No abstract

Bacteria produce a wide range of exopolysaccharides which are synthesized via different biosynthesis pathways. The genes responsible for synthesis are often clustered within the genome of the respective production organism. A better understanding of the fundamental processes involved in exopolysaccharide biosynthesis and the regulation of these processes is critical toward genetic, metabolic and protein-engineering approaches to produce tailor-made polymers. These designer polymers will exhibit superior material properties targeting medical and industrial applications. Exploiting the natural design space for production of a variety of biopolymer will open up a range of new applications. Here, we summarize the key aspects of microbial exopolysaccharide biosynthesis and highlight the latest engineering approaches toward the production of tailor-made variants with the potential to be used as valuable renewable and high-performance products for medical and industrial applications.

The limited supply of fossil resources demands the development of renewable alternatives to petroleum-based products. Here, biobased higher alcohols such as isobutanol are versatile platform molecules for the synthesis of chemical commodities and fuels. Currently, their fermentation-based production is limited by the low tolerance of microbial production systems to the end products and also by the low substrate flux into cell metabolism. We developed an innovative cell-free approach, utilizing an artificial minimized glycolytic reaction cascade that only requires one single coenzyme. Using this toolbox the cell-free production of ethanol and isobutanol from glucose was achieved. We also confirmed that these streamlined cascades functioned under conditions at which microbial production would have ceased. Our system can be extended to an array of industrially-relevant molecules. Application of solvent-tolerant biocatalysts potentially allows for high product yields, which significantly simplifies downstream product recovery.

No abstract

Cascade reactions catalyzed by multienzymatic systems have strongly moved into the focus of researchers in the field of biocatalysis because of their unique potential for the environmentally benign production of chemicals and materials. Inspired by Nature's ingenuity, considerable progress has been made in recent years to develop multistep reactions that combine the synthetic power of several enzymes in one pot. In addition, the combination of this biocatalytic power with the potential of chemical reactions, man-made transition-metal catalysts, and metalloenzymes has even further widened the repertoire of possible catalyzed reaction schemes. In this Review, we describe recent developments with respect to major challenges and solutions in the field of multienzyme cascade reactions, covering recent concurrent and sequential approaches with three or more enzymes in linear sequence as well as chemo-enzymatic reactions that combine a chemical step with at least two different enzymes over the last six years.

Laccase from Myceliophthora thermophila (MtL) was expressed in functional form in Saccharomyces cerevisiae. Directed evolution improved expression eightfold to the highest yet reported for a laccase in yeast (18 mg/liter). Together with a 22-fold increase in k cat , the total activity was enhanced 170-fold. Specific activities of MtL mutants toward 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine indicate that substrate specificity was not changed by the introduced mutations. The most effective mutation (10-fold increase in total activity) introduced a Kex2 protease recognition site at the C-terminal processing site of the protein, adjusting the protein sequence to the different protease specificities of the heterologous host. The C terminus is shown to be important for laccase activity, since removing it by a truncation of the gene reduces activity sixfold. Mutations accumulated during nine generations of evolution for higher activity decreased enzyme stability. Screening for improved stability in one generation produced a mutant more stable than the heterologous wild type and retaining the improved activity. The molecular mass of MtL expressed in S. cerevisiae is 30% higher than that of the same enzyme expressed in M. thermophila (110 kDa versus 85 kDa). Hyperglycosylation, corresponding to a 120-monomer glycan on one N-glycosylation site, is responsible for this increase. This S. cerevisiae expression system makes MtL available for functional tailoring by directed evolution.Directed evolution by random mutagenesis and recombination followed by screening or selection is a valuable tool for the engineering of enzymes (2,3,16,61). Functional gene expression in a suitable host is a prerequisite for directed evolution. Considering transformation efficiency, stability of plasmid DNA, and growth rate, Escherichia coli and Saccharomyces cerevisiae are best suited for these experiments. Heterologous expression in these hosts, however, is often limited by differences in the expression systems from the native organism (50). Different codon usage, missing chaperones, and posttranslational modifications such as disulfide bridges or glycosylation can all cause low expression levels and misfolded proteins that are degraded or driven into inclusion bodies (20). Finding the bottlenecks of a specific expression system requires consideration of many possibilities whose impact is hard to predict. Some incompatibilities between the expressed gene and heterologous host, such as codon usage or the recognition of signal sequences, can be overcome by changing the gene sequence. Thus, achieving functional expression is a good target for directed evolution (13,42,43).Laccases, like other ligninolytic enzymes, are notoriously difficult to express in nonfungal systems. The laccase from Myceliophthora thermophila (MtL) used in this work was previously expressed only in Aspergillus oryzae (6). Expression in S. cerevisiae has been reported for other laccase genes (11,33,34,60). Kojima et al. demonstrated expression of...

We describe a method for the stabilization of proteins that links the protease resistance of stabilized variants of a protein with the infectivity of a filamentous phage. A repertoire of variants of the protein to be stabilized is inserted between two domains (N2 and CT) of the gene-3-protein of the fd phage. The infectivity of fd phage is lost when the three domains are disconnected by the proteolytic cleavage of unstable protein inserts. Rounds of in vitro proteolysis, infection, and propagation can thus be performed to enrich those phage containing the most stable variants of the protein insert. This strategy discriminates between variants of a model protein (ribonuclease T1) differing in conformational stability and selects from a large repertoire variants that are only marginally more stable than others. Because fd phage are exceptionally stable and the proteolysis in the selection step takes place in vitro a wide range of solvent conditions can be used, tailored for the protein to be stabilized.

Class I terpene synthases generate the structural core of bioactive terpenoids. Deciphering structure-function relationships in the reactive closed complex and targeted engineering is hampered by highly dynamic carbocation rearrangements during catalysis. Available crystal structures, however, represent the open, catalytically inactive form or harbor nonproductive substrate analogs. Here, we present a catalytically relevant, closed conformation of taxadiene synthase (TXS), the model class I terpene synthase, which simulates the initial catalytic time point. In silico modeling of subsequent catalytic steps allowed unprecedented insights into the dynamic reaction cascades and promiscuity mechanisms of class I terpene synthases. This generally applicable methodology enables the active-site localization of carbocations and demonstrates the presence of an active-site base motif and its dominating role during catalysis. It additionally allowed in silico-designed targeted protein engineering that unlocked the path to alternate monocyclic and bicyclic synthons representing the basis of a myriad of bioactive terpenoids.computational biology | closed complex modeling | protein engineering | terpene synthases | terpene synthase catalysis