From carbon dioxide to starch: no plants required Many plants turn glucose from photosynthesis into polymers that form insoluble starch granules ideal for long-term energy storage in roots and seeds. Cai et al . developed a hybrid system in which carbon dioxide is reduced to methanol by an inorganic catalyst and then converted by enzymes first to three and six carbon sugar units and then to polymeric starch. This artificial starch anabolic pathway relies on engineered recombinant enzymes from many different source organisms and can be tuned to produce amylose or amylopectin at excellent rates and efficiencies relative to other synthetic carbon fixation systems—and, depending on the metric used, even to field crops. —MAF

Ionic liquids (ILs) are attractive (co-)solvents for biocatalysis. However, in high concentration (>10 % IL), enzymes usually show decreased activity. No general principles have been discovered to improve IL resistance of enzymes by protein engineering. We present a systematic study to elucidate general engineering principles by site saturation mutagenesis on the complete gene bsla. Screening in presence of four [BMIM]-based ILs revealed two unexpected lessons on directed evolution: 1) resistance improvement was obtainable at 50-69 % of all amino acid positions, thus explaining the success of small sized random mutant libraries; 2) 6-13 % of substitutions led to improved resistance. Among these, 66-95 % were substitutions by chemically different amino acids (e.g., aromatic to polar/aliphatic/charged amino acids), thus indicating that mutagenesis methods introducing such changes should, at least for lipases like BSLA, be favored to improve IL resistance.

Directed evolution has matured to a routinely applied algorithm to tailor enzyme properties to meet the demands in various applications. In order to free directed enzyme evolution from methodological restraints and to efficiently explore its potential, many different strategies have been used in directed evolution campaigns. Analysis of directed evolution campaigns reveals that traditional approaches, in which several iterative rounds of diversity generation and screening are performed, are gradually replaced by strategies which require less time, less screening efforts, and generate a molecular understanding of the targeted properties. In this review, conceptual advances in knowledge generating directed evolution strategies are summarized, compared to each other and to traditional directed evolution strategies. Finally, a 'KnowVolution' (knowledge gaining directed evolution) termed strategy is proposed.