Development of engineered Candida tropicalis strain for efficient corncob-based xylitol-ethanol biorefinery.

BACKGROUND: Xylitol has a wide range of applications in the pharmaceuticals, cosmetic, food and beverage industry. Microbial xylitol production reduces the risk of contamination and is considered as environment friendly and sustainable compared to the chemical method. In this study, random mutagenesis and genetic engineering approaches were employed to develop Candida tropicalis strains with reduced xylitol dehydrogenase (XDH) activity to eliminate co-substrate requirement for corn cob-based xylitol-ethanol biorefinery. RESULTS: The results suggest that when pure xylose (10% w/v) was fermented in bioreactor, the Ethyl methane sulfonate (EMS) mutated strain (C. tropicalis K2M) showed 9.2% and XYL2 heterozygous (XYL2/xyl2Δ::FRT) strain (C. tropicalis K21D) showed 16% improvement in xylitol production compared to parental strain (C. tropicalis K2). Furthermore, 1.5-fold improvement (88.62 g/L to 132 g/L) in xylitol production was achieved by C. tropicalis K21D after Response Surface Methodology (RSM) and one factor at a time (OFAT) applied for media component optimization. Finally, corncob hydrolysate was tested for xylitol production in biorefinery mode, which leads to the production of 32.6 g/L xylitol from hemicellulosic fraction, 32.0 g/L ethanol from cellulosic fraction and 13.0 g/L animal feed. CONCLUSIONS: This work, for the first time, illustrates the potential of C. tropicalis K21D as a microbial cell factory for efficient production of xylitol and ethanol via an integrated biorefinery framework by utilising lignocellulosic biomass with minimum waste generation.

MICROBIAL isoprene production: an overview.

Isoprene, a volatile C5 hydrocarbon, is a precursor of synthetic rubber and an important building block for a variety of natural products, solely being produced by petrochemical routes. To mitigate the ever-increasing contribution of petrochemical industry to global warming through significant carbon (CO2) evolution, bio-based process for isoprene production using microbial cell factories have been explored. Highly efficient fermentation-based processes have been studied for little over a decade now with extensive research on the rational strain development for creating robust strains for commercial isoprene production. Most of these studies involved sugars as feedstocks and using naturally occurring isoprene pathways viz., mevalonate and methyl erythritol pathway in E. coli. Recent advances, driven by efforts in reducing environmental pollution, have focused on utilization of inorganic CO2 by cyanobacteria or syngas from waste gases by acetogens for isoprene production. This review endeavors to capture the latest relevant progress made in rational strain development, metabolic engineering and synthetic biology strategies used, challenges in fermentation process development at lab and commercial scale production of isoprene along with a future perspective pertaining to this area of research.

Advances and prospects in metabolic engineering of Escherichia coli for L-tryptophan production.

As an important raw material for pharmaceutical, food and feed industry, highly efficient production of L-tryptophan by Escherichia coli has attracted a considerable attention. However, there are complicated and multiple layers of regulation networks in L-tryptophan biosynthetic pathway and thus have difficulty to rewrite the biosynthetic pathway for producing L-tryptophan with high efficiency in E. coli. This review summarizes the biosynthetic pathway of L-tryptophan and highlights the main regulatory mechanisms in E. coli. In addition, we discussed the latest metabolic engineering strategies achieved in E. coli to reconstruct the L-tryptophan biosynthetic pathway. Moreover, we also review a few strategies that can be used in E. coli to improve robustness and streamline of L-tryptophan high-producing strains. Lastly, we also propose the potential strategies to further increase L-tryptophan production by systematic metabolic engineering and synthetic biology techniques.

Efficient breeding of industrial brewing yeast strains using CRISPR/Cas9-aided mating-type switching.

