Microbial Secondary Metabolites via Fermentation Approaches for Dietary Supplementation Formulations.

Food supplementation formulations refer to products that are designed to provide additional nutrients to the diet. Vitamins, dietary fibers, minerals and other functional compounds (such as antioxidants) are concentrated in dietary supplements. Specific amounts of dietary compounds are given to the body through food supplements, and these include as well so-called non-essential compounds such as secondary plant bioactive components or microbial natural products in addition to nutrients in the narrower sense. A significant social challenge represents how to moderately use the natural resources in light of the growing world population. In terms of economic production of (especially natural) bioactive molecules, ways of white biotechnology production with various microorganisms have recently been intensively explored. In the current review other relevant dietary supplements and natural substances (e.g., vitamins, amino acids, antioxidants) used in production of dietary supplements formulations and their microbial natural production via fermentative biotechnological approaches are briefly reviewed. Biotechnology plays a crucial role in optimizing fermentation conditions to maximize the yield and quality of the target compounds. Advantages of microbial production include the ability to use renewable feedstocks, high production yields, and the potential for cost-effective large-scale production. Additionally, it can be more environmentally friendly compared to chemical synthesis, as it reduces the reliance on petrochemicals and minimizes waste generation. Educating consumers about the benefits, safety, and production methods of microbial products in general is crucial. Providing clear and accurate information about the science behind microbial production can help address any concerns or misconceptions consumers may have.

Biopolishing of denim by the recombinant xylanase II of Caulobacter crescentus.

Denim, also known as jeans, is a fabric made up of braided cotton threads dyed indigo blue, whose fibers contain approximately 10% of non-cellulosic impurities that reduce its commercial value. Microbial enzymes can act in the cleaning and desizing processes of jeans, improving their color, softness, and covering capacity. The recombinant Xylanase II (XynA2) from the aquatic bacterial Caulobacter crescentus (C. crescentus), previously characterized in terms of its biochemical features, was applied to the biotreatment of jeans to clean and degum it. The biotreatment performance was evaluated in terms of tissue weight loss, amount of reducing sugars released and analysis of the images obtained by scanning electron microscopy (SEM). Biotreated tissues, at 12 and 24 h, showed a dry weight loss of 4.9 and 6.6%, respectively. The reducing sugars amount released after XynA2 action over the jean's fibers showed statistically significant values when compared with each other and with their respective controls. SEM images clearly shown that the fabric treated for 12 h presented a smooth and polished surface, while the fabric treated for 24 h showed the cotton fibers broken, displaying severe damage to the textile. The best treatment for the jeans was in the presence of 1 U mg-1 XynA2 at pH 8 and 60 °C during 12 h. In conclusion, XynA2 of C. crescentus was satisfactorily applied for the biopolishing of denim jeans being a more sustainable alternative to the use of chemical and abrasive processes to obtain the same effects.

Yarrowia lipolytica Strains and Their Biotechnological Applications: How Natural Biodiversity and Metabolic Engineering Could Contribute to Cell Factories Improvement.

Among non-conventional yeasts of industrial interest, the dimorphic oleaginous yeast Yarrowia lipolytica appears as one of the most attractive for a large range of white biotechnology applications, from heterologous proteins secretion to cell factories process development. The past, present and potential applications of wild-type, traditionally improved or genetically modified Yarrowia lipolytica strains will be resumed, together with the wide array of molecular tools now available to genetically engineer and metabolically remodel this yeast. The present review will also provide a detailed description of Yarrowia lipolytica strains and highlight the natural biodiversity of this yeast, a subject little touched upon in most previous reviews. This work intends to fill this gap by retracing the genealogy of the main Yarrowia lipolytica strains of industrial interest, by illustrating the search for new genetic backgrounds and by providing data about the main publicly available strains in yeast collections worldwide. At last, it will focus on exemplifying how advances in engineering tools can leverage a better biotechnological exploitation of the natural biodiversity of Yarrowia lipolytica and of other yeasts from the Yarrowia clade.

Methyl Jasmonate and Methyl-ß-Cyclodextrin Individually Boost Triterpenoid Biosynthesis in Chlamydomonas Reinhardtii UVM4.

