Direct production of L-lysine from raw corn starch by Corynebacterium glutamicum secreting Streptococcus bovis alpha-amylase using cspB promoter and signal sequence

Corynebacterium glutamicum is an important microorganism in the industrial production of amino acids. We engineered a strain of C. glutamicum that secretes α-amylase from Streptococcus bovis 148 (AmyA) for the efficient utilization of raw starch. Among the promoters and signal sequences tested, those of cspB from C. glutamicum possessed the highest expression level. The fusion gene was introduced into the homoserine dehydrogenase gene locus on the chromosome by homologous recombination. L-Lysine fermentation was conducted using C. glutamicum secreting AmyA in the growth medium containing 50 g/l of raw corn starch as the sole carbon source at various temperatures in the range 30 to 40°C. Efficient L-lysine production and raw starch degradation were achieved at 34 and 37°C, respectively. The α-amylase activity using raw corn starch was more than 2.5 times higher than that using glucose as the sole carbon source during L-lysine fermentation. AmyA expression under the control of cspB promoter was assumed to be induced when raw starch was used as the sole carbon source. These results indicate that efficient simultaneous saccharification and fermentation of raw corn starch to L-lysine were achieved by C. glutamicum secreting AmyA using the cspB promoter and signal sequence.     

Family of the major cold-shock protein, CspA (CS7.4), of Escherichia coli, whose members show a high sequence similarity with the eukaryotic Y-box binding proteins

The cspA is a gene of Escherichia coli, whose expression is specifically induced at low temperatures to a level of 13% of total protein synthesis. The CspA protein consisting of 70 amino add residues has high sequence similarity with eukaryotic Y-box DNA-binding proteins. We found two independent clones from the Kohara miniset phage collection, which hybridized with a DNA fragment containing cspA. DNA sequencing of these clones confirmed that the two genes are highly homologous to cspA. One designated cspB is mapped at 35 min on the E. coli chromosome and encodes a 71-residue protein with 79% identity to CspA, while the other, cspC, is mapped at 40 min and encodes a 69-residue protein with 70% identity. In addition, a DNA sequence upstream of the clpA gene at 19 mm published elsewhere contains an open reading frame for a 74-residue protein with 45% identity to CspA. All csp genes were fused in the coding regions with the lacZ gene, and the expression of β-galactosidase was examined for these hybrid genes upon cold shock. A similar cold-shock induction to cspA was observed for cspB but not cspC and cspD. These results Indicate that E. coli has a family of the cspA gene, some of which are induced by cold shock.     

Development of novel cell surface display in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> using porin

We have developed a novel cell surface display in Corynebacterium glutamicum using porin proteins as anchor proteins. Porins are localized at C. glutamicum mycolic acid layer and exist as a hexamer. We used α-amylase from Streptococcus bovis 148 (AmyA) as a model protein to be displayed on the C. glutamicum cell surface. AmyA was fused to the C terminus of the porins PorB, PorC, or PorH. Expression vectors using fused proteins under the control of the cspB promoter were constructed and introduced into the C. glutamicum Cm strain. Immunostaining microscopy and flow cytometric analysis revealed that PorB-AmyA, PorC-AmyA, and PorH-AmyA were displayed on the C. glutamicum cell surface. AmyA activity was only detected in the cell fraction of C. glutamicum cells that displayed AmyA fused to PorB, PorC or PorH and AmyA activity was not detected in the supernatants of C. glutamicum culture broths after 72 h cultivation. Thus, we have demonstrated that C. glutamicum porins are very efficient anchor proteins for protein display in C. glutamicum.     

A Corynebacterium glutamicum rnhA recG Double Mutant Showing Lysozyme- sensitivity,Temperature-sensitive Growth,and UV-Sensitivity

Corynebacterium glutamicum mutant KY9707 was originally isolated for lysozyme-sensitivity, and showed temperature-sensitive growth. Two DNA fragments from a wild-type C. glutamicum chromosomal library suppressed the temperature-sensitivity of KY9707. These clones also rescued the lysozyme-sensitivity of KY9707, although partially. One of them encodes a protein of 382 amino acid residues, the N-terminal domain of which was homologous to RNase HI. This gene suppressed the temperature-sensitive growth of an Escherichia coli rnhA rnhB double mutant. We concluded that this gene encodes a functional RNase HI of C. glutamicum and designated it as rnhA. The other gene encodes a protein of 707 amino acid residues highly homologous to RecG protein. The C. glutamicum recG gene complemented the UV-sensitivity of E. coli recG258::kan mutant. KY9707 showed increased UV-sensitivity, which was partially rescued by either the recG or rnhA gene of C. gluamicum. Point mutations were found in both recG and rnhA genes in KY9707. These suggest that temperature-sensitive growth, UV-sensitivity, and probably lysozyme-sensitivity also, of KY9707 were caused by mutations in the genes encoding RNase HI and RecG.     

