Systematic metabolic engineering of Zymomonas mobilis for ß-farnesene production.

Zymomonas mobilis is an ethanologenic bacterium that can produce hopanoids using farnesyl pyrophosphate (FPP), which can be used as the precursor by ß-farnesene synthase for ß-farnesene production. To explore the possibility and bottlenecks of developing Z. mobilis for ß-farnesene production, five heterologous ß-farnesene synthases were selected and screened, and AaBFS from Artemisia annua had the highest ß-farnesene titer. Recombinant strains with AaBFS driven by the strong constitutive promoter Pgap (Pgap-AaBFS) doubled its ß-farnesene production to 25.73 ± 0.31 mg/L compared to the recombinant strain with AaBFS driven by Ptet (Ptet-AaBFS), which can be further improved by overexpressing the Pgap-AaBFS construct using the strategies of multiple plasmids (41.00 ± 0.40 mg/L) or genomic multi-locus integration (48.33 ± 3.40 mg/L). The effect of cofactor NADPH balancing on ß-farnesene production was also investigated, which can be improved only in zwf-overexpressing strains but not in ppnK-overexpressing strains, indicating that cofactor balancing is important and sophisticated. Furthermore, the ß-farnesene titer was improved to 73.30 ± 0.71 mg/L by overexpressing dxs, ispG, and ispH. Finally, the ß-farnesene production was increased to 159.70 ± 7.21 mg/L by fermentation optimization, including the C/N ratio, flask working volume, and medium/dodecane ratio, which was nearly 13-fold improved from the parental strain. This work thus not only generated a recombinant ß-farnesene production Z. mobilis strain but also unraveled the bottlenecks to engineer Z. mobilis for farnesene production, which will help guide the future rational design and construction of cell factories for terpenoid production in non-model industrial microorganisms.

Systematic investigation of TetR-family transcriptional regulators and their roles on lignocellulosic inhibitor acetate tolerance in Zymomonas mobilis.

TetR-family transcriptional regulators are widely distributed among bacteria and involved in various cellular processes such as multidrug and inhibitor resistance. Zymomonas mobilis is a industrial bacterium for lignocellulosic ethanol production. Although TetR-family regulators and their associated RND-family efflux pumps in Z. mobilis have been identified to be differentially expressed under various inhibitors and stressful conditions, there are no systematic investigation yet. In this study, bioinformatic analyses indicated that there are three TetR-family transcriptional regulators (ZMO0281, ZMO0963, ZMO1547) and two RND-family efflux pumps (ZMO0282-0285, ZMO0964-0966) adjacent to corresponding TetR-family regulators of ZMO0281 and ZMO0963 in Z. mobilis. Genetics studies were then carried out with various mutants of TetR-family regulators constructed, and ZMO0281 was characterized to be related to acetate tolerance. Combining transcriptomics and dual-reporter gene system, this study demonstrated that three TetR-family regulators repressed their adjacent genes specifically. Moreover, TetR-family regulator ZMO0281 might also be involved in other cellular processes in the presence of acetate. In addition, the upregulation of RND-family efflux pumps due to ZMO0281 deletion might lead to an energy imbalance and decreased cell growth in Z. mobilis under acetate stress. The systematic investigation of all three TetR-family regulators and their roles on a major lignocellulosic inhibitor acetate tolerance in Z. mobilis thus not only unravels the molecular mechanisms of TetR-family regulators and their potential cross-talks on regulating RND-family efflux pumps and other genes in Z. mobilis, but also provides guidance on understanding the roles of multiple regulators of same family in Z. mobilis and other microorganisms for efficient lignocellulosic biochemical production.

Metabolic engineering of an industrial bacterium Zymomonas mobilis for anaerobic l-serine production.

Due to the complicated metabolic and regulatory networks of l-serine biosynthesis and degradation, microbial cell factories for l-serine production using non-model microorganisms have not been reported. In this study, a combination of synthetic biology and process optimization were applied in an ethanologenic bacterium Zymomonas mobilis for l-serine production. By blocking the degradation pathway while introducing an exporter EceamA from E. coli, l-serine titer in recombinant Z. mobilis was increased from 15.30 mg/L to 62.67 mg/L. It was further increased to 260.33 mg/L after enhancing the l-serine biosynthesis pathway. Then, 536.70 mg/L l-serine was achieved by removing feedback inhibition with a SerA mutant, and an elevated titer of 687.67 mg/L was further obtained through increasing serB copies while enhancing the precursors. Finally, 855.66 mg/L l-serine can be accumulated with the supplementation of the glutamate precursor. This work thus not only constructed an l-serine producer to help understand the bottlenecks limiting l-serine production in Z. mobilis for further improvement, but also provides guidance on engineering non-model microbes to produce biochemicals with complicated pathways such as amino acids or terpenoids.

