Pamela Peralta-Yahya

Identification and microbial production of a terpene-based advanced biofuel

Rising petroleum costs, trade imbalances and environmental concerns have stimulated efforts to advance the microbial production of fuels from lignocellulosic biomass. Here we identify a novel biosynthetic alternative to D2 diesel fuel, bisabolane, and engineer microbial platforms for the production of its immediate precursor, bisabolene. First, we identify bisabolane as an alternative to D2 diesel by measuring the fuel properties of chemically hydrogenated commercial bisabolene. Then, via a combination of enzyme screening and metabolic engineering, we obtain a more than tenfold increase in bisabolene titers in Escherichia coli to >900 mg l−1. We produce bisabolene in Saccharomyces cerevisiae (>900 mg l−1), a widely used platform for the production of ethanol. Finally, we chemically hydrogenate biosynthetic bisabolene into bisabolane. This work presents a framework for the identification of novel terpene-based advanced biofuels and the rapid engineering of microbial farnesyl diphosphate-overproducing platforms for the production of biofuels.

Advanced biofuel production in microbes.

The cost‐effective production of biofuels from renewable materials will begin to address energy security and climate change concerns. Ethanol, naturally produced by microorganisms, is currently the major biofuel in the transportation sector. However, its low energy content and incompatibility with existing fuel distribution and storage infrastructure limits its economic use in the future. Advanced biofuels, such as long chain alcohols and isoprenoid‐ and fatty acid‐based biofuels, have physical properties that more closely resemble petroleum‐derived fuels, and as such are an attractive alternative for the future supplementation or replacement of petroleum‐derived fuels. Here, we review recent developments in the engineering of metabolic pathways for the production of known and potential advanced biofuels by microorganisms. We concentrate on the metabolic engineering of genetically tractable organisms such as Escherichia coli and Saccharomyces cerevisiae for the production of these advanced biofuels.

Synthesis of three advanced biofuels from ionic liquid-pretreated switchgrass using engineered Escherichia coli.

One approach to reducing the costs of advanced biofuel production from cellulosic biomass is to engineer a single microorganism to both digest plant biomass and produce hydrocarbons that have the properties of petrochemical fuels. Such an organism would require pathways for hydrocarbon production and the capacity to secrete sufficient enzymes to efficiently hydrolyze cellulose and hemicellulose. To demonstrate how one might engineer and coordinate all of the necessary components for a biomass-degrading, hydrocarbon-producing microorganism, we engineered a microorganism naïve to both processes, Escherichia coli, to grow using both the cellulose and hemicellulose fractions of several types of plant biomass pretreated with ionic liquids. Our engineered strains express cellulase, xylanase, beta-glucosidase, and xylobiosidase enzymes under control of native E. coli promoters selected to optimize growth on model cellulosic and hemicellulosic substrates. Furthermore, our strains grow using either the cellulose or hemicellulose components of ionic liquid-pretreated biomass or on both components when combined as a coculture. Both cellulolytic and hemicellulolytic strains were further engineered with three biofuel synthesis pathways to demonstrate the production of fuel substitutes or precursors suitable for gasoline, diesel, and jet engines directly from ionic liquid-treated switchgrass without externally supplied hydrolase enzymes. This demonstration represents a major advance toward realizing a consolidated bioprocess. With improvements in both biofuel synthesis pathways and biomass digestion capabilities, our approach could provide an economical route to production of advanced biofuels.

Redesigning photosynthesis to sustainably meet global food and bioenergy demand

The world’s crop productivity is stagnating whereas population growth, rising affluence, and mandates for biofuels put increasing demands on agriculture. Meanwhile, demand for increasing cropland competes with equally crucial global sustainability and environmental protection needs. Addressing this looming agricultural crisis will be one of our greatest scientific challenges in the coming decades, and success will require substantial improvements at many levels. We assert that increasing the efficiency and productivity of photosynthesis in crop plants will be essential if this grand challenge is to be met. Here, we explore an array of prospective redesigns of plant systems at various scales, all aimed at increasing crop yields through improved photosynthetic efficiency and performance. Prospects range from straightforward alterations, already supported by preliminary evidence of feasibility, to substantial redesigns that are currently only conceptual, but that may be enabled by new developments in synthetic biology. Although some proposed redesigns are certain to face obstacles that will require alternate routes, the efforts should lead to new discoveries and technical advances with important impacts on the global problem of crop productivity and bioenergy production.

Microbial synthesis of pinene.

The volumetric heating values of todays biofuels are too low to power energy-intensive aircraft, rockets, and missiles. Recently, pinene dimers were shown to have a volumetric heating value similar to that of the tactical fuel JP-10. To provide a sustainable source of pinene, we engineered Escherichia coli for pinene production. We combinatorially expressed three pinene synthases (PS) and three geranyl diphosphate synthases (GPPS), with the best combination achieving ~28 mg/L of pinene. We speculated that pinene toxicity was limiting production; however, toxicity should not be limiting at current titers. Because GPPS is inhibited by geranyl diphosphate (GPP) and to increase flux through the pathway, we combinatorially constructed GPPS-PS protein fusions. The Abies grandis GPPS-PS fusion produced 32 mg/L of pinene, a 6-fold improvement over the highest titer previously reported in engineered E. coli. Finally, we investigated the pinene isomer ratio of our pinene-producing microbe and discovered that the isomer profile is determined not only by the identity of the PS used but also by the identity of the GPPS with which the PS is paired. We demonstrated that the GPP concentration available to PS for cyclization alters the pinene isomer ratio.

