芽胞杆菌中重组蛋白分泌表达的优化策略及研究进展

芽胞杆菌属具有良好的蛋白表达和分泌能力,在工业酶的生产中被广泛应用,是理想的工业宿主菌,但实现蛋白分泌表达的普遍高效性还存在许多瓶颈。本文综述了芽胞杆菌的蛋白分泌表达策略,从启动子、信号肽、分泌途径、宿主和培养条件这5个方面总结了提高芽胞杆菌中分泌表达重组蛋白的方法,对芽胞杆菌高效生产工业酶有一定的参考价值,最后展望了优化芽胞杆菌分泌表达的研究方向,各种新型生物技术的发展必将推进芽胞杆菌在分泌表达领域有更深入的应用。     

Isolation, culture and characterization of a new strain of Euglena gracilis

A new strain of Euglena gracilis Klebs has been isolated from a highly polluted river; it was named MAT. Strain growth in different culture media was evaluated under heterotrophic and autotrophic conditions. Total lipid, sugar, protein and chlorophyll a production were studied. Results obtained for MAT were compared with data obtained for a UTEX Culture Collection strain. Likewise, cells from both strains were bleached using streptomycin, and grown in the same media used for green samples. Both MAT and UTEX showed clear differences in their biochemical composition and growth rate depending on the media used. They also exhibited different growth patterns. E. gracilis medium proved to be the best culture environment for both strains either in autotrophic or heterotrophic conditions. Results show that basal contents of lipids, sugars, proteins and chlorophyll a vary depending on the strain, and thus values obtained for one strain do not apply to another. Moreover, strain origin may have an influence on the mechanisms of adaptation or defense developed by each sample.     

A low-sodium-salt formulation for the fermentation of salinosporamides by <Emphasis Type="Italic">Salinispora tropica</Emphasis> strain NPS21184

In this paper, we described the development of a potassium-chloride-based-salt formulation containing low sodium concentrations (5.0 to 11 mM) to support the growth of Salinispora tropica strain NPS21184 and its production of salinosporamide A (NPI-0052). The sodium present in the media was essentially derived from the complex nitrogen sources Hy Soy, yeast extract, and peptone used in the media. We demonstrated that good growth rate and yield of S. tropica strain NPS21184 were detected in both agar and liquid media containing the potassium-chloride-based-salt formulation with sodium concentration as low as 5.0 mM, significantly less than the critical seawater-growth requirement concentration of 50 mM sodium for a marine microorganism. We also observed good production of NPI-0052 (176 to 243 mg/l) by S. tropica strain NPS21184 grown in production media containing the potassium chloride-based-salt formulation. The production of deschloro analog, salinosporamide B (NPI-0047), was significantly lower in the low-sodium-salt-formulation medium than in the high-sodium-salt-formulation media. We demonstrated that while S. tropica strain NPS21184 is a novel marine actinomycete that requires high salt content for growth, it does not require sodium-chloride-based seawater-type media for growth and production of NPI-0052.     

Increased osmotolerance of genetically modified ethanol producing strains of Saccharomyces Sp.

Summary A stable spheroplast fusion product of the polyploid brewing strain Saccharomyces uvarum (cares bergensis) , strain 21 and a genetically constructed diploid Saccharomyces diastaticus, strain 1384 has been shown to have improved ethanol producing capability in defined media (Panchal et al., 1982). This fusion product, strain 1400 was further subjected to fermentations in defined media containing glucose substrate and varying concentrations of the non-metabolized sugars sorbitol or mannitol.While the fermentation efficiencies of all the three strains decreased with increasing osmotic pressure imparted by sorbitol or mannitol, the detrimental effect was least apparent with the fusion product than with either of the fusion partners. This attribute of the stable fusion product has major significance in relation to its potential for industrial ethanol production.     

Production of Bacillus sphaericus larvicide on industrial peptones

Summary A range of industrial peptones in combination with other carbon and nitrogen sources were investigated in order to define a medium for production of insecticide from Bacillus sphaericus, strain 2362. Industrial protein hydrolysates with an average peptide chain length above 30 were found to be poor substrates. Fermentation of B. sphaericus on protein-based media supplemented with glycerol resulted in production of 4–5x10³ units of larvicide per ml fermentation broth, and toxicity of the larvicide up to 10⁴ units per mg. Equally high larvicide production was obtained, when peptone was substituted with 30 mM l-glutamate and 2 mM proline. Addition of 2 mM l-arginine to this medium prevented sporulation and larvicide production.     

