Significant production of vanillin and in vitro amplification of ech gene in local bacterial isolates / Produção significativa de vanilina e amplificação in vitro do gene ech em isolados bacterianos locais

Abstract Vanillin is the major component which is responsible for flavor and aroma of vanilla extract and is produced by 3 ways: natural extraction from vanilla plant, chemical synthesis and from microbial transformation. Current research was aimed to study bacterial production of vanillin from native natural sources including sewage and soil from industrial areas. The main objective was vanillin bio-production by isolating bacteria from these native sources. Also to adapt methodologies to improve vanillin production by optimized fermentation media and growth conditions. 47 soil and 13 sewage samples were collected from different industrial regions of Lahore, Gujranwala, Faisalabad and Kasur. 67.7% bacterial isolates produced vanillin and 32.3% were non-producers. From these 279 producers, 4 bacterial isolates selected as significant producers were; A3, A4, A7 and A10. These isolates were identified by ribotyping as A3 Pseudomonas fluorescence (KF408302), A4 Enterococcus faecium (KT356807), A7 Alcaligenes faecalis (MW422815) and A10 Bacillus subtilis (KT962919). Vanillin producers were further tested for improved production of vanillin and were grown in different fermentation media under optimized growth conditions for enhanced production of vanillin. The fermentation media (FM) were; clove oil based, rice bran waste (residues oil) based, wheat bran based and modified isoeugenol based. In FM5, FM21, FM22, FM23, FM24, FM30, FM31, FM32, FM34, FM35, FM36, and FM37, the selected 4 bacterial strains produced significant amounts of vanillin. A10 B. subtilis produced maximum amount of vanillin. This strain produced 17.3 g/L vanillin in FM36. Cost of this fermentation medium 36 was 131.5 rupees/L. This fermentation medium was modified isoeugenol based medium with 1% of isoeugenol and 2.5 g/L soybean meal. ech gene was amplified in A3 P. fluorescence using ech specific primers. As vanillin use as flavor has increased tremendously, the bioproduction of vanillin must be focused.

Resumo A vanilina é o principal componente responsável pelo sabor e aroma do extrato de baunilha e é produzida de três formas: extração natural da planta da baunilha, síntese química e transformação microbiana. A pesquisa atual teve como objetivo estudar a produção bacteriana de vanilina a partir de fontes naturais nativas, incluindo esgoto e solo de áreas industriais. O objetivo principal era a bioprodução de vanilina por meio do isolamento de bactérias dessas fontes nativas. Também para adaptar metodologias para melhorar a produção de vanilina por meio de fermentação otimizada e condições de crescimento. Foram coletadas 47 amostras de solo e 13 de esgoto de diferentes regiões industriais de Lahore, Gujranwala, Faisalabad e Kasur; 67,7% dos isolados bacterianos produziram vanilina e 32,3% eram não produtores. Desses 279 produtores, 4 isolados bacterianos selecionados como produtores significativos foram: A3, A4, A7 e A10. Esses isolados foram identificados por ribotipagem como fluorescência A3 Pseudomonas (KF408302), A4 Enterococcus faecium (KT356807), A7 Alcaligenes faecalis (MW422815) e A10 Bacillus subtilis (KT962919). Os produtores de vanilina foram posteriormente testados para produção aprimorada de vanilina e foram cultivados em diferentes meios de fermentação sob condições de crescimento otimizadas para produção aprimorada de vanilina. Os meios de fermentação (FM) foram: à base de óleo de cravo, à base de resíduos de farelo de arroz (resíduos de óleo), à base de farelo de trigo e à base de isoeugenol modificado. Em FM5, FM21, FM22, FM23, FM24, FM30, FM31, FM32, FM34, FM35, FM36 e FM37, as 4 cepas bacterianas selecionadas produziram quantidades significativas de vanilina. A10 B. subtilis produziu quantidade máxima de vanilina. Essa cepa produziu 17,3 g / L de vanilina em FM36. O custo desse meio de fermentação 36 foi de 131,5 rúpias / L. Esse meio de fermentação foi um meio à base de isoeugenol modificado com 1% de isoeugenol e 2,5 g / L de farelo de soja. O gene ech foi amplificado em A3 P. fluorescence usando primers específicos para ech. Como o uso da vanilina como sabor aumentou tremendamente, a bioprodução da vanilina deve ser focada.

