Microbial Production Processes Research Articles

Screening of novel β-carotene hydroxylases for the production of β-cryptoxanthin and zeaxanthin and the impact of enzyme localization and crowding on their production in Yarrowia lipolytica

Zeaxanthin, a vital dietary carotenoid, is naturally synthesized by plants, microalgae, and certain microorganisms. Large-scale zeaxanthin production can be achieved through plant extraction, chemical synthesis, or microbial fermentation. The environmental and health implications of the first two methods have made microbial fermentation an appealing alternative for natural zeaxanthin production despite the challenges in scaling up the bioprocess. An intermediate between β-carotene and zeaxanthin, β-cryptoxanthin, is found only in specific fruits and vegetables and has several important functions for human health. The low concentration of β-cryptoxanthin in these sources results in low extraction yields, making biotechnological production a promising alternative for achieving higher yields. Currently, there is no industrially relevant microbial fermentation process for β-cryptoxanthin production, primarily due to the lack of identified enzymes that specifically convert β-carotene to β-cryptoxanthin without further conversion to zeaxanthin. In this study, we used genetic engineering to leverage the oleaginous yeast Yarrowia lipolytica as a bio-factory for zeaxanthin and β-cryptoxanthin production. We screened 22 β-carotene hydroxylases and identified eight novel enzymes with β-carotene hydroxylating activity: six producing zeaxanthin and two producing only β-cryptoxanthin. By introducing the β-carotene hydroxylase from the bacterium Chondromyces crocatus (CcBCH), a β-cryptoxanthin titer of 24 ± 6 mg/L was achieved, representing the highest reported titer of sole β-cryptoxanthin in Y. lipolytica to date. By targeting zeaxanthin-producing β-carotene hydroxylase to the endoplasmic reticulum and peroxisomes, we increased the production of zeaxanthin by 54% and 66%, respectively, compared to untargeted enzyme. The highest zeaxanthin titer of 412 ± 34 mg/L was achieved by targeting β-carotene hydroxylases to peroxisomes. In addition, by constructing multienzyme scaffold-free complexes with short peptide tags RIDD and RIAD, we observed a 39% increase in the zeaxanthin titer and a 28% increase in the conversion rate compared to the strain expressing unmodified enzyme. The zeaxanthin titers obtained in this study are not the highest reported; however, our goal was to demonstrate that specific approaches can enhance both titer and conversion rate, rather than to achieve the maximum titer. These findings underscore the potential of Y. lipolytica as a promising platform for carotenoid production and provide a foundation for future research, where further optimization is required to maximize production.

Multi-stage process for mixed microbial culture production of polyhydroxyalkanoates from sugarcane stillage: Assessment of external nutrient supplementation

Sugarcane stillage is an abundant wastewater from ethanol production. It has drawn considerable interest as a potential feedstock for biotechnological processes aiming at the recovery of energy and value-added products. In this study we explored the use of stillage for the production of polyhydroxyalkanoates (PHA), which are microbial bioplastics with potential to replace conventional plastics in specific applications. As stillage is nutrient deficient, we investigated the impact of external nutrient supplementation on the selection of PHA-producing mixed cultures. Cultures selected under four different total-chemical-oxygen-demand-to-nitrogen ratios (COD.t/N) (15, 30, 40 and 80) were evaluated according to their PHA production performance and microbial composition. The experimental results demonstrated that the most favorable PHA production was achieved for the biomass selected under strict carbon-limiting conditions, with a COD.t/N ratio of 15. This resulted in an overall PHA volumetric productivity of 2.10 g PHA L−1 d−1, a maximum intracellular PHA content of 0.65 g PHA g VSS−1 and a PHA storage yield of 0.71 g CODPHA g CODH.Org−1. The resulting PHA was a copolymer of 3-hydroxybutyrate (73 %mol) and 3-hydroxyvalerate (27 %mol) monomers. The microbial consortium was enriched by members of the Brevundimonas genus, known PHA producers, with a relative abundance of 26.4 %.

