Production of biofuels from renewable resources such as cellulosic biomass provides a source of liquid transportation fuel to replace petroleum-based fuels. This endeavor requires the conversion of cellulosic biomass into simple sugars, and the conversion of simple sugars into biofuels. Recently, microorganisms have been engineered to convert simple sugars into several types of biofuels, such as alcohols, fatty acid alkyl esters, alkanes, and terpenes, with high titers and yields. Here, we review recently engineered biosynthetic pathways from the well-characterized microorganisms Escherichia coli and Saccharomyces cerevisiae for the production of several advanced biofuels.

Metabolic flux analysis via (13)C labeling ((13)C MFA) quantitatively tracks metabolic pathway activity and determines overall enzymatic function in cells. Three core techniques are necessary for (13)C MFA: (1) a steady state cell culture in a defined medium with labeled-carbon substrates; (2) precise measurements of the labeling pattern of targeted metabolites; and (3) evaluation of the data sets obtained from mass spectrometry measurements with a computer model to calculate the metabolic fluxes. In this review, we summarize recent advances in the (13)C-flux analysis technologies, including mini-bioreactor usage for tracer experiments, isotopomer analysis of metabolites via high resolution mass spectrometry (such as GC-MS, LC-MS, or FT-ICR), high performance and large-scale isotopomer modeling programs for flux analysis, and the integration of fluxomics with other functional genomics studies. It will be shown that there is a significant value for (13)C-based metabolic flux analysis in many biological research fields.

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A family of engineered endopeptidases has been created that is capable of cleaving a diverse array of peptide sequences with high selectivity and catalytic efficiency (k cat /K M > 10 4 M −1 s −1 ). By screening libraries with a selection-counterselection substrate method, protease variants were programmed to recognize amino acids having altered charge, size and hydrophobicity properties adjacent to the scissile bond of the substrate, including Glu↓Arg, a specificity that to our knowledge has not been observed among natural proteases. Members of this artificial protease family resulted from a relatively small number of amino acid substitutions that (at least in one case) proved to be epistatic.Around 2% of the mammalian genome encodes for enzymes involved in protein degradation, underscoring the fundamental role of proteolysis in living organisms 1 . Unregulated proteolysis in vivo is lethal, and hence there is a critical requirement for precise sequence specificity as well as temporal and spatial control over protease activity 2,3 . The generalizable ability to engineer a protease to cleave any desired peptide sequence in an exquisitely selective manner and with high catalytic efficiency is of substantial interest for analytical, biotechnological and even therapeutic applications [4][5][6][7] . Although the utility of structure-guided mutagenesis to swap substrate preferences between homologous proteases has been previously demonstrated8 , 9, the systematic engineering of protease specificity to accommodate a diverse set of substrate sequences while maintaining high catalytic activity has remained elusive. Furthermore, typical directed evolution efforts to modify substrate preferences have given rise to enzymes exhibiting either relaxed selectivity or lower turnover for the new substrate10 ,11 . Herein we report the surprisingly general ability to program a family of highly selective and active proteases with new substrate specificities at both the P1 and P1′ positions, including sequences not recognized by the wild-type (WT) enzyme. Using a dual-substrate selection-counterselection flow cytometric assay to screen libraries derived from various diversification methods, protease variants were engineered to be specific for scissile bonds comprised of amino acids having altered charge, size, and NIH Public Access RESULTS Selection and counterselection strategiesThe Escherichia coli surface endopeptidase OmpT likely plays multiple roles in virulence 12 and exhibits a strong preference for cleavage between pairs of the basic amino acids lysine and arginine (especially the latter), cleaving these pairs with high catalytic efficiency 13 . Importantly, OmpT is not active until incorporated into the E. coli outer membrane, minimizing host lethality 14 . A quantitative, single cell-based assay optimized for dynamic range and sensitivity was developed to isolate only those OmpT variants capable of cleaving a desired selection peptide substrate (SelSub), but not a counterselection substrate (CtsSub) (Fig. 1a, ref...

BackgroundGlycolysis breakdowns glucose into essential building blocks and ATP/NAD(P)H for the cell, occupying a central role in its growth and bio-production. Among glycolytic pathways, the Entner Doudoroff pathway (EDP) is a more thermodynamically favorable pathway with fewer enzymatic steps than either the Embden–Meyerhof–Parnas pathway (EMPP) or the oxidative pentose phosphate pathway (OPPP). However, Escherichia coli do not use their native EDP for glucose metabolism.ResultsOverexpression of edd and eda in E. coli to enhance EDP activity resulted in only a small shift in the flux directed through the EDP (~20 % of glycolysis flux). Disrupting the EMPP by phosphofructokinase I (pfkA) knockout increased flux through OPPP (~60 % of glycolysis flux) and the native EDP (~14 % of glycolysis flux), while overexpressing edd and eda in this ΔpfkA mutant directed ~70 % of glycolytic flux through the EDP. The downregulation of EMPP via the pfkA deletion significantly decreased the growth rate, while EDP overexpression in the ΔpfkA mutant failed to improve its growth rates due to metabolic burden. However, the reorganization of E. coli glycolytic strategies did reduce glucose catabolite repression. The ΔpfkA mutant in glucose medium was able to cometabolize acetate via the citric acid cycle and gluconeogenesis, while EDP overexpression in the ΔpfkA mutant repressed acetate flux toward gluconeogenesis. Moreover, 13C-pulse experiments in the ΔpfkA mutants showed unsequential labeling dynamics in glycolysis intermediates, possibly suggesting metabolite channeling (metabolites in glycolysis are pass from enzyme to enzyme without fully equilibrating within the cytosol medium).ConclusionsWe engineered E. coli to redistribute its native glycolytic flux. The replacement of EMPP by EDP did not improve E. coli glucose utilization or biomass growth, but alleviated catabolite repression. More importantly, our results supported the hypothesis of channeling in the glycolytic pathways, a potentially overlooked mechanism for regulating glucose catabolism and coutilization of other substrates. The presence of channeling in native pathways, if proven true, would affect synthetic biology applications and metabolic modeling.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0630-y) contains supplementary material, which is available to authorized users.