![]() Many prokaryotes and eukaryotes display strong preference for certain codons over their synonymous alternatives 15, 16 (Fig. The 20 proteinogenic amino acids are encoded by 61 codons, and with up to six different codons specifying the same amino acid according to standard code table 14. Therefore, it is of great need to develop alternative approaches that provide accuracy, sensitivity, and high-throughput simultaneously. ![]() Third, proper analogues for specific amino acids are limited 5. Therefore, it is necessary to test the selected mutations individually to verify their amino acid productivities. The analogues could also be degraded to nontoxic forms or be incorporated into the bacterial proteome after evolution 13. Specifically, the analogues could be blocked by amino acid transporters with increased selectivity, or pumped outside of the cells by enhanced efflux 6 (Fig. Second, cells may escape from the selection pressure of an analogue by developing detoxification mechanisms. Thus, mutants that have enhanced amino acid productivities may not survive these side effects. For example, it could jeopardize cell growth by disrupting the nucleus regions 7, affecting the structure of cellular membranes 11, inhibiting purine and pyrimidine biosynthesis or decreasing the level of ATP 7, 11, 12. First, an analogue could interfere with cellular activities beyond the protein synthesis. However, the use of analogues faces severe disadvantages that could compromise the selection results. For instance, a high concentration of 4-azaleucine has been successfully applied to select l-leucine overproduction strains 8, 9, 10. Cells overproducing an amino acid might produce enough functional proteins to survive the stresses from the analogue of that amino acid 7 and could be selected. The losing of functional proteins could result in growth retardation or even death 6. Once inserted into any polypeptide, the analogue could disrupt the synthesis or function of that polypeptide. An analogue would compete with its corresponding amino acid for the finite tRNAs in the process of protein biosynthesis 4, 5. The traditional strategy for screening amino acid overproducers took the advantage of toxic analogues, which has similar size, structure, and charge properties as the proteinogenic amino acids. Theoretically, the retarded protein expression should be restored by increased intracellular concentrations of the corresponding amino acids coli) that pair with the low-abundance tRNAs would dramatically slow down protein expression (lower box). On the contrary, the rare tRNAs have lower chances to be charged with the corresponding amino acids, switching to the rare alternatives (e.g. leucine codon) with synonymous ones that are recognized by the most abundant tRNAs for a specific host would typically improve the expression of the desired protein (upper box). d For an exogenous gene, replacing its codons (e.g. coli W1485, a K strain derivative at a growth rate of 0.4 doublings h –1. The fraction of individual tRNA out of the total tRNA was derived from E. c Codon usage and the fraction of tRNAs (bubble diameter) in E. The analogues could be blocked (ii) or pumped outside of the cells (iii). b After taken up by the cells (i), the amino acid analogues (orange square) compete with the corresponding natural amino acids (blue hexagon) for the finite tRNAs, a step catalyzed by the aminoacyl-tRNA synthetase (aaRS). a Global productions of amino acids (left), the annual productions (right, represented by color intensity), and the fermentation titer (right, represented by bar height) for nine selected amino acids. This strategy sheds new light on obtaining and understanding amino acid overproduction strains.Īmino acid productions and codon usage. The system is also applied to Corynebacterium glutamicum to screen out l-arginine overproducers. As a proof-of-concept, Escherichia coli strains overproducing l-leucine, l-arginine or l-serine are successfully selected from random mutation libraries. Results show that integration of rare codons can inhibit gene translations in a frequency-dependent manner. ![]() Inspired by this assumption, we develop a screening or selection system for obtaining overproducers of a target amino acid by replacing its common codons with the corresponding synonymous rare alternative in the coding sequence of selected reporter proteins or antibiotic-resistant markers. Theoretically, disrupted or retarded translation caused by the lack of charged rare tRNAs can be partially restored by feeding or intracellular synthesis of the corresponding amino acids. The translation of rare codons relies on their corresponding rare tRNAs, which could not be fully charged under amino acid starvation.
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