Effects of pleiotropic ompR and envZ alleles of Escherichia coli on envelope stress and antibiotic sensitivity.

The EnvZ-OmpR two-component system of Escherichia coli regulates the expression of the ompF and ompC porin genes in response to medium osmolarity. However, certain mutations in envZ confer pleiotropy by affecting the expression of genes of the iron and maltose regulons not normally controlled by EnvZ-OmpR. In this study, we obtained two novel envZ and ompR pleiotropic alleles, envZT15P and ompRL19Q, among revertants of a mutant with heightened envelope stress and an outer membrane (OM) permeability defect. Unlike envZ, pleiotropic mutations in ompR have not been described previously. The mutant alleles reduced the expression of several outer membrane proteins (OMPs), overcame the temperature-sensitive growth defect of a protease-deficient (ΔdegP) strain, and lowered envelope stress and OM permeability defects in a background lacking the BamB protein of an essential ß-barrel assembly machinery complex. Biochemical analysis showed OmpRL19Q, like wild-type OmpR, is readily phosphorylated by EnvZ, but the EnvZ-dependent dephosphorylation of OmpRL19Q~P was drastically impaired compared to wild-type OmpR. This defect would lead to a prolonged half-life for OmpRL19Q~P, an outcome remarkably similar to what we had previously described for EnvZR397L, resulting in pleiotropy. By employing null alleles of the OMP genes, it was determined that the three pleiotropic alleles lowered envelope stress by reducing OmpF and LamB levels. The absence of LamB was principally responsible for lowering the OM permeability defect, as assessed by the reduced sensitivity of a ΔbamB mutant to vancomycin and rifampin. Possible mechanisms by which novel EnvZ and OmpR mutants influence EnvZ-OmpR interactions and activities are discussed.IMPORTANCEMaintenance of the outer membrane (OM) integrity is critical for the survival of Gram-negative bacteria. Several envelope homeostasis systems are activated when OM integrity is perturbed. Through the isolation and characterization of novel pleiotropic ompR/envZ alleles, this study highlights the involvement of the EnvZ-OmpR two-component system in lowering envelope stress and the OM permeability defect caused by the loss of proteins that are involved in OM biogenesis, envelope homeostasis, and structural integrity.

Effect of nitrogen management in cultivation on the stability and microbial community of post-harvest Monoraphidium sp. algae biomass.

Long-term storage is necessary to mitigate for seasonal variation in algae productivity, to preserve biomass quality and to guarantee a constant biomass supply to a conversion facility. While ensiling has shown promise as a solution, biomass attributes for successful storage are poorly understood. Storage studies of Monoraphidium sp. biomass indicate a strong correlation between nitrogen management in algae cultivation and stability of post-harvest algae biomass. Algae cultivated with periodic nitrogen addition were stored poorly (>20% loss, dry basis) compared to biomass from nitrogen depleted cultivation (8% loss, dry basis). A follow-up study compared the post-harvest stability of Monoraphidium biomass cultivated in nitrogen-deplete or nitrogen-replete conditions. Replete biomass experienced the largest degradation (24%, dry basis), while deplete biomass experienced the least (10%, dry basis). Dry matter loss experienced among blends of each correlated positively with nitrogen-replete biomass content. The composition of the post-storage algae microbial community was also affected by cultivation conditions, with Clostridia species being more prevalent in stored biomass obtained from nitrogen-replete cultivations. Nitrogen management has long been known to influence algae biomass productivity and biochemical composition; here, we demonstrate that it also strongly influences the stability of post-harvest algae biomass in anaerobic storage.

Roles of the EnvZ/OmpR Two-Component System and Porins in Iron Acquisition in Escherichia coli.

