Lactate dehydrogenase gene variability among predominant lactate utilizing ruminal bacteria

The inter- and intraspecies variability of lactate dehydrogenase (ldh) gene was determined among the predominant ruminal lactate utilizing bacteria. Nearly complete nucleotide sequences of ldh gene, encoding NAD-dependent lactate dehydrogenase of three Megasphaera elsdenii and six Selenomonas ruminantium strains, were obtained and compared. Phylogenetic analyses revealed a limited variability between the ldh sequences studied. The majority of differences observed were silent mutations at the 3rd position of codons. Surprisingly, the intraspecies diversity of the ldh gene among S. ruminantium isolates was higher than the interspecies level between S. ruminantium and M. elsdenii, which strongly suggests the possibility of acquisition of this gene by horizontal gene transfer.     

Cloning, Sequence, and Expression of the L-(+) Lactate Dehydrogenase of Streptococcus bovis

The ldh gene encoding the fructose-1,6-diphosphate-dependent L-(+) lactate dehydrogenase from the ruminal bacterium Streptococcus bovis was cloned and sequenced. A genomic library of S. bovis JB1 DNA was constructed in lambda ZAP II and screened by use of a heterologous probe derived from the cloned Streptococcus mutans ldh gene. Several clones were isolated that contained a common 2.9-kb fragment as determined by restriction analysis. Nucleotide sequence analysis revealed a 987-bp open reading frame with extensive homology to Streptococcus thermophilus and S. mutans ldh nucleic acid and amino acid sequences. Expression of the cloned S. bovis ldh gene in Escherichia coli was confirmed by the ability to complement the ldh mutation of E. coli FMJ39, by using an in-gel activity screen and by enzymatic assay. Increased LDH activity was observed in S. bovis JB1 containing the cloned ldh genes on a multicopy plasmid. Received: 15 October 1996 / Accepted: 3 December 1996     

Metabolic Engineering of Bacillus subtilis for Ethanol Production: Lactate Dehydrogenase Plays a Key Role in Fermentative Metabolism

Wild-type Bacillus subtilis ferments 20 g/liter glucose in 48 h, producing lactate and butanediol, but not ethanol or acetate. To construct an ethanologenic B. subtilis strain, homologous recombination was used to disrupt the native lactate dehydrogenase (LDH) gene (ldh) by chromosomal insertion of the Zymomonas mobilis pyruvate decarboxylase gene (pdc) and alcohol dehydrogenase II gene (adhB) under the control of the ldh native promoter. The values of the intracellular PDC and ADHII enzymatic activities of the engineered B. subtilis BS35 strain were similar to those found in an ethanologenic Escherichia coli strain. BS35 produced ethanol and butanediol; however, the cell growth and glucose consumption rates were reduced by 70 and 65%, respectively, in comparison to those in the progenitor strain. To eliminate butanediol production, the acetolactate synthase gene (alsS) was inactivated. In the BS36 strain (BS35 ΔalsS), ethanol production was enhanced, with a high yield (89% of the theoretical); however, the cell growth and glucose consumption rates remained low. Interestingly, kinetic characterization of LDH from B. subtilis showed that it is able to oxidize NADH and NADPH. The expression of the transhydrogenase encoded by udhA from E. coli allowed a partial recovery of the cell growth rate and an early onset of ethanol production. Beyond pyruvate-to-lactate conversion and NADH oxidation, an additional key physiological role of LDH for glucose consumption under fermentative conditions is suggested. Long-term cultivation showed that 8.9 g/liter of ethanol can be obtained using strain BS37 (BS35 ΔalsS udhA⁺). As far as we know, this is the highest ethanol titer and yield reported with a B. subtilis strain.     

Double mutation of the <Emphasis Type="Italic">PDC1</Emphasis> and <Emphasis Type="Italic">ADH1</Emphasis> genes improves lactate production in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing the bovine lactate dehydrogenase gene

Expression of a heterologous l-lactate dehydrogenase (l-ldh) gene enables production of optically pure l-lactate by yeast Saccharomyces cerevisiae. However, the lactate yields with engineered yeasts are lower than those in the case of lactic acid bacteria because there is a strong tendency for ethanol to be competitively produced from pyruvate. To decrease the ethanol production and increase the lactate yield, inactivation of the genes that are involved in ethanol production from pyruvate is necessary. We conducted double disruption of the pyruvate decarboxylase 1 (PDC1) and alcohol dehydrogenase 1 (ADH1) genes in a S. cerevisiae strain by replacing them with the bovine l-ldh gene. The lactate yield was increased in the pdc1/adh1 double mutant compared with that in the single pdc1 mutant. The specific growth rate of the double mutant was decreased on glucose but not affected on ethanol or acetate compared with in the control strain. The aeration rate had a strong influence on the production rate and yield of lactate in this strain. The highest lactate yield of 0.75 g lactate produced per gram of glucose consumed was achieved at a lower aeration rate.     

