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This review explores the production of the fungal polysaccharide pullulan by mutants and natural isolates of Aureobasidium species using strain improvement. Pullulan is a neutral polysaccharide gum whose structure is a maltotriose-containing glucan. This polysaccharide gum has applications in the fields of food, pharmaceuticals, biomedical and wastewater treatment. The strain improvement of Aureobasidium species has focused on the pullulan production process, including the isolation of strains exhibiting reduced pigmentation, polysaccharide overproduction, the production of pullulan with variable molecular weight, and increased osmotolerant strains promoting pullulan production at high carbon source concentrations and pullulan production on hemicellulosic substrates. The majority of studies have emphasized the isolation of reduced pigmentation and pullulan hyperproducer strains since the goal of large-scale commercial pullulan production is to synthesize non-pigmented polysaccharides. A promising area of strain improvement is the isolation of strains that synthesize authentic pullulan from hemicellulosic substrates. If strain improvement in this area is successful, the goal of commercially producing pullulan at a competitive cost will eventually be achieved. [ABSTRACT FROM AUTHOR]

Background: Oritavancin is a new generation of semi-synthetic glycopeptide antibiotics against Gram-positive bacteria, which served as the first and only antibiotic with a single-dose therapeutic regimen to treat ABSSSI. A naturally occurring glycopeptide A82846B is the direct precursor of oritavancin. However, its application has been hampered by low yields and homologous impurities. This study established a multi-step combinatorial strategy to rationally construct a high-quality and high-efficiency biosynthesis system for A82846B and systematically optimize its fermentation process to break through the bottleneck of microbial fermentation production. Results: Firstly, based on the genome sequencing and analysis, we deleted putative competitive pathways and constructed a better A82846B-producing strain with a cleaner metabolic background, increasing A82846B production from 92 to 174 mg/L. Subsequently, the PhiC31 integrase system was introduced based on the CRISPR-Cas12a system. Then, the fermentation level of A82846B was improved to 226 mg/L by over-expressing the pathway-specific regulator StrR via the constructed PhiC31 system. Furthermore, overexpressing glycosyl-synthesis gene evaE enhanced the production to 332 mg/L due to the great conversion of the intermediate to target product. Finally, the scale-up production of A82846B reached 725 mg/L in a 15 L fermenter under fermentation optimization, which is the highest reported yield of A82846B without the generation of homologous impurities. Conclusion: Under approaches including blocking competitive pathways, inserting site-specific recombination system, overexpressing regulator, overexpressing glycosyl-synthesis gene and optimizing fermentation process, a multi-step combinatorial strategy for the high-level production of A82846B was developed, constructing a high-producing strain AO-6. The combinatorial strategies employed here can be widely applied to improve the fermentation level of other microbial secondary metabolites, providing a reference for constructing an efficient microbial cell factory for high-value natural products. [ABSTRACT FROM AUTHOR]

Strain improvement via chemical mutagen could impart traits with better enzyme production or improved characteristics. The present study sought to investigate the physicochemical properties of pullulanase produced from the wild Bacillus sp and the mutant. The pullulanases produced from the wild and the mutant Bacillus sp. (obtained via induction with ethyl methyl sulfonate) were purified in a-three step purification procedure and were also characterized. The wild and mutant pullulanases, which have molecular masses of 40 and 43.23 kDa, showed yields of 2.3% with 6.0-fold purification and 2.0% with 5.0-fold purification, respectively, and were most active at 50 and 40 °C and pH 7 and 8, respectively. The highest stability of the wild and mutant was between 40 and 50 °C after 1 h, although the mutant retained greater enzymatic activity between pH 6 and 9 than the wild. The mutant had a decreased Km of 0.03 mM as opposed to the wild type of 1.6 mM. In comparison to the wild, the mutant demonstrated a better capacity for tolerating metal ions and chelating agents. These exceptional characteristics of the mutant pullulanase may have been caused by a single mutation, which could improve its utility in industrial and commercial applications. [ABSTRACT FROM AUTHOR]

Microfluidic systems have fundamentally transformed the realm of adaptive laboratory evolution (ALE) for microorganisms by offering unparalleled control over environmental conditions, thereby optimizing mutant generation and desired trait selection. This review summarizes the substantial influence of microfluidic technologies and their design paradigms on microbial adaptation, with a primary focus on leveraging spatial stressor concentration gradients to enhance microbial growth in challenging environments. Specifically, microfluidic platforms tailored for scaled-down ALE processes not only enable highly autonomous and precise setups but also incorporate novel functionalities. These capabilities encompass fostering the growth of biofilms alongside planktonic cells, refining selection gradient profiles, and simulating adaptation dynamics akin to natural habitats. The integration of these aspects enables shaping phenotypes under pressure, presenting an unprecedented avenue for developing robust, stress-resistant strains, a feat not easily attainable using conventional ALE setups. The versatility of these microfluidic systems is not limited to fundamental research but also offers promising applications in various areas of stress resistance. As microfluidic technologies continue to evolve and merge with cutting-edge methodologies, they possess the potential not only to redefine the landscape of microbial adaptation studies but also to expedite advancements in various biotechnological areas. Key points: • Microfluidics enable precise microbial adaptation in controlled gradients. • Microfluidic ALE offers insights into stress resistance and distinguishes between resistance and persistence. • Integration of adaptation-influencing factors in microfluidic setups facilitates efficient generation of stress-resistant strains. [ABSTRACT FROM AUTHOR]

Microfluidic systems have fundamentally transformed the realm of adaptive laboratory evolution (ALE) for microorganisms by offering unparalleled control over environmental conditions, thereby optimizing mutant generation and desired trait selection. This review summarizes the substantial influence of microfluidic technologies and their design paradigms on microbial adaptation, with a primary focus on leveraging spatial stressor concentration gradients to enhance microbial growth in challenging environments. Specifically, microfluidic platforms tailored for scaled-down ALE processes not only enable highly autonomous and precise setups but also incorporate novel functionalities. These capabilities encompass fostering the growth of biofilms alongside planktonic cells, refining selection gradient profiles, and simulating adaptation dynamics akin to natural habitats. The integration of these aspects enables shaping phenotypes under pressure, presenting an unprecedented avenue for developing robust, stress-resistant strains, a feat not easily attainable using conventional ALE setups. The versatility of these microfluidic systems is not limited to fundamental research but also offers promising applications in various areas of stress resistance. As microfluidic technologies continue to evolve and merge with cutting-edge methodologies, they possess the potential not only to redefine the landscape of microbial adaptation studies but also to expedite advancements in various biotechnological areas. Key points: • Microfluidics enable precise microbial adaptation in controlled gradients. • Microfluidic ALE offers insights into stress resistance and distinguishes between resistance and persistence. • Integration of adaptation-influencing factors in microfluidic setups facilitates efficient generation of stress-resistant strains. [ABSTRACT FROM AUTHOR]