Metabolic engineering of Zymomonas mobilis for co-production of D-lactic acid and ethanol using waste feedstocks of molasses and corncob residue hydrolysate.

Lactate is the precursor for polylactide. In this study, a lactate producer of Z. mobilis was constructed by replacing ZMO0038 with LmldhA gene driven by a strong promoter PadhB, replacing ZMO1650 with native pdc gene driven by Ptet, and replacing native pdc with another copy of LmldhA driven by PadhB to divert carbon from ethanol to D-lactate. The resultant strain ZML-pdc-ldh produced 13.8 ± 0.2 g/L lactate and 16.9 ± 0.3 g/L ethanol using 48 g/L glucose. Lactate production of ZML-pdc-ldh was further investigated after fermentation optimization in pH-controlled fermenters. ZML-pdc-ldh produced 24.2 ± 0.6 g/L lactate and 12.9 ± 0.8 g/L ethanol as well as 36.2 ± 1.0 g/L lactate and 40.3 ± 0.3 g/L ethanol, resulting in total carbon conversion rate of 98.3% ± 2.5% and 96.2% ± 0.1% with final product productivity of 1.9 ± 0.0 g/L/h and 2.2 ± 0.0 g/L/h in RMG5 and RMG12, respectively. Moreover, ZML-pdc-ldh produced 32.9 ± 0.1 g/L D-lactate and 27.7 ± 0.2 g/L ethanol as well as 42.8 ± 0.0 g/L D-lactate and 53.1 ± 0.7 g/L ethanol with 97.1% ± 0.0% and 99.1% ± 0.8% carbon conversion rate using 20% molasses or corncob residue hydrolysate, respectively. Our study thus demonstrated that it is effective for lactate production by fermentation condition optimization and metabolic engineering to strengthen heterologous ldh expression while reducing the native ethanol production pathway. The capability of recombinant lactate-producer of Z. mobilis for efficient waste feedstock conversion makes it a promising biorefinery platform for carbon-neutral biochemical production.

Identification and Characterization of Genes Related to Ampicillin Antibiotic Resistance in Zymomonas mobilis.

Antibiotics can inhibit or kill microorganisms, while microorganisms have evolved antibiotic resistance strategies to survive antibiotics. Zymomonas mobilis is an ideal industrial microbial chassis and can tolerate multiple antibiotics. However, the mechanisms of antibiotic resistance and genes associated with antibiotic resistance have not been fully analyzed and characterized. In this study, we investigated genes associated with antibiotic resistance using bioinformatic approaches and examined genes associated with ampicillin resistance using CRISPR/Cas12a-based genome-editing technology. Six ampicillin-resistant genes (ZMO0103, ZMO0893, ZMO1094, ZMO1650, ZMO1866, and ZMO1967) were identified, and five mutant strains ZM4∆0103, ZM4∆0893, ZM4∆1094, ZM4∆1650, and ZM4∆1866 were constructed. Additionally, a four-gene mutant ZM4∆ARs was constructed by knocking out ZMO0103, ZMO0893, ZMO1094, and ZMO1650 continuously. Cell growth, morphology, and transformation efficiency of mutant strains were examined. Our results show that the cell growth of ZM4∆0103 and ZM4∆ARs was significantly inhibited with 150 µg/mL ampicillin, and cells changed to a long filament shape from a short rod shape. Moreover, the transformation efficiencies of ZM4∆0103 and ZM4∆ARs were decreased. Our results indicate that ZMO0103 is the key to ampicillin resistance in Z. mobilis, and other ampicillin-resistant genes may have a synergetic effect with it. In summary, this study identified and characterized genes related to ampicillin resistance in Z. mobilis and laid a foundation for further study of other antibiotic resistance mechanisms.

Microbial Lipid Production from High Concentration of Volatile Fatty Acids via Trichosporon cutaneum for Biodiesel Preparation.

