Xenomicrobiology: a roadmap for genetic code engineering

Abstract

Biology is an analytical and informational science that is becoming increasingly dependent on chemical synthesis. One example is the high-throughput and low-cost synthesis of DNA, which is a foundation for the research field of synthetic biology (SB). The aim of SB is to provide biotechnological solutions to health, energy and environmental issues as well as unsustainable manufacturing processes in the frame of naturally existing chemical building blocks. Xenobiology (XB) goes a step further by implementing non-natural building blocks in living cells. In this context, genetic code engineering respectively enables the re-design of genes/genomes and proteins/proteomes with non-canonical nucleic (XNAs) and amino (ncAAs) acids. Besides studying information flow and evolutionary innovation in living systems, XB allows the development of new-to-nature therapeutic proteins/peptides, new biocatalysts for potential applications in synthetic organic chemistry and biocontainment strategies for enhanced biosafety. In this perspective, we provide a brief history and evolution of the genetic code in the context of XB. We then discuss the latest efforts and challenges ahead for engineering the genetic code with focus on substitutions and additions of ncAAs as well as standard amino acid reductions. Finally, we present a roadmap for the directed evolution of artificial microbes for emancipating rare sense codons that could be used to introduce novel building blocks. The development of such xenomicroorganisms endowed with a 'genetic firewall' will also allow to study and understand the relation between code evolution and horizontal gene transfer.

Figure 1

Experimental approaches in xenobiology to re‐design the flow of genetic information. Left: the central dogma of molecular biology is shown. DNA is replicated and transmitted to the descendants or transcribed into RNA. Translation subsequently gives rise to proteins, making up the functional proteome. Recent advances show that the introduction of artificial base pairs as XNA can also replicate and proliferate (Malyshev et al., 2014). To date, however, this information cannot be transcribed or translated in vivo. Incorporation of ncAAs into the proteome was shown to be feasible if evolutionary pressure is applied (Hoesl et al., 2015). Right: Methods and levels of the genetic code re‐design that enables the partial or full re‐design of informational flow in biology according to Budisa (2014).

Figure 2

The genetic code structure in the RNA format in a radial representation. Chart from inside to outside: a triplet of mRNA (5′→3′) is assigned to one of the 20 canonical amino acids or a stop codon. The natural expansion of the genetic code of selenocysteine (SeC) at opal and pyrrolysine (Pyl) at the amber and opal stop codons is depicted (Courtesy provided by Dr Stefan Oehm) (Oehm, 2016).

Figure 3

The codon capture and ambiguous intermediate theory for codon reassignment in the historical development of the genetic code. While in codon capture (left) the endogenous tRNA disappears due to the removal of its designated codon, in ambiguous intermediate theory (right) the exogenous tRNA is in direct competition with the cognate one. In the end, both theories end up in a reassigned codon. Figure modified according Santos et al. (2004) and Budisa (2006a).

Figure 4

Flow chart presentations of two basic in vivo approaches for incorporating non‐canonical amino acids into proteins. (A) Auxotrophy‐based selective pressure incorporation (SPI) method exploits the endogenous translational system to load an isostructural analogue onto a canonical tRNA leading to residue‐specific incorporation, i.e. cAA→ncAA substitution. (B) Stop codon suppression (SCS) usually endows the host with a new orthogonal aaRS:tRNA pair in charge of introducing a given ncAA or more at one or more stop codons. For more details see Hoesl and Budisa (2012).