Multi-omics network model reveals key genes associated with p-coumaric acid stress response in an industrial yeast strain.

The production of ethanol from lignocellulosic sources presents increasingly difficult issues for the global biofuel scenario, leading to increased production costs of current second-generation (2G) ethanol when compared to first-generation (1G) plants. Among the setbacks encountered in industrial processes, the presence of chemical inhibitors from pre-treatment processes severely hinders the potential of yeasts in producing ethanol at peak efficiency. However, some industrial yeast strains have, either naturally or artificially, higher tolerance levels to these compounds. Such is the case of S. cerevisiae SA-1, a Brazilian fuel ethanol industrial strain that has shown high resistance to inhibitors produced by the pre-treatment of cellulosic complexes. Our study focuses on the characterization of the transcriptomic and physiological impact of an inhibitor of this type, p-coumaric acid (pCA), on this strain under chemostat cultivation via RNAseq and quantitative physiological data. It was found that strain SA-1 tend to increase ethanol yield and production rate while decreasing biomass yield when exposed to pCA, in contrast to pCA-susceptible strains, which tend to decrease their ethanol yield and fermentation efficiency when exposed to this substance. This suggests increased metabolic activity linked to mitochondrial and peroxisomal processes. The transcriptomic analysis also revealed a plethora of differentially expressed genes located in co-expressed clusters that are associated with changes in biological pathways linked to biosynthetic and energetical processes. Furthermore, it was also identified 20 genes that act as interaction hubs for these clusters, while also having association with altered pathways and changes in metabolic outputs, potentially leading to the discovery of novel targets for metabolic engineering toward a more robust industrial yeast strain.

Genotypic and phenotypic characterization of industrial autochthonous Saccharomyces cerevisiae for the selection of well-adapted bioethanol-producing strains.

In northwestern Argentina, sugarcane-derived industrial fermentation is being extensively used for bioethanol production, where highly adaptive native strains compete with the baker's yeast Saccharomyces cerevisiae traditionally used as starter culture. Yeast populations of 10 distilleries from Tucumán (Argentina) were genotypic and phenotypic characterized to select well-adapted bioethanol-producing autochthonous strains to be used as starter cultures for the industrial production of bioethanol fuel. From the 192 isolates, 69.8% were identified as S. cerevisiae, 25.5% as non-Saccharomyces, and 4.7% as Saccharomyces sp. wild yeasts. The majority of S. cerevisiae isolates (68.5%) were non-flocculating yeasts, while the flocculating strains were all obtained from the only continuous fermentation process included in the study. Simple Sequence Repeat analysis revealed a high genetic diversity among S. cerevisiae genotypes, where all of them were very different from the original baker's strain used as starter. Among these, 38 strains multi-tolerant to stress by ethanol (8%), temperature (42.5 °C) and pH (2.0) were obtained. No major differences were found among these strains in terms of ethanol production and residual sugars in batch fermentation experiments with cell recycling. However, only 10 autochthonous strains maintained their viability (more than 80%) throughout five consecutive cycles of sugarcane-based fermentations. In summary, 10 autochthonous isolates were found to be superior to baker's yeast used as starter culture (S. cerevisiae Calsa) in terms of optimal technological, physiological and ecological properties. The knowledge generated on the indigenous yeast populations in industrial fermentation processes of bioethanol-producing distilleries allowed the selection of well-adapted bioethanol-producing strains.

Digital models in biotechnology: Towards multi-scale integration and implementation.