Escherichia coli secretes high-affinity Fe3+ chelators to solubilize and transport chelated Fe3+ via specific outer membrane receptors. In microaerobic and anaerobic growth environments, where the reduced Fe2+ form is predominant, ferrous transport systems fulfill the bacterial need for iron. Expression of genes coding for iron metabolism is controlled by Fur, which when bound to Fe2+ acts as a repressor. Work carried out here shows that the constitutively activated EnvZ/OmpR two-component system, which normally controls expression of the ompC and ompF porin genes, dramatically increases the intracellular pool of accessible iron, as determined by whole-cell electron paramagnetic resonance spectroscopy, by inducing the OmpC/FeoB-mediated ferrous transport pathway. Elevated levels of intracellular iron in turn activated Fur, which inhibited the ferric transport pathway but not the ferrous transport pathway. The data show that the positive effect of constitutively activated EnvZ/OmpR on feoB expression is sufficient to overcome the negative effect of activated Fur on feoB In a tonB mutant, which lacks functional ferric transport systems, deletion of ompR severely impairs growth on rich medium not supplemented with iron, while the simultaneous deletion of ompC and ompF is not viable. These data, together with the observation of derepression of the Fur regulon in an OmpC mutant, show that the porins play an important role in iron homeostasis. The work presented here also resolves a long-standing paradoxical observation of the effect of certain mutant envZ alleles on iron regulon.IMPORTANCE The work presented here solved a long-standing paradox of the negative effects of certain missense alleles of envZ, which codes for kinase of the EnvZ/OmpR two-component system, on the expression of ferric uptake genes. The data revealed that the constitutive envZ alleles activate the Feo- and OmpC-mediated ferrous uptake pathway to flood the cytoplasm with accessible ferrous iron. This activates the ferric uptake regulator, Fur, which inhibits ferric uptake system but cannot inhibit the feo operon due to the positive effect of activated EnvZ/OmpR. The data also revealed the importance of porins in iron homeostasis.

Preservation of Microalgae, Lignocellulosic Biomass Blends by Ensiling to Enable Consistent Year-Round Feedstock Supply for Thermochemical Conversion to Biofuels.

Seasonal variation in microalgae productivity is a significant barrier to economical production of algae biofuels and chemicals. Summer production can be 3-5 times higher than in the winter resulting in uneven feedstock supplies at algae biorefineries. A portion of the summer production must be preserved for conversion in the winter in order to maintain a biorefinery running at capacity. Ensiling, a preservation process that utilizes lactic acid fermentation to limit microbial degradation, has been demonstrated to successfully stabilize algae biomass (20% solids) and algae-lignocellulosic blends (40% algae-60% lignocellulosic biomass, dry basis) for over 6 months, resulting in fuel production cost savings with fewer emissions. Preservation of algae as blends could be beneficial to biorefineries that utilize thermochemical approaches to fuel production as co-processing of algae and lignocellulosic biomass has been observed to enhance biocrude yield and improve oil quality. This study conducts a resource assessment of biomass residues in the southern United States to identify materials available during peak algae productivity and in sufficient quantity to meet the algae storage needs of an algae biofuel industry. Eight feedstocks met the quantity threshold but only three, distillers grains, haylage, and yard waste, were also available in season. Storage experiments utilizing both freshwater and marine strains of microalgae - Scenedesmus acutus, Chlorella vulgaris, Chlorella zofingiensis, Nannochloropsis gaditana, and Porphyridium purpureum - and yard waste were conducted for 30 days. Storage losses were less than 10% in all but one case, and the pH of all but one blend was reduced to less than 4.7, indicating that yard waste is a suitable feedstock for blending with algae prior to storage. To better understand whether the benefits to conversion realized by processing blends might be affected by storage, elemental analysis and bomb calorimetry of pre- and post-storage algae-yard waste blends were conducted to characterize changes occurring during storage. Storing algae biomass as blends with lignocellulosic biomass could be an effective method of mitigating seasonal variability in algae biomass production while retaining the synergistic effect of co-processing algae blends in thermochemical conversion.

Astaxanthin Is Ketolated from Zeaxanthin Independent of Fatty Acid Synthesis in Chromochloris zofingiensis.