Phosphate limitation enhances malic acid production on nitrogen-rich molasses with Ustilago trichophora

BackgroundAn important step in replacing petrochemical products with sustainable, cost-effective alternatives is the use of feedstocks other than, e.g., pure glucose in the fermentative production of platform chemicals. Ustilaginaceae offer the advantages of a wide substrate spectrum and naturally produce a versatile range of value-added compounds under nitrogen limitation. A promising candidate is the dicarboxylic acid malic acid, which may be applied as an acidulant in the food industry, a chelating agent in pharmaceuticals, or in biobased polymer production. However, fermentable residue streams from the food and agricultural industry with high nitrogen content, e.g., sugar beet molasses, are unsuited for processes with Ustilaginaceae, as they result in low product yields due to high biomass and low product formation.ResultsThis study uncovers challenges in evaluating complex feedstock applicability for microbial production processes, highlighting the role of secondary substrate limitations, internal storage molecules, and incomplete assimilation of these substrates. A microliter-scale screening method with online monitoring of microbial respiration was developed using malic acid production with Ustilago trichophora on molasses as an application example. Investigation into nitrogen, phosphate, sulphate, and magnesium limitations on a defined minimal medium demonstrated successful malic acid production under nitrogen and phosphate limitation. Furthermore, a reduction of nitrogen and phosphate in the elemental composition of U. trichophora was revealed under the respective secondary substrate limitation. These adaptive changes in combination with the intricate metabolic response hinder mathematical prediction of product formation and make the presented screening methodology for complex feedstocks imperative. In the next step, the screening was transferred to a molasses-based complex medium. It was determined that the organism assimilated only 25% and 50% of the elemental nitrogen and phosphorus present in molasses, respectively. Due to the overall low content of bioavailable phosphorus in molasses, the replacement of the state-of-the-art nitrogen limitation was shown to increase malic acid production by 65%.ConclusionThe identification of phosphate as a superior secondary substrate limitation for enhanced malic acid production opens up new opportunities for the effective utilization of molasses as a more sustainable and cost-effective substrate than, e.g., pure glucose for biobased platform chemical production.

Antibiotic fermentation residue for biohydrogen production: Inhibitory mechanisms of the inherent antibiotic

Antibiotic fermentation residue, which is generated from the microbial antibiotic production process, has been a troublesome waste faced by the pharmaceutical industry. Dark fermentation is a potential technology to treat antibiotic fermentation residue in terms of renewable H2 generation and waste management. However, the inherent antibiotic in antibiotic fermentation residue may inhibit its dark fermentation performance, and current understanding on this topic is limited. This investigation examined the impact of the inherent antibiotic on the dark H2 fermentation of Cephalosporin C (CEPC) fermentation residue, and explored the mechanisms from the perspectives of bacterial communities and functional genes. It was found that CEP-C in the antibiotic fermentation residue significantly inhibited the H2 production, with the H2 yield decreasing from 17.2 mL/g-VSadded to 12.5 and 9.6 mL/g-VSadded at CEP-C concentrations of 100 and 200 mg/L, respectively. CEP-C also prolonged the H2-producing lag period. Microbiological analysis indicated that CEP-C remarkably decreased the abundances of high-yielding H2-producing bacteria, as well as downregulated the genes involved in hydrogen generation from the“pyruvate pathway” and“NADH pathway”, essentially leading to the decline of H2 productivity. The present work gains insights into how cephalosporin antibiotics influence the dark H2 fermentation, and provide guidance for mitigating the inhibitory effects.

Nonsterile Process for Biohydrogen Production: Recent Updates, Challenges, and Opportunities.

