[Show abstract][Hide abstract] ABSTRACT: The draft genome sequence of the oleaginous yeast Yarrowia lipolytica stain PO1f, a commonly used metabolic engineering host, is presented here. The approximately 20.3-Mb genome sequence of PO1f
will greatly facilitate research efforts in metabolic engineering of Yarrowia lipolytica for value-added chemical production.
[Show abstract][Hide abstract] ABSTRACT: Saccharomyces cerevisiae can serve as a key production platform for biofuels, nutraceuticals, industrial compounds, and therapeutic proteins. Over the recent years, synthetic biology tools and libraries have expanded in yeast to provide newfound control over regulation and synthetic circuits. This review provides an update on the status of the synthetic biology toolbox in yeast for use as a cell factory. Specifically, we discuss the impact of plasmid selection and composition, promoter, terminator, transcription factor, and aptamer selection. In doing so, we highlight documented interactions between these components, current states of development, and applications that demonstrate the utility of these parts with a particular focus on synthetic gene expression control.This article is protected by copyright. All rights reserved.
Preview · Article · Jul 2014 · FEMS Yeast Research
[Show abstract][Hide abstract] ABSTRACT: Model-based design of biological parts is a critical goal of synthetic biology, especially for eukaryotes. Here we demonstrate that nucleosome architecture can have a role in defining yeast promoter activity and utilize a computationally-guided approach that can enable both the redesign of endogenous promoter sequences and the de novo design of synthetic promoters. Initially, we use our approach to reprogram native promoters for increased expression and evaluate their performance in various genetic contexts. Increases in expression ranging from 1.5- to nearly 6-fold in a plasmid-based system and up to 16-fold in a genomic context were obtained. Next, we demonstrate that, in a single design cycle, it is possible to create functional, purely synthetic yeast promoters that achieve substantial expression levels (within the top sixth percentile among native yeast promoters). In doing so, this work establishes a unique DNA-level specification of promoter activity and demonstrates predictive design of synthetic parts.
Preview · Article · May 2014 · Nature Communications
[Show abstract][Hide abstract] ABSTRACT: Heterologous gene expression is an important tool for synthetic biology that enables metabolic engineering and the production of non-natural biologics in a variety of host organisms. The translational efficiency of heterologous genes can often be improved by optimizing synonymous codon usage to better match the host organism. However, traditional approaches for optimization neglect to take into account many factors known to influence synonymous codon distributions.
Here we define an alternative approach for codon optimization that utilizes systems level information and codon context for the condition under which heterologous genes are being expressed. Furthermore, we utilize a probabilistic algorithm to generate multiple variants of a given gene. We demonstrate improved translational efficiency using this condition-specific codon optimization approach with two heterologous genes, the fluorescent protein-encoding eGFP and the catechol 1,2-dioxygenase gene CatA, expressed in S. cerevisiae. For the latter case, optimization for stationary phase production resulted in nearly 2.9-fold improvements over commercial gene optimization algorithms.
Codon optimization is now often a standard tool for protein expression, and while a variety of tools and approaches have been developed, they do not guarantee improved performance for all hosts of applications. Here, we suggest an alternative method for condition-specific codon optimization and demonstrate its utility in Saccharomyces cerevisiae as a proof of concept. However, this technique should be applicable to any organism for which gene expression data can be generated and is thus of potential interest for a variety of applications in metabolic and cellular engineering.
Preview · Article · Mar 2014 · BMC Systems Biology
[Show abstract][Hide abstract] ABSTRACT: Economic feasibility of biosynthetic fuel and chemical production hinges upon harnessing metabolism to achieve high titre and yield. Here we report a thorough genotypic and phenotypic optimization of an oleaginous organism to create a strain with significant lipogenesis capability. Specifically, we rewire Yarrowia lipolytica's native metabolism for superior de novo lipogenesis by coupling combinatorial multiplexing of lipogenesis targets with phenotypic induction. We further complete direct conversion of lipid content into biodiesel. Tri-level metabolic control results in saturated cells containing upwards of 90% lipid content and titres exceeding 25 g l(-1) lipids, which represents a 60-fold improvement over parental strain and conditions. Through this rewiring effort, we advance fundamental understanding of lipogenesis, demonstrate non-canonical environmental and intracellular stimuli and uncouple lipogenesis from nitrogen starvation. The high titres and carbon-source independent nature of this lipogenesis in Y. lipolytica highlight the potential of this organism as a platform for efficient oleochemical production.
No preview · Article · Jan 2014 · Nature Communications
[Show abstract][Hide abstract] ABSTRACT: Utilization of exogenous sugars found in lignocellulosic biomass hydrolysates, such as xylose, must be improved before yeast can serve as an efficient biofuel and biochemical production platform. In particular, the first step in this process, the molecular transport of xylose into the cell, can serve as a significant flux bottleneck and is highly inhibited by other sugars. Here we demonstrate that sugar transport preference and kinetics can be rewired through the programming of a sequence motif of the general form G-G/F-XXX-G found in the first transmembrane span. By evaluating 46 different heterologously expressed transporters, we find that this motif is conserved among functional transporters and highly enriched in transporters that confer growth on xylose. Through saturation mutagenesis and subsequent rational mutagenesis, four transporter mutants unable to confer growth on glucose but able to sustain growth on xylose were engineered. Specifically, Candida intermedia gxs1 Phe(38)Ile(39)Met(40), Scheffersomyces stipitis rgt2 Phe(38) and Met(40), and Saccharomyces cerevisiae hxt7 Ile(39)Met(40)Met(340) all exhibit this phenotype. In these cases, primary hexose transporters were rewired into xylose transporters. These xylose transporters nevertheless remained inhibited by glucose. Furthermore, in the course of identifying this motif, novel wild-type transporters with superior monosaccharide growth profiles were discovered, namely S. stipitis RGT2 and Debaryomyces hansenii 2D01474. These findings build toward the engineering of efficient pentose utilization in yeast and provide a blueprint for reprogramming transporter properties.
Preview · Article · Dec 2013 · Proceedings of the National Academy of Sciences
[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.
No preview · Article · Dec 2013 · ACS Synthetic Biology
[Show abstract][Hide abstract] 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.
No preview · Article · Dec 2013 · Biotechnology Journal
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
No preview · Article · Nov 2013 · Chemical Engineering Science
[Show abstract][Hide abstract] 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.
No preview · Article · Jul 2013 · Metabolic Engineering
[Show abstract][Hide abstract] 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.
No preview · Article · Jul 2013 · Biotechnology Journal
[Show abstract][Hide abstract] 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.
Preview · Article · May 2013 · Biotechnology Journal
[Show abstract][Hide abstract] ABSTRACT: Systems Metabolic Enginering, edited by Christoph Wittmann and Sang Yup Lee “sits at the crossroads of being an introductory book providing an overview of the field and a handy desk-reference for state-of-the-art case studies for the expert metabolic engineer”. Read this book review by Hal Alper.
Preview · Article · May 2013 · Biotechnology Journal
[Show abstract][Hide abstract] 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.
No preview · Article · Apr 2013 · Journal of Biotechnology
[Show abstract][Hide abstract] 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.
No preview · Article · Mar 2013 · Current Opinion in Biotechnology