The biosynthesis of astaxanthin, a high-value keto-carotenoid with broad industrial applications, remains unambiguous in algae. Here, we dissected the astaxanthin biosynthetic pathway and the coordination between astaxanthin and triacylglycerol (TAG) biosynthesis in the emerging model alga Chromochloris zofingiensis In vivo and in vitro experiments demonstrated that astaxanthin, utilizing the methylerythritol phosphate pathway-derived isopentenyl diphosphate as the building block, was synthesized from ß-carotenoid ketolase-mediated ketolation of zeaxanthin rather than ß-carotenoid hydroxylase-mediated hydroxylation of canthaxanthin, thus leading to the buildup of astaxanthin and canthaxanthin as end products in C. zofingiensis The synthesized astaxanthin, stored in TAG-filled lipid droplets, was esterified mainly with the fatty acid C18:1, which was not catalyzed by any acyltransferase previously proposed. Astaxanthin accumulated in a well-coordinated manner with TAG, supported by the coordinated up-regulation of both biosynthetic pathways at the transcriptional level. Nevertheless, astaxanthin and TAG showed no interdependence: inhibition of de novo fatty acid biosynthesis severely attenuated TAG biosynthesis but promoted the accumulation of astaxanthin, particularly in the diester form, leading to a fivefold increase in the astaxanthin/TAG ratio; however, inhibition of astaxanthin biosynthesis showed little effect on TAG accumulation. Our data suggest that an increase in astaxanthin accumulation following inhibition of de novo fatty acid biosynthesis, which is not regulated at the transcriptional level, is likely derived from the conversion of other carotenoids rather than from a shunt of carbon flux from lipid biosynthesis. Combined, these findings further our understanding of astaxanthin biosynthesis and provide a feasible strategy for promoting astaxanthin content and purity in algae.

Assessing the stability and techno-economic implications for wet storage of harvested microalgae to manage seasonal variability.

BACKGROUND: Seasonal variation in microalgae production is a significant challenge to developing cost-competitive algae biofuels. Summer production can be three to five times greater than winter production, which could result in winter biomass shortages and summer surpluses at algae biorefineries. While the high water content (80%, wet basis) of harvested microalgae biomass makes drying an expensive approach to preservation, it is not an issue for ensiling. Ensiling relies on lactic acid fermentation to create anaerobic acidic conditions, which limits further microbial degradation. This study explores the feasibility of preserving microalgae biomass through wet anaerobic storage ensiling over 30 and 180 days of storage, and it presents a techno-economic analysis that considers potential cost implications. RESULTS: Harvested Scenedesmus acutus biomass untreated (anaerobic) or supplemented with 0.5% sulfuric acid underwent robust lactic acid fermentation (lactic acid content of 6-9%, dry basis) lowering the pH to 4.2. Dry matter losses after 30 days ranged from 10.8 to 15.5% depending on the strain and treatment without additional loss over the next 150 days. Long-term storage of microalgae biomass resulted in lactic acid concentrations that remained high (6%, dry basis) with a low pH (4.2-4.6). Detailed biochemical composition revealed that protein and lipid content remained unaffected by storage while carbohydrate content was reduced, with greater dry matter loss associated with greater reduction in carbohydrate content, primarily affecting glucan content. Techno-economic analysis comparing wet storage to drying and dry storage demonstrated the cost savings of this approach. The most realistic dry storage scenario assumes a contact drum dryer and aboveground carbon steel storage vessels, which translates to a minimum fuel selling price (MFSP) of $3.72/gallon gasoline equivalent (GGE), whereas the most realistic wet storage scenario, which includes belowground, covered wet storage pits translates to an MFSP of $3.40/GGE. CONCLUSIONS: Microalgae biomass can be effectively preserved through wet anaerobic storage, limiting dry matter loss to below 10% over 6 months with minimal degradation of carbohydrates and preservation of lipids and proteins. Techno-economic analysis indicates that wet storage can reduce overall biomass and fuel costs compared to drying and dry storage.

