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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]

CRISPR/Cas9 system is a cutting-edge gene-editing tool that has recently been introduced for the strain improvement of fungi to enhance their cellulolytic activity. Cellulase is an enzyme complex that hydrolyzes cellulosic fraction of lignocellulosic biomass into glucose which can be converted subsequently into biofuels and other value-added products. Among diverse microbial communities, fungi are considered the most potent cellulolytic candidates but the naturally low cellulase titers have been the major bottleneck in using them for industrial and biotechnological applications. However, CRISPR/Cas9 mediated fungal strain engineering has opened up new avenues to overcome this challenge by enabling precise and efficient genome editing which induces the overexpression of cellulase genes, deletion of cellulase repressors, and alterations in the regulatory elements governing the overall cellulase gene expression. The effective CRISPR/Cas9 systems have already been designed for the functional genome editing of industrially competent cellulolytic fungal strains such as Aspergillus strains, Trichoderma reesei , and Penicillium chrysogenum , however, the technological advancements to establish it across the fungal kingdom are still desired. The employment of CRISPR/Cas9 in fungal strain improvement poses various limitations such as off-target effects, the need for designing suitable delivery methods, and the appropriate selection markers, therefore, future research should focus on addressing these issues and further refining the CRISPR/Cas9 system for fungal strain improvement. Conclusively, this RNA-directed DNA endonuclease system is a promising future tool for improving cellulase production in fungi leading to the development of numerous industrially competent strains, thus, supporting the sustainable development goal (SDG 7) of affordable and clean energy through efficient bioconversion of lignocellulosic biomass into biofuels coupled with other value-added products. [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]

Nowadays, there is an increasing demand for novel feedstocks and alternative protein sources to meet global needs. Because of their rich nutritional profiles and high protein contents, microalgae-based food products and supplements are being developed. Nonetheless, these products present organoleptic characteristics such as taste, smell and colour that are often considered unpleasant by human and animal consumers. To address this constraint, strain improvement approaches such as random mutagenesis have been used, which combined with the right selection strategy, lead to more appealing microalgal biomass. In this work, a novel selection strategy using oxyfluorfen, an inhibitor of the chlorophyll synthesis pathway, was applied for the first time to isolate chlorophyll-deficient strains of Scenedesmus rubescens and Chlorella vulgaris upon treatment with ethyl methanesulfonate (EMS). With this approach, one S. rubescens brownish (37Y01) mutant strain, as well as two C. vulgaris mutant strains, one yellow (31Y15) and one white (31W62), were obtained. S. rubescens 37Y01 displayed a reduced protein content of 19.1% dry weight (DW) compared to that of the wildtype, which presented a protein content of 25.0% DW. C. vulgaris wildtype and mutants exhibited higher protein contents, in the 42.8‐44.3% DW range, compared to Scenedesmus rubescens (p < 0.05). The selective pressure of this inhibitor allowed the selection of S. rubescens and C. vulgaris mutants displaying 55% and 95% decrease in chlorophyll content, respectively. The reduced chlorophyll content greatly improves the sensory properties and consumer acceptance of established mutants, increasing the potential of both strains as feedstocks to develop novel food products. [Display omitted] • Strain improvement enabled the development of appealing microalgal products. • A novel selection method to screen randomly-generated mutants is proposed. • Oxyfluorfen was used to select mutants with up to 55–95% less chlorophyll. • A brownish mutant of S. rubescens was isolated with 19% DW of protein content. • Yellow and white mutants of C. vulgaris with protein of 43–44% DW were isolated. [ABSTRACT FROM AUTHOR]

To minimize the cost of algae-based biofuels and bioproducts, boosting biomass productivity and enhancing the stability of cultures in outdoor cultivation systems are imperative. Strain improvement strategies, mainly adaptive evolution, random mutagenesis, and genetic engineering, focus on perturbing the metabolism of algae to develop desired phenotypes. However, the aforementioned techniques are time- and labor-intensive with a possibility of reversion of the mutated/adapted phenotypes back to wild type. In this regard, the chemical genetics approach has emerged as a rapid and powerful approach to preferentially direct the metabolism of algae towards desired target products with the incorporation of small chemical modulators in a dose-dependent manner. In this review, we critically asses the use of chemical modulators to tweak the metabolism of several algal species and enhance biomass production, bioproduct yield, and resilience to abiotic stressors. The review highlights important categories of chemical modulators and the techniques employed to identify and screen such small effective molecules. Chemical modulators enable the re-design and fine-tuning of targeted algal metabolic pathways rendering chemical genetics a promising approach for enhancing algal productivity particularly in large-scale cultivation. • Use of chemical modulators helps generate desired algal phenotypes. • High throughput screening can identify potential chemical modulators. • Phytohormones, amino acids, inhibitors, and metabolites boost algal productivity. • Exogenous addition of chemical modulators alleviates oxidative stress in algae. • Chemical genetics can facilitate development of algal biofuels and bioproducts. [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]

