Microbial glycoside hydrolases play a dominant role in the biochemical conversion of cellulosic biomass to high-value biofuels. Anaerobic cellulolytic bacteria are capable of producing multicomplex catalytic subunits containing cell-adherent cellulases, hemicellulases, xylanases, and other glycoside hydrolases to facilitate the degradation of highly recalcitrant cellulose and other related plant cell wall polysaccharides. Clostridium thermocellum is a cellulosome producing bacterium that couples rapid reproduction rates to highly efficient degradation of crystalline cellulose. Herein, we have developed and applied a suite of difluoromethylphenyl aglycone, N-halogenated glycosylamine, and 2-deoxy-2-fluoroglycoside activity-based protein profiling (ABPP) probes to the direct labeling of the C. thermocellum cellulosomal secretome. These activity-based probes (ABPs) were synthesized with alkynes to harness the utility and multimodal possibilities of click chemistry, and to increase enzyme active site inclusion for LC-MS analysis. We directly analyzed ABP-labeled and unlabeled global MS data, revealing ABP selectivity for glycoside hydrolase (GH) enzymes, in addition to a large collection of integral cellulosome-containing proteins. By identifying reactivity and selectivity profiles for each ABP, we demonstrate our ability to widely profile the functional cellulose degrading machinery of the bacterium. Derivatization of the ABPs, including reactive groups, acetylation of the glycoside binding groups, and mono- and disaccharide binding groups, resulted in considerable variability in protein labeling. Our probe suite is applicable to aerobic and anaerobic microbial cellulose degrading systems, and facilitates a greater understanding of the organismal role associated with biofuel development.

Biological engineering of microorganisms to produce value-added chemicals is a promising route to sustainable manufacturing. However, overproduction of metabolic intermediates at high titer, rate, and yield from inexpensive substrates is challenging in non-model systems where limited information is available regarding metabolic flux and its control in production conditions. Integrated multi-omic analyses of engineered strains offers an in-depth look at metabolites and proteins directly involved in growth and production of target and non-target bioproducts. Here we applied multi-omic analyses to overproduction of the polymer precursor 3-hydroxypropionic acid (3HP) in the filamentous fungus Aspergillus pseudoterreus. A synthetic pathway consisting of aspartate decarboxylase, beta-alanine pyruvate transaminase, and 3HP dehydrogenase was designed and built for A. pseudoterreus. Strains with single- and multi-copy integration events were isolated and multi-omics analysis consisting of intracellular and extracellular metabolomics and targeted and global proteomics was used to interrogate the strains in shake-flask and bioreactor conditions. Production of a variety of co-products (organic acids and glycerol) and oxidative degradation of 3HP were identified as metabolic pathways competing with 3HP production. Intracellular accumulation of nitrogen as 2,4-diaminobutanoate was identified as an off-target nitrogen sink that may also limit flux through the engineered 3HP pathway. Elimination of the high-expression oxidative 3HP degradation pathway by deletion of a putative malonate semialdehyde dehydrogenase improved the yield of 3HP by 3.4 × after 10 days in shake-flask culture. This is the first report of 3HP production in a filamentous fungus amenable to industrial scale biomanufacturing of organic acids at high titer and low pH.

There is little published data on the performance of biological indicator tests and immunoassays that could be used by first responders to determine if a suspicious powder contains a potential biothreat agent. We evaluated a range of biological indicator tests, including 3 protein tests, 2 ATP tests, 1 DNA test, and 1 FTIR spectroscopy instrument for their ability to screen suspicious powders for Bacillus anthracis (B. anthracis) spores and ricin. We also evaluated 12 immunoassays (mostly lateral flow immunoassays) for their ability to screen for B. anthracis and ricin. We used a cost-effective, statistically based test plan that allows instruments to be evaluated at performance levels ranging from 0.85 to 0.95 lower confidence bound of the probability of detection at confidence levels of 80% to 95%. We also assessed interference with 22 common suspicious powders encountered in the field. The detection reproducibility for the biological indicators was evaluated at 108 B. anthracis spores and 62.5 μg ricin, and the immunoassay detection reproducibility was evaluated at 107 spores/mL (B. anthracis) and 0.1 μg/mL (ricin). Seven out of 12 immunoassays met our most stringent criteria for B. anthracis detection, while 9 out of 12 met our most stringent test criteria for ricin detection. Most of the immunoassays also detected ricin in 3 different crude castor seed preparations. Our testing results varied across products and sample preparations, indicating the importance of reviewing performance data for specific instruments and sample types of interest for the application in order to make informed decisions regarding the selection of biodetection equipment for field use.