Multiomics analysis reveals a distinct mechanism of oleaginousness in the emerging model alga Chromochloris zofingiensis.

Chromochloris zofingiensis, featured due to its capability to simultaneously synthesize triacylglycerol (TAG) and astaxanthin, is emerging as a leading candidate alga for production uses. To better understand the oleaginous mechanism of this alga, we conducted a multiomics analysis by systematically integrating time-resolved transcriptomes, lipidomes and metabolomes in response to nitrogen deprivation. The data analysis unraveled the distinct mechanism of TAG accumulation, which involved coordinated stimulation of multiple biological processes including supply of energy and reductants, carbon reallocation from protein and starch, and 'pushing' and 'pulling' carbon to TAG synthesis. Unlike the model alga Chlamydomonas, de novo fatty acid synthesis in C. zofingiensis was promoted, together with enhanced turnover of both glycolipids and phospholipids, supporting the drastic need of acyls for TAG assembly. Moreover, genomewide analysis identified many key functional enzymes and transcription factors that had engineering potential for TAG modulation. Two genes encoding glycerol-3-phosphate acyltransferase (GPAT), the first committed enzyme for TAG assembly, were found in the C. zofingiensis genome; in vivo functional characterization revealed that extrachloroplastic GPAT instead of chloroplastic GPAT played a central role in TAG synthesis. These findings illuminate distinct oleaginousness mechanisms in C. zofingiensis and pave the way towards rational manipulation of this alga to becone an emerging model for trait improvements.

Novel combination of feed enzymes to improve the degradation of Chlorella vulgaris recalcitrant cell wall.

In this study, a rational combination of 200 pre-selected Carbohydrate-Active enzymes (CAZymes) and sulfatases were tested, individually or combined, according to their ability to degrade Chlorella vulgaris cell wall to access its valuable nutritional compounds. The disruption of microalgae cell walls by a four-enzyme mixture (Mix) in comparison with the control, enabled to release up to 1.21 g/L of reducing sugars (p < 0.001), led to an eight-fold increase in oligosaccharides release (p < 0.001), and reduced the fluorescence intensity by 47% after staining with Calcofluor White (p < 0.001). The Mix treatment was successful in releasing proteins (p < 0.001), some MUFA (p < 0.05), and the beneficial 18:3n-3 fatty acid (p < 0.05). Even if no variation was detected for chlorophylls (p > 0.05), total carotenoids were increased in the supernatant (p < 0.05) from the Mix treatment, relative to the control. Taken together, these results indicate that this four-enzyme Mix displays an effective capacity to degrade C. vulgaris cell wall. Thus, these enzymes may constitute a good approach to improve the bioavailability of C. vulgaris nutrients for monogastric diets, in particular, and to facilitate the cost-effective use of microalgae by the feed industry, in general.

Down-Selection and Outdoor Evaluation of Novel, Halotolerant Algal Strains for Winter Cultivation.

Algae offer promising feedstocks for the production of renewable fuel and chemical intermediates. However, poor outdoor winter cultivation capacity currently limits deployment potential. In this study, 300 distinct algal strains were screened in saline medium to determine their cultivation suitability during winter conditions in Mesa, Arizona. Three strains, from the genera Micractinium, Chlorella, and Scenedesmus, were chosen following laboratory evaluations and grown outdoors in 1000 L raceway ponds during the winter. Strains were down-selected based on doubling time, lipid and carbohydrate amount, final biomass accumulation capacity, cell size and phylogenetic diversity. Algal biomass productivity and compositional analysis for lipids and carbohydrates show successful outdoor deployment and cultivation under winter conditions for these strains. Outdoor harvest-yield biomass productivities ranged from 2.9 to 4.0 g/m2/day over an 18 days winter cultivation trial, with maximum productivities ranging from 4.0 to 6.5 g/m2/day, the highest productivities reported to date for algal winter strains grown in saline media in open raceway ponds. Peak fatty acid levels ranged from 9 to 26% percent of biomass, and peak carbohydrate levels ranged from 13 to 34% depending on the strain. Changes in the lipid and carbohydrate profile throughout outdoor growth are reported. This study demonstrates that algal strain screening under simulated outdoor environmental conditions in the laboratory enables identification of strains with robust biomass productivity and biofuel precursor composition. The strains isolated here represent promising winter deployment candidates for seasonal algal biomass production when using crop rotation strategies.

