Microbial production of nattokinase: current progress,challenge and prospect

Nattokinase (EC 3.4.21.62) is a profibrinolytic serine protease with a potent fibrin-degrading activity, and it has been produced by many host strains. Compared to other fibrinolytic enzymes (urokinase, t-PA and streprokinase), nattokinase shows the advantages of having no side effects, low cost and long life-time, and it has the potential to be used as a drug for treating cardiovascular disease and served as a functional food additive. In this review, we focused on screening of producing strains, genetic engineering, fermentation process optimization for microbial nattokinase production, and the extraction and purification of nattokinase were also discussed in this particular chapter. The selection of optimal nattokinase producing strain was the crucial starting element for improvement of nattokinase production. Genetic engineering, protein engineering, fermentation optimization and process control have been proved to be the effective strategies for enhancement of nattokinase production. Also, extraction and purification of nattokinase are critical for the quality evaluation of nattokinase. Finally, the prospect of microbial nattokinase production was also discussed regarding the recent progress, challenge, and trends in this field.     

The art of strain improvement of industrial lactic acid bacteria without the use of recombinant DNA technology

The food industry is constantly striving to develop new products to fulfil the ever changing demands of consumers and the strict requirements of regulatory agencies. For foods based on microbial fermentation, this pushes the boundaries of microbial performance and requires the constant development of new starter cultures with novel properties. Since the use of ingredients in the food industry is tightly regulated and under close scrutiny by consumers, the use of recombinant DNA technology to improve microbial performance is currently not an option. As a result, the focus for improving strains for microbial fermentation is on classical strain improvement methods. Here we review the use of these techniques to improve the functionality of lactic acid bacteria starter cultures for application in industrial-scale food production. Methods will be described for improving the bacteriophage resistance of specific strains, improving their texture forming ability, increasing their tolerance to stress and modulating both the amount and identity of acids produced during fermentation. In addition, approaches to eliminating undesirable properties will be described. Techniques include random mutagenesis, directed evolution and dominant selection schemes.     

Criteria for lactic acid bacteria screening to enhance silage quality

The main challenge of ensiling is conserving the feed through a fermentative process that results in high nutritional and microbiological quality while minimizing fermentative losses. This challenge is of growing interest to farmers, industry and research and involves the use of additives to improve the fermentation process and preserve the ensiled material. Most studies involved microbial additives; lactic acid bacteria (LAB) have been the focus of much research and have been widely used. Currently, LABs are used in modern and sustainable agriculture because of their considerable potential for enhancing human and animal health. Although the number of studies evaluating LABs in silages has increased, the potential use of these micro-organisms in association with silage has not been adequately studied. Fermentation processes using the same strain produce very different results depending on the unique characteristics of the substrate, so the choice of silage inoculant for different starting substrates is of extreme importance to maximize the nutritional quality of the final product. This review describes the current scenario of the bioprospecting and selection process for choosing the best LAB strain as an inoculant for ensiling. In addition, we analyse developments in the fermentation process and strategies and methods that will assist future studies on the selection of new strains of LAB as a starter culture or inoculant.     

Genetic improvement of processes yielding microbial products

Although microorganisms are extremely good in presenting us with an amazing array of valuable products, they usually produce them only in amounts that they need for their own benefit; thus, they tend not to overproduce their metabolites. In strain improvement programs, a strain producing a high titer is usually the desired goal. Genetics has had a long history of contributing to the production of microbial products. The tremendous increases in fermentation productivity and the resulting decreases in costs have come about mainly by mutagenesis and screening/selection for higher producing microbial strains and the application of recombinant DNA technology.     

High-throughput screening for high-efficiency small-molecule biosynthesis

Systems metabolic engineering faces the formidable task of rewiring microbial metabolism to cost-effectively generate high-value molecules from a variety of inexpensive feedstocks for many different applications. Because these cellular systems are still too complex to model accurately, vast collections of engineered organism variants must be systematically created and evaluated through an enormous trial-and-error process in order to identify a manufacturing-ready strain. The high-throughput screening of strains to optimize their scalable manufacturing potential requires execution of many carefully controlled, parallel, miniature fermentations, followed by high-precision analysis of the resulting complex mixtures. This review discusses strategies for the design of high-throughput, small-scale fermentation models to predict improved strain performance at large commercial scale. Established and promising approaches from industrial and academic groups are presented for both cell culture and analysis, with primary focus on microplate- and microfluidics-based screening systems.     

