Engineering of glycoside hydrolase family 7 cellobiohydrolases directed by natural diversity screening.

Protein engineering and screening of processive fungal cellobiohydrolases (CBHs) remain challenging due to limited expression hosts, synergy-dependency, and recalcitrant substrates. In particular, glycoside hydrolase family 7 (GH7) CBHs are critically important for the bioeconomy and typically difficult to engineer. Here, we target the discovery of highly active natural GH7 CBHs and engineering of variants with improved activity. Using experimentally assayed activities of genome mined CBHs, we applied sequence and structural alignments to top performers to identify key point mutations linked to improved activity. From ∼1500 known GH7 sequences, an evolutionarily diverse subset of 57 GH7 CBH genes was expressed in Trichoderma reesei and screened using a multiplexed activity screening assay. Ten catalytically enhanced natural variants were identified, produced, purified, and tested for efficacy using industrially relevant conditions and substrates. Three key amino acids in CBHs with performance comparable or superior to Penicillium funiculosum Cel7A were identified and combinatorially engineered into P. funiculosum cel7a, expressed in T. reesei, and assayed on lignocellulosic biomass. The top performer generated using this combined approach of natural diversity genome mining, experimental assays, and computational modeling produced a 41% increase in conversion extent over native P. funiculosum Cel7A, a 55% increase over the current industrial standard T. reesei Cel7A, and 10% improvement over Aspergillus oryzae Cel7C, the best natural GH7 CBH previously identified in our laboratory.

A multi-plex protein expression system for production of complex enzyme formulations in Trichoderma reesei.

Heterologous protein production has been challenging in the hyper-cellulolytic fungus, Trichoderma reesei as the species is known for poor transformation efficiency, low homologous recombination frequency, and marginal screening systems for the identification of successful transformants. We have applied the 2A-peptide multi-gene expression system to co-express four proteins, which include three cellulases: a cellobiohydrolase (CBH1), an endoglucanase (EG1), and a ß-D-glucosidase (BGL1), as well as the enhanced green fluorescent protein (eGFP) marker protein. We designed a new chassis vector, pTrEno-4X-2A, for this work. Expression of these cellulase enzymes was confirmed by real-time quantitative reverse transcription PCR and immunoblot analysis. The activity of each cellulase was assessed using chromogenic substrates, which confirmed the functionality of the enzymes. Expression and activity of these enzymes were proportional to the level of eGFP fluorescence, thereby validating the reliability of this screening technique. An 18-fold differencein protein expression was observed between the first and third genes within the 2A-peptide construct. The availability of this new multi-gene expression and screening tool is expected to greatly impact multi-enzyme applications, such as the production of complex commercial enzyme formulations and metabolic pathway enzymes, especially those destined for cell-free applications.

Characterization of the Biomass Degrading Enzyme GuxA from Acidothermus cellulolyticus.

Microbial conversion of biomass relies on a complex combination of enzyme systems promoting synergy to overcome biomass recalcitrance. Some thermophilic bacteria have been shown to exhibit particularly high levels of cellulolytic activity, making them of particular interest for biomass conversion. These bacteria use varying combinations of CAZymes that vary in complexity from a single catalytic domain to large multi-modular and multi-functional architectures to deconstruct biomass. Since the discovery of CelA from Caldicellulosiruptor bescii which was identified as one of the most active cellulase so far identified, the search for efficient multi-modular and multi-functional CAZymes has intensified. One of these candidates, GuxA (previously Acel_0615), was recently shown to exhibit synergy with other CAZymes in C. bescii, leading to a dramatic increase in growth on biomass when expressed in this host. GuxA is a multi-modular and multi-functional enzyme from Acidothermus cellulolyticus whose catalytic domains include a xylanase/endoglucanase GH12 and an exoglucanase GH6, representing a unique combination of these two glycoside hydrolase families in a single CAZyme. These attributes make GuxA of particular interest as a potential candidate for thermophilic industrial enzyme preparations. Here, we present a more complete characterization of GuxA to understand the mechanism of its activity and substrate specificity. In addition, we demonstrate that GuxA exhibits high levels of synergism with E1, a companion endoglucanase from A. cellulolyticus. We also present a crystal structure of one of the GuxA domains and dissect the structural features that might contribute to its thermotolerance.

