Engineering nonphosphorylative metabolism to generate lignocellulose-derived products.

Conversion of lignocellulosic biomass into value-added products provides important environmental and economic benefits. Here we report the engineering of an unconventional metabolism for the production of tricarboxylic acid (TCA)-cycle derivatives from D-xylose, L-arabinose and D-galacturonate. We designed a growth-based selection platform to identify several gene clusters functional in Escherichia coli that can perform this nonphosphorylative assimilation of sugars into the TCA cycle in less than six steps. To demonstrate the application of this new metabolic platform, we built artificial biosynthetic pathways to 1,4-butanediol (BDO) with a theoretical molar yield of 100%. By screening and engineering downstream pathway enzymes, 2-ketoacid decarboxylases and alcohol dehydrogenases, we constructed E. coli strains capable of producing BDO from D-xylose, L-arabinose and D-galacturonate. The titers, rates and yields were higher than those previously reported using conventional pathways. This work demonstrates the potential of nonphosphorylative metabolism for biomanufacturing with improved biosynthetic efficiencies.

Engineered biosynthesis of medium-chain esters in Escherichia coli.

Medium-chain esters such as isobutyl acetate (IBAc) and isoamyl acetate (IAAc) are high-volume solvents, flavors and fragrances. In this work, we engineered synthetic metabolic pathways in Escherichia coli for the total biosynthesis of IBAc and IAAc directly from glucose. Our pathways harnessed the power of natural amino acid biosynthesis. In particular, the native valine and leucine pathways in E. coli were utilized to supply the precursors. Then alcohol acyltransferases from various organisms were investigated on their capability to catalyze esterification reactions. It was discovered that ATF1 from Saccharomyces cerevisiae was the best enzyme for the formation of both IBAc and IAAc in E. coli. In vitro biochemical characterization of ATF1 confirmed the fermentation results and provided rational guidance for future enzyme engineering. We also performed strain improvement by removing byproduct pathways (Δldh, ΔpoxB, Δpta) and increased the production of both target chemicals. Then the best IBAc producing strain was used for scale-up fermentation in a 1.3-L benchtop bioreactor. 36g/L of IBAc was produced after 72h fermentation. This work demonstrates the feasibility of total biosynthesis of medium-chain esters as renewable chemicals.

Scalable production of mechanically tunable block polymers from sugar.

Development of sustainable and biodegradable materials is essential for future growth of the chemical industry. For a renewable product to be commercially competitive, it must be economically viable on an industrial scale and possess properties akin or superior to existing petroleum-derived analogs. Few biobased polymers have met this formidable challenge. To address this challenge, we describe an efficient biobased route to the branched lactone, ß-methyl-δ-valerolactone (ßMδVL), which can be transformed into a rubbery (i.e., low glass transition temperature) polymer. We further demonstrate that block copolymerization of ßMδVL and lactide leads to a new class of high-performance polyesters with tunable mechanical properties. Key features of this work include the creation of a total biosynthetic route to produce ßMδVL, an efficient semisynthetic approach that employs high-yielding chemical reactions to transform mevalonate to ßMδVL, and the use of controlled polymerization techniques to produce well-defined PLA-PßMδVL-PLA triblock polymers, where PLA stands for poly(lactide). This comprehensive strategy offers an economically viable approach to sustainable plastics and elastomers for a broad range of applications.

Two-stage transcriptional reprogramming in Saccharomyces cerevisiae for optimizing ethanol production from xylose.