Yeast breeding is a powerful tool for developing and improving brewing yeast in a number of industry-relevant respects. However, breeding of industrial brewing yeast can be challenging, as strains are typically sterile and have large complex genomes. To facilitate breeding, we used the CRISPR/Cas9 system to generate double-stranded breaks in the MAT locus, generating transformants with a single specified mating type. The single mating type remained stable even after loss of the Cas9 plasmid, despite the strains being homothallic, and these strains could be readily mated with other brewing yeast transformants of opposite mating type. As a proof of concept, we applied this technology to generate yeast hybrids with an aim to increase ß-lyase activity for fermentation of beer with enhanced hop flavour. First, a genetic and phenotypic pre-screening of 38 strains was carried out in order to identify potential parent strains with high ß-lyase activity. Mating-competent transformants of eight parent strains were generated, and these were used to generate over 60 hybrids that were screened for ß-lyase activity. Selected phenolic off-flavour positive (POF +) hybrids were further sporulated to generate meiotic segregants with high ß-lyase activity, efficient wort fermentation, and lack of POF, all traits that are desirable in strains for the fermentation of modern hop-forward beers. Our study demonstrates the power of combining the CRISPR/Cas9 system with classic yeast breeding to facilitate development and diversification of brewing yeast. KEY POINTS: • CRISPR/Cas9-based mating-type switching was applied to industrial yeast strains. • Transformed strains could be readily mated to form intraspecific hybrids. • Hybrids exhibited heterosis for a number of brewing-relevant traits.

Novel mutagenesis and screening technologies for food microorganisms: advances and prospects.

Microorganisms are indispensable in the food industry, but wild-type strains hardly meet the current industrial demands due to several undesirable traits. Therefore, microbial strain improvement offers a critical solution to enhance the food industry. Traditional techniques for food microbial improvement, such as the use of chemical mutagens and manual isolation/purification, are inefficient, time-consuming, and laborious, restricting further progress in the area of food fermentation. In this review, the applications of novel mutagenesis and screening technologies used for the improvement of food microbes were summarized, including random mutagenesis based on physical irradiation, microbial screening facilitated by a microtiter plate, fluorescence-activated cell or droplet sorting, and microscaled fermentation in a microtiter plate or microbioreactor. In comparison with conventional methods, these new tools have the potential in accelerating microbial strain improvement and their combined applications could create a new trend for strain development. However, several problems that could affect its potential application may include the following: the lack of specific mutagenesis devices and biosensing systems, the insufficient improvement of the mixed culture system, the low efficiency when using filamentous fungi and flocculating bacteria, and the insufficient safety assessment on harnessing genome-editing technology. Therefore, future works on strain improvement remain challenging for the food industry.

Genome shuffling improves pigment and other bioactive compound production in Monascus purpureus.

Monascus purpureus, a pigment-producing ascomycetous fungus, has been traditionally used for red rice preparation using solid-state fermentation. The objective of this study was to develop an improved pigment-producing strain of M. purpureus MTCC 1090 through genome shuffling followed by detailed analytical estimations of pigments and other bioactive compounds produced by the fusant. Protoplast formation was optimum with 12 h-old mycelia incubated at 30 °C with cellulase, lyticase, and chitinase (40:1:1) for 5 h. Four UV-induced mutants that produced 13.1-39.5% higher amount of yellow, orange, and red pigments in fermented low-grade (cheap) broken rice were used as parents for genome shuffling. After the first round of fusion, four fusants with 35.9-60.52% higher pigment production capabilities were fused again, and finally the fusant F2-19 with distinct culture characteristic was selected under multi-selection pressure. It consistently produced 67%, 70%, and 76% higher content of yellow, orange, and red pigments respectively as compared to the wild-type. High-performance liquid chromatography (HPLC) analysis also reveals clear variation in pigment productions between wild-type and the fusant. Furthermore, HPLC analysis of F2-19 fermented rice extract confirms the production of 186 ± 8.71 and 3810 ± 29.81 mg/kg mevinolin and gamma-aminobutyric acid respectively. Citrinin was not detected. F2-19 fermented rice also has high antioxidant activity (7.92 ± 0.32 mg/g trolox equivalent), with good amount of phenolics (18.0 ± 0.95 mg/g gallic acid equivalent) and flavonoids (2.7 ± 0.26 mg/g quercetin equivalent). Thus, genome shuffling was successfully implemented on M. purpureus for the first time to develop a citrinin-free, better-performing fusant that holds future biotechnological potential. KEY POINTS: • Genome shuffling was performed by recursive protoplast fusion in Monascus purpureus. • The selected fusant, F2-19, was used in solid-state fermentation using low-grade rice. • It produced 67-76% higher content of yellow, orange, and red pigments than the wild-type. • HPLC detected 186 mg/kg mevinolin and 3810 mg/kg γ-aminobutyric acid, but no citrinin. • F2-19 shows high antioxidant activity with good amount of phenolics and flavonoids. Graphical abstract.

