Hal S Alper

University of Texas at Austin, Austin, Texas, United States

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Publications (75)455.12 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Reduction of endogenous gene expression is a fundamental operation of metabolic engineering, yet current methods for gene knockdown (i.e. genome editing) remain laborious and slow, especially in yeast. In contrast, RNA interference allows facile and tunable gene knockdown via a simple plasmid transformation step, enabling metabolic engineers to rapidly prototype knockdown strategies in multiple strains before expending significant cost to undertake genome editing. Although RNAi is naturally present in a myriad of eukaryotes, it has only been recently implemented in S. cerevisiae as a heterologous pathway and so has not yet been optimized as a metabolic engineering tool. In this study, we elucidate a set of design principles for the construction of hairpin RNA expression cassettes in yeast and implement RNA interference to quickly identify routes for improvement of itaconic acid production in this organism. The approach developed here enables rapid prototyping of knockdown strategies and thus accelerates and reduces the cost of the design-build-test cycle in yeast.
    ACS Synthetic Biology 12/2013; 3(5). DOI:10.1021/sb4001432 · 3.95 Impact Factor
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    ABSTRACT: Cellular systems can be engineered into factories that produce high-value chemicals from renewable feedstock. Such an approach requires an expanded toolbox for metabolic engineering. Recently, protein engineering and directed evolution strategies have started to play a growing and critical role within metabolic engineering. This review focuses on the various ways in which directed evolution can be applied in conjunction with metabolic engineering to improve product yields. Specifically, we discuss the application of directed evolution on both catalytic and non-catalytic traits of enzymes, on regulatory elements, and on whole genomes in a metabolic engineering context. We demonstrate how the goals of metabolic pathway engineering can be achieved in part through evolving cellular parts as opposed to traditional approaches that rely on gene overexpression and deletion. Finally, we discuss the current limitations in screening technology that hinder the full implementation of a metabolic pathway-directed evolution approach.
    Biotechnology Journal 12/2013; 8(12). DOI:10.1002/biot.201300021 · 3.71 Impact Factor
  • Sun-Mi Lee, Hal Alper
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    ABSTRACT: Biofuels production from lignocellulosic biomass can be both sustainable and economical when all available carbon sources are completely utilized. Pentose sugars, such as xylose and arabinose, constitute significant portion of lignocellulosic biomass hydrolysates. However, these sugars are poorly utilized by the yeast Saccharomyces cerevisiae despite decades of research. Here, we discuss the advantages of both combinatorial and evolutionary engineering approaches for the improvement of pentose sugar utilization. Specifically, we discuss the directed evolution of a xylose isomerase-based pathway to achieve a 61 fold improvement in growth rate and 8 fold improvement in ethanol production from xylose. We next discuss combinatorial and evolutionary engineering efforts to establish functional pathways. In particular, key catabolic enzymes were either randomly mutated from a previously reported enzyme or newly obtained from a pentose sugar utilizing fungi. We show that alternative pentose sugar catabolic pathways in S. cerevisiae can perform better compared with previously reported pentose sugar pathways. Moreover, key design principles for catabolic enzyme pathways can be extracted from these studies. These strains can be further improved through adaptive or evolutionary engineering efforts. Finally, these studies describe an effective combinatorial engineering strategy to develop efficient heterologous pathway in S. cerevisiae.
    13 AIChE Annual Meeting; 11/2013
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    ABSTRACT: Concerns about energy security, the global petroleum supply and climate change have increased interest in the production of sustainable and renewable biofuels. The oleaginous yeast Yarrowia lipolytica naturally possesses moderate lipid (biodiesel precursor) production and grows on different kinds of biomass (and organic waste). However, production from native, un-engineered strains is not sufficient for an industrial process. Here, we report on a rational and combinatorial metabolic engineering approach to establish Y. lipolytica as a premier platform for industrial-level, high lipid production. Specifically, several rational gene targets were combined to uncover potential synergistic genetic influencing lipogenesis. This study resulted in the largest collection of genetically modified strains of Y. lipolytica. The lipid content in the best engineered strain exceeded 80% of its dry weight. In parallel with these efforts, an inverse, combinatorial metabolic engineering approach was used to isolate improved lipid production strains. Whole genome re-sequencing of isolated strains revealed a novel lipid enhancer element. Further improvements of lipid production were achieved by combining these two approaches. Through this effort, we enabled extremely high lipid production in Y. lipolytica. This project helps elucidate the mechanism of lipogenesis and establish Y. lipolytica as an oleochemicals platform strain.