Background Efficient and economically viable production of biofuels from lignocellulosic biomass is dependent on mechanical and chemical pretreatment and enzymatic hydrolysis of plant material. These processing steps yield simple sugars as well as plant-derived and process-added organic acids, sugar-derived dehydration products, aldehydes, phenolics and other compounds that inhibit the growth of many microorganisms. Lipomyces starkeyi is an oleaginous yeast capable of robust growth on a variety of sugars and lipid accumulation on pretreated lignocellulosic substrates making it attractive as an industrial producer of biofuels. Here, we examined gene expression during batch growth and lipid accumulation in a 20-L bioreactor with either a blend of pure glucose and xylose or pretreated corn stover (PCS) that had been enzymatically hydrolyzed as the carbon sources. Results We monitored sugar and ammonium utilization as well as biomass accumulation and found that growth of L. starkeyi is inhibited with PCS hydrolysate as the carbon source. Both acetic acid and furfural are present at concentrations toxic to L. starkeyi in PCS hydrolysate. We quantified gene expression at seven time-points for each carbon source during batch growth and found that gene expression is similar at physiologically equivalent points. Analysis of promoter regions revealed that gene expression during the transition to lipid accumulation is regulated by carbon and nitrogen catabolite repression, regardless of carbon source and is associated with decreased expression of the translation machinery and suppression of the cell cycle. We identified 73 differentially expressed genes during growth phase in the bioreactor that may be involved in detoxification of corn stover hydrolysate. Conclusions Growth of L. starkeyi is inhibited by compounds present in PCS hydrolysate. Here, we monitored key metabolites to establish physiologically equivalent comparisons during a batch bioreactor run comparing PCS hydrolysate and purified sugars. L. starkeyi ’s response to PCS hydrolysate is primarily at the beginning of the run during growth phase when inhibitory compounds are presumably at their highest concentration and inducing the general detoxification response by L. starkeyi . Differentially expressed genes identified herein during growth phase will aid in the improvement of industrial strains capable of robust growth on substrates containing various growth inhibitory compounds. Electronic supplementary material The online version of this article (10.1186/s13068-019-1510-z) contains supplementary material, which is available to authorized users.

The objective of this study was to disrupt the non-homologous end-joining (NHEJ) pathway gene (Lsku70Δ) and evaluate the effects of selected gene deletions related to glycogen synthesis (LsGSY1) and lipid degradation (LsMFE1, LsPEX10, and LsTGL4) on lipid production in the oleaginous yeast Lipomyces starkeyi. Disruption of the NHEJ pathway to reduce the rate of non-homologous recombination is a common approach used to overcome low-efficiency targeted deletion or insertion in various organisms. Here, the homologue of the LsKU70 gene was identified and disrupted in L. starkeyi NRRL Y-11558. The LsGSY1, LsMFE1, LsPEX10, LsTGL4, and LsURA3 genes were then replaced with a resistance marker in the Lsku70Δ strain and several site-specific insertions were assessed for targeted over-expression of selected genes. The targeted disruption efficiency of five selected genes (LsGSY1, LsMFE1, LsPEX10, LsTGL4, and LsURA3) was increased from 0 to 10% in the parent to 50-100% of transformants screened in the Lsku70Δ strain with 0.8-1.4 kb homologous flanking sequences, while the efficiency of site-specific gene insertion with the β-glucuronidase reporter gene was 100% in the locus near the 3'-end coding (LsKU70) and non-coding (LsGSY1, LsMFE1, and LsPEX10) regions. Disruption of LsKU70 in isolation and in conjunction with LsGSY1, LsMFE1, LsPEX10, or LsTGL4 did not affect lipid production in L. starkeyi. Furthermore, β-glucuronidase reporter gene activity was similar in strains containing site-specific targeted insertions. Therefore, over-expression of genes related to lipid synthesis at targeted loci can be further examined for improvement of total lipid production in L. starkeyi.

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Lignocellulosic biomass has great promise as a highly abundant and renewable source for the production of biofuels. However, the recalcitrant nature of lignocellulose toward hydrolysis into soluble sugars remains a significant challenge to harnessing the potential of this source of bioenergy. A primary method for deconstructing lignocellulose is via chemical treatments, high temperatures, and hydrolytic enzyme cocktails, many of which are derived from the fungus Trichoderma reesei. Herein, we use an activity-based probe for glycoside hydrolases to rapidly identify optimal conditions for maximum enzymatic lignocellulose deconstruction. We also demonstrate that subtle changes to enzyme composition and activity in various strains of T. reesei can be readily characterized by our probe approach. The approach also permits multimodal measurements, including fluorescent gel-based analysis of activity in response to varied conditions and treatments, and mass spectrometry-based quantitative identification of labelled proteins. We demonstrate the promise this probe approach holds to facilitate rapid production of enzyme cocktails for high-efficiency lignocellulose deconstruction to accommodate high-yield biofuel production.