A type-I diacylglycerol acyltransferase modulates triacylglycerol biosynthesis and fatty acid composition in the oleaginous microalga, Nannochloropsis oceanica.

BACKGROUND: Photosynthetic oleaginous microalgae are considered promising feedstocks for biofuels. The marine microalga, Nannochloropsis oceanica, has been attracting ever-increasing interest because of its fast growth, high triacylglycerol (TAG) content, and available genome sequence and genetic tools. Diacylglycerol acyltransferase (DGAT) catalyzes the last and committed step of TAG biosynthesis in the acyl-CoA-dependent pathway. Previous studies have identified 13 putative DGAT-encoding genes in the genome of N. oceanica, but the functional role of DGAT genes, especially type-I DGAT (DGAT1), remains ambiguous. RESULTS: Nannochloropsis oceanica IMET1 possesses two DGAT1 genes: NoDGAT1A and NoDGAT1B. Functional complementation demonstrated the capability of NoDGAT1A rather than NoDGAT1B to restore TAG synthesis in a TAG-deficient yeast strain. In vitro DGAT assays revealed that NoDGAT1A preferred saturated/monounsaturated acyl-CoAs and eukaryotic diacylglycerols (DAGs) for TAG synthesis, while NoDGAT1B had no detectable enzymatic activity. Assisted with green fluorescence protein (GFP) fusion, fluorescence microscopy analysis indicated the localization of NoDGAT1A in the chloroplast endoplasmic reticulum (cER) of N. oceanica. NoDGAT1A knockdown caused ~25% decline in TAG content upon nitrogen depletion, accompanied by the reduced C16:0, C18:0, and C18:1 in TAG sn-1/sn-3 positions and C18:1 in the TAG sn-2 position. NoDGAT1A overexpression, on the other hand, led to ~39% increase in TAG content upon nitrogen depletion, accompanied by the enhanced C16:0 and C18:1 in the TAG sn-1/sn-3 positions and C18:1 in the TAG sn-2 position. Interestingly, NoDGAT1A overexpression also promoted TAG accumulation (by ~2.4-fold) under nitrogen-replete conditions without compromising cell growth, and TAG yield of the overexpression line reached 0.49 g L-1 at the end of a 10-day batch culture, 47% greater than that of the control line. CONCLUSIONS: Taken together, our work demonstrates the functional role of NoDGAT1A and sheds light on the underlying mechanism for the biosynthesis of various TAG species in N. oceanica. NoDGAT1A resides likely in cER and prefers to transfer C16 and C18 saturated/monounsaturated fatty acids to eukaryotic DAGs for TAG assembly. This work also provides insights into the rational genetic engineering of microalgae by manipulating rate-limiting enzymes such as DGAT to modulate TAG biosynthesis and fatty acid composition for biofuel production.

Proteomic analysis of astaxanthin biosynthesis in Xanthophyllomyces dendrorhous in response to low carbon levels.