Long-chain polyunsaturated fatty acids (LC-PUFA) are nutritionally and pharmacologically well-recognized beneficial compounds widely used as nutrient supplements for various applications. As the human body cannot accomplish de novo synthesis of PUFA, we depend on exogenous bioresources for the supply of these essential PUFA for day-to-day activities. The PUFA global market is expected to reach about US$ 9.7 billion by 2032. The recent drop in natural fish catch and global climate change severely hamper the global PUFA supply chain. As a sustainable, promising resource, marine zoosporic microorganisms are considered to be safe and alternative primary producers of beneficial PUFAs for direct and indirect human consumption. However, by considering the limitations associated with strain efficiency, final product yield and production cost, more viable low-cost production strategies and establishment of highly efficient, stable, high production cell lines specific for marine zoosporic microbes are essential to meet global PUFA market demand. The exploration of the latest techniques for research and development could shape the dynamics better for the global market for marine zoosporic organism-based PUFA products. Therefore, this review first summarizes low-cost production and strain improvement strategies, and later highlights the advantages and commercial prospects for marine zoosporic microbe-derived PUFA. [ABSTRACT FROM AUTHOR]

Brevibacillus sp. SPR20 produced potentially antibacterial substances against methicillin-resistant Staphylococcus aureus (MRSA). The synthesis of these substances is controlled by their biosynthetic gene clusters. Several mutagenesis methods are used to overcome the restriction of gene regulations when genetic information is absent. Atmospheric and room temperature plasma (ARTP) is a powerful technique to initiate random mutagenesis for microbial strain improvement. This study utilized an argon-based ARTP to conduct the mutations on SPR20. The positive mutants of 40% occurred. The M27 mutant exhibited an increase in anti-MRSA activity when compared to the wild-type strain, with the MIC values of 250–500 and 500 μg/mL, respectively. M27 had genetic stability because it exhibited constant activity throughout fifteen generations. This mutant had similar morphology and antibiotic susceptibility to the wild type. Comparative proteomic analysis identified some specific proteins that were upregulated in M27. These proteins were involved in the metabolism of amino acids, cell structure and movement, and catalytic enzymes. These might result in the enhancement of the anti-MRSA activity of the ARTP-treated SPR20 mutant. This study supports the ARTP technology designed to increase the production of valuable antibacterial agents. [ABSTRACT FROM AUTHOR]

In the present study, Aspergillus uvarum was subjected to random mutagenesis, by treatment with chemical mutagen i.e., Ethyl methanesulfonate (EMS). Among the various mutant strains of A. uvarum, the strain that was obtained after treatment with EMS at 12% v/v concentration and 60 min of exposure time showed 1.8-fold and 1.4-fold increased cellulase activity using submerged (SmF) and solid state fermentation (SSF), respectively, as compared to the parent wild strain. Partial purification of the cellulase by ammonium sulphate fractionation and dialysis showed 1.46 and 2.02 fold purification, respectively. Among the several concentrations of sodium alginate and calcium chloride used for immobilization, partially purified cellulase demonstrated maximum activity with 2% w/v concentration of sodium alginate and 0.2 M concentration of calcium chloride. The optimal temperature and pH for immobilized cellulase were observed to be 60 °C and 7, respectively. Furthermore, the immobilized enzyme presented increased reusability, having 18.5% relative activity after five consecutive uses, and was more stable, maintaining 64.5 and 48.7% of its initial activity at 4 and 30 °C, respectively, after 30 days of storage. [ABSTRACT FROM AUTHOR]

Strains' improvement technology plays an essential role in enhancing the quality of industrial strains. Several traditional methods and modern techniques have been used to further improve strain engineering programs. The advances stated in strain engineering and the increasing demand for microbial metabolites leads to the invention of the genome shuffling technique, which ensures a specific phenotype improvement through inducing mutation and recursive protoplast fusion. In such technique, the selection of multi-parental strains with distinct phenotypic traits is crucial. In addition, as this evolutionary strain improvement technique involves combinative approaches, it does not require any gene sequence data for genome alteration and, therefore, strains developed by this elite technique will not be considered as genetically modified organisms. In this review, the different stages involved in the genome shuffling technique and its wide applications in various phenotype improvements will be addressed. Taken together, data discussed here highlight that the use of genome shuffling for strain improvement will be a plus for solving complex phenotypic traits and in promoting the rapid development of other industrially important strains. [ABSTRACT FROM AUTHOR]

Propionic acid (PA) is a 3-carbon carboxylic acid widely used in the chemical, food and pharmaceutical industries. Current production of PA is primarily via petrochemical routes and alternative sources using renewable feedstocks are desirable. The goal of the present study was to develop bacterial strains for efficient PA production by fermentation of sugarcane-derived saccharides in inverted sugarcane syrup. Adaptive evolution and random chemical mutagenesis of Propionibacterium acidipropionici were individually evaluated. Although evolutionary engineering proved to be a suitable strategy for producing strains tolerant to acid stress, chemical mutagenesis coupled with a novel screening methodology proved to be a superior strategy for strain improvement. The selected strain presented high PA productivity (0.55 g L−1 h−1) and titer (40 g L−1) in relation to the parental strain (0.38 g L−1 h−1 and 27 g L−1, respectively), in fermentations using inverted sugarcane syrup. [Display omitted] • P. acidipropionici is more tolerant to propionate than other propionibacteria. • Acid tolerance did not correlate with higher propionate production. • Random mutagenesis coupled to rounds of selection results in improved strains. • The M8 strain ferments high test molasses with a 60% production rate improvement. [ABSTRACT FROM AUTHOR]

Studies on the microorganisms used in food production are of interest because microbial genotypes are reflected in food qualities such as taste, flavor, and yield. However, several microbes are nonmodel organisms, and their analysis is often limited by the lack of genetic tools. Tetragenococcus halophilus, a halophilic lactic acid bacterium used in soy sauce fermentation starter culture, is one such microorganism. The lack of DNA transformation techniques for T. halophilus makes gene complementation and disruption assays difficult. Here, we report that the endogenous insertion sequence ISTeha4, belonging to the IS4 family, is translocated at an extremely high frequency in T. halophilus and causes insertional mutations at various loci. We developed a method named targeting spontaneous insertional mutations in genomes (TIMING), which combines high-frequency insertional mutations and efficient PCR screening, enabling the isolation of gene mutants of interest from a library. The method provides a reverse genetics and strain improvement tool, does not require the introduction of exogenous DNA constructs, and enables the analysis of nonmodel microorganisms lacking DNA transformation techniques. Our results highlight the important role of insertion sequences as a source of spontaneous mutagenesis and genetic diversity in bacteria. IMPORTANCE Genetic and strain improvement tools to manipulate a gene of interest are required for the nontransformable lactic acid bacterium Tetragenococcus halophilus. Here, we demonstrate that an endogenous transposable element, ISTeha4, is transposed into the host genome at an extremely high frequency. A genotype-based and non-genetically engineered screening system was constructed to isolate knockout mutants using this transposable element. The method described enables a better understanding of the genotype-phenotype relationship and serves as a tool to develop food-grade-appropriate mutants of T. halophilus. [ABSTRACT FROM AUTHOR]

