Molecular control mechanisms of lysine and threonine biosynthesis in amino acid-producing corynebacteria: Redirecting carbon flow

Abstract Threonine and lysine are two of the economically most important essential amino acids. They are produced industrially by species of the genera Corynebacterium and Brevibacterium . The branched biosynthetic pathway of these amino acids in corynebacteria is unusual in gene organization and in the control of key enzymatic steps with respect to other microorganisms. This article reviews the molecular control mechanisms of the biosynthetic pathways leading to threonine and lysine in corynebacteria, and their implications in the production of these amino acids. Carbon flux can be redirected at branch points by gene disruption of the competing pathways for lysine or threonine. Removal of bottlenecks has been achieved by amplification of genes which encode feedback resistant aspartokinase and homoserine dehydrogenase (obtained by in vitro directed mutagenesis).     

Efflux permease CgAcr3-1 of Corynebacterium glutamicum is an arsenite-specific antiporter

Resistance to arsenite (As(III)) by cells is generally accomplished by arsenite efflux permeases from Acr3 or ArsB unrelated families. We analyzed the function of three Acr3 proteins from Corynebacterium glutamicum, CgAcr3-1, CgAcr3-2, and CgAcr3-3. CgAcr3-1 conferred the highest level of As(III) resistance and accumulation in vivo. CgAcr3-1 was also the most active when everted membranes vesicles from Escherichia coli or C. glutamicum mutants were assayed for efflux with different energy sources. As(III) and antimonite (Sb(III)) resistance and accumulation studies using E. coli or C. glutamicum arsenite permease mutants clearly show that CgAcr3-1 is specific for As(III). In everted membrane vesicles expressing CgAcr3-1, dissipation of either the membrane potential or the pH gradient of the proton motive force did not prevent As(III) uptake, whereas dissipation of both components eliminated uptake. Further, a mutagenesis study of CgAcr3-1 suggested that a conserved cysteine and glutamate are involved in active transport. Therefore, we propose that CgAcr3-1 is an antiporter that catalyzes arsenite-proton exchange with residues Cys129 and Glu305 involved in efflux.     

Identification, characterization, and chromosomal organization of the ftsZ gene from Brevibacterium lactofermentum

The ftsZ gene was cloned from the chromosomal DNA of Brevibacterium lactofermentum by the polymerase chain reaction (PCR) using two oligonucleotides designed from two conserved regions found in most of the previously cloned and sequenced ftsZ genes from other microorganisms. ftsZ is a single-copy gene in corynebacteria and is located downstream from ftsQ and murC, indicating linkage between genes involved in peptidoglycan synthesis (mur genes) and genes involved in cell division (fts genes). The organisation of the cluster is similar to that in Streptomyces and different from those of Escherichia coli or Bacillus subtilis because ftsA is not located upstream of ftsZ. The gene was expressed in E. coli using the T7 expression system; the calculated molecular weight of the expressed protein was 50 kDa. Expression of the B. lactofermentum ftsZ gene in E. coli inhibited cell division and led to filamentation. The ftsZ gene of this organism does not complement ftsZ mutations or deletions in E. coli, when cloned on low or high-copy-number vectors. Received: 14 January 1998 / Accepted: 31 March 1998     

Expression of Streptomyces genes encoding extracellular enzymes in Brevibacterium lactofermentum: secretion proceeds by removal of the same leader peptide as in Streptomyces lividans

The -amylase gene (amy) from Streptomyces griseus IMRU 3570 and the -galactosidase gene (lac) from S. lividans were subcloned into Brevibacterium lactofermentum or B. lactofermentum/Escherichia coli shuttle vectors. The amy gene was not expressed in B. lactofermentum from its own promoter but was efficiently expressed when the promoter of the kanamycin resistance gene (kan) was inserted upstream of the promoterless amylase gene. The lac gene from S. lividans was subcloned without its native promoter and was expressed when placed downstream of pBL1 promoters P₂ or P₃. The -amylase was secreted extracellularly by removal of the same 28-amino acid leader peptide as in S. lividans. The amy and lac genes provide useful markers for selection of transformants and will facilitate the study of protein secretion in B. lactofermentum. Correspondence to: J. F. Martín     

O-Mycoloylated Proteins from Corynebacterium: AN UNPRECEDENTED POST-TRANSLATIONAL MODIFICATION IN BACTERIA*