The commercialisation of valuable plant triterpenoids faces major challenges, including low abundance in natural hosts and costly downstream purification procedures. Endeavours to produce these compounds at industrial scale using microbial systems are gaining attention. Here, we report on a strategy to enrich the biomass of the biotechnologically-relevant Chlamydomonas reinhardtii strain UVM4 with valuable triterpenes, such as squalene and (S)-2,3-epoxysqualene. C. reinhardtii UVM4 was subjected to the elicitor compounds methyl jasmonate (MeJA) and methyl-ß-cyclodextrine (MßCD) to increase triterpene yields. MeJA treatment triggered oxidative stress, arrested growth, and altered the photosynthetic activity of the cells, while increasing squalene, (S)-2,3-epoxysqualene, and cycloartenol contents. Applying MßCD to cultures of C. reinhardtii lead to the sequestration of the two main sterols (ergosterol and 7-dehydroporiferasterol) into the growth medium and the intracellular accumulation of the intermediate cycloartenol, without compromising cell growth. When MßCD was applied in combination with MeJA, it counteracted the negative effects of MeJA on cell growth and physiology, but no synergistic effect on triterpene yield was observed. Together, our findings provide strategies for the triterpene enrichment of microalgal biomass and medium.

Response surface methodology-mediated improvement of the irradiated endophytic fungal strain, Alternaria brassicae AGF041 for Huperzine A-hyperproduction.

Huperzine A (HupA) is an anti-Alzheimer's therapeutic and a dietary supplement for memory boosting that is extracted mainly from Huperziacae plants. Endophytes represent the upcoming refuge to protect the plant resource from distinction but their HupA yield is still far from commercialization. In this context, UV and gamma radiation mutagenesis of the newly isolated HupA-producing Alternaria brassicae AGF041 would be applied in this study for improving the endophytic HupA yield. Compared to non-irradiated cultures, UV (30-40 min, exposure) and γ (0·5 KGy, dose) irradiated cultures, each separately, showed a significant higher HupA yield (17·2 and 30·3%, respectively). While, application of a statistically optimized compound irradiation (0·70 KGy of γ treatment and 42·49 min of UV exposure, sequentially) via Response Surface Methodology (RSM) resulted in 53·1% production increase. Moreover, a stable selected mutant strain CM003 underwent batch cultivation using a 6·6 l bioreactor for the first time and was successful for scaling up the HupA production to 261·6 µg l-1 . Findings of this research are demonstrated to be valuable as the employed batch fermentation represents a successful starting step towards the promising endophytic HupA production at an industrial scale.

Engineering microbial pathways for production of bio-based chemicals from lignocellulosic sugars: current status and perspectives.

Lignocellulose is the most abundant biomass on earth with an annual production of about 2 × 1011 tons. It is an inedible renewable carbonaceous resource that is very rich in pentose and hexose sugars. The ability of microorganisms to use lignocellulosic sugars can be exploited for the production of biofuels and chemicals, and their concurrent biotechnological processes could advantageously replace petrochemicals' processes in a medium to long term, sustaining the emerging of a new economy based on bio-based products from renewable carbon sources. One of the major issues to reach this objective is to rewire the microbial metabolism to optimally configure conversion of these lignocellulosic-derived sugars into bio-based products in a sustainable and competitive manner. Systems' metabolic engineering encompassing synthetic biology and evolutionary engineering appears to be the most promising scientific and technological approaches to meet this challenge. In this review, we examine the most recent advances and strategies to redesign natural and to implement non-natural pathways in microbial metabolic framework for the assimilation and conversion of pentose and hexose sugars derived from lignocellulosic material into industrial relevant chemical compounds leading to maximal yield, titer and productivity. These include glycolic, glutaric, mesaconic and 3,4-dihydroxybutyric acid as organic acids, monoethylene glycol, 1,4-butanediol and 1,2,4-butanetriol, as alcohols. We also discuss the big challenges that still remain to enable microbial processes to become industrially attractive and economically profitable.

A New Synthetic Pathway for the Bioproduction of Glycolic Acid From Lignocellulosic Sugars Aimed at Maximal Carbon Conservation.

