An extracellular glucose sensor for substrate-dependent secretion and display of cellulose-degrading enzymes.

The efficient hydrolysis of lignocellulosic biomass into fermentable sugars is key for viable economic production of biofuels and biorenewable chemicals from second-generation feedstocks. Consolidated bioprocessing (CBP) combines lignocellulose saccharification and chemical production in a single step. To avoid wasting valuable resources during CBP, the selective secretion of enzymes (independent or attached to the surface) based on the carbon source available is advantageous. To enable enzyme expression and secretion based on extracellular glucose levels, we implemented a G-protein-coupled receptor (GPCR)-based extracellular glucose sensor; this allows the secretion and display of cellulases in the presence of the cellulosic fraction of lignocellulose by leveraging cellobiose-dependent signal amplification. We focused on the glucose-responsiveness of the HXT1 promoter and engineered PHXT1 by changing its core to that of the strong promoter PTHD3 , increasing extracellular enzyme activity by 81%. We then demonstrated glucose-mediated expression and cell-surface display of the ß-glucosidase BglI on the surface of Saccharomyces cerevisiae. The display system was further optimized by re-directing fatty acid pools from lipid droplet synthesis toward formation of membrane precursors via knock-out of PAH1. This resulted in an up to 4.2-fold improvement with respect to the baseline strain. Finally, we observed cellobiose-dependent signal amplification of the system with an increase in enzymatic activity of up to 3.1-fold when cellobiose was added.

Assembly of Metabolons in Yeast Using Cas6-Mediated RNA Scaffolding.

Cells often localize pathway enzymes in close proximity to reduce substrate loss via diffusion and to ensure that carbon flux is directed toward the desired product. To emulate this strategy for the biosynthesis of heterologous products in yeast, we have taken advantage of the highly specific Cas6-RNA interaction and the predictability of RNA hybridizations to demonstrate Cas6-mediated RNA-guided protein assembly within the yeast cytosol. The feasibility of this synthetic scaffolding technique for protein localization was first demonstrated using a split luciferase reporter system with each part fused to a different Cas6 protein. In Saccharomyces cerevisiae, the luminescence signal increased 3.6- to 20-fold when the functional RNA scaffold was also expressed. Expression of a trigger RNA, designed to prevent the formation of a functional scaffold by strand displacement, decreased the luminescence signal by nearly 2.3-fold. Temporal control was also possible, with induction of scaffold expression resulting in an up to 11.6-fold increase in luminescence after 23 h. Cas6-mediated assembly was applied to create a two-enzyme metabolon to redirect a branch of the violacein biosynthesis pathway. Localizing VioC and VioE together increased the amount of deoxyviolacein (desired) relative to prodeoxyviolacein (undesired) by 2-fold. To assess the generality of this colocalization method in other yeast systems, the split luciferase reporter system was evaluated in Kluyveromyces marxianus; RNA scaffold expression resulted in an increase in the luminescence signal of up to 1.9-fold. The simplicity and flexibility of the design suggest that this strategy can be used to create metabolons in a wide range of recombinant hosts of interest.

Enhanced production of acetyl-CoA-based products via peroxisomal surface display in Saccharomyces cerevisiae.

