Production Of High-value Compounds Research Articles

Marine Purple Photosynthetic Bacteria as Sustainable Microbial Production Hosts.

Photosynthetic microorganisms can serve as the ideal hosts for the sustainable production of high-value compounds. Purple photosynthetic bacteria are typical anoxygenic photosynthetic microorganisms and are expected to be one of the suitable microorganisms for industrial production. Purple photosynthetic bacteria are reported to produce polyhydroxyalkanoate (PHA), extracellular nucleic acids and hydrogen gas. We characterized PHA production as a model compound in purple photosynthetic bacteria, especially focused on marine strains. PHA is a family of biopolyesters synthesized by a variety of microorganisms as carbon and energy storage materials. PHA have recently attracted attention as an alternative to conventional petroleum-based plastics. Production of extracellular nucleic acids have been studied in Rhodovulum sulfidophilum, a marine purple non-sulfur bacterium. Several types of artificial RNAs have been successfully produced in R. sulfidophilum. Purple photosynthetic bacteria produce hydrogen via nitrogenase, and genetic engineering strategies have been investigated to enhance the hydrogen production. This mini review describes the microbial production of these high-value compounds using purple photosynthetic bacteria as the host microorganism.

Orthogonal monoterpenoid biosynthesis in yeast constructed on an isomeric substrate

Synthetic biology efforts for the production of valuable chemicals are frequently hindered by the structure and regulation of the native metabolic pathways of the chassis. This is particularly evident in the case of monoterpenoid production in Saccharomyces cerevisiae, where the canonical terpene precursor geranyl diphosphate is tightly coupled to the biosynthesis of isoprenoid compounds essential for yeast viability. Here, we establish a synthetic orthogonal monoterpenoid pathway based on an alternative precursor, neryl diphosphate. We identify structural determinants of isomeric substrate selectivity in monoterpene synthases and engineer five different enzymes to accept the alternative substrate with improved efficiency and specificity. We combine the engineered enzymes with dynamic regulation of metabolic flux to harness the potential of the orthogonal substrate and improve the production of industrially-relevant monoterpenes by several-fold compared to the canonical pathway. This approach highlights the introduction of synthetic metabolism as an effective strategy for high-value compound production.

A Multi-Omics Extraction Method for the In-Depth Analysis of Synchronized Cultures of the Green Alga Chlamydomonas reinhardtii.

Microalgae have been the focus of research for their applications in the production of high value compounds, food and fuel. Moreover, they are valuable photosynthetic models facilitating the understanding of the basic cellular processes. System wide studies enable comprehensive and in-depth understanding of molecular functions of the organisms. However, multiple independent samples and protocols are required for proteomics, lipidomics and metabolomics studies introducing higher error and variability. A robust high throughput extraction method for the simultaneous extraction of chlorophyll, lipids, metabolites, proteins and starch from a single sample of the green alga Chlamydomonas reinhardtii is presented here. The illustrated experimental setup is for Chlamydomonas cultures synchronized using 12 h/12 h light/dark conditions. Samples were collected over a 24 h cell cycle to demonstrate that the metabolites, lipids and starch data obtained using various analytical platforms are well conformed. Furthermore, protein samples collected using the same extraction protocol were used to conduct detailed proteomics analysis to evaluate their quality and reproducibility. Based on the data, it can be inferred that the illustrated method provides a robust and reproducible approach to advance understanding of various biochemical pathways and their functions with greater confidence for both basic and applied research.

Metabolic Engineering of the MEP Pathway in Bacillus subtilis for Increased Biosynthesis of Menaquinone-7.

Vitamin K is essential for blood coagulation and plays important roles in bone and cardiovascular health. Menaquinone-7 (MK-7) is one form of vitamin K that is especially useful due to its long half-life in the circulation. MK-7 is difficult to make via organic synthesis, and is thus commonly produced by fermentation. This study aimed to genetically modify Bacillus subtilis cultures to increase their MK-7 yield and reduce production costs. We constructed 12 different strains of B.subtilis 168 by overexpressing different combinations of the rate-limiting enzymes Dxs, Dxr, Idi, and MenA. We observed an 11-fold enhancement of production in the best-performing strain, resulting in 50 mg/L MK-7. Metabolite analysis revealed new bottlenecks in the pathway at IspG and IspH, which suggest avenues for further optimization. This work highlights the usefulness of Bacillus subtilis for industrial production of high value compounds.