Composition and yield of non-cellulosic and cellulosic sugars in soluble and particulate fractions during consolidated bioprocessing of poplar biomass by Clostridium thermocellum.

BACKGROUND: Terrestrial plant biomass is the primary renewable carbon feedstock for enabling transition to a sustainable bioeconomy. Consolidated bioprocessing (CBP) by the cellulolytic thermophile Clostridium thermocellum offers a single step microbial platform for production of biofuels and biochemicals via simultaneous solubilization of carbohydrates from lignocellulosic biomass and conversion to products. Here, solubilization of cell wall cellulosic, hemicellulosic, and pectic polysaccharides in the liquor and solid residues generated during CBP of poplar biomass by C. thermocellum was analyzed. RESULTS: The total amount of biomass solubilized in the C. thermocellum DSM1313 fermentation platform was 5.8, 10.3, and 13.7% of milled non-pretreated poplar after 24, 48, and 120 h, respectively. These results demonstrate solubilization of 24% cellulose and 17% non-cellulosic sugars after 120 h, consistent with prior reports. The net solubilization of non-cellulosic sugars by C. thermocellum (after correcting for the uninoculated control fermentations) was 13 to 36% of arabinose (Ara), xylose (Xyl), galactose (Gal), mannose (Man), and glucose (Glc); and 15% and 3% of fucose and glucuronic acid, respectively. No rhamnose was solubilized and 71% of the galacturonic acid (GalA) was solubilized. These results indicate that C. thermocellum may be selective for the types and/or rate of solubilization of the non-cellulosic wall polymers. Xyl, Man, and Glc were found to accumulate in the fermentation liquor at levels greater than in uninoculated control fermentations, whereas Ara and Gal did not accumulate, suggesting that C. thermocellum solubilizes both hemicelluloses and pectins but utilizes them differently. After five days of fermentation, the relative amount of Rha in the solid residues increased 21% indicating that the Rha-containing polymer rhamnogalacturonan I (RG-I) was not effectively solubilized by C. thermocellum CBP, a result confirmed by immunoassays. Comparison of the sugars in the liquor versus solid residue showed that C. thermocellum solubilized hemicellulosic xylan and mannan, but did not fully utilize them, solubilized and appeared to utilize pectic homogalacturonan, and did not solubilize RG-I. CONCLUSIONS: The significant relative increase in RG-I in poplar solid residues following CBP indicates that C. thermocellum did not solubilize RG-I. These results support the hypothesis that this pectic glycan may be one barrier for efficient solubilization of poplar by C. thermocellum.

Towards sustainable production and utilization of plant-biomass-based nanomaterials: a review and analysis of recent developments.

Plant-biomass-based nanomaterials have attracted great interest recently for their potential to replace petroleum-sourced polymeric materials for sustained economic development. However, challenges associated with sustainable production of lignocellulosic nanoscale polymeric materials (NPMs) need to be addressed. Producing materials from lignocellulosic biomass is a value-added proposition compared with fuel-centric approach. This report focuses on recent progress made in understanding NPMs-specifically lignin nanoparticles (LNPs) and cellulosic nanomaterials (CNMs)-and their sustainable production. Special attention is focused on understanding key issues in nano-level deconstruction of cell walls and utilization of key properties of the resultant NPMs to allow flexibility in production to promote sustainability. Specifically, suitable processes for producing LNPs and their potential for scaled-up production, along with the resultant LNP properties and prospective applications, are discussed. In the case of CNMs, terminologies such as cellulose nanocrystals (CNCs) and cellulose nanofibrils (CNFs) used in the literature are examined. The term cellulose nano-whiskers (CNWs) is used here to describe a class of CNMs that has a morphology similar to CNCs but without specifying its crystallinity, because most applications of CNCs do not need its crystalline characteristic. Additionally, progress in enzymatic processing and drying of NPMs is also summarized. Finally, the report provides some perspective of future research that is likely to result in commercialization of plant-based NPMs.

Coexpression of a ß-d-Xylosidase from Thermotoga maritima and a Family 10 Xylanase from Acidothermus cellulolyticus Significantly Improves the Xylan Degradation Activity of the Caldicellulosiruptor bescii Exoproteome.