Industrial biotechnology encompasses a large area of multi-scale and multi-disciplinary research activities. With the recent megatrend of digitalization sweeping across all industries, there is an increased focus in the biotechnology industry on developing, integrating and applying digital models to improve all aspects of industrial biotechnology. Given the rapid development of this field, we systematically classify the state-of-art modelling concepts applied at different scales in industrial biotechnology and critically discuss their current usage, advantages and limitations. Further, we critically analyzed current strategies to couple cell models with computational fluid dynamics to study the performance of industrial microorganisms in large-scale bioprocesses, which is of crucial importance for the bio-based production industries. One of the most challenging aspects in this context is gathering intracellular data under industrially relevant conditions. Towards comprehensive models, we discuss how different scale-down concepts combined with appropriate analytical tools can capture intracellular states of single cells. We finally illustrated how the efforts could be used to develop digitals models suitable for both cell factory design and process optimization at industrial scales in the future.

Automated Evolutionary Engineering of Yeasts.

Evolutionary engineering of microbes provides a powerful tool for untargeted optimization of (engineered) cell factories and identification of genetic targets for further research. Directed evolution is an intrinsically time-intensive effort, and automated methods can significantly reduce manual labor. Here, design considerations for various evolutionary engineering methods are described, and generic workflows for batch-, chemostat-, and accelerostat-based evolution in automated bioreactors are provided. These methods can be used to evolve yeast cultures for >1000 generations and are designed to require minimal manual intervention.

Metabolic and biotechnological insights on the analysis of the Pdh bypass and acetate production in the yeast Dekkera bruxellensis.

The advancement of knowledge about the physiology of Dekkera bruxellensis has shown its potential for the production of fuel ethanol very close to the conventional fermenting yeast S. cerevisiae. However, some aspects of its metabolism remain uncovered. In the present study, the respiro-fermentative parameters of D. bruxellensis GDB 248 were evaluated under different cultivation conditions. The results showed that sucrose was more efficiently converted to ethanol than glucose, regardless the nitrogen source, which points out for the industrial efficiency of this yeast in sucrose-based substrate. The blockage of the cytosolic acetate production incremented the yeast fermentative efficiency by 27% (in glucose) and 14% (in sucrose). On the other hand, the presence of nitrate as inducer of acetate production reducing the production of ethanol. Altogether, these results settled the hypothesis that acetate metabolism is the main constraint for ethanol production. Besides, this acetate-generating pathway seems to exert some regulatory action on the flux and distribution of the carbon flowing through the central metabolism. These physiological aspects were corroborated by the relative expression analysis of key genes in the crossroad to ethanol, acetate and biomass formation. All the results were discussed in the light of the industrial potential of this yeast.

Quantification of Microbial Robustness in Yeast.

Stable cell performance in a fluctuating environment is essential for sustainable bioproduction and synthetic cell functionality; however, microbial robustness is rarely quantified. Here, we describe a high-throughput strategy for quantifying robustness of multiple cellular functions and strains in a perturbation space. We evaluated quantification theory on experimental data and concluded that the mean-normalized Fano factor allowed accurate, reliable, and standardized quantification. Our methodology applied to perturbations related to lignocellulosic bioethanol production showed that the industrial bioethanol producing strain Saccharomyces cerevisiae Ethanol Red exhibited both higher and more robust growth rates than the laboratory strain CEN.PK and industrial strain PE-2, while a more robust product yield traded off for lower mean levels. The methodology validated that robustness is function-specific and characterized by positive and negative function-specific trade-offs. Systematic quantification of robustness to end-use perturbations will be important to analyze and construct robust strains with more predictable functions.

Mandarin essential oil as an antimicrobial in ethanolic fermentation: Effects on Limosilactobacillus fermentum and Saccharomyces cerevisiae.

