates remarkably close to thermodynamic equilibrium, with an overall drop in Gibbs free energy 5-fold lower than that of T. saccharolyticum or anaerobically grownEscherichia coli. The limited thermodynamic driving force of glycolysis in C. thermocellum could be attributed in large part<a href='#' onclick='$(this).hide().next().show().next().show();return false;' style='margin-left:10px;'>more »</a><span style='display:none;'> to the small free energy of the phosphofructokinase reaction producing fructose bisphosphate. The ethanol fermentation pathway was also substantially more reversible in C. thermocellummthan in T. saccharolyticum. These observations help explain the comparatively low ethanol titers of C. thermocellum and suggest engineering interventions that can be used to increase its ethanol productivity and glycolytic rate. In addition to thermodynamic analysis, we used our isotope tracer data to reconstruct the T. saccharolyticum central metabolic network, revealing exclusive use of the Embden-Meyerhof-Parnas (EMP) pathway for glycolysis, a bifurcated tricarboxylic acid (TCA) cycle, and a sedoheptulose bisphosphate bypass active within the pentose phosphate pathway. Thermodynamics constitutes a key determinant of flux and enzyme efficiency in metabolic networks. Here, we provide new insights into the divergent thermodynamics of the glycolytic pathways of C. thermocellum and T. saccharolyticum, two industrially relevant thermophilic bacteria whose metabolism still is not well understood. We report that while the glycolytic pathway in T. saccharolyticum is as thermodynamically favorable as that found in model organisms, such asE. coliorSaccharomyces cerevisiae, the glycolytic pathway of C. thermocellum operates near equilibrium. The use of a near-equilibrium glycolytic pathway, with potentially increased ATP yield, by this cellulolytic microbe may represent an evolutionary adaptation to growth on cellulose, but it has the drawback of being highly susceptible to product feedback inhibition. The results of this study will facilitate future engineering of high-performance strains capable of transforming cellulosic biomass to biofuels at high yields and titers.</span><a href='#' onclick='$(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 1<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.00736-19" target="_blank" rel="noopener" title="Link to document DOI" data-ostiid="1604827" data-product-type="Journal Article" data-product-subtype="PA" >10.1128/mSystems.00736-19</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="5" /><div class="item-info"> <h2 class="title" itemprop="name headline"><a href="/biblio/842949-role-glycolytic-intermediate-regulation-improving-lycopene-production-escherichia-coli-engineering-metabolic-control" itemprop="url">Role of glycolytic intermediate in regulation: Improving lycopene production in Escherichia coli by engineering metabolic control</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">Farmer, W R</span> ; <span class="author">Liao, J C</span> <span class="text-muted pubdata"></span> </span> </div> <div class="abstract">Metabolic engineering in the postgenomic era is expected to benefit from a full understanding of the biosynthetic capability of microorganisms as a result of the progress being made in bioinformatics and functional genomics. The immediate advantage of such information is to allow the rational design of novel pathways and the elimination of native reactions that are detrimental or unnecessary for the desired purpose. However, with the ability to manipulate metabolic pathways becoming more effective, metabolic engineering will need to face a new challenge: the reengineering of the regulatory hierarchy that controls gene expression in those pathways. In addition to constructing<a href='#' onclick='$(this).hide().next().show().next().show();return false;' style='margin-left:10px;'>more »</a><span style='display:none;'> the genetic composition of a metabolic pathway, they propose that it will become just as important to consider the dynamics of pathways gene expression. It has been widely observed that high-level induction of a recombinant protein or pathway leads to growth retardation and reduced metabolic activity. These phenotypic characteristics result from the fact that the constant demands of production placed upon the cell interfere with its changing requirements for growth. They believe that this common situation in metabolic engineering can be alleviated by designing a dynamic controller that is able to sense the metabolic state of the cell and regulate the expression of the recombinant pathway accordingly. This approach, which is termed metabolic control engineering, involves redesigning the native regulatory circuits and applying them to the recombinant pathway. The general goal of such an effort will be to control the flux to the recombinant pathway adaptively according to the cell's metabolic state. The dynamically