Lactococcus lactis NZ9010 in which the las operon-encoded ldh gene was replaced with an erythromycin resistance gene cassette displayed a stable phenotype when grown under aerobic conditions, and its main end products of fermentation under these conditions were acetate and acetoin. However, under anaerobic conditions, the growth of these cells was strongly retarded while the main end products of fermentation were acetate and ethanol. Upon prolonged subculturing of this strain under anaerobic conditions, both the growth rate and the ability to produce lactate were recovered after a variable number of generations. This recovery was shown to be due to the transcriptional activation of a silent ldhB gene coding for an Ldh protein (LdhB) with kinetic parameters different from those of the native las operon-encoded Ldh protein. Nevertheless, cells producing LdhB produced mainly lactate as the end product of fermentation. The mechanism underlying the ldhB gene activation was primarily studied in a single-colony isolate of the recovered culture, designated L. lactis NZ9015. Integration of IS981 in the upstream region of ldhB was responsible for transcription activation of the ldhB gene by generating an IS981-derived ؊35 promoter region at the correct spacing with a natively present ؊10 region. Subsequently, analysis of 10 independently isolated lactate-producing derivatives of L. lactis NZ9010 confirmed that the ldhB gene is transcribed in all of them. Moreover, characterization of the upstream region of the ldhB gene in these derivatives indicated that site-specific and directional IS981 insertion represents the predominant mechanism of the observed recovery of the ability to produce lactate.Homolactic fermentation by lactic acid bacteria involves the classical Embden-Meyerhoff-Parnas pathway leading to pyruvate, which is converted to lactic acid by lactate dehydrogenase. This enzyme and the gene that encodes it have been studied in many lactic acid bacteria, including Lactococcus lactis (11, 34), Streptococcus thermophilus (19), and various lactobacilli (2,15,47,51). L. lactis is the best-studied representative of this group, and the complete and partial genomes of several strains have been determined (4, 29). The gene encoding L. lactis Ldh was identified and characterized by Llanos and coworkers in 1992 (33, 34). The ldh gene is the last gene of the so-called lactic acid synthesis or las operon, which also encodes the glycolytic enzymes phosphofructokinase and pyruvate kinase. Transcription of the las operon was shown to yield a polycistronic transcript encompassing all three genes. But under some conditions, transcripts representing only two genes (pfk and pyk or pyk and ldh) or even a single gene (ldh) of the operon were also detected, which probably resulted from RNA processing upstream of the pyk and ldh genes (38). It has been shown that the las operon is subject to CcpAmediated carbon catabolite transcriptional activation, and a CcpA target site (cre sequence) was found within the las promoter region (38). The...

Acetoin reductase (ACR) catalyzes the conversion of acetoin to 2,3-butanediol. Under certain conditions, Clostridium acetobutylicum ATCC 824 (and strains derived from it) generates both D-and L-stereoisomers of acetoin, but because of the absence of an ACR enzyme, it does not produce 2,3-butanediol. A gene encoding ACR from Clostridium beijerinckii NCIMB 8052 was functionally expressed in C. acetobutylicum under the control of two strong promoters, the constitutive thl promoter and the late exponential adc promoter. Both ACR-overproducing strains were grown in batch cultures, during which 89 to 90% of the natively produced acetoin was converted to 20 to 22 mM D-2,3-butanediol. The addition of a racemic mixture of acetoin led to the production of both D-2,3-butanediol and meso-2,3-butanediol. A metabolic network that is in agreement with the experimental data is proposed. Native 2,3-butanediol production is a first step toward a potential homofermentative 2-butanol-producing strain of C. acetobutylicum.

Aldolases are enzymes with potential applications in biosynthesis, depending on their activity, specificity and stability. In the present study, the genomes of Sulfolobus species were screened for aldolases. Two new KDGA [2-keto-3-deoxygluconate (2-oxo-3-deoxygluconate) aldolases] from Sulfolobus acidocaldarius and Sulfolobus tokodaii were identified, overexpressed in Escherichia coli and characterized. Both enzymes were found to have biochemical properties similar to the previously characterized S. solfataricus KDGA, including the condensation of pyruvate and either D,L-glyceraldehyde or D,L-glyceraldehyde 3-phosphate. The crystal structure of S. acidocaldarius KDGA revealed the presence of a novel phosphate-binding motif that allows the formation of multiple hydrogen-bonding interactions with the acceptor substrate, and enables high activity with glyceraldehyde 3-phosphate. Activity analyses with unnatural substrates revealed that these three KDGAs readily accept aldehydes with two to four carbon atoms, and that even aldoses with five carbon atoms are accepted to some extent. Water-mediated interactions permit binding of substrates in multiple conformations in the spacious hydrophilic binding site, and correlate with the observed broad substrate specificity.