Glycolic acid is a two-carbon α-hydroxy acid with many applications in industrial sectors including packaging, fine chemistry, cosmetics, and pharmaceutics. Currently, glycolic acid is chemically manufactured from fossil resources. This chemical mode of production is raising some concerns regarding its use in health for personal care. Microbial production of GA stands as a remarkable challenge to meet these concerns, while responding to the increasing demand to produce bio-sourced products from renewable carbon resources. We here report on the design and expression of a novel non-natural pathway of glycolic acid in E. coli. The originality of this new pathway, termed "glycoptimus" relies on two pillars. On the one hand, it requires the overexpression of three naturally occurring E. coli genes, namely kdsD encoding a D-arabinose-5-P isomerase, fsaA encoding a class 1 aldolase that cleaves D-arabinose-5-P into glyceraldehyde-3-P and glycolaldehyde, and aldA coding for an aldehyde dehydrogenase that oxidizes glycoladehyde in glycolate. These three genes constitute the "glycoptimus module." On the other hand, the expression of these genes together with a reshaping of the central carbon metabolism should enable a production of glycolic acid from pentose and hexose at a molar ratio of 2.5 and 3, respectively, which corresponds to 50% increase as compared to the existing pathways. We demonstrated the 'in vivo' potentiality of this pathway using an E. coli strain, which constitutively expressed the glycoptimus module and whose carbon flow in glycolysis was blocked at the level of glyceraldehyde-3-P dehydrogenase reaction step. This engineered strain was cultivated on a permissive medium containing malate and D-glucose. Upon exhaustion of malate, addition of either D-glucose, D-xylose or L-arabinose led to the production of glycolic acid reaching about 30% of the maximum molar yield. Further improvements at the level of enzymes, strains and bioprocess engineering are awaited to increase yield and titer, rendering the microbial production of glycolic acid affordable for a cost-effective industrial process.

Microbial Nanotechnology: Challenges and Prospects for Green Biocatalytic Synthesis of Nanoscale Materials for Sensoristic and Biomedical Applications.

Nanomaterials are increasingly being used in new products and devices with a great impact on different fields from sensoristics to biomedicine. Biosynthesis of nanomaterials by microorganisms is recently attracting interest as a new, exciting approach towards the development of 'greener' nanomanufacturing compared to traditional chemical and physical approaches. This review provides an insight about microbial biosynthesis of nanomaterials by bacteria, yeast, molds, and microalgae for the manufacturing of sensoristic devices and therapeutic/diagnostic applications. The last ten-year literature was selected, focusing on scientific works where aspects like biosynthesis features, characterization, and applications have been described. The knowledge, challenges, and potentiality of microbial-mediated biosynthesis was also described. Bacteria and microalgae are the main microorganism used for nanobiosynthesis, principally for biomedical applications. Some bacteria and microalgae have showed the ability to synthetize unique nanostructures: bacterial nanocellulose, exopolysaccharides, bacterial nanowires, and biomineralized nanoscale materials (magnetosomes, frustules, and coccoliths). Yeasts and molds are characterized by extracellular synthesis, advantageous for possible reuse of cell cultures and reduced purification processes of nanomaterials. The intrinsic variability of the microbiological systems requires a greater protocols standardization to obtain nanomaterials with increasingly uniform and reproducible chemical-physical characteristics. A deeper knowledge about biosynthetic pathways and the opportunities from genetic engineering are stimulating the research towards a breakthrough development of microbial-based nanosynthesis for the future scaling-up and possible industrial exploitation of these promising 'nanofactories'.

Lipasas en síntesis de polímeros: avances y contribución a la química verde de polímeros / Lipases in polymer synthesis: advantages and its contribution to green polymer chemistry

RESUMEN Las lipasas han sido usadas en las últimas décadas como catalizadores eficientes en la síntesis enzimática de polímeros y gracias a características como alta selectividad, reciclabilidad, inocuidad y fácil separación/purificación se han convertido en una herramienta importante en el campo de los polímeros. En este trabajo se recopilan los desarrollos más importantes en el área y a su vez se muestra la tendencia actual de este campo de investigación.