Direct bioconversion of high concentration of volatile fatty acids (VFAs) into microbial lipid is challenging due to the aggravated cytotoxicity of VFAs at high loadings. Herein, a robust oleaginous yeast Trichosporon cutaneum was screened for lipogenesis from high concentration of VFAs using a regular batch culture. Biomass and lipid content of 8.9 g/L and 49.1%, respectively, were attained from 50 g/L acetic acid with 90.9% of which assimilated within 10 days. The blend of VFAs (50 g/L), with mass ratio of acetic, propionic, and butyric acids of 6:3:1, was found superior to acetic acid for lipogenesis. Biomass and lipid titer increased by 16.9% and 18.2%, respectively, with the three VFAs completely consumed within 8 days. Butyric acid was assimilated simultaneously with acetic acid at the beginning of the culture. Heptadecanoic acid (C17:0) and heptadecenoic acid (C17:1) were produced when propionic acid co-existed with acetic and butyric acids. The estimation of biodiesel properties indicated that lipid prepared from VFA blend showed superiority to acetic acid for high-quality biodiesel production. This study strongly supported that T. cutaneum permitted high concentration of VFA mixture for lipid production.

Cysteine supplementation enhanced inhibitor tolerance of Zymomonas mobilis for economic lignocellulosic bioethanol production.

Inhibitors in lignocellulosic hydrolysates are toxic to Zymomonas mobilis and reduce its bioethanol production. This study revealed cysteine supplementation enhanced furfural tolerance in Z. mobilis with a 2-fold biomass increase. Transcriptomic study illustrated that cysteine biosynthesis pathway was down-regulated while cysteine catabolism was up-regulated with cysteine supplementation. Mutants for genes involved in cysteine metabolism were constructed, and metabolites in cysteine metabolic pathway including methionine, glutathione, NaHS, glutamate, and pyruvate were supplemented into media. Cysteine supplementation boosted glutathione synthesis or H2S release effectively in Z. mobilis leading to the reduced accumulation of reactive oxygen species (ROS) induced by furfural, while pyruvate and glutamate produced in the H2S generation pathway promoted cell growth by serving as the carbon or nitrogen source. Finally, cysteine supplementation was confirmed to enhance Z. mobilis tolerance against ethanol, acetate, and corncob hydrolysate with an enhanced ethanol productivity from 0.38 to 0.55 g-1∙L-1∙h-1.

Development and characterization of efficient xylose utilization strains of Zymomonas mobilis.

BACKGROUND: Efficient use of glucose and xylose is a key for the economic production of lignocellulosic biofuels and biochemicals, and different recombinant strains have been constructed for xylose utilization including those using Zymomonas mobilis as the host. However, the xylose utilization efficiency still needs to be improved. In this work, the strategy of combining metabolic engineering and adaptive laboratory evolution (ALE) was employed to develop recombinant Z. mobilis strains that can utilize xylose efficiently at high concentrations, and NGS-based genome resequencing and RNA-Seq transcriptomics were performed for strains evolved after serial transfers in different media to understand the impact of xylose and differences among strains with different xylose-utilization capabilities at molecular level. RESULTS: Heterologous genes encoding xylose isomerase and xylulokinase were evaluated, which were then introduced into xylose-utilizing strain Z. mobilis 8b to enhance its capacity of xylose utilization. The results demonstrated that the effect of three xylose isomerases on xylose utilization was different, and the increase of copy number of xylose metabolism genes can improve xylose utilization. Among various recombinant strains constructed, the xylose utilization capacity of the recombinant strain 8b-RsXI-xylB was the best, which was further improved through continuous adaption with 38 transfers over 100 days in 50 g/L xylose media. The fermentation performances of the parental strain 8b, the evolved 8b-S38 strain with the best xylose utilization capability, and the intermediate strain 8b-S8 in different media were compared, and the results showed that only 8b-S38 could completely consume xylose at 50 g/L and 100 g/L concentrations. In addition, the xylose consumption rate of 8b-S38 was faster than that of 8b at different xylose concentrations from 50 to 150 g/L, and the ethanol yield increased by 16 ~ 40%, respectively. The results of the mixed-sugar fermentation also demonstrated that 8b-S38 had a higher xylose consumption rate than 8b, and its maximum ethanol productivity was 1.2 ~ 1.4 times higher than that of 8b and 8b-S8. Whole-genome resequencing identified three common genetic changes in 8b-S38 compared with 8b and 8b-S8. RNA-Seq study demonstrated that the expression levels of genes encoding chaperone proteins, ATP-dependent proteases, phage shock proteins, ribosomal proteins, flagellar operons, and transcriptional regulators were significantly increased in xylose media in 8b-S38. The up-regulated expression of these genes may therefore contribute to the efficient xylose utilization of 8b-S38 by maintaining the normal cell metabolism and growth, repairing cellular damages, and rebalancing cellular energy to help cells resist the stressful environment. CONCLUSIONS: This study provides gene candidates to improve xylose utilization, and the result of expressing an extra copy of xylose isomerase and xylulokinase improved xylose utilization also provides a direction for efficient xylose-utilization strain development in other microorganisms. In addition, this study demonstrated the necessity to combine metabolic engineering and ALE for industrial strain development. The recombinant strain 8b-S38 can efficiently metabolize xylose for ethanol fermentation at high xylose concentrations as well as in mixed sugars of glucose and xylose, which could be further developed as the microbial biocatalyst for the production of lignocellulosic biofuels and biochemicals.

