Fungal Fermentation of Lignocellulosic Biomass for Itaconic and Fumaric Acid Production.

The production of high-value chemicals from natural resources as an alternative for petroleum-based products is currently expanding in parallel with biorefinery. The use of lignocellulosic biomass as raw material is promising to achieve economic and environmental sustainability. Filamentous fungi, particularly Aspergillus species, are already used industrially to produce organic acid as well as many enzymes. The production of lignocellulose-degrading enzymes opens the possibility for direct fungal fermentation towards organic acids such as itaconic acid (IA) and fumaric acid (FA). These acids have wide-range applications and potentially addressable markets as platform chemicals. However, current technologies for the production of these compounds are mostly based on submerged fermentation. This work showed the capacity of two Aspergillus species (A. terreus and A. oryzae) to yield both acids by solid-state fermentation and simultaneous saccharification and fermentation. FA was optimally produced at by A. oryzae in simultaneous saccharification and fermentation (0.54 mg/g wheat bran). The yield of 0.11 mg IA/g biomass by A. oryzae is the highest reported in the literature for simultaneous solid-state fermentation without sugar supplements.

HstK, a cyanobacterial protein with both a serine/threonine kinase domain and a histidine kinase domain: implication for the mechanism of signal transduction.

Two distinct families of protein kinases are involved in signal transduction: Ser, Thr and Tyr kinases, which are predominantly found among eukaryotes, and His kinases, as part of bacterial two-component signalling systems. Genetic studies in Arabidopsis and Saccharomyces have demonstrated that bacterial-type two-component systems may act upstream of Ser/Thr kinases in the same signalling pathway, but how this coupling is accomplished remains unclear. In the present study, we report the characterization of a protein kinase, HstK, from the N(2)-fixing cyanobacterium Anabaena sp. PCC 7120, that possesses both a Ser/Thr kinase domain and a His kinase domain. Proteins with a structural architecture similar to that of HstK can be found in the eukaryote, Schizosaccharomyces pombe, and the bacterium, Rhodococcus sp. M5. HstK was present in cells grown with NH(4)(+) or N(2) as the nitrogen source, but was absent in cells grown with NO(3)(-). The hstK gene was inactivated and the mutant phenotype was characterized. The catalytic domain of the Ser/Thr kinase of HstK functionally replaced that of Hog1p, a well-characterized protein kinase required for the response to high osmolarity in the S. cerevisiae heterologous system. The unusual multidomain structure of HstK suggests that a two-component system could be directly coupled to Ser/Thr kinases in the same signal transduction pathway.

Characterization of PknC, a Ser/Thr kinase with broad substrate specificity from the cyanobacterium Anabaena sp. strain PCC 7120.

Eukaryotic-like protein Ser/Thr and Tyr kinases have only recently been discovered in prokaryotes. In most cases, their biochemical properties have been poorly characterized. The nitrogen-fixing and heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 houses a family of eukaryotic-like Ser/Thr kinases. Some of these enzymes are required for cell growth or development under certain conditions. None of them, however, has been shown experimentally to possess Ser/Thr kinase activity. A gene, pknC, encoding a novel putative Ser/Thr kinase was isolated from Anabaena sp. PCC 7120. The recombinant PknC was shown to be phosphorylated on a Thr residue. This phosphorylation was probably due to the autophosphorylation activity of PknC itself because mutation of two amino acid residues within the subdomain II of its catalytic domain eliminated the phosphorylation of PknC. PknC displayed also a Ser kinase activity towards several nonspecific substrates, and the two residues needed for PknC autophosphorylation was equally required for the phosphorylation of other substrates. PknC is thus a Ser/Thr kinase with broad substrate specificity. The activity of PknC is likely to be regulated in vivo in order to limit the spectrum of its substrate specificity.

Cloning of Schizosaccharomyces pombe bio2 by heterologous complementation of a Saccharomyces cerevisiae mutant.

A Saccharomyces cerevisiae mutant affected in the last step of the biotin biosynthesis pathway was isolated by using a transposon mutagenesis method. The gene BIO2, encoding a biotin synthase, is shown to be interrupted in this mutant. Heterologous complementation experiment allowed the cloning and the characterization of a novel bio gene: bio2, encoding biotin synthase from Schizosaccharomyces pombe.

Characterization of the biotin biosynthesis pathway in Saccharomyces cerevisiae and evidence for a cluster containing BIO5, a novel gene involved in vitamer uptake.

