Carlos G. Acevedo-Rocha

Reducing Codon Redundancy and Screening Effort of Combinatorial Protein Libraries Created by Saturation Mutagenesis

Saturation mutagenesis probes define sections of the vast protein sequence space. However, even if randomization is limited this way, the combinatorial numbers problem is severe. Because diversity is created at the codon level, codon redundancy is a crucial factor determining the necessary effort for library screening. Additionally, due to the probabilistic nature of the sampling process, oversampling is required to ensure library completeness as well as a high probability to encounter all unique variants. Our trick employs a special mixture of three primers, creating a degeneracy of 22 unique codons coding for the 20 canonical amino acids. Therefore, codon redundancy and subsequent screening effort is significantly reduced, and a balanced distribution of codon per amino acid is achieved, as demonstrated exemplarily for a library of cyclohexanone monooxygenase. We show that this strategy is suitable for any saturation mutagenesis methodology to generate less-redundant libraries.

From essential to persistent genes: a functional approach to constructing synthetic life

A central undertaking in synthetic biology (SB) is the quest for the ‘minimal genome’. However, ‘minimal sets’ of essential genes are strongly context-dependent and, in all prokaryotic genomes sequenced to date, not a single protein-coding gene is entirely conserved. Furthermore, a lack of consensus in the field as to what attributes make a gene truly essential adds another aspect of variation. Thus, a universal minimal genome remains elusive. Here, as an alternative to defining a minimal genome, we propose that the concept of gene persistence can be used to classify genes needed for robust long-term survival. Persistent genes, although not ubiquitous, are conserved in a majority of genomes, tend to be expressed at high levels, and are frequently located on the leading DNA strand. These criteria impose constraints on genome organization, and these are important considerations for engineering cells and for creating cellular life-like forms in SB.

Lipase Congeners Designed by Genetic Code Engineering

Classical enzyme optimization exploits the chemistry confined to the 20 canonical amino acids encoded by the standard genetic code. ‘Genetic code engineering’ allows the global substitution of particular residues with synthetic analogues, endowing proteins with chemical diversity not found in nature. These proteins are congeners of the parent protein because they originate from the same gene sequence, but contain a fraction of noncanonical amino acids. Global substitutions of methionine, proline, phenylalanine, and tyrosine have been carried out with related analogues in Thermoanaerobacter thermohydrosulfuricus lipase. This study represents the first extensive report of an important biocatalyst substituted with a high number of noncanonical amino acids. The generated lipase congeners displayed special features such as enhanced activation, elevated enzyme activity (by up to 25 %) and substrate tolerance (by up to 40 %), and changes in optimal temperature (by up to 20 °C) and pH (by up to 3). These emergent features achieved by genetic code engineering might be important not only for academic research, but also for numerous economical applications in the food, detergent, chemical, pharmaceutical, leather, textile, cosmetic, and paper industries.

Biocatalysis with Unnatural Amino Acids: Enzymology Meets Xenobiology

The goal of xenobiology is to design biological systems endowed with unusual biochemical functions, whereas enzymology concerns the study of enzymes, the workhorses of biocatalysis. Biocatalysis employs enzymes and organisms to perform useful biotransformations in synthetic chemistry and biotechnology. During the past few years, the effects of incorporating noncanonical amino acids (ncAAs) into enzymes with potential applications in biocatalysis have been increasingly investigated. In this Review, we provide an overview of the effects of new chemical functionalities that have been introduced into proteins to improve various facets of enzymatic catalysis. We also discuss future research avenues that will complement unnatural mutagenesis with standard protein engineering to produce novel and versatile biocatalysts with applications in synthetic organic chemistry and biotechnology.

Directed evolution of stereoselective enzymes based on genetic selection as opposed to screening systems.