2-Phenylethanol (2- PE) is an aromatic alcohol with wide applications, but there is still no efficient microbial cell factory for 2-PE based on Escherichia coli. In this study, we constructed a metabolically engineered E. coli capable of de novo synthesis of 2-PE from glucose. Firstly, the heterologous styrene-derived and Ehrlich pathways were individually constructed in an L-Phe producer. The results showed that the Ehrlich pathway was better suited to the host than the styrene-derived pathway, resulting in a higher 2-PE titer of ∼0.76 ± 0.02 g/L after 72 h of shake flask fermentation. Furthermore, the phenylacetic acid synthase encoded by feaB was deleted to decrease the consumption of 2-phenylacetaldehyde, and the 2-PE titer increased to 1.75 ± 0.08 g/L. As phosphoenolpyruvate (PEP) is an important precursor for L-Phe synthesis, both the crr and pykF genes were knocked out, leading to ∼35% increase of the 2-PE titer, which reached 2.36 ± 0.06 g/L. Finally, a plasmid-free engineered strain was constructed based on the Ehrlich pathway by integrating multiple ARO10 cassettes (encoding phenylpyruvate decarboxylases) and overexpressing the yjgB gene. The engineered strain produced 2.28 ± 0.20 g/L of 2-PE with a yield of 0.076 g/g glucose and productivity of 0.048 g/L/h. To our best knowledge, this is the highest titer and productivity ever reported for the de novo synthesis of 2-PE in E. coli. In a 5-L fermenter, the 2-PE titer reached 2.15 g/L after 32 h of fermentation, suggesting that the strain has the potential to efficiently produce higher 2-PE titers following further fermentation optimization. [ABSTRACT FROM AUTHOR]

Transposons are mobile genetic elements that can give rise to gene mutation and genome rearrangement. Due to their mobility, transposons have been exploited as genetic tools for modification of plants, animals, and microbes. Although a plethora of reviews have summarized families of transposons, the transposons from fermentation bacteria have not been systematically documented, which thereby constrain the exploitation for metabolic engineering and synthetic biology purposes. In this review, we summarize the transposons from the most used fermentation bacteria including Escherichia coli, Bacillus subtilis, Lactococcus lactis, Corynebacterium glutamicum, Klebsiella pneumoniae, and Zymomonas mobilis by literature retrieval and data mining from GenBank and KEGG. We also outline the state-of-the-art advances in basic research and industrial applications especially when allied with other genetic tools. Overall, this review aims to provide valuable insights for transposon-mediated strain improvement. Key points: • The transposons from the most-used fermentation bacteria are systematically summarized. • The applications of transposons in strain improvement are comprehensively reviewed. [ABSTRACT FROM AUTHOR]

Antibiotic resistance is a major health concern worldwide. In our previous study, some bacterial isolates exhibited antibacterial activity against Staphylococcus aureus and methicillin-resistant S. aureus (MRSA). However, the production of antibacterial substances by native microorganisms is limited by biosynthetic genes. This study aimed to improve the antibacterial activity of SPR19 using atmospheric and room temperature plasma mutagenesis (ARTP). The results showed that SPR19 belonged to the Brevibacillus genus. The growth curves and production kinetics of antibacterial substances were investigated. Argon-based ARTP was applied to SPR19, and the 469 mutants were preliminarily screened using agar overlay method. The remaining 25 mutants were confirmed by agar well diffusion assay against S. aureus TISTR 517 and MRSA isolates 142, 1096, and 2468. M285 exhibited the highest activity compared to the wild-type strain (10.34–13.59%) and this mutant was stable to produce the active substances throughout 15 generations consistently. The antibacterial substances from M285 were tolerant to various conditions (heat, enzyme, surfactant, and pH) while retaining more than 90% of their activities. Therefore, Brevibacillus sp. SPR19 is a potential source of antibacterial substances. ARTP mutagenesis is a powerful method for strain improvement that can be utilized to treat MRSA infection in the future. [ABSTRACT FROM AUTHOR]

D -arabitol, being a xylitol enantiomer with a caloric value (0.2 kcal/g) much lower than sucrose occurs typically in yeasts and higher fungi when these organisms are under environmentally stressed conditions. Credited with such beneficial properties, the United States Department of Energy in 2004 has subsumed it as one among the twelve useful building-block chemicals for biorefinery. Current industrial production employs chemical synthesis which includes two-step hydrogenation process using arabinoic acid and lactones as a starting material. Due to its limited availability in natural sources, maneuvering strategies for D -arabitol production via biological process might cater to a scalable yield in the future. Herein, a detailed summary on strain improvement , where the scope of metabolic and genetic engineering methods to engender yeasts strains for improved arabitol production is highlighted. Further, the importance of understanding the metabolic pathway that regulates D -arabitol synthesis and its significant influence in optimizing the fermentation process is also briefly summarized. Producing D -arabitol by yeast chiefly involves fermenting glucose and glycerol. Elucidating the D -arabitol biosynthesis pathways and regulatory mechanism has elevated the researcher's interest in engineering the host strain by mutagenesis, genome shuffling, metabolic engineering, and regulators for increasing D -arabitol titers. Nevertheless, rapid progress in the past half-decade culminating in the synthesis of D -arabitol from cheap renewable sources and byproducts that arise during biodiesel production are reviewed. In the future, along with the approaches mentioned above, works focusing more on building prokaryotic hosts (E. coli , B. subtilis , etc.) via synthetic biology will drive the D -arabitol production closer to the industries that would benefit humanity. • 1. D -arabitol is one among the top-twelve useful building blocks for biorefinery. • Yeasts serve as an important microbial host for D -arabitol biosynthesis. • Genome and metabolic engineering help the yeast to enhance the D -arabitol yields. • Feasibility of xylose, arabinose and glycerol for D -arabitol synthesis are highlighted. • Fermentation and pentose phosphate pathway regulation influence D -arabitol yields. [ABSTRACT FROM AUTHOR]

Milbemycins, a group of 16-membered macrocylic lactones with excellent acaricidal, insecticidal and anthelmintic activities, can be produced by several Streptomyces species. For the reason that they have low toxicity in mammals, milbemycins and their derivatives are widely used in agricultural, medical and veterinary industries. Streptomyces bingchenggensis, one of milbemycin-producing strains, has been sequenced and intensively investigated in the past decades. In this mini-review, we comprehensively revisit the progress that has been made in research efforts to elucidate the biosynthetic pathways and regulatory networks for the cellular production of milbemycins. The advances in the development of production strains for milbemycin and its derivatives are discussed along the strain-generation technical approaches of random mutagenesis, metabolic engineering and combinatorial biosynthesis. The research progress made so far indicates that strain improvement and generation of novel milbemycin derivatives will greatly benefit from future development of enabling technologies and deeper understanding of the fundamentals of biosynthesis of milbemycin and the regulation of its production in S. bingchenggensis. This mini-review also proposes that the overproduction of milbemycins could be greatly enhanced by genome minimization, systematical metabolic engineering and synthetic biology approaches in the future. [ABSTRACT FROM AUTHOR]

