Characterization of a secondary uptake system for l-glutamate in Corynebacterium glutamicum

Abstract A new transport system for the uptake of l-glutamate was characterized in Corynebacterium glutamicum strain Δ glu, in which the previously described binding protein-dependent glutamate uptake system is not present. Kinetic characterization revealed a highly specific secondary transport system, dependent on sodium ions. Glutamate uptake showed Michaelis-Menten kinetics, with a K _m of 0.6 mM and a V _max of 15 nmol min⁻¹ (mg dw)⁻¹. For the co-transported sodium ions, a relatively low K _m of 3.3 mM was determined.     

Functional expression of the glutamate uptake system from Corynebacterium glutamicum in Escherichia coli

Abstract Glutamate uptake in the Gram-positive Corynebacterium glutamicum is mediated via a binding protein-dependent transport system, which is encoded by the gluABCD gene cluster. Cloning of these genes in an expression vector and subsequent transformation of the resulting plasmid allows different strains of the Gram-negative bacterium Escherichia coli to grow on glutamate as sole carbon and nitrogen source. However, overexpression of the glutamate uptake system results in growth inhibitory effects, probably due to the particular topology of the binding protein.     

Carrier-mediated acetate uptake in Corynebacterium glutamicum

Acetate is effectively taken up by whole cells of Corynebacterium glutamicum via a specific carrier with a pH optimum of 8. The K _m of acetate uptake was 50 μM and the V _max 25–35 nmol/mg dw min. The activation energy was determined to be 70 kJ/mol. Acetate uptake was competitively inhibited by propionate with a K _i of about 30 μM and blocked by addition of sulfhydryl reagents. The transport activity was clearly dependent on the membrane potential, but independent of the presence of Na⁺-ions. It is concluded that uptake of acetate proceeds by a secondary, proton coupled mechanism.     

Isoleucine uptake in Corynebacterium glutamicum ATCC 13032 is directed by the brnQ gene product

By complementation analysis of an isoleucine-uptake-deficient Escherichia coli strain, it was shown that a 1.6-kb HindIII-StuI fragment of Corynebacterium glutamicum ATCC 13032, located downstream of the aecD gene, encodes an isoleucine uptake system. Sequence analysis revealed that the complementing fragment carried an open reading frame, termed brnQ, that encodes a protein with sequence similarities to branched-chain amino acid carriers of gram-positive and gram-negative bacteria. The brnQ gene specifies a predominantly hydrophobic protein of 426 amino acid residues with a calculated molecular mass of 44.9 kDa. A topology prediction by neural network computer analysis suggests the existence of 12 hydrophobic segments that most probably form transmembrane α-helices. A C. glutamicum mutant strain harboring a defined deletion of brnQ in the chromosome showed a considerably lower isoleucine uptake rate of 0.04 nmol min^–1 mg (dry mass)^–1 as compared to the wild-type strain rate of 1.2 nmol min^–1 mg (dry mass)^–1. Overexpression of brnQ by means of a tac promotor resulted in an elevated uptake rate for isoleucine of 11.3 nmol min^–1 mg (dry mass)^–1. Evidently, the brnQ gene encodes the only transport system in C. glutamicum directing isoleucine uptake. Received: 16 October 1997 / Accepted: 27 November 1997     

Myo-inositol facilitators IolT1 and IolT2 enhance d-mannitol formation from d-fructose in Corynebacterium glutamicum

