Microbial biosurfactants production, applications and future potential

Microorganisms synthesise a wide range of surface-active compounds (SAC), generally called biosurfactants. These compounds are mainly classified according to their molecular weight, physico-chemical properties and mode of action. The low-molecular-weight SACs or biosurfactants reduce the surface tension at the air/water interfaces and the interfacial tension at oil/water interfaces, whereas the high-molecular-weight SACs, also called bioemulsifiers, are more effective in stabilising oil-in-water emulsions. Biosurfactants are attracting much interest due to their potential advantages over their synthetic counterparts in many fields spanning environmental, food, biomedical, and other industrial applications. Their large-scale application and production, however, are currently limited by the high cost of production and by limited understanding of their interactions with cells and with the abiotic environment. In this paper, we review the current knowledge and the latest advances in biosurfactant applications and the biotechnological strategies being developed for improving production processes and future potential.     

Biotechnological application of Streptomyces for the production of clinical drugs and other bioactive molecules

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Recent application of metagenomic approaches toward the discovery of antimicrobials and other bioactive small molecules

Bacteria grown in pure culture have been the starting point for the discovery of many of the antibacterials now in use. Metagenomics, which utilizes culture-independent methods to access the collective genomes of natural bacterial populations, provides a means of exploring the antimicrobials produced by the large collections of bacteria that are known to be present in the environment but remain recalcitrant to culturing. Both novel small molecule antibiotics and new antibacterially active proteins have been identified using metagenomic approaches. The recent application of metagenomics to the discovery of bioactive small molecules, small molecule biosynthetic gene clusters and antibacterially active enzymes is discussed here.     

Biotechnological potential of microbial consortia and future perspectives

Design of a microbial consortium is a newly emerging field that enables researchers to extend the frontiers of biotechnology from a pure culture to mixed cultures. A microbial consortium enables microbes to use a broad range of carbon sources. It provides microbes with robustness in response to environmental stress factors. Microbes in a consortium can perform complex functions that are impossible for a single organism. With advancement of technology, it is now possible to understand microbial interaction mechanism and construct consortia. Microbial consortia can be classified in terms of their construction, modes of interaction, and functions. Here we discuss different trends in the study of microbial functions and interactions, including single-cell genomics (SCG), microfluidics, fluorescent imaging, and membrane separation. Community profile studies using polymerase chain-reaction denaturing gradient gel electrophoresis (PCR-DGGE), amplified ribosomal DNA restriction analysis (ARDRA), and terminal restriction fragment-length polymorphism (T-RFLP) are also reviewed. We also provide a few examples of their possible applications in areas of biopolymers, bioenergy, biochemicals, and bioremediation.     

Recent applications of biosurfactants as biological and immunological molecules

The interest in microbial biosurfactants has steadily increased during the past decade. In addition to the classical application as emulsifiers of hydrocarbons, they can be used in environmental protection, crude-oil recovery, food-processing industries and in various fields of biomedicine. Biosurfactants have several advantages over chemical surfactants including lower toxicity and higher biodegradability, and are likely to become molecules of the future in areas such as biomedicine and therapeutics. Here, we discuss the role and applications of biosurfactants (mainly glycolipids and lipopeptides) focusing on medicinal and therapeutic perspectives.     

Binase and other microbial RNases as potential anticancer agents

Some RNases possess preferential cytotoxicity against malignant cells. The best known of these RNases, onconase, was isolated from frog oocytes and is in clinical trials as anticancer therapy. Here we propose an alternative platform for anticancer therapy based on T1 RNases of microbial origin, in particular binase from Bacillus intermedius and RNase Sa from Streptomyces aureofaciens. We discuss their advantages and the most promising directions of research for their potential clinical applications.     

Widespread occurrence of secondary lipid biosynthesis potential in microbial lineages

Bacterial production of long-chain omega-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), is constrained to a narrow subset of marine γ-proteobacteria. The genes responsible for de novo bacterial PUFA biosynthesis, designated pfaEABCD, encode large, multi-domain protein complexes akin to type I iterative fatty acid and polyketide synthases, herein referred to as "Pfa synthases". In addition to the archetypal Pfa synthase gene products from marine bacteria, we have identified homologous type I FAS/PKS gene clusters in diverse microbial lineages spanning 45 genera representing 10 phyla, presumed to be involved in long-chain fatty acid biosynthesis. In total, 20 distinct types of gene clusters were identified. Collectively, we propose the designation of "secondary lipids" to describe these biosynthetic pathways and products, a proposition consistent with the "secondary metabolite" vernacular. Phylogenomic analysis reveals a high degree of functional conservation within distinct biosynthetic pathways. Incongruence between secondary lipid synthase functional clades and taxonomic group membership combined with the lack of orthologous gene clusters in closely related strains suggests horizontal gene transfer has contributed to the dissemination of specialized lipid biosynthetic activities across disparate microbial lineages.     