Actinomycetes are recognized as excellent producers of microbial natural products, which have a wide range of applications, especially in medicine, agriculture and stockbreeding. The three main indexes of industrialization (titer, purity and stability) must be taken into overall consideration in the manufacturing process of natural products. Over the past decades, synthetic biology techniques have expedited the development of industrially competitive strains with excellent performances. Here, we summarize various rational engineering strategies for upgrading the performance of industrial actinomycetes, which include enhancing the yield of natural products, eliminating the by-products and improving the genetic stability of engineered strains. Furthermore, the current challenges and future perspectives for optimizing the industrial strains more systematically through combinatorial engineering strategies are also discussed. [Display omitted] • Actinomycetes are the important producers of microbial natural products. • Strategies for improving the productivity of industrial microbes are summarized. • Strategies for ameliorating the quality of microbial natural products are showcased. • Available engineering strategies for retarding strain degeneration are highlighted. [ABSTRACT FROM AUTHOR]

Antibiotic-resistant infection is a major health problem, and a limited number of drugs are currently approved as antibiotics. Soil bacteria are promising sources in the search for novel antibiotics. The aim of the present study is to isolate and assess soil bacteria with anti-MRSA activity and improve their capabilities by UV mutagenesis. Soil samples from the upper south of Thailand were screened for antibacterial activity using the cross-streak method. Agar well diffusion was used to examine the activity of isolates against a spectrum of human pathogens. The most active isolate was identified by 16S rRNA sequencing, and the production kinetics and stability were investigated. The most promising isolate was mutated by UV radiation, and the resulting activity and strain stability were studied. The results show that isolates from the cross-streak method could inhibit Staphylococcus aureus TISTR 517 (94 isolates) and Escherichia coli TISTR 887 (67 isolates). Nine isolates remained active against S. aureus TISTR 517 and MRSA, and eight isolates inhibited the growth of E. coli TISTR 887 as assessed using agar well diffusion. The most active strain was Brevibacillus sp. SPR-20, which had the highest activity at 24 h of incubation. The active substances in culture supernatants exhibited more than 90% activity when subjected to treatments involving various heat, enzymes, surfactants, and pH conditions. The mutant M201 showed significantly higher activity (109.88-120.22%) and strain stability compared to the wild-type strain. In conclusion, we demonstrate that soil Brevibacillus sp. is a potential resource that can be subjected to UV mutagenesis as a useful approach for improving the production of anti-MRSA in the era of antibiotic resistance. [ABSTRACT FROM AUTHOR]

Microalgae have potential for environmental remediation, but they must better tolerate stress. Adaptive laboratory evolution (ALE) is effective to construct evolved strains, but its efficiency is low. Highly efficient ALE relies on selecting suitable environmental stress, original strain selection, and optimizing initial cell density and stress strategy. [ABSTRACT FROM AUTHOR]

• Bioprocess of long-chain fatty acids accumulating new isolate Micractinium sp. • Optimized FACS conditions in combination with fluorescent dyes for cell sorting. • Established of FACS-based adaptive evolution strategy for strain improvement. • Achieved improved phenotypes with high biomass and desirable fatty acids. composition A new green microalga isolate Micractinium sp. GA001 was found to accumulate long-chain fatty acids, and the strain was subjected to flow cytometry-based adaptive evolution approach to produce improved phenotypes. At first, original phenotype of new isolate GA001 was well characterized followed by establishment of flow cytometry conditions in combination with fluorescent dyes BODIPY and Nile Red, to screen intracellular long-chain fatty acids in GA001. Fluorescent dyes staining and flow cytometry analysis revealed the progressive accumulation of desirable lipid components in GA001. Further, a flow cytometry-based strategy was used to selectively isolate and enrich particular GA001 phenotypes with higher accumulation of long-chain fatty acids, under nitrogen-depletion and –repletion conditions. This strategy yielded an improved population with high lipid content than original population. Micractinium sp. GA001 was proved to be a promising strain with improved phenotypes for the production of large-scale target-specific long-chain fatty acids. [ABSTRACT FROM AUTHOR]

Background: Activation of silent biosynthetic gene clusters (BGCs) in marine-derived actinomycete strains is a feasible strategy to discover bioactive natural products. Actinoalloteichus sp. AHMU CJ021, isolated from the seashore, was shown to contain an intact but silent caerulomycin A (CRM A) BGC-cam in its genome. Thus, a genome mining work was preformed to activate the strain's production of CRM A, an immunosuppressive drug lead with diverse bioactivities. Results: To well activate the expression of cam, ribosome engineering was adopted to treat the wild type Actinoalloteichus sp. AHMU CJ021. The initial mutant strain XC-11G with gentamycin resistance and CRM A production titer of 42.51 ± 4.22 mg/L was selected from all generated mutant strains by gene expression comparison of the essential biosynthetic gene-camE. The titer of CRM A production was then improved by two strain breeding methods via UV mutagenesis and cofactor engineering-directed increase of intracellular riboflavin, which finally generated the optimal mutant strain XC-11GUR with a CRM A production titer of 113.91 ± 7.58 mg/L. Subsequently, this titer of strain XC-11GUR was improved to 618.61 ± 16.29 mg/L through medium optimization together with further adjustment derived from response surface methodology. In terms of this 14.6 folds increase in the titer of CRM A compared to the initial value, strain XC-GUR could be a well alternative strain for CRM A development. Conclusions: Our results had constructed an ideal CRM A producer. More importantly, our efforts also had demonstrated the effectiveness of abovementioned combinatorial strategies, which is applicable to the genome mining of bioactive natural products from abundant actinomycetes strains. [ABSTRACT FROM AUTHOR]

The global energy crisis and limited supply of petroleum fuels have rekindled the interest in utilizing a sustainable biomass to produce biofuel. Butanol, an advanced biofuel, is a superior renewable resource as it has a high energy content and is less hygroscopic than other candidates. At present, the biobutanol route, employing acetone–butanol–ethanol (ABE) fermentation in Clostridium species, is not economically competitive due to the high cost of feedstocks, low butanol titer, and product inhibition. Based on an analysis of the physiological characteristics of solventogenic clostridia, current advances that enhance ABE fermentation from strain improvement to product separation were systematically reviewed, focusing on: (1) elucidating the metabolic pathway and regulation mechanism of butanol synthesis; (2) enhancing cellular performance and robustness through metabolic engineering, and (3) optimizing the process of ABE fermentation. Finally, perspectives on engineering and exploiting clostridia as cell factories to efficiently produce various chemicals and materials are also discussed. [ABSTRACT FROM AUTHOR]

Strain development is frequently used to improve the performance and functionality of industrially important microbes. As traditional mutagenesis screen is especially utilized by the food industry to improve strains used in food fermentation, high-throughput and cost-effective screening tools are important in mutant selection. The emerging droplet-based microfluidics technology miniaturizes the volume for cell cultivation and phenotype interrogation down to the picoliter scales, which facilitates screening of microbes for improved phenotypical properties tremendously. In this mini review, we present recent application of the droplet-based microfluidics in microbial strain improvement with a focus on its potential use in the screening of lactic acid bacteria. [ABSTRACT FROM AUTHOR]