High-throughput selection for cellulase catalysts using chemical complementation.

Efficient enzymatic hydrolysis of lignocellulosic material remains one of the major bottlenecks to cost-effective conversion of biomass to ethanol. Improvement of glycosylhydrolases, however, is limited by existing medium-throughput screening technologies. Here, we report the first high-throughput selection for cellulase catalysts. This selection was developed by adapting chemical complementation to provide a growth assay for bond cleavage reactions. First, a URA3 counter selection was adapted to link chemical dimerizer activated gene transcription to cell death. Next, the URA3 counter selection was shown to detect cellulase activity based on cleavage of a tetrasaccharide chemical dimerizer substrate and decrease in expression of the toxic URA3 reporter. Finally, the utility of the cellulase selection was assessed by isolating cellulases with improved activity from a cellulase library created by family DNA shuffling. This application provides further evidence that chemical complementation can be readily adapted to detect different enzymatic activities for important chemical transformations for which no natural selection exists. Because of the large number of enzyme variants that selections can now test as compared to existing medium-throughput screens for cellulases, this assay has the potential to impact the discovery of improved cellulases and other glycosylhydrolases for biomass conversion from libraries of cellulases created by mutagenesis or obtained from natural biodiversity.

PCRless library mutagenesis via oligonucleotide recombination in yeast

The directed evolution of biomolecules with new functions is largely performed in vitro, with PCR mutagenesis followed by high‐throughput assays for desired activities. As synthetic biology creates impetus for generating biomolecules that function in living cells, new technologies are needed for performing mutagenesis and selection for directed evolution in vivo. Homologous recombination, routinely exploited for targeted gene alteration, is an attractive tool for in vivo library mutagenesis, yet surprisingly is not routinely used for this purpose. Here, we report the design and characterization of a yeast‐based system for library mutagenesis of protein loops via oligonucleotide recombination. In this system, a linear vector is co‐transformed with single‐stranded mutagenic oligonucleotides. Using repair of nonsense codons engineered in three different active‐site loops in the selectable marker TRP1 as a model system, we first optimized the recombination efficiency. Single‐loop recombination was highly efficient, averaging 5%, or 4.0 × 105 recombinants. Multiple loops could be simultaneously mutagenized, although the efficiencies dropped to 0.2%, or 6.0 × 103 recombinants, for two loops and 0.01% efficiency, or 1.5 × 102 recombinants, for three loops. Finally, the utility of this system for directed evolution was tested explicitly by selecting functional variants from a mock library of 1:106 wild‐type:nonsense codons. Sequencing showed that oligonucleotide recombination readily covered this large library, mutating not only the target codon but also encoded silent mutations on either side of the library cassette. Together these results establish oligonucleotide recombination as a simple and powerful library mutagenesis technique and advance efforts to engineer the cell for fully in vivo directed evolution.

Metabolic engineering strategies to bio-adipic acid production

Adipic acid is the most industrially important dicarboxylic acid as it is a key monomer in the synthesis of nylon. Today, adipic acid is obtained via a chemical process that relies on petrochemical precursors and releases large quantities of greenhouse gases. In the last two years, significant progress has been made in engineering microbes for the production of adipic acid and its immediate precursors, muconic acid and glucaric acid. Not only have the microbial substrates expanded beyond glucose and glycerol to include lignin monomers and hemicellulose components, but the number of microbial chassis now goes further than Escherichia coli and Saccharomyces cerevisiae to include microbes proficient in aromatic degradation, cellulose secretion and degradation of multiple carbon sources. Here, we review the metabolic engineering and nascent protein engineering strategies undertaken in each of these chassis to convert different feedstocks to adipic, muconic and glucaric acid. We also highlight near term prospects and challenges for each of the metabolic routes discussed.

Characterization of a new glycosynthase cloned by using chemical complementation.