Caldicellulosiruptor species are hyperthermophilic, Gram-positive anaerobes and the most thermophilic cellulolytic bacteria so far described. They have been engineered to convert switchgrass to ethanol without pretreatment and represent a promising platform for the production of fuels, chemicals, and materials from plant biomass. Xylooligomers, such as xylobiose and xylotriose, that result from the breakdown of plant biomass more strongly inhibit cellulase activity than do glucose or cellobiose. High concentrations of xylobiose and xylotriose are present in C. bescii fermentations after 90 h of incubation, and removal or breakdown of these types of xylooligomers is crucial to achieving high conversion of plant biomass to product. In previous studies, the addition of exogenous ß-d-xylosidase substantially improved the performance of glucanases and xylanases in vitro. ß-d-Xylosidases are, in fact, essential enzymes in commercial preparations for efficient deconstruction of plant biomass. In addition, the combination of xylanase and ß-d-xylosidase is known to exhibit synergistic action on xylan degradation. In spite of its ability to grow efficiently on xylan substrates, no extracellular ß-d-xylosidase was identified in the C. bescii genome. Here, we report that the coexpression of a thermal stable ß-d-xylosidase from Thermotoga maritima and a xylanase from Acidothermus cellulolyticus in a C. bescii strain containing the A. cellulolyticus E1 endoglucanase significantly increased the activity of the exoproteome as well as growth on xylan substrates. The combination of these enzymes also resulted in increased growth on crystalline cellulose in the presence of exogenous xylan. IMPORTANCECaldicellulosiruptor species are bacteria that grow at extremely high temperature, more than 75°C, and are the most thermophilic bacteria so far described that are capable of growth on plant biomass. This native ability allows the use of unpretreated biomass as a growth substrate, eliminating the prohibitive cost of preprocessing/pretreatment of the biomass. They only grow under strictly anaerobic conditions, and the combination of high temperature and the lack of oxygen reduces the cost of fermentation and contamination by other microbes. They have been genetically engineered to convert switchgrass to ethanol without pretreatment and represent a promising platform for the production of fuels, chemicals, and materials from plant biomass. In this study, we introduced genes from other cellulolytic bacteria and identified a combination of enzymes that improves growth on plant biomass. An important feature of this study is that it measures growth, validating predictions made from adding enzyme mixtures to biomass.

Iron incorporation both intra- and extra-cellularly improves the yield and saccharification of switchgrass (Panicum virgatum L.) biomass.

BACKGROUND: Pretreatments are commonly used to facilitate the deconstruction of lignocellulosic biomass to its component sugars and aromatics. Previously, we showed that iron ions can be used as co-catalysts to reduce the severity of dilute acid pretreatment of biomass. Transgenic iron-accumulating Arabidopsis and rice plants exhibited higher iron content in grains, increased biomass yield, and importantly, enhanced sugar release from the biomass. RESULTS: In this study, we used intracellular ferritin (FerIN) alone and in combination with an improved version of cell wall-bound carbohydrate-binding module fused iron-binding peptide (IBPex) specifically targeting switchgrass, a bioenergy crop species. The FerIN switchgrass improved by 15% in height and 65% in yield, whereas the FerIN/IBPex transgenics showed enhancement up to 30% in height and 115% in yield. The FerIN and FerIN/IBPex switchgrass had 27% and 51% higher in planta iron accumulation than the empty vector (EV) control, respectively, under normal growth conditions. Improved pretreatability was observed in FerIN switchgrass (~ 14% more glucose release than the EV), and the FerIN/IBPex plants showed further enhancement in glucose release up to 24%. CONCLUSIONS: We conclude that this iron-accumulating strategy can be transferred from model plants and applied to bioenergy crops, such as switchgrass. The intra- and extra-cellular iron incorporation approach improves biomass pretreatability and digestibility, providing upgraded feedstocks for the production of biofuels and bioproducts.

Intracellular pathways for lignin catabolism in white-rot fungi.