The antibacterial activity of citrus essential oils (EOs) in the context of combating Limosilactobacillus fermentum, one of the most important bacterial contaminants in the bioethanol production industry, has never been explored previously. Industrial processes usually utilize sulfuric acid for cell treatment to decrease bacterial contamination. However, due to the hazardous nature of sulfuric acid, an alternative to it is highly desirable. Therefore, in the present study, the efficacy of Fremont IAC 543 mandarin EO against a strain of L. fermentum (ATCC® 9338™) was evaluated under proliferative/nonproliferative conditions, in both pure culture and co-culture with an industrial strain of Saccharomyces cerevisiae. The mandarin EO exhibited higher effectiveness against L. fermentum compared to that against S. cerevisiae under nonproliferative conditions (added to water rather than to culture medium). At the concentration of 0·05%, the EO was as effective as the acid solution with pH 2·0 in reducing the count of L. fermentum almost 5 log CFU ml-1 cycles, while the concentration of 0·1% led to the complete loss of bacterial culturability. When L. fermentum was co-cultured with S. cerevisiae, the efficacy of the EO against the bacterial strain was reduced. However, despite this reduced efficacy in co-culture, mandarin EO may be considered effective in combating L. fermentum and could be applied in processes where this bacterium proves to be unfavourable and does not interact with S. cerevisiae.

High-level production of the agmatine in engineered Corynebacterium crenatum with the inhibition-releasing arginine decarboxylase.

BACKGROUND: Agmatine is a member of biogenic amines and is an important medicine which is widely used to regulate body balance and neuroprotective effects. At present, the industrial production of agmatine mainly depends on the chemical method, but it is often accompanied by problems including cumbersome processes, harsh reaction conditions, toxic substances production and heavy environmental pollution. Therefore, to tackle the above issues, arginine decarboxylase was overexpressed heterologously and rationally designed in Corynebacterium crenatum to produce agmatine from glucose by one-step fermentation. RESULTS: In this study, we report the development in the Generally Regarded as Safe (GRAS) L-arginine-overproducing C. crenatum for high-titer agmatine biosynthesis through overexpressing arginine decarboxylase based on metabolic engineering. Then, arginine decarboxylase was mutated to release feedback inhibition and improve catalytic activity. Subsequently, the specific enzyme activity and half-inhibitory concentration of I534D mutant were increased 35.7% and 48.1%, respectively. The agmatine production of the whole-cell bioconversion with AGM3 was increased by 19.3% than the AGM2. Finally, 45.26 g/L agmatine with the yield of 0.31 g/g glucose was achieved by one-step fermentation of the engineered C. crenatum with overexpression of speAI534D. CONCLUSIONS: The engineered C. crenatum strain AGM3 in this work was proved as an efficient microbial cell factory for the industrial fermentative production of agmatine. Based on the insights from this work, further producing other valuable biochemicals derived from L-arginine by Corynebacterium crenatum is feasible.

Robustness: linking strain design to viable bioprocesses.

Microbial cell factories are becoming increasingly popular for the sustainable production of various chemicals. Metabolic engineering has led to the design of advanced cell factories; however, their long-term yield, titer, and productivity falter when scaled up and subjected to industrial conditions. This limitation arises from a lack of robustness - the ability to maintain a constant phenotype despite the perturbations of such processes. This review describes predictable and stochastic industrial perturbations as well as state-of-the-art technologies to counter process variability. Moreover, we distinguish robustness from tolerance and discuss the potential of single-cell studies for improving system robustness. Finally, we highlight ways of achieving consistent and comparable quantification of robustness that can guide the selection of strains for industrial bioprocesses.

Blocking Mitophagy Does Not Significantly Improve Fuel Ethanol Production in Bioethanol Yeast Saccharomyces cerevisiae.