Interspecies variation of Kitasatospora recifensis endophytic from yam bean producing thermostable amylases in alternative media

An endophytic actinomycete isolated from tubers of yam beam (Pachyrhizus erosus L. Urban) was classified as a novel species nominated Kitasatospora recifensis based in phenotypic and genotypic analysis (16S rDNA gene sequence). Monosporic culture using specific ISP2 media revealed three interspecies, which were identified by DNA southern hybridization (Wild strain 13817 W, Aerial Mycelium strain 13817 AM and Vegetative Mycelium strain 13817 VM). The strains were tested for the production of amylolitic enzymes in alternative media. Maximum yields for both enzymes were observed in starch-casein. Higher α-amylase was obtained with strain 13817 W in starch-urea, and amyloglucosidase with strain 13817 AM in starch-ammonium that are economic sources and may be important for industrial purposes. Type strain (DAUFPE 13817 ^T  = KCTC 9972^T = DSM 44943^T).     

Extension of cell membrane boosting squalene production in the engineered Escherichia coli

Squalene is a lipophilic and non-volatile triterpene with many industrial applications for food, pharmaceuticals, and cosmetics. Metabolic engineering focused on optimization of the production pathway suffer from little success in improving titers because of a limited space of the cell membrane accommodating the lipophilic product. Extension of cell membrane would be a promising approach to overcome the storage limitation for successful production of squalene. In this study, Escherichia coli was engineered for squalene production by overexpression of some membrane proteins. The highest production of 612 mg/L was observed in the engineered E. coli with overexpression of Tsr, a serine chemoreceptor protein, which induced invagination of inner membrane to form multilayered structure. It was also observed an increase in unsaturated fatty acid in membrane lipids composition, suggesting cellular response to maintain membrane fluidity against squalene accumulation in the engineered strain. This study potentiates the capability of E. coli for squalene production and provides an effective strategy for the enhanced production of such compounds.     

Effect of Simazine on the production of lysine and methionine byAzotobacter chroococcum andAzotobacter vinelandii

Summary Production of lysine and methionine byAzotobacter chroococcum strain H23 andA. vinelandii strain ATCC 12837 was studied in chemically-defined medium and dialysed-soil medium, amended with different concentrations of Simazine. Responses on production due to Simazine were different for each strain and were fairly conditioned by culture media composition. Quantitative production of amino acids was significantly affected by the xenobiotic only at higher doses (50–100,g/ml). The effect of Simazine on methionine production by strain H23 was very pronounced when bacteria were grown in dialysed-soil medium, which was specially formulated to reproduce the natural habitat of the organisms.     

Detection of Homoserine Lactone-Like Quorum Sensing Molecules in Bradyrhizobium Strains

One hundred and forty-two Bradyrhizobium strains were screened for their ability to produce N-acyl homoserine lactone-like molecules (AHLs) by using an Agrobacterium tumefaciens biosensor strain containing a traI-lacZ fusion. Approximately 22% (31 of 142) of the tested strains produced AHLs that induced moderate to elevated β-galactosidase activity levels in the biosensor strain. Bradyrhizobium japonicum and Bradyrhizobium elkanii strains were both shown to produce AHLs. Age of culture, and media composition were each shown to influence production of AHL(s), with greater production occurring in 2 day-old cultures grown in rich media. Reverse-phase high-performance liquid chromatography and thin-layer chromatography analyses indicated that the B. japonicum strain USDA 290 produced at least two types of AHLs. Our results indicate that the production AHL-like autoinducers is widespread among both B. japonicum and B. elkanii strains.     