Production of microbial chitinases – A revisit

Production of microbial chitinases have received increased attention in recent years due to its potential application in various fields. There has been an all around effort to increase the production of chitinases by using genetically engineered organisms and by incorporating modern fermentation techniques. This review presents a brief outline on the production of microbial chitinases which includes information on random screening and selection of chitinolytic organisms and the rationale behind the use of genetically engineered organisms. The problems and perspectives involved in large scale production of chitinases is discussed with special reference to bioreactor studies and new fermentation methods. The present limitations in the understanding of the chitinase fermentation process is also discussed and the scope for future investigation is outlined in this communication.     

长链二元酸发酵菌种创制和工艺研究进展

长链二元酸作为合成多种高附加值化学品的原料,已广泛应用于化工、农业和医药等领域,目前全球对于长链二元酸的需求呈逐年增长态势。化学法合成长链二元酸对反应条件要求严苛且工艺复杂,而微生物发酵合成在经济性和难易度等方面具有无可比拟的优势。本文综述了长链二元酸的合成方法,包括化学合成法和微生物发酵法,分子工程选育高产菌株的进展以及生物发酵法生产长链二元酸的产业化现状,并就其存在的问题进行了探讨,最后对合成生物学创制长链二元酸高产菌株进行了总结和展望。     

产顺,顺-粘康酸细胞工厂的构建与优化

顺,顺-粘康酸是重要的平台化学品。目前,生物合成顺,顺-粘康酸还缺乏高性能菌株,已报道的主要工程菌株不仅需要诱导表达,遗传不稳定,而且发酵培养基组分复杂,不利于大规模工业化生产。构建能利用简单无机盐培养基、遗传稳定且不需要诱导表达的新型工程菌受到人们的关注。本研究在实验室前期构建的产三脱氢莽草酸工程菌株WJ060中,整合合成顺,顺-粘康酸的3个外源基因(aro Z、aro Y、cat A),并且利用3个不同强度的组成型启动子进行组合调控,成功构建了27株顺,顺-粘康酸工程菌,得到的最优工程菌MA30的产量达到1.7 g/L。为了进一步提高顺,顺-粘康酸工程菌的生产能力,利用基因组复制工程构建突变体库,结合高通量筛选方法,经过两轮筛选,成功筛选到了顺,顺-粘康酸产量提高超过8%的大肠杆菌MA30-G2。利用5 L发酵罐进行分批补料发酵,MA30-G2的顺,顺-粘康酸产量达到了11.5 g/L。本研究采用组合调控和高通量筛选相结合的策略不仅促进了顺,顺-粘康酸的生物合成,同时也为其他生物基化学品的生物制造提供了重要参考。     

Microbial formation, biotechnological production and applications of 1,2-propanediol

This short review covers metabolic pathways, genetics and metabolic engineering of 1,2-propanediol formation in microbes. 1,2-Propanediol production by bacteria and yeasts has been known for many years and two general pathways are recognized. One involves the metabolism of deoxyhexoses, where lactaldehyde is formed during the glycolytic reactions and is then reduced to 1,2-propanediol. The second pathway derives from the formation of methylglyoxal from dihydroxyacetonephosphate and its subsequent reduction to 1,2-propanediol. The enzymes involved in the reduction of methylglyoxal can generate isomers of lactaldehyde or acetol, which can be further reduced by specific reductases, giving chiral 1,2-propanediol as the product. The stereospecificity of the enzymes catalyzing the two reduction steps is important in deriving a complete pathway. Through genetic engineering, appropriate combinations of enzymes have been brought together in Escherichia coli and yeast to generate 1,2-propanediol from glucose. The optimization of these strains may yield microbial processes for the production of this widely used chemical. Received: 25 May 2000 / Received revision: 24 July 2000 / Accepted: 25 July 2000     

Large-scale production of chlamydospores of Gliocladium virens strain GL-21 in submerged culture

SoilGard™ is a commercial microbial fungicide containing chlamydospores of Gliocladium virens strain GL-21. The formulation is registrated with the US Environmental Protection Agency (EPA) by Thermo Trilogy Corporation, Columbia, Maryland. The biocontrol agent was developed in cooperation with the Biocontrol of Plant Diseases Laboratory (BPDL), US Department of Agriculture (USDA), and is targeted for controlling damping-off diseases caused by Rhizoctonia solani and Pythium ultimum in vegetable and ornamental greenhouses. Formulation requires production of chlamydospore biomass in liquid fermentation which was carried out in 20-L, 1500-L and 4000-L submerged liquid fermentors. Key economic process parameters, fermentation kinetics and the reproducibility of the fermentation at this scale were evaluated and analyzed. Starter culture quality and maturity of chlamydospores were identified as key issues for obtaining successful fermentations. Received 07 February 1997/ Accepted in revised form 27 May 1997     