Lignin is a biopolymer found in plant cell walls that accounts for 30% of the organic carbon in the biosphere. White-rot fungi (WRF) are considered the most efficient organisms at degrading lignin in nature. While lignin depolymerization by WRF has been extensively studied, the possibility that WRF are able to utilize lignin as a carbon source is still a matter of controversy. Here, we employ 13C-isotope labeling, systems biology approaches, and in vitro enzyme assays to demonstrate that two WRF, Trametes versicolor and Gelatoporia subvermispora, funnel carbon from lignin-derived aromatic compounds into central carbon metabolism via intracellular catabolic pathways. These results provide insights into global carbon cycling in soil ecosystems and furthermore establish a foundation for employing WRF in simultaneous lignin depolymerization and bioconversion to bioproducts-a key step toward enabling a sustainable bioeconomy.

Chimeric cellobiohydrolase I expression, activity, and biochemical properties in three oleaginous yeast.

Consolidated bioprocessing using oleaginous yeast is a promising modality for the economic conversion of plant biomass to fuels and chemicals. However, yeast are not known to produce effective biomass degrading enzymes naturally and this trait is essential for efficient consolidated bioprocessing. We expressed a chimeric cellobiohydrolase I gene in three different oleaginous, industrially relevant yeast: Yarrowia lipolytica, Lipomyces starkeyi, and Saccharomyces cerevisiae to study the biochemical and catalytic properties and biomass deconstruction potential of these recombinant enzymes. Our results showed differences in glycosylation, surface charge, thermal and proteolytic stability, and efficacy of biomass digestion. L. starkeyi was shown to be an inferior active cellulase producer compared to both the Y. lipolytica and S. cerevisiae enzymes, whereas the cellulase expressed in S. cerevisiae displayed the lowest activity against dilute-acid-pretreated corn stover. Comparatively, the chimeric cellobiohydrolase I enzyme expressed in Y. lipolytica was found to have a lower extent of glycosylation, better protease stability, and higher activity against dilute-acid-pretreated corn stover.

Disruption of the Snf1 Gene Enhances Cell Growth and Reduces the Metabolic Burden in Cellulase-Expressing and Lipid-Accumulating Yarrowia lipolytica.

Yarrowia lipolytica is known to be capable of metabolizing glucose and accumulating lipids intracellularly; however, it lacks the cellulolytic enzymes needed to break down cellulosic biomass directly. To develop Y. lipolytica as a consolidated bioprocessing (CBP) microorganism, we previously expressed the heterologous CBH I, CBH II, and EG II cellulase enzymes both individually and collectively in this microorganism. We concluded that the coexpression of these cellulases resulted in a metabolic drain on the host cells leading to reduced cell growth and lipid accumulation. The current study aims to build a new cellulase coexpressing platform to overcome these hinderances by (1) knocking out the sucrose non-fermenting 1 (Snf1) gene that represses the energetically expensive lipid and protein biosynthesis processes, and (2) knocking in the cellulase cassette fused with the recyclable selection marker URA3 gene in the background of a lipid-accumulating Y. lipolytica strain overexpressing ATP citrate lyase (ACL) and diacylglycerol acyltransferase 1 (DGA1) genes. We have achieved a homologous recombination insertion rate of 58% for integrating the cellulases-URA3 construct at the disrupted Snf1 site in the genome of host cells. Importantly, we observed that the disruption of the Snf1 gene promoted cell growth and lipid accumulation and lowered the cellular saturated fatty acid level and the saturated to unsaturated fatty acid ratio significantly in the transformant YL163t that coexpresses cellulases. The result suggests a lower endoplasmic reticulum stress in YL163t, in comparison with its parent strain Po1g ACL-DGA1. Furthermore, transformant YL163t increased in vitro cellulolytic activity by 30%, whereas the "total in vivo newly formed FAME (fatty acid methyl esters)" increased by 16% in comparison with a random integrative cellulase-expressing Y. lipolytica mutant in the same YNB-Avicel medium. The Snf1 disruption platform demonstrated in this study provides a potent tool for the further development of Y. lipolytica as a robust host for the expression of cellulases and other commercially important proteins.

Phylogenetics-based identification and characterization of a superior 2,3-butanediol dehydrogenase for Zymomonas mobilis expression.