The ratio of carbon to nitrogen (C/N) in media plays a crucial role in the production of microbial carotenoids. However, the effects of a high C/N ratio on carotenoid production are ambiguous, and the mechanism of how C/N ratio affects astaxanthin accumulation in X. dendrorhous is unclear. In this study, the influence of C/N ratio on astaxanthin biosynthesis in X. dendrorhous at a fixed nitrogen concentration was investigated, and comparative proteomics were applied to address how C/N ratio affects cell growth and astaxanthin accumulation in X. dendrorhous. The results showed that cell growth and astaxanthin accumulation in X. dendrorhous were strongly related to the ratio of carbon to nitrogen with increasing C/N ratio in medium. However, the astaxanthin content per cell showed an inverse relationship, decreasing with an increasing C/N ratio. Differential proteomics showed the proteins with highest degree of change in expression under varying C/N ratios were mainly involved in carbohydrate metabolic pathways and carotenogenesis metabolism. In addition, several redox- and stress-associated proteins were up-regulated along with the carotenogenesis proteins, implying the environmental stress may affect metabolism and astaxanthin synthesis. A possible regulatory mechanism in response to glucose in X. dendrorhous is discussed.

Rapid Characterization of Microalgae and Microalgae Mixtures Using Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS).

Current molecular methods to characterize microalgae are time-intensive and expensive. Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) may represent a rapid and economical alternative approach. The objectives of this study were to determine whether MALDI-TOF MS can be used to: 1) differentiate microalgae at the species and strain levels and 2) characterize simple microalgal mixtures. A common protein extraction sample preparation method was used to facilitate rapid mass spectrometry-based analysis of 31 microalgae. Each yielded spectra containing between 6 and 56 peaks in the m/z 2,000 to 20,000 range. The taxonomic resolution of this approach appeared higher than that of 18S rDNA sequence analysis. For example, two strains of Scenedesmus acutus differed only by two 18S rDNA nucleotides, but yielded distinct MALDI-TOF mass spectra. Mixtures of two and three microalgae yielded relatively complex spectra that contained peaks associated with members of each mixture. Interestingly, though, mixture-specific peaks were observed at m/z 11,048 and 11,230. Our results suggest that MALDI-TOF MS affords rapid characterization of individual microalgae and simple microalgal mixtures.

Screening and characterization of oleaginous Chlorella strains and exploration of photoautotrophic Chlorella protothecoides for oil production.

The growth and oil production of nine Chlorella strains were comparatively assessed and Chlorellaprotothecoides CS-41 demonstrated the greatest lipid production potential. The effects of different nitrogen forms and concentrations, phosphorus concentrations and light intensities on growth and oil production were studied in laboratory columns. C. protothecoides CS-41 accumulated lipids up to 55% of dry weight, with triacylglycerol and oleic acid being 71% of total lipids and 59% of total fatty acids, respectively. High biomass and lipid productivities were achieved in outdoor panel PBRs, up to 1.25 and 0.59 g L(-1) day(-1), or 44. 1 and 16.1 g m(-2) day(-1), respectively. A two-stage cultivation strategy was proposed to enhance the algal biomass and lipid production. This is the first comprehensive investigation of both indoor and outdoor photoautotrophic C. protothecoides cultures for oil production, and C. protothecoides CS-41 represents a promising biofuel feedstock worthy of further exploration.

Furfural and hydroxymethylfurfural tolerance in Escherichia coli ΔacrR regulatory mutants.

The presence of the highly toxic furfural and hydroxymethylfurfural (HMF) in the hydrolysate of lignocellulosic biomass prompted the investigation of the Escherichia coli ΔacrR regulatory mutant for higher tolerance to these compounds, to facilitate the production of biofuels and biochemicals, and further biocatalytic conversions. In comparison with the parental strain, the regulatory mutant with the upregulated efflux pump AcrAB-TolC produced moderately better growth and higher tolerance to concentrations of furfural and HMF between 1 and 2 g L(-1) .

Highly-efficient enzymatic conversion of crude algal oils into biodiesel.