O-Acylation of proteins was known only in a few eukaryotic proteins but never in bacteria. We demonstrate, using a combination of protein chemistry and mass spectrometry, the occurrence of three O-acylated polypeptides in Corynebacterium glutamicum, PorA, PorH, and an unknown small protein. The three polypeptides are O-substituted by mycolic acids, long chain α-alkyl and β-hydroxy fatty acids specifically produced by members of the Corynebacterineae suborder. To date these acids were described only as esterifying trehalose and arabinogalactan, and less frequently glycerol, important components of the highly impermeable outer barrier of Corynebacterineae. We show that the post-translational mycoloylation of PorA occurs at Ser-15 and is necessary for the pore-forming activity of C. glutamicum.     

从白喉棒状杆菌PW8株中筛选高产毒株

Corynebacterium diphtheria PW8-weissensee was grown on different mediafor screening high toxin produce. Through six times of screening (selecting butyrousbrilliant smooth small colonies and teshng their toxin production capacity) fromoriginal strain producing toxin l60Lf/ ml, a high toxin- producing descendandculture was obtained, which produced 79% more toxin than the original strain did.     

Interspecies electro-transformation in Corynebacteria

Plasmid DNA was efficiently electro-transformed into intact cells of nine Corynebacteria strains belonging to Brevibacterium lactofermentum, Brevibacterium flavum, Corynebacterium glutamicum and Corynebacterium melassecola. Relationships were explored between transformation efficiency and parameters such as electric field strength and pulse length, DNA concentration, physiological state and concentration of the cells. In optimal conditions, more than 10(7) transformants per microgram of DNA could be obtained. Electro-transformation with plasmid DNA isolated from different sources indicates that DNA modification may play a role in transformation efficiency.     

Production of malodorous steroids from androsta-5,16-dienes and androsta-4,16-dienes by Corynebacteria and other human axillary bacteria

The biotransformations of a number of steroids, chiefly 5,6,16,17-tetradehydro-androstanes, are reported. The strains investigated were Corynebacteria sp. G38, G40, G41, B, Brevis sp. CW5 and Micrococcus sp. M-DH2. Corynebacterium sp. G41 proved remarkably efficient in effecting oxidative isomerisation of 5-ene-3-sterols into the corresponding 4-en-3-ones. The main biochemical reactions involved were oxidation at C-3; no reduction processes were observed. Conversions of 3β-sterols into the C-3 oxo-steroids were high, but were correspondingly low for the 3-sterol epimers. Androsta-4,16-dien-3-one and 5β-androsta-16-en-3-one are crucial to the formation of malodour. The rate of formation of these compounds was measured over 72 h incubation periods using three substrates: androsta-5,16-dien-3β-ol, androsta-4,16-dien-3β-ol and androsta-5,16-dien-3-one. Induction studies of the transformation of the androsta-5,16-dien-3β-ol into the very odorous compound androsta-4,16-dien-3-one showed that cells incubated with a mixture of antibiotics displayed the same extent of biotransformation as normal cells if the concentration of antibiotic was low (1, 3, 5 and 7 μg/ml), although at concentrations higher than 10 μg/ml, biotransformation yields were reduced. Pre-incubation with a 3β-fluoro-steroid inhibited the formation of the odorous androsta-4,16-dien-3-one.     

钝齿棒杆菌CD945丙酮酸羧化酶基因的克隆、序列分析及表达

以钝齿棒杆菌（Corynebacterium crenatum）突变株CD945的基因组为模板,运用PCR方法,扩增出丙酮酸羧化酶的基因片段。核苷酸序列分析结果表明,该片段全长3657bp,以GTG为起始密码子,编码一个ORF。该ORF的核酸序列与Corynebacterium glutamicum,Mycobacterium smegmatis以及Saccharomyces cerevisiae的丙酮酸羧化酶结构基因相比,相似性分别为98.22%、62.41%和49.61%。由ORF推导出的氨基酸序列与上述属种的丙酮酸羧化酶相比,同源性分别是99.30%、64.65%和44.04%。经证实对于酶的催化活性至关重要的一些保守区域,如ATP和生物素的结合位点等,在该氨基酸序列中都存在。将该基因片段转化钝齿棒杆菌（C. crenatum） CD945,利用CTAB处理细胞和苹果酸脱氢酶偶联测定相结合的方法,进行酶活力的分析,结果表明,重组子与供体菌相比,丙酮酸羧化酶的活力提高5倍。