Reduction of d -fructose to d -mannitol by whole-cell biotransformation with recombinant resting cells of Corynebacterium glutamicum ATCC13032 requires the coexpression of mdh and fdh , which encode mannitol and formate dehydrogenases, respectively. However, d -mannitol formation is limited by the uptake of d -fructose in its unphosphorylated form, because additional expression of the sugar facilitator from Zymomonas mobilis resulted in a significantly increased productivity. Here we identified similarities of the myo -inositol transporters IolT1 and IolT2 of C. glutamicum to the sugar facilitator of Z. mobilis . The myo -inositol transporter genes were both individually overexpressed and deleted in recombinants expressing mdh and fdh . Biotransformation experiments showed that the presence and absence, respectively, of IolT1 and IolT2 significantly influenced d -mannitol formation, indicating a d -fructose transport capability of these transporters. For further evidence, a C. glutamicum Δ ptsF mutant unable to grow with d -fructose was complemented with a heterologous fructokinase gene. This resulted in restoration of growth with d -fructose. Using overexpressed iolT1, mdh and fdh , d -mannitol formation obtained with C. glutamicum was 34.2 g L⁻¹, as opposed to 16 g L⁻¹ formed by the strain overexpressing only mdh and fdh , showing the suitability of myo -inositol transporters for d -fructose uptake to obtain d -mannitol formation by whole-cell biotransformation with C. glutamicum .     

Characterization of phosphoenolpyruvate carboxykinase from Corynebacterium glutamicum

Abstract Phosphoenolpyruvate (PEP) carboxykinase is present in crude extracts of Corynebacterium glutamicum grown on both glucose and lactate. Preparation of PEP carboxykinase free from interfering PEP carboxylase and oxaloacetate decarboxylase showed an absolute dependence on divalent manganese and IDP for activity in the oxaloacetate (OAA) formation. Other diphosphate nucleotides could not substitute for IDP. The enzyme activity displayed Michaelis-Menten kinetics for the substrates PEP, IDP, KHCO₃, OAA and ITP with a K _m of 0.7 mM, 0.4 mM, 12 mM, 1.0 mM, and 0.5 mM, respectively. At the optimum pH of 6.6, 850 nmol of OAA were formed per min per mg of protein. ATP inhibited PEP carboxykinase in the OAA forming reaction for 60% at 0.1 mM, indicating that the enzyme mainly functions in gluconeogenesis.     

Isolation of the putP gene of Corynebacterium glutamicum and characterization of a low-affinity uptake system for compatible solutes

Corynebacterium glutamicum accumulates the compatible solutes proline, glycine betaine, and ectoine under conditions of high osmolality. Uptake of proline is mediated by both a high-affinity and a low-affinity secondary transport system. The low-affinity uptake system also accepts glycine betaine and ectoine as substrates. In the present study, the gene encoding the high-affinity proline uptake system PutP was isolated by heterologous complementation of Escherichia coli mutant strain WG389, which lacks the transport systems BetT, PutP, ProP, and ProU and is unable to synthesize proline and glycine betaine. This gene (putP) encodes a protein of 524 amino acids that shares identity with the proline transport systems PutP of E. coli, Staphylococcus aureus, Salmonella typhimurium, Haemophilus influenzae, and Klebsiella pneumoniae. Functional studies of PutP synthesized in E. coli mutant strain MKH13, which also lacks the transport systems for compatible solutes and is unable to synthesize glycine betaine, revealed that this carrier system is not regulated by the external osmolality on the level of activity. K _m values of 7.6 mM for proline and 1.3 mM for sodium as cotransported ion were determined. Deletion of the putP gene allowed the functional characterization of another proline uptake system with low affinity. Received: 27 February 1997 / Accepted: 24 April 1997     

代谢木糖重组谷氨酸棒杆菌的构建

为了使谷氨酸棒杆菌较好地利用木糖生产有机酸,将来自Escherichia coli K-12的木糖异构酶基因xylA构建到表达载体pXMJ19中,导入Corynebacterium glutamicum ATCC13032Δldh中,成功表达了该酶基因。结果表明:重组菌株在以木糖为唯一C源进行发酵时,木糖的消耗速率为0.54 g/(L·h),木糖异构酶比酶活约为0.54 U/mL;在以木糖和葡萄糖的混合糖为C源进行发酵时,菌株优先利用葡萄糖,在葡萄糖完全消耗后,菌株开始有效利用木糖;以木糖为唯一C源进行两阶段发酵时,琥珀酸的收率可达(0.62±0.003)g/g。     