ABSTRACT Lipases have been used in last decades as efficient catalysts in enzimatic polymer chemistry, thanks to its high selectivity, recyclabil-ity, safety and easy recovery/ separation, lipases have become in an important tool in polymer field. In this paper we compile the most important achievements in the enzimatic polymer chemistry and, at the same, time we show the current trend in this field of research.

Industrializing a Bacterial Strain for l-Serine Production through Translation Initiation Optimization.

Turning a proof-of-concept synthetic biology design into a robust, high performing cell factory is a major time and money consuming task, which severely limits the growth of the white biotechnology sector. Here, we extend the use of tunable antibiotic resistance markers for synthetic evolution (TARSyn), a workflow for screening translation initiation region (TIR) libraries with antibiotic selection, to generic pathway engineering, and transform a proof-of-concept synbio design into a process that performs at industrially relevant levels. Using a combination of rational design and adaptive evolution, we recently engineered a high-performing bacterial strain for production of the important building block biochemical l-serine, based on two high-copy pET vectors facilitating expression of the serine biosynthetic genes serA, serC, and serB from three independent transcriptional units. Here, we prepare the bacterial strain for industrial scale up by transferring and reconfiguring the three genes into an operon encoded on a single low-copy plasmid. Not surprisingly, this initially reduces production titers considerably. We use TARSyn to screen both experimental and computational optimization designs resulting in high-performing synthetic serine operons and reach industrially relevant production levels of 50 g/L in fed-batch fermentations, the highest reported so far for serine production.

Metabolic engineering of Yarrowia lipolytica for the biosynthesis of crotonic acid.

In this study, Y. lipolytica was engineered to produce crotonic acid via the butanol-forming route. Firstly, the crotonase and 3-hydroxybutyryl-CoA dehydrogenase genes from Clostridium beijerinckii, and the thioesterase gene from Bacteroides thetaiotaomicron were heterologously expressed in Y. lipolytica, the engineered strain LZJ001 accumulated 62.3 ±â€¯4.2 mg/L of crotonic acid. Secondly, the acetyl-CoA acetyltransferase from Saccharomyces cerevisiae was overexpressed, the derived recombinant strain LZJ002 produced 123.5 ±â€¯6.8 mg/L of crotonic acid. Finally, the pyruvate dehydrogenase from Escherichia coli was additionally expressed, giving the fully engineered strain LZJ004 that produced 220.0 ±â€¯8.2 mg/L of crotonic acid in shaking-flask culture, which represents a 3.5-fold increase over LZJ001 strain. The approach described here paves the way for environmentally friendly and large-scale industrial production of crotonic acid.

Characterization of a novel lytic bacteriophage from an industrial Escherichia coli fermentation process and elimination of virulence using a heterologous CRISPR-Cas9 system.

Bacterial-bacteriophage interactions are a well-studied and ecologically-important aspect of microbiology. Many commercial fermentation processes are susceptible to bacteriophage infections due to the use of high-density, clonal cell populations. Lytic infections of bacterial cells in these fermentations are especially problematic due to their negative impacts on product quality, asset utilization, and fouling of downstream equipment. Here, we report the isolation and characterization of a novel lytic bacteriophage, referred to as bacteriophage DTL that is capable of rapid lytic infections of an Escherichia coli K12 strain used for commercial production of 1,3-propanediol (PDO). The bacteriophage genome was sequenced and annotated, which identified 67 potential open-reading frames (ORF). The tail fiber ORF, the largest in the genome, was most closely related to bacteriophage RTP, a T1-like bacteriophage reported from a commercial E. coli fermentation process in Germany. To eliminate virulence, both a fully functional Streptococcus thermophilus CRISPR3 plasmid and a customized S. thermophilus CRISPR3 plasmid with disabled spacer acquisition elements and seven spacers targeting the bacteriophage DTL genome were constructed. Both plasmids were separately integrated into a PDO production strain, which was subsequently infected with bacteriophage DTL. The native S. thermophilus CRISPR3 operon was shown to decrease phage susceptibility by approximately 96%, while the customized CRISPR3 operon provided complete resistance to bacteriophage DTL. The results indicate that the heterologous bacteriophage-resistance system described herein is useful in eliminating lytic infections of bacteriophage DTL, which was prevalent in environment surrounding the manufacturing facility.