Challenges in the microbial production of flavonoids

As flavonoids have beneficial health effects on humans, they are gaining increasing interest from pharmaceutical and health industries. However, current production methods, such as plant extraction and chemical synthesis, are inadequate to meet the demand. Therefore, microbial production might offer a promising alternative. During recent years, microbial strains able to produce flavonoids to a certain extent have been developed. However production titers are limited to the mg l^?1 range, hampering the industrial exploitation of these strains. The latter will not be achieved by simply introducing the heterologous pathway in the production host and optimizing the fermentation process, but will depend on the interaction of different aspects of metabolic engineering and process engineering to overcome the current limitations. Next to engineering the production strain to optimize the availability of precursors, the pathway itself also requires intensive engineering. Currently utilized strategies result in a wide variety of different production strains, requiring high-throughput screening methods to identify optimal performing strains. As more and more organisms are being characterized, each with their own specific properties which might be beneficial for the heterologous production of flavonoids, the choice of the production host is another important aspect. Finally, the use of co-cultures might offer an alternative in which different parts of the process are performed by different organisms. This review aims to provide an overview of the research that has been done on these separate aspects. The work presented here could be used as a framework for further research.     

Consolidated bioprocessing and simultaneous saccharification and fermentation of lignocellulose to ethanol with thermotolerant yeast strains

Consolidated bioprocessing (CBP), which integrates enzyme production, saccharification and fermentation into a single process, is a promising strategy for effective ethanol production from lignocellulosic materials because of the resulting reduction in utilities, the substrate and other raw materials and simplification of operation. CBP requires a highly engineered microbial strain capable of hydrolyzing biomass with enzymes produced on its own and producing high-titer ethanol. Recently, heterologous production of cellulolytic enzymes has been pursued with yeast hosts, which has realized direct conversion of cellulose to ethanol. Specifically, the development of cell surface engineering, which provides a display of cellulolytic enzymes on the yeast cell surface, facilitates effective biomass hydrolysis concomitantly with ethanol production. On the other hand, the difference in optimum temperature between saccharification and fermentation is a drawback of efficient ethanol production in the simultaneous saccharification and fermentation (SSF). The application of thermotolerant yeast strains engineered to the SSF process would overcome the drawback by performing hydrolysis and fermentation at elevated temperature. In this review, we focus on the recent advances in the application of thermotolerant yeast to CBP and SSF of lignocellulosic material to ethanol. The development of thermotolerant and ethanologenic yeast strains with the ability to hydrolyze lignocellulosic materials is emphasized for high-temperature CBP.     

Bacteriocin Production: a Probiotic Trait?

Bacteriocins are an abundant and diverse group of ribosomally synthesized antimicrobial peptides produced by bacteria and archaea. Traditionally, bacteriocin production has been considered an important trait in the selection of probiotic strains, but until recently, few studies have definitively demonstrated the impact of bacteriocin production on the ability of a strain to compete within complex microbial communities and/or positively influence the health of the host. Although research in this area is still in its infancy, there is intriguing evidence to suggest that bacteriocins may function in a number of ways within the gastrointestinal tract. Bacteriocins may facilitate the introduction of a producer into an established niche, directly inhibit the invasion of competing strains or pathogens, or modulate the composition of the microbiota and influence the host immune system. Here we review the role of bacteriocin production in complex microbial communities and their potential to enhance human health.     

生物菌剂用于玉米浆液贮运的实验研究

目的:筛选用于玉米浆液态贮运的生物菌剂和应用技术。方法:利用筛选到的优势菌系进行组合制成复合菌剂添加到玉米浆液中,模拟自然条件发酵培养并定期取样和观察。结果:玉米浆液贮运过程中添加适量的(0.1%)生物菌剂能有效保持和改善玉米浆液理化和生物性状,其中还原糖在40h时下降23%;菌数达1.5×108cfu/mL。结论:该研究证实玉米浆液贮运过程中应用生物菌剂的可行性。     

Bioconversion of lutein to products with aroma

A residual mud sample from the marigold flower dehydration process was screened and 19 putative colonies were isolated for their ability to degrade lutein in a chemically defined medium supplemented with marigold flower flour as a carbon source. Among the colonies isolated, two generated volatile compounds in fermentation and one was chosen for further study for its ability to produce a strong tobacco smell. This colony contained two microorganisms, identified as Geotrichum sp. and Bacillus sp. The aroma production requires the presence of both microorganisms and lutein. Using gas chromatography coupled to mass spectrometry (GC/MS), four compounds were identified: 7,8-dihydro- β-ionol, β-ionone, 7,8-dihydro-β-ionone, and 3-hydroxy-β-ionone, in proportions of 84.2%, 9.4%, 3.5%, and 2.9%, respectively. Received: 30 November 1999 / Received revision: 18 April 2000 / Accepted: 1 May 2000     

Isolation by genetic and physiological characteristics of a fuel-ethanol fermentative Saccharomyces cerevisiae strain with potential for genetic manipulation

