L-phenylalanine (L-Phe) is an aromatic amino acid with diverse commercial applications. Technologies for industrial microbial synthesis of L-Phe using glucose as a starting raw material currently achieve a relatively low conversion yield (Y(Phe/Glc)). The purpose of this work was to study the effect of PTS (phosphotransferase transport system) inactivation and overexpression of different versions of feedback inhibition resistant chorismate mutase-prephenate dehydratase (CM-PDT) on the yield (Y(Phe/Glc)) and productivity of L-Phe synthesized from glucose. The E. coli JM101 strain and its mutant derivative PB12 (PTS(-)Glc(+) phenotype) were used as hosts. PB12 has an inactive PTS, but is capable of transporting and phosphorylating glucose by using an alternative system constituted by galactose permease (GalP) and glucokinase activities (Glk). JM101 and PB12 were transformed with three plasmids, harboring genes that encode for a feedback inhibition resistant DAHP synthase (aroG(fbr)), transketolase (tktA) and either a truncated CM-PDT (pheA(fbr)) or its derived evolved genes (pheA(ev1) or pheA(ev2)). Resting-cells experiments with these engineered strains showed that JM101 and PB12 strains expressing either pheA(ev1) or pheA(ev2) genes produced l-Phe from glucose with Y(Phe/Glc) of 0.21 and 0.33 g/g, corresponding to 38 and 60% of the maximum theoretical yield (0.55 g/g), respectively. In addition, in both engineered strains the reached q(Phe) high levels of 40 mg/g-dcw.h. The metabolic engineering strategy followed in this work, including a strain with an inactive PTS, resulted in a positive impact over the Y(Phe/Glc), enhancing it nearly 57% compared with its PTS(+) counterpart. This is the first report wherein PTS inactivation was a successful strategy to improve the Y(Phe/Glc).

The expression of genes transcribed by the RNA polymerase with the alternative sigma factor os4 ( E d 4 ) is absolutely dependent on activator proteins that bind to enhancer-like sites, located far upstream from the promoter. These unique prokaryotic proteins, known as enhancer-binding proteins (EBP), mediate open promoter complex formation in a reaction dependent on NTP hydrolysis. The best characterized proteins of this family of regulators are NtrC and NifA, which activate genes required for ammonia assimilation and nitrogen fixation, respectively. In a recent IRBM course ("Frontiers of protein structure prediction," IRBM, Pomezia, Italy, 1995; see web site http://www.mrc-cpe.cam.uk/ irbm-course95/), one of us (J.O.) participated in the elaboration of the proposal that the Central domain of the EBPs might adopt the classical mononucleotide-binding fold. This suggestion was based on the results of a new protein fold recognition algorithm (Map) and in the mapping of correlated mutations calculated for the sequence family on the same mononucleotide-binding fold topology. In this work, we present new data that support the previous conclusion. The results from a number of different secondary structure prediction programs suggest that the Central domain could adopt an alp topology. The fold recognition programs ProFIT 0.9, 3D PROFILE combined with secondary structure prediction, and 123D suggest a mononucleotide-binding fold topology for the Central domain amino acid sequence. Finally, and most importantly, three of five reported residue alterations that impair the Central domain ATPase activity of the E d 4 activators are mapped to polypeptide regions that might be playing equivalent roles as those involved in nucleotidebinding in the mononucleotide-binding proteins. Furthermore, the known residue substitutions that alter the function of the Eos4 activators, leaving intact the Central domain ATPase activity, are mapped on a region proposed to play an equivalent role as the effector region of the GTPase superfamily.

Circular permutation of proteins is a powerful technique to explore the importance of the polypeptide secondary structure order for attaining the final three-dimensional structure. Here, we designed a circular permutation of the TEM beta-lactamase in order to produce a new domain-forming amino acid arrangement in the polypeptide sequence. Closing the normal N- and C-termini with the connecting peptide GGS and creating new N- and C-termini at position 216, produces a severely impaired permuted protein. Introduction of a connector with random components allows the isolation of enzymes with better activities and indicates a selection for a potential helix-stop signal at the new super-secondary motif. We applied several directed-evolution cycles, starting from permuted enzymes with each of the two different connecting peptides, and selecting for antibiotic resistance and isolated several mutants with resistance levels close to those of the wild-type enzyme. We also analyze some of the data collected on the outcomes and paths of these evolutionary experiments. A purified sixth cycle variant with connector peptide GGS showed catalytic efficiency values approximately 8% of the natural enzyme.

A method to delete in-phase codons throughout a defined target region of a gene has been developed. This approach, named the codon-based random deletion (COBARDE) method, is able to delete complete codons in a random and combinatorial mode. Robustness, automation and fine-tuning of the mutagenesis rate are essential characteristics of the method, which is based on the assembly of oligonucleotides and on the use of two transient orthogonal protecting groups during the chemical synthesis. The performance of the method for protein function evolution was demonstrated by changing the substrate specificity of TEM-1 beta-lactamase. Functional ceftazidime-resistant beta-lactamase variants containing several deleted residues inside the catalytically important omega-loop region were found. The results show that the COBARDE method is a useful new molecular tool to access previously unexplorable sequence space.

BackgroundThe effect of single and multiple amino acid substitutions in the green fluorescent protein (GFP) from Aequorea victoria has been extensively explored, yielding several proteins of diverse spectral properties. However, the role of amino acid deletions in this protein -as with most proteins- is still unknown, due to the technical difficulties involved in generating combinatorial in-phase amino acid deletions on a target region.ResultsIn this study, the region I129-L142 of superglo GFP (sgGFP), corresponding to the longest loop of the protein and located far away from the central chromophore, was subjected to a random amino acid deletion approach, employing an in-house recently developed mutagenesis method termed Codon-Based Random Deletion (COBARDE). Only two mutants out of 16384 possible variant proteins retained fluorescence: sgGFP-Δ I129 and sgGFP-Δ D130. Interestingly, both mutants were thermosensitive and at 30°C sgGFP-Δ D130 was more fluorescent than the parent protein. In contrast with deletions, substitutions of single amino acids from residues F131 to L142 were well tolerated. The substitution analysis revealed a particular importance of residues F131, G135, I137, L138, H140 and L142 for the stability of the protein.ConclusionThe behavior of GFP variants with both amino acid deletions and substitutions demonstrate that this loop is playing an important structural role in GFP folding. Some of the amino acids which tolerated any substitution but no deletion are simply acting as "spacers" to localize important residues in the protein structure.