Hydrogen (H2), a clean and versatile energy carrier, has recently gained significant attention as a potential solution for reducing carbon emissions and promoting sustainable energy systems. The yield and efficiency of the biological H2 production process primarily depend on sterilization conditions. Various strategies, such as heat inactivation and membrane-based sterilization, have been used to achieve desirable yields via microbial fermentation. Almost every failed biotransformation process is linked to nonsterile conditions at any reaction stage. Therefore, the production of renewable biofuels as alternatives to fossil fuels is more attractive. Pure sugars have been widely documented as a costly feedstock for H2 production under sterile conditions. Biotransformation under nonsterile conditions is more desirable for stable and sustainable operation. Low-cost feeds, such as biowaste, are considered suitable alternatives, but they require appropriate sterilization to overcome the limitations of inherited or contaminating microbes during H2 production. This article describes the status of microbial fermentative processes for H2 production under nonsterile conditions and discusses strategies to improve such processes for sustainable, cleaner production.

Polyglutamate: Unleashing the Versatility of a Biopolymer for Cosmetic Industry Applications

Polyglutamic acid (PGA), a biopolymer comprising repeating units of glutamic acid, has garnered significant attention owing to its versatile applications. In recent years, microbial production processes have emerged as promising methods for the large-scale synthesis of PGA, offering advantages such as sustainability, efficiency, and tailored molecular properties. Beyond its industrial applications, PGA exhibits unique properties that render it an attractive candidate for use in the cosmetic industry. The biocompatibility, water solubility, and film-forming characteristics of PGA make it an ideal ingredient for cosmetic formulations. This article explores the extensive potential cosmetic applications of PGA, highlighting its multifaceted role in skincare, haircare, and various beauty products. From moisturizing formulations to depigmentating agents and sunscreen products, PGA offers a wide array of benefits. Its ability to deeply hydrate the skin and hair makes it an ideal ingredient for moisturizers, conditioners, and hydrating masks. Moreover, PGA’s depigmentating properties contribute to the reduction in hyperpigmentation and uneven skin tone, enhancing the overall complexion. As the demand for sustainable and bio-derived cosmetic ingredients escalates, comprehending the microbial production and cosmetic benefits of PGA becomes crucial for driving innovation in the cosmetic sector.

From Saccharomyces cerevisiae to Ethanol: Unlocking the Power of Evolutionary Engineering in Metabolic Engineering Applications.

Increased human population and the rapid decline of fossil fuels resulted in a global tendency to look for alternative fuel sources. Environmental concerns about fossil fuel combustion led to a sharp move towards renewable and environmentally friendly biofuels. Ethanol has been the primary fossil fuel alternative due to its low carbon emission rates, high octane content and comparatively facile microbial production processes. In parallel to the increased use of bioethanol in various fields such as transportation, heating and power generation, improvements in ethanol production processes turned out to be a global hot topic. Ethanol is by far the leading yeast output amongst a broad spectrum of bio-based industries. Thus, as a well-known platform microorganism and native ethanol producer, baker's yeast Saccharomyces cerevisiae has been the primary subject of interest for both academic and industrial perspectives in terms of enhanced ethanol production processes. Metabolic engineering strategies have been primarily adopted for direct manipulation of genes of interest responsible in mainstreams of ethanol metabolism. To overcome limitations of rational metabolic engineering, an alternative bottom-up strategy called inverse metabolic engineering has been widely used. In this context, evolutionary engineering, also known as adaptive laboratory evolution (ALE), which is based on random mutagenesis and systematic selection, is a powerful strategy to improve bioethanol production of S. cerevisiae. In this review, we focus on key examples of metabolic and evolutionary engineering for improved first- and second-generation S. cerevisiae bioethanol production processes. We delve into the current state of the field and show that metabolic and evolutionary engineering strategies are intertwined and many metabolically engineered strains for bioethanol production can be further improved by powerful evolutionary engineering strategies. We also discuss potential future directions that involve recent advancements in directed genome evolution, including CRISPR-Cas9 technology.