[Progress and perspective on development of non-model industrial bacteria as chassis cells for biochemical production in the synthetic biology era].

The development and implement of microbial chassis cells can provide excellent cell factories for diverse industrial applications, which help achieve the goal of environmental protection and sustainable bioeconomy. The synthetic biology strategy of Design-Build-Test-Learn (DBTL) plays a crucial role on rational and/or semi-rational construction or modification of chassis cells to achieve the goals of "Building to Understand" and "Building for Applications". In this review, we briefly comment on the technical development of the DBTL cycle and the research progress of a few model microorganisms. We mainly focuse on non-model bacterial cell factories with potential industrial applications, which possess unique physiological and biochemical characteristics, capabilities of utilizing one-carbon compounds or of producing platform compounds efficiently. We also propose strategies for the efficient and effective construction and application of synthetic microbial cell factories securely in the synthetic biology era, which are to discover and integrate the advantages of model and non-model industrial microorganisms, to develop and deploy intelligent automated equipment for cost-effective high-throughput screening and characterization of chassis cells as well as big-data platforms for storing, retrieving, analyzing, simulating, integrating, and visualizing omics datasets at both molecular and phenotypic levels, so that we can build both high-quality digital cell models and optimized chassis cells to guide the rational design and construction of microbial cell factories for diverse industrial applications.

Metabolic engineering of Zymomonas mobilis for anaerobic isobutanol production.

BACKGROUND: Biofuels and value-added biochemicals derived from renewable biomass via biochemical conversion have attracted considerable attention to meet global sustainable energy and environmental goals. Isobutanol is a four-carbon alcohol with many advantages that make it attractive as a fossil-fuel alternative. Zymomonas mobilis is a highly efficient, anaerobic, ethanologenic bacterium making it a promising industrial platform for use in a biorefinery. RESULTS: In this study, the effect of isobutanol on Z. mobilis was investigated, and various isobutanol-producing recombinant strains were constructed. The results showed that the Z. mobilis parental strain was able to grow in the presence of isobutanol below 12 g/L while concentrations greater than 16 g/L inhibited cell growth. Integration of the heterologous gene encoding 2-ketoisovalerate decarboxylase such as kdcA from Lactococcus lactis is required for isobutanol production in Z. mobilis. Moreover, isobutanol production increased from nearly zero to 100-150 mg/L in recombinant strains containing the kdcA gene driven by the tetracycline-inducible promoter Ptet. In addition, we determined that overexpression of a heterologous als gene and two native genes (ilvC and ilvD) involved in valine metabolism in a recombinant Z. mobilis strain expressing kdcA can divert pyruvate from ethanol production to isobutanol biosynthesis. This engineering improved isobutanol production to above 1 g/L. Finally, recombinant strains containing both a synthetic operon, als-ilvC-ilvD, driven by Ptet and the kdcA gene driven by the constitutive strong promoter, Pgap, were determined to greatly enhance isobutanol production with a maximum titer about 4.0 g/L. Finally, isobutanol production was negatively affected by aeration with more isobutanol being produced in more poorly aerated flasks. CONCLUSIONS: This study demonstrated that overexpression of kdcA in combination with a synthetic heterologous operon, als-ilvC-ilvD, is crucial for diverting pyruvate from ethanol production for enhanced isobutanol biosynthesis. Moreover, this study also provides a strategy for harnessing the valine metabolic pathway for future production of other pyruvate-derived biochemicals in Z. mobilis.