Autolysis of Pichia pastoris induced by cold.

The production of recombinant biopharmaceutical proteins is a multi-billion dollar market. Protein recovery represents a major part of the production costs. Pichia pastoris is one of the microbial systems most used for the production of heterologous proteins. The use of a cold-induced promoter to express lytic enzymes in the yeast after the growth stage could reduce protein recovery costs. This study shows that a cold-shock can be applied to induce lysis of the yeast cells. A strain of P. pastoris was constructed in which the endogenous eng gene encoding a putative endo-ß-1,3-glucanase was overexpressed using the cold-shock induced promoter of the cctα gene from Saccharomyces cerevisiae. In the transgenic P. pastoris, the expression of eng increased 3.6-fold after chilling the cells from 30 to 4 °C (cold-shock stage) followed by incubation for 6 h (eng expression stage). The culture was heated to 30 °C for 6 h (ENG synthesis stage) and kept at 37 °C for 24 h (lysis stage). After this procedure the cell morphology changed, spheroplasts were obtained and cellular lysis was observed. Thus, a clone of P. pastoris was obtained, which undergoes autolysis after a cold-shock.

Citric acid: emerging applications of key biotechnology industrial product.

Owing to new biotechnological production units mostly located in China, global supply of citric acid in the course of the last two decades rose from less than 0.5 to more than 2 million tonnes becoming the single largest chemical obtained via biomass fermentation and the most widely employed organic acid. Critically reviewing selected research achievements and production trends, we identify the reasons for which this polycarboxylic acid will become a key chemical in the emerging bioeconomy.Graphical abstractPalermo's Fabbrica Chimica Italiana Goldenberg today. In 1930 it was Europe's largest citric acid plant (photo courtesy of Aldo Ferrande).

Tracking bio-hydrogen-mediated production of commodity chemicals from carbon dioxide and renewable electricity.

This study reveals that reduction of carbon dioxide (CO2) to commodity chemicals can be functionally compartmentalized in bioelectrochemical systems. In the present example, a syntrophic consortium composed by H2-producers (Rhodobacter sp.) in the biofilm is combined with carboxidotrophic Clostridium species, mainly found in the bulk liquid. The performance of these H2-mediated electricity-driven systems could be tracked by the activity of a biological H2 sensory protein identified at cathode potentials between -0.2V and -0.3V vs SHE. This seems to point out that such signal is not strain specific, but could be detected in any organism containing hydrogenases. Thus, the findings of this work open the door to the development of a biosensor application or soft sensors for monitoring such systems.

Degradation and half-life of DNA present in biomass from a genetically-modified organism during land application.

White biotechnology has made a positive impact on the chemical industry by providing safer, more efficient chemical manufacturing processes that have reduced the use of toxic chemicals, harsh reaction conditions, and expensive metal catalysts, which has improved alignment with the principles of Green Chemistry. The genetically-modified (GM) biocatalysts that are utilized in these processes are typically separated from high-value products and then recycled, or eliminated. Elimination routes include disposal in sanitary landfills, incineration, use as a fuel, animal feed, or reuse as an agricultural soil amendment or other value-added products. Elimination routes that have the potential to impact the food chain or environment have been more heavily scrutinized for the fate and persistence of biological products. In this study, we developed and optimized a method for monitoring the degradation of strain-specific DNA markers from a genetically-modified organism (GMO) used for the commercial production of 1,3-propanediol. Laboratory and field tests showed that a marker for heterologous DNA in the GM organism was no longer detectable by end-point polymerase chain reaction (PCR) after 14 days. The half-life of heterologous DNA was increased by 17% (from 42.4 to 49.7 h) after sterilization of the soil from a field plot, which indicated that abiotic factors were important in degradation of DNA under field conditions. There was no evidence for horizontal transfer of DNA target sequences from the GMO to viable organisms present in the soil.

Utilization of protein-rich residues in biotechnological processes.