Dissolved Organic Matter Fluorescence as a Tracer of Upwelling and Microbial Activities in Two Cyclonic Eddies in the Eastern Tropical North Atlantic

AbstractMesoscale eddies are frequently observed in the Eastern Tropical North Atlantic (ETNA), yet their effects on the transport and distribution of biogeochemical solutes, and specifically on the production and remineralization of dissolved organic matter (DOM) remain difficult to elucidate. Here, we investigated the submesoscale variability of chromophoric DOM (CDOM) and fluorescent DOM (FDOM) together with microbial production and remineralization processes in two cyclonic eddies (CEs) in the ETNA during summer and winter 2019. One CE, formed near the coast off Mauritania during the post‐upwelling season, was sampled along a ∼900 km zonal corridor between Mauritania and the Cape Verde Islands. The other CE, formed nearby Brava Island, was out of coastal influence. Four fluorescent components were identified with parallel factor analysis, two humic‐like, and two protein‐like components. Humic‐like FDOM components correlated to optode‐based community respiration and were also good indicators of upwelling associated with the Brava Island CE as they correlated to physical parameters (e.g., temperature) and to dissolved inorganic nitrogen. The tryptophan‐like FDOM components correlated with the carbon and nitrogen content of semi‐labile DOM, phytoplankton biomass, community respiration, and bacterial production. Overall, our study revealed that DOM optical properties are suitable for tracing freshly produced organic matter and the transport of remineralized DOM within offshore eddies.

Engineering the methylotrophic yeast Ogataea polymorpha for lactate production from methanol.

Introduction: Lactate has gained increasing attention as a platform chemical, particularly for the production of the bioplastic poly-lactic acid (PLA). While current microbial lactate production processes primarily rely on the use of sugars as carbon sources, it is possible to envision a future where lactate can be produced from sustainable, non-food substrates. Methanol could be such a potential substrate, as it can be produced by (electro)chemical hydrogenation from CO2. Methods: In this study, the use of the methylotrophic yeast Ogataea polymorpha as a host organism for lactate production from methanol was explored. To enable lactate production in Ogataea polymorpha, four different lactate dehydrogenases were expressed under the control of the methanol-inducible MOX promoter. The L-lactate dehydrogenase of Lactobacillus helveticus performed well in the yeast, and the lactate production of this engineered strain could additionally be improved by conducting methanol fed-batch experiments in shake flasks. Further, the impact of different nitrogen sources and the resulting pH levels on production was examined more closely. In order to increase methanol assimilation of the lactate-producing strain, an adaptive laboratory evolution experiment was performed. Results and Discussion: The growth rate of the lactate-producing strain on methanol was increased by 55%, while at the same time lactate production was preserved. The highest lactate titer of 3.8g/L in this study was obtained by cultivating this evolved strain in a methanol fed-batch experiment in shake flasks with urea as nitrogen source. This study provides a proof of principle that Ogataea polymorpha is a suitable host organism for the production of lactate using methanol as carbon source. In addition, it offers guidance for the engineering of methylotrophic organisms that produce platform chemicals from CO2-derived substrates. With reduced land use, this technology will promote the development of a sustainable industrial biotechnology in the future.

Microbial production of trans-aconitic acid

Trans-aconitic acid (TAA) is a promising bio-based chemical with the structure of unsaturated tricarboxylic acid, and also has the potential to be a non-toxic nematicide as a potent inhibitor of aconitase. However, TAA has not been commercialized because the traditional production processes of plant extraction and chemical synthesis cannot achieve large-scale production at a low cost. The availability of TAA is a serious obstacle to its widespread application. In this study, we developed an efficient microbial synthesis and fermentation production process for TAA. An engineered Aspergillus terreus strain producing cis-aconitic acid and TAA was constructed by blocking itaconic acid biosynthesis in the industrial itaconic acid-producing strain. Through heterologous expression of exogenous aconitate isomerase, we further designed a more efficient cell factory to specifically produce TAA. Subsequently, the fermentation process was developed and scaled up step-by-step, achieving a TAA titer of 60 g L−1 at the demonstration scale of a 20 m3 fermenter. Finally, the field evaluation of the produced TAA for control of the root-knot nematodes was performed in a field trial, effectively reducing the damage of the root-knot nematode. Our work provides a commercially viable solution for the green manufacturing of TAA, which will significantly facilitate biopesticide development and promote its widespread application as a bio-based chemical.