CspC and CspD are essential for Caulobacter crescentus stationary phase survival

The cold shock response in bacteria involves the expression of low-molecular weight cold shock proteins (CSPs) containing a nucleic acid-binding cold shock domain (CSD), which are known to destabilize secondary structures on mRNAs, facilitating translation at low temperatures. Caulobacter crescentus cspA and cspB are induced upon cold shock, while cspC and cspD are induced during stationary phase. In this work, we determined a new coding sequence for the cspC gene, revealing that it encodes a protein containing two CSDs. The phenotypes of C. crescentus csp mutants were analyzed, and we found that cspC is important for cells to maintain viability during extended periods in stationary phase. Also, cspC and cspCD strains presented altered morphology, with frequent non-viable filamentous cells, and cspCD also showed a pronounced cell death at late stationary phase. In contrast, the cspAB mutant presented increased viability in this phase, which is accompanied by an altered expression of both cspC and cspD, but the triple cspABD mutant loses this characteristic. Taken together, our results suggest that there is a hierarchy of importance among the csp genes regarding stationary phase viability, which is probably achieved by a fine tune balance of the levels of these proteins.     

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.     

Molecular cloning, sequencing, and expression in Escherichia coli of the gene encoding a novel 5-oxoprolinase without ATP-hydrolyzing activity from Alcaligenes faecalis N-38A

The gene encoding a novel 5-oxoprolinase without ATP-hydrolyzing activity from Alcaligenes faecalis N-38A was cloned and characterized. The coding region of this gene is 1,299 bp long. The predicted primary protein is composed of 433 amino acid residues, with a 31-amino-acid signal peptide. The mature protein is composed of 402 amino acid residues with a molecular mass of 46,163 Da. The derived amino acid sequence of the enzyme showed no significant sequence similarity to any other proteins reported so far. The 5-oxoprolinase gene was expressed in Escherichia coli by using a regulatory expression system with an isopropyl-beta-D-thiogalactopyranoside-inducible tac promoter, and its expression level was approximately 16 mg per liter. The purified enzyme has the same characteristics as the authentic enzyme, except for the amino terminus, which has three additional amino acids. The enzyme was markedly inhibited by p-chloromercuribenzoic acid, EDTA, o-phenanthroline, HgCl(2), and CuSO(4). The EDTA-inactivated enzyme was completely restored by the addition of Zn(2+) or Co(2+). In addition, the enzyme was found to contain 1 g-atom of zinc per mol of protein. These results suggest that the 5-oxoprolinase produced by A. faecalis N-38A is a zinc metalloenzyme.     

Cloning and nucleotide sequence of the csp1 gene encoding PS1, one of the two major secreted proteins of Corynebacterium glutamicum: the deduced N-terminal region of PS1 is similar to the Mycobacterium antigen 85 complex

Two proteins, PS1 and PS2, were detected in the culture medium of Corynebacterium glutamicum and are the major proteins secreted by this bacterium. No enzymatic activity was identified for either of the two proteins. Immunologically cross-reacting proteins were found in a variety of C. glutamicum strains but not in the coryneform Arthrobacter aureus. The gene encoding PS1, csp1, was cloned in lambda gt11 using polyclonal antibodies raised against PS1 to screen for producing clones. The csp1 gene was expressed in Escherichia coli, presumably from its own promoter, and directed the synthesis of two proteins recognized by anti-PS1 antibodies. The major protein band, of lower M(r), was detected in the periplasmic fraction. It had the same M(r) as the PS1 protein band detected in the supernatant of C. glutamicum cultures and presumably corresponds to the mature form of PS1. The minor protein band appears to be the precursor form of PS1. The nucleotide sequence of the csp1 gene was determined and contained an open reading frame encoding a polypeptide with a calculated molecular weight of 70,874, with a putative signal peptide with a molecular weight of 4411. This is consistent with the M(r) determined for PS1 from C. glutamicum culture supernatant and E. coli whole-cell extracts. The NH2-half of the deduced amino acid is similar (about 33% identical residues and 52% including similar residues) to the secreted antigen 85 protein complex of Mycobacterium. The csp1 gene in C. glutamicum was disrupted without any apparent effect on growth or viability.     

Molecular cloning and expression of a Tetrahymena pyriformis ubiquitin fusion gene coding for a 53-amino-acid extension protein.