Effect of growth temperature and media composition on the fatty acid composition of Bacillus stearothermophilus AN 002

The influence of growth temperature, media composition and cell age on the chemical composition of Bacillus stearothermophilus strain AN 002 has been determined. The total cellular protein decreased and the free amino acid content increased with growth temperature, in both exponential and stationary growth phase. The protein and free amino acid contents of cells were higher in the stationary phase than in the exponential phase, irrespective of growth temperature and media composition. The RNA content was only reduced in cells grown at 55° C. No significant variations were observed in the DNA and carbohydrate contents with respect to growth temperature and cell age. The total lipid and fatty acid compositions on the other hand varied as a function of growth temperature, cell age and media composition. Differences in the relative concentrations of even, odd and branched chain fatty acids were noticed. Novariation was observed in the antiiso and unsaturated fatty acids with respect to growth temperature. The unique variations in the fatty acid composition and total lipids at the growth temperature of 50° C and their variations in the stationary growth phase seem to be characteristic for B. stearothermophilus AN 002.     

The expression of the Cuphea palustris thioesterase CpFatB2 in Yarrowia lipolytica triggers oleic acid accumulation

The conversion of industrial by‐products into high‐value added compounds is a challenging issue. Crude glycerol, a by‐product of the biodiesel production chain, could represent an alternative carbon source for the cultivation of oleaginous yeasts. Here, we developed five minimal synthetic glycerol‐based media, with different C/N ratios, and we analyzed the production of biomass and fatty acids by Yarrowia lipolytica Po1g strain. We identified two media at the expense of which Y. lipolytica was able to accumulate ～5 g L^?1 of biomass and 0.8 g L^?1 of fatty acids (0.16 g of fatty acids per g of dry weight). These optimized media contained 0.5 g L^?1 of urea or ammonium sulfate and 20 g L^?1 of glycerol, and were devoid of yeast extract. Moreover, Y. lipolytica was engineered by inserting the FatB2 gene, coding for the CpFatB2 thioesterase from Cuphea palustris, in order to modify the fatty acid composition towards the accumulation of medium‐chain fatty acids. Contrary to the expected, the expression of the heterologous gene increased the production of oleic acid, and concomitantly decreased the level of saturated fatty acids. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:26–35, 2016     

枯草芽胞杆菌蛋白质表达分泌系统发展及展望

枯草芽胞杆菌作为革兰阳性模式菌株是基础研究和工业应用的常用宿主细胞。介绍了枯草芽胞杆菌中蛋白合成和分泌过程中的重要步骤及重要调控位点。在枯草芽胞杆菌蛋白表达及分泌系统中,可以针对目标基因在体内的转录、翻译、折叠、转运和菌株改造等方面对表达分泌系统进行优化改良,针对不同的目标蛋白,可进行不同优化模块的组装和拼搭,以达到针对目标蛋白产物定制化地提高产量和分泌量的目的。在未来,随着基因编辑和合成生物技术的发展,菌株改良策略的不断优化,枯草芽胞杆菌将会在工业生产蛋白质制品领域发挥更大的应用价值。     

Isolation and Characterization of Three Porin-Like Proteins from Vibrio vulnificus: Effect of Different Growth Media on Their Production

The effect of the culture media on the composition of the outer membrane protein of Vibrio vulnificus strain 393 from human blood was examined. Only one major outer membrane protein, with an apparent molecular weight of 37,000 (37K protein) and 34,000 (34K protein), was formed in the cells grown in 3% NaCl-BHI broth and chemically defined medium, respectively. The production of one major outer membrane protein was also observed in other isolates from humans and asari clam when they were grown in 3% NaCl-BHI broth. On the other hand, three major outer membrane proteins, with apparent molecular weights of 48,000 (48K protein), 37,000 (37K protein), and 34,000 (34K protein), were produced in the cells grown in 3% NaCl-nutrient broth. Three proteins, 48K, 37K, and 34K from strain 393, were purified and the amino acid compositions were determined. Although there was a little difference in the composition of amino acid among three proteins, the amino acid compositions of the three porin-like proteins showed characteristic properties of the porins of Escherichia coli and Salmonella typhimurium. Immunoblot analysis of the outer membrane proteins from four vibrios, E. coli, and S. typhimurium using monospecific antisera against these three porin-like proteins showed that only the antiserum against 37K protein cross-reacted with the outer membrane proteins from all the strains tested.     