Production of fuel ethanol from Vitellaria paradoxa fruit pulp by simultaneous saccharification and fermentation

Biofuels have become the world’s greatest, safest, cleanest and cheapest alternative to fossil fuels. Transport sector continues to receive influx of cars and general machinery every year worldwide leading to an overwhelming use of fossil fuel. The fossil fuel sources are gradually becoming depleted. Sustainable bioethanol recovered from inexhaustible sources are constantly been used to replace fossil fuels in the transport sector. The shea nut pulp (SNP) is a cheap source of carbon, nitrogen and calcium for the production of bioethanol. This study has employed the shea nut pulp as substrate, Saccharomyces cerevisiae-the Baker’s Yeast strain-as the fermentation microbe, Alpha Amylase enzyme for hydrolysis of the substrate and supported the simultaneous fermentation process with nutrient supplements. The data were subjected to statistical analysis. Approximately 37.8 and 40.0 g/L of ethanol after 16 and 20 h, were produced from without-α-amylase and with-α-amylase treatments, respectively. Fermentation efficiencies of 58.6 and 65.0 % were observed ethanol yields for without-α-amylase and with-α-amylase, respectively when compared to the theoretical yield. This research is useful in harnessing the potentials of the shea nut pulp as industrially relevant substrate for use independently or in combination with other substrates in microbial fermentation processes for ethanol production. An assessment of the fermentation process has revealed that the SNP is a good source of bioethanol going into the future. The research therefore recommends further work on scale-up of the bioethanol production process from the shea nut pulp.

Valorization of food wastes with a sequential two-step process for microbial β-carotene production: A zero waste approach

Here, two consecutive β-carotene fermentation processes were carried out with Rhodotorula glutinis yeast in the growth media obtained from orange and grape wastes. Firstly, waste biomasses were subjected to hot water extraction. Effects of waste type, drying pretreatment, particle size and solid/liquid ratio on the total concentration and yield of sugars recovered were tested. The highest sugar concentration was obtained by the hot water extraction of fresh grape pomace as 61.2 g total reducing sugars (TRS)/L at a solid/liquid ratio of 100 g/L. In the first fermentation process, effect of solid/liquid ratio (initial TRS concentration) on β-carotene production pattern of R. glutinis was investigated in the media obtained directly by hot water extraction of the wastes. Microorganism and β-carotene concentrations increased with increasing solid/liquid ratio (range 10–100 g/L), and the microbial growth data fit the Monod model well for all cases. Maximum β-carotene concentration in the growth medium obtained from hot water extraction of 100 g/L of grape pomace was determined as 5988.6 mg/L. In the second fermentation process, β-carotene was produced in the acid hydrolysate of extraction residues. 10.1 g/L and 6.7 g/L of TRS was obtained after acid hydrolysis of orange and grape residues, respectively, and the highest β-carotene concentration of 370.0 mg/L was found in the medium of hydrolyzed orange peel extraction residue. Total β-carotene production increased to 1777.1 and 3279.6 mg/L (26% and 4.9% of increase) after the second fermentation step. 85.3% and 80.2% of reduction in orange and grape waste weights were observed at the end of the process, which was an indicator of efficient waste biomass disposal. Two sequential β-carotene fermentation steps offered significant advantages in terms of both efficiency and a zero waste approach.