Variations and drivers of methane fluxes from a rice-wheat rotation agroecosystem in eastern China at seasonal and diurnal scales.

The paddy rice fields act as an important anthropogenic source of methane (CH4) to the atmosphere. The study of pattern, magnitude, and environmental controls of CH4 emissions are still insufficient due to limited measurements and understand of underlying drivers for variations of CH4 fluxes at different temporal scales. In this study, CH4 fluxes from a rice-wheat rotation agroecosystem in eastern China were continuously measured using the eddy covariance technique. The diurnal and seasonal variations of CH4 flux and potential controlling factors in 2016 were analyzed using wavelet coherence, conditional Granger causality, correlation analysis and path analysis methods. CH4 fluxes showed distinguishable diurnal variations with single peaks during 13: 00-16: 00 local time. At the diurnal timescale, gross primary productivity (GPP) regulates CH4 fluxes after accounting for the effects of latent heat flux (LE), air temperature (TA), and soil temperature (TS) on CH4 fluxes. LE mirrored the diurnal pattern of CH4 fluxes when the effects of TA and TS on CH4 fluxes were considered. Daily CH4 fluxes exhibited large seasonal variations, with the largest daily CH4 flux of 1191.78 mg C-CH4 m-2 d-1 on 29 July 2016. The daily CH4 fluxes were continuously low in the growing season of wheat, and sharply increased from very low values in late June to peaks in late July and early August, and then gradually decreased to low values at the end of the rice growing season in late November and early December. Correlation analysis and path analysis showed that seasonal variations of soil temperature, air temperature, and GPP had strong effects on daily CH4 fluxes during pre-panicle initiation of the rice growing season, while soil temperature and leaf area index (LAI) had very strong effects on daily CH4 fluxes during the post-panicle initiation stage. The total of CH4 fluxes from the rice-wheat rotation agroecosystem into the atmosphere amounted to 58.08 ±â€¯9.87 g C m-2 in 2016, and the annual net carbon (C) budget and greenhouse gas (GHG) budget were 163.50 ±â€¯9.87 g C m-2 and 2322.53 ±â€¯329.00 g CO2eq m-2, respectively. This study represents a comprehensive assessment of fluxes and drivers of CH4 from a rice-wheat rotation agroecosystem at different timescales. Additionally, the consecutive data of CH4 emission in this region will also useful for model calibration and validation.

Oleaginicity of the yeast strain Saccharomyces cerevisiae D5A.

BACKGROUND: The model yeast, Saccharomyces cerevisiae, is not known to be oleaginous. However, an industrial wild-type strain, D5A, was shown to accumulate over 20% storage lipids from glucose when growth is nitrogen-limited compared to no more than 7% lipid accumulation without nitrogen stress. METHODS AND RESULTS: To elucidate the mechanisms of S. cerevisiae D5A oleaginicity, we compared physiological and metabolic changes; as well as the transcriptional profiles of the oleaginous industrial strain, D5A, and a non-oleaginous laboratory strain, BY4741, under normal and nitrogen-limited conditions using analytic techniques and next-generation sequencing-based RNA-Seq transcriptomics. Transcriptional levels for genes associated with fatty acid biosynthesis, nitrogen metabolism, amino acid catabolism, as well as the pentose phosphate pathway and ethanol oxidation in central carbon (C) metabolism, were up-regulated in D5A during nitrogen deprivation. Despite increased carbon flux to lipids, most gene-encoding enzymes involved in triacylglycerol (TAG) assembly were expressed at similar levels regardless of the varying nitrogen concentrations in the growth media and strain backgrounds. Phospholipid turnover also contributed to TAG accumulation through increased precursor production with the down-regulation of subsequent phospholipid synthesis steps. Our results also demonstrated that nitrogen assimilation via the glutamate-glutamine pathway and amino acid metabolism, as well as the fluxes of carbon and reductants from central C metabolism, are integral to the general oleaginicity of D5A, which resulted in the enhanced lipid storage during nitrogen deprivation. CONCLUSION: This work demonstrated the disequilibrium and rebalance of carbon and nitrogen contribution to the accumulation of lipids in the oleaginous yeast S. cerevisiae D5A. Rather than TAG assembly from acyl groups, the major switches for the enhanced lipid accumulation of D5A (i.e., fatty acid biosynthesis) are the increases of cytosolic pools of acetyl-CoA and NADPH, as well as alternative nitrogen assimilation.