Fuel ethanol fermentation process is a complex environment with an intensive succession of yeast strains. The population stability depends on the use of a well-adapted strain that can fit to a particular industrial plant. This stability helps to keep high level of ethanol yield and it is absolutely required when intending to use recombinant strains. Yeast strains have been previously isolated from different distilleries in Northeast Brazil and clustered in genetic strains by PCR-fingerprinting. In this report we present the isolation and selection of a novel Saccharomyces cerevisiae strain by its high dominance in the yeast population. The new strain, JP1 strain, presented practically the same fermentative capacity and stress tolerance like the most used commercial strains, with advantages of being highly adapted to different industrial units in Northeast Brazil that used sugar cane juice as substrate. Moreover, it presented higher transformation efficiency that pointed out its potential for genetic manipulations. The importance of this strain selection programme for ethanol production is discussed.     

Biofuels – challenges & chances: How biofuel development can benefit from advanced process technology

The presented article highlights the process of biofuel production with a special focus on bioethanol. After a short introduction to the “problems” of biofuels – the “first generation” biofuels (in regards to their competition to feed and food production) and the “second generation” biofuels (in regards to the required more complex process technology) − the different steps in the process from natural resources towards the final product are presented and the underlying biotechnological challenges discussed: the pre-treatment of the natural resources followed by the biotechnological processes of hydrolysis and fermentation. Topics such as enzyme screening for efficient or even multi-step hydrolysis as well as microbial strain selection under process conditions and the optimization of the anaerobic fermentative conversion of the saccharides to bioethanol are discussed. Optimizing the production of bioethanol to be competitive with petrochemical fuels is the main challenge for the underlying process development.     

Screening and selection of lactic acid bacteria strains suitable for ensiling grass

AIM: Lactic acid bacteria (LAB) strains shown to have broad-spectrum antimicrobial activity were screened for potential as grass silage inoculants. The strains capable of rapidly lowering the pH of the grass matrix and with low proteolytic activity were assessed in laboratory-scale silos in a grass matrix containing natural microbial flora. METHODS AND RESULTS: Screening of nine candidate strains was performed first in a grass extract medium. The four most promising strains were selected on the basis of growth rate in the medium, capacity to reduce pH and ability to limit the formation of ammonia-N. The efficiency of the selected strains was further assessed in a laboratory-scale ensiling experiment. Untreated (no additive) and formic acid served as controls. All tested inoculants improved silage quality compared with untreated. With one exception (Pediococcus parvulus E315) the fermentation losses in the inoculated silages were even lower than in the acid-treated control silage. Pure lactic acid fermentation was obtained in the timothy-meadow fescue silage with all inoculants. The results obtained in the ensiling experiments were consistent with those of the screening procedure, which appeared to predict correctly the potential of LAB as silage inoculants. The strains with a low ammonia production rate in the grass extract medium behaved similarly in the silage. Especially in this respect the strain Lactobacillus plantarum E76 was superior to the other candidates. CONCLUSIONS: The screening method using grass extract proved to be useful in strain selection. SIGNIFICANCE AND IMPACT OF THE STUDY: The rapid screening method developed for the LAB strains provides a useful tool for more systematic product development of commercial inoculant preparations. Time consuming and laborious ensiling experiments can be limited only to the most promising strains.     

微生物过氧化氢酶的发酵生产及其在纺织工业的应用

微生物过氧化氢酶是一种重要的工业酶制剂,可以催化分解过氧化氢生成水和氧气。这一酶制剂在食品、纺织、医药等领域表现出广泛的应用潜力。生物工程和基因工程技术的进步推动了微生物过氧化氢酶的发酵生产。以下综述了微生物过氧化氢酶发酵生产的进展及其在纺织工业中的应用,同时讨论了微生物过氧化氢酶的发酵生产和纺织工业应用的未来趋势。     

Recombinant expression analysis of natural and synthetic bovine alpha-casein in Escherichia coli

As a prelude to developing a yeast-based fermentation process for the production of phenylalanine-free alpha-casein as a foodstuff for patients suffering from phenylketonuria, we cloned the gene encoding bovine alpha-casein. We synthesised a modified gene sequence encoding the same, but devoid of phenylalanine codons and with a codon bias similar to that of naturally occurring highly expressed genes in Saccharomyces cerevisiae. The results show that both gene sequences are readily expressed in Escherichia coli when cloned in an E. coli bacteriophage T7 promoter-driven plasmid vector. In this host, the natural and synthetic casein proteins were produced at levels equating to 18.0% and 7.6% of the cell's soluble protein, respectively. Received: 18 January 2000 / Received revision: 22 May 2000 / Accepted: 26 May 2000