Production of optically pure D-lactic acid in mineral salts medium by metabolically engineered Escherichia coli W3110.

The resistance of polylactide to biodegradation and the physical properties of this polymer can be controlled by adjusting the ratio of L-lactic acid to D-lactic acid. Although the largest demand is for the L enantiomer, substantial amounts of both enantiomers are required for bioplastics. We constructed derivatives of Escherichia coli W3110 (prototrophic) as new biocatalysts for the production of D-lactic acid. These strains (SZ40, SZ58, and SZ63) require only mineral salts as nutrients and lack all plasmids and antibiotic resistance genes used during construction. D-Lactic acid production by these new strains approached the theoretical maximum yield of two molecules per glucose molecule. The chemical purity of this D-lactic acid was approximately 98% with respect to soluble organic compounds. The optical purity exceeded 99%. Competing pathways were eliminated by chromosomal inactivation of genes encoding fumarate reductase (frdABCD), alcohol/aldehyde dehydrogenase (adhE), and pyruvate formate lyase (pflB). The cell yield and lactate productivity were increased by a further mutation in the acetate kinase gene (ackA). Similar improvements could be achieved by addition of 10 mM acetate or by an initial period of aeration. All three approaches reduced the time required to complete the fermentation of 5% glucose. The use of mineral salts medium, the lack of antibiotic resistance genes or plasmids, the high yield of D-lactate, and the high product purity should reduce costs associated with nutrients, purification, containment, biological oxygen demand, and waste treatment.

N-terminal truncations in the FhlA protein result in formate- and MoeA-independent expression of the hyc (formate hydrogenlyase) operon of Escherichia coli.

The formate hydrogenlyase complex of Escherichia coli catalyses the cleavage of formate to CO2 and H2 and consists of a molybdoenzyme formate dehydrogenase-H, hydrogenase 3 and intermediate electron carriers. The structural genes of this enzyme complex are activated by the FhlA protein in the presence of both formate and molybdate; ModE-Mo serves as a secondary activator. Mutational analysis of the FhlA protein established that the unique N-terminal region of this protein was responsible for formate- and molybdenum-dependent transcriptional control of the hyc operon. Analysis of the N-terminal sequence of the FhlA protein revealed a unique motif (amino acids 7-37), which is also found in ATPases associated with several members of the ABC-type transporter family. A deletion derivative of FhlA lacking these amino acids (FhlA9-2) failed to activate the hyc operon in vivo, although the FhlA9-2 did bind to hyc promoter DNA in vitro. The ATPase activity of the FhlA9-2-DNA-formate complex was at least three times higher than that of the native protein-DNA-formate complex, and this degree of activity was achieved at a lower formate level. Extending the deletion to amino acid 117 (FhlA167) not only reversed the FhlA(-) phenotype of FhlA9-2, but also led to both molybdenum- and formate-independence. Deleting the entire N-terminal domain (between amino acids 5 and 374 of the 692 amino acid protein) also led to an effector-independent transcriptional activator (FhlA165), which had a twofold higher level of hyc operon expression than the native protein. Both FhlA165 and FhlA167 still required ModE-Mo as a secondary activator for an optimal level of hyc-lac expression. The FhlA165 protein also had a twofold higher affinity to hyc promoter DNA than the native FhlA protein, while the FhlA167 protein had a significantly lower affinity for hyc promoter DNA in vitro. Although the ATPase activity of the native protein was increased by formate, the ATPase activity of neither FhlA165 or FhlA167 responded to formate. Removal of the first 117 amino acids of the FhlA protein appears to result in a constitutive, effector-independent activation of transcription of the genes encoding the components of the formate hydrogenlyase complex. The sequence similarity to ABC-ATPases, combined with the properties of the FhlA deletion proteins, led to the proposal that the N-terminal region of the native FhlA protein interacts with formate transport proteins, both as a formate transport facilitator and as a cytoplasmic acceptor.

Molybdate transport.

In both bacteria and archaea, molybdate is transported by an ABC-type transporter comprising three proteins, ModA (periplasmic binding protein), ModB (membrane protein) and ModC, the ATPase. The modABC operon expression is controlled by ModE-Mo. In the absence of the high-affinity molybdate transporter, molybdate is also transported by another ABC transporter which transports sulfate/thiosulfate as well as by a nonspecific anion transporter. Comparative analysis of the molybdate transport proteins in various bacteria and archaea is the focus of this review.

Transcriptional regulation of the moe (molybdate metabolism) operon of Escherichia coli.