    13 AIChE Annual Meeting; 11/2013
  • Nathan Crook, Hal S. Alper
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    ABSTRACT: Synthetic biology brings engineering tools and perspectives to the design of living systems. In contrast to classical cell engineering approaches, synthetic biology enables cellular networks to be understood as a combination of modular elements in much the same way as unit operations combine to describe a chemical plant. Consequently, models for the behavior of these designed systems are inspired by frameworks developed for traditional chemical engineering design. There are direct analogies between cellular metabolism and reaction networks in a chemical process. As examples, thermodynamic and kinetic models of chemical reaction networks have been used to simulate fluxes within living systems and predict the performance of synthetic parts. Concepts from process control have been brought to bear on the design of transcriptional and translational regulatory networks. Such engineering frameworks have greatly aided the design and understanding of living systems and have enabled the design of cells exhibiting complex dynamic behavior and high productivity of desirable compounds. This review summarizes efforts to quantitatively model cellular behavior (both endogenous and synthetic), especially as related to the design of living systems.
    Chemical Engineering Science 11/2013; 103:2-11. DOI:10.1016/j.ces.2012.12.022 · 2.61 Impact Factor
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    ABSTRACT: Control of gene and protein expression of both endogenous and heterologous genes is a key component of metabolic engineering. While a large amount of work has been published characterizing promoters for this purpose, less effort has been exerted to elucidate the role of terminators in yeast. In this study, we characterize over 30 terminators for use in metabolic engineering applications in Saccharomyces cerevisiae and determine mRNA half-life changes to be the major cause of the varied protein and transcript expression level. We demonstrate that the difference in transcript level can be over 6.5-fold even for high strength promoters. The influence of terminator selection is magnified when coupled with a low-expression promoter, with a maximum difference in protein expression of 11-fold between a high-capacity terminator and the parent plasmid terminator and over 35-fold difference when compared with a no-terminator baseline. This is the first time that terminators have been investigated in the context of multiple promoters spanning orders of magnitude in activity. Finally, we demonstrate the utility of terminator selection for metabolic engineering by using a mutant xylose isomerase gene as a proof-of-concept. Through pairing a high-capacity terminator with a low-expression promoter, we were able to achieve the same phenotypic result as with a promoter considerably higher in strength. Moreover, we can further boost the phenotype of the high-strength promoter by pairing it with a high-capacity terminator. This work highlights how terminator elements can be used to control metabolic pathways in the same way that promoters are traditionally used in yeast. Together, this work demonstrates that terminators will be an important part of heterologous gene expression and metabolic engineering for yeast in the future.
    Metabolic Engineering 07/2013; 19. DOI:10.1016/j.ymben.2013.07.001 · 8.26 Impact Factor
  • Amanda M Lanza, Do Soon Kim, Hal S Alper
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    ABSTRACT: Selection markers are common genetic elements used in recombinant cell line development. While several selection systems exist for use in mammalian cell lines, no previous study has comprehensively evaluated their performance in the isolation of recombinant populations and cell lines. Here we examine four antibiotics, hygromycin, neomycin, puromycin, and Zeocin, and their corresponding selector genes, using a green fluorescent protein (GFP) as a reporter in two model cell lines, HT1080 and HEK293. We identify Zeocin as the best selection agent for cell line development in human cells. In comparison to the other selection systems, Zeocin is able to identify populations with higher fluorescence levels, which in turn leads to the isolation of better clonal populations and less false positives. Further, Zeocin-resistant populations exhibit better transgene stability in the absence of selection pressure compared to other selection agents. All isolated Zeocin-resistant clones, regardless of cell type, exhibited GFP expression. By comparison, only 79% of hygromycin-resistant, 47% of neomycin-resistant and 14% of puromycin-resistant clones expressed GFP. Based on these results, we would rank Zeocin > hygromycin ∼ puromycin > neomycin for cell line development in human cells. Furthermore, this study demonstrates that selection marker choice does impact cell line development.