Colocalization of enzymes is a proven approach to increase pathway flux and the synthesis of nonnative products. Here, we develop a method for enzyme colocalization using the yeast peroxisomal membrane as an anchor point. Pathway enzymes were fused to the native Pex15 anchoring motif to enable display on the surface of the peroxisome facing the cytosol. The peroxisome is the sole location of ß-oxidation in Saccharomyces cerevisiae, and acetyl-CoA is a by-product that is exported in the form of acetyl-carnitine. To access this untapped acetyl-CoA pool, we surface-anchored the native peroxisomal/mitochondrial enzyme Cat2 to convert acetyl-carnitine to acetyl-CoA directly upon export across the peroxisomal membrane; this increased acetyl-CoA levels 3.7-fold. Subsequent surface attachment of three pathway enzymes - Cat2, a high stability Acc1 (for conversion of acetyl-CoA to malonyl-CoA), and the type III PKS 2-pyrone synthase - demonstrated the success of peroxisomal surface display for both enzyme colocalization and access to acetyl-CoA from exported acetyl-carnitine. Synthesis of the polyketide triacetic acid lactone increased by 21% over cytosolic expression at low gene copy number, and an additional 11-fold (to 766 mg/L) after further optimization. Finally, we explored increasing peroxisomal membrane area through overexpression of the peroxisomal biogenesis protein Pex11. Our findings establish peroxisomal surface display as an efficient strategy for enzyme colocalization and for accessing the peroxisomal acetyl-CoA pool to increase synthesis of acetyl-CoA-based products.

Successful Enzyme Colocalization Strategies in Yeast for Increased Synthesis of Non-native Products.

Yeast cell factories, particularly Saccharomyces cerevisiae, have proven valuable for the synthesis of non-native compounds, ranging from commodity chemicals to complex natural products. One significant challenge has been ensuring sufficient carbon flux to the desired product. Traditionally, this has been addressed by strategies involving "pushing" and "pulling" the carbon flux toward the products by overexpression while "blocking" competing pathways via downregulation or gene deletion. Colocalization of enzymes is an alternate and complementary metabolic engineering strategy to control flux and increase pathway efficiency toward the synthesis of non-native products. Spatially controlling the pathway enzymes of interest, and thus positioning them in close proximity, increases the likelihood of reaction along that pathway. This mini-review focuses on the recent developments and applications of colocalization strategies, including enzyme scaffolding, construction of synthetic organelles, and organelle targeting, in both S. cerevisiae and non-conventional yeast hosts. Challenges with these techniques and future directions will also be discussed.

Developing a broad-range promoter set for metabolic engineering in the thermotolerant yeast Kluyveromyces marxianus.

Kluyveromyces marxianus is an emerging host for metabolic engineering. This thermotolerant yeast is the fastest growing eukaryote, has high flux through the TCA cycle, and can metabolize a broad range of C5, C6, and C12 carbon sources. In comparison to the common host Saccharomyces cerevisiae, this non-conventional yeast suffers from a lack of metabolic engineering tools to control gene expression over a wide transcriptional range. To address this issue, we designed a library of 25 native-derived promoters from K. marxanius CBS6556 that spans 87-fold transcriptional strength under glucose metabolism. Six promoters from the library were further characterized in both glucose and xylose as well as across various temperatures from 30 to 45 â€‹°C. The temperature study revealed that in most cases EGFP expression decreased with elevating temperature; however, two promoters, P SSA3 and P ADH1 , increased expression above 40 â€‹°C in both xylose and glucose. The six-promoter set was also validated in xylose for triacetic acid lactone (TAL) production. By controlling the expression level of heterologous 2-pyrone synthase (2-PS), the specific TAL titer increased over 8-fold at 37 â€‹°C. Cultures at 41 â€‹°C exhibited a similar TAL biosynthesis capability, while at 30 â€‹°C TAL levels were lower. Taken together, these results advance the metabolic engineering tool set in K. marxianus and further develop this new host for chemical biosynthesis.

Engineering the early secretory pathway for increased protein secretion in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is a valuable host for the production of heterologous proteins with a wide array of applications, ranging from cellulose saccharification enzymes to biopharmaceuticals. Efficient protein secretion may be critical for economic viability; however previous efforts have shown limited improvements that are often protein-specific. By enhancing transit through the early secretory pathway, we have successfully improved extracellular levels of three different proteins from variety of origins: a bacterial endoglucanase (CelA), a fungal ß-glucosidase (BglI) and a single-chain antibody fragment (4-4-20 scFv). Efficient co-translational translocation into the endoplasmic reticulum (ER) was achieved via secretion peptide engineering and the novel use of a 3'-untranslated region, improving extracellular activity or fluorescence 2.2-5.4-fold. We further optimized the pathway using a variety of new strategies including: i) increasing secretory pathway capacity by expanding the ER, ii) limiting ER-associated degradation, and iii) enhancing exit from the ER. By addressing these additional ER processing steps, extracellular activity/fluorescence increased by 3.5-7.1-fold for the three diverse proteins. The optimal combination of pathway interventions varied, and the highest overall increases ranged from 5.8 to 11-fold. These successful strategies should prove effective for improving the secretion of a wide range of heterologous proteins.