Effects of temperature and its combination with high light intensity on lipid production of Monoraphidium dybowskii Y2 from semi-arid desert areas.

Temperature and light intensity are important environmental factors influencing microalgae for biodiesel production. The aim of present work was to study the effects of temperature (15 °C, 25 °C, and 35 °C) and its combination with high light intensity (HL, 400 µmol photon m-2 s-1) on lipid production of Monoraphidium dybowskii Y2 which was isolated from desert. The results demonstrated that algal growth was only inhibited at 15 °C. Promoted lipid content and decreased Fv/Fm were observed in 15 °C and 35 °C. Cellular carbohydrate, protein conversion and membrane lipid (MGDG, DGDG and SQDG) remodeling contributes for lipid accumulation. Stress combined temperatures with HL are benefit for lipid production, especially desired neutral lipid productivity all exceed 40 mg L-1 d-1. Fatty acids compositions of C16:0 and C18:1 were further promoted under 15 °C or 35 °C combined with HL. Thus, M. dybowskii Y2 will well-adapted to outdoors cultivation for biodiesel production.

Feasibility of biodiesel production and CO2 emission reduction by Monoraphidium dybowskii LB50 under semi-continuous culture with open raceway ponds in the desert area.

BACKGROUND: Compared with other general energy crops, microalgae are more compatible with desert conditions. In addition, microalgae cultivated in desert regions can be used to develop biodiesel. Therefore, screening oil-rich microalgae, and researching the algae growth, CO2 fixation and oil yield in desert areas not only effectively utilize the idle desertification lands and other resources, but also reduce CO2 emission. RESULTS: Monoraphidium dybowskii LB50 can be efficiently cultured in the desert area using light resources, and lipid yield can be effectively improved using two-stage induction and semi-continuous culture modes in open raceway ponds (ORPs). Lipid content (LC) and lipid productivity (LP) were increased by 20% under two-stage industrial salt induction, whereas biomass productivity (BP) increased by 80% to enhance LP under semi-continuous mode in 5 m2 ORPs. After 3 years of operation, M. dybowskii LB50 was successfully and stably cultivated under semi-continuous mode for a month during five cycles of repeated culture in a 200 m2 ORP in the desert area. This culture mode reduced the supply of the original species. The BP and CO2 fixation rate were maintained at 18 and 33 g m-2 day-1, respectively. Moreover, LC decreased only during the fifth cycle of repeated culture. Evaporation occurred at 0.9-1.8 L m-2 day-1, which corresponded to 6.5-13% of evaporation loss rate. Semi-continuous and two-stage salt induction culture modes can reduce energy consumption and increase energy balance through the energy consumption analysis of life cycle. CONCLUSION: This study demonstrates the feasibility of combining biodiesel production and CO2 fixation using microalgae grown as feedstock under culture modes with ORPs by using the resources in the desert area. The understanding of evaporation loss and the sustainability of semi-continuous culture render this approach practically viable. The novel strategy may be a promising alternative to existing technology for CO2 emission reduction and biofuel production.

Advances and prospects in metabolic engineering of Zymomonas mobilis.

Biorefinery of biomass-based biofuels and biochemicals by microorganisms is a competitive alternative of traditional petroleum refineries. Zymomonas mobilis is a natural ethanologen with many desirable characteristics, which makes it an ideal industrial microbial biocatalyst for commercial production of desirable bioproducts through metabolic engineering. In this review, we summarize the metabolic engineering progress achieved in Z. mobilis to expand its substrate and product ranges as well as to enhance its robustness against stressful conditions such as inhibitory compounds within the lignocellulosic hydrolysates and slurries. We also discuss a few metabolic engineering strategies that can be applied in Z. mobilis to further develop it as a robust workhorse for economic lignocellulosic bioproducts. In addition, we briefly review the progress of metabolic engineering in Z. mobilis related to the classical synthetic biology cycle of "Design-Build-Test-Learn", as well as the progress and potential to develop Z. mobilis as a model chassis for biorefinery practices in the synthetic biology era.

Culture modes and financial evaluation of two oleaginous microalgae for biodiesel production in desert area with open raceway pond.

Cultivation modes of autotrophic microalgae for biodiesel production utilizing open raceway pond were analyzed in this study. Five before screened good microalgae were tested their lipid productivity and biodiesel quality