Abstract Metabolically engineered bacterial strains are fast becoming the center of attraction in the biosynthesis of various bio-based ingredients. Currently, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) with its associated RNA guided endonuclease (Cas9), is considered as the most promising tool that has brought a breakthrough in the genome editing approach. Although this versatile system originates from the immune systems of bacteria, its application in bacterial genome editing as an approach of metabolic engineering is still not so extensive. This review paper discusses the challenges, opportunities and application of CRISPR/Cas system in bacterial genome editing for biosynthesis of various value-added ingredients. The content of this paper mainly emphasizes Type II CRISPR/Cas9 system used for knock-in and knock-out of various target genes in the bacterial strains as a pre-requisite for increasing the production of bio-based materials. [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]

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]

Microalgal biotechnology has gained increasing attention over the last few decades as a next-generation driver for obtaining food, feed and biofuels and to carry out bioremediation of effluents and CO 2 mitigation. Flow cytometry (FC) and fluorescence-activated cell sorting (FACS) have recently acquired outstanding importance in the development of high-throughput methodologies. For example, bioprospecting novel species using FACS widened the current portfolio of available strains for drug discovery and biomass production in large-scale production systems. Moreover, FACS has recently prompted several approaches for the effective improvement of microalgal strains by means of genetic engineering, serial selection and random mutagenesis. In the upcoming years, routine implementation of FC and FACS is expected to further bring forward the field of microalgal biotechnological research as occurred with mammalian cells in biomedical sciences. This review highlights the recent developments of FACS applications to different biotechnological goals, as well as the principles and details of FACS-based microalgal analysis. In addition, the future perspectives of novel and innovative approaches of FACS applications in microalgal biotechnology are suggested and discussed. [ABSTRACT FROM AUTHOR]

Our goal in the present study was to evaluate the potential for lipid production of two cell populations of the marine microalgae Chlorococcum littorale under different climate conditions. We selected, in a previous study and via fluorescence activated cell sorting (FACS), a new cell population of Chlorococcum littorale , namely S5. S5 showed a stable doubled triacylglycerol (TAG) productivity in comparison with the original population. A previously developed model was expanded to include day:night cycles and validated to predict biomass and outdoor TAG productivities at different locations. Four different locations were chosen to simulate the response of C. littorale to different day lengths and light intensities (the Netherlands, Norway, Brazil and Spain). Indoor experiments (simulated summer) were carried out with Original and S5, showing that S5 had a doubled TAG productivity under N-starvation. Finally, simulations of biomass and TAG productivities of Original and S5 at different locations were performed. At locations with lower light intensities, Norway and the Netherlands, biomass productivities were higher than at locations with higher light intensities, Brazil/Spain. Such results might be associated with light-saturation effects. TAG productivities, however, showed no effect of local light intensity. Locations at higher latitudes, Norway/Netherlands, cannot sustain phototrophic year-round production, hence, the yearly average TAG productivities were doubled in Brazil/Spain (from 1.4–1.6 to 3.0–3.2 g m − 2 d − 1 ). Likewise, C. littorale S5 was simulated with doubled TAG productivities when compared with Original, at all locations (2.5–2.7 (low light) to 4.7–5.2 g m − 2 d − 1 (high light)). The present results confirm the industrial potential of Chlorococcum littorale , both Original and S5, as a source of TAG. Furthermore, our results can be used for comparison and to estimate future production scenarios. [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]

Kinetic models of cellular metabolism are important tools for the rational design of metabolic engineering strategies and to explain properties of complex biological systems. The recent developments in high-throughput experimental data are leading to new computational approaches for building kinetic models of metabolism. Herein, we briefly survey the available databases, standards and software tools that can be applied for kinetic models of metabolism. In addition, we give an overview about recently developed ordinary differential equations (ODE)-based kinetic models of metabolism and some of the main applications of such models are illustrated in guiding metabolic engineering design. Finally, we review the kinetic modeling approaches of large-scale networks that are emerging, discussing their main advantages, challenges and limitations. [ABSTRACT FROM AUTHOR]