Deletion of <Emphasis Type="Italic">ldh</Emphasis>A and <Emphasis Type="Italic">ald</Emphasis>H genes in <Emphasis Type="Italic">Klebsiella</Emphasis><Emphasis Type="Italic">pneumoniae</Emphasis> to enhance 1,3-propanediol production

Objectives

To improve 1,3-propanediol (1,3-PD) production and reduce byproduct concentration during the fermentation of Klebsiella pneumonia.

Results

Klebsiella. pneumonia 2-1ΔldhA, K. pneumonia 2-1ΔaldH and K. pneumonia 2-1ΔldhAΔaldH mutant strains were obtained through deletion of the ldhA gene encoding lactate dehydrogenase required for lactate synthesis and the aldH gene encoding acetaldehyde dehydrogenase involved in the synthesis of ethanol. After fed-batch fermentation, the production of 1,3-PD from glycerol was enhanced and the concentrations of byproducts were reduced compared with the original strain K. pneumonia 2-1. The maximum yields of 1,3-PD were 85.7, 82.5 and 87.5 g/l in the respective mutant strains.

Conclusion

Deletion of either aldH or ldhA promoted 1,3-PD production in K. pneumonia.

     

Native xylose-inducible promoter expands the genetic tools for the biomass-degrading,extremely thermophilic bacterium Caldicellulosiruptor bescii

Regulated control of both homologous and heterologous gene expression is essential for precise genetic manipulation and metabolic engineering of target microorganisms. However, there are often no options available for inducible promoters when working with non-model microorganisms. These include extremely thermophilic, cellulolytic bacteria that are of interest for renewable lignocellulosic conversion to biofuels and chemicals. In fact, improvements to the genetic systems in these organisms often cease once transformation is achieved. This present study expands the tools available for genetically engineering Caldicellulosiruptor bescii, the most thermophilic cellulose-degrader known growing up to 90 °C on unpretreated plant biomass. A native xylose-inducible (P _xi ) promoter was utilized to control the expression of the reporter gene (ldh) encoding lactate dehydrogenase. The P _xi -ldh construct resulted in a both increased ldh expression (20-fold higher) and lactate dehydrogenase activity (32-fold higher) in the presence of xylose compared to when glucose was used as a substrate. Finally, lactate production during growth of the recombinant C. bescii strain was proportional to the initial xylose concentration, showing that tunable expression of genes is now possible using this xylose-inducible system. This study represents a major step in the use of C. bescii as a potential platform microorganism for biotechnological applications using renewable biomass.

     

Modulation of Gene Expression Made Easy

A new approach for modulating gene expression, based on randomization of promoter (spacer) sequences, was developed. The method was applied to chromosomal genes in Lactococcus lactis and shown to generate libraries of clones with broad ranges of expression levels of target genes. In one example, overexpression was achieved by introducing an additional gene copy into a phage attachment site on the chromosome. This resulted in a series of strains with phosphofructokinase activities from 1.4 to 11 times the wild-type activity level. In this example, the pfk gene was cloned upstream of a gusA gene encoding β-glucuronidase, resulting in an operon structure in which both genes are transcribed from a common promoter. We show that there is a linear correlation between the expressions of the two genes, which facilitates screening for mutants with suitable enzyme activities. In a second example, we show that the method can be applied to modulating the expression of native genes on the chromosome. We constructed a series of strains in which the expression of the las operon, containing the genes pfk, pyk, and ldh, was modulated by integrating a truncated copy of the pfk gene. Importantly, the modulation affected the activities of all three enzymes to the same extent, and enzyme activities ranging from 0.5 to 3.5 times the wild-type level were obtained.     

Engineering Lactococcus lactis for Production of Mannitol: High Yields from Food-Grade Strains Deficient in Lactate Dehydrogenase and the Mannitol Transport System