Regulation of transcription of the Escherichia coli moe operon, which codes for proteins connecting molybdate metabolism, molybdopterin synthesis, and apomolybdoenzyme synthesis, was investigated. Expression of the moe operon was independent of genes coding for molybdate transport and Mo-cofactor biosynthesis. Expression of moeA-lacZ increased during anaerobic growth (2.5-fold over the aerobic value) and in the presence of nitrate and trimethylamine N-oxide (3.5- and 1.5-fold, respectively). The nitrate-dependent increase in moe expression required the NarL protein, while the anaerobiosis-dependent increase in moeA-lacZ expression required Arc proteins. ArcA-phosphate and not ArcA bound to the DNA upstream of moe, shifted the electrophoretic mobility of moe promoter DNA, and protected the DNA from DNase I hydrolysis. Nitrate-independent transcription of moeA-lacZ was repressed by the FNR protein, which also protected moe operator DNA from DNase I hydrolysis. These results show that ArcA-phosphate and FNR have opposite effects on the transcriptional regulation of the moe operon, and the combined action of the two redox regulators modulate the level of Mo-cofactor in the cell. Apparently, the control of synthesis of Mo-cofactor and the apomolybdoenzymes nitrate reductase and trimethylamine N-oxide reductase are coupled at the level of the moe operon.

Long-term immunological memory induced by recombinant oral Salmonella vaccine vectors.

We have previously shown that Salmonella enterica serovar Typhimurium expressing the hagB hemagglutinin gene from Porphyromonas gingivalis can induce primary and recall immune responses in serum and secretions in mice; however, the longevity of memory induced by oral Salmonella carriers has not been adequately demonstrated. In this study, we examined the capacity of mice to mount a recall response 52 weeks after primary immunization. Recall responses were seen in serum immunoglobulin G (IgG) and IgA following boosting at week 52, and in most cases, they were equal to or greater than the primary responses. Significant mucosal IgA recall responses in saliva and vaginal wash were also detected following boosting at week 52. In addition, there was a considerable residual response in secretions at week 51, prior to boosting. These results indicate that oral Salmonella vectors can induce long-term memory to recombinant HagB and are particularly effective at inducing long-lasting mucosal responses as well as at inducing the capacity for mucosal recall responses.

Molybdate-dependent transcription of hyc and nar operons of Escherichia coli requires MoeA protein and ModE-molybdate.

In Escherichia coli, ModE-molybdate, a repressor of modABCD operon (molybdate transport), was previously shown to be an additional transcriptional activator of hyc operon (formate hydrogenlyase) and narGHJI operon (respiratory nitrate reductase). However, in a modE mutant, both operons were expressed at about 50% of the wild-type level in a molybdate-dependent manner. This ModE-independent, molybdate-dependent, expression of hyc, narG and narK operons required MoeA protein. An E. coli modE, moeA double mutant failed to produce formate hydrogenlyase or respiratory nitrate reductase activity irrespective of the growth medium. Tungstate substituted for molybdate in the activation of transcription of hyc and nar operons by ModE could not replace molybdate for MoeA-dependent expression. It is proposed that the MoeA-catalyzed product, an activated form of molybdate, interacts with a transcriptional activator/regulator other than ModE and regulates hyc and nar operons.

Physiological and genetic analyses leading to identification of a biochemical role for the moeA (molybdate metabolism) gene product in Escherichia coli.

A unique class of chlorate-resistant mutants of Escherichia coli which produced formate hydrogenlyase and nitrate reductase activities only when grown in medium with limiting amounts of sulfur compounds was isolated. These mutants failed to produce the two molybdoenzyme activities when cultured in rich medium or glucose-minimal medium. The mutations in these mutants were localized in the moeA gene. Mutant strains with polar mutations in moeA which are also moeB did not produce active molybdoenzymes in any of the media tested. moeA mutants with a second mutation in either cysDNCJI or cysH gene lost the ability to produce active molybdoenzyme even when grown in medium limiting in sulfur compounds. The CysDNCJIH proteins along with CysG catalyze the conversion of sulfate to sulfide. Addition of sulfide to the growth medium of moeA cys double mutants suppressed the MoeA- phenotype. These results suggest that in the absence of MoeA protein, the sulfide produced by the sulfate activation/reduction pathway combines with molybdate in the production of activated molybdenum. Since hydrogen sulfide is known to interact with molybdate in the production of thiomolybdate, it is possible that the MoeA-catalyzed activated molybdenum is a form of thiomolybdenum species which is used in the synthesis of molybdenum cofactor from Mo-free molybdopterin.