Ethanolic fermentation is frequently performed under conditions of low nitrogen. In Saccharomyces cerevisiae, nitrogen limitation induces macroautophagy, including the selective removal of mitochondria, also called mitophagy. Previous research showed that blocking mitophagy by deletion of the mitophagy-specific gene ATG32 increased the fermentation performance during the brewing of Ginjo sake. In this study, we tested if a similar strategy could enhance alcoholic fermentation in the context of fuel ethanol production from sugarcane in Brazilian biorefineries. Conditions that mimic the industrial fermentation process indeed induce Atg32-dependent mitophagy in cells of S. cerevisiae PE-2, a strain frequently used in the industry. However, after blocking mitophagy, no significant differences in CO2 production, final ethanol titers, or cell viability were observed after five rounds of ethanol fermentation, cell recycling, and acid treatment, which is commonly performed in sugarcane biorefineries. To test if S. cerevisiae's strain background influenced this outcome, cultivations were carried out in a synthetic medium with strains PE-2, Ethanol Red (industrial), and BY (laboratory) with and without a functional ATG32 gene and under oxic and oxygen restricted conditions. Despite the clear differences in sugar consumption, cell viability, and ethanol titers, among the three strains, we did not observe any significant improvement in fermentation performance related to the blocking of mitophagy. We concluded, with caution, that the results obtained with Ginjo sake yeast were an exception and cannot be extrapolated to other yeast strains and that more research is needed to ascertain the role of autophagic processes during fermentation. IMPORTANCE Bioethanol is the largest (per volume) ever biobased bulk chemical produced globally. The fermentation process is well established, and industries regularly attain nearly 85% of maximum theoretical yields. However, because of the volume of fuel produced, even a small improvement will have huge economic benefits. To this end, besides already implemented process improvements, various free energy conservation strategies have been successfully exploited at least in laboratory strains to increase ethanol yields and decrease byproduct formation. Cellular housekeeping processes have been an almost unexplored territory in strain improvement. It was previously reported that blocking mitophagy by deletion of the mitophagy receptor gene ATG32 in Saccharomyces cerevisiae led to a 2.1% increase in final ethanol titers during Japanese sake fermentation. We found in two commercially used bioethanol strains (PE-2 and Ethanol Red) that ATG32 deficiency does not lead to a significant improvement in cell viability or ethanol levels during fermentation with molasses or in a synthetic complete medium. More research is required to ascertain the role of autophagic processes during fermentation conditions.

Machine learning-informed and synthetic biology-enabled semi-continuous algal cultivation to unleash renewable fuel productivity.

Algal biofuel is regarded as one of the ultimate solutions for renewable energy, but its commercialization is hindered by growth limitations caused by mutual shading and high harvest costs. We overcome these challenges by advancing machine learning to inform the design of a semi-continuous algal cultivation (SAC) to sustain optimal cell growth and minimize mutual shading. An aggregation-based sedimentation (ABS) strategy is then designed to achieve low-cost biomass harvesting and economical SAC. The ABS is achieved by engineering a fast-growing strain, Synechococcus elongatus UTEX 2973, to produce limonene, which increases cyanobacterial cell surface hydrophobicity and enables efficient cell aggregation and sedimentation. SAC unleashes cyanobacterial growth potential with 0.1 g/L/hour biomass productivity and 0.2 mg/L/hour limonene productivity over a sustained period in photobioreactors. Scaling-up the SAC with an outdoor pond system achieves a biomass yield of 43.3 g/m2/day, bringing the minimum biomass selling price down to approximately $281 per ton.

Application of Corynebacterium glutamicum engineering display system in three generations of biorefinery.

The fermentation production of platform chemicals in biorefineries is a sustainable alternative to the current petroleum refining process. The natural advantages of Corynebacterium glutamicum in carbon metabolism have led to C. glutamicum being used as a microbial cell factory that can use various biomass to produce value-added platform chemicals and polymers. In this review, we discussed the use of C. glutamicum surface display engineering bacteria in the three generations of biorefinery resources, and analyzed the C. glutamicum engineering display system in degradation, transport, and metabolic network reconstruction models. These engineering modifications show that the C. glutamicum engineering display system has great potential to become a cell refining factory based on sustainable biomass, and further optimizes the inherent properties of C. glutamicum as a whole-cell biocatalyst. This review will also provide a reference for the direction of future engineering transformation.

Biotechnology approach using watermelon rind for optimization of α-amylase enzyme production from Trichoderma virens using response surface methodology under solid-state fermentation.

