谷氨酸棒杆菌碱基编辑的条件优化

近年来,基于CRISPR/Cas9的碱基编辑技术因其具有不产生DNA双链断裂、无需外源DNA模板、不依赖宿主同源重组修复的优势,已经逐渐发展成为一种强大的基因组编辑工具,在动物、植物、酵母和细菌中得到了开发和应用。研究团队前期已在重要的工业模式菌株谷氨酸棒杆菌中开发了一种多元自动化的碱基编辑技术MACBETH,为进一步优化该方法,提高碱基编辑技术在谷氨酸棒杆菌中的应用效率,本研究首先在谷氨酸棒杆菌中构建了基于绿色荧光蛋白(GFP)的检测系统:将GFP基因的起始密码子ATG人工突变为ACG,GFP无法正常表达,当该密码子的C经编辑后恢复为T,即实现GFP蛋白的复活,结合流式细胞仪分析技术,可快速衡量编辑效率。然后,构建针对靶标位点的碱基编辑工具,经测试,该位点可成功被编辑,在初始编辑条件下碱基编辑效率为(13.11±0.21)%。在此基础上,通过对不同培养基类型、诱导初始OD600、诱导时间、诱导物浓度进行优化,确定最优编辑条件是:培养基为CGXII,初始OD600为0.05,诱导时间为20 h,IPTG浓度为0.01 mmol/L。经过优化,编辑效率达到(30.35±0.75)%,较初始条件提高了1.3倍。最后,选取原编辑条件下编辑效率较低的位点,进行了优化后编辑条件下的编辑效率评估,结果显示,不同的位点在最优编辑条件下的编辑效率提高了1.7–2.5倍,进一步证实该优化条件的有效性及通用性。研究结果为碱基编辑技术在谷氨酸棒杆菌中更好的应用提供了重要的参考价值。     

Large-scale engineering of the Corynebacterium glutamicum genome

The engineering of Corynebacterium glutamicum is important for enhanced production of biochemicals. To construct an improved C. glutamicum genome, we developed a precise genome excision method based on the Cre/loxP recombination system and successfully deleted 11 distinct genomic regions identified by comparative analysis of C. glutamicum genomes. Despite the loss of several predicted open reading frames, the mutant cells exhibited normal growth under standard laboratory conditions. With a total of 250 kb (7.5% of the genome), the 11 genomic regions were loaded with cryptic prophages, transposons, and genes of unknown function which were dispensable for cell growth, indicating recent horizontal acquisitions to the genome. This provides an interesting background for functional genomic studies and can be used in the improvement of cell traits.     

Cre/loxP-mediated deletion system for large genome rearrangements in Corynebacterium glutamicum

Genome rearrangement is an increasingly important technique to facilitate the understanding of genome functions. A Cre/loxP-mediated deletion system for large-scale genome rearrangements in Corynebacterium glutamicum was developed. By comparative analysis of C. glutamicum R and C. glutamicum 13032 genomes, distinct 14.5-kb and 56-kb regions not essential for cell survival were identified and targeted for deletion. By homologous recombination, loxP sites were integrated at each end of the target region. Deletions between the two chromosomal loxP sites in the presence of Cre recombinase were highly efficient. Accurate deletion was observed in all 96 Cre-expressing strains tested. These deletions represent the largest genomic excisions in C. glutamicum reported to date. Despite the loss of 11 and 58 predicted ORF(s), respectively, upon the deletion of the14.5-kb and 56-kb regions, the cells still exhibited normal growth under standard laboratory conditions. Based on the precision of its deletion, the Cre/loxP system provides a new, efficient genome rearrangement technique for studying C. glutamicum.     

利用蛋白质序列模式识别改善谷氨酸棒杆菌基因组注释

即使细菌基因组的基因结构较为简单,但在注释过程中也可能出现基因遗漏的现象。当潜在基因在高质量数据库中没有显著同源序列时,基于知识库的基因预测方法就会遇到困难。本文希望通过系统扫描基因组所有可能ORF的蛋白质序列模式来搜索遗漏基因。为验证该方法的可行性,作者系统分析了重要的工业发酵微生物谷氨酸棒杆菌的基因组,发现了25个候选疑似基因。它们具有显著的蛋白质序列模式,但在Swiss-Prot中元显著同源序列,并且在GenBank中仍未注释。深入分析发现,25个候选疑似基因中19个为可能基因,3个为可能假基因,3个为疑似基因序列。这些结果说明本文的分析方法可以有效地用于无显著同源序列基因的搜索。     

Cloning vector system for Corynebacterium glutamicum.

