Successful co-infection of two different baculovirus species in the same cell line reveals a potential strategy for large in vitro production.

Baculoviruses have been applied for biocontrol of agricultural pests, such as velvetbean caterpillar (Anticarsia gemmatalis) and fall armyworm (Spodoptera frugiperda). Cell culture is an interesting approach for large-scale production of these viruses. Co-infection of a host cell with two distinct viruses can contribute to reduce costs due to saving cell culture media, bioreactor space and the resulting co-occluded polyhedra may help to reduce final biopesticide costs. The baculovirus Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) and Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) were chosen to test a model for in vitro co-infection in SF21 cells. Different proportions of SfMNPV/AgMNPV were evaluated along three in vitro passages by optical microscopy analysis of cells and real-time PCR (qPCR) of DNA obtained from budded viruses (BVs) and occlusion bodies (OBs). The kinetics of viral protein synthesis was carried out for analysis of the co-infection in first passage and bioassays with the resulting OBs were performed against A. gemmatalis and S. frugiperda larvae. The results demonstrated successful co-infection in these cells. The quantity of SfMNPV and AgMNPV in supernatants and sediments tends to be maintained stable during the three passages, although the amount of AgMNPV was higher than SfMPNV in most of the experiments. Analysis of the kinetics of radiolabed proteins showed that the cell protein synthesis was shut off and two distinct bands of about 30 kDa, regarded to be the polyhedrin of each virus, were strongly detected at 48 and 72 hp.i. Although the pathogenicity of the produced viruses was not completely satisfactory, the bioassays confirmed occurrence of co-infected larvae with disproportional amount of each virus.

Bio-conversion of CO2 into biofuels and other value-added chemicals via metabolic engineering.

Carbon dioxide (CO2) occurs naturally in the atmosphere as a trace gas, which is produced naturally as well as by anthropogenic activities. CO2 is a readily available source of carbon that in principle can be used as a raw material for the synthesis of valuable products. The autotrophic organisms are naturally equipped to convert CO2 into biomass by obtaining energy from sunlight or inorganic electron donors. This autotrophic CO2 fixation has been exploited in biotechnology, and microbial cell factories have been metabolically engineered to convert CO2 into biofuels and other value-added bio-based chemicals. A variety of metabolic engineering efforts for CO2 fixation ranging from basic copy, paste, and fine-tuning approaches to engineering and testing of novel synthetic CO2 fixing pathways have been demonstrated. In this paper, we review the current advances and innovations in metabolic engineering for bio-conversion of CO2 into bio biofuels and other value-added bio-based chemicals.

Myxobacteria and their products: current trends and future perspectives in industrial applications.

Myxobacteria belong to a group of bacteria that are known for their well-developed communication system and synchronized or coordinated movement. This typical behavior of myxobacteria is mediated through secondary metabolites. They are capable of producing secondary metabolites belonging to several chemical classes with unique and wide spectrum of bioactivities. It is predominantly significant that myxobacteria specialize in mechanisms of action that are very rare with other producers. Most of the metabolites have been explored for their medical and pharmaceutical values while a lot of them are still unexplored. This review is an attempt to understand the role of potential metabolites produced by myxobacteria in different applications. Different myxobacterial metabolites have demonstrated antibacterial, antifungal, and antiviral properties along with cytotoxic activity against various cell lines. Beside their metabolites, these myxobacteria have also been discussed for better exploitation and implementation in different industrial sectors.

Synthetic biology approaches to actinomycete strain improvement.

Their biochemical versatility and biotechnological importance make actinomycete bacteria attractive targets for ambitious genetic engineering using the toolkit of synthetic biology. But their complex biology also poses unique challenges. This mini review discusses some of the recent advances in synthetic biology approaches from an actinomycete perspective and presents examples of their application to the rational improvement of industrially relevant strains.

From baker's yeast to genetically modified budding yeasts: the scientific evolution of bioethanol industry from sugarcane.

The peculiarities of Brazilian fuel ethanol fermentation allow the entry of native yeasts that may dominate over the starter strains of Saccharomyces cerevisiae and persist throughout the sugarcane harvest. The switch from the use of baker's yeast as starter to selected budding yeasts obtained by a selective pressure strategy was followed by a wealth of genomic information that enabled the understanding of the superiority of selected yeast strains. This review describes how the process of yeast selection evolved in the sugarcane-based bioethanol industry, the selection criteria and recent advances in genomics that could advance the fermentation process. The prospective use of genetically modified yeast strains, specially designed for increased robustness and product yield, with special emphasis on those obtained by the CRISPR (clustered regularly interspaced palindromic repeats)-Cas9 (CRISPR-associated protein 9) genome-editing approach, is discussed as a possible solution to confer higher performance and stability to the fermentation process for fuel ethanol production.