    Biotechnology Journal 07/2013; 8(7). DOI:10.1002/biot.201200364 · 3.71 Impact Factor
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    Hal S. Alper
    Biotechnology Journal 05/2013; 8(5). DOI:10.1002/biot.201200307 · 3.71 Impact Factor
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    Hal S Alper, Christoph Wittmann
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    ABSTRACT: Systems metabolic engineering is becoming a widely-evoked paradigm for industrial strain design and optimization. Specifically, systems wide experimental and computational analyses of cells and their environments enable guide metabolic engineers to quickly parse the genome and creating desirable overproduction phenotypes.
    Biotechnology Journal 05/2013; 8(5):506-7. DOI:10.1002/biot.201300167 · 3.71 Impact Factor
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    ABSTRACT: The complete biosynthetic replacement of petroleum transportation fuels requires a metabolic pathway capable of producing short chain n-alkanes. Here, we report and characterize a proof-of-concept pathway that enables microbial production of the C5n-alkane, pentane. This pathway utilizes a soybean lipoxygenase enzyme to cleave linoleic acid to pentane and a tridecadienoic acid byproduct. Initial expression of the soybean lipoxygenase enzyme within a Yarrowia lipolytica host yielded 1.56mg/L pentane. Efforts to improve pentane yield by increasing substrate availability and strongly overexpressing the lipoxygenase enzyme successfully increased pentane production three-fold to 4.98mg/L. This work represents the first-ever microbial production of pentane and demonstrates that short chain n-alkane synthesis is conceivable in model cellular hosts. In this regard, we demonstrate the potential pliability of Y. lipolytica towards the biosynthetic production of value-added molecules from its generous fatty acid reserves.
    Journal of Biotechnology 04/2013; 165(3-4). DOI:10.1016/j.jbiotec.2013.04.003 · 2.88 Impact Factor
  • Leqian Liu, Heidi Redden, Hal S Alper
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    ABSTRACT: Microbial systems provide an attractive, renewable route to produce desired organic molecules such as fuels and chemicals. While attention within the field of metabolic engineering has mostly focused on Escherichia coli, yeast is a potent host and growing host for industrial products and has many outstanding, biotechnologically desirable native traits. Thus, there has been a recent shift in focus toward yeast as production hosts to replace E. coli. As such, products have diversified in yeast beyond simply ethanol. Additionally, nonconventional yeasts have been considered to move beyond Saccharomyces cerevisiae. This review highlights recent advances in metabolic engineering of yeasts for producing value-added chemical compounds including alcohols, sugar derivatives, organic acids, fats, terpenes, aromatics, and polyketides. Furthermore, we will also discuss the future direction of metabolic engineering of yeasts.
    Current Opinion in Biotechnology 03/2013; 24(6). DOI:10.1016/j.copbio.2013.03.005 · 8.04 Impact Factor
  • John Blazeck, Hal S Alper
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    ABSTRACT: Synthetic control of gene expression is critical for metabolic engineering efforts. Specifically, precise control of key pathway enzymes (heterologous or native) can help maximize product formation. The fundamental level of transcriptional control takes place at promoter elements that drive gene expression. Endogenous promoters are limited in that they do not fully sample the complete continuum of transcriptional control, and do not maximize the transcription levels achievable within an organism. To address this issue, several attempts at promoter engineering have shown great promise both in expanding the cell-wide transcriptional capacity of an organism and in enabling tunable levels of gene expression. Thus, this review highlights the recent advances and approaches for altering gene expression control at the promoter level. Furthermore, we propose that recent advances in the understanding of transcription factors and their DNA-binding sites will enable rational and predictive control of gene expression.