Energy-intensive chemical conversion of crude algal oils into biodiesel is a major barrier for cost-effective algal biofuel production. To overcome this problem, we developed an enzyme-based platform for conversion of crude algal oils into fatty acid methyl esters. Crude algal oils were extracted from the oleaginous microalga Nannochloropsis oceanica IMET1 and converted by an immobilized lipase from Candida antarctica. The effects of different acyl acceptors, t-butanol as a co-solvent, oil to t-butanol ratio, oil to methanol ratio, temperature and reaction time on biodiesel conversion efficiency were studied. The conversion efficiency reached 99.1% when the conversion conditions were optimized, i.e., an oil to t-butanol weight ratio of 1:1, an oil to methanol molar ratio of 1:12, and a reaction time of 4h at 25°C. The enzymatic conversion process developed in this study may hold a promise for low energy consumption, low wastewater-discharge biochemical conversion of algal feedstocks into biofuels.

Ultrastructure and composition of the Nannochloropsis gaditana cell wall.

Marine algae of the genus Nannochloropsis are promising producers of biofuel precursors and nutraceuticals and are also harvested commercially for aquaculture feed. We have used quick-freeze, deep-etch electron microscopy, Fourier transform infrared spectroscopy, and carbohydrate analyses to characterize the architecture of the Nannochloropsis gaditana (strain CCMP 526) cell wall, whose recalcitrance presents a significant barrier to biocommodity extraction. The data indicate a bilayer structure consisting of a cellulosic inner wall (~75% of the mass balance) protected by an outer hydrophobic algaenan layer. Cellulase treatment of walls purified after cell lysis generates highly enriched algaenan preparations without using the harsh chemical treatments typically used in algaenan isolation and characterization. Nannochloropsis algaenan was determined to comprise long, straight-chain, saturated aliphatics with ether cross-links, which closely resembles the cutan of vascular plants. Chemical identification of >85% of the isolated cell wall mass is detailed, and genome analysis is used to identify candidate biosynthetic enzymes.

Chlorella zofingiensis as an alternative microalgal producer of astaxanthin: biology and industrial potential.

Astaxanthin (3,3'-dihydroxy-ß,ß-carotene-4,4'-dione), a high-value ketocarotenoid with a broad range of applications in food, feed, nutraceutical, and pharmaceutical industries, has been gaining great attention from science and the public in recent years. The green microalgae Haematococcus pluvialis and Chlorella zofingiensis represent the most promising producers of natural astaxanthin. Although H. pluvialis possesses the highest intracellular astaxanthin content and is now believed to be a good producer of astaxanthin, it has intrinsic shortcomings such as slow growth rate, low biomass yield, and a high light requirement. In contrast, C. zofingiensis grows fast phototrophically, heterotrophically and mixtrophically, is easy to be cultured and scaled up both indoors and outdoors, and can achieve ultrahigh cell densities. These robust biotechnological traits provide C. zofingiensis with high potential to be a better organism than H. pluvialis for mass astaxanthin production. This review aims to provide an overview of the biology and industrial potential of C. zofingiensis as an alternative astaxanthin producer. The path forward for further expansion of the astaxanthin production from C. zofingiensis with respect to both challenges and opportunities is also discussed.

Genetic engineering of the green alga Chlorella zofingiensis: a modified norflurazon-resistant phytoene desaturase gene as a dominant selectable marker.

The unicellular green alga Chlorella zofingiensis has been proposed as a promising producer of natural astaxanthin, a commercially important ketocarotenoid. But the genetic toolbox for this alga is not available. In the present study, an efficient transformation system was established for C. zofingiensis. The transformation system utilized a modified norflurazon-resistant phytoene desaturase (PDS-L516F, with an leucine-phenylalanine change at position 516) as the selectable marker. Three promoters from endogenous PDS, nitrate reductase (NIT), and ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RBCS) genes were tested, with the RBCS promoter demonstrating the highest transformation efficiency. Inclusion of the first intron of the PDS gene further enhanced the efficiency by 91 %. Both particle bombardment and electroporation methods were examined, and the latter gave a fourfold higher transformation efficiency. The introduction of PDS-L516F, which exhibited a 33 % higher desaturation activity than the unaltered enzyme, enabled C. zofingiensis to produce 32.1 % more total carotenoids (TCs) and 54.1 % more astaxanthin. The enhanced accumulation of astaxanthin in transformants was revealed to be related to the increase in the transcripts of PDS, ß-carotenoid ketolase (BKT), and hydroxylase (CHYb) genes. Our study clearly shows that the modified PDS gene is a dominant selectable marker for the transformation of C. zofingiensis and possibly for the genetic engineering of the carotenoid biosynthetic pathway. In addition, the engineered C. zofingiensis might serve as an improved source of natural astaxanthin.