Screening of Isochrysis strains for simultaneous production of docosahexaenoic acid and fucoxanthin

Microalgae are capable of synthesizing a wide range of value-added compounds such as docosahexaenoic acid (DHA) and fucoxanthin. In the present study, the potential for simultaneous production of DHA and fucoxanthin by the marine alga Isochrysis was investigated via strain screening and culture optimization. A comparative analysis of sixteen Isochrysis strains revealed that CCMP1324 was superior with regard to growth and production of DHA and fucoxanthin. In response to various nutritional and environmental cues, CCMP1324 accumulated DHA and fucoxanthin in a coordinated and growth-dependent manner, which reached ~17.1 and 14.1 mg g−1, respectively. A semi-continuous culture strategy leading to high productivities of DHA (9.05 mg L−1 day−1) and fucoxanthin (7.96 mg L−1 day−1) was developed. Taken together, our study represents a pioneering work studying the accumulation coordination between DHA and fucoxanthin in Isochrysis. These findings provide valuable insights into future exploration for the integrated production of high-value compounds.

Enhanced protopanaxadiol production from xylose by engineered Yarrowia lipolytica

BackgroundAs renewable biomass, lignocellulose remains one of the major choices for most countries in tackling global energy shortage and environment pollution. Efficient utilization of xylose, an important monosaccharide in lignocellulose, is essential for the production of high-value compounds, such as ethanol, lipids, and isoprenoids. Protopanaxadiol (PPD), a kind of isoprenoids, has important medical values and great market potential.ResultsThe engineered protopanaxadiol-producing Yarrowia lipolytica strain, which can use xylose as the sole carbon source, was constructed by introducing xylose reductase (XR) and xylitol dehydrogenase (XDH) from Scheffersomyces stipitis, overexpressing endogenous xylulose kinase (ylXKS) and heterologous PPD synthetic modules, and then 18.18 mg/L of PPD was obtained. Metabolic engineering strategies such as regulating cofactor balance, enhancing precursor flux, and improving xylose metabolism rate via XR (K270R/N272D) mutation, the overexpression of tHMG1/ERG9/ERG20 and transaldolase (TAL)/transketolase (TKL)/xylose transporter (TX), were implemented to enhance PPD production. The final Y14 strain exhibited the greatest PPD titer from xylose by fed-batch fermentation in a 5-L fermenter, reaching 300.63 mg/L [yield, 2.505 mg/g (sugar); productivity, 2.505 mg/L/h], which was significantly higher than the titer of glucose fermentation [titer, 167.17 mg/L; yield, 1.194 mg/g (sugar); productivity, 1.548 mg/L/h].ConclusionThe results showed that xylose was more suitable for PPD synthesis than glucose due to the enhanced carbon flux towards acetyl-CoA, the precursor for PPD biosynthetic pathway. This is the first report to produce PPD in Y. lipolytica with xylose as the sole carbon source, which developed a promising strategy for the efficient production of high-value triterpenoid compounds.

Screening for heterotrophy in microalgae of various taxonomic positions and potential of mixotrophy for production of high-value compounds

We tested 37 photoautotrophic microalgal strains of various taxonomic positions for their heterotrophic growth on glucose. We showed that facultative heterotrophy was quite common among certain groups of green algae (Chlorellales or Sphaeropleales) and of Xanthophyceae, whereas it was completely absent from Eustigmatophyceae. For the first time, we found heterotrophy in some members of green algae (Botryosphaerella sudetica, Bracteacoccus sp., Dictyosphaerium spp., Lemmermannia sp., Parachlorella kessleri). Of the tested facultative heterotrophs, three strains, Scenedesmus sp., Dictyosphaerium chlorelloides, and Tribonema aequale, were selected to investigate, in detail, their mixotrophic growth and production of specific metabolites, carotenoids, exopolysaccharides, and eicosapentaenoic acid, respectively. Among them only T. aequale showed significantly higher production of the target compound, eicosapentaenoic acid, under the mixotrophic cultivation mode than photoautotrophically. Together with the ease of biomass harvest, Tribonema microalgae are therefore proposed as a possible source of high-quality ω-3 polyunsaturated fatty acids.