Summary The genome of Tetrahymena pyriformis has been shown to contain a ubiquitin multigene family consisting of several polyubiquitin genes and at least one ubiquitin fusion gene. We report here the isolation and characterization of one genomic clone (pTUl1), that encodes a ubiquitin extension protein. A comparison of the predicted amino acid sequence of the ubiquitin extension protein gene of T. pyriformis with those from other organisms indicated a high degree of homology. However, the Tetrahymena ubiquitin extension protein contains 53 and not 52 amino acids. This feature is different from all ubiquitin 52-amino-acid extension protein genes thus far sequenced. Furthermore, we found an array of four cysteine residues similar to those found in nucleic acid binding proteins. Also, the C-terminal sequence possesses a conserved motif which may represent a nuclear translocation signal. The ubiquitin 53-amino-acid extension protein gene encodes the smallest class of ubiquitin mRNAs in T. pyriformis.     

Organization of the outer layers of the cell envelope of Corynebacterium glutamicum: A combined freeze-etch electron microscopy and biochemical study

The cell surface of Corynebacterium glutamicum grown on solid medium was totally covered with a highly ordered, hexagonal surface layer. Also, freeze-fracture revealed two fracture surfaces which were totally covered with ordered arrays displaying an hexagonal arrangement and the same unit cell dimension as the surface layer. The ordered arrays on the concave fracture surface, closest to the cell surface, were due to the presence of particles while those on the convex fracture surface were their imprints. The same cells grown on liquid medium displayed a cell surface and fracture surfaces only partially covered with ordered arrays. In this case, the ordered regions had the same relative position on the cell surface and on the fracture surfaces. All ordered arrays were totally absent in a mutant for cspB, the gene encoding PS2, one of the two major cell wall proteins. Treatment of the cells with proteinase K caused the gradual alteration of PS2 into a slightly lower molecular mass form. This was accompanied by a concomitant disappearance of the ordered fracture surfaces followed by the detachment of the ordered surface layer from the cell as large ordered patches displaying the same lattice symmetry and dimension as those of the surface layer. The ordered patches were isolated. They contained the totality of PS2 initially associated with the cell. We conclude that the highly ordered surface layer of the intact cell was composed of PS2 interacting strongly with some cell wall material leading to its organization. This organized cell wall material produced the ordered fracture surfaces. We show that in the absence of intact PS2 protein on the cell wall, the same cell wall material was not organized and formed a structureless smooth layer.     

Metabolic engineering of 1,2-propanediol pathways in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

We analyzed 1,2-propanediol (1,2-PD) production in metabolically engineered Corynebacterium glutamicum. Wild-type C. glutamicum produced 93 μM 1,2-PD after 132 h incubation under aerobic conditions. No gene encoding the methylglyoxal synthase (MGS) which catalyzes the first step of 1,2-PD synthesis from the glycolytic pathway was detected on the C. glutamicum genome, but several genes annotated as encoding putative aldo-keto reductases (AKRs) were present. AKR functions as a methylglyoxal reductase in the 1,2-PD synthesis pathway. Expressing Escherichia coli mgs gene in C. glutamicum increased 1,2-PD yield 100-fold, suggesting that wild-type C. glutamicum carries the genes downstream of MGS in the 1,2-PD synthesis pathway. Furthermore, simultaneous overexpression of mgs and cgR_2242, one of the genes annotated as AKRs, enhanced 1,2-PD production to 24 mM. This work establishes that 1,2-PD synthesis by C. glutamicum, previously unknown, is possible.     

The glycosylated cell surface protein Rpf2, containing a resuscitation-promoting factor motif,is involved in intercellular communication of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

The genome of Corynebacterium glutamicum ATCC 13032 contains two genes, rpf1 and rpf2, encoding proteins with similarities to the essential resuscitation-promoting factor (Rpf) of Micrococcus luteus. Both the Rpf1 (20.4 kDa) and Rpf2 (40.3 kDa) proteins share the so-called Rpf motif, a highly conserved protein domain of approximately 70 amino acids, which is also present in Rpf-like proteins of other gram-positive bacteria with a high G+C content of the chromosomal DNA. Purification of the C. glutamicum Rpf2 protein from concentrated supernatants, SDS-PAGE and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identified modified Rpf2 variants with increased or reduced mobility when compared with the calculated size of Rpf2. A Western blot-based enzyme immunoassay demonstrated glycosylation of the Rpf2 variants with higher molecular masses. Galactose and mannose were identified as two components of the oligosaccharide portion of the Rpf2 glycoprotein by capillary gas chromatography coupled to mass spectrometry. The Rpf2 protein was localized on the surface of C. glutamicum with the use of immuno-fluorescence microscopy. C. glutamicum strains with defined deletions in the rpf1 or rpf2 gene or simultaneous deletions in both rpf genes were constructed, indicating that the rpf genes are neither individually nor collectively essential for C. glutamicum. The C. glutamicum rpf double mutant displayed slower growth and a prolonged lag phase after transfer of long-stored cells into fresh medium. The addition of supernatant from exponentially growing cultures of the rpf double mutant, the wild type or C. glutamicum strains with increased expression of the rpf1 or rpf2 gene significantly reduced the lag phase of long-stored wild-type and rpf single mutant strains, but addition of purified His-tagged Rpf1 or Rpf2 did not. In contrast, the lag phase of the C. glutamicum rpf double mutant was not affected upon addition of these culture supernatants.     

