Hal S. Alper

Engineering yeast transcription machinery for improved ethanol tolerance and production

Global transcription machinery engineering (gTME) is an approach for reprogramming gene transcription to elicit cellular phenotypes important for technological applications. Here we show the application of gTME to Saccharomyces cerevisiae for improved glucose/ethanol tolerance, a key trait for many biofuels programs. Mutagenesis of the transcription factor Spt15p and selection led to dominant mutations that conferred increased tolerance and more efficient glucose conversion to ethanol. The desired phenotype results from the combined effect of three separate mutations in the SPT15 gene [serine substituted for phenylalanine (Phe177Ser) and, similarly, Tyr195His, and Lys218Arg]. Thus, gTME can provide a route to complex phenotypes that are not readily accessible by traditional methods.

Construction of lycopene-overproducing E. coli strains by combining systematic and combinatorial gene knockout targets.

Identification of genes that affect the product accumulation phenotype of recombinant strains is an important problem in industrial strain construction and a central tenet of metabolic engineering. We have used systematic (model-based) and combinatorial (transposon-based) methods to identify gene knockout targets that increase lycopene biosynthesis in strains of Escherichia coli. We show that these two search strategies yield two distinct gene sets, which affect product synthesis either through an increase in precursor availability or through (largely unknown) kinetic or regulatory mechanisms, respectively. Exhaustive exploration of all possible combinations of the above gene sets yielded a unique set of 64 knockout strains spanning the metabolic landscape of systematic and combinatorial gene knockout targets. This included a global maximum strain exhibiting an 8.5-fold product increase over recombinant K12 wild type and a twofold increase over the engineered parental strain. These results were further validated in controlled culture conditions.

Engineering for biofuels: exploiting innate microbial capacity or importing biosynthetic potential?

The ideal microorganism for biofuel production will possess high substrate utilization and processing capacities, fast and deregulated pathways for sugar transport, good tolerance to inhibitors and product, and high metabolic fluxes and will produce a single fermentation product. It is unclear whether such an organism will be engineered using a native, isolated strain or a recombinant, model organism as the starting point. The choice between engineering natural function and importing biosynthetic capacity is affected by current progress in metabolic engineering and synthetic biology. This Review highlights some of the factors influencing the above decision, in light of current advances.

Harnessing Yarrowia lipolytica lipogenesis to create a platform for lipid and biofuel production

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 lipolyticas 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.

Engineering of Promoter Replacement Cassettes for Fine-Tuning of Gene Expression in Saccharomyces cerevisiae

ABSTRACT The strong overexpression or complete deletion of a gene gives only limited information about its control over a certain phenotype or pathway. Gene function studies based on these methods are therefore incomplete. To effect facile manipulation of gene expression across a full continuum of possible expression levels, we recently created a library of mutant promoters. Here, we provide the detailed characterization of our yeast promoter collection comprising 11 mutants of the strong constitutive Saccharomyces cerevisiae TEF1 promoter. The activities of the mutant promoters range between about 8% and 120% of the activity of the unmutated TEF1 promoter. The differences in reporter gene expression in the 11 mutants were independent of the carbon source used, and real-time PCR confirmed that these differences were due to varying levels of transcription (i.e., caused by varying promoter strengths). In addition to a CEN/ARS plasmid-based promoter collection, we also created promoter replacement cassettes. They enable genomic integration of our mutant promoter collection upstream of any given yeast gene, allowing detailed genotype-phenotype characterizations. To illustrate the utility of the method, the GPD1 promoter of S. cerevisiae was replaced by five TEF1 promoter mutants of different strengths, which allowed analysis of the impact of glycerol 3-phosphate dehydrogenase activity on the glycerol yield.

Tuning Gene Expression in Yarrowia lipolytica by a Hybrid Promoter Approach

ABSTRACT The development of strong and tunable promoter elements is necessary to enable metabolic and pathway engineering applications for any host organism. Here, we have expanded and generalized a hybrid promoter approach to produce libraries of high-expressing, tunable promoters in the nonconventional yeast Yarrowia lipolytica. These synthetic promoters are comprised of two modular components: the enhancer element and the core promoter element. By exploiting this basic promoter architecture, we have overcome native expression limitations and provided a strategy for both increasing the native promoter capacity and producing libraries for tunable gene expression in a cellular system with ill-defined genetic tools. In doing so, this work has created the strongest promoters ever reported for Y. lipolytica. Furthermore, we have characterized these promoters at the single-cell level through the use of a developed fluorescence-based assay as well as at the transcriptional and whole-cell levels. The resulting promoter libraries exhibited a range of more than 400-fold in terms of mRNA levels, and the strongest promoters in this set had 8-fold-higher fluorescence levels than those of typically used endogenous promoters. These results suggest that promoters in Y. lipolytica are enhancer limited and that this limitation can be partially or fully alleviated through the addition of tandem copies of upstream activation sequences (UASs). Finally, this work illustrates that tandem copies of UAS regions can serve as synthetic transcriptional amplifiers that may be generically used to increase the expression levels of promoters.

Exploiting biological complexity for strain improvement through systems biology

Cellular complexity makes it difficult to build a complete understanding of cellular function but also offers innumerable possibilities for modifying the cellular machinery to achieve a specific purpose. The exploitation of cellular complexity for strain improvement has been a challenging goal for applied biological research because it requires the coordinated understanding of multiple cellular processes. It is therefore pursued most efficiently in the framework of systems biology. Progress in strain improvement will depend not only on advances in technologies for high-throughput measurements but, more importantly, on the development of theoretical methods that increase the information content of these measurements and, as such, facilitate the elucidation of mechanisms and the identification of genetic targets for modification.

Metabolic engineering of muconic acid production in Saccharomyces cerevisiae

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.

Controlling promoter strength and regulation in Saccharomyces cerevisiae using synthetic hybrid promoters.

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, PGPD, 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.

Improvement of Xylose Uptake and Ethanol Production in Recombinant Saccharomyces cerevisiae through an Inverse Metabolic Engineering Approach

ABSTRACT We used an inverse metabolic engineering approach to identify gene targets for improved xylose assimilation in recombinant Saccharomyces cerevisiae. Specifically, we created a genomic fragment library from Pichia stipitis and introduced it into recombinant S. cerevisiae expressing XYL1 and XYL2. Through serial subculturing enrichment of the transformant library, 16 transformants were identified and confirmed to have a higher growth rate on xylose. Sequencing of the 16 plasmids isolated from these transformants revealed that the majority of the inserts (10 of 16) contained the XYL3 gene, thus confirming the previous finding that XYL3 is the consensus target for increasing xylose assimilation. Following a sequential search for gene targets, we repeated the complementation enrichment process in a XYL1 XYL2 XYL3 background and identified 15 fast-growing transformants, all of which harbored the same plasmid. This plasmid contained an open reading frame (ORF) designated PsTAL1 based on a high level of homology with S. cerevisiae TAL1. To further investigate whether the newly identified PsTAL1 ORF is responsible for the enhanced-growth phenotype, we constructed an expression cassette containing the PsTAL1 ORF under the control of a constitutive promoter and transformed it into an S. cerevisiae recombinant expressing XYL1, XYL2, and XYL3. The resulting recombinant strain exhibited a 100% increase in the growth rate and a 70% increase in ethanol production (0.033 versus 0.019 g ethanol/g cells · h) on xylose compared to the parental strain. Interestingly, overexpression of PsTAL1 did not cause growth inhibition when cells were grown on glucose, unlike overexpression of the ScTAL1 gene. These results suggest that PsTAL1 is a better gene target for engineering of the pentose phosphate pathway in recombinant S. cerevisiae.