Exploring industrial and natural Saccharomyces cerevisiae strains for the bio-based economy from biomass: the case of bioethanol.

Saccharomyces cerevisiae is the preferred microorganism for the production of bioethanol from biomass. Industrial strain development for first-generation ethanol from sugar cane and corn mostly relies on the historical know-how from high gravity beer brewing and alcohol distilleries. However, the recent design of yeast platforms for the production of second-generation biofuels and green chemicals from lignocellulose exposes yeast to different environments and stress challenges. The industrial need for increased productivity, wider substrate range utilization, and the production of novel compounds leads to renewed interest in further extending the use of current industrial strains by exploiting the immense, and still unknown, potential of natural yeast strains. This review describes key metabolic engineering strategies tailored to develop efficient industrial and novel natural yeast strains towards bioethanol production from biomass. Furthermore, it shapes how proof-of-concept studies, often advanced in academic settings on natural yeast, can be upgraded to meet the requirements for industrial applications. Academic and industrial research should continue to cooperate on both improving existing industrial strains and developing novel phenotypes by exploring the vast biodiversity available in nature on the road to establish yeast biorefineries where a range of biomass substrates are converted into valuable compounds.

Metabolic engineering of l-leucine production in Escherichia coli and Corynebacterium glutamicum: a review.

l-Leucine, as an essential branched-chain amino acid for humans and animals, has recently been attracting much attention because of its potential for a fast-growing market demand. The applicability ranges from flavor enhancers, animal feed additives and ingredients in cosmetic to specialty nutrients in pharmaceutical and medical fields. Microbial fermentation is the major method for producing l-leucine by using Escherichia coli and Corynebacterium glutamicum as host bacteria. This review gives an overview of the metabolic pathway of l-leucine (i.e. production, import and export systems) and highlights the main regulatory mechanisms of operons in E. coli and C. glutamicum l-leucine biosynthesis. We summarize here the current trends in metabolic engineering techniques and strategies for manipulating l-leucine producing strains. Finally, future perspectives to construct industrially advantageous strains are considered with respect to recent advances in biology.

Biotechnology in ciliates: an overview.

Since their description and classification in the 19th century, ciliates have played an important role in science, leading to several fundamental discoveries in the areas of cellular and molecular biology. During the last decades, with the emergence of biotechnology, many new developments are also coming to light. In this review, we describe a range of applications in which ciliates have found a niche, ranging from the production of a vast array of proteins, lipids, metabolites, and antigens to their use in toxicity screening, biocontrol, bioremediation, and biotransformation of substrates into more valuable products. We highlight the benefits and drawbacks of their use in biotechnology, the latest developments in large-scale culture and state-of-the-art molecular-genetic techniques, as well as the estimations on the exploitation areas with better potential, i.e., the production of complex membrane proteins, and those less interesting or with less chances of success.

Experimental evolution: its principles and applications in developing stress-tolerant yeasts.

Stress tolerance and resistance in industrial yeast strains are important attributes for cost-effective bioprocessing. The source of stress-tolerant yeasts ranges from extremophilic environments to laboratory engineered strains. However, industrial stress-tolerant yeasts are very rare in nature as the natural environment forces them to evolve traits that optimize survival and reproduction and not the ability to withstand harsh habitat-irrelevant industrial conditions. Experimental evolution is a frequent method used to uncover the mechanisms of evolution and microbial adaption towards environmental stresses. It optimizes biological systems by means of adaptation to environmental stresses and thus has immense power of development of robust stress-tolerant yeasts. This mini-review briefly outlines the basics and implications of evolution experiments and their applications to industrial biotechnology. This work is meant to serve as an introduction to those new to the field of experimental evolution, and as a guide to biologists working in the field of yeast stress response. Future perspectives of experimental evolution for potential biotechnological applications have also been elucidated.

Strategy for improving L-isoleucine production efficiency in Corynebacterium glutamicum.