Recent Advances in the Microbial Synthesis of Hemoglobin.

Hemoglobin is a cofactor-containing protein with heme that plays important roles in transporting and storing oxygen. Hemoglobins have been widely applied as acellular oxygen carriers, bioavailable iron-supplying agents, and food-grade coloring and flavoring agents. To meet increasing demands and overcome the drawbacks of chemical extraction, the biosynthesis of hemoglobin has become an attractive alternative. Several hemoglobins have recently been synthesized by various microorganisms through metabolic engineering and synthetic biology. In this review, we summarize the novel strategies that have been used to biosynthesize hemoglobin. These strategies can also serve as references for producing other heme-binding proteins.

Thraustochytrid Cell Factories for Producing Lipid Compounds.

Thraustochytrids can accumulate over 150 g/l biomass, containing up to 55% lipids, without any genetic modification. Their broad substrate utilization capacity, several effective key metabolic pathways, and a well-developed suite of bioprocess engineering strategies all point toward great promise for the future development of these marine protists.

Research on endophytic fungi for producing huperzine A on a large-scale.

Huperzine A (HupA) is an effective inhibitor of acetylcholinesterase and has attracted great interest as a therapeutic candidate for Alzheimer's disease. However, the use of HupA is limited by resource scarcity as well as by its low yields from Huperzia serrata, its primary plant source. Recent studies have shown that this compound is produced by various endophytic fungi, thereby providing a promising alternative source, as fungi are much more amenable than plants owing to their simpler genetics and the ease of manipulation. In this review, we summarize the progress in research on the methods to increase HupA production, including fermentation conditions, fungal elicitors, gene expression, and the activation of key enzymes. This review provides guidance for further studies on HupA-producing endophytic fungi aimed at efficient HupA synthesis and accumulation, and offers new approaches for studies on the regulation of high-value bioactive secondary metabolites.

Recent Advances in Microbial Production of cis,cis-Muconic Acid.

cis,cis-Muconic acid (MA) is a valuable C6 dicarboxylic acid platform chemical that is used as a starting material for the production of various valuable polymers and drugs, including adipic acid and terephthalic acid. As an alternative to traditional chemical processes, bio-based MA production has progressed to the establishment of de novo MA pathways in several microorganisms, such as Escherichia coli, Corynebacterium glutamicum, Pseudomonas putida, and Saccharomyces cerevisiae. Redesign of the metabolic pathway, intermediate flux control, and culture process optimization were all pursued to maximize the microbial MA production yield. Recently, MA production from biomass, such as the aromatic polymer lignin, has also attracted attention from researchers focusing on microbes that are tolerant to aromatic compounds. This paper summarizes recent microbial MA production strategies that involve engineering the metabolic pathway genes as well as the heterologous expression of some foreign genes involved in MA biosynthesis. Microbial MA production will continue to play a vital role in the field of bio-refineries and a feasible way to complement various petrochemical-based chemical processes.

Membrane transporters in the bioproduction of organic acids: state of the art and future perspectives for industrial applications.

Organic acids such as monocarboxylic acids, dicarboxylic acids or even more complex molecules such as sugar acids, have displayed great applicability in the industry as these compounds are used as platform chemicals for polymer, food, agricultural and pharmaceutical sectors. Chemical synthesis of these compounds from petroleum derivatives is currently their major source of production. However, increasing environmental concerns have prompted the production of organic acids by microorganisms. The current trend is the exploitation of industrial biowastes to sustain microbial cell growth and valorize biomass conversion into organic acids. One of the major bottlenecks for the efficient and cost-effective bioproduction is the export of organic acids through the microbial plasma membrane. Membrane transporter proteins are crucial elements for the optimization of substrate import and final product export. Several transporters have been expressed in organic acid-producing species, resulting in increased final product titers in the extracellular medium and higher productivity levels. In this review, the state of the art of plasma membrane transport of organic acids is presented, along with the implications for industrial biotechnology.

[Advances in gene editing of Corynebacterium glutamate].

Corynebacterium glutamicum, an important microorganism to produce amino acids and organic acids, has been widely applied in food and medicine fields. Therefore, using editing tools to study the function of unknown genes in C. glutamicum has great significance for systematic development of industrial strain with efficient and novel production capability. Recently, gene editing has been greatly developed. Traditional gene editing based on homologous recombination and gene editing mediated by nuclease are successfully applied in C. glutamicum. Among these, the CRISPR system has been developed to be a main tool used for gene knockout of C. glutamicum due to its advantages of efficiency, simplicity and good target specificity. However, more efficient and reliable knockout system is still urgently demanded, to help develop high-performing strains in industrial application.