Mannitol is a sugar polyol claimed to have health-promoting properties. A mannitol-producing strain of Lactococcus lactis was obtained by disruption of two genes of the phosphoenolpyruvate (PEP)-mannitol phosphotransferase system (PTS^Mtl). Genes mtlA and mtlF were independently deleted by double-crossover recombination in strain L. lactis FI9630 (a food-grade lactate dehydrogenase-deficient strain derived from MG1363), yielding two mutant (ΔldhΔmtlA and ΔldhΔmtlF) strains. The new strains, FI10091 and FI10089, respectively, do not possess any selection marker and are suitable for use in the food industry. The metabolism of glucose in nongrowing cell suspensions of the mutant strains was characterized by in vivo ¹³C-nuclear magnetic resonance. The intermediate metabolite, mannitol-1-phosphate, accumulated intracellularly to high levels (up to 76 mM). Mannitol was a major end product, one-third of glucose being converted to this hexitol. The double mutants, in contrast to the parent strain, were unable to utilize mannitol even after glucose depletion, showing that mannitol was taken up exclusively by PEP-PTS^Mtl. Disruption of this system completely blocked mannitol transport in L. lactis, as intended. In addition to mannitol, approximately equimolar amounts of ethanol, 2,3-butanediol, and lactate were produced. A mixed-acid fermentation (formate, ethanol, and acetate) was also observed during growth under controlled conditions of pH and temperature, but mannitol production was low. The reasons for the alteration in the pattern of end products under nongrowing and growing conditions are discussed, and strategies to improve mannitol production during growth are proposed.     

Properties and Role of Glyceraldehyde-3-Phosphate Dehydrogenase in the Control of Fermentation Pattern and Growth in a Ruminal Bacterium, <Emphasis Type="Italic">Streptococcus bovis</Emphasis>

To clarify the control of glycolysis and the fermentation pattern in Streptococcus bovis, the molecular and enzymatic properties of NAD⁺-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were examined. The GAPDH gene (gapA) was found to cluster with several others, including those that encode phosphoglycerate kinase and translation elongation factor G, however, gapA was transcribed in a monocistronic fashion. Since biochemical properties, such as optimal pH and affinity for glyceraldehyde-3-phosphate (GAP), were not very different between GAPDH- and NADP⁺-specific glyceraldehyde-3-phosphate dehydrogenase (GAPN), the flux from GAP may be greatly influenced by the relative amounts of these two enzymes. Using S. bovis JB1 as a parent, JB1gapA and JB1ldh, which overproduce GAPDH and lactate dehydrogenase (LDH), respectively, were constructed to examine the control of the glycolytic flux and lactate production. There were no significant differences in growth rates and formate-to-lactate ratios among JB1, JB1gapA, and JB1ldh grown on glucose. When grown on lactose, JB1ldh showed a much lower formate-to-lactate ratio than JB1gapA, which showed the highest NADH-to-NAD⁺ ratio. However, growth rates did not differ among JB1, JB1gapA, and JB1ldh. These results suggest that GAPDH is not involved in the control of the glycolytic flux and that lactate production is mainly controlled by LDH activity.     

Identification of the [FeFe]-Hydrogenase Responsible for Hydrogen Generation in Thermoanaerobacterium saccharolyticum and Demonstration of Increased Ethanol Yield via Hydrogenase Knockout

Three putative hydrogenase enzyme systems in Thermoanaerobacterium saccharolyticum were investigated at the genetic, mRNA, enzymatic, and phenotypic levels. A four-gene operon containing two [FeFe]-hydrogenase genes, provisionally termed hfs (hydrogenase-Fe-S), was found to be the main enzymatic catalyst of hydrogen production. hfsB, perhaps the most interesting gene of the operon, contains an [FeFe]-hydrogenase and a PAS sensory domain and has several conserved homologues among clostridial saccharolytic, cellulolytic, and pathogenic bacteria. A second hydrogenase gene cluster, hyd, exhibited methyl viologen-linked hydrogenase enzymatic activity, but hyd gene knockouts did not influence the hydrogen yield of cultures grown in closed-system batch fermentations. This result, combined with the observation that hydB contains NAD(P)+ and FMN binding sites, suggests that the hyd genes are specific to the transfer of electrons from NAD(P)H to hydrogen ions. A third gene cluster, a putative [NiFe]-hydrogenase with homology to the ech genes, did not exhibit hydrogenase activity under any of the conditions tested. Deletion of the hfs and hydA genes resulted in a loss of detectable methyl viologen-linked hydrogenase activity. Strains with a deletion of the hfs genes exhibited a 95% reduction in hydrogen and acetic acid production. A strain with hfs and ldh deletions exhibited an increased ethanol yield from consumed carbohydrates and represents a new strategy for engineering increased ethanol yields in T. saccharolyticum.Thermophilic anaerobic bacteria have long been of interest for studies of cellulosic biomass conversion due to their native hydrolytic and fermentative abilities (5, 33). However, all thermophilic anaerobes isolated to date have branched fermentation pathways which produce organic acids in addition to solvents such as ethanol (12). For cellulosic fuel production, significant yield loss is likely to be economically unfeasible (11).In their natural environments, saccharolytic fermentative bacteria participate in interspecies hydrogen transfer, producing hydrogen from carbohydrates that is rapidly consumed by methanogens and sulfate-reducing bacteria (30). As a result, the hydrogen partial pressure remains exceedingly low, allowing hydrogen (E₀′, −414 mV) to be produced not only from ferredoxin (E₀′, ∼−400 mV) but also from the less favorable electron source NAD(P)H (E₀′, −320 mV). Fermentative bacteria benefit from hydrogen production, because they are able to coproduce acetic acid and an additional ATP via acetate kinase (23). When grown in pure culture in a closed fermentation vessel, hydrogen is generated primarily from reduced ferredoxin, since generation from NAD(P)H becomes less favorable as the concentration of hydrogen increases (7).We have recently demonstrated high-yield ethanol production in the thermophilic anaerobe Thermoanaerobacterium saccharolyticum JW/SL-YS485 through deletion of the l-lactate dehydrogenase (ldh), phosphate acetyltransferase (pta), and acetate kinase (ack) genes (20). In addition to producing ethanol at high yield, this strain produced significantly less hydrogen, as is required to balance end product electron stoichiometry, although hydrogenase activity in cell extracts remained high. In this study, we used gene knockout to identify gene clusters that are implicated in hydrogenase activity in T. saccharolyticum and to identify the hfs gene operon, which is required for hydrogen production. The hfs operon contains a protein with [FeFe]-hydrogenase and PAS sensory domains that is conserved among a few members of the genera Clostridium and Thermoanaerobacter. Strains with hfs deletions showed decreased acetic acid production, and a strain with hfs and ldh deletions produced ethanol at an increased yield.     