Fermented Solids and Their Application in the Production of Organic Compounds of Biotechnological Interest.

We review the application of dry fermented solids (DFS) containing naturally immobilized enzymes as catalysts in synthesis and in hydrolysis reactions. The most studied application is the use of DFS containing lipases in the synthesis of biodiesel esters, by transesterification of oils or by esterification of fatty acids with short-chain alcohols in solvent-free reaction media. Other applications of DFS that have been studied include the use of DFS containing pectinases to liberate D-galacturonic acid from pectin and the production of high-value compounds by DFS containing lipases, such as the synthesis of sugar esters and the production of pure enantiomers by resolution of racemic mixtures. To date, studies are limited to proof of concept, and there are still many challenges to be faced in the development of industrial-scale processes using DFS as catalysts. A key challenge is the relatively low activity of DFS compared to commercial enzyme preparations. Attention needs to be given to scale up, not only of the bioreactor for the application of the DFS but also for the production of the fermented solids. There is also a need for economic feasibility studies to determine whether the production of DFS and their use as catalysts can be competitive at industrial scale. Graphical Abstract.

Genome Editing for the Production of Natural Products in Escherichia coli

AbstractNatural products such as secondary metabolites (e.g., plant terpenoids) are found to be a major source of bioactive compounds. These natural products accumulate as complex mixtures with other related compounds and this chemical complexity adds cost to the downstream recovery and purification of natural products from plant biomass. One aim of synthetic biology and metabolic engineering programmes is to produce such compounds from synthetic gene clusters in heterologous hosts and thereby achieve more targeted and affordable production. Both fungi and bacteria are common hosts for metabolic engineering in industry. Fungal hosts include Penicillium chrysogenum, Saccharomyces cerevisiae, Aspergillus niger and the bacterial hosts Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum. E. coli is often selected as a host given the ease of its genetic manipulation and the long history of using this organism in laboratory‐based bioengineering. The bioengineering of E. coli extends also to feedstock pathways to interface and optimize the production of high value compounds from widely available and inexpensive carbon sources. Genome editing is important in these microbial bioengineering programmes and is needed to isolate stable strains and to optimize production. Herein, this review discusses frequently used methods for genome editing in E. coli in relation to the production of natural compounds and chemicals.

Modification of membrane lipid compositions in single-celled organisms - From basics to applications.

All intact cells, and their organelles, are surrounded by a ∼30 Å hydrophobic film that typically separates the interior from the environment. This film is composed of lipid bilayers that form from a pool of structurally highly diverse, amphipathic lipids. The specific composition and nature of these lipids strongly contributes to many different processes in the cell by influencing membrane structures, membrane protein sorting and functionalities. In this review, we discuss strategies to alter membrane lipid compositions of organelles and plasma membranes in different organisms, focusing on microbial cells. Reflecting the many essential roles of lipids in cellular regulation, we delineate diverse cellular processes affected by membrane lipid modifications and discuss possible applications in a biotechnological and biomedical context. A major motivation for membrane lipid engineering has been the improvement of expression, translocation and activity of heterologous membrane proteins, which can facilitate the biochemical and structural characterization of this challenging class of proteins. Additionally, better expression of membrane proteins or membrane lipid engineering - or a combination of both - led to improved production of high-value compounds and food additives, e.g. polyunsaturated fatty acids and glycolipids, in diverse hosts. More recently it has been shown that diverse cellular pathologies such as cancer and Alzheimer's disease are associated with lipid alterations. Hence, the progress in our understanding of membrane structure, function and protein-lipid interactions, and the resulting possibilities regarding the engineering of membrane lipid composition clearly enable novel nutraceutical and pharmaceutical interventions to be developed. Significant progress in this important area of research is highlighted in this review.