Synthesis of polyketides from low cost substrates by the thermotolerant yeast Kluyveromyces marxianus.

Kluyveromyces marxianus is a promising nonconventional yeast for biobased chemical production due to its rapid growth rate, high TCA cycle flux, and tolerance to low pH and high temperature. Unlike Saccharomyces cerevisiae, K. marxianus grows on low-cost substrates to cell densities that equal or surpass densities in glucose, which can be beneficial for utilization of lignocellulosic biomass (xylose), biofuel production waste (glycerol), and whey (lactose). We have evaluated K. marxianus for the synthesis of polyketides, using triacetic acid lactone (TAL) as the product. The 2-pyrone synthase (2-PS) was expressed on a CEN/ARS plasmid in three different strains, and the effects of temperature, carbon source, and cultivation strategy on TAL levels were determined. The highest titer was obtained in defined 1% xylose medium at 37°C, with substantial titers at 41 and 43°C. The introduction of a high-stability 2-PS mutant and a promoter substitution increased titer four-fold. 2-PS expression from a multi-copy pKD1-based plasmid improved TAL titers a further five-fold. Combining the best plasmid, promoter, and strain resulted in a TAL titer of 1.24 g/L and a yield of 0.0295 mol TAL/mol carbon for this otherwise unengineered strain in 3 ml tube culture. This is an excellent titer and yield (on xylose) before metabolic engineering or fed-batch culture relative to other hosts (on glucose), and demonstrates the promise of this rapidly growing and thermotolerant yeast species for polyketide production.

Tailoring Collagen to Engineer the Cellular Microenvironment.

Collagen is the most abundant protein in the extracellular matrix (ECM), and it can direct the behavior of the neighboring cells. By customizing properties of collagen, it is possible to control the cells that interact with it. Utilizing a bottom-up strategy, modular gene fragments are assembled and recombinantly processed to create collagen-mimetic variants that modulate proteolytic degradation, cell adhesion, and mechanical characteristics. The removal of the native MMP cleavage site results in MMP-1 resistant collagen. By introducing additional MMP-susceptible sequences, the degradation characteristics of collagen molecules are modified. Additional non-native functionality is also introduced into the collagen, including the IKVAV sequence, which has been implicated in neurite outgrowth. This mutation, which disrupts the Gly-X-Y tripeptide repeat of collagen, does not prevent the formation of triple-helical collagen. Non-native cysteines and the integrin binding sequence GFOGER are combined in the collagen, and encapsulation of normal human lung fibroblasts within collagen hydrogels are tested. Cells remain spherical, when encapsulated within hydrogels of collagen variants in which the native integrin binding sites are removed, but cell adhesion is restored with the introduction of non-native GFOGER binding sequences. This modular collagen system allows for the combination of multiple functionalities, and it enables the production of biomimetic scaffolds with customizable characteristics to modulate cellular microenvironments.

A coupled in vitro/in vivo approach for engineering a heterologous type III PKS to enhance polyketide biosynthesis in Saccharomyces cerevisiae.