As one of the three branched-chain amino acids essential for human body, L-isoleucine is widely used in food, medicine, and feed industries. At present, L-isoleucine is mainly produced by microbial fermentation, and the main production strain is Corynebacterium glutamicum. The biosynthetic pathway of L-isoleucine in C. glutamicum is complex, and the activity of key enzymes and the transcription of key genes in the pathway are strictly regulated. The intracellularly synthesized L-isoleucine is secreted by transporters, and the activity of the transporters is also regulated. These intricate regulatory mechanisms increase the difficulty to engineer the L-isoleucine-producing C. glutamicum. This article focuses on the mechanism of L-isoleucine biosynthesis, secretion, and regulation in C. glutamicum and reviews the various metabolic engineering strategies for improving L-isoleucine production efficiency in C. glutamicum.

Constraint-based modeling in microbial food biotechnology.

Genome-scale metabolic network reconstruction offers a means to leverage the value of the exponentially growing genomics data and integrate it with other biological knowledge in a structured format. Constraint-based modeling (CBM) enables both the qualitative and quantitative analyses of the reconstructed networks. The rapid advancements in these areas can benefit both the industrial production of microbial food cultures and their application in food processing. CBM provides several avenues for improving our mechanistic understanding of physiology and genotype-phenotype relationships. This is essential for the rational improvement of industrial strains, which can further be facilitated through various model-guided strain design approaches. CBM of microbial communities offers a valuable tool for the rational design of defined food cultures, where it can catalyze hypothesis generation and provide unintuitive rationales for the development of enhanced community phenotypes and, consequently, novel or improved food products. In the industrial-scale production of microorganisms for food cultures, CBM may enable a knowledge-driven bioprocess optimization by rationally identifying strategies for growth and stability improvement. Through these applications, we believe that CBM can become a powerful tool for guiding the areas of strain development, culture development and process optimization in the production of food cultures. Nevertheless, in order to make the correct choice of the modeling framework for a particular application and to interpret model predictions in a biologically meaningful manner, one should be aware of the current limitations of CBM.

Random mutagenesis of super Koji (Aspergillus oryzae): improvement in production and thermal stability of α-amylases for maltose syrup production.

BACKGROUND: Alpha-amylases hydrolyze 1,4 α-glycosidic bonds of starch and produce malto-oligosaccharides. It is an important enzyme generally applied in textile, food and brewing industries. Enhancement in thermal stability and productivity of enzymes are the two most sought after properties for industrial use. The Aspergillus oryzae (Koji) has Generally Recognized as Safe (GRAS) status and safe for use in food industry. Hence, Koji strain's development for the screening of potent mutants, hyper producer of thermostable α-amylases, with desired attributes is the need of the time. RESULTS: A process has been developed to improve super Koji (A. oryzae cmc1) strain through γ-rays treatment. The doses i.e. 0.60, 0.80, 1.00, 1.20 & 1.40 KGy gave more than 3.0 log kill. Initially, 52 Koji mutants resistant to 1% (w/v) Triton X-100 were selected. 2nd screening was based on α-amylases hyper production and 23 mutants were sorted out by measuring clearing zones index (CI). Afterwards nine potent mutants, resistant to 2-deoxy D-glucose, were screened based on CI. These were further analyzed for thermal stability and productivity of α-amylase under submerged conditions. The mutants' M-80(10), M-100(6) & M-120(5) gave about four fold increases in α-amylases productivity. The half life of M-100(6) α-amylase at 55 °C was 52 min and was highest among the mutants. Liquid Chromatography-Mass Spectrometry (LC-MS) analysis confirmed that mutants did not produce aflatoxins. Field Emission Scanning Electron Microscopy (FESEM) of Koji mycelia depicted that exposure to gamma rays increased rigidity of the mycelium. The potent Koji mutant M-100(6) was grown on soluble starch in 10L fermenter and produced 40.0 IU ml-1 of α-amylases with specific activity of 2461 IU mg-1 protein. Growth kinetic parameters were: µ = Specific growth rate= 0.069 h-1, td = Biomass doubling time= 10.0 h, Yp/x = Product yield coefficient with respect to cell mass = 482 U g-1; qp= Specific rate of product formation= 33.29 U g-1 h-1. CONCLUSION: It was concluded that the developed five step screening process has great potential to generate potent mutants for the hyper production of thermostable enzymes through γ-rays mediated physical mutagenesis. The developed thermostable α-amylases of super Koji mutantM-100(6) has immense potential for application in saccharification process for maltose syrup production. Moreover, the developed five step strain's development process may be used for the simultaneous improvement in productivity and thermal stability of other microbial enzymes.