Several lactic acid bacterial (LAB) strains have probiotic properties. A crucial requirement for probiotics to benefit the host's health is their ability to adhere to the intestinal lining enriched with glycosylated mucins. Many intestinal diseases display marked changes in mucin glycosylation such as reduced sialylation and fucosylation. Such alterations have been shown to allow for the lesser adhesion of the probiotic bacteria in vitro. Although probiotics are often used in intestinal diseases, this phenomenon might reduce their in vivo intestinal adhesion, which could be a reason for the ineffectiveness of some probiotics in such conditions as reported in a few studies. Adaptive laboratory evolution (ALE) offers an intuitive way of improving bacterial properties without performing deliberate genetic modifications. In this paper, it is hypothesized that probiotic strains with enhanced adhesion to the mucin altered in intestinal diseases would be better candidates to alleviate the symptoms of these diseases, and such strains can be created through ALE. This hypothesis is supported by a few earlier studies showing the ability of certain other bacteria to mutate themselves to enhance their adhesion to biotic and abiotic environments. The steps involved in testing the hypothesis would be improving the in vitro adhesion of lactobacilli to the intestinal mucus obtained from patients with intestinal diseases by daily adhesion and passaging of the adherent bacteria for several weeks. The improved strains can be subjected to animal studies and human clinical trials in the second part. Since some commensals make short-chain fatty acids from dietary fiber, which stimulate mucin production by the intestinal cells, the clinical trials can be performed under a dietary regime. Apart from providing novel probiotic candidates, such a study can also reveal yet the novel mechanism of adhesion of probiotics to the intestinal environment. [ABSTRACT FROM AUTHOR]

Following its introduction in 1967, rifampicin has become a mainstay of therapy in the treatment of tuberculosis, leprosy and many other widespread diseases. Its potent antibacterial activity is due to specific inhibition of bacterial RNA polymerase. However, resistance to rifampicin was reported shortly after its introduction in the medical practice. Studies in the model organism Escherichia coli helped to define the molecular mechanism of rifampicin-resistance demonstrating that resistance is mostly due to chromosomal mutations in rpoB gene encoding the RNA polymerase β chain. These studies also revealed the amazing potential of the molecular genetics to elucidate the structure–function relationships in bacterial RNA polymerase. The scope of this paper is to illustrate how rifampicin-resistance has been recently exploited to better understand the regulatory mechanisms that control bacterial cell physiology and virulence, and how this information has been used to maneuver, on a global scale, gene expression in bacteria of industrial interest. In particular, we reviewed recent literature regarding: (i) the effects of rpoB mutations conferring rifampicin-resistance on transcription dynamics, bacterial fitness, physiology, metabolism and virulence; (ii) the occurrence in nature of “mutant-type” or duplicated rifampicin-resistant RNA polymerases; and (iii) the RNA polymerase genetic engineering method for strain improvement and drug discovery. [ABSTRACT FROM AUTHOR]

The potential for Chlamydomonas reinhardtii to be utilized for biofuel production was strengthened by developing it for growth in elevated salinity via the selective breeding method of genome shuffling. A population was constructed via random mutagenesis and subjected to multiple rounds of sex and growth in increasing salinity. This sexual line was capable of growth in up to 700 mM NaCl, unlike its progenitor, which could only grow in 300 mM NaCl. An asexual control line was capable of growth in 500 mM NaCl. Palmelloid aggregations increased in size and the concentration of final biomass decreased as a function of NaCl concentration, which poses considerations for future strain development. The sexual line maintained sexual efficiencies of up to 50% over the course of selection. This investigation achieved significant strain improvement of C. reinhardtii and demonstrated the clear advantage of its ability to participate in laboratory controlled and reproducible high efficiency sex. [ABSTRACT FROM AUTHOR]

Highlights: [•] Genetic engineering tools appear interesting for strain improvement for clostridia for CBP. [•] Genes encoding enzymes of solvent pathways have been cloned and sequenced for clostridia. [•] Their expression has been correlated with the pattern of end-product formation in fermentations. [•] CBP offers potential benefits for the production of biofuels from lignocellulose. [•] Clostridia appear the best choice for CBP. [ABSTRACT FROM AUTHOR]

The use of renewable plant biomass, lignocellulose, to produce biofuels and biochemicals using microbial cell factories plays a fundamental role in the future bioeconomy. The development of cell factories capable of efficiently fermenting complex biomass streams will improve the cost-effectiveness of microbial conversion processes. At present, inhibitory compounds found in hydrolysates of lignocellulosic biomass substantially influence the performance of a cell factory and the economic feasibility of lignocellulosic biofuels and chemicals. Here, we present and statistically analyze data on Saccharomyces cerevisiae mutants engineered for altered tolerance towards the most common inhibitors found in lignocellulosic hydrolysates: acetic acid, formic acid, furans, and phenolic compounds. We collected data from 7971 experiments including single overexpression or deletion of 3955 unique genes. The mutants included in the analysis had been shown to display increased or decreased tolerance to individual inhibitors or combinations of inhibitors found in lignocellulosic hydrolysates. Moreover, the data included mutants grown on synthetic hydrolysates, in which inhibitors were added at concentrations that mimicked those of lignocellulosic hydrolysates. Genetic engineering aimed at improving inhibitor or hydrolysate tolerance was shown to alter the specific growth rate or length of the lag phase, cell viability, and vitality, block fermentation, and decrease product yield. Different aspects of strain engineering aimed at improving hydrolysate tolerance, such as choice of strain and experimental set-up are discussed and put in relation to their biological relevance. While successful genetic engineering is often strain and condition dependent, we highlight the conserved role of regulators, transporters, and detoxifying enzymes in inhibitor tolerance. The compiled meta-analysis can guide future engineering attempts and aid the development of more efficient cell factories for the conversion of lignocellulosic biomass. • Data mining of 3955 genes involved in tolerance to lignocellulosic hydrolysates. • Most mutants screened for tolerance towards acetic acid or specific hydrolysates. • Laboratory strains are often used even though they are very sensitive to inhibitors. • The success of strain engineering attempts is often strain and condition dependent. [ABSTRACT FROM AUTHOR]