Polyketides are attractive compounds for uses ranging from biorenewable chemical precursors to high-value therapeutics. In many cases, synthesis in a heterologous host is required to produce these compounds in industrially relevant quantities. The type III polyketide synthase 2-pyrone synthase (2-PS) from Gerbera hybrida was used for the production of triacetic acid lactone (TAL) in Saccharomyces cerevisiae. Initial in vitro characterization of 2-PS led to the identification of active site variants with improved kinetic properties relative to wildtype. Further in vivo evaluation in S. cerevisiae suggested certain 2-PS mutations altered enzyme stability during fermentation. In vivo experiments also revealed beneficial cysteine to serine mutations that were not initially explored due to their distance from the active site of 2-PS, leading to the design of additional 2-PS enzymes. While these variants showed varying catalytic efficiencies in vitro, they exhibited up to 2.5-fold increases in TAL production when expressed in S. cerevisiae. Coupling of the 2-PS variant [C35S,C372S] to an engineered S. cerevisiae strain led to over 10 g/L TAL at 38% of theoretical yield following fed-batch fermentation, the highest reported to date. Our studies demonstrate the success of a coupled in vitro/in vivo approach to engineering enzymes and provide insight on cysteine-rich enzymes and design principles toward their use in non-native microbial hosts.

Recombinant collagen scaffolds as substrates for human neural stem/progenitor cells.

Adhesion to the microenvironment profoundly affects stem cell functions, including proliferation and differentiation, and understanding the interaction of stem cells with the microenvironment is important for controlling their behavior. In this study, we investigated the effects of the integrin binding epitopes GFOGER and IKVAV (natively present in collagen I and laminin, respectively) on human neural stem/progenitor cells (hNSPCs). To test the specificity of these epitopes, GFOGER or IKVAV were placed within the context of recombinant triple-helical collagen III engineered to be devoid of native integrin binding sites. HNSPCs adhered to collagen that presented GFOGER as the sole integrin-binding site, but not to IKVAV-containing collagen. For the GFOGER-containing collagens, antibodies against the ß1 integrin subunit prevented cellular adhesion, antibodies against the α1 subunit reduced cell adhesion, and antibodies against α2 or α3 subunits had no significant effect. These results indicate that hNSPCs primarily interact with GFOGER through the α1ß1 integrin heterodimer. These GFOGER-presenting collagen variants also supported differentiation of hNSPCs into neurons and astrocytes. Our findings show, for the first time, that hNSPCs can bind to the GFOGER sequence, and they provide motivation to develop hydrogels formed from recombinant collagen variants as a cell delivery scaffold. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1363-1372, 2018.

Molecular tools for pathway engineering in Saccharomyces cerevisiae.

Molecular tools for the regulation of protein expression in Saccharomyces cerevisiae have contributed to rapid advances in pathway engineering for this yeast. This review considers new and enhanced additions to this toolbox, focusing on experimental approaches to modulate enzyme synthesis and enzyme fate. Methods for genome engineering, regulation of transcription, post-translational protein localization, and combinatorial screening and sensing in S. cerevisiae are highlighted, and promising new approaches are introduced.

Engineering Saccharomyces cerevisiae for high-level synthesis of fatty acids and derived products.

Fatty acids and fatty acid derivatives are important biorenewable products, as well as precursors for further transformation via chemical catalysis. This minireview focuses on recent advances in increasing the production of fatty acids and derived products in the yeast Saccharomyces cerevisiae. The engineering of upstream pathways to increase levels of the required precursors, fatty acid synthase systems to increase expression and to modify chain length, and downstream pathways to produce free fatty acids, fatty acid ethyl esters, fatty alcohols and alkanes are highlighted, and current challenges are discussed.

Engineering Saccharomyces cerevisiae fatty acid composition for increased tolerance to octanoic acid.