Performance of an active disturbance rejection control on a simulated continuous microalgae photobioreactor

Microalgae are used for the industrial production of high value compounds. The aim in continuous bioreactors is to obtain the highest biomass production. It is necessary to guarantee that the bioprocesses attain and maintain the optimal reference biomass CX*(t), despite endogenous and exogenous disturbances. This paper describes the numerical simulation of the application of active disturbance rejection control (ADRC) to control the dilution rate (D(t)) in a continuous culture of the microalga Chlorella vulgaris. To reduce the bioprocess to a “SISO” system, the authors chose the dilution rate, D(t), to be the only control signal. The control proposal was illustrated and evaluated through a numerical simulation using MATLAB/Simulink™ environment. The performance of the ADRC was tested by the application of external perturbations and variation of parameters over a nominal case. At nominal conditions, D(t) was always maintained within the physical limits imposed by the bioprocess. Step and smooth type signals, at 96.4%·|Dmax(t)|, were imposed as external perturbation on the control signal input, D(t). The controller response kept the output signal CX(t) within an insignificant 0.0043%·|CXmax(t)|. The algal culture had a strongly asymmetric response to variations of the ideal maximum growth rate, μmax(t) ± 30%·|μmax(t)|, and of the nominal light intensity, Iin(t) ± 30%·|Iin(t)|. Nonetheless, the controller promptly returned the output signal to its reference value, CX(t)*. The numerical test of the control proposal, in summary, showed that the ADRC strategy ensures excellent reference tracking capability and robustness towards parametric uncertainties, un-modeled dynamics, and external disturbances.

Establishment of Novel Biosynthetic Pathways for the Production of Salicyl Alcohol and Gentisyl Alcohol in Engineered Escherichia coli

Salicyl alcohol and gentisyl alcohol are two important phenolic alcohols that possess significant biological activities and pharmaceutical properties. Here, we report establishment of novel biosynthetic pathways for microbial production of salicyl alcohol and gentisyl alcohol from renewable feedstocks. We first examined the promiscuity of the carboxylic acid reductase CAR toward salicylic acid and 2,5-DHBA, which enabled efficient synthesis of salicyl alcohol and gentisyl alcohol. Then, we employed a novel salicylic acid 5-hydroxylase to achieve 2,5-DHBA production from salicylic acid. After that, the de novo biosynthetic pathways were assembled and optimized by programming the carbon flux into the shikimate pathway. The final titers of salicyl alcohol and gentisyl alcohol reached to 594.4 mg/L and 30.1 mg/L, respectively. To our knowledge, this work achieved microbial production of salicyl alcohol and gentisyl alcohol for the first time. Our present study also demonstrated application of enzyme promiscuity to establish non-natural biosynthetic pathways for the production of high-value compounds.

Interactions of microalgae and other microorganisms for enhanced production of high-value compounds.

The cultivation of microalgae for the production of biomass and associated valuable compounds has gained increasing interest not only within the scientific community but also at the industrial level. Microalgae cells are capable of producing high-value compounds that are widely used in food, feed, pharmaceutical, medical, nutraceutical, cosmeceutical, and aquaculture industries. For example, lipids produced by algae can be converted to biodiesel, other fuels and bio-products. Hence, high oil content algal biomass has been regarded as a potential alternative feedstock to replace terrestrial crops for sustainable production of bio-products. It has been reported that the interaction of microalgae and other microorganisms greatly enhances the efficiency of microalgal biomass production and its chemical composition. Microalgae-bacteria interaction with an emphasis on the nature of symbiotic relationship in mutualisitc and parasitic consortia has been extensively studied. For instance, it is well documented that production of vitamins or growth promoting factors by bacteria enhances the growth of microalgae. Little attention has been paid to the consortia formed by microalgae and other microorganisms such as other microalgae strains, cyanobacteria, fungi, and yeasts. Hence, the aim of this review is to investigate the impact of the microalgae-other microorganism interactions on the production of high value compounds.