[Progress in biological utilization of formic acid].

The use of microbial cell factories to achieve efficient conversion of raw materials and synthesis of target substances is one of the important research directions of synthetic biology. Traditional industrial microorganisms have mainly used sugar-based raw materials as fermentation substrates. How to adopt cheaper carbon resources and realize their efficient use has been widely concerned. Formic acid is an important organic one-carbon source and widely used in industrial manufacturing of pesticides, leather, dyes, medicine and rubber. In recent years, due to the demand fluctuation in downstream industries, formic acid production is facing the dilemma of overcapacity, and therefore, requiring new conversion paths for expansion and extension of the related industrial chain. Biological route is one of the important options. However, natural formate-utilizing microorganisms generally grow slowly when metabolizing formic acid, and moreover, are difficult to be artificially modified by the absence of effective genetic tools. Construction of non-natural formate-utilizing microorganisms is another alternative strategy, but still in its infancy and has a huge space for further improvements. Here, we briefly summarize the recent research progress of biological utilization of formic acid, and also propose the future research focus and direction.

[Advances in microbial degradation of chlorinated hydrocarbons].

Chlorinated hydrocarbons (CAHs) threaten human health and the ecological environment due to their strong carcinogenic, teratogenic, mutagenic and heritable properties. Heterotrophic assimilation degradation can completely and effectively degrade CAHs, without secondary pollution. However, it is crucial to comprehensively understand the heterotrophic assimilation process of CAHs for its application. Therefore, we review here the characteristics and advantages of heterotrophic assimilation degradation of CAHs. Moreover, we systematically summarize current research status of heterotrophic assimilation of CAHs. Furthermore, we analyze bacterial genera and metabolism, key enzymes and characteristic genes involved in the metabolic process. Finally, we indicate existing problems of heterotrophic assimilation research and future research needs.

[Progress in metabolic engineering of biosynthesis of 3-hydroxypropionic acid].

As an important platform compound, 3-hydroxypropionic acid (3-HP) can be used as a substrate to synthesize a variety of biological products with commercial potential. The titer of 3-HP by wild-type bacteria is low, which severely limits the large-scale application and production of 3-HP. By modifying the genes related to the metabolic pathway, engineered bacteria using cheap substrates as carbon sources are constructed, the aim of reducing production cost and increasing output is realized. In this paper, the recent progress in the synthesis of 3-HP by metabolic engineering at home and abroad is reviewed. The advantages and disadvantages of glycerol pathway, malonyl-CoA pathway and beta-alanine pathway for synthesis of 3-HP are also summarized and analyzed, and the future development of 3-HP is prospected.

Applications of CRISPR-Cas systems in lactic acid bacteria.

As a phenotypically and phylogenetically diverse group, lactic acid bacteria are found in a variety of natural environments and occupy important roles in medicine, biotechnology, food and agriculture. The widespread use of lactic acid bacteria across these industries fuels the need for new and functionally diverse strains that may be utilized as starter cultures or probiotics. Originally characterized in lactic acid bacteria, CRISPR-Cas systems and derived molecular machines can be used natively or exogenously to engineer new strains with enhanced functional attributes. Research on CRISPR-Cas biology and its applications has exploded over the past decade with studies spanning from the initial characterization of CRISPR-Cas immunity in Streptococcus thermophilus to the use of CRISPR-Cas for clinical gene therapies. Here, we discuss CRISPR-Cas classification, overview CRISPR biology and mechanism of action, and discuss current and future applications in lactic acid bacteria, opening new avenues for their industrial exploitation and manipulation of microbiomes.

Potential applications of extracellular enzymes from Streptomyces spp. in various industries.

Extracellular enzymes produced from Streptomyces have the potential to replace toxic chemicals that are being used in various industries. The endorsement of this replacement has not received a better platform in developing countries. In this review, we have discussed the impact of chemicals and conventional practices on environmental health, and the role of extracellular enzymes to replace these practices. Burning of fossil fuels and agriculture residue is a global issue, but the production of biofuel using extracellular enzymes may be the single key to solve all these issues. We have discussed the replacement of hazardous chemicals with the use of xylanase, cellulase, and pectinase in food industries. In paper industries, delignification was done by the chemical treatment, but xylanase and laccase have the efficient potential to remove the lignin from pulp. In textile industries, the conventional method includes the chemicals which affect the nervous system and other organs. The use of xylanase, cellulase, and pectinase in different processes can give a safe and environment-friendly option to textile industries. Hazardous chemical pesticides can be replaced by the use of chitinase as an insecticide and fungicide in agricultural practices.