Evaluation of promoter sequences for the secretory production of a Clostridium thermocellum cellulase in Paenibacillus polymyxa.

Due to their high secretion capacity, Gram-positive bacteria from the genus Bacillus are important expression hosts for the high-yield production of enzymes in industrial biotechnology; however, to date, strains from only few Bacillus species are used for enzyme production at industrial scale. Herein, we introduce Paenibacillus polymyxa DSM 292, a member of a different genus, as a novel host for secretory protein production. The model gene cel8A from Clostridium thermocellum was chosen as an easily detectable reporter gene with industrial relevance to demonstrate heterologous expression and secretion in P. polymyxa. The yield of the secreted cellulase Cel8A protein was increased by optimizing the expression medium and testing several promoter sequences in the expression plasmid pBACOV. Quantitative mass spectrometry was used to analyze the secretome in order to identify promising new promoter sequences from the P. polymyxa genome itself. The most abundantly secreted host proteins were identified, and the promoters regulating the expression of their corresponding genes were selected. Eleven promoter sequences were cloned and tested, including well-characterized promoters from Bacillus subtilis and Bacillus megaterium. The best result was achieved with the promoter for the hypothetical protein PPOLYM_03468 from P. polymyxa. In combination with the optimized expression medium, this promoter enabled the production of 5475 U/l of Cel8A, which represents a 6.2-fold increase compared to the reference promoter PaprE. The set of promoters described in this work covers a broad range of promoter strengths useful for heterologous expression in the new host P. polymyxa.

Production of L-valine from metabolically engineered Corynebacterium glutamicum.

L-Valine is one of the three branched-chain amino acids (valine, leucine, and isoleucine) essential for animal health and important in metabolism; therefore, it is widely added in the products of food, medicine, and feed. L-Valine is predominantly produced through microbial fermentation, and the production efficiency largely depends on the quality of microorganisms. In recent years, continuing efforts have been made in revealing the mechanisms and regulation of L-valine biosynthesis in Corynebacterium glutamicum, the most utilitarian bacterium for amino acid production. Metabolic engineering based on the metabolic biosynthesis and regulation of L-valine provides an effective alternative to the traditional breeding for strain development. Industrially competitive L-valine-producing C. glutamicum strains have been constructed by genetically defined metabolic engineering. This article reviews the global metabolic and regulatory networks responsible for L-valine biosynthesis, the molecular mechanisms of regulation, and the strategies employed in C. glutamicum strain engineering.

Recent advances in microbial production of mannitol: utilization of low-cost substrates, strain development and regulation strategies.

Mannitol has been widely used in fine chemicals, pharmaceutical industries, as well as functional foods due to its excellent characteristics, such as antioxidant protecting, regulation of osmotic pressure and non-metabolizable feature. Mannitol can be naturally produced by microorganisms. Compared with chemical manufacturing, microbial production of mannitol provides high yield and convenience in products separation; however the fermentative process has not been widely adopted yet. A major obstacle to microbial production of mannitol under industrial-scale lies in the low economical efficiency, owing to the high cost of fermentation medium, leakage of fructose, low mannitol productivity. In this review, recent advances in improving the economical efficiency of microbial production of mannitol were reviewed, including utilization of low-cost substrates, strain development for high mannitol yield and process regulation strategies for high productivity.

Yeast-bacteria competition induced new metabolic traits through large-scale genomic rearrangements in Lachancea kluyveri.