Directed evolution of stereoselective enzymes provides a means to generate useful biocatalysts for asymmetric transformations in organic chemistry and biotechnology. Almost all of the numerous examples reported in the literature utilize high-throughput screening systems based on suitable analytical techniques. Since the screening step is the bottleneck of the overall procedure, researchers have considered the use of genetic selection systems as an alternative to screening. In principle, selection would be the most elegant and efficient approach because it is based on growth advantage of host cells harboring stereoselective mutants, but devising such selection systems is very challenging. They must be designed so that the host organism profits from the presence of an enantioselective variant. Progress in this intriguing research area is summarized in this review, which also includes some examples of display systems designed for enantioselectivity as assayed by fluorescence-activated cell sorting (FACS). Although the combination of display systems and FACS is a powerful approach, we also envision innovative ideas combining metabolic engineering and genetic selection systems with protein directed evolution for the development of highly selective and efficient biocatalysts.

Speeding up Directed Evolution: Combining the Advantages of Solid-Phase Combinatorial Gene Synthesis with Statistically Guided Reduction of Screening Effort

Efficient and economic methods in directed evolution at the protein, metabolic, and genome level are needed for biocatalyst development and the success of synthetic biology. In contrast to random strategies, semirational approaches such as saturation mutagenesis explore the sequence space in a focused manner. Although several combinatorial libraries based on saturation mutagenesis have been reported using solid-phase gene synthesis, direct comparison with traditional PCR-based methods is currently lacking. In this work, we compare combinatorial protein libraries created in-house via PCR versus those generated by commercial solid-phase gene synthesis. Using descriptive statistics and probabilistic distributions on amino acid occurrence frequencies, the quality of the libraries was assessed and compared, revealing that the outsourced libraries are characterized by less bias and outliers than the PCR-based ones. Afterward, we screened all libraries following a traditional algorithm for almost complete library coverage and compared this approach with an emergent statistical concept suggesting screening a lower portion of the protein sequence space. Upon analyzing the biocatalytic landscapes and best hits of all combinatorial libraries, we show that the screening effort could have been reduced in all cases by more than 50%, while still finding at least one of the best mutants.

On the road towards chemically modified organisms endowed with a genetic firewall.