BACKGROUND: Zymomonas mobilis has recently been shown to be capable of producing the valuable platform biochemical, 2,3-butanediol (2,3-BDO). Despite this capability, the production of high titers of 2,3-BDO is restricted by several physiological parameters. One such bottleneck involves the conversion of acetoin to 2,3-BDO, a step catalyzed by 2,3-butanediol dehydrogenase (Bdh). Several Bdh enzymes have been successfully expressed in Z. mobilis, although a highly active enzyme is yet to be identified for expression in this host. Here, we report the application of a phylogenetic approach to identify and characterize a superior Bdh, followed by validation of its structural attributes using a mutagenesis approach. RESULTS: Of the 11 distinct bdh genes that were expressed in Z. mobilis, crude extracts expressing Serratia marcescens Bdh (SmBdh) were found to have the highest activity (8.89 µmol/min/mg), when compared to other Bdh enzymes (0.34-2.87 µmol/min/mg). The SmBdh crystal structure was determined through crystallization with cofactor (NAD+) and substrate (acetoin) molecules bound in the active site. Active SmBdh was shown to be a tetramer with the active site populated by a Gln247 residue contributed by the diagonally opposite subunit. SmBdh showed a more extensive supporting hydrogen-bond network in comparison to the other well-studied Bdh enzymes, which enables improved substrate positioning and substrate specificity. This protein also contains a short α6 helix, which provides more efficient entry and exit of molecules from the active site, thereby contributing to enhanced substrate turnover. Extending the α6 helix to mimic the lower activity Enterobacter cloacae (EcBdh) enzyme resulted in reduction of SmBdh function to nearly 3% of the total activity. In great contrast, reduction of the corresponding α6 helix of the EcBdh to mimic the SmBdh structure resulted in ~ 70% increase in its activity. CONCLUSIONS: This study has demonstrated that SmBdh is superior to other Bdhs for expression in Z. mobilis for 2,3-BDO production. SmBdh possesses unique structural features that confer biochemical advantage to this protein. While coordinated active site formation is a unique structural characteristic of this tetrameric complex, the smaller α6 helix and extended hydrogen network contribute towards improved activity and substrate promiscuity of the enzyme.

Overview and Future Directions.

Modern microbial and enzyme engineering and their advancement are increasingly dependent on the marriage of a wide range of sophisticated technologies. For students entering the field of biotechnology, the outlook is indeed daunting. Expertise at levels beyond that of simple familiarity will be needed to conduct competitive research. It goes without saying that to be competitive, all new researchers in this field will need basic preparation in molecular biology, biochemistry, and genetics. Further, experience working with concepts and experimental tools in enzyme biochemistry and kinetics, gene editing, computational metabolic pathway modeling, experimental pathway flux analysis, and computational clustering tools to process complex data sets will be vital for success. We speculate that most biotechnology researchers in early career at this time will build teams of collaborators to address these disparate science fields rather than attempt to become experts in one lab.

Genetics of Unstudied Thermophiles for Industry.

Thermophilic organisms hold great potential for industry due to their numerous advantages in biotechnological applications such as higher reaction rate, higher substrate loading, decreased susceptibility to reaction contamination, energy savings in industrial fermentations, and ability to express thermostable proteins that can be utilized in many important industrial processes. Bioprospecting for thermophiles will continue to reveal new enzymatic and metabolic paradigms with industrial applicability. In order to translate these paradigms to production scale, routine methods for microbial genetic engineering are needed, yet remain to be developed in many newly isolated thermophiles. Major challenges and recent developments in the establishment of reliable genetic systems in thermophiles are discussed. Here, we use a hyperthermophilic, cellulolytic bacterium, Caldicellulosiruptor bescii, as a case study to demonstrate the development of a genetic system for an industrially useful thermophile, describing in detail methods for transformation, genetic tool utilization, and chromosomal modification using targeted gene deletion and insertion techniques.

Methods for Metabolic Engineering of a Filamentous Trichoderma reesei.

In this work, we describe genetic tools and techniques for engineering Trichoderma reesei for the production of farnesene. Enhanced production of farnesene was used as an example of this methodology; as were the overexpression of a key enzyme, HMGS, in the MVA pathway.