controlled recombinant pathway can potentially lead to enhanced production, minimized growth retardation, and reduced toxic by-product formation. The regulation of gene expression in response to the physiological state is also essential to the success of gene therapy. Here they illustrate an application of this approach for the enhanced production of lycopene in Escherichia coli. The chose lycopene as a model compound because of its potential beneficial effects on human health. Lycopene, being an effective antioxidant, has been proposed as a possible treatment for some cancers and other degenerative human conditions. As a result, the in vivo synthesis of lycopene and related carotenoids has received increasing attention, and a number of reports have described their production in recombinant microorganism.</span><a href='#' onclick='$(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/10.2172/842949" target="_blank" rel="noopener" title="Link to document DOI" data-ostiid="842949" data-product-type="Technical Report" data-product-subtype="" >10.2172/842949</a></span></li> <li class="pure-menu-item"><span class="item-info-ftlink"><a class="misc fulltext-link " href="/servlets/purl/842949" title="Link to document media" target="_blank" rel="noopener" data-ostiid="842949" 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="6" /><div class="item-info"> <h2 class="title" itemprop="name headline"><a href="/biblio/1069258-genomics-investigation-partitioning-among-flavonoid-derived-condensed-tannins-carbon-sequestration-populus" itemprop="url">A genomics investigation of partitioning into and among flavonoid-derived condensed tannins for carbon sequestration in Populus</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">Harding, Scott, A</span> ; <span class="author">Tsai, Chung-jui</span> ; <span class="author">Lindroth, Richard, L</span> <span class="text-muted pubdata"></span> </span> </div> <div class="abstract">The project set out to use comparative (genotype and treatment) and transgenic approaches to investigate the determinants of condensed tannin (CT) accrual and chemical variability in Populus. CT type and amount are thought to effect the decomposition of plant detritus in the soil, and thereby the sequestering of carbon in the soil. The stated objectives were: 1. Genome-wide transcriptome profiling (microarrays) to analyze structural gene, transcription factor and metabolite control of CT partitioning; 2. Transcriptomic (microarray) and chemical analysis of ontogenetic effects on CT and PG partitioning; and 3. Transgenic manipulation of flavonoid biosynthetic pathway genes to modify the control<a href='#' onclick='$(this).hide().next().show().next().show();return false;' style='margin-left:10px;'>more »</a><span style='display:none;'> of CT composition. Objective 1: A number of approaches for perturbing CT content and chemistry were tested in Objective 1, and those included nitrogen deficit, leaf wounding, drought, and salicylic acid spraying. Drought had little effect on CTs in the genotypes we used. Plants exhibited unpredictability in their response to salicylic acid spraying, leading us to abandon its use. Reduced plant nitrogen status and leaf wounding caused reproducible and magnitudinally striking increases in leaf CT content. Microarray submissions to NCBI from those experiments are the following: GSE ID 14515: Comparative transcriptomics analysis of Populus leaves under nitrogen limitation: clone 1979. Public on Jan 04, 2010; Contributor(s) Harding SA, Tsai C GSE ID 14893: Comparative transcriptomics analysis of Populus leaves under nitrogen limitation: clone 3200. Public on Feb 19, 2009; Contributor(s) Harding SA, Tsai C GSE ID 16783 Wound-induced gene expression changes in Populus: 1 week; clone RM5. Status Public on Dec 01, 2009; Contributor(s) Harding SA, Tsai C GSE ID 16785 Wound-induced gene expression changes in Populus: 90 hours; clone RM5 Status Public on Dec 01, 2009; Contributor(s) Harding SA, Tsai C Although CT amount changed in response to treatments, CT composition was essentially conserved. Overall phenylpropanoid composition exhibited changes due to large effects on phenolic glycosides containing a salicin moiety. There were no effects on lignin content. Efforts to publish this work continue, and depend on additional data which we are still collecting. This ongoing work is expected to strengthen our most provocative metabolic profiling data which suggests as yet unreported links controlling the balance between the two major leaf phenylpropanoid sinks, the CTs and the salicin-PGs. Objective 2: Ontogenic effects on leaf CT accrual and phenylpropanoid complexity (Objective 2) have been reported in the past and we contributed two manuscripts on how phenylpropanoid sinks in roots and stems could have an increasing effect on leaf CT as plants grow larger and plant proportions of stem, root and leaf change. Tsai C.