High 1-butanol titer has been achieved in a transgenic Escherichia coli strain JCL299FT with a heterologous 1-butanol pathway by deleting competing pathways, balancing of cofactor and resolving free CoA imbalance. However, further improvement of 1-butanol production is still possible in the highest producing strain JCL299FT as indicated by the accumulation of acetate, a major undesired by-product during bio-production by microorganisms that competes with 1-butanol production for the available acetyl-CoA and inhibits protein synthesis resulting in poor growth. In this study, liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based metabolome analysis was performed to identify new rate limiting steps in the 1-butanol production pathway of E. coli strain JCL299FT. The results of metabolome analysis showed increased amounts of glyoxylate in JCL299FT compared to the previous highest-producing strain JCL299F. Knocking out aceA successfully decreased the amount of glyoxylate and reduced acetate accumulation, resulting in the increased levels of TCA cycle and 1-butanol pathway metabolites. These observations indicated that there was a redirection of flux from acetate to TCA cycle and 1-butanol producing pathway, which led to better growth of the 1-butanol producing strain. Consequently, 1-butanol production titer was improved by 39% and the production yield was improved by 12% in M9 medium supplemented with yeast extract. This study is the first report of using the knockout of aceA, the first gene in the glyoxylate shunt that encodes isocitrate lyase, as an effective strategy to reduce acetate overflow in 1-butanol producing E. coli. Highlights • Knockout of aceA improved 1-butanol production in Escherichia coli. • The aceA knockout strain showed decreased acetate production and increased growth. • 1-Butanol production titer was improved by 39% in the aceA knockout strain. [ABSTRACT FROM AUTHOR]

L-Lysine is widely used as a nutrition supplement in feed, food, and beverage industries as well as a chemical intermediate. At present, great efforts are made to further decrease the cost of lysine to make it more competitive in the markets. Furthermore, lysine also shows potential as a feedstock to produce other high-value chemicals for active pharmaceutical ingredients, drugs, or materials. In this review, the current biomanufacturing of lysine is first presented. Second, the production of novel derivatives from lysine is discussed. Some chemicals like L-pipecolic acid, cadaverine, and 5-aminovalerate already have been obtained at a lab scale. Others like 6-aminocaproic acid, valerolactam, and caprolactam could be produced through a biological and chemical coupling pathway or be synthesized by a hypothetical pathway. This review demonstrates an active and expansive lysine industry, and these green biomanufacturing strategies could also be applied to enhance the competitiveness of other amino acid industry. [ABSTRACT FROM AUTHOR]

In industry, filamentous fungi have a prominent position as producers of economically relevant primary or secondary metabolites. Particularly, the advent of genetic engineering of filamentous fungi has led to a growing number of molecular tools to adopt filamentous fungi for biotechnical applications. Here, we summarize recent developments in fungal biology, where fungal host systems were genetically manipulated for optimal industrial applications. Firstly, available inducible promoter systems depending on carbon sources are mentioned together with various adaptations of the Tet-Off and Tet-On systems for use in different industrial fungal host systems. Subsequently, we summarize representative examples, where diverse expression systems were used for the production of heterologous products, including proteins from mammalian systems. In addition, the progressing usage of genomics and functional genomics data for strain improvement strategies are addressed, for the identification of biosynthesis genes and their related metabolic pathways. Functional genomic data are further used to decipher genomic differences between wild-type and high-production strains, in order to optimize endogenous metabolic pathways that lead to the synthesis of pharmaceutically relevant end products. Lastly, we discuss how molecular data sets can be used to modify products for optimized applications. [ABSTRACT FROM AUTHOR]

The growing demand for biotechnological products against limited metabolic capacity of industrially used microorganisms has led to an increased interest on strain-improvement over the last several decades, which aimed to enhance metabolite yield, substrate uptake and tolerance of the strains. Among a few techniques of strain-improvement, genome shuffling is the most recent and promising approach used for rapid strain-improvement that can yield a new strain by combining whole genomes of multi-parental microorganisms using the principles of protoplast fusion. Genome shuffling has brought a major breakthrough in the strain-improvement concept as it is found to be effective and reliable for expressing complex phenotypes. This review will discuss the technical aspects and applications of genome shuffling for various industrial strains to present its current status and recent progress. In the concluding remarks, a summary will be presented focusing on the major challenges and future outlooks of this technology. [ABSTRACT FROM AUTHOR]

Beer production is one of the oldest known traditional biotechnological processes, but is nowadays facing increasing demands not only for enhanced product quality, but also for improved production economics. Targeted genetic modification of a yeast strain is one way to increase beer quality and to improve the economics of beer production. In this review we will present current knowledge on traditional approaches for improving brewing strains and for rational metabolic engineering. These research efforts will, in the near future, lead to the development of a wider range of industrial strains that should increase the diversity of commercial beers. [ABSTRACT FROM AUTHOR]

Can we approach theoretical lipid yields in microalgae? Yes: we can substantially reduce the gap between current and theoretical maximum yield. A realistic maximum is approximately 0.5 g triacylglycerol (TAG) per mol photons, about five times higher than what is currently achieved in outdoor cultivation. Achieving this realistic maximum will require several breakthroughs. First, outdoor operation typically has low yields, mainly caused by fluctuating insolation. Future adaptive control models will help increase these yields. Additionally, the lipid production capacity of currently used strains needs to increase. Powerful strain-specific molecular toolboxes are being developed, shifting the bottleneck towards understanding metabolism and identifying target genes. Finally, strains and processes should be improved concurrently to fully exploit the potential lipid production from microalgae. [ABSTRACT FROM AUTHOR]

Glycopeptide antibiotics are drugs of last resort for treating severe infections caused by multi-drug resistant Gram-positive pathogens. First-generation glycopeptides (vancomycin and teicoplanin) are produced by soil-dwelling actinomycetes. Second-generation glycopeptides (dalbavancin, oritavancin, and telavancin) are semi-synthetic derivatives of the progenitor natural products. Herein, we cover past and present biotechnological approaches for searching for and producing old and new glycopeptide antibiotics. We review the strategies adopted to increase microbial production (from classical strain improvement to rational genetic engineering), and the recent progress in genome mining, chemoenzymatic derivatization, and combinatorial biosynthesis for expanding glycopeptide chemical diversity and tackling the never-ceasing evolution of antibiotic resistance. [ABSTRACT FROM AUTHOR]

The bleomycins (BLMs) are important clinical drugs extensively used in combination chemotherapy for the treatment of various cancers. Dose-dependent lung toxicity and the development of drug resistance have restricted their wide applications. 6′-Deoxy-BLM Z, a recently engineered BLM analogue with improved antitumor activity, has the potential to be developed into the next-generation BLM anticancer drug. However, its low titer in the recombinant strain Streptomyces flavoviridis SB9026 has hampered current efforts, which require sufficient compound, to pursue preclinical studies and subsequent clinical development. Here, we report the strain improvement by combined UV mutagenesis and ribosome engineering, as well as the fermentation optimization, for enhanced 6′-deoxy-BLM production. A high producer, named S. flavoviridis G-4F12, was successfully isolated, producing 6′-deoxy-BLM at above 70 mg/L under the optimized fermentation conditions, representing a sevenfold increase in comparison with that of the original producer. These findings demonstrated the effectiveness of combined empirical breeding methods in strain improvement and set the stage for sustainable production of 6′-deoxy-BLM via pilot-scale microbial fermentation. [ABSTRACT FROM AUTHOR]