Expression and export: recombinant protein production systems for Aspergillus

Several Aspergillus species, in particular Aspergillus niger and Aspergillus oryzae, are widely used as protein production hosts in various biotechnological applications. In order to improve the expression and secretion of recombinant proteins in these filamentous fungi, several novel genetic engineering strategies have been developed in recent years. This review describes state-of-the-art genetic manipulation technologies used for strain improvement, as well as recent advances in designing the most appropriate engineering strategy for a particular protein production process. Furthermore, current developments in identifying bottlenecks in the protein production and secretion pathways are described and novel approaches to overcome these limitations are introduced. An appropriate combination of expression vectors and optimized host strains will provide cell factories customized for each production process and expand the great potential of Aspergilli as biotechnology workhorses to more complex multi-step industrial applications.     

Comparative proteome analysis of robust <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> insights into industrial continuous and batch fermentation

A robust Saccharomyces cerevisiae strain has been widely applied in continuous and batch/fed-batch industrial fermentation. However, little is known about the molecular basis of fermentative behavior of this strain in the two realistic fermentation processes. In this paper, we presented comparative proteomic profiling of the industrial yeast in the industrial fermentation processes. The expression levels of most identified protein were closely interrelated with the different stages of fermentation processes. Our results indicate that, among the 47 identified protein spots, 17 of them belonging to 12 enzymes were involved in pentose phosphate, glycolysis, and gluconeogenesis pathways and glycerol biosynthetic process, indicating that a number of pathways will need to be inactivated to improve ethanol production. The differential expressions of eight oxidative response and heat-shock proteins were also identified, suggesting that it is necessary to keep the correct cellular redox or osmotic state in the two industrial fermentation processes. Moreover, there are significant differences in changes of protein levels between the two industrial fermentation processes, especially these proteins associated with the glycolysis and gluconeogenesis pathways. These findings provide a molecular understanding of physiological adaptation of industrial strain for optimizing the performance of industrial bioethanol fermentation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Inhibition of E. coli Host RNA Polymerase Allows Efficient Extracellular Recombinant Protein Production by Enhancing Outer Membrane Leakiness

With the growing interest in continuous cultivation of Escherichia coli, secretion of product to the medium is not only a benefit, but a necessity in future bioprocessing. In this study, it is shown that induced decoupling of growth and heterologous gene expression in the E. coli X-press strain (derived from BL21(DE3)) facilitates extracellular recombinant protein production. The effect of the process parameters temperature and specific glucose consumption rate (q_S) on growth, productivity, lysis and leakiness, is investigated, to find the parameter space allowing extracellular protein production. Two model proteins are used, Protein A (SpA) and a heavy-chain single-domain antibody (VHH), and performance is compared to the industrial standard strain BL21(DE3). It is shown that inducible growth repression in the X-press strain greatly mitigates the effect of metabolic burden under different process conditions. Furthermore, temperature and q_S are used to control productivity and leakiness. In the X-press strain, extracellular SpA and VHH titer reach up to 349 and 19.6 mg g⁻¹, respectively, comprising up to 90% of the total soluble product, while keeping cell lysis at a minimum. The findings demonstrate that the X-press strain constitutes a valuable host for extracellular production of recombinant protein with E. coli.     

Effects of potassium chloride on ethanol production by an osmotolerant mutant of Zymomonas mobilis

The effect of increasing the KCl concentration in the culture medium of an alcoholic fermentation of glucose using the bacterium Zymomonas mobilis was investigated. Data obtained with the wild-type strain (ZM4, ATCC 31821) and with a newly isolated osmotolerant mutant (SBE15) were compared. It was observed that, at high salt concentration, inhibition of growth occured (specific growth rate and biomass yield) while ethanol production (specific ethanol productivity and ethanol yield) was unaffected. In contrast, the specific rate of in-vitro ethanol production, using either cell-free extract or washed cells, was strongly inhibited by increasing the KCl concentration in the incubation mixture. Therefore, it was concluded that the intracellular concentration of KCl was maintained below the inhibitory concentration by an active transport system. In addition, the fermentation performances of the osmotolerant mutant strain were higher than those of the parent strain at all the KCl concentrations tested, suggesting the utility of the former to run ethanolic fermentations in crude industrial media with a high salt content. Furthermore, the fermentation data on media containing added KCl agreed well with those obtained on molasses media, suggesting that the inhibition observed on these media was due to their high osmolality. Correspondence to: J. Baratti     