A protoplast transformation system has been developed for Corynebacterium glutamicum by using a C. glutamicum-Bacillus subtilis chimeric vector. The chimera was constructed by joining a 3.0-kilobase cryptic C. glutamicum plasmid and the B. subtilis plasmid pBD10. The neomycin resistance gene on the chimera, pHY416, was expressed in C. glutamicum, although the chloramphenicol resistance gene was not. The various parameters in the transformation protocol were analyzed separately and optimized. The resulting transformation system is simple and routinely yields 10(4) transformants per microgram of plasmid DNA.     

The Corynebacterium glutamicum genome: features and impacts on biotechnological processes

Corynebacterium glutamicum has played a principal role in the progress of the amino acid fermentation industry. The complete genome sequence of the representative wild-type strain of C. glutamicum, ATCC 13032, has been determined and analyzed to improve our understanding of the molecular biology and physiology of this organism, and to advance the development of more efficient production strains. Genome annotation has helped in elucidation of the gene repertoire defining a desired pathway, which is accelerating pathway engineering. Post genome technologies such as DNA arrays and proteomics are currently undergoing rapid development in C. glutamicum. Such progress has already exposed new regulatory networks and functions that had so far been unidentified in this microbe. The next goal of these studies is to integrate the fruits of genomics into strain development technology. A novel methodology that merges genomics with classical strain improvement has been developed and applied for the reconstruction of classically derived production strains. How can traditional fermentation benefit from the C. glutamicum genomic data? The path from genomics to biotechnological processes is presented.     

Functional analysis of all aminotransferase proteins inferred from the genome sequence of Corynebacterium glutamicum

Twenty putative aminotransferase (AT) proteins of Corynebacterium glutamicum, or rather pyridoxal-5'-phosphate (PLP)-dependent enzymes, were isolated and assayed among others with L-glutamate, L-aspartate, and L-alanine as amino donors and a number of 2-oxo-acids as amino acceptors. One outstanding AT identified is AlaT, which has a broad amino donor specificity utilizing (in the order of preference) L-glutamate > 2-aminobutyrate > L-aspartate with pyruvate as acceptor. Another AT is AvtA, which utilizes L-alanine to aminate 2-oxo-isovalerate, the L-valine precursor, and 2-oxo-butyrate. A second AT active with the L-valine precursor and that of the other two branched-chain amino acids, too, is IlvE, and both enzyme activities overlap partially in vivo, as demonstrated by the analysis of deletion mutants. Also identified was AroT, the aromatic AT, and this and IlvE were shown to have comparable activities with phenylpyruvate, thus demonstrating the relevance of both ATs for L-phenylalanine synthesis. We also assessed the activity of two PLP-containing cysteine desulfurases, supplying a persulfide intermediate. One of them is SufS, which assists in the sulfur transfer pathway for the Fe-S cluster assembly. Together with the identification of further ATs and the additional analysis of deletion mutants, this results in an overview of the ATs within an organism that may not have been achieved thus far.     

Protein secretion in Corynebacterium glutamicum

Corynebacterium glutamicum, a Gram-positive bacterium, has been widely used for the industrial production of amino acids, such as glutamate and lysine, for decades. Due to several characteristics – its ability to secrete properly folded and functional target proteins into culture broth, its low levels of endogenous extracellular proteins and its lack of detectable extracellular hydrolytic enzyme activity – C. glutamicum is also a very favorable host cell for the secretory production of heterologous proteins, important enzymes, and pharmaceutical proteins. The target proteins are secreted into the culture medium, which has attractive advantages over the manufacturing process for inclusion of body expression – the simplified downstream purification process. The secretory process of proteins is complicated and energy consuming. There are two major secretory pathways in C. glutamicum, the Sec pathway and the Tat pathway, both have specific signal peptides that mediate the secretion of the target proteins. In the present review, we critically discuss recent progress in the secretory production of heterologous proteins and examine in depth the mechanisms of the protein translocation process in C. glutamicum. Some successful case studies of actual applications of this secretory expression host are also evaluated. Finally, the existing issues and solutions in using C. glutamicum as a host of secretory proteins are specifically addressed.     

Ethanol catabolism in Corynebacterium glutamicum

Corynebacterium glutamicum grows on a variety of carbohydrates and organic acids as single or combined sources of carbon and energy. Here we show the ability of C. glutamicum to grow on ethanol with growth rates up to 0.24 h(-1) and biomass yields up to 0.47 g dry weight (g ethanol)(-1). Mutants of C. glutamicum deficient in phosphotransacetylase (PTA), isocitrate lyase (ICL) and malate synthase (MS) were unable to grow on ethanol, indicating that acetate activation and the glyoxylate cycle are essential for utilization of this substrate. In accordance, the expression profile of ethanol-grown C. glutamicum cells compared to that of glucose-grown cells revealed an increased expression of genes encoding acetate kinase (AK), PTA, ICL and MS. Furthermore, the specific activities of these four enzymes as well as those of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) were found to be high in ethanol-grown and low in glucose-grown cells. Growth of C. glutamicum on a mixture of glucose and ethanol led to a biphasic growth behavior, which was due to the sequential utilization of glucose before ethanol. Accordingly, the specific activities of ADH, ALDH, AK, PTA, ICL and MS in cells grown in medium containing both substrates were as low as in glucose-grown cells in the first growth phase, but increased 5- to 100-fold during the second growth phase. The results indicate that ethanol catabolism in C. glutamicum is subject to carbon source-dependent regulation, i.e., to a carbon catabolite control.     