Bakery industries are thriving to augment the diverse properties of Saccharomyces cerevisiae to increase its flavor, texture and nutritional parameters to attract the more consumers. The improved technologies adopted for quality improvement of baker's yeast are attracting the attention of industry and it is playing a pivotal role in redesigning the quality parameters. Modern yeast strain improvement tactics revolve around the use of several advanced technologies such as evolutionary engineering, systems biology, metabolic engineering, genome editing. The review mainly deals with the technologies for improving S. cerevisiae, with the objective of broadening the range of its industrial applications. [ABSTRACT FROM AUTHOR]

Acetoin and 2,3-butanediol (2,3-BD) have a large number of industrial applications. The production of acetoin and 2,3-BD has traditionally relied on oil supplies. Microbial production of acetoin and 2,3-BD will alleviate the dependence on oil. Acetoin and 2,3-BD are neighboring metabolites in the 2,3-BD metabolic pathway of bacteria. This review summarizes metabolic engineering strategies for improvement of microbial acetoin and 2,3-BD production. We also propose enhancements to current acetoin and 2,3-BD production strategies, by offering a metabolic engineering approach that is guided by systems biology and synthetic biology. [ABSTRACT FROM PUBLISHER]

The filamentous fungus Trichoderma reesei is found predominantly in the tropics but also in more temperate regions, such as Europe, and is widely known as a producer of large amounts of plant cell wall-degrading enzymes. We sequenced the genome of the sexually competent isolate CBS999.97, which is phenotypically different from the female sterile strain QM6a but can cross sexually with QM6a. Transcriptome data for growth on cellulose showed that entire carbohydrate-active enzyme (CAZyme) families are consistently differentially regulated between these strains. We evaluated backcrossed strains of both mating types, which acquired female fertility from CBS999.97 but maintained a mostly QM6a genetic background, and we could thereby distinguish between the effects of strain background and female fertility or mating type. We found clear regulatory differences associated with female fertility and female sterility, including regulation of CAZyme and transporter genes. Analysis of carbon source utilization, transcriptomes, and secondary metabolites in these strains revealed that only a few changes in gene regulation are consistently correlated with different mating types. Different strain backgrounds (QM6a versus CBS999.97) resulted in the most significant alterations in the transcriptomes and in carbon source utilization, with decreased growth of CBS999.97 on several amino acids (for example proline or alanine), which further correlated with the downregulation of genes involved in the respective pathways. In combination, our findings support a role of fertility-associated processes in physiology and gene regulation and are of high relevance for the use of sexual crossing in combining the characteristics of two compatible strains or quantitative trait locus (QTL) analysis. IMPORTANCE Trichoderma reesei is a filamentous fungus with a high potential for secretion of plant cell wall-degrading enzymes. We sequenced the genome of the fully fertile field isolate CBS999.97 and analyzed its gene regulation characteristics in comparison with the commonly used laboratory wild-type strain QM6a, which is not female fertile. Additionally, we also evaluated fully fertile strains with genotypes very close to that of QM6a in order to distinguish between strain-specific and fertilityspecific characteristics. We found that QM6a and CBS999.97 clearly differ in their growth patterns on different carbon sources, CAZyme gene regulation, and secondary metabolism. Importantly, we found altered regulation of 90 genes associated with female fertility, including CAZyme genes and transporter genes, but only minor mating type-dependent differences. Hence, when using sexual crossing in research and for strain improvement, it is important to consider female fertile and female sterile strains for comparison with QM6a and to achieve optimal performance. [ABSTRACT FROM AUTHOR]

Jatropha curcas seedcake (JSC), a byproduct of the biodiesel industry was utilized for the production of protease. To enhance the protease production, the parent strain of Aspergillus terreus CJS-127 subjected to UV (15, 30, 60, 90, 120 and 150 min) and chemical mutagenesis by using ethyl methanesulphonate (EMS-125, 250 and 500 mg). Two mutant strains designated as UV-6 and EMS-5 selected for optimization of protease production using JSC medium by submerged fermentation. Under the optimized conditions of pH 7.0, temperature 30°C, incubation period 72 h, inoculum 105/ml spores, 150 rpm agitation, JSC at 2% level, the maximum protease production was 172.32 and 163.57 U/ml for EMS-5 and UV-6 mutant strains respectively. The addition of maltose (2%) was found to enhance the protease production in the mutant strains. The protease activity increased by 2.30 and 2.41 folds for UV 6 and EMS 5 mutant strains after optimization of cultural conditions. [ABSTRACT FROM AUTHOR]

The recent start-up of several full-scale 'second generation' ethanol plants marks a major milestone in the development of Saccharomyces cerevisiae strains for fermentation of lignocellulosic hydrolysates of agricultural residues and energy crops. After a discussion of the challenges that these novel industrial contexts impose on yeast strains, this minireview describes key metabolic engineering strategies that have been developed to address these challenges. Additionally, it outlines how proof-of-concept studies, often developed in academic settings, can be used for the development of robust strain platforms that meet the requirements for industrial application. Fermentation performance of current engineered industrial S. cerevisiae strains is no longer a bottleneck in efforts to achieve the projected outputs of the first large-scale second-generation ethanol plants. Academic and industrial yeast research will continue to strengthen the economic value position of second-generation ethanol production by further improving fermentation kinetics, product yield and cellular robustness under process conditions. [ABSTRACT FROM AUTHOR]

The ability to produce polysaccharides with diverse biological functions is widespread in bacteria. In lactic acid bacteria (LAB), production of polysaccharides has long been associated with the technological, functional and health-promoting benefits of these microorganisms. In particular, the capsular polysaccharides and exopolysaccharides have been implicated in modulation of the rheological properties of fermented products. For this reason, screening and selection of exocellular polysaccharide-producing LAB has been extensively carried out by academia and industry. To further exploit the ability of LAB to produce polysaccharides, an in-depth understanding of their biochemistry, genetics, biosynthetic pathways, regulation and structure-function relationships is mandatory. Here, we provide a critical overview of the latest advances in the field of glycosciences in LAB. Surprisingly, the understanding of the molecular processes involved in polysaccharide synthesis is lagging behind, and has not accompanied the increasing commercial value and application potential of these polymers. Seizing the natural diversity of polysaccharides for exciting new applications will require a concerted effort encompassing in-depth physiological characterization of LAB at the systems level. Combining high-throughput experimentation with computational approaches, biochemical and structural characterization of the polysaccharides and understanding of the structure-function-application relationships is essential to achieve this ambitious goal. [ABSTRACT FROM AUTHOR]