海产品嗜冷菌波罗的海希瓦氏菌中3个冷激蛋白功能分析

【目的】波罗的海希瓦氏菌是冷藏海产品中常见的腐败菌,而该菌中关于冷激蛋白的功能研究尚未见报道。本研究从分子生物学角度分析波罗的海希瓦氏菌中3个冷激蛋白各自的功能。【方法】采用BEAST软件分析γ-变形菌纲中部分食源性微生物的冷激蛋白进化时间,接着利用实时荧光定量PCR方法检测波罗的海希瓦氏菌3个冷激蛋白基因的表达规律,进而构建3个冷激蛋白的基因敲除株,分析敲除株在不同温度和不同环境胁迫条件下的生长状况、群体感应现象以及致腐能力,最后构建3个冷激蛋白的异源表达菌株并分析它们在不同温度和不同环境胁迫条件下的生长状况。【结果】波罗的海希瓦氏菌中鉴定到3个冷激蛋白,分别为cspC、cspD、cspG。所有γ-变形菌纲的cspD基因单独聚成一支,并于1 109.6百万年前与其他csp基因相分离,波罗的海希瓦氏菌的cspC和cspG在858.8百万年前互相分开。cspG基因是波罗的海希瓦氏菌低温生存的必需基因,且广泛响应环境胁迫条件;cspC基因对cspG基因功能的实施起辅助作用;cspD不响应冷激,但却会随生长阶段的变化而发生变化。此外,cspC基因和cspG基因在低温条件下与细菌的致腐能力相关。【结论】波罗的海希瓦氏菌3个冷激蛋白基因各有不同,且cspC基因和cspG基因与该菌致腐能力有关,这为今后研究腐败菌的冷适应和致腐机制提供了新思路。     

Nucleotide Sequence of the Gene Encoding β-Isopropylmalate Dehydrogenase (leuB) from Bacteroides fragilis

The complete nucleotide sequence of the gene (leuB) coding for β-isopropylmaiate dehydrogenase of Bacteroides fragilis was determined. An open reading frame of 1,061 nucleotides was detected that could encode a polypeptide of 353 amino acid residues with a calculated molecular mass of 39,179 Da. The deduced amino acid sequence of the β-isopropylmalate dehydrogenase from B. fragilis showed substantial sequence similarity with the β-isopropylmalate dehydrogenases from other bacteria.     

Identification of new secreted proteins and secretion of heterologous amylase by <Emphasis Type="Italic">C. glutamicum</Emphasis>

In this study, secreted Corynebacterium glutamicum proteins were investigated by two-dimensional gel electrophoresis. Around 100 spots observed in the pH range 4.5–5.5 had molecular masses that varied from 10 to 50 kDa. Upon N-terminal amino acid sequence analysis by Edman degradation, two of them were hits to two hypothetical proteins encoded by cgR_1176 and cgR_2070 on C. glutamicum R genome, respectively. Active-form α-amylase derived from Geobacillus stearothermophilus was successfully secreted by using the predicted cgR_1176 and cgR_2070 signal sequences, indicating that these hypothetical proteins were secreted proteins. Analysis using a disruption mutant of the twin-arginine translocation (Tat) export pathway machinery of C. glutamicum suggested that one is Tat pathway dependent secretion while the other is independent of the pathway. Our results demonstrate that C. glutamicum can secrete exoproteins by using its own signal sequences, indicating its potential as a host for protein productions.     

Expression of recombinant protein using Corynebacterium Glutamicum: progress,challenges and applications

Corynebacterium glutamicum (C. glutamicum) is a highly promising alternative prokaryotic host for recombinant protein expression, as it possesses several significant advantages over Escherichia coli (E. coli), the currently leading bacterial protein expression system. During the past decades, several experimental techniques and vector components for genetic manipulation of C. glutamicum have been developed and validated, including strong promoters for tightly regulating target gene expression, various types of plasmid vectors, protein secretion systems and methods of genetically modifying the host strain genome to improve protein production potential. This review critically discusses current progress in establishing C. glutamicum as a host for recombinant protein expression, and examines, in depth, some successful case studies of actual application of this expression system. The established “expression tool box” for developing novel constructs based on C. glutamicum as a host are also evaluated. Finally, the existing issues and solutions in process development with C. glutamicum as a host are specifically addressed.