Production of amylases by fungi under solid-state fermentation is considered the best methodology for commercial scaling that addresses the ever-escalating needs of the worldwide enzyme market. Here response surface methodology (RSM) was used for the optimization of process variables for α-amylase enzyme production from Trichoderma virens using watermelon rinds (WMR) under solid-state fermentation (SSF). The statistical model included four variables, each detected at two levels, followed by model development with partial purification and characterization of α-amylase. The partially purified α-amylase was characterized with regard to optimum pH, temperature, kinetic constant, and substrate specificity. The results indicated that both pH and moisture content had a significant effect (P < 0.05) on α-amylase production (880 U/g) under optimized process conditions at a 3-day incubation time, moisture content of 50%, 30 °C, and pH 6.98. Statistical optimization using RSM showed R2 values of 0.9934, demonstrating the validity of the model. Five α-amylases were separated by using DEAE-Sepharose and characterized with a wide range of optimized pH values (pH 4.5-9.0), temperature optima (40-60 °C), low Km values (2.27-3.3 mg/mL), and high substrate specificity toward large substrates. In conclusion, this study presents an efficient and green approach for utilization of agro-waste for production of the valuable α-amylase enzyme using RSM under SSF. RSM was particularly beneficial for the optimization and analysis of the effective process parameters.

Applicability and information value of biocalorimetry for the monitoring of fungal solid-state fermentation of lignocellulosic agricultural by-products.

The applicability of biocalorimetry for monitoring fungal conversion of lignocellulosic agricultural by-products during solid-state fermentation (SSF) was substantiated through linking the non-invasive measurement of metabolic heat fluxes to conventional invasive determination of fungal activity (growth, substrate degradation, enzyme activity) parameters. For this, the fast-growing, cellulose-utilising ascomycete Stachybotrys chlorohalonata and the comparatively slow-growing litter-decay basidiomycete Stropharia rugosoannulata were investigated as model organisms during growth on solid wheat straw. Both biocalorimetric and non-calorimetric data may suggest R (ruderal)- and C (combative)-selected life history strategies in S. chlorohalonata and S. rugosoannulata, respectively. For both species, a strong linear correlation of the released metabolic heat with the corresponding fungal biomass was observed. Species-specific YQ/X values (metabolic heat released per fungal biomass unit) were obtained, which potentially enable use of biocalorimetric signals for the quantification of fungal biomass during single-species SSF processes. Moreover, YQ/X values may also indicate different fungal life history strategies and therefore be considered as useful parameters aiding fungal ecology research.

A multi-omics study to boost continuous bolaform sophorolipid production.

Biodegradable and biobased surface active agents are renewable and environmentally friendly alternatives to petroleum derived or oleochemical surfactants. However, they are accompanied by relatively high production costs. In this study, the aim was to reduce the production costs for an innovative type of microbial biosurfactant: bolaform sophorolipids, produced by the yeast Starmerella bombicola ΔsbleΔat. A novel continuous retentostat set-up was performed whereby continuous broth microfiltration retained the biomass in the bioreactor while performing an in situ product separation of bolaform sophorolipids. Although a mean volumetric productivity of 0.56 g L-1 h-1 was achieved, it was not possible to maintain this productivity, which collapsed to almost 0 g L-1 h-1. Therefore, two process adaptations were evaluated, a sequential batch strategy and a phosphate limitation alleviation strategy. The sequential batch set-up restored the mean volumetric productivity to 0.66 g L-1 h-1 for an additional 132 h but was again followed by a productivity decline. A similar result was obtained with the phosphate limitation alleviation strategy where a mean volumetric productivity of 0.54 g L-1 h-1 was reached, but a productivity decline was also observed. Whole genome variant analysis uncovered no evidence for genomic variations for up to 1306 h of retentostat cultivation. Untargeted metabolomics analysis identified 8-hydroxyguanosine, a biomarker for oxidative RNA damage, as a key metabolite correlating with high bolaform sophorolipid productivity. This study showcases the application of a retentostat to increase bolaform sophorolipid productivity and lays the basis of a multi-omics platform for in depth investigation of microbial biosurfactant production with S. bombicola.

The impact of biomass withdrawal strategy on the biomass selection and polyhydroxyalkanoates accumulation of mixed microbial cultures.

