Mutations to gene regulatory networks can be maladaptive or a source of evolutionary novelty. Epistasis confounds our understanding of how mutations impact the expression patterns of gene regulatory networks, because such nonlinearities make it difficult to predict the combined phenotypic effects of mutations based on knowledge of the mutations' individual effects. This challenge is exacerbated by the dependence of epistasis on the environment, which is particularly germane to gene regulatory networks that interpret signals in space or time. To help fill this knowledge gap, we used the toolkit of synthetic biology to systematically assay the effects of pairwise and triplet combinations of mutant genotypes on the expression pattern of a gene regulatory network expressed in Escherichia coli that interprets an inducer gradient across a spatial domain. We uncovered a preponderance of epistasis in both pairwise and triplet combinations that can switch in magnitude and sign across the inducer gradient to produce a greater diversity of expression pattern phenotypes than would be possible in the absence of such environment-dependent epistasis. We discuss our findings in the context of the evolution of hybrid incompatibilities and evolutionary novelties, arguing that environment-dependent epistasis is likely an important cause of both phenomena in gene regulatory networks.

Mutations to gene regulatory networks can be maladaptive or a source of evolutionary novelty. Epistasis confounds our understanding of how mutations affect the expression patterns of gene regulatory networks, a challenge exacerbated by the dependence of epistasis on the environment. We used the toolkit of synthetic biology to systematically assay the effects of pairwise and triplet combinations of mutant genotypes on the expression pattern of a gene regulatory network expressed in Escherichia coli that interprets an inducer gradient across a spatial domain. We uncovered a preponderance of epistasis that can switch in magnitude and sign across the inducer gradient to produce a greater diversity of expression pattern phenotypes than would be possible in the absence of such environment-dependent epistasis. We discuss our findings in the context of the evolution of hybrid incompatibilities and evolutionary novelties.

Bispecific antibodies (bsAbs) are often composed of several polypeptide chains that have to be expressed adequately to enable optimal assembly and yield of the bsAb. κλ bodies are a bispecific format with a native IgG structure, composed of two different light chains that pair with a common heavy chain. Introduction of non-optimal codons into the sequence of a particular polypeptide is an effective strategy for down modulating its expression. Here we applied this strategy but restricted the modification of the codon content to the constant domain of one light chain. This approach facilitates parallel optimization of several bsAbs by using the same modified constant domains. Partial sequence de-optimization reduced expression of the targeted polypeptide. Stable cell pools could be isolated displaying increased bispecific antibody titers as well as changes in the abundance of undesired by-products that require elimination during downstream processing. Thus, modulating the relative expression of polypeptides can have a significant impact on bsAb titer and product related impurities; which are important factors for large scale manufacturing for clinical supply.

Lausannevirus belongs to the family Marseilleviridae within the group of nucleocytoplasmic large DNA viruses (NCLDVs). These giant viruses exhibit unique features, including a large genome, ranging from 100 kb to 2.5 Mb and including from 150 to more than 2,500 genes, as well as the presence of genes coding for proteins involved in transcription and translation. The large majority of Lausannevirus open reading frames have unknown functions. Interestingly, a bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) is encoded in the Lausannevirus genome. The enzyme plays central roles in DNA precursor biosynthesis. DHFR is the pharmacological target of antifolates, such as trimethoprim, pyrimethamine, and proguanil. First, the functionality of Lausannevirus DHFR-TS was demonstrated by the successful complementation of a DHFR-deficient Saccharomyces cerevisiae strain with a plasmid expressing the heterologous gene. Additionally, using this heterologous expression system, we demonstrated the in vitro susceptibility of Lausannevirus DHFR-TS to proguanil and its resistance to pyrimethamine and trimethoprim. Proguanil may provide a unique and useful treatment if Lausannevirus proves to be a human pathogen. To our knowledge, this is the first time that a DHFR-TS has been described and characterized in an NCLDV.