Using engineered initiator tRNA for precise control of protein translation within cells has great promise within future orthogonal translation systems to decouple housekeeping protein metabolism from that of engineered genetic systems. Previously, E. coli strain C321.ΔA.exp lacking all UAG stop codons was created, freeing this "amber" stop codon for other purposes. An engineered "amber initiator" tRNA CUA fMet that activates translation at UAG codons is available, but little is known about this tRNA's orthogonality. Here, we combine for the first time the amber initiator tRNA CUA fMet in C321.ΔA.exp and measure its cellular effects. We found that the tRNA CUA fMet expression resulted in a nearly 200-fold increase in fluorescent reporter expression with a unimodal population distribution and no apparent cellular fitness defects. Proteomic analysis revealed upregulated ribosome-associated, tRNA degradation, and amino acid biosynthetic proteins, with no evidence for off-target translation initiation. In contrast to previous work, we show that UAG-initiated proteins carry N-terminal methionine, but have no evidence for glutamine. Together, our results identify beneficial features of using the amber initiator tRNA CUA fMet to control gene expression while also revealing fundamental challenges to using engineered initiator tRNAs as the basis for orthogonal translation initiation systems.

Using engineered initiator tRNA for precise control of protein translation within cells has great promise within future orthogonal translation systems to decouple housekeeping protein metabolism from that of engineered genetic systems. Previously, E. coli strain C321.ΔA exp lacking all UAG stop codons was created, freeing this 'amber' stop codon for other purposes. An engineered 'amber initiator' tRNACUAfMet that activates translation at UAG codons is available, but little is known about this tRNA's orthogonality. Here, we combine for the first time the amber initiator tRNACUAfMet in C321.ΔA.exp and measure its cellular effects. We found that the tRNACUAfMet expression resulted in a nearly 200-fold increase in fluorescent reporter expression with a unimodal population distribution and no apparent cellular fitness defects. Proteomic analysis revealed upregulated ribosome-associated, tRNA degradation, and amino acid biosynthetic proteins, with no evidence for off-target translation initiation. In contrast to previous work, we show that UAG-initiated proteins carry N-terminal methionine exclusively. Together, our results identify beneficial features of using the amber initiator tRNACUAfMet to control gene expression while also revealing fundamental challenges to using engineered initiator tRNAs as the basis for orthogonal translation initiation systems.

Translation initiation is a sequential process involving interactions between the 30S small ribosomal subunit, initiation factors and initiator tRNA. The Escherichia coli K-12 strain is unique in the Escherichia because it has two different initiator tRNA sequences, tRNA fMet1 encoded by the metZWV genes and tRNA fMet2 encoded by the metY gene. A mutant of the metY gene was previously made where the anticodon sequence, responsible for specifying the start codon where translation initiation begins, was changed so that it bound to the amber stop codon UAG instead of the usual AUG start codon [1]. This amber initiator tRNA has already been shown to be functional in the K-12 strain [1] [2], but it is unclear whether it would function in other strains normally lacking the tRNA fMet2 variant. In this work, we transformed E. coli K-12, and four other generally regarded as safe (GRAS) laboratory strains, with a plasmid expressing the amber initiator tRNA and evaluated its functionality and growth effects on the bacteria. We performed these tests because, despite these strains all belonging to E. coli phylogenetic group A, it is well known that there is significant variation between even closely related E. coli strains in their metabolism [3] [4] [5], transcriptional response to exogenous DNA expression [6] and rates of amber stop codon suppression [7]. We found that the amber initiator functions similarly across the five strains, effectively initiating translation at the orthogonal UAG start codon and that it had modest growth-slowing effects in the Crooks, W, and K-12 strains. The five tested E. coli strains in this work (K-12, B, C, W, and Crooks) are important workhorses of academic and industrial research and development. The path is now clear to deploy the amber initiator tRNA into these five strains to precisely control gene expression.

Lignin is a complex natural polysaccharide primarily present in secondary wood or secondary xylem and phloem elements of the plant body. It constitutes one-fourth to one-third of the dry mass of wood and also, provides rigidity and strength. Lignin lacks a defined primary structure and is a heterogeneous biopolymer. Lignin-degradation is a major challenge because it can be a potential source of edible polysaccharide including glucose. In this investigation, commercial coir was considered as the source of isolating lignin-degrading fungus. A simple bioassay was carried out in coconut fibre (coir) and wood. In case of the fungal sample, the coir was inoculated in dry and wet conditions which resulted in 5.63% and 48.35% degradation respectively. On the basis of this, different lignin-degrading enzymes were assayed and purified. The fungus was identified as Microascus sp. on the basis of colony morphology, spore structure and perithecium formation. Further studies were conducted on the degraded coir and scanning electron microscope (SEM) images were taken. In future, these organisms can be a potential source of ligninolytic enzymes useful in different activities.