An Improved Leaf Protoplast System for Highly Efficient Transient Expression in Switchgrass (Panicum virgatum L.).

As a robust perennial C4-type monocot plant and a native species to North America, switchgrass (Panicum virgatum) has been evaluated and designated as a strong candidate bioenergy crop by the U.S. DOE. Although genetic modifications of switchgrass have been used to successfully reduce the recalcitrance of switchgrass biomass for biofuel production, the generation of transgenic switchgrass is still a slow and laborious process. A transient protoplast system can provide an excellent platform to accelerate the selection of genes-of-interest for tailoring switchgrass biomass. However, partially due to the lack of the complete genomic information, the attempts to optimize the transient protoplast system for switchgrass remain scarce. In this chapter, we provide an improved protocol for switchgrass protoplast isolation, increased transformation efficiency using CsCl gradient ultracentrifugation-derived plasmid DNA and extended application of the transient switchgrass protoplast system to analyze protein expression using western blot.

Crystallography of Metabolic Enzymes.

The metabolic enzymes like any enzymes generally display globular architecture where secondary structure elements and interactions between them preserve the spatial organization of the protein. A typical enzyme features a well-defined active site, designed for selective binding of the reaction substrate and facilitating a chemical reaction converting the substrate into a product. While many chemical reactions could be facilitated using only the functional groups that are found in proteins, the large percentage or intracellular reactions require use of cofactors, varying from single metal ions to relatively large molecules like numerous coenzymes, nucleotides and their derivatives, dinucleotides or hemes. Quite often these large cofactors become important not only for the catalytic function of the enzyme but also for the structural stability of it, as those are buried deep in the enzyme.

Molecular Mechanism of Polysaccharide Acetylation by the Arabidopsis Xylan O-acetyltransferase XOAT1.

Xylans are a major component of plant cell walls. O-Acetyl moieties are the dominant backbone substituents of glucuronoxylan in dicots and play a major role in the polymer-polymer interactions that are crucial for wall architecture and normal plant development. Here, we describe the biochemical, structural, and mechanistic characterization of Arabidopsis (Arabidopsis thaliana) xylan O-acetyltransferase 1 (XOAT1), a member of the plant-specific Trichome Birefringence Like (TBL) family. Detailed characterization of XOAT1-catalyzed reactions by real-time NMR confirms that it exclusively catalyzes the 2-O-acetylation of xylan, followed by nonenzymatic acetyl migration to the O-3 position, resulting in products that are monoacetylated at both O-2 and O-3 positions. In addition, we report the crystal structure of the catalytic domain of XOAT1, which adopts a unique conformation that bears some similarities to the α/ß/α topology of members of the GDSL-like lipase/acylhydrolase family. Finally, we use a combination of biochemical analyses, mutagenesis, and molecular simulations to show that XOAT1 catalyzes xylan acetylation through formation of an acyl-enzyme intermediate, Ac-Ser-216, by a double displacement bi-bi mechanism involving a Ser-His-Asp catalytic triad and unconventionally uses an Arg residue in the formation of an oxyanion hole.

Ferrous and Ferric Ion-Facilitated Dilute Acid Pretreatment of Lignocellulosic Biomass under Anaerobic or Aerobic Conditions: Observations of Fe Valence Interchange and the Role of Fenton Reaction.

Ferrous ion co-catalyst enhancement of dilute-acid (DA) pretreatment of biomass is a promising technology for increasing the release of sugars from recalcitrant lignocellulosic biomass. However, due to the reductive status of ferrous ion and its susceptibility to oxidation with exposure to atmosphere, its effective application presumably requires anaerobic aqueous conditions created by nitrogen gas-purging, which adds extra costs. The objective of this study was to assess the effectiveness of oxidative iron ion, (i.e., ferric ion) as a co-catalyst in DA pretreatment of biomass, using an anaerobic chamber to strictly control exposure to oxygen during setup and post-pretreatment analyses. Remarkably, the ferric ions were found to be as efficient as ferrous ions in enhancing sugar release during DA pretreatment of biomass, which may be attributed to the observation that a major portion of the initial ferric ions were converted to ferrous during pretreatment. Furthermore, the detection of hydrogen peroxide in the liquors after DA/Fe ion pretreatment suggests that Fenton reaction chemistry was likely involved in DA/Fe ion pretreatments of biomass, contributing to the observed ferric and ferrous interchanges during pretreatment. These results help define the extent and specification requirements for applying iron ions as co-catalysts in DA pretreatments of biomass.