The demand for renewable and sustainable energy has generated considerable interest in the conversion of cellulosic biomass into liquid fuels such as ethanol using a filamentous fungus. While attempts have been made to study cellulose metabolism through the use of knock-out mutants, there have been no systematic effort to characterize natural variation for cellulose metabolism in ecotypes adapted to different habitats. Here, we characterized natural variation in saccharification of cellulose and fermentation in 73 ecotypes and 89 laboratory strains of the model fungus Neurospora crassa. We observed significant variation in both traits among natural and laboratory generated populations, with some elite strains performing better than the reference strain. In the F1 population N345, 15% of the population outperformed both parents with the top performing strain having 10% improvement in ethanol production. These results suggest that natural alleles can be exploited through fungal breeding for developing elite industrial strains for bioethanol production. [ABSTRACT FROM AUTHOR]

Chromosomal copy number variation (CCNV) plays a key role in evolution and health of eukaryotes. The unicellular yeast Saccharomyces cerevisiae is an important model for studying the generation, physiological impact, and evolutionary significance of CCNV. Fundamental studies of this yeast have contributed to an extensive set of methods for analyzing and introducing CCNV. Moreover, these studies provided insight into the balance between negative and positive impacts of CCNV in evolutionary contexts. A growing body of evidence indicates that CCNV not only frequently occurs in industrial strains of Saccharomyces yeasts but also is a key contributor to the diversity of industrially relevant traits. This notion is further supported by the frequent involvement of CCNV in industrially relevant traits acquired during evolutionary engineering. This review describes recent developments in genome sequencing and genome editing techniques and discusses how these offer opportunities to unravel contributions of CCNV in industrial Saccharomyces strains as well as to rationally engineer yeast chromosomal copy numbers and karyotypes. [ABSTRACT FROM AUTHOR]

Rapamycin is a polyketide with a 31-membered macrolide ring that possesses powerful immunosuppressant activity. In this study, we firstly obtained a mutant, shikimate-resistant Streptomyces hygroscopicus strain UV-II, which displayed about 3.20-fold higher rapamycin production (305.9 mg/L) than the wild-type S. hygroscopicus ATCC29253 (95.5 mg/L). Under optimal conditions, with the addition of 2 g/L shikimic acid, the strain's rapamycin production was further increased by approximately 34.9%, to 412.6 mg/L. To gain deeper insights into the effects of shikimic acid resistance and supplementation, the fermentation properties, metabolite concentrations, and transcriptional levels of relevant genes were analyzed and evaluated for differences between this improved mutant and its parental strain. The results showed that most of the metabolic modules involved in rapamycin biosynthesis were upregulated in the mutant strain. Analysis of metabolic pathways and gene expression levels further revealed that shikimic acid metabolism plays a crucial role in the synthesis of rapamycin, and identified the rapK gene as a potential target for genetic manipulation to obtain rapamycin-producing strains with improved product yield. Consequently, the rapK gene was overexpressed in the UV-II strain, which to our delight further improved rapamycin production to 457.3 mg/L. These findings thus provide a theoretical basis for further improvements in the production of not only rapamycin, but also of other, analogous macrolide compounds. [ABSTRACT FROM AUTHOR]

High titer 1-butanol production in Escherichia coli has previously been achieved by overexpression of a modified clostridial 1-butanol production pathway and subsequent deletion of native fermentation pathways. This strategy couples growth with production as 1-butanol pathway offers the only available terminal electron acceptors required for growth in anaerobic conditions. With further inclusion of other well-established metabolic engineering principles, a titer of 15 g/L has been obtained. In achieving this titer, many currently existing strategies have been exhausted, and 1-butanol toxicity level has been surpassed. Therefore, continued engineering of the host strain for increased production requires implementation of alternative strategies that seek to identify non-obvious targets for improvement. In this study, a metabolomics-driven approach was used to reveal a CoA imbalance resulting from a pta deletion that caused undesirable accumulation of pyruvate, butanoate, and other CoA-derived compounds. Using metabolomics, the reduction of butanoyl-CoA to butanal catalyzed by alcohol dehydrogenase AdhE2 was determined as a rate-limiting step. Fine-tuning of this activity and subsequent release of free CoA restored the CoA balance that resulted in a titer of 18.3 g/L upon improvement of total free CoA levels using cysteine supplementation. By enhancing AdhE2 activity, carbon flux was directed towards 1-butanol production and undesirable accumulation of pyruvate and butanoate was diminished. This study represents the initial report describing the improvement of 1-butanol production in E. coli by resolving CoA imbalance, which was based on metabolome analysis and rational metabolic engineering strategies. [ABSTRACT FROM AUTHOR]

An industrial clavulanic acid (CA) overproducer Streptomyces clavuligerus strain, namely DEPA, was engineered to further enhance its CA production. Single or multiple copies of ccaR, claR (pathway-specific activators), and cas2 (CA synthase) genes under the control of different promoters were introduced into this strain. CA titers of the resulting recombinants were analyzed by HPLC in a dynamic fashion and compared to the vector-only controls and a wild-type strain of S. clavuligerus while their growth was monitored throughout fermentation. The addition of an extra copy of ccaR, under control of its own promoter or constitutive ermE* promoter (PermE*), led to 7.6- and 2.3-fold increased volumetric levels of CA in respective recombinants, namely the AK9 and ID3 strains. Its highly stable multicopy expression by the glpF promoter (PglpF) provided up to 25.9-fold enhanced volumetric CA titers in the respective recombinant, IDG3. claR expression controlled with its own promoter or ermE* and glpF-mediated amplification in an industrial strain brought about only about 1.2-fold increase in the volumetric CA titers. An extra copy of cas2 integration with PermE* into the S. clavuligerus DEPA genome led to 3.8-fold higher volumetric CA production by GV61. Conclusively, multicopy expression of ccaR under PglpF can result in significantly improved industrial high-titer CA producers. [ABSTRACT FROM AUTHOR]

Background: Cephalosporins and penicillins are the most frequently used β-lactam antibiotics for the treatment of human infections worldwide. The main industrial producers of these antibiotics are Acremonium chrysogenum and Penicillium chrysogenum, two taxonomically unrelated fungi. Both were subjects of long-term strain development programs to reach economically relevant antibiotic titers. It is so far unknown, whether equivalent changes in gene expression lead to elevated antibiotic titers in production strains. Results: Using the sequence of PcbC, a key enzyme of β-lactam antibiotic biosynthesis, from eighteen different pro- and eukaryotic microorganisms, we have constructed a phylogenetic tree to demonstrate the distant relationship of both fungal producers. To address the question whether both fungi have undergone similar genetic adaptions, we have performed a comparative gene expression analysis of wild-type and production strains. We found that strain improvement is associated with the remodeling of the transcriptional landscape in both fungi. In P. chrysogenum, 748 genes showed differential expression, while 1572 genes from A. chrysogenum are differentially expressed in the industrial strain. Common in both fungi is the upregulation of genes belonging to primary and secondary metabolism, notably those involved in precursor supply for β-lactam production. Other genes not essential for β-lactam production are downregulated with a preference for those responsible for transport processes or biosynthesis of other secondary metabolites. Transcriptional regulation was shown to be an important parameter during strain improvement in different organisms. We therefore investigated deletion strains of the major transcriptional regulator velvet from both production strains. We identified 567 P. chrysogenum and 412 A. chrysogenum Velvet target genes. In both deletion strains, approximately 50% of all secondary metabolite cluster genes are differentially regulated, including β-lactam biosynthesis genes. Most importantly, 35-57% of Velvet target genes are among those that showed differential expression in both improved industrial strains. Conclusions: The major finding of our comparative transcriptome analysis is that strain improvement programs in two unrelated fungal β-lactam antibiotic producers alter the expression of target genes of Velvet, a global regulator of secondary metabolism. From these results, we conclude that regulatory alterations are crucial contributing factors for improved β-lactam antibiotic titers during strain improvement in both fungi. [ABSTRACT FROM AUTHOR]