Experimental Study On Production of Biogas from Kitchen Waste

In our institute we have four hostels and all having their own individual mess, where daily a large amount of kitchen waste is obtained which can be utilized for better purposes. Biogas production requires Anaerobic digestion. Project was to Create an Organic Processing Facility to create biogas which will be more cost effective, eco-friendly, cut down on land fill waste, generate a high-quality renewable fuel, and reduce carbon dioxide & methane emissions. Overall, by creating a biogas reactor on campus in the backyard of our hostels will be beneficial. Kitchen (food waste) was collected from different hostels of PACE IT & S Mess as feed stock for our reactor which works as an aerobic digester system to produce biogas energy. The anaerobic digestion of kitchen waste produces biogas, a valuable energy resource Anaerobic digestion is a microbial process for production of biogas, which consist of Primarily methane (CH4) & carbon dioxide (CO2). Biogas can be used as energy source and also for numerous purposes. But any possible applications require knowledge & information about the composition and quantity of constituents in the biogas produced. The continuously-fed digester requires addition of sodium hydroxide (NaOH) to maintain the alkalinity and pH to 7. For this reactor we have prepared our Inoculum than we installed batch reactors, to which inoculum of previous cow dung slurry along with the kitchen waste was added to develop our own Inoculum. A combination of these mixed inoculum was used for biogas production at 37°C in laboratory (small scale) reactor (20L capacity) In our study, the production of biogas and methane is done from the starch-rich and sugary material and is determined at laboratory scale using the simple digesters.

Nonmonotonic effect of CuO nanoparticles on medium-chain carboxylates production from waste activated sludge

The growing applications of CuO nanoparticles (NPs) in industrial and agriculture has increased their concentrations in wastewater and subsequently accumulated in waste activated sludge (WAS), raising concerns about their impact on reutilization of WAS, especially on the medium-chain carboxylates (MCCs) production from anaerobic fermentation of WAS. Here we showed that CuO NPs at 10–50 mg/g-TS can significantly inhibit MCCs production, and reactive oxygen species generation was revealed to be the key factor linked to the phenomena. At lower CuO NPs concentrations (0.5–2.5 mg/g-TS), however, MCCs production was enhanced, with a maximum level of 37% compared to the control. The combination of molecular approaches and metaproteomic analysis revealed that although low dosage CuO NPs (2.5 mg/g-TS) weakly inhibited chain elongation process, they displayed contributive characteristics both in WAS solubilization and transport/metabolism of carbohydrate. These results demonstrated that the complex microbial processes for MCCs production in the anaerobic fermentation of WAS can be affected by CuO NPs in a dosage-dependent manner via regulating microbial protein expression level. Our findings can provide new insights into the influence of CuO NPs on anaerobic fermentation process and shed light on the treatment option for the resource utilization of CuO NPs polluted WAS.

Identification of a Potential Vaccine against Treponema pallidum Using Subtractive Proteomics and Reverse-Vaccinology Approaches

Syphilis, a sexually transmitted infection, is a deadly disease caused by Treponema pallidum. It is a Gram-negative spirochete that can infect nearly every organ of the human body. It can be transmitted both sexually and perinatally. Since syphilis is the second most fatal sexually transmitted disease after AIDS, an efficient vaccine candidate is needed to establish long-term protection against infections by T. pallidum. This study used reverse-vaccinology-based immunoinformatic pathway subtractive proteomics to find the best antigenic proteins for multi-epitope vaccine production. Six essential virulent and antigenic proteins were identified, including the membrane lipoprotein TpN32 (UniProt ID: O07950), DNA translocase FtsK (UniProt ID: O83964), Protein Soj homolog (UniProt ID: O83296), site-determining protein (UniProt ID: F7IVD2), ABC transporter, ATP-binding protein (UniProt ID: O83930), and Sugar ABC superfamily ATP-binding cassette transporter, ABC protein (UniProt ID: O83782). We found that the multiepitope subunit vaccine consisting of 4 CTL, 4 HTL, and 11 B-cell epitopes mixed with the adjuvant TLR-2 agonist ESAT6 has potent antigenic characteristics and does not induce an allergic response. Before being docked at Toll-like receptors 2 and 4, the developed vaccine was modeled, improved, and validated. Docking studies revealed significant binding interactions, whereas molecular dynamics simulations demonstrated its stability. Furthermore, the immune system simulation indicated significant and long-lasting immunological responses. The vaccine was then reverse-transcribed into a DNA sequence and cloned into the pET28a (+) vector to validate translational activity as well as the microbial production process. The vaccine developed in this study requires further scientific consensus before it can be used against T. pallidum to confirm its safety and efficacy.