Highlights: [•] Demonstrated a novel directed evolution approach to obtain biocatalysts with improved desulfurization specific activity. [•] Modified the desulfurization operon by including a gene encoding a sulfur-rich polypeptide to increase the nutritional requirement for sulfur. [Copyright &y& Elsevier]

Abstract: The growing concern about the safety of food and dairy additives and the increasing costs of petroleum-based chemicals have rekindled the interest in the fermentation processes for fumaric acid production. The key problems of the industrial production of microbial fumaric acid are reviewed in this paper. Various strategies, including strain improvement, morphology control, substrate choice, fermentation process and separation process, are summarized and compared, and their economical possibilities for industrial processes are discussed. The market prospects and technological strategies for value-added fumaric acid derivatives are also addressed. The future prospects of microbial fumaric acid production are proposed at the end of this article. [Copyright &y& Elsevier]

Abstract: The biopharmaceutical market has come a long way since 1982, when the first biopharmaceutical product, recombinant human insulin, was launched. Just over 200 biopharma products have already gained approval. The global market for biopharmaceuticals which is currently valued at over US$99 billion has been growing at an impressive compound annual growth rate over the previous years. To produce these biopharmaceuticals and other industrially important heterologous proteins, different prokaryotic and eukaryotic expression systems are used. All expression systems have some advantages as well as some disadvantages that should be considered in selecting which one to use. Choosing the best one requires evaluating the options – from yield to glycosylation, to proper folding, to economics of scale-up. No host cell from which all the proteins can be universally expressed in large quantities has been found so far. Therefore, it is important to provide a variety of host–vector expression systems in order to increase the opportunities to screen for the most suitable expression conditions or host cell. In this overview, we focus on Streptomyces lividans, a Gram-positive bacterium with a proven excellence in secretion capacity, as host for heterologous protein production. We will discuss its advantages and disadvantages, and how with systems biology approaches strains can be developed to better producing cell factories. [Copyright &y& Elsevier]

Abstract: Sucrose isomerase (SI) activity is used industrially for the conversion of sucrose into isomers, particularly isomaltulose or trehalulose, which have properties advantageous over sucrose for some food uses. All of the known microbial SIs are TIM barrel proteins that convert sucrose without need for any cofactors, with varying kinetics and product specificities. The current analysis was undertaken to bridge key gaps between the information in patents and scientific publications about the microbes and enzymes useful for sucrose isomer production. This analysis shows that microbial SIs can be considered in 5 structural classes with corresponding functional distinctions that broadly align with the taxonomic differences between producing organisms. The most widely used bacterial strain for industrial production of isomaltulose, widely referred to as “Protaminobacter rubrum” CBS 574.77, is identified as Serratia plymuthica. The strain producing the most structurally divergent SI, with a high product specificity for trehalulose, widely referred to as “Pseudomonas mesoacidophila” MX-45, is identified as Rhizobium sp. Each tested SI-producer is shown to have a single SI gene and enzyme, so the properties reported previously for the isolated proteins can reasonably be associated with the products of the genes subsequently cloned from the same isolates and SI classes. Some natural isolates with potent SI activity do not catabolize the isomer under usual production conditions. The results indicate that their industrial potential may be further enhanced by selection for variants that do not catabolize the sucrose substrate. [Copyright &y& Elsevier]

Abstract: Present study introduces linseed oil cake as a novel substrate for phytase production by Rhizopus oryzae. Statistical approach was employed to optimize various medium components under solid state fermentation (SSF). An overall 8.41-fold increase in phytase production was achieved at the optimum concentrations (w/w, mannitol, 2.05%; ammonium sulfate, 2.84% and phosphate, 0.38%). Further enhancement by 59% was observed due to a novel strain improvement approach. Purified phytase (∼34kDa) showed optimal temperature of 45°C, dual pH optima at 1.5 and 5.5 and possesses high catalytic efficiency (2.38×106 M−1 s−1). Characterization study demonstrates the phytase as highly thermostable and resistant to proteolysis, heavy metal ions, etc. Furthermore, an improved HPLC method was introduced to confirm the ability of phytase to degrade phytic acid completely and was found to be an efficient method. [Copyright &y& Elsevier]