Chimpanzee Malaria Parasites Related to Plasmodium ovale in Africa

Since the 1970''s, the diversity of Plasmodium parasites in African great apes has been neglected. Surprisingly, P. reichenowi, a chimpanzee parasite, is the only such parasite to have been molecularly characterized. This parasite is closely phylogenetically related to P. falciparum, the principal cause of the greatest malaria burden in humans. Studies of malaria parasites from anthropoid primates may provide relevant phylogenetic information, improving our understanding of the origin and evolutionary history of human malaria species. In this study, we screened 130 DNA samples from chimpanzees (Pan troglodytes) and gorillas (Gorilla gorilla) from Cameroon for Plasmodium infection, using cytochrome b molecular tools. Two chimpanzees from the subspecies Pan t. troglodytes presented single infections with Plasmodium strains molecularly related to the human malaria parasite P. ovale. These chimpanzee parasites and 13 human strains of P. ovale originated from a various sites in Africa and Asia were characterized using cytochrome b and cytochrome c oxidase 1 mitochondrial partial genes and nuclear ldh partial gene. Consistent with previous findings, two genetically distinct types of P. ovale, classical and variant, were observed in the human population from a variety of geographical locations. One chimpanzee Plasmodium strain was genetically identical, on all three markers tested, to variant P. ovale type. The other chimpanzee Plasmodium strain was different from P. ovale strains isolated from humans. This study provides the first evidence of possibility of natural cross-species exchange of P. ovale between humans and chimpanzees of the subspecies Pan t. troglodytes.     

Diacetyl and acetoin production from whey permeate using engineered <Emphasis Type="Italic">Lactobacillus casei</Emphasis>

The capability of Lactobacillus casei to produce the flavor-related compounds diacetyl and acetoin from whey permeate has been examined by a metabolic engineering approach. An L. casei strain in which the ilvBN genes from Lactococcus lactis, encoding acetohydroxyacid synthase, were expressed from the lactose operon was mutated in the lactate dehydrogenase gene (ldh) and in the pdhC gene, which codes for the E2 subunit of the pyruvate dehydrogenase complex. The introduction of these mutations resulted in an increased capacity to synthesize diacetyl/acetoin from lactose in whey permeate (1,400 mg/l at pH 5.5). The results showed that L. casei can be manipulated to synthesize added-value metabolites from dairy industry by-products.     

Metabolic engineering of Geobacillus thermoglucosidasius for high yield ethanol production

We describe the metabolic engineering of two strains of Geobacillus thermoglucosidasius to divert their fermentative carbon flux from a mixed acid pathway, to one in which ethanol becomes the major product. This involved elimination of the lactate dehydrogenase and pyruvate formate lyase pathways by disruption of the ldh and pflB genes, respectively, together with upregulation of expression of pyruvate dehydrogenase. Unlike the situation in Escherichia coli, pyruvate dehydrogenase is active under anaerobic conditions in thermophilic bacilli, but expressed sub-optimally for a role as the primary fermentation pathway. Mutants were initially characterised in batch culture using glucose as carbon substrate and strains with all three modifications shown to form ethanol efficiently and rapidly at temperatures in excess of 60 °C in yields in excess of 90% of theoretical. The strain containing the 3 modifications, TM242, was also shown to efficiently ferment cellobiose and a mixed hexose and pentose feed.