A drawback of biotechnological processes, where microorganisms convert biomass constituents, such as starch, cellulose, hemicelluloses, lipids, and proteins, into wanted products, is the economic feasibility. Particularly the cost of nitrogen sources in biotechnological processes can make up a large fraction of total process expenses. To further develop the bioeconomy, it is of considerable interest to substitute cost-intensive by inexpensive nitrogen sources. The aim of this mini-review was to provide a comprehensive insight of utilization methods of protein-rich residues, such as fish waste, green biomass, hairs, and food waste. The methods described include (i) production of enzymes, (ii) recovery of bioactive compounds, and/or (iii) usage as nitrogen source for microorganisms in biotechnological processes. In this aspect, the utilization of protein-rich residues, which are conventionally considered as waste, allows the development of value-adding processes for the production of bioactive compounds, biomolecules, chemicals, and materials.

Biocatalysts: application and engineering for industrial purposes.

Enzymes are widely applied in various industrial applications and processes, including the food and beverage, animal feed, textile, detergent and medical industries. Enzymes screened from natural origins are often engineered before entering the market place because their native forms do not meet the requirements for industrial application. Protein engineering is concerned with the design and construction of novel enzymes with tailored functional properties, including stability, catalytic activity, reaction product inhibition and substrate specificity. Two broad approaches have been used for enzyme engineering, namely, rational design and directed evolution. The powerful and revolutionary techniques so far developed for protein engineering provide excellent opportunities for the design of industrial enzymes with specific properties and production of high-value products at lower production costs. The present review seeks to highlight the major fields of enzyme application and to provide an updated overview on previous protein engineering studies wherein natural enzymes were modified to meet the operational conditions required for industrial application.

White biotechnology: State of the art strategies for the development of biocatalysts for biorefining.

White biotechnology is a term that is now often used to describe the implementation of biotechnology in the industrial sphere. Biocatalysts (enzymes and microorganisms) are the key tools of white biotechnology, which is considered to be one of the key technological drivers for the growing bioeconomy. Biocatalysts are already present in sectors such as the chemical and agro-food industries, and are used to manufacture products as diverse as antibiotics, paper pulp, bread or advanced polymers. This review proposes an original and global overview of highly complementary fields of biotechnology at both enzyme and microorganism level. A certain number of state of the art approaches that are now being used to improve the industrial fitness of biocatalysts particularly focused on the biorefinery sector are presented. The first part deals with the technologies that underpin the development of industrial biocatalysts, notably the discovery of new enzymes and enzyme improvement using directed evolution techniques. The second part describes the toolbox available by the cell engineer to shape the metabolism of microorganisms. And finally the last part focuses on the 'omic' technologies that are vital for understanding and guide microbial engineering toward more efficient microbial biocatalysts. Altogether, these techniques and strategies will undoubtedly help to achieve the challenging task of developing consolidated bioprocessing (i.e. CBP) readily available for industrial purpose.

Discovery and molecular and biocatalytic properties of hydroxynitrile lyase from an invasive millipede, Chamberlinius hualienensis.

Hydroxynitrile lyase (HNL) catalyzes the degradation of cyanohydrins and causes the release of hydrogen cyanide (cyanogenesis). HNL can enantioselectively produce cyanohydrins, which are valuable building blocks for the synthesis of fine chemicals and pharmaceuticals, and is used as an important biocatalyst in industrial biotechnology. Currently, HNLs are isolated from plants and bacteria. Because industrial biotechnology requires more efficient and stable enzymes for sustainable development, we must continuously explore other potential enzyme sources for the desired HNLs. Despite the abundance of cyanogenic millipedes in the world, there has been no precise study of the HNLs from these arthropods. Here we report the isolation of HNL from the cyanide-emitting invasive millipede Chamberlinius hualienensis, along with its molecular properties and application in biocatalysis. The purified enzyme displays a very high specific activity in the synthesis of mandelonitrile. It is a glycosylated homodimer protein and shows no apparent sequence identity or homology with proteins in the known databases. It shows biocatalytic activity for the condensation of various aromatic aldehydes with potassium cyanide to produce cyanohydrins and has high stability over a wide range of temperatures and pH values. It catalyzes the synthesis of (R)-mandelonitrile from benzaldehyde with a 99% enantiomeric excess, without using any organic solvents. Arthropod fauna comprise 80% of terrestrial animals. We propose that these animals can be valuable resources for exploring not only HNLs but also diverse, efficient, and stable biocatalysts in industrial biotechnology.