Engineering microbial cell factories for biosynthesis of isoprenoid molecules: beyond lycopene

The isoprenoid superfamily of compounds holds great potential for delivering commercial therapeutics, neutraceuticals and fine chemicals. As such, it has attracted widespread attention and prompted research aimed at metabolic engineering of the pathway for isoprenoid overproduction. The carotenoids in particular, because of their convenient colorimetric screening properties, have facilitated the investigation of new tools for pathway optimization. Because all isoprenoids share common metabolic precursors, genetic platforms resulting from work with carotenoids can be applied to the biosynthesis of other valuable products. In this review we summarize the many tools and methods that have been developed for isoprenoid pathway engineering, and the potential of these technologies for producing other molecules of this family, especially terpenoids.     

Directed evolution and synthetic biology applications to microbial systems

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Biological control of microbial infections and cancer in humans: Historical use to future potential

Prior to the advent of antibiotics, live organisms were used directly in attempts to control microbial infections and cure cancers. Examples of such biological control included bacteriotherapy, bacteriophage therapy, malaria therapy, probiotics and the use of living maggots. In all cases, the organisms themselves, rather than products of their metabolism, were used as the potentially curative agents. The history of the use of biocontrol agents in the treatment of human infections and cancer is discussed here in relation to more recent examples of the use of this approach. Modern studies suggest that the use of biological control in the treatment of human infections may be worth re‐evaluating in the light of the increasing world‐wide occurrence of antibiotic‐resistant bacteria and the opportunities provided by recent developments in gene technology.     

启动子和细胞全局转录机制的定向进化在微生物代谢工程中的应用

通过随机突变和定向选择而进行的定向进化(又称分子进化或人工进化)在改造酶的催化特性和稳定性、扩展酶的底物范围等方面具有广泛的应用。近年来,定向进化也开始应用在对结构基因的启动子区域和具有调节功能的蛋白如转录因子等进行代谢工程改造,并成功选育了对环境胁迫因素具有较强耐受性,以及发酵效率提高的微生物菌种。以下着重介绍近年来启动子的定向进化,包括启动子的强度和调节功能的分子进化,以及细胞全局转录工程等技术在微生物代谢工程中的应用,这些定向进化技术使人们可以更精细地调节基因表达水平,并可同时改变细胞内多个基因的转录水平,是代谢工程研究新的有力工具。     

甲壳素酶Chisb的定向进化及生物转化合成几丁寡糖

甲壳素酶具有广泛的工业应用前景,如可将虾壳、蟹壳和其他甲壳废物降解成以几丁寡糖为主的高附加值产品,但野生型甲壳素酶催化效率低,大大限制了几丁寡糖的生产。笔者在前期研究中表达了一个具有较高效催化效率的甲壳素酶Chisb,并对其酶学性质进行了初步研究。为进一步提高甲壳素酶Chisb的催化效率,以R13NprB-C-SP-H为亲本,采用易错PCR(Error-pronePCR)技术构建随机突变体文库,对甲壳素酶Chisb进行定向进化。经过96孔板初筛和摇瓶复筛,获得了两个催化效率进一步提高的突变体C43D和E336R。对突变体的酶学性质进行分析, C43D和E336R的最适催化温度为55℃, C43D的最适pH为5.0,E336R的最适pH为9.0;其催化效率相比对照分别提高了1.35倍和1.57倍;而E336R和C43D催化产几丁寡糖的含量分别为2.53 g/L和2.06 g/L,相比对照(0.89 g/L)分别提高了2.84倍和2.31倍;底物转化率分别为84.3%和68.7%,相比对照(29.7%)分别提高了54.6%和39%。研究表明,通过易错PCR引入随机突变的方法能够有效提高甲壳素酶Chisb的催化效率。上述研究获得的催化效率提高的正向突变体及其酶学性质分析对生物转化合成几丁寡糖具有重要研究意义和应用价值。     

The application of a high throughput analysis method for the screening of potential biosurfactants from natural sources

The presence of biosurfactants in growth media can be evaluated by a variety of methods, none of which are suitable for high throughput studies. The method described here is based on the effect of meniscus shape on the image of a grid viewed through the wells of a 96-well plate. The efficacy of the method was demonstrated by the selection of a bacterium (producing a biosurfactant able to reduce the surface tension of pure water from 72 to 28.75 mN m^− 1) from a culture collection isolated from aviation fuel-contaminated land. The assay was found to be more sensitive, rapid and easy to perform than other published methods. It does not need specialised equipment or chemicals and excludes the bias which results from the surfactant properties of medium used for bacterial growth.     