Construction of a cellulose-metabolizing Komagataella phaffii (Pichia pastoris) by co-expressing glucanases and β-glucosidase.

Cellulose is a highly available and renewable carbon source in nature. However, it cannot be directly metabolized by most microbes including Komagataella phaffii (formerly Pichia pastoris), which is a frequently employed host for heterologous protein expression and production of high-value compounds. A K. phaffii strain was engineered that constitutively co-expresses an endoglucanase and a β-glucosidase both from Aspergillus niger and an exoglucanase from Trichoderma reesei under the control of bidirectional promoters. This engineered strain was able to grow on cellobiose and carboxymethyl cellulose (CMC) but not on Avicel. However, the detected release of cellobiose from Avicel by using the produced mixture of endoglucanase and exoglucanase as well as the released glucose from Avicel by using the produced mixture of all three cellulases at 50°C indicated the production of exoglucanase under the liquid culture conditions. The successful expression of three cellulases in K. phaffii demonstrated the feasibility to enable K. phaffii to directly use cellulose as a carbon source for producing recombinant proteins or other high-value compounds.

Optimal decision curve of light intensity to maximize the biomass concentration in a batch culture

This paper proposes a method to find an optimal decision curve to manage the incident light intensity (q0) that is applied to microalgae cultivation to maximize biomass concentration (CX). Microalgae are characterized by the production of high value compounds of interest to industry; the challenge is to obtain the highest biomass concentration. Optimization of the performance of microalgae culture systems is important to guarantee the viability of the economical process. The advantages of this optimization proposal are to attain to CX maximum productivities, as well as its simplicity and its robustness against perturbations. The optimization proposal is illustrated and evaluated on a numerical simulation in a batch culture of microalga Chlamydomonas reinhardtii. Additionally, it was compared to a conventional constant light operation and with an optimization approach based on finding the ratio between q0 and CX (light-to-microalga ratio). In the analysis, the performance of the optimal decision curve was contemplated in presence of the perturbations and variation of parameters. The simulations of proposal in this paper shows an optimal behavior in terms of a maximum production CX · In addition, this would have a better behavior in front of robustness and disturbance rejection capabilities, compared to both, constant light operation and light-to-microalga ratio.

The Potential of a Brown Microalga Cultivated in High Salt Medium for the Production of High-Value Compounds.

Amphora sp. was isolated from the Sfax Solar Saltern and cultivated under hypersaline conditions. It contains moderate rates of proteins, lipids, sugars, and minerals and a prominent content of bioactive compounds: polyphenols, chlorophyll a, carotenoids, and fatty acids. The analysis of fatty acids with GC/MS showed that the C16 series accounted for about 75% of Amphora sp. lipids. Saturated fatty acids whose palmitic acid was the most important (27.41%) represented 41.31%. Amphora sp. was found to be rich in monounsaturated fatty acids with dominance of palmitoleic acid. It also contains a significant percentage of polyunsaturated fatty acids with a high amount of eicosapentaenoic acid (2.36%). Among the various solvents used, ethanol at 80% extracted the highest amounts of phenols and flavonoids that were 38.27 mg gallic acid equivalent and 17.69 mg catechin equivalent g−1 of dried extract, respectively. Using various in vitro assays including DPPH and ABTS radicals methods, reducing power assay, and β-carotene bleaching assay, the 80% ethanolic extract showed high antioxidant activity. A strong antibacterial activity was checked against Gram-positive bacteria (Staphylococcus aureus and Micrococcus luteus) and Gram-negative bacteria (Klebsiella pneumoniae and Salmonella enterica). These results are in favor of Amphora sp. valorization in aquaculture and food and pharmaceutical industries.