谷氨酸棒杆菌1dh基因的敲除

谷氨酸棒杆菌中ldh基因编码乳酸脱氢酶,可催化丙酮酸转化生成乳酸.利用重叠延伸PCR的方法,获得中间缺失部分序列的dldh基因片段,将其与载体pk 18mobsacB连接,转化大肠杆菌感受态,筛选出阳性转化子后,转化谷氨酸棒杆菌ATCC 13032感受态细胞.分别在卡那霉素抗性平板及10％蔗糖平板上进行两次筛选,利用PCR方法鉴定,成功获得ldh基因缺失的谷氨酸棒杆菌突变株ATCC 13032-(4)ldh.应用荧光定量PCR检测,ATCC 13032-(z)ldh中的ldh基因在转录水平与野生型菌株ATCC 13032相比,相对表达量为O.ldh基因的敲除对菌株的生长造成了一定的影响.     

Molecular and biochemical characterization of mechanosensitive channels in Corynebacterium glutamicum

Database searches in the Corynebacterium glutamicum genome sequence revealed homologs of the mechanosensitive channels MscL and YggB of Escherichia coli. To elucidate the physiological role of these putative channels deletion mutants were constructed. Betaine efflux induced by osmotic downshock of the mscL deletion mutant was nearly identical to that of the wild-type, whereas the yggB deletion mutant showed a reduced efflux rate. Interestingly, the double deletion strain, which was expected to have an even more decreased capability of betaine excretion, had only a slightly reduced efflux rate compared to the wild-type and did not show an increased mortality after osmotic downshift. These results led to the hypothesis that C. glutamicum may possess a third type of mechanosensitive channel not related to the MscL and YggB/KefA families. Furthermore it is unlikely that an MscM-like activity is responsible for the betaine efflux, because of the high transport capacity detected in the double deletion mutant.     

Molecular Analysis of the Corynebacterium glutamicum Transketolase Gene

Transketolase is important in production of the aromatic amino acids in Corynebacterium glutamicum. The complete nucleotide sequence of the C. glutamicum transketolase gene has been identified. The DNA-derived protein sequence is highly similar to the transketolase of Mycobacterium tuberculosis, taxonomically related to C. glutamicum. The alignment of the N-terminus regions between both transketolases showed TTG to be the most probable start codon. Potential ribosomal binding and promoter regions were situated upstream from the TTG. The deduced amino acid sequence consists of 700 residues with a calculated molecular mass of 75 kDa, and contains all amino acid residues involved in cofactor and substrate binding in the well-characterized yeast transketolase sequence.     

L-精氨酸生物合成的代谢流量分析

建立并完善了谷氨酸棒杆菌GWY020及其2个逐步叠加不同遗传标记的突变株HUI821和GUI089合成L-精氨酸的中心代谢网络.分别测定了它们在特定培养时段(50 h～52 h)L-精氨酸等代谢物的胞外浓度,由此计算这一时段这些代谢物在发酵液中积累(或消耗)的速率,分别作出这3株菌在拟稳态下的代谢流量分布图,进而研究育种过程中不同遗传标记的叠加对代谢网络中L-精氨酸合成流量分布的影响.结果表明遗传标记的引入使流量分配发生了重大变化,节点处的流量分配朝着有利于L-精氨酸合成的方向改变.从代谢流量分析角度上,证明结构类似物抗性和敏感性突变是代谢流导向和设计育种的有效手段,代谢流量分析将成为设计育种的提供新思路.     

L-精氨酸生物合成的代谢流量分析

建立并完善了谷氨酸棒杆菌GWY020及其2个逐步叠加不同遗传标记的突变株HUI821和GUI089合成L-精氨酸的中心代谢网络。分别测定了它们在特定培养时段(50 h~52 h)L-精氨酸等代谢物的胞外浓度, 由此计算这一时段这些代谢物在发酵液中积累(或消耗)的速率, 分别作出这3株菌在拟稳态下的代谢流量分布图, 进而研究育种过程中不同遗传标记的叠加对代谢网络中L-精氨酸合成流量分布的影响。结果表明遗传标记的引入使流量分配发生了重大变化, 节点处的流量分配朝着有利于L-精氨酸合成的方向改变。从代谢流量分析角度上, 证明结构类似物抗性和敏感性突变是代谢流导向和设计育种的有效手段, 代谢流量分析将成为设计育种的提供新思路。     