Vanillate Metabolism in Corynebacterium glutamicum

Corynebacterium glutamicum, a Gram-positive soil bacterium belonging to the mycolic acids-containing actinomycetes, is able to use the lignin degradation products ferulate, vanillate, and protocatechuate as sole carbon sources. The gene cluster responsible for vanillate catabolism was identified and characterized. The vanAB genes encoding vanillate demethylase are organized in an operon together with the vanK gene, coding for a transport system most likely responsible for protocatechuate uptake. While gene disruption mutagenesis revealed that vanillate demethylase is indispensable for ferulate and vanillate utilization, a vanK mutation does not lead to a complete growth arrest but to a decreased growth rate on protocatechuate, indicating that one or more additional protocatechuate transporter(s) are present in C. glutamicum.     

New multiple-deletion method for the Corynebacterium glutamicum genome, using a mutant lox sequence

Due to the difficulty of multiple deletions using the Cre/loxP system, a simple, markerless multiple-deletion method based on a Cre/mutant lox system combining a right-element (RE) mutant lox site with a left-element (LE) mutant lox site was employed for large-scale genome rearrangements in Corynebacterium glutamicum. Eight distinct genomic regions that had been identified previously by comparative analysis of C. glutamicum R and C. glutamicum 13032 genomes were targeted for deletion. By homologous recombination, LE and RE mutant lox sites were integrated at each end of a target region. Highly efficient and accurate deletions between the two chromosomal mutant lox sites in the presence of Cre recombinase were realized. A deletion mutant lacking 190 kb of chromosomal regions, encoding a total of 188 open reading frames (ORFs), was obtained. These deletions represent the largest genomic excisions in C. glutamicum reported to date. Despite the loss of numerous predicted ORFs, the mutant exhibited normal growth under standard laboratory conditions. The Cre/loxP system using a pair of mutant lox sites provides a new, efficient genome rearrangement technique for C. glutamicum. It should facilitate the understanding of genome functions of microorganisms.     

In vitro analysis of the two-component system MtrB-MtrA from Corynebacterium glutamicum

The two-component system MtrBA is involved in the osmostress response of Corynebacterium glutamicum. MtrB was reconstituted in a functionally active form in liposomes and showed autophosphorylation and phosphatase activity. In proteoliposomes, MtrB activity was stimulated by monovalent cations used by many osmosensors for the detection of hypertonicity. Although MtrB was activated by monovalent cations, they lead in vitro to a general stabilization of histidine kinases and do not represent the stimulus for MtrB to sense hyperosmotic stress.     

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.     

Evolutionary engineering of Corynebacterium glutamicum

A unique feature of biotechnology is that we can harness the power of evolution to improve process performance. Rational engineering of microbial strains has led to the establishment of a variety of successful bioprocesses, but it is hampered by the overwhelming complexity of biological systems. Evolutionary engineering represents a straightforward approach for fitness‐linked phenotypes (e.g., growth or stress tolerance) and is successfully applied to select for strains with improved properties for particular industrial applications. In recent years, synthetic evolution strategies have enabled selection for increased small molecule production by linking metabolic productivity to growth as a selectable trait. This review summarizes the evolutionary engineering strategies performed with the industrial platform organism Corynebacterium glutamicum. An increasing number of recent studies highlight the potential of adaptive laboratory evolution (ALE) to improve growth or stress resistance, implement the utilization of alternative carbon sources, or improve small molecule production. Advances in next‐generation sequencing and automation technologies will foster the application of ALE strategies to streamline microbial strains for bioproduction and enhance our understanding of biological systems.     

Large-Scale Engineering of the Corynebacterium glutamicum Genome

The engineering of Corynebacterium glutamicum is important for enhanced production of biochemicals. To construct an improved C. glutamicum genome, we developed a precise genome excision method based on the Cre/loxP recombination system and successfully deleted 11 distinct genomic regions identified by comparative analysis of C. glutamicum genomes. Despite the loss of several predicted open reading frames, the mutant cells exhibited normal growth under standard laboratory conditions. With a total of 250 kb (7.5% of the genome), the 11 genomic regions were loaded with cryptic prophages, transposons, and genes of unknown function which were dispensable for cell growth, indicating recent horizontal acquisitions to the genome. This provides an interesting background for functional genomic studies and can be used in the improvement of cell traits.     

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.     

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.