Abstract: Bioprocesses are of growing importance as an avenue to produce chemicals. Microorganisms containing only desired catalytic and replication capabilities in their metabolic pathways are expected to offer efficient processes for chemical production. Realizing such minimal cells is the holy grail of metabolic engineering. In this paper, we propose a new method that combines graph-theoretic approaches with mixed-integer liner programming (MILP) to design metabolic networks with minimal reactions. Existing MILP based computational approaches are computationally complex especially for large networks. The proposed graph-theoretic approach offers an efficient divide-and-conquer strategy using the MILP formulation on sub-networks rather than considering the whole network monolithically. In addition to the resulting improvement in computational complexity, the proposed method also aids in identifying the key reactions to be knocked-out in order to achieve the minimal cell. The efficacy of the proposed approach is demonstrated using three case studies from two organisms, Escherichia coli and Saccharomyces cerevisiae. [Copyright &y& Elsevier]

Abstract: A strategy has been developed for creating Saccharomyces cerevisiae strains with a high RNA content by following a three-step breeding procedure. In the first step, an S. cerevisiae disruptant of the RRN10 gene, one of the components of the UAF (upstream activation factor) complex of rRNA transcription, was constructed and showed severely slow growth. In the second step, seven suppressors were isolated that restored the slow growth of the Δrrn10 disruptant. Genetic analysis revealed that each of the seven suppressors that were isolated appeared to have dominant and multiple mutations. The specific growth rate of those suppressors was increased approximately two-fold as compared with the Δrrn10 parental strain. The absolute RNA content showed that the suppressors had an RNA content 32–56% higher than that of the Δrrn10 parental strain. In the last step, the RRN10 wild-type gene was integrated into chromosome V of each of the original suppressors. The total RNA content of the integrants was also 1.4- to 2.3-fold higher than the wild-type strain. In conclusion, since yeast RNA is the source of 5′-IMP and 5′-GMP that enhance the delicious taste in certain types of food, like soups and sauces, the strategy taken in this study provides effective approach to breed S. cerevisiae strains producing a higher content of RNA that will contribute to yeast food biotechnology. [Copyright &y& Elsevier]

Abstract: 2,3-Butanediol is a promising bulk chemical due to its extensive industry applications. The state-of-the-art nature of microbial 2,3-butanediol production is reviewed in this paper. Various strategies for efficient and economical microbial 2,3-butanediol production, including strain improvement, substrate alternation, and process development, are reviewed and compared with regard to their pros and cons. This review also summarizes value added derivatives of biologically produced 2,3-butanediol and different strategies for downstream processing. The future prospects of microbial 2,3-butanediol production are discussed in light of the current progress, challenges, and trends in this field. Guidelines for future studies are also proposed. [Copyright &y& Elsevier]

Abstract: The production of riboflavin from vegetable oil was increased using a mutant strain of Ashbya gossypii. This mutant was generated by treating the wild-type strain with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Riboflavin production was 10-fold higher in the mutant compared to the wild-type strain. The specific intracellular catalase activity after 3 d of culture was 6-fold higher in the mutant than in the wild-type strain. For the mutant, riboflavin production in the presence of 40 mM hydrogen peroxide was 16% less than that in the absence of hydrogen peroxide, whereas it was 56% less for the wild-type strain. The isocitrate lyase (ICL) activity of the mutant was 0.26 mU/mg of protein during the active riboflavin production phase, which was 2.6-fold higher than the wild-type strain. These data indicate that the mutant utilizes the carbon flux from the TCA cycle to the glyoxylate cycle more efficiently than the wild-type strain, resulting in enhanced riboflavin production. This novel mutant has the potential to be of use for industrial-scale riboflavin production from waste-activated bleaching earth (ABE), thereby transforming a useless material into a valuable bioproduct. [Copyright &y& Elsevier]

• Current process developments for microbial polyol production are presented. • Yeasts are favorable cell factories for sugar alcohols. • Cell factory design to improve yeast strains for polyol production is addressed. • Agro-industrial side streams are studied for sugar alcohol production. • Lignocellulosic feedstocks and glycerol are the most studied substrates. Sugar alcohols are widely marketed compounds. They are useful building block chemicals and of particular value as low- or non-calorigenic sweeteners, serving as sugar substitutes in the food industry. To date most sugar alcohols are produced by chemical routes using pure sugars, but a transition towards the use of renewable, non-edible feedstocks is anticipated. Several yeasts are naturally able to convert renewable feedstocks, such as lignocellulosic substrates, glycerol and molasses, into sugar alcohols. These bioconversions often face difficulties to obtain sufficiently high yields and productivities necessary for industrialization. This review provides insight into the most recent studies on utilizing yeasts for the conversion of renewable feedstocks to diverse sugar alcohols, including xylitol, erythritol, mannitol and arabitol. Moreover, metabolic approaches are highlighted that specifically target shortcomings of sugar alcohol production by yeasts from these renewable substrates. [ABSTRACT FROM AUTHOR]