Biorenewable chemicals such as short and medium chain fatty acids enable functional or direct substitution of petroleum-derived building blocks, allowing reduction of anthropogenic greenhouse gases while meeting market needs of high-demand products like aliphatic alcohols and alpha olefins. However, producing these fatty acids in microorganisms can be challenging due to toxicity issues. Octanoic acid (C8) can disrupt the integrity of the cell membrane in yeast, and exogenous supplementation of oleic acid has been shown to help alleviate this. We recently engineered the Saccharomyces cerevisiae enzyme acetyl-CoA carboxylase by replacing serine residue 1157 with alanine to prevent deactivation by phosphorylation. Expression of Acc1S1157A in S. cerevisiae resulted in an increase in total fatty acid production, with the largest increase for oleic acid. In this study, we evaluated the effect of this modified lipid profile on C8 toxicity to the yeast. Expression of Acc1S1157A in S. cerevisiae BY4741 increased the percentage of oleic acid 3.1- and 1.6-fold in the absence and presence of octanoic acid challenge, respectively. Following exposure to 0.9 mM of C8 for 24 h, the engineered yeast had a 10-fold higher cell density relative to the baseline strain. Moreover, overexpressing Acc1S1157A allowed survival at C8 concentrations that were lethal for the baseline strain. This marked reduction of toxicity was shown to be due to higher membrane integrity as an 11-fold decrease in leakage of intracellular magnesium was observed. Due to the increase in oleic acid, this approach has the potential to reduce toxicity of other valuable bioproducts such as shorter chain aliphatic acids and alcohols and other membrane stressors. In an initial screen, increased resistance to n-butanol, 2-propanol, and hexanoic acid was demonstrated with cell densities 3.2-, 1.8-, and 29-fold higher than the baseline strain, respectively. Biotechnol. Bioeng. 2017;114: 1531-1538. © 2017 Wiley Periodicals, Inc.

Engineering cofactor and transport mechanisms in Saccharomyces cerevisiae for enhanced acetyl-CoA and polyketide biosynthesis.

Synthesis of polyketides at high titer and yield is important for producing pharmaceuticals and biorenewable chemical precursors. In this work, we engineered cofactor and transport pathways in Saccharomyces cerevisiae to increase acetyl-CoA, an important polyketide building block. The highly regulated yeast pyruvate dehydrogenase bypass pathway was supplemented by overexpressing a modified Escherichia coli pyruvate dehydrogenase complex (PDHm) that accepts NADP(+) for acetyl-CoA production. After 24h of cultivation, a 3.7-fold increase in NADPH/NADP(+) ratio was observed relative to the base strain, and a 2.2-fold increase relative to introduction of the native E. coli PDH. Both E. coli pathways increased acetyl-CoA levels approximately 2-fold relative to the yeast base strain. Combining PDHm with a ZWF1 deletion to block the major yeast NADPH biosynthesis pathway resulted in a 12-fold NADPH boost and a 2.2-fold increase in acetyl-CoA. At 48h, only this coupled approach showed increased acetyl-CoA levels, 3.0-fold higher than that of the base strain. The impact on polyketide synthesis was evaluated in a S. cerevisiae strain expressing the Gerbera hybrida 2-pyrone synthase (2-PS) for the production of the polyketide triacetic acid lactone (TAL). Titers of TAL relative to the base strain improved only 30% with the native E. coli PDH, but 3.0-fold with PDHm and 4.4-fold with PDHm in the Δzwf1 strain. Carbon was further routed toward TAL production by reducing mitochondrial transport of pyruvate and acetyl-CoA; deletions in genes POR2, MPC2, PDA1, or YAT2 each increased titer 2-3-fold over the base strain (up to 0.8g/L), and in combination to 1.4g/L. Combining the two approaches (NADPH-generating acetyl-CoA pathway plus reduced metabolite flux into the mitochondria) resulted in a final TAL titer of 1.6g/L, a 6.4-fold increase over the non-engineered yeast strain, and 35% of theoretical yield (0.16g/g glucose), the highest reported to date. These biological driving forces present new avenues for improving high-yield production of acetyl-CoA derived compounds.

Disrupted short chain specific ß-oxidation and improved synthase expression increase synthesis of short chain fatty acids in Saccharomyces cerevisiae.