The production of polyhydroxyalkanoates (PHA) by mixed microbial cultures (MMC) has been studied as an alternative to pure cultures in order to reduce the price of PHA through use of open systems and low-cost substrates, such as agro-industrial sub-products. However, the widespread applicability of this process depends on the optimization of operational factors impacting PHA productivity. This study addresses the impact of biomass withdrawal strategy on the performance of MMC selection reactors and consequently on biomass productivity and global PHA productivity. Two selection reactors were operated in parallel under similar conditions, except for the timing of biomass withdrawal, at the end of the famine phase (Reactor 1, R1) versus at the end of the feast phase (Reactor 2, R2) at an organic loading rate of 100 Cmmol.L-1.d-1 and solids retention time of 4 days. The biomass selected in both conditions had similar PHA storing capacity as shown by the similar yields of PHA per substrate obtained in the accumulation assays; however, R1 reached a higher biomass productivity (about 4-fold higher than R2). This study demonstrated that removing the excess biomass at the end of the famine phase resulted in a much higher global PHA productivity and that the key parameter affecting the global PHA productivity of the 2-stage system was the volumetric biomass productivity. Results obtained provide important insight into how MMC systems can be best operated to maximize PHA productivity.

Fucoxanthin and docosahexaenoic acid production by cold-adapted Tisochrysis lutea.

Tisochrysis lutea is an important microalgal species for fucoxanthin and docosahexaenoic acid (DHA) production with an optimum cultivation temperature of approximately 30 °C. The aim of the present work was to develop a winter strain with high productivity at 15 °C. The response of the original strain to a decrease in temperature from 30 °C to 15 °C was investigated in continuous turbidostat experiments. This was followed by adaptation for >180 days at 15 °C and 2 rounds of sorting for cells with high chlorophyll fluorescence (top 5%) using fluorescence-activated cell sorting (FACS). For the original strain the productivity of biomass, fucoxanthin, and DHA decreased by 92 %, 98 % and 85 % respectively when decreasing the temperature from 30 °C to 15 °C. In the sorted cold-adapted 'winter strain', biomass, fucoxanthin, and DHA productivities were similar to those at 30 °C. In addition, the fucoxanthin concentration increased from 1.11 to 4.24 mg g-1 dry weight and the polar lipid fraction in total fatty acids increased from 21 % to 55 %. The winter strain showed a robust and stable phenotype after one year of cultivation, expanding the outdoor fucoxanthin and lipid production seasons for this species.

Physiological limitations and opportunities in microbial metabolic engineering.

Metabolic engineering can have a pivotal role in increasing the environmental sustainability of the transportation and chemical manufacturing sectors. The field has already developed engineered microorganisms that are currently being used in industrial-scale processes. However, it is often challenging to achieve the titres, yields and productivities required for commercial viability. The efficiency of microbial chemical production is usually dependent on the physiological traits of the host organism, which may either impose limitations on engineered biosynthetic pathways or, conversely, boost their performance. In this Review, we discuss different aspects of microbial physiology that often create obstacles for metabolic engineering, and present solutions to overcome them. We also describe various instances in which natural or engineered physiological traits in host organisms have been harnessed to benefit engineered metabolic pathways for chemical production.

A fattening factor to quantify the accumulation ability of microorganisms under N-starvation.

Many microorganisms can accumulate biomass in the form of lipids and polysaccharides, which can be used for biofuels, bioplastics, food and feed. Some innovative bioprocesses exploit the competitive advantage provided by such accumulation ability, mainly under N-starvation, to select high-accumulating strains against biological contaminants, by using uncoupled nutrient feeding. However, there is no general and easily comparable parameter available to compare biomass accumulation ability among different microbial strains, which could measure the competitive advantage. Here, a parameter termed "fattening factor" (ηx) is described to quantify such strain-specific biomass accumulation ability in bacteria, yeasts and microalgae. This parameter measures how many fold a microbial population can increase its biomass just as the result of accumulation. It is derived from considerations about the main metabolic aspects of cells' response to N-starvation, which induces variations in cell cycle, biomass production and biochemical composition. The fattening factor described here should be easily estimatable in N-starvation for every culturable microbial strain, by measuring the amount of accumulated biomass.