Question

DNA shuffling is a method for in vitro homologous recombination of pools of selected mutant genes by random fragmentation and polymerase chain reaction (PCR) reassembly. In our class, we went through a paper describing the evolution of four cephalosporinase from various species. The paper showed that a single cycle of shuffling yielded 8-fold improvements from the four separately evolved genes, versus a 270- to 540-fold improvement from the four genes shuffled together (so called family shuffling). Discuss how this result come out.

Four cephalosporinase ; Citrobacter freundii, Enterbacter cloacae, Yersinia enterclitica, Klebsiella pneumoniae

Answer 1

ans;-) DNA shuffling is a powerful process for directed evolution, which generates diversity by recombination1,2, combining useful mutations from individual genes. Libraries of chimaeric genes can be generated by random fragmentation of a pool of related genes, followed by reassembly of the fragments in a self-priming polymerasereaction. Template switching causes crossovers in areas of sequence homology. Our previous studies used single genes and random point mutations as the source of diversity3–6. An alternative source of diversity is naturally occurring homologous genes, which provide ‘functional diversity’.

Clones originating from the four single gene libraries showed up to eightfold increases in moxalactam resistance as compared to those expressing the wild-type genes (0.38 to 3.0mg ml-1 for the Klebsiella and Yersinia genes and 0.75 to 6.0mg ml-1 for the Citrobacter and Enterobacter genes). In contrast, the best clone (A) originating from the gene family library showed a 540-fold increase in resistance (0.38 to 200mg ml-1) compared to the wildtype
Klebsiella and Yersinia genes and a 270-fold increase (0.75 to 200 mg ml-1) compared to the wild-type Enterobacter and Citrobacter genes (Fig. 2a). Thus ‘family shuffling’ accelerated the rate of functional enzyme improvement 34- to 68-fold in a single cycle.A second round of shuffling was performed using the pool of
colonies selected in the first round. Plating of about 5 3 104 colonies on a range of concentrations of moxalactam yielded three clones which had a further 3.5-fold increased moxalactam resistance over the most resistant clone for the first cycle (clone A). The reduction in the rate of improvement is expected given the limited dynamic range of the bioassay. Based on our experiencewith other genes3–6, a second round of intra-species shuffling of the selected pools containing the eightfold improved genes would clearly not have achieved the degree of improvement we obtained by two rounds of inter-species shuffling.

The two most resistant clones (A and B) obtained in the first round of family shuffling also showed increased resistance against other b-lactam antibiotics. Clone A was resistant to cefoxitine, carbenicillin and cephafloridine at 100 mg ml-1 each, an improvement over the four wild-type enzymes of 4–16-fold. This was unexpected because in previous results for single gene shuffling of other enzymes the activity for the original substrate was decreased
after evolution for increased activity on a new substrate3,4. Plasmid transfer experiments into E. coli NM522 showed that all of the increased resistance was conferred by the plasmid. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis of periplasmic extracts showed that the expression level of the four
wild-type cephalosporinases was indistinguishable from that of the mutant clones A and B (data not shown). Antibiotic bioassays showed that all of the moxalactam was degraded by cells expressingchimaeric enzyme A or B, but not by cells expressing any of the four wild-type enzymes. This indicates that the specific activity of the
chimaeric moxalactamases was improved. However, our attempts to obtain and compare the kinetic profiles of the wild-type and chimaeric enzymes failed because of the very low activity of the
wild-type enzymes. The genes encoding the new moxalactamases from these two clones were sequenced. Clones A and B were both found to be chimaeras of the genes from Citrobacter, Enterobacter and Klebsiella. Both clones had a similar overall structure containing eight segments resulting from seven crossovers. Because of local DNA
homology the crossover location could not be defined more exactly than indicated by the grey segments shown in The crossovers occurred in areas where the two genes had 14–37 base pairs (bp) of nearly identical sequence (average 57% GC). In addition,chimaera A had 47 DNA point mutations, 6 of which were silent, resulting in 33 amino-acid substitutions scattered throughout the gene that did not exist in any of the four parental enzymes.