Saccharomyces yeasts have been used for millennia for the production of beer, wine, bread, and other fermented products. Long-term 'unconscious' selection and domestication led to the selection of hundreds of strains with desired production traits having significant phenotypic and genetic differences from their wild ancestors. This review summarizes the results of recent research in deciphering the genomes of wine Saccharomyces strains, the use of comparative genomics methods to study the mechanisms of yeast genome evolution under conditions of artificial selection, and the use of genomic and postgenomic approaches to identify the molecular nature of the important characteristics of commercial wine strains of Saccharomyces. Succinctly, data concerning metagenomics of microbial communities of grapes and wine and the dynamics of yeast and bacterial flora in the course of winemaking is provided. A separate section is devoted to an overview of the physiological, genetic, and biochemical features of sherry yeast strains used to produce biologically aged wines. The goal of the review is to convince the reader of the efficacy of new genomic and postgenomic technologies as tools for developing strategies for targeted selection and creation of new strains using 'classical' and modern techniques for improving wine-making technology. [ABSTRACT FROM AUTHOR]

Many high-value secondary metabolites are assembled by very large multifunctional polyketide synthases or non-ribosomal peptide synthetases encoded by giant genes, for instance, natamycin production in an industrial strain of Streptomyces gilvosporeus . In this study, a large operon reporter-based selection system has been developed using the selectable marker gene neo to report the expression both of the large polyketide synthase genes and of the entire gene cluster, thereby facilitating the selection of natamycin-overproducing mutants by iterative random mutagenesis breeding. In three successive rounds of mutagenesis and selection, the natamycin titer was increased by 110%, 230%, and 340%, respectively, and the expression of the whole biosynthetic gene cluster was correspondingly increased. An additional copy of the natamycin gene cluster was found in one overproducer. These findings support the large operon reporter-based selection system as a useful tool for the improvement of industrial strains utilized in the production of polyketides and non-ribosomal peptides. [ABSTRACT FROM AUTHOR]

Background: Despite extensive research in the last decades, microalgae are still only economically feasible for high valued markets. Strain improvement is a strategy to increase productivities, hence reducing costs. In this work, we focus on microalgae selection: taking advantage of the natural biological variability of species to select variations based on desired characteristics. We focused on triacylglycerol (TAG), which have applications ranging from biodiesel to high-value omega-3 fatty-acids. Hence, we demonstrated a strategy to sort microalgae cells with increased TAG productivity. Results: 1. We successfully identified sub-populations of cells with increased TAG productivity using Fluorescence assisted cell sorting (FACS). 2. We sequentially sorted cells after repeated cycles of N-starvation, resulting in five sorted populations (S1-S5). 3. The comparison between sorted and original populations showed that S5 had the highest TAG productivity [0.34 against 0.18 g l-1 day-1 (original), continuous light]. 4. Original and S5 were compared in lab-scale reactors under simulated summer conditions confirming the increased TAG productivity of S5 (0.4 against 0.2 g l-1 day-1). Biomass composition analyses showed that S5 produced more biomass under N-starvation because of an increase only in TAG content and, flow cytometry showed that our selection removed cells with lower efficiency in producing TAGs. Conclusions: All combined, our results present a successful strategy to improve the TAG productivity of Chlorococcum littorale, without resourcing to genetic manipulation or random mutagenesis. Additionally, the improved TAG productivity of S5 was confirmed under simulated summer conditions, highlighting the industrial potential of S5 for microalgal TAG production. [ABSTRACT FROM AUTHOR]

Pleurotus pulmonarius has gained popularity as an edible mushroom in many parts of the world due to its numerous advantageous properties. However, there have been very few instances of successful breeding of any of the various species of Pleurotus. The current study attempted to generate improved strains of P. pulmonarius by establishing a systematic scheme using genetics and hybrid breeding. After assessments of the sample collections, three principal parents, and in turn 22 single spore isolates, were nominated for crossing. Of the 231 crosses done, 88 were compatible, and 46 hybrids were able to form basidiocarps in the first cycle of cultivation (CC1). The significant genetic variations among the crosses moved the study in the direction of diallel design, to gain a greater insight into the heritability of the targeted traits and the general and specific combining ability (GCA and SCA) of the parents; and at the same time to estimate their genetic variance components. Mean and heterosis analyses were used to score the obtained hybrids, leading to the selection of four hybrids for CC2, and ultimately three superior hybrids for CC3. The GCA indicated that the Thai strain was the best combiner, while the highest values of SCA were observed in combinations between the Thai and PL27 strains. The genetic variance components and heritability analyses indicated that the bulk of the total genetic variations observed for most traits were associated with genes which were dominant in their effects. The newly generated hybrids were identified by both morphological and molecular fingerprinting (PCR-RFLP). This research involved a successful initial attempt to use a broad range of analyses and the systematic integration of genetics and breeding to assist in generating superior hybrids of P. pulmonarius. [ABSTRACT FROM AUTHOR]

Lignocellulose is the most plentiful non-food biomass and one of the most inexhaustible renewable resources on the planet, which is an alternative sustainable energy source for the production of second generation biofuels. Lignocelluloses are composed of cellulose, hemicellulose and lignin, in which the sugar polymers account for a large portion of the biomass. Cellulases belong to the glycoside hydrolase family and catalyze the hydrolysis of glyosidic linkages depolymerizing cellulose to fermentable sugars. They are multi-enzymatic complex proteins and require the synergistic action of three key enzymes: endoglucanase (E.C. 3.2.1.4), exoglucanase (E.C. 3.2.1.176) (E.C. 3.2.1.91) and β-glucosidase (E.C. 3.2.1.21) for the depolymerization of cellulose to glucose. Solid state fermentation, which holds growth of microorganisms on moist solid substrates in the absence of free flowing water, has gained considerable attention of late due its several advantages over submerged fermentation. The review summarizes the critical analysis of recent literature covering production of cellulase in solid state fermentation using advance technologies such as consolidated bioprocessing, metabolic engineering and strain improvement, and circumscribes the strategies to improve the enzyme yield. [ABSTRACT FROM AUTHOR]