    Biotechnology Journal 01/2013; 8(1). DOI:10.1002/biot.201200120 · 3.71 Impact Factor
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    ABSTRACT: The dicarboxylic acid muconic acid has garnered significant interest due to its potential use as a platform chemical for the production of several valuable consumer bio-plastics including nylon-6,6 and polyurethane (via an adipic acid intermediate) and polyethylene terephthalate (PET) (via a terephthalic acid intermediate). Many process advantages (including lower pH levels) support the production of this molecule in yeast. Here, we present the first heterologous production of muconic acid in the yeast Saccharomyces cerevisiae. A three-step synthetic, composite pathway comprised of the enzymes dehydroshikimate dehydratase from Podospora anserina, protocatechuic acid decarboxylase from Enterobacter cloacae, and catechol 1,2-dioxygenase from Candida albicans was imported into yeast. Further genetic modifications guided by metabolic modeling and feedback inhibition mitigation were introduced to increase precursor availability. Specifically, the knockout of ARO3 and overexpression of a feedback-resistant mutant of aro4 reduced feedback inhibition in the shikimate pathway, and the zwf1 deletion and over-expression of TKL1 increased flux of necessary precursors into the pathway. Further balancing of the heterologous enzyme levels led to a final titer of nearly 141mg/L muconic acid in a shake-flask culture, a value nearly 24 fold higher than the initial strain. Moreover, this strain has the highest titer and second highest yield of any reported shikimate and aromatic amino acid-based molecule in yeast in a simple batch condition. This work collectively demonstrates that yeast has the potential to be a platform for the bioproduction of muconic acid and suggests an area that is ripe for future metabolic engineering efforts.
    Metabolic Engineering 11/2012; 15. DOI:10.1016/j.ymben.2012.10.003 · 8.26 Impact Factor
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    ABSTRACT: A dynamic range of well-controlled constitutive and tunable promoters are essential for metabolic engineering and synthetic biology applications in all host organisms. Here, we apply a synthetic hybrid promoter approach for the creation of strong promoter libraries in the model yeast, Saccharomyces cerevisiae. Synthetic hybrid promoters are composed of two modular components-the enhancer element, consisting of tandem repeats or combinations of upstream activation sequences (UAS), and the core promoter element. We demonstrate the utility of this approach with three main case studies. First, we establish a dynamic range of constitutive promoters and in doing so expand transcriptional capacity of the strongest constitutive yeast promoter, P(GPD) , by 2.5-fold in terms of mRNA levels. Second, we demonstrate the capacity to impart synthetic regulation through a hybrid promoter approach by adding galactose activation and removing glucose repression. Third, we establish a collection of galactose-inducible hybrid promoters that span a nearly 50-fold dynamic range of galactose-induced expression levels and increase the transcriptional capacity of the Gal1 promoter by 15%. These results demonstrate that promoters in S. cerevisiae, and potentially all yeast, are enhancer limited and a synthetic hybrid promoter approach can expand, enhance, and control promoter activity. Biotechnol. Bioeng. 2012; 109: 2884-2895. © 2012 Wiley Periodicals, Inc.
    Biotechnology and Bioengineering 11/2012; 109(11):2884-95. DOI:10.1002/bit.24552 · 4.16 Impact Factor
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    ABSTRACT: Highlights ► Demand for speed of development and low cost drive the need for cell line development technologies. ► Advancements in genome targeting enable precise and robust transgene expression and deletions. ► cis-Acting and trans-acting elements enable control of gene expression and complex circuit design. ► These technologies enable more complex metabolic and pathway engineering of mammalian systems. ► Advancements in these areas will drive advances in cell line development.