Current study was focused on the development of a non-fastidious lactic acid producing strain having better growth rate, low pH tolerance and good productivity by genome shuffling of a mutant strain of Lactobacillus delbrueckii NCIM 2025 and an amylase producing non-fastidious Bacillus amyloliquefaciens ATCC 23842. After the third cycle of the protoplast fusion, lactic acid production by few fusants was monitored and the best fusant was selected for further studies. Optimization of the important process parameters for lactic acid production was conducted using Plackett–Burman design and response surface methodology. Selected fusant could utilize the liquefied cassava bagasse starch directly with minimum nutrient supplementation for lactic acid production. During validation, 40g/L of lactic acid was obtained (∼96% conversion of starch to lactic acid) by using fusant inoculum (3%, v/v) from 83g/L cassava bagasse (starch content 50% w/w) supplemented with yeast extract and peptone (0.2% each, w/v) and the buffering agent (2% CaCO3, w/v). [Copyright &y& Elsevier]

Abstract: Mutations induced by classical whole-cell mutagenesis using N-methyl-N′-nitro-N-nitrosoguanidine (NTG) were determined for all genes of pathways from glucose to l-lysine in an industrial l-lysine producer of Corynebacterium glutamicum. A total of 50 mutations with a genome-wide distribution were identified and characterized for mutational types and mutagenic specificities. Those mutations were all point mutations with single-base substitutions and no deletions, frame shifts, and insertions were found. Among six possible types of base substitutions, the mutations consisted of only two types: 47 G·C→A·T transitions and three A·T→G·C transitions with no transversion. The findings indicate a limited repertoire of amino acid substitutions by classical NTG mutagenesis and thus raise a new possibility of further improving industrial strains by optimizing key mutations through PCR-mediated site-directed mutagenesis. [Copyright &y& Elsevier]

Abstract: Gene targeting frequencies in Aspergillus niger are often very low and hamper efficient functional genomics in this biotechnologically important fungus. Deletion of the A. niger kusA gene encoding the ortholog of the Ku70 protein in other eukaryotes, dramatically improved homologous integration efficiency and reached more than 80% compared to 7% in the wild-type background, when 500bp homologous flanks were used. Furthermore, the use of the ΔkusA strain resulted in a high frequency of heterokaryon formation (70%) in primary transformants in the case disrupting an essential gene. Deletion of kusA had no obvious effect on the growth of the fungus, but renders the ΔkusA strain 10 times more sensitive to X-ray irradiation and two to three times more sensitive to UV exposure. The highly efficient gene targeting in combination with the A. niger genome sequence allows a systematic approach to generate gene knockouts and will help in improving the capacities of A. niger as producer of commercially interesting proteins and metabolites. [Copyright &y& Elsevier]

Abstract: By introducing defined deletions in recA and an essential sporulation gene (spoIV), stable mutant strains of Bacillus licheniformis were obtained which are totally asporogenous and severely affected in DNA repair, and thus being UV-hypersensitive. Studies on growth in various liquid media as well as on amylase production revealed no differences of the mutants when compared to the wild type. Hence, such genes appear to be suitable disruption targets for achieving passive biological containment in this industrially exploited species. [Copyright &y& Elsevier]

Abstract: Fermentation processes have been developed for producing most of the essential amino acids. Methionine is one exception. Although microbial production of methionine has been attempted, no commercial bioproduction exists. Here, we discuss the prospects of producing methionine by fermentation. A detailed account is given of methionine biosynthesis and its regulation in some potential producer microorganisms. Problems associated with isolation of methionine overproducing strains are discussed. Approaches to selecting microorganism having relaxed and complex regulatory control mechanisms for methionine biosynthesis are examined. The importance of fermentation media composition and culture conditions for methionine production is assessed and methods for recovering methionine from fermentation broth are considered. [Copyright &y& Elsevier]

Strain improvement has been conventionally achieved through mutation and selection. However, the genetic and metabolic profiles of mutant strains were poorly characterized and mutagenesis remained a random process. Metabolic flux analysis (MFA) of mutants will enable us to better understand the change of flux profile in vivo and the mechanism of mutagenesis. A l(+)-lactic acid over-producing mutant, Rhizopus oryzae R1021, was isolated by mutagenizing the parent strain (R. oryzae R3017) with UV, diethyl sulfate (DES) and 60Co. Starting with a concentration of 120 g/l corn starch, mutant R1021 produced 79.4 g/l l(+)-lactic acid after 60 h in flasks, 52% higher than that produced by the parent strain. The l(+)-lactic acid purity was 99.05% by weight based on the amount of total lactic acid. The mutant R1021 was also morphologically different from the parent strain. The results of carbon flux analysis of the parent strain and mutants showed that the pyruvate node in the metabolic model was the principle and flexible node. The results show that the key steps of the pathways in parent strain where most carbon is lost from lactic acid formation are the reactions form pyruvate to acetyl-CoA, oxaloacetate and ethanol. Although the fractions of the carbon from pyruvate to ethanol and acetyl-CoA were reduced in mutants, these two pathways are still the steps that are most likely to be further targeted to reroute the pyruvate metabolism to improve lactic acid production. [Copyright &y& Elsevier]