An engineered mutant of Saccharomyces cerevisiae affected in biotin biosynthesis has been isolated. This mutant allowed the characterization of a bio cluster (BIO3-4-5). We demonstrate that BIO3 (YNR058w) and BIO4 (YNR057c) encode, respectively, a 7, 8-diaminopelargonic acid aminotransferase and a dethiobiotin synthase, involved in the biotin biosynthesis pathway. A novel gene, BIO5 (YNR056c), is present immediately downstream from BIO4. This gene encodes Bio5p, a protein with 11 putative transmembrane regions. Uptake experiments performed with labeled 7-keto 8-aminopelargonic acid indicate that Bio5p is responsible for transport into the cell of 7-keto 8-aminopelargonic acid.

Survey, analysis and genetic organization of genes encoding eukaryotic-like signaling proteins on a cyanobacterial genome.

Bacteria usually use two-component systems for signal transduction, while eukaryotic organisms employ Ser/Thr and Tyr kinases and phosphatases for the same purpose. Many prokaryotes turn out to harbor Ser/Thr and Tyr kinases, Ser/Thr and Tyr phosphatases, and their accessory components as well. The sequence determination of the genome of the cyanobacterium Synechocystis sp. strain PCC 6803 offers the possibility to survey the extent of such molecules in a prokaryotic organism. This cyanobacterium possesses seven Ser/Thr kinases, seven Ser/Thr and Tyr phosphatases, one protein kinase interacting protein, one protein kinase regulatory subunit and several WD40-repeat-containing proteins. The majority of the protein phosphatases presented in this study were previously reported as hypothetical proteins. We analyze here the structure and genetic organization of these ORFs in the hope of providing a guidance for their functional analysis. Unlike their eukaryotic counterparts, many of these genes are clustered on the chromosome, and this genetic organization offers the opportunity to study their possible interaction. In several cases, genes of two-component transducers are found within the same cluster as those encoding a Ser/Thr kinase or a Ser/Thr phosphatase; the implication for signal transduction mechanism will be discussed.

Diacetyl and acetoin production from the co-metabolism of citrate and xylose by Leuconostoc mesenteroides subsp. mesenteroides.

The co-metabolism of citrate plus xylose by Leuconostoc mesenteroides subsp. mesenteroides results in a growth stimulation, an increase in D-lactate and acetate production and repression of ethanol production. This correlated well with the levels of key enzymes involved. A partial repression of alcohol dehydrogenase and a marked stimulation of acetate kinase were observed. High citrate bioconversion yields in diacetyl plus acetoin were obtained at pH 5.2 in batch (11.5%) or in chemostat (up to 17.4%) culture. In contrast, no diacetyl or acetoin was detected in citrate plus glucose fermentation.

Purification, properties and DNA sequence of the D-lactate dehydrogenase from Leuconostoc mesenteroides subsp. cremoris.

The complete sequence of the D-lactate dehydrogenase (D-ldh) gene from Leuconostoc mesenteroides subsp. cremoris, cloned in Escherichia coli, were determined. The deduced amino acid sequence showed homologies with all members of the D-specific-2-hydroxyacid dehydrogenase family. Furthermore, the essential residues detected so far as being involved in catalysis were also conserved. Purification of the enzyme revealed physico-chemical properties corresponding to those predicted from the sequence. The active enzyme was a dimer of 40-kDa subunits. The Km values for pyruvate, lactate, NADH and NAD were 0.3, 19, 0.03 and 0.16 mM, indicating that the enzyme reduced pyruvate in vivo. Besides the D-LDH activity, L. mesenteroides subsp. cremoris also displayed HicDH enzymatic activity, catalysing the reduction of pyruvate analogs. The purified D-LDH displayed low HicDH-type activity; therefore, differences in specificity profiles between the crude extract and the purified enzyme suggested the occurrence of a specific HicDH.

Purification and properties of the alpha-acetolactate decarboxylase from Lactococcus lactis subsp. lactis NCDO 2118.

alpha-Acetolactate decarboxylase from Lactococcus lactis subsp. lactis NCDO 2118 was expressed at low levels in cell extracts and was also unstable. The purification was carried out from E. coli in which the enzyme was expressed 36-fold higher. The specific activity was 24-fold enhanced after purification. The main characteristics of alpha-acetolactate decarboxylase were: (i) activation by the three branched chain amino acids leucine, valine and isoleucine; (ii) allosteric properties displayed in absence and Michaelis kinetics in the presence of leucine. The enzyme is composed of six identical subunits of 26,500 Da.