Living systems are chemical “machines” controlled by a genetic program with standardized compounds and controlled chemical processes. One of the greatest challenges for scientists is finding a way to expand the standard chemical repertoire of living cells. This can be achieved by directed artificial evolution of organisms with novel chemical compositions. The resultant cells are expected to be viable and robust for growth as well as replication for an unlimited time in genetic isolation from natural species. However, this is not a trivial task, as the organization of metabolic pathways and information processing as well as a standard set of macromolecules (nucleic acids, proteins, fatty acids) are common features of all currently known cells. These building blocks include the standard repertoire of 20 canonical amino acids for protein biosynthesis and, in the case when DNA is the genetic material, four types of canonical nucleotides: adenine (A), thymine (T), guanine (G), and cytosine (C). In addition, the universality of the genetic code enables the horizontal transfer of genes across biological taxa. As a consequence of this high degree of standardization and interconnectivity, fundamental chemical changes within living systems generally tend to be lethal. The breakthrough by Marli re et al. is the first successful attempt to cross the canonical/noncanonical chemical barrier by artificially evolving bacteria with a chlorinated DNA genome. They showed that T could be “transliterated” to its noncanonical analogue 5-chorouracil (c) in the genome of Escherichia coli, whose progeny retained the ability to use c. This was achieved by extensively modifying the T biosynthetic pathway and by the successful selection of robust E. coli variants with the ability to grow on c. The choice of c is based on earlier reports that show that: 1) c has long been known to be incorporated into DNA; 2) c forms stable base pairs with A, and 3) c is readily metabolized by the components of the T pathway in E. coli. Another advantage is that T is the only DNA base not used in RNA metabolism. In addition, Marli re et al. were already able to generate extremely oligotropic E. coli cells capable of growing on any spare T by using a method to evolve new life forms. The available intracellular thymine is normally metabolically converted into thymidine triphosphate (dTTP) as a building block for DNA. However, this pathway can also be used by c (as a replacement for T) to generate chlorodeoxyuridine triphosphate (dcTP), which is enzymatically polymerized into chlorinated DNA. Evolution of the E. coli strain THY1 (a derivative of the E. coli K12 strain MG1655) in the presence of c under “harsh” and “gentle” experimental conditions yielded the CLU2 and CLU4 isolates, respectively. The experiment was stopped after the exclusive consumption of c and concomitant generation of CLU2 and CLU4 (after 164 and 166 days, respectively); subsequent analysis of the DNA composition revealed 90 % deoxychlorouridine (dc) in both strains. The residual 10% deoxythimidine (dT) came from a rather unconventional source: In most tRNAs, the uracil (unmethylated T) at position 54 is methylated by the S-AdoMetdependent 5-MeU-54-tRNA methyltransferase (encoded by the trmA gene). After knocking out this gene locus in the THY4 strain, the resulting THY5 variant was capable of growing on c, thereby giving rise to the variant CLU5, whose genomic DNA contained only traces (1.6%) of dT. This is most probably due to other RNAor DNA-modifying enzymes. Figure 1 presents the morphology of the native genomic DNA-containing strains as well as the one with a chlorinated genome. When an organism is exposed to a novel environment, it can only adapt through massive modification of the enzymes and proteins that originally evolved to respond to quite different regimes. Long-term cultivation experiments with fast-growing asexual bacterial cells—pioneered by Richard Lenski and co-workers—are the approach of choice to study these processes. In fact, they succeeded in cultivating up to 2000, 10 000 , 20000, and 40 000 E. coli generations by serial dilution. However, this is technically tedious and time-consuming, because contaminations and discontinuous growth are difficult to avoid. In conventional continuous culture setups, on the other hand, undesired dilution-resistant adhesive strain variants usually stick to surfaces of the device. [*] Dr. C. G. Acevedo-Rocha, Prof. Dr. N. Budisa Biocatalysis Group, Institute of Chemistry Berlin Institute of Technology/TU Berlin Franklinstrasse 29, 10587 Berlin (Germany) E-mail: budisa@biocat.tu-berlin.de Homepage: http://www.biocat.tu-berlin.de

Non-canonical amino acids as a useful synthetic biological tool for lipase-catalysed reactions in hostile environments

The incorporation of several non-canonical amino acids into the Thermoanaerobacter thermohydrosulfuricus lipase confers not only activity enhancement upon treatment with organic solvents (by up to 450%) and surfactants (resp. 1630%), but also protective effects against protein reducing (resp. 140%), alkylating (resp. 160%), and denaturing (resp.190%) agents as well as inhibitors (resp. 40%). This approach offers novel chemically diversified biocatalysts for hostile environments.

Economical analysis of saturation mutagenesis experiments

Saturation mutagenesis is a powerful technique for engineering proteins, metabolic pathways and genomes. In spite of its numerous applications, creating high-quality saturation mutagenesis libraries remains a challenge, as various experimental parameters influence in a complex manner the resulting diversity. We explore from the economical perspective various aspects of saturation mutagenesis library preparation: We introduce a cheaper and faster control for assessing library quality based on liquid media; analyze the role of primer purity and supplier in libraries with and without redundancy; compare library quality, yield, randomization efficiency, and annealing bias using traditional and emergent randomization schemes based on mixtures of mutagenic primers; and establish a methodology for choosing the most cost-effective randomization scheme given the screening costs and other experimental parameters. We show that by carefully considering these parameters, laboratory expenses can be significantly reduced.

Bioorthogonal Enzymatic Activation of Caged Compounds

Engineered cytochrome P450 monooxygenase variants are reported as highly active and selective catalysts for the bioorthogonal uncaging of propargylic and benzylic ether protected substrates, including uncaging in living E. coli. observed selectivity is supported by induced-fit docking and molecular dynamics simulations. This proof-of-principle study points towards the utility of bioorthogonal enzyme/protecting group pairs for applications in the life sciences.