The two Dps proteins, NpDps2 and NpDps5, are involved in light-induced oxidative stress tolerance in the N2-fixing cyanobacterium Nostoc punctiforme

Cyanobacteria are photosynthetic prokaryotes that are considered biotechnologically prominent organisms for production of high-value compounds. Cyanobacteria are subject to high-light intensities, which is a challenge that needs to be addressed in design of efficient bio-engineered photosynthetic organisms. Dps proteins are members of the ferritin superfamily and are omnipresent in prokaryotes. They play a major role in oxidative stress protection and iron homeostasis. The filamentous, heterocyst-forming Nostoc punctiforme, has five Dps proteins. In this study we elucidated the role of these Dps proteins in acclimation to high light intensity, the gene loci organization and the transcriptional regulation of all five dps genes in N. punctiforme was revealed, and dps-deletion mutant strains were used in physiological characterization. Two mutants defective in Dps2 and Dps5 activity displayed a reduced fitness under increased illumination, as well as a differential Photosystem (PS) stoichiometry, with an elevated Photosystem II to Photosystem I ratio in the dps5 deletion strain. This work establishes a Dps-mediated link between light tolerance, H2O2 detoxification, and iron homeostasis, and provides further evidence on the non-redundant role of multiple Dps proteins in this multicellular cyanobacterium.

Dynamic flux balance modeling to increase the production of high-value compounds in green microalgae.

BackgroundPhotosynthetic organisms can be used for renewable and sustainable production of fuels and high-value compounds from natural resources. Costs for design and operation of large-scale algae cultivation systems can be reduced if data from laboratory scale cultivations are combined with detailed mathematical models to evaluate and optimize the process.ResultsIn this work we present a flexible modeling formulation for accumulation of high-value storage molecules in microalgae that provides quantitative predictions under various light and nutrient conditions. The modeling approach is based on dynamic flux balance analysis (DFBA) and includes regulatory models to predict the accumulation of pigment molecules. The accuracy of the model predictions is validated through independent experimental data followed by a subsequent model-based fed-batch optimization. In our experimentally validated fed-batch optimization study we increase biomass and upbeta-carotene density by factors of about 2.5 and 2.1, respectively.ConclusionsThe analysis shows that a model-based approach can be used to develop and significantly improve biotechnological processes for biofuels and pigments.

Bioprocess engineering of microalgae to optimize lipid production through nutrient management

Microalgae have been used commercially as a feedstock for the production of high-value compounds, pigments, cosmetics, and nutritional supplements. In addition, because of their rapid growth rates, high photosynthetic efficiency, and high lipid and protein content, commodity products including biodiesel, feed supplements, and polyunsaturated fatty acids derived from algal biomass are of current interest. Since microalgae lack non-photosynthetic structures and float in water, they do not need massive amounts of structural cellulose found in land plants. Thus, under optimal culture conditions, some oleaginous species can allocate up to 70 % of their biomass to lipids. Lipid production and its regulation in microalgae are species-specific and influenced by environmental conditions. Various strategies have been developed to improve lipid productivity and fatty acid composition to meet specific production goals. Manipulation of physiochemical parameters, trophic modes, and nutrient levels, known as process engineering, is a simple approach that leads to desired alterations in the biochemical composition of algal biomass, including lipid quantity and quality. In this paper, we review the effects of manipulating biochemical parameters such as necessary nutrients (C, N, P, S, Fe, and Si), NaCl concentration, and pH of culture medium to optimize lipid content and profile in some algae strains with commercial potential.

Biotechnological Approaches for Production of High Value Compounds from Bread Waste

A growing global population has led to an increasing demand for food processing industries and consequently the generation of large amounts of food waste. This problem has intensified due to slow progress in the development of effective waste treatment and disposal strategies. Food waste such as bread waste is a reservoir of complex carbohydrates, proteins and lipids which have the potential to be reused in fermentation processes. In this regard, bread waste has been used for the production of a variety of bio-products including ethanol, methane, lactic acid, succinic acid, amylase and protease. This review provides an overview of the previous works in the literature on the utilization of bread waste and the different bio processing approaches to produce a higher value products. Recommendations are also provided for areas of future research.