    11/2012; 1(4):403–410. DOI:10.1016/j.coche.2012.09.005
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    ABSTRACT: Many metabolic engineering and genetic engineering applications in yeast rely on the use of plasmids. Despite their pervasive use and the diverse collections available, there is a fundamental lack of understanding of how commonly used DNA plasmids affect the cell's ability to grow and how the choice of plasmid components can influence plasmid load and burden. In this study, we characterized the major attributes of the 2μ and centromeric plasmids typically used in yeast by examining the impact of choice of selection marker, promoter, origin of replication, and strain ploidy on conferred growth rates and plasmid copy number. We conclude that the "plasmid burden," as demonstrated by a reduced growth rate, is primarily due to the choice of selection marker, especially when auxotrophic markers are utilized. The plasmid burden traditionally attributed to replication and maintenance of plasmid DNA plays only a minor role in haploid yeast yet is much more significant in diploid strains. The selection marker can also significantly change plasmid copy number. In fact, plasmid copy number can be influenced to some extent by all of the parameters tested. The information presented in this study will allow for more rational design and selection of plasmids for engineering applications. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
    FEMS Yeast Research 10/2012; 13(1). DOI:10.1111/1567-1364.12016 · 2.44 Impact Factor
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    Hal S Alper
    10/2012; 3(4):e201210001. DOI:10.5936/csbj.201210001
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    ABSTRACT: Both varied and strong promoters are essential for metabolic and pathway engineering applications in any host organism. To enable this capacity, here we demonstrate a generalizable method for the de novo construction of strong, synthetic hybrid promoter libraries. Specifically, we demonstrate how promoter truncation and fragment dissection analysis can be utilized to identify both novel upstream activating sequences (UAS) and core promoters-the two components required to generate hybrid promoters. As a base case, the native TEF promoter in Yarrowia lipolytica was examined to identify putative UAS elements that serve as modular synthetic transcriptional activators. Resulting synthetic promoters containing a core promoter region activated by between one and twelve tandem repeats of the newly isolated, 230 nucleotide UAS(TEF)#2 element showed promoter strengths 3- to 4.5-fold times the native TEF promoter. Further analysis through transcription factor binding site abrogation revealed the GCR1p binding site to be necessary for complete UAS(TEF)#2 function. These various promoters were tested for function in a variety of carbon sources. Finally, by combining disparate UAS elements (in this case, UAS(TEF) and UAS1B), we developed a high-strength promoter with for Y. lipolytica with an expression level of nearly sevenfold higher than that of the strong, constitutive TEF promoter. Thus, the general strategy described here enables the efficient, de novo construction of synthetic promoters to both increase native expression capacity and to produce libraries for tunable gene expression.
    Applied Microbiology and Biotechnology 09/2012; 97(7). DOI:10.1007/s00253-012-4421-5 · 3.81 Impact Factor
  • Hal Alper, Wilfried Weber
    Current Opinion in Biotechnology 07/2012; 23(5):641-3. DOI:10.1016/j.copbio.2012.07.001 · 8.04 Impact Factor
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    ABSTRACT: Cre recombinase is a commonly-used genome editing tool suitable for site-specific integrations in mammalian genomes; however, the efficiency of transgenic swapping events compared to excision remains limited. Here we sought to identify important parameters and limiting factors that influence swapping propensity in this system, especially when using one wild-type loxP site. To modulate and increase the occurrence of swapping events, we identified two novel parameters. First, we identified the loxFAS-loxP pairing, a sequence never before used in mammalian systems, as the best choice for increasing swapping events in human cell lines. Second, for the first time we implicate the importance of delayed introduction of Cre DNA for optimal swapping efficiency. This same modification could potentially be of use to other systems catalyzing trimolecular reactions such as ΦC31 integrase and FLP recombinase where we hypothesize that transport of the exchange cassette is likewise initially rate limiting. The total number of recombination events, but not the ratio of swapping to excision, was found to be influenced by the quantity of Cre DNA transfected. Through this study, we were able to obtain Cre-mediated swapping frequencies of 8-12% without antibiotic enrichment, which represents nearly an order of magnitude increase over prior reports in the literature.
    Biotechnology Journal 07/2012; 7(7):898-908. DOI:10.1002/biot.201200034 · 3.71 Impact Factor

Publication Stats

3k Citations
455.12 Total Impact Points

Institutions

  • 2009–2015
    • University of Texas at Austin
      • Department of Chemical Engineering
      Austin, Texas, United States
  • 2012
    • Korea Institute of Science and Technology
      • Clean Energy Research Center
      Sŏul, Seoul, South Korea
  • 2005–2009
    • Massachusetts Institute of Technology
      • Department of Chemical Engineering
      Cambridge, Massachusetts, United States
  • 2006
    • Technische Universität Berlin
      Berlín, Berlin, Germany