One major mission of microbial cell factory is overproduction of desired chemicals. To this end, it is necessary to orchestrate enzymes that affect metabolic fluxes. However, only modification of a small number of enzymes in most cases cannot maximize desired metabolites, and global regulation is required. Of myriad enzymes influencing global regulation, RNA polymerase (RNAP) may be the most versatile enzyme in biological realm because it not only serves as the workhorse of central dogma but also participates in a plethora of biochemical events. In fact, recent years have witnessed extensive exploitation of RNAPs for phenotypic engineering. While a few impressive reviews showcase the structures and functionalities of RNAPs, this review not only summarizes the state-of-the-art advance in the structures of RNAPs but also points out their enormous potentials in metabolic engineering and synthetic biology. This review aims to provide valuable insights for strain improvement. [ABSTRACT FROM AUTHOR]

Microbial fermentation is currently still the major way to produce structural complicated clinical drugs. Yet, the low productivity and genetic instability of producing strains remain the bottlenecks in microbial pharmaceutical industry. Fidaxomicin is a microbial drug against the Clostridium difficile infection. Here, a genome-based combinatorial engineering strategy was established to improve both fidaxomicin production and the genetic stability of Actinoplanes deccanensis YP-1. Guided by genomic analysis, several genetic instability-associated elements were cumulatively deleted, generating a more genetically stable mutant. Further rational engineering approaches including elimination of a pigment pathway, duplication of the fidaxomicin gene cluster, overexpression of a positive regulator and optimization of the fermentation medium, led to an overall 27-folds improvement in fidaxomicin production. Taken together, the genome-based rational combinatorial engineering strategy was efficient to enhance the fidaxomicin production and ameliorate the genetic stability of YP-1, it can also be widely used in other industrial actinomycetes for strain improvement. [ABSTRACT FROM AUTHOR]

Originally developed in bacterial and fungal models, Adaptive Laboratory Evolution (ALE) experimentally selects for targeted phenotypes by allowing cells containing beneficial random genetic mutations to replicate more quickly in the presence of environmental stress. When applied to microalgae, this technique has been used to increase growth rate, stress tolerance, substrate utilization, and product yield. This review discusses ALE experimental design parameters, and summarizes key results and lessons learned from recent microalgal ALE studies. By documenting the current state of the field, we provide insight on the ways microalgal ALE might be used to develop commercially viable biotechnology strains. • Adaptive Laboratory Evolution can be successfully used to improve strain fitness. • Strains with improved growth, salt tolerance and CO 2 utilization have been reported. • Photobioreactors are being used for algal strain improvement. [ABSTRACT FROM AUTHOR]

Advancement of microalgae for use as biofuel feedstock depends on bioprospecting novel strains and then applying classical strain improvement techniques to enhance desirable traits. Here we successfully demonstrate this approach using two novel strains of green algae, identified as Desmodesmus armatus and Chlorella vulgaris, to improve biomass and lipid yields. UV irradiation was utilized to generate random mutations and the resulting populations were analyzed via flow cytometry to quantify lipid fluorescence while enriching for high lipid variants using fluorescence activated cell sorting. Secondary screening was performed via in situ fluorescence staining to identify the single highest lipid producing mutants from each strain. This approach produced a Desmodesmus armatus mutant that exhibited both increased biomass productivity and higher lipid content, which doubled lipid yield from the culture. The resulting Chlorella vulgaris mutant exhibited an increase in growth only, resulting in a 33% increase in lipid yield. • Generated novel UV-mutants of Desmodesmus armatus and Chlorella vulgaris. • Mutants displayed increased biomass and fatty acid productivities and yields. • Characterized growth and lipid composition of wild type and mutant strains. • Nile Red fluorescence did not consistently correlate with lipid content in mutants. [ABSTRACT FROM AUTHOR]

• PHB production using different substrates critically reviewed. • Oil and starch based substrates seem potential sources for higher PHB production. • Agro-industrial residues offer cost benefits for PHB production. • Bacillus sp., Ralstonia sp. and Cupriavidus sp. are efficient microorganisms for PHB production. • Only limited information available on scale-up for PHB production processes. Polyhydroxybutyrates (PHBs) are a class of biopolymers produced by different microbial species and are biodegradable and biocompatible in nature as opposed to petrochemically derived plastics. PHBs have advanced applications in medical sector, packaging industries, nanotechnology and agriculture, among others. PHB is produced using various feedstocks such as glycerol, dairy wastes, agro-industrial wastes, food industry waste and sugars. Current focus on PHB research has been primarily on reducing the cost of production and, on downstream processing to isolate PHB from cells. Recent advancements to improve the productivity and quality of PHB include genetic modification of producer strain and modification of PHB by blending to develop desirable properties suited to diversified applications. Selection of feedstock plays a critical role in determining the economic feasibility and sustainability of the process. This review provides a bird's eye view of the suitability of different waste resources for producing polyhydroxybutyrate; providing state-of the art information and analysis. [ABSTRACT FROM AUTHOR]