Threonine diffusion and threonine transport in Corynebacterium glutamicum and their role in threonine production

Transmembrane threonine fluxes (i.e., uptake, diffusion, and carrier-mediated excretion) all contribut-ing to threonine production by a recombinant strain of Corynebacterium glutamicum, were analyzed and quantitated. A threonine-uptake carrier that transports threonine in symport with sodium ions was identified. Under production conditions (i.e., when internal threonine is high), this uptake system catalyzed predominantly threonine/threonine exchange. Threonine export via the uptake system was excluded. Threonine efflux from the cells was shown to comprise both carrier-mediated excretion and passive diffusion. The latter process was analyzed after inhibition of all carrier-mediated fluxes. Threonine diffusion was found to proceed with a first-order rate constant of 0.003 min^–1 or 0.004 μl min^–1 (mg dry wt.)^–1, which corresponds to a permeability of 8 × 10^–10 cm s^–1. According to this permeability, less than 10% of the efflux observed under optimal conditions takes place via diffusion, and more than 90% must result from the activity of the excretion carrier. In addition, the excretion carrier was identified by (1) inhibition of its activity by amino acid modifying reagents and (2) its dependence on metabolic energy in the form of the membrane potential. Activity of the excretion system depended on the membrane potential, but not on the presence of sodium ions. Threonine export in antiport against protons is proposed. Received: 25 August 1995 / Accepted: 18 October 1995     

High efficiency electroporation of intact Corynebacterium glutamicum cells

High-frequency electroporation of whole Corynebacterium glutamicum cells without enzymatic pretreatment was achieved. Under optimized conditions concerning growth stage, washing of cells, cell concentration and pulse parameter transformation efficiencies of far more than 10(7) transformants per microgram pWST4B plasmid DNA were reached. Using electroporation, linearised and subsequently religated plasmid as well as chimeric ligase reaction products were directly introduced into C. glutamicum with reasonable efficiencies. Electrotransformation efficiency was reduced about 10(5)-fold for plasmid DNA cycled through E. coli JM83. Restriction deficient mutants of C. glutamicum were isolated which could be efficiently transformed with foreign DNA.     

溶氧量与谷氨酸棒杆菌代谢

正自从1957年Kinoshita等首次描述谷氨酸棒杆菌(Corynebacterium glutamicum)为谷氨酸产生菌[1]以来,其已成为用于氨基酸生产的主要菌株。目前,全世界每年利用谷氨酸棒杆菌生产约100万t L-谷氨酸用于食品调味剂和约45万t L-赖氨酸用作食品添加剂[2]。通过谷氨酸棒状杆菌发酵获得谷氨酸的发酵水平已较高,通过进一步优化工艺来提高产量具有较大困难[3]。     

Structure of a GTP-dependent bacterial PEP-carboxykinase from Corynebacterium glutamicum

GTP-dependent phosphoenolpyruvate carboxykinase (PCK) is the key enzyme that controls the blood glucose level during fasting in higher animals. Here we report the first substrate-free structure of a GTP-dependent phosphoenolpyruvate (PEP) carboxykinase from a bacterium, Corynebacterium glutamicum (CgPCK). The protein crystallizes in space group P2₁ with four molecules per asymmetric unit. The 2.3 Å resolution structure was solved by molecular replacement using the human cytosolic PCK (hcPCK) structure (PDB ID: 1KHF) as the starting model. The four molecules in the asymmetric unit pack as two dimers, and is an artifact of crystal packing. However, the P-loop and the guanine binding loop of the substrate-free CgPCK structure have different conformations from the other published GTP-specific PCK structures, which all have bound substrates and/or metal ions. It appears that a change in the P-loop and guanine binding loop conformation is necessary for substrate binding in GTP-specific PCKs, as opposed to overall domain movement in ATP-specific PCKs.