Hydrolysis of glucosinolates using nylon-immobilized myrosinase to produce pure bioactive molecules

Bioactive compounds were produced from natural glucosinolates, secondary plant metabolites, using myrosinase (thioglucoside glucohydrolase EC 3.2.3.1) isolated from ripe seeds of Sinapis alba. The enzyme was immobilized on granular nylon 6.6 with the crosslinking technique. Immobilized myrosinase displayed extraordinary operational and storage stability. Using a small thermostatted continuous packed-bed bioreactor, the enzyme activity was unchanged after 15 days of continuous use at 37 degrees C and after >1 year of storage at room temperature. The bioreactor was particularly efficient in producing pure isothiocyanates, but it was less efficient for pure nitrile production.     

Potent anti-mycobacterial and immunomodulatory activity of some bioactive molecules of Indian ethnomedicinal plants that have the potential to enter in TB management

Tuberculosis (TB) is one of the deadliest infectious diseases of human civilization. Approximately one-third of global population is latently infected with the TB pathogen Mycobacterium tuberculosis (M.tb). The discovery of anti-TB antibiotics leads to decline in death rate of TB. However, the evolution of antibiotic-resistant M.tb-strain and the resurgence of different immune-compromised diseases re-escalated the death rate of TB. WHO has already cautioned about the chances of pandemic situation in TB endemic countries until the discovery of new anti-tubercular drugs, that is, the need of the hour. Analysing the pathogenesis of TB, it was found that M.tb evades the host by altering the balance of immune response and affects either by killing the cells or by creating inflammation. In the pre-antibiotic era, traditional medicines were only therapeutic measures for different infectious diseases including tuberculosis. The ancient literatures of India or ample Indian traditional knowledge and ethnomedicinal practices are evidence for the treatment of TB using different indigenous plants. However, in the light of modern scientific approach, anti-TB effects of those plants and their bioactive molecules were not established thoroughly. In this review, focus has been given on five bioactive molecules of different traditionally used Indian ethnomedicinal plants for treatment of TB or TB-like symptom. These compounds are also validated with proper identification and their mode of action with modern scientific approaches. The effectiveness of these molecules for sensitive or drug-resistant TB pathogen in clinical or preclinical studies was also evaluated. Thus, our specific aim is to highlight such scientifically validated bioactive compounds having anti-mycobacterial and immunomodulatory activity for future use as medicine or adjunct-therapeutic molecule for TB management.     

Directed assembly of DNA molecules via simultaneous ligation and digestion

DNA ligation is a routine laboratory practice, yet the yield of the desired product is often very low due to competing off-pathway reactions. The sensitivity of subsequent manipulations (e.g., selection via bacterial transformation) often obviates the need for a high yield of correctly ligated products. However the ability to perform high-yield, preparative-scale DNA ligations would benefit a number of downstream applications ranging from standard molecular cloning to biophysics and DNA computing. We describe here a ligation technique that specifically converts off-pathway ligation products back into substrate. We term this second-chance strategy enzymatic ligation assisted by nucleases (ELAN) and demonstrate the ordered assembly of four DNA fragments via simultaneous ligation and digestion in the presence of eight restriction enzymes. Use of ELAN increased the yield of the desired product by more than 30-fold.     

Identification and validation of protein targets of bioactive small molecules

Identification and validation of protein targets of bioactive small molecules is an important problem in chemical biology and drug discovery. Currently, no single method is satisfactory for this task. Here, we provide an overview of common methods for target identification and validation that historically were most successful. We have classified for the first time the existing methods into two distinct and complementary types, the 'top-down' and 'bottom-up' approaches. In a typical top-down approach, the cellular phenotype is used as a starting point and the molecular target is approached through systematic narrowing down of possibilities by taking advantage of the detailed existing knowledge of cellular pathways and processes. In contrast, the bottom-up approach entails the direct detection and identification of the molecular targets using affinity-based or genetic methods. A special emphasis is placed on target validation, including correlation analysis and genetic methods, as this area is often ignored despite its importance.