Large-scale chromosomal rearrangements are an important source of evolutionary novelty that may have reshaped the genomes of existing yeast species. They dramatically alter genome organization and gene expression fueling a phenotypic leap in response to environmental constraints. Although the emergence of such signatures of genetic diversity is thought to be associated with human exploitation of yeasts, less is known about the driving forces operating in natural habitats. Here we hypothesize that an ecological battlefield characteristic of every autumn when fruits ripen accounts for the genomic innovations in natural populations. We described a long-term cross-kingdom competition experiment between Lachancea kluyveri and five species of bacteria. Now, we report how we further subjected the same yeast to a sixth species of bacteria, Pseudomonas fluorescens, resulting in the appearance of a fixed and stably inherited large-scale genomic rearrangement in two out of three parallel evolution lines. The 'extra-banded' karyotype, characterized by a higher fitness and an elevated fermentative capacity, conferred the emergence of new metabolic traits in most carbon sources and osmolytes. We tracked down the event to a duplication and translocation event involving a 261-kb segment. Such an experimental setup described here is an attractive method for developing industrial strains without genetic engineering strategies.

A new source of resistance to 2-furaldehyde from Scheffersomyces (Pichia) stipitis for sustainable lignocellulose-to-biofuel conversion.

Aldehyde inhibitory compounds derived from lignocellulosic biomass pretreatment have been identified as a major class of toxic chemicals that interfere with microbial growth and subsequent fermentation for advanced biofuel production. Development of robust next-generation biocatalyst is a key for a low-cost biofuel production industry. Scheffersomyces (Pichia) stipitis is a naturally occurring C-5 sugar utilization yeast; however, little is known about the genetic background underlying its potential tolerance to biomass conversion inhibitors. We investigated and identified five uncharacterized putative aryl-alcohol dehydrogenase genes (SsAADs) from this yeast as a new source of resistance against biomass fermentation inhibitor 2-furaldehyde (furfural) by gene expression, gene cloning, and direct enzyme assay analysis using partially purified proteins. All five proteins from S. stipitis showed furfural reduction using cofactor NADH. An optimum active temperature was observed at 40 °C for SsAad1p; 30 °C for SsAad3p, SsAad4p, and SsAad5p; and 20 °C for SsAad2p. SsAad2p, SsAad3p, and SsAad4p showed tolerance to a wide range of pH from 4.5 to 8, but SsAad1p and SsAad5p were sensitive to pH changes beyond 7. Genes SsAAD2, SsAAD3, and SsAAD4 displayed significantly enhanced higher levels of expression in response to the challenge of furfural. Their encoding proteins also showed higher levels of specific activity toward furfural and were suggested as core functional enzymes contributing aldehyde resistance in S. stipitis.

Progress and perspectives on improving butanol tolerance.

Fermentative production of butanol for use as a biofuel or chemical feedstock is regarded as a promising renewable technology that reduces greenhouse gas emissions and has the potential to become a substitute for non-sustainable chemical production route. However, butanol toxicity to the producing microbes remains a barrier to achieving sufficiently high titers for cost-effective butanol fermentation and recovery. Investigations of the external stress of high butanol concentration on butanol-producing microbial strains will aid in developing improved microbes with increased tolerance to butanol. With currently available molecular tool boxes, researchers have aimed to address and understand how butanol affects different microbes. This review will cover the individual organism's inherent responses to surrounding butanol levels, and the collective efforts by researchers to improve production and tolerance. The specific microorganisms discussed here include the native butanol producer Clostridium species, the fermentation industrial model Saccharomyces cerevisiae and the photosynthetic cyanobacteria, the genetic engineering workhorse Escherichia coli, and also the butanol-tolerant lactic acid bacteria that utilize diverse substrates. The discussion will help to understand the physiology of butanol resistance and to identify specific butanol tolerance genes that will lead to informed genetic engineering strategies for new strain development.

Biofuels and bio-based chemicals from lignocellulose: metabolic engineering strategies in strain development.

Interest in developing a sustainable technology for fuels and chemicals has unleashed tremendous creativity in metabolic engineering for strain development over the last few years. This is driven by the exceptionally recalcitrant substrate, lignocellulose, and the necessity to keep the costs down for commodity products. Traditional methods of gene expression and evolutionary engineering are more effectively used with the help of synthetic biology and -omics techniques. Compared to the last biomass research peak during the 1980s oil crisis, a more diverse range of microorganisms are being engineered for a greater variety of products, reflecting the broad applicability and effectiveness of today's gene technology. We review here several prominent and successful metabolic engineering strategies with emphasis on the following four areas: xylose catabolism, inhibitor tolerance, synthetic microbial consortium, and cellulosic oligomer assimilation.