Production of a novel bioflocculant MNXY1 by Klebsiella pneumoniae strain NY1 and application in precipitation of cyanobacteria and municipal wastewater treatment

Aims: To isolate and characterize the novel bioflocculant‐producing bacteria, to optimize the bioflocculant production and to evaluate its potential applications. Methods and Results: Klebsiella pneumoniae strain NY1, a bacterium that produces a novel bioflocculant (MNXY1), was selected on the chemically defined media. It was classified according to the 16S rRNA gene sequence, morphological and microscopic characteristics. MNXY1 was characterized to contain 26% protein and 66% total sugar. The constituent sugar monomers of MNXY1, revealed by NMR analysis, are glucose, galactose and quinovose. Favourable culture conditions for MNXY1 production were determined. Strain NY1 produces a high level (14.9 g l^?1) of MNXY1. MNXY1 is thermostable and tolerant to the extreme pH. It precipitated 54% of cyanobacteria from laboratory culture and 72% of the total suspended solids from raw wastewater. Conclusions: Strain NY1 was identified to produce a novel bioflocculant MNXY1. The outstanding performance of MNXY1 in practical applications and its availability in copious amounts make it attractive for further investigation and development for industrial scale applications. Significance and Impact of the Study: This is first report for the identification of a quinovose‐containing bioflocculant and application of a protein–polysaccharide complex bioflocculant in precipitation of cyanobacteria. These findings suggest that MNXY1 holds great potential for use in management of harmful algae and city wastewater treatment.     

Biosynthesis of protease by Pseudomonas aeruginosa MN7 grown on fish substrate

Fish powders and fish protein hydrolysates (FPH) from sardinella (Sardinella aurita) were prepared and tested as growth media for alkaline protease production by Pseudomonas aeruginosa MN7. Cultivated in fish substrate as carbon source, the strain exhibited a slightly greater protease production (about 7800 U ml^–1) than that obtained with commercial peptones (about 7222 U ml^–1). Furthermore, P. aeruginosa MN7 produced the same amount of protease when cultivated in medium containing only fish substrate or that containing all ingredients, indicating that the strain can obtain its carbon and nitrogen requirements directly from whole fish proteins. Moreover, it was found that extensive hydrolysis of fish proteins did not increase protease formation. Protease production in media containing only FPH prepared by Alcalase was about 70% of those obtained with MN7 protease digest of fish protein or with meat-fish powder. These results indicate that sardinella substrates are an excellent carbon and nitrogen source for the growth of P. aeruginosa MN7 and the production of protease.     

Isolation of a Leptothrix Strain,OUMS1, from Ocherous Deposits in Groundwater

Leptothrix species in aquatic environments produce uniquely shaped hollow microtubules composed of aquatic inorganic and bacterium-derived organic hybrids. Our group termed this biologically derived iron oxide as “biogenous iron oxide (BIOX)”. The artificial synthesis of most industrial iron oxides requires massive energy and is costly while BIOX from natural environments is energy and cost effective. The BIOX microtubules could potentially be used as novel industrial functional resources for catalysts, adsorbents and pigments, among others if effective and efficient applications are developed. For these purposes, a reproducible system to regulate bacteria and their BIOX productivity must be established to supply a sufficient amount of BIOX upon industrial demand. However, the bacterial species and the mechanism of BIOX microtubule formation are currently poorly understood. In this study, a novel Leptothrix sp. strain designated OUMS1 was successfully isolated from ocherous deposits in groundwater by testing various culture media and conditions. Morphological and physiological characters and elemental composition were compared with those of the known strain L. cholodnii SP-6 and the differences between these two strains were shown. The successful isolation of OUMS1 led us to establish a basic system to accumulate biological knowledge of Leptothrix and to promote the understanding of the mechanism of microtubule formation. Additional geochemical studies of the OUMS1-related microstructures are expected provide an attractive approach to study the broad industrial application of bacteria-derived iron oxides.