• Bacteria are an irrefutable competitor for existing carotenoid source. • Carotenoid are associated with diverse health benefits. • Stress triggers selective carotenoid accumulation as defence response. • SWOT analysis targeting perils and promises of bacterial carotenoid. Carotenoid has gained a reputation amongst researchers for its robust antioxidant capabilities. This review reasons the inevitability of carotenoid for a larger role than just dietary supplements. In an era dominated by chemical or plant-derived carotenoid, bacterial carotenoid provides a compelling forte to be exploited as a promising alternative. Bacteria are amazingly resourceful beings; however, their low carotenoid content makes them undesirable for commercial applications. Here, we have reviewed their applications as a strong prophylactic agent in the health sector with a myriad of applications. Additionally, various measures for augmenting carotenoid yield through new-age technologies, like sequential nutrition starvation, the induction of carotenoid accumulation in microbial cells by employing several stress factors, and the construction of hyper-carotenoid producing strains through genetic engineering for creating proficient producers have been offered. Finally, SWOT (Strengths, Weaknesses, Opportunities and Threats) analysis has been presented to perceive the significance of four major components involved in its commercialisation. [ABSTRACT FROM AUTHOR]

Vitamin K2, also called menaquinone, is an essential lipid-soluble vitamin that plays a critical role in blood clotting and prevention of osteoporosis. It has become a focus of research in recent years and has been widely used in the food and pharmaceutical industries. This review will briefly introduce the functions and applications of vitamin K2 first, after which the biosynthesis pathways and enzymes will be analyzed in-depth to highlight the bottlenecks facing the microbial vitamin K2 production on the industrial scale. Then, various strategies, including strain mutagenesis and genetic modification, different cultivation modes, fermentation and separation processes, will be summarized and discussed. The future prospects and perspectives of microbial menaquinone production will also be discussed finally. [ABSTRACT FROM AUTHOR]

• This review provides an overview of metabolic pathways for fumaric acid production. • Different fumaric acid producers and strain improvement strategies involved were introduced. • Alternative substrates for fumaric acid production and their fermentation performances were shown. • The optimization of fermentation conditions was discussed. Fumaric acid is an industrially important platform chemical, which has been widely used in food, chemicals, agriculture and pharmaceutical industries. Due to the rising price of raw materials in traditional petrochemical method and demand for sustainable development, fermentative fumaric acid production has attracted great attention. In this context, various strains with enhanced fumaric acid production or elimination of by-products were developed through mutagenesis or metabolic engineering. In addition, renewable organic wastes together with the fermentation process optimization have also been widely investigated. Accordingly, this review will comprehensively summarize the achievements of fermentative fumaric acid production in recent years and major obstacles occurring in industrialization to provide insights and perspective for future microbial fermentation of fumaric acid. [ABSTRACT FROM AUTHOR]

Algae biotechnology holds great promise for the production of sustainable and environmentally friendly products, however more robust strains of microalgae must be developed before algae can be used as a commercially relevant production platform. Here we demonstrate a timely and effective approach to significantly improve a desired phenotype in algae through a combination of mutagenesis and genome shuffling. In this study, we engineered a strain of Chlamydomonas reinhardtii to produce recombinant green fluorescent protein in the chloroplast and then subjected the nuclear genome to various rounds of UV-induced mutagenesis and genome shuffling via sexual recombination, followed by isolation of strains with enhanced recombinant protein expression using fluorescence-activated cell sorting (FACS). The greatest success came from first UV-mutagenizing the host strain to create mutations in the nuclear genome, followed by FACS to isolate the best GFP-expressing mutant, then shuffling the nuclear genome of that mutant by mating it with selected wild-type strains, and then again isolating the best progeny via FACS. Using this strategy, a novel strain was evolved in <3 months to express GFP over 2% total soluble protein (TSP), a 15-fold increase from the initial recombinant strain which expressed GFP at 0.14% TSP. This significant increase in GFP expression from the chloroplast was accomplished without altering the GFP gene itself or reducing the growth rate of the strain, demonstrating the effectiveness of this strategy to enhance a specific phenotype and significantly improve product production within an algal species in a commercially relevant timeframe. • Transformed C. reinhardtii to express GFP in the chloroplast • Caused mutations to nuclear genome with UV-irradiation • Shuffled nuclear genome by mating transgenic strain with wild-type strains • Used FACS to isolate high-GFP expressing progeny • Mutagenesis then genome shuffling resulted in a 15-fold increase in GFP production [ABSTRACT FROM AUTHOR]