Biologically derived fatty acids have gained tremendous interest as an alternative to petroleum-derived fuels and chemical precursors. We previously demonstrated the synthesis of short chain fatty acids in Saccharomyces cerevisiae by introduction of the Homo sapiens fatty acid synthase (hFAS) with heterologous phosphopantetheine transferases and heterologous thioesterases. In this study, short chain fatty acid production was improved by combining a variety of novel enzyme and metabolic engineering strategies. The use of a H. sapiens-derived thioesterase and phosphopantetheine transferase were evaluated. In addition, strains were engineered to disrupt either the full ß-oxidation (by deleting FAA2, PXA1, and POX1) or short chain-specific ß-oxidation (by deleting FAA2, ANT1, and PEX11) pathways. Prohibiting full ß-oxidation increased hexanoic and octanoic acid levels by 8- and 79-fold relative to the parent strain expressing hFAS. However, by targeting only short chain ß-oxidation, hexanoic and octanoic acid levels increased further to 31- and 140-fold over the parent. In addition, an optimized hFAS gene increased hexanoic, octanoic, decanoic and total short chain fatty acid levels by 2.9-, 2.0-, 2.3-, and 2.2-fold, respectively, relative to the non-optimized counterpart. By combining these unique enzyme and metabolic engineering strategies, octanoic acid was increased more than 181-fold over the parent strain expressing hFAS.

Functional replacement of the Saccharomyces cerevisiae fatty acid synthase with a bacterial type II system allows flexible product profiles.

The native yeast type I fatty acid synthase (FAS) is a complex, rigid enzyme, and challenging to engineer for the production of medium- or short-chain fatty acids. Introduction of a type II FAS is a promising alternative as it allows expression control for each discrete enzyme and the addition of heterologous thioesterases. In this study, the native Saccharomyces cerevisiae FAS was functionally replaced by the Escherichia coli type II FAS (eFAS) system. The E. coli acpS + acpP (together), fabB, fabD, fabG, fabH, fabI, fabZ, and tesA were expressed in individual S. cerevisiae strains, and enzyme activity was confirmed by in vitro activity assays. Eight genes were then integrated into the yeast genome, while tesA or an alternate thioesterase gene, fatB from Ricinus communis or TEII from Rattus novergicus, was expressed from a multi-copy plasmid. Native FAS activity was eliminated by knocking out the yeast FAS2 gene. The strains expressing only the eFAS as de novo fatty acid source grew without fatty acid supplementation demonstrating that this type II FAS is able to functionally replace the native yeast FAS. The engineered strain expressing the R. communis fatB thioesterase increased total fatty acid titer 1.7-fold and shifted the fatty acid profile towards C14 production, increasing it from <1% in the native strain to more than 30% of total fatty acids, and reducing C18 production from 39% to 8%.

Tuning cellular response by modular design of bioactive domains in collagen.

Collagen's ability to direct cellular behavior suggests that redesigning it at the molecular level could enable manipulation of cells residing in an engineered microenvironment. However, the fabrication of full-length collagen mimics of specified sequence de novo has been elusive, and applications still rely on material from native tissues. Using a bottom-up strategy, we synthesized modular genes and expressed recombinant human collagen variants in Saccharomyces cerevisiae. The resulting biopolymers contained prescribed cell-interaction sites that can direct and tune cellular responses, with retention of the important triple-helical self-assembled structure. Removal of the native integrin-binding sites GROGER, GAOGER, GLOGEN, GLKGEN, and GMOGER in human collagen III yielded collagen that did not support adhesion of mammalian cells. Introduction of GFOGER sequences to this scaffold at specified locations and densities resulted in varying degrees of cellular attachment. The recruitment of focal adhesion complexes on the different collagens ranged from a 96% reduction to a 56% increase over native collagen I. Adhesion to the GFOGER-containing variants was entirely dependent and partially dependent on the ß1 and α2 subunits of integrin, respectively, with cell adhesion on average reduced by 86% with anti-ß1 and 38% with anti-α2 integrin antibody incubation. Results support the importance of local context in collagen-cell interactions. The investigation demonstrates the flexibility of this approach to introduce targeted changes throughout the collagen polymer for producing fully-prescribed variants with tailored properties.