Novel Strategies and Platforms for Industrial Isoprenoid Engineering.

Metabolic engineering has emerged as an important tool for reconstructing heterologous isoprenoid metabolic pathways in microbial hosts. Here, we provide an overview of promising engineering strategies that have proven to be successful for the high-yield production of isoprenoids. Besides 'conventional' approaches, such as the 'push-pull' and protein engineering to optimize the isoprenoid flux and limited catalytic activity of enzymes, we review emerging strategies in the field, including compartmentalization between synthetic consortia members, novel bypass pathways for isoprenoid synthesis, cell-free systems, and improvement of the lipid content to overcome storage isoprenoid limitations. Pitfalls, along with lessons learned from the application of these strategies, will be addressed with the hope of guiding future efforts toward cost-effective and sustainable production of isoprenoids.

Technically relevant enzymes and proteins produced by LAB suitable for industrial and biological activity.

Lactic acid bacteria (LAB) are a unique subset of microorganisms that have co-evolved with humans since the beginning of agricultural practices and animal domestication and throughout our never-ending quest for food preservation, digestibility, and flavor enhancement. LAB have historically played a preponderant role in our foods. In this review, we focus on the enzymatic activities and current or potential applications of LAB in our lives. A description of each of the enzymatic systems in LAB is included. Glycosidases, which hydrolyze the most abundant food molecules and as sources of carbon, sustain the lives of organisms on Earth as well as ensure microbial innocuity by the production of lactic acid from the uniquely mammalian carbohydrate, lactose. Lipases and proteases or proteinases are of fundamental importance in food fermentations and in dairy foods for flavor development. Bacteriocins and peptidoglycan hydrolases are part of the enzymatic system of LAB that has evolved to make these bacteria fierce competitors in various microbiomes, which are highly important for the human gut. In this review, we also present an explanation on how the versatility of the genetics of LAB can adapt to the matrix where they are placed with the advantage of not having any toxicity to humans. The systematic study of LAB enzymes has allowed for some unique applications in foods and biopharmaceutical industries. Here, we summarize how different enzyme systems in LAB are classified, and thus, facilitate much-needed further studies to understand the fundamentals and translate them into applications to improve our lives.

Haloferax volcanii for biotechnology applications: challenges, current state and perspectives.

Haloferax volcanii is an obligate halophilic archaeon with its origin in the Dead Sea. Simple laboratory culture conditions and a wide range of genetic tools have made it a model organism for studying haloarchaeal cell biology. Halophilic enzymes of potential interest to biotechnology have opened up the application of this organism in biocatalysis, bioremediation, nanobiotechnology, bioplastics and the biofuel industry. Functionally active halophilic proteins can be easily expressed in a halophilic environment, and an extensive genetic toolkit with options for regulated protein overexpression has allowed the purification of biotechnologically important enzymes from different halophiles in H. volcanii. However, corrosion mediated damage caused to stainless-steel bioreactors by high salt concentrations and a tendency to form biofilms when cultured in high volume are some of the challenges of applying H. volcanii in biotechnology. The ability to employ expressed active proteins in immobilized cells within a porous biocompatible matrix offers new avenues for exploiting H. volcanii in biotechnology. This review critically evaluates the various application potentials, challenges and toolkits available for using this extreme halophilic organism in biotechnology.

Aerobic nitrogen-fixing bacteria for hydrogen and ammonium production: current state and perspectives.

Biological nitrogen fixation (BNF) is accomplished through the action of the oxygen-sensitive enzyme nitrogenase. One unique caveat of this reaction is the inclusion of hydrogen gas (H2) evolution as a requirement of the reaction mechanism. In the absence of nitrogen gas as a substrate, nitrogenase will reduce available protons to become a directional ATP-dependent hydrogenase. Aerobic nitrogen-fixing microbes are of particular interest, because these organisms have evolved to perform these reactions with oxygen-sensitive enzymes in an environment surrounded by oxygen. The ability to maintain a functioning nitrogenase in aerobic conditions facilitates the application of these organisms under conditions where most anaerobic nitrogen fixers are excluded. In recent years, questions related to the potential yields of the nitrogenase-derived products ammonium and H2 have grown more approachable to experimentation based on efforts to construct increasingly more complicated strains of aerobic nitrogen fixers such as the obligate aerobe Azotobacter vinelandii. This mini-review provides perspectives of recent and historical efforts to understand and quantify the yields of ammonium and H2 that can be obtained through the model aerobe A. vinelandii, and outstanding questions that remain to be answered to fully realize the potential of nitrogenase in these applications with model aerobic bacteria.