-J., El Kayal W., Harding S.A. (2006) Populus, the new model system for investigating phenylpropanoid complexity. International Journal of Applied Science and Engineering 4: 221-233. We presented evidence that flavonoid precursors of CT rapidly decline in roots under conditions that favor CT accrual in leaves. Harding SA, Jarvie MM, Lindroth RL, Tsai C-J (2009) A comparative analysis of phenylpropanoid metabolism, N utilization and carbon partitioning in fast- and slow-growing Populus hybrid clones. Journal of Experimental Botany. 60:3443-3452. We presented evidence that nitrogen delivery to leaves as a fraction of nitrogen taken up by the roots is lower in high leaf CT genotypes. We presented a hypothesis from our data that N was sequestered in proportion to lignin content in stem tissues. Low leaf N content and high leaf CT in genotypes with high stem lignin was posited to be a systemic outcome of N demand in lignifiying stem tissues. Thereby, stem lignin and leaf CT accrual might be systemically linked, placing control of leaf phenylpropanoids under systemic rather than solely organ specific determinants. Analyses of total structural and non-structural carbohydrates contributed to the model presented. Harding SA, Xue L, Du L, Nyamdari B, Sykes R, Davis M, Lindroth RL, Tsai CJ (submitted March 2013) Condensed tannin biosynthesis in leaves conditions carbon use, defense and growth in Populus. (Invited submission to Tree Physiology) MS abstract: Condensed tannins (CT) are flavonoid end products that can comprise a large fraction of leaf, bark and root biomass in Populus species. CT accrual was investigated in relation to metabolic carbon and nitrogen use in young leaves and shoot tips (ST) where CT biosynthesis was most active. A slow-growing genotype (SG) and a fast-growing genotype (FG) were compared. Both genotypes exhibited the capacity to accrue similarly large reserves of salicortin a phenolic glycoside (PG), but the slow-growing line also produced CT. PG accrual was developmentally delayed in the slow-growing line, SG. Irrigation with low-N nutrients promoted PG accrual in FG plants, but PG accrual was suspended in CT-producing SG plants. In addition, the low C:N amide asparagine accumulated and glucose was depleted in ST and expanding leaves of SG plants. The monoamine phenylethylamine (PEA) was abundant in SG leaves and absent in FG leaves. Leaf metabolite and gene expression differences were observed between SG and FG that would be expected to impinge upon glycolysis, acetyl-CoA production and flavonoid production. A model that integrates PEA with those activities and CT accrual was developed. Briefly, the data support a model in which flavonoid biosynthesis depleted the acetyl-CoA pool, thereby promoting glycolytic and shikimic pathway fluxes in SG plants. PEA results from decarboxylation of the shikimic pathway end-product phenylalanine, and is proposed to have facilitated CT polymerization, thereby promoting the continued biosynthesis of flavonoid CT precursors in SG leaves. The leaf differentials described here were absent in young roots, as was PEA. The potential contribution of PEA to CT polymerization constituted a metabolic carbon drain in developing leaves that was not observed in the roots. We propose that PEA, in addition to other factors, including flavonoid pathway Myb transcription factors, is an important contributor to carbon management and plant defense in Populus. Objective 3: From work related to the first two objectives, it appeared that CT chemistry, at least in terms of the proportions of mono, di and tri hydroxylation at the phenylpropanoid-derived B-ring, changed little if at all when CT accrual per unit time was increased. A large number of transgenic Populus plants with alterations in the expression of flavonoid pathway genes and the potential to produce B-ring, chemically altered CT were generated during the project. Transgenic lines of Populus tremula Michx. Populus alba L. clone 717-1B4, a low CT producer, were produced that over- or under-express several mid and late flavonoid pathway genes including dihydroxyflavonol reductase (DFR-2 isoforms), leucoanthocyanidin reductase (LAR-3 isoforms), anthocyanidin reductase (ANR-2 isoforms), flavonol synthase (FLS-2 isoforms). A large number of additional transformation constructs (chalcone synthases, flavone synthases, and flavanol hydroxylases) were developed that failed to result in transgenic plants. We have purified CT from several of the successful lines and have obtained evidence from pyrolysis GC-MS that CT chemical composition was altered in transgenic lines harboring overexpression constructs for one of the two DFR isoforms. We have also observed increased CT levels in leaves of those lines, but the increases vary substantially in magnitude from experiment to experiment which has led to ongoing efforts to understand the variation before attempting to publish the findings. Preliminary results from some of the transgenic work were presented: An C*, Luo