Enzymatic cell wall degradation of Chlorella vulgaris and other microalgae for biofuels production.

Cell walls of microalgae consist of a polysaccharide and glycoprotein matrix providing the cells with a formidable defense against its environment. We characterized enzymes that can digest the cell wall and weaken this defense for the purpose of protoplasting or lipid extraction. A growth inhibition screen demonstrated that chitinase, lysozyme, pectinase, sulfatase, ß-glucuronidase, and laminarinase had the broadest effect across the various Chlorella strains tested and also inhibited Nannochloropsis and Nannochloris strains. Chlorella is typically most sensitive to chitinases and lysozymes, both enzymes that degrade polymers containing N-acetylglucosamine. Using a fluorescent DNA stain, we developed rapid methodology to quantify changes in permeability in response to enzyme digestion and found that treatment with lysozyme in conjunction with other enzymes has a drastic effect on cell permeability. Transmission electron microscopy of enzymatically treated Chlorella vulgaris indicates that lysozyme degrades the outer surface of the cell wall and removes hair-like fibers protruding from the surface, which differs from the activity of chitinase. This action on the outer surface of the cell causes visible protuberances on the cell surface and presumably leads to the increased settling rate when cells are treated with lysozyme. We demonstrate radical ultrastructural changes to the cell wall in response to treatment with various enzyme combinations which, in some cases, causes a greater than twofold increase in the thickness of the cell wall. The enzymes characterized in this study should prove useful in the engineering and extraction of oils from microalgae.

Reversal of the ΔdegP phenotypes by a novel rpoE allele of Escherichia coli.

RseA sequesters RpoE (σ(E)) to the inner membrane of Escherichia coli when envelope stress is low. Elevated envelope stress triggers RseA cleavage by the sequential action of two membrane proteases, DegS and RseP, releasing σ(E) to activate an envelope stress reducing pathway. Revertants of a ΔdegP ΔbamB strain, which fails to grow at 37°C due to high envelope stress, harbored mutations in the rseA and rpoE genes. Null and missense rseA mutations constitutively hyper-activated the σ(E) regulon and significantly reduced the major outer membrane protein (OMP) levels. In contrast, a novel rpoE allele, rpoE3, resulting from the partial duplication of the rpoE gene, increased σ(E) levels greater than that seen in the rseA mutant background but did not reduce OMP levels. A σ(E)-dependent RybB::LacZ construct showed only a weak activation of the σ(E) pathway by rpoE3. Despite this, rpoE3 fully reversed the growth and envelope vesiculation phenotypes of ΔdegP. Interestingly, rpoE3 also brought down the modestly activated Cpx envelope stress pathway in the ΔdegP strain to the wild type level, showing the complementary nature of the σ(E) and Cpx pathways. Through employing a labile mutant periplasmic protein, AcrA(L222Q), it was determined that the rpoE3 mutation overcomes the ΔdegP phenotypes, in part, by activating a σ(E)-dependent proteolytic pathway. Our data suggest that a reduction in the OMP levels is not intrinsic to the σ(E)-mediated mechanism of lowering envelope stress. They also suggest that under extreme envelope stress, a tight homeostasis loop between RseA and σ(E) may partly be responsible for cell death, and this loop can be broken by mutations that either lower RseA activity or increase σ(E) levels.