Triacetic acid lactone production in industrial Saccharomyces yeast strains.

Triacetic acid lactone (TAL) is a potential platform chemical that can be produced in yeast. To evaluate the potential for industrial yeast strains to produce TAL, the g2ps1 gene encoding 2-pyrone synthase was transformed into 13 industrial yeast strains of varied genetic background. TAL production varied 63-fold between strains when compared in batch culture with glucose. Ethanol, acetate, and glycerol were also tested as potential carbon sources. Batch cultures with ethanol medium produced the highest titers. Therefore, fed-batch cultivation with ethanol feed was assayed for TAL production in bioreactors, producing our highest TAL titer, 5.2 g/L. Higher feed rates resulted in a loss of TAL and subsequent production of additional TAL side products. Finally, TAL efflux was measured and TAL is actively exported from S. cerevisiae cells. Percent yield for all strains was low, indicating that further metabolic engineering of the strains is required.

Overproduction and secretion of free fatty acids through disrupted neutral lipid recycle in Saccharomyces cerevisiae.

The production of fuels and chemicals from biorenewable resources is important to alleviate the environmental concerns, costs, and foreign dependency associated with the use of petroleum feedstock. Fatty acids are attractive biomolecules due to the flexibility of their iterative biosynthetic pathway, high energy content, and suitability for conversion into other secondary chemicals. Free fatty acids (FFAs) that can be secreted from the cell are particularly appealing due to their lower harvest costs and straightforward conversion into a broad range of biofuel and biochemical products. Saccharomyces cerevisiae was engineered to overproduce extracellular FFAs by targeting three native intracellular processes. ß-oxidation was disrupted by gene knockouts in FAA2, PXA1 and POX1, increasing intracellular fatty acids levels up to 55%. Disruptions in the acyl-CoA synthetase genes FAA1, FAA4 and FAT1 allowed the extracellular detection of free fatty acids up to 490mg/L. Combining these two disrupted pathways, a sextuple mutant (Δfaa1 Δfaa4 Δfat1 Δfaa2 Δpxa1 Δpox1) was able to produce 1.3g/L extracellular free fatty acids. Further diversion of carbon flux into neutral lipid droplet formation was investigated by the overexpression of DGA1 or ARE1 and by the co-overexpression of a compatible lipase, TGL1, TGL3 or TGL5. The sextuple mutant overexpressing the diacylglycerol acyltransferase, DGA1, and the triacylglycerol lipase, TGL3, yielded 2.2g/L extracellular free fatty acids. This novel combination of pathway interventions led to 4.2-fold higher extracellular free fatty acid levels than previously reported for S. cerevisiae.

Engineering catalyst microenvironments for metal-catalyzed hydrogenation of biologically derived platform chemicals.

It is shown that microenvironments formed around catalytically active sites mitigate catalyst deactivation by biogenic impurities that are present during the production of biorenewable chemicals from biologically derived species. Palladium and ruthenium catalysts are inhibited by the presence of sulfur-containing amino acids; however, these supported metal catalysts are stabilized by overcoating with poly(vinyl alcohol) (PVA), which creates a microenvironment unfavorable for biogenic impurities. Moreover, deactivation of Pd catalysts by carbon deposition from the decomposition of highly reactive species is suppressed by the formation of bimetallic PdAu nanoparticles. Thus, a PVA-overcoated PdAu catalyst was an order of magnitude more stable than a simple Pd catalyst in the hydrogenation of triacetic acid lactone, which is the first step in the production of biobased sorbic acid. A PVA-overcoated Ru catalyst showed a similar improvement in stability during lactic acid hydrogenation to propylene glycol in the presence of methionine.