K, El Kayal W, Harding SA, Tsai C-J (2009) Transgenic manipulation of condensed tannins in Populus. IUFRO Tree Biotechnology Conference, Whistler, BC, Canada Work on the design of some of the constructs for the CT transgenics work has been published: Luo K, Harding SA, Tsai C-J (2008) A modified T-vector for simplified assembly of hairpin RNAi constructs. Biotechnology Letters 30: 1271-1274. DOE support from this project was also acknowledged in a book chapter: Douglas CJ, Ehlting J, Harding SA (2009) Phenylpropanoid and Phenolic Metabolism in Populus: Gene Family Structure and Comparative and Functional Genomics In Joshi, C.P., and S.P. DiFazio (eds). Genetics, Genomics and Breeding of Crop Plants: Poplar. Science Publishers, Enfield, New Hampshire. Pp. 304-326 Other work directly related to and supported in part by this project include: Qin H, Feng T, Harding SA, Tsai C-J, Zhang S (2008) An efficient method to identify differentially expressed genes in microarray experiments. Bioinformatics 24: 1583-1589. Tsai C-J, Ranjan P, DiFazio SP, Tuskan GA, Johnson V (2011) Poplar genome microarrays. In: Joshi CP, DiFazio SP and Kole C (eds), Genetics, Genomics and Breeding of Poplars. Science Publishers, Enfield, NH. pp. 112-127. Street N, Tsai C-J (2010) Populus resources and bioinformatics. In: Jansson S, Bhalerao R, and Groover AT (eds), Genetics and Genomics of Populus. Plant Genetics and Genomics: Crops and Models book series. Springer, New York, pp. 135-152.</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/10.2172/1069258" target="_blank" rel="noopener" title="Link to document DOI" data-ostiid="1069258" data-product-type="Technical Report" data-product-subtype="" >10.2172/1069258</a></span></li> <li class="pure-menu-item"><span class="item-info-ftlink"><a class="misc fulltext-link " href="/servlets/purl/1069258" title="Link to document media" target="_blank" rel="noopener" data-ostiid="1069258" 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="7" /><div class="item-info"> <h2 class="title" itemprop="name headline"><a href="/pages/biblio/1213363-production-long-chain-alcohols-alkanes-upon-coexpression-acyl-acp-reductase-aldehyde-deformylating-oxgenase-bacterial-type-fatty-acid-synthase-coli" itemprop="url">Production of long chain alcohols and alkanes upon coexpression of an acyl-ACP reductase and aldehyde-deformylating oxgenase with a bacterial type-I fatty acid synthase in <i>E. coli</i></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">Coursolle, Dan</span> ; <span class="author">Shanklin, John</span> ; <span class="author">Lian, Jiazhang</span> ; <span class="author">...</span> <span class="text-muted pubdata"> - Molecular BioSystems</span> </span> </div> <div class="abstract">Microbial long chain alcohols and alkanes are renewable biofuels that could one day replace petroleum-derived fuels. Here we report a novel pathway for high efficiency production of these products in <i>Escherichia coli</i> strain BL21(DE3). We first identified the acyl-ACP reductase/aldehyde deformylase combinations with the highest activity in this strain. Next, we used catalase coexpression to remove toxic byproducts and increase the overall titer. Finally, by introducing the type-I fatty acid synthase from <i>Corynebacterium ammoniagenes</i>, we were able to bypass host regulatory mechanisms of fatty acid synthesis that have thus far hampered efforts to optimize the yield of acyl-ACP-derived products in<a href='#' onclick='$(this).hide().next().show().next().show();return false;' style='margin-left:10px;'>more »</a><span style='display:none;'> BL21(DE3). When all these engineering strategies were combined with subsequent optimization of fermentation conditions, we were able to achieve a final titer around 100 mg/L long chain alcohol/alkane products including a 57 mg/L titer of pentadecane, the highest titer reported in <i>E. coli</i> BL21(DE3) to date. The expression of prokaryotic type-I fatty acid synthases offer a unique strategy to produce fatty acid-derived products in <i>E. coli</i> that does not rely exclusively on the endogenous type-II fatty acid synthase system.</span><a href='#' onclick='$(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 7<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/10.1039/C5MB00268K" target="_blank" rel="noopener" title="Link to document DOI" data-ostiid="1213363" data-product-type="Journal Article" data-product-subtype="AM" >10.1039/C5MB00268K</a></span></li> <li class="pure-menu-item"><span class="item-info-ftlink"><a class="misc fulltext-link " href="/pages/servlets/purl/1213363" title="Link to document media" target="_blank" rel="noopener" data-ostiid="1213363" 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.200912.1307.css" rel="stylesheet"> <script src="/pages/js/pages.200912.1307.js"></script><noscript></noscript> <script defer src="/pages/js/pages.biblio.200912.1307.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.200912.1307 --> </html>