Acetoin reductase is an important enzyme for the fermentative production of 2,3-butanediol, a chemical compound with a very broad industrial use. Here, we report on the discovery and characterization of an acetoin reductase from Clostridium beijerinckii NCIMB 8052. An in silico screen of the C. beijerinckii genome revealed eight potential acetoin reductases. One of them (CBEI_1464) showed substantial acetoin reductase activity after expression in Escherichia coli. The purified enzyme (C. beijerinckii acetoin reductase [Cb-ACR]) was found to exist predominantly as a homodimer. In addition to acetoin (or 2,3-butanediol), other secondary alcohols and corresponding ketones were converted as well, provided that another electronegative group was attached to the adjacent C-3 carbon. Optimal activity was at pH 6.5 (reduction) and 9.5 (oxidation) and around 68°C. Cb-ACR accepts both NADH and NADPH as electron donors; however, unlike closely related enzymes, NADPH is preferred (K m , 32 M). Cb-ACR was compared to characterized close homologs, all belonging to the "threonine dehydrogenase and related Zndependent dehydrogenases" (COG1063). Metal analysis confirmed the presence of 2 Zn 2؉ atoms. To gain insight into the substrate and cofactor specificity, a structural model was constructed. The catalytic zinc atom is likely coordinated by Cys 37 , His 70 , and Glu 71 , while the structural zinc site is probably composed of Cys 100 , Cys 103 , Cys 106 , and Cys 114 . Residues determining NADP specificity were predicted as well. The physiological role of Cb-ACR in C. beijerinckii is discussed.

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Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase (SacKdgA) displays optimal activity at 95 degrees C and is studied as a model enzyme for aldol condensation reactions. For application of SacKdgA at lower temperatures, a library of randomly generated mutants was screened for improved synthesis of 2-keto-3-deoxygluconate from pyruvate and glyceraldehyde at the suboptimal temperature of 50 degrees C. The single mutant SacKdgA-V193A displayed a threefold increase in activity compared with wild type SacKdgA. The increased specific activity at 40-60 degrees C of this mutant was observed, not only for the condensation of pyruvate with glyceraldehyde, but also for several unnatural acceptor aldehydes. The optimal temperature for activity of SacKdgA-V193A was lower than for the wild type enzyme, but enzymatic stability of the mutant was similar to that of the wild type, indicating that activity and stability were uncoupled. Valine193 has Van der Waals interactions with Lysine153, which covalently binds the substrate during catalysis. The mutation V193A introduced space close to this essential residue, and the increased activity of the mutant presumably resulted from increased flexibility of Lysine153. The increased activity of SacKdgA-V193A with unaffected stability demonstrates the potential for optimizing extremely thermostable aldolases for synthesis reactions at moderate temperatures.

Solvent-producing clostridia have attracted renewed scientific interest because of their potential to produce biofuels. Clostridium acetobutylicum ATCC824 and Clostridium beijerinckii NCIMB 8052 are the best-studied solventogenic species, and the availability of their entire genome sequences offered the possibility to compare both solventogenic species at the gene level. General genome features as well as COG (clusters of orthologous groups of proteins) categorizations were summarized. All genes coding for the various proteins of the main catabolic pathway were discussed and compared, including paralogous and orthologous genes. Whenever possible, the most likely catabolic gene was identified, based on available experimental data. Overall, for most enzymatic steps comparable genes were found in both genomes, although C. beijerinckii in general harbors more paralogs, resulting also in a relatively large genome size. Essential differences were found as well. C. acetobutylicum contains a pyruvate decarboxylase, while C. beijerinckii contains an Rnf cluster and a trimeric bifurcating hydrogenase. Moreover, in C. acetobutylicum most of the solventogenic Systems Biology of Clostridium Downloaded from www.worldscientific.com by NANYANG TECHNOLOGICAL UNIVERSITY on 08/26/15. For personal use only.