Carbon/nitrogen ratio as a tool to enhance the lipid production in Rhodosporidium toruloides-1588 using C5 and C6 wood hydrolysates

The interest in microbial lipids has recently increased because of their wide use to produce several value-added compounds in the biofuel, pharmaceutical and food industries. Oleaginous yeast such as Rhodosporidium toruloides could be an efficient option because of its ability to consume five-carbon sugars, high lipid accumulation, and tolerance to toxic compounds such as furans, phenolic compounds, and organic acids. The present study aims to investigate the effect of different initial sugar ratios, in combination with different carbon/nitrogen ratios, and the use of dibasic sodium phosphate (Na2HPO4) as an inducer on cell biomass production, sugar consumption, and lipid accumulation by Rhodosporidium toruloides-1588. The investigation showed a maximum lipid accumulation of 5.35 gL-1 (0.28 g of lipids/g of sugar) under the culture conditions of initial glucose: xylose ratio of 1:1, C/N ratio of 70, and Na2HPO4 concentration of 1.05 gL-1. The predominant lipids composition was palmitic, stearic, oleic, and linoleic acids which could be used as a suitable feedstock for biofuel production. Additionally, under the optimal conditions (initial glucose: xylose ratio of 1:1, 1.19 gL-1 of Na2HPO4 and C/N ratio of 70.50) an increase of 10.5% and 7.5% in lipid accumulation was observed, compared with control treatments (glucose and xylose, respectively). In addition, the study shows the ability of R. toruloides-1588 to tolerate inhibitors, a feature that could be a promising alternative to increase the feasibility of the microbial lipid production process using undetoxified wood hydrolysate as a sustainable culture media.

Scheffersomyces stipitis ability to valorize different residual biomasses for vitamin B9 production.

Sugar beet pulp (SBP), sugar beet molasses (SBM) and unfermented grape marcs (UGM) represent important waste in the agro-food sector. If suitably pre-treated, hexose and pentose sugars can be released in high quantities and can subsequently be used by appropriate cell factories as growth media and for the production of (complex) biomolecules, accomplishing the growing demand for products obtained from sustainable resources. One example is vitamin B9 or folate, a B-complex vitamin currently produced by chemical synthesis, almost exclusively in the oxidized form of folic acid (FA). It is therefore desirable to develop novel competitive strategies for replacing its current fossil-based production with a sustainable bio-based process. In this study, we assessed the production of natural folate by the yeast Scheffersomyces stipitis, investigating SBM, SBP and UGM as potential growth media. Pre-treatment of SBM and SBP had previously been optimized in our laboratory; thus, here we focused only on UGM pre-treatment and hydrolysis strategies for the release of fermentable sugars. Then, we optimized the growth of S. stipitis on the three media formulated from those biomasses, working on inoculum pre-adaptation, oxygen availability and supplementation of necessary nutrients to support the microorganism. Folate production, measured with a microbiological assay, reached 188.2 ± 24.86 μg/L on SBM, 130.6 ± 1.34 μg/L on SBP and 101.9 ± 6.62 μg/L on UGM. Here, we demonstrate the flexibility of S.stipitis in utilizing different residual biomasses as growth media. Moreover, we assessed the production of folate from waste, and to the best of our knowledge, we obtained the highest production of folate from residual biomasses ever reported, providing the first indications for the future development of this microbial production process.

Heterologous Lignan Production in Stirred-Tank Reactors—Metabolomics-Assisted Bioprocess Development for an In Vivo Enzyme Cascade