Glycosylation of hyperthermostable designer cellulosome components yields enhanced stability and cellulose hydrolysis.

Biomass deconstruction remains integral for enabling second-generation biofuel production at scale. However, several steps necessary to achieve significant solubilization of biomass, notably harsh pretreatment conditions, impose economic barriers to commercialization. By employing hyperthermostable cellulase machinery, biomass deconstruction can be made more efficient, leading to milder pretreatment conditions and ultimately lower production costs. The hyperthermophilic bacterium Caldicellulosiruptor bescii produces extremely active hyperthermostable cellulases, including the hyperactive multifunctional cellulase CbCel9A/Cel48A. Recombinant CbCel9A/Cel48A components have been previously produced in Escherichia coli and integrated into synthetic hyperthermophilic designer cellulosome complexes. Since then, glycosylation has been shown to be vital for the high activity and stability of CbCel9A/Cel48A. Here, we studied the impact of glycosylation on a hyperthermostable designer cellulosome system in which two of the cellulosomal components, the scaffoldin and the GH9 domain of CbCel9A/Cel48A, were glycosylated as a consequence of employing Ca. bescii as an expression host. Inclusion of the glycosylated components yielded an active cellulosome system that exhibited long-term stability at 75 °C. The resulting glycosylated designer cellulosomes showed significantly greater synergistic activity compared to the enzymatic components alone, as well as higher thermostability than the analogous nonglycosylated designer cellulosomes. These results indicate that glycosylation can be used as an essential engineering tool to improve the properties of designer cellulosomes. Additionally, Ca. bescii was shown to be an attractive candidate for production of glycosylated designer cellulosome components, which may further promote the viability of this bacterium both as a cellulase expression host and as a potential consolidated bioprocessing platform organism.

Metabolic engineering of Zymomonas mobilis for anaerobic isobutanol production.

BACKGROUND: Biofuels and value-added biochemicals derived from renewable biomass via biochemical conversion have attracted considerable attention to meet global sustainable energy and environmental goals. Isobutanol is a four-carbon alcohol with many advantages that make it attractive as a fossil-fuel alternative. Zymomonas mobilis is a highly efficient, anaerobic, ethanologenic bacterium making it a promising industrial platform for use in a biorefinery. RESULTS: In this study, the effect of isobutanol on Z. mobilis was investigated, and various isobutanol-producing recombinant strains were constructed. The results showed that the Z. mobilis parental strain was able to grow in the presence of isobutanol below 12 g/L while concentrations greater than 16 g/L inhibited cell growth. Integration of the heterologous gene encoding 2-ketoisovalerate decarboxylase such as kdcA from Lactococcus lactis is required for isobutanol production in Z. mobilis. Moreover, isobutanol production increased from nearly zero to 100-150 mg/L in recombinant strains containing the kdcA gene driven by the tetracycline-inducible promoter Ptet. In addition, we determined that overexpression of a heterologous als gene and two native genes (ilvC and ilvD) involved in valine metabolism in a recombinant Z. mobilis strain expressing kdcA can divert pyruvate from ethanol production to isobutanol biosynthesis. This engineering improved isobutanol production to above 1 g/L. Finally, recombinant strains containing both a synthetic operon, als-ilvC-ilvD, driven by Ptet and the kdcA gene driven by the constitutive strong promoter, Pgap, were determined to greatly enhance isobutanol production with a maximum titer about 4.0 g/L. Finally, isobutanol production was negatively affected by aeration with more isobutanol being produced in more poorly aerated flasks. CONCLUSIONS: This study demonstrated that overexpression of kdcA in combination with a synthetic heterologous operon, als-ilvC-ilvD, is crucial for diverting pyruvate from ethanol production for enhanced isobutanol biosynthesis. Moreover, this study also provides a strategy for harnessing the valine metabolic pathway for future production of other pyruvate-derived biochemicals in Z. mobilis.