Despite their fundamental role in biological processes and potential use as therapeutics, it still remains difficult to synthesize carbohydrates. In the past two decades, there has been tremendous progress in the chemical synthesis of complex carbohydrates. However, chemical synthesis is still limited by the need for differentially protected intermediates and reactant-dependent coupling yields and stereocontrol. Enzymes, with their control of both regioand stereochemistry, provide an obvious alternative to traditional small-molecule chemistry for the synthesis of oligosaccharides. 9] Recently, Withers and co-workers demonstrated that retaining glycosidases can be engineered to glycosynthases simply by mutating the nucleophilic Glu residue at the base of the active site to a small hydrophobic residue and using an a-glycosyl fluoride as the donor substrate. This strategy is based on extensive characterization of the mechanism of retaining glycosidases. Mutation of the active-site nucleophile to a small residue both accommodates the glycosyl fluoride donor and inactivates the hydrolytic activity of the enzyme, allowing the reaction to proceed in the reverse direction. This approach was first demonstrated with the Agrobacterium sp. b-glucosidase/galactosidase (Abg). The active-site nucleophile Glu358 was mutated to Ala. This Abg:E358A variant was shown to accept both galactosyl fluoride and glucosyl fluoride as donors to form glycosidic bonds with several monoand disaccharides. This result opened a new route for carbohydrate synthesis, and already several retaining glycosidases have been successfully converted to glycosynthases with this strategy. Directed evolution would offer an obvious route to improve the activity and alter the substrate selectivity of these enzymes, except that there is no intrinsic way to screen or select for glycosynthase activity. Mayer et al. developed a coupled enzyme assay using an endo-cellullase that can be used to screen for glycosynthase mutants with improved activity. This screen, however, can only be used for glycosynthases that synthesize products that are substrates of the endo-cellulase. Screens only allow relatively small libraries, about 10 variants, to be assayed. Thus, our laboratory applied “chemical complementation”, a general, high-throughput assay for enzyme catalysis of bond formation and cleavage reactions, to the directed evolution of glycosynthases. In chemical complementation, glycosynthase activity is linked to reporter gene transcription and hence cell survival through covalent coupling of a methoACHTUNGTRENNUNGtrexACHTUNGTRENNUNGate ACHTUNGTRENNUNG(Mtx)-disaccharide-fluoride donor and a dexa ACHTUNGTRENNUNGmethaACHTUNGTRENNUNGsoneACHTUNGTRENNUNG(Dex)-disaccharide acceptor, such that Dex-tetrasaccharide-Mtx effectively reconstitutes the transcriptional activator and increases transcription of a downstream reporter gene. Use of the reporter gene LEU2 allows for a growth selection in the ACHTUNGTRENNUNGabsence of leucine (Figure 1). Using the LEU2 selection, we previously demonstrated that chemical complementation can be used to read-out glycosynthase activity, and a Humicola insolens Cel7B:E197S variant with a fivefold increase in glycosynthase activity was selected from a Glu197 saturation library. Having established chemical complementation as a selection for glycosynthase activity, we then sought a glycosynthase that would provide a robust scaffold for the directed evolution of glycosynthase variants with altered substrate specificities. In our initial publication, the H. insolens CeL7B:E197A glycosynthase was employed because it was the only reported endoglycosynthase at that time. However, this enzyme has poor expression properties and does not present an obvious scaffold for protein engineering. Thus, we sought to develop an endo-glycosynthase derived from a family 5 glycosidase. The in vitro activities and substrate specificities of many family 5 glycosidases have been extensively characterized, and several of these enzymes have shown good expression in E. coli. Moreover, family 5 retaining glycosidases are monomeric triose-phosphate isomerase (TIM) barrel enzymes, an appealing scaffold for enzyme engineering given that TIM barrels arguably are a “privileged” scaffold for enzyme catalysis of diverse chemical transformations. In this paper, we report the cloning and characterization of a new glycosynthase from a family 5 glycosidase using a chemical complementation LEU2 enrichment assay. Given that not all retaining glycosidases provide efficient glycosynthases upon mutation of the active-site nucleophile, we adapted our LEU2 selection as an enrichment assay to clone the new glycosynthase. Using this assay, the family 5 TIM-barrel glycosynthase was cloned by screening the activesite E:G and E:S variants of known family 5 glycosidases. Specifically, three family 5 glycosidases, Clostridium cellulolyticum Cel5A, Clostridium thermocellum CelG and Clostridium cellulolyticum Cel5N, were tested. Among these genes, Cel5A and CelG have been overexpressed and purified from E. coli, while the catalytic domains of CelG and Cel5N share high sequence identity. The high-resolution structure of the Cel5A catalytic domain shows a classic TIM barrel fold, with Glu170 as the [a] Dr. H. Tao, P. Peralta-Yahya, Dr. J. Decatur, Prof. V. W. Cornish Department of Chemistry, Columbia University New York, NY 10027 (USA) Fax: (+1)212-932-1289 E-mail : vc114@columbia.edu [b] Dr. H. Tao Current address: Genomics Institute of the Novartis Research Foundation San Diego, CA 92121 (USA) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author: Experimental procedures and NMR data.

Accelerating the semisynthesis of alkaloid-based drugs through metabolic engineering

Alkaloid-derived pharmaceuticals are commonly semisynthesized from plant-extracted starting materials, which often limits their availability and final price. Recent advances in synthetic biology have enabled the introduction of complete plant pathways into microbes for the production of plant alkaloids. Microbial production of modified alkaloids has the potential to accelerate the semisynthesis of alkaloid-derived drugs by providing advanced intermediates that are structurally closer to the final pharmaceuticals and could be used as advanced intermediates for the synthesis of novel drugs. Here, we analyze the scientific and engineering challenges that must be overcome to generate microbes to produce modified plant alkaloids that can provide more suitable intermediates to US Food and Drug Administration-approved pharmaceuticals. We highlight modified alkaloids that currently could be produced by leveraging existing alkaloid microbial platforms with minor variations to accelerate the semisynthesis of seven pharmaceuticals on the market.