Conversion of lignocellulosic material to ethanol is a major challenge in second generation bio-fuel production by yeast Saccharomyces cerevisiae. This report describes a novel strategy named "two-stage transcriptional reprogramming (TSTR)" in which key gene expression at both glucose and xylose fermentation phases is optimized in engineered S. cerevisiae. Through a combined genome-wide screening of stage-specific promoters and the balancing of the metabolic flux, ethanol yields and productivity from mixed sugars were significantly improved. In a medium containing 50g/L glucose and 50g/L xylose, the top-performing strain WXY12 rapidly consumed glucose within 12h and within 84h it consistently achieved an ethanol yield of 0.48g/g total sugar, which was 94% of the theoretical yield. WXY12 utilizes a KGD1 inducible promoter to drive xylose metabolism, resulting in much higher ethanol yield than a reference strain using a strong constitutive PGK1 promoter. These promising results validate the TSTR strategy by synthetically regulating the xylose assimilation pathway towards efficient xylose fermentation.

A bio-catalytic approach to aliphatic ketones.

Depleting oil reserves and growing environmental concerns have necessitated the development of sustainable processes to fuels and chemicals. Here we have developed a general metabolic platform in E. coli to biosynthesize carboxylic acids. By engineering selectivity of 2-ketoacid decarboxylases and screening for promiscuous aldehyde dehydrogenases, synthetic pathways were constructed to produce both C5 and C6 acids. In particular, the production of isovaleric acid reached 32 g/L (0.22 g/g glucose yield), which is 58% of the theoretical yield. Furthermore, we have developed solid base catalysts to efficiently ketonize the bio-derived carboxylic acids such as isovaleric acid and isocaproic acid into high volume industrial ketones: methyl isobutyl ketone (MIBK, yield 84%), diisobutyl ketone (DIBK, yield 66%) and methyl isoamyl ketone (MIAK, yield 81%). This hybrid "Bio-Catalytic conversion" approach provides a general strategy to manufacture aliphatic ketones, and represents an alternate route to expanding the repertoire of renewable chemicals.

Alteration of xylose reductase coenzyme preference to improve ethanol production by Saccharomyces cerevisiae from high xylose concentrations.

A K270R mutation of xylose reductase (XR) was constructed by site-direct mutagenesis. Fermentation results of the F106X and F106KR strains, which carried wild type XR and K270R respectively, were compared using different substrate concentrations (from 55 to 220 g/L). After 72 h, F106X produced less ethanol than xylitol, while F106KR produced ethanol at a constant yield of 0.36 g/g for all xylose concentrations. The xylose consumption rate and ethanol productivity increased with increasing xylose concentrations in F106KR strain. In particular, F106KR produced 77.6g/L ethanol from 220 g/L xylose and converted 100 g/L glucose and 100g/L xylose into 89.0 g/L ethanol in 72h, but the corresponding values of F106X strain are 7.5 and 65.8 g/L. The ethanol yield of F106KR from glucose and xylose was 0.42 g/g, which was 82.3% of the theoretical yield. These results suggest that the F106KR strain is an excellent producer of ethanol from xylose.

Afr1p mediates activation of the Slt2p MAP kinase induced by pheromone, heat shock and hypo-osmotic shock in Saccharomyces cerevisiae.

In this study, we reinvestigated the role of Afr1p in the regulation of pheromone signaling and demonstrated that pheromone signaling was not regulated by Afr1p because neither deletion nor overexpression of AFR1 affected alpha-factor-induced transcription induction of the FUS1 gene. The enhanced alpha-factor resistance resulting from overexpression of AFR1 was dependent on the Slt2p mitogen-activated protein kinase. We also found that alpha-factor-induced activation of Slt2p required Afr1p. In the absence of Afr1p, activation of Slt2p was significantly reduced and, in a strain overexpressing Afr1p, the level of Slt2p activation was enhanced, indicating that Afr1p may function to mediate cross-talks between the mating pathway and the cell integrity pathway. Further, Afr1p was also required for adequate activation of Slt2p in cells exposed to heat shock and hypo-osmotic shock. These results indicated that, in addition to its role in establishing pheromone-induced morphogenesis, Afr1p may act as a 'sensor' to transduce cell wall stress resulting from different stimuli to the cell integrity pathway, leading to Slt2p activation.