Towards establishing a prospective industrial microbial lignan production process, we set up and investigated the biotransformation of coniferyl alcohol to secoisolariciresinol with recombinant Escherichia coli in a stirred-tank reactor (STR). Initially, we tested different cofactor concentrations and antifoam additions in shake flasks. Next, we designed an STR batch bioprocess and tested aeration rates, pH regulation, and substrate-feeding strategies. Targeted metabolomics of phenylpropanoids and lignans assisted the bioprocess development by monitoring the lignan pathway activity. We found that the copper concentration and the substrate-feeding strategy had considerable impact on lignan production. Furthermore, time-resolved monitoring of pathway metabolites revealed two maximal intracellular lignan concentrations, the first shortly after induction of gene expression and the second after the cells entered the stationary growth phase. During STR cultivation, a maximal intracellular titer of 130.4 mg L−1 secoisolariciresinol was achieved, corresponding to a yield coefficient of 26.4 mg g−1 and a space–time yield of 2.6 mg L−1 h−1. We report for the first time the in-depth evaluation of microbially produced lignans in a well-controlled STR bioprocess. Monitoring of the lignan pathway activity showed that lignan accumulation is highly dynamic during the cultivation and points towards the need for a more efficient coniferyl alcohol dimerization system for optimal microbial production conditions.

Light-regulated gene expression in Bacteria: Fundamentals, advances, and perspectives.

Numerous photoreceptors and genetic circuits emerged over the past two decades and now enable the light-dependent i.e., optogenetic, regulation of gene expression in bacteria. Prompted by light cues in the near-ultraviolet to near-infrared region of the electromagnetic spectrum, gene expression can be up- or downregulated stringently, reversibly, non-invasively, and with precision in space and time. Here, we survey the underlying principles, available options, and prominent examples of optogenetically regulated gene expression in bacteria. While transcription initiation and elongation remain most important for optogenetic intervention, other processes e.g., translation and downstream events, were also rendered light-dependent. The optogenetic control of bacterial expression predominantly employs but three fundamental strategies: light-sensitive two-component systems, oligomerization reactions, and second-messenger signaling. Certain optogenetic circuits moved beyond the proof-of-principle and stood the test of practice. They enable unprecedented applications in three major areas. First, light-dependent expression underpins novel concepts and strategies for enhanced yields in microbial production processes. Second, light-responsive bacteria can be optogenetically stimulated while residing within the bodies of animals, thus prompting the secretion of compounds that grant health benefits to the animal host. Third, optogenetics allows the generation of precisely structured, novel biomaterials. These applications jointly testify to the maturity of the optogenetic approach and serve as blueprints bound to inspire and template innovative use cases of light-regulated gene expression in bacteria. Researchers pursuing these lines can choose from an ever-growing, versatile, and efficient toolkit of optogenetic circuits.

Improved xylitol production by the novel inhibitor-tolerant yeast Candida tropicalis K2

ABSTRACT Production of potential value-added products from different lignocellulosic biomass is becoming more common due to the availability of the feedstocks in abundance and the environment- friendly nature of the microbial production process. Due to the large array of its applications in the pharmaceutical and food sectors, xylitol is considered as potential value-added compound for production. In this study, organic waste samples were collected from various habitats and screened for potential yeast isolates for xylitol production. Among 124 tested isolates, Candida tropicalis K2 showed the highest potential for xylitol production as well as inhibitors tolerance (Furfural, 5-hydroxymethyl furfural and acetic acid) phenotypes. C. tropicalis K2 produced 90 g/L of xylitol in batch fermentation (100 g/L xylose supplemented with 20 g/L of glycerol as co-substrate) with the yield and productivity of 0.90 g/g and 1.5 g/L.h, respectively, at pH 5.5 and 30°C temperature. Together, >10% higher xylitol yield was achieved when glycerol was used as a co-substrate with pure xylose. Moreover, with non-detoxified corncob and Albizia pod hydrolysates, C. tropicalis K2 isolate produced 0.62 and 0.69 g/g of xylitol yields and 1.04 and 0.75 g/L.h xylitol productivities, respectively. Thus, C. tropicalis K2 isolate could be considered as promising candidate for xylitol production from different lignocellulosic biomass. HIGHLIGHTS Candia tropicalis K2 isolate was screened from natural sites of biomass degradation and characterized for xylitol production. Non-detoxified Albizia pod and corncob hydrolysates were explored for xylitol production using selected C. tropicalis K2 isolate. A maximum of 0.90 g/g yield and 1.07 g/L.h xylitol productivity was achieved with pure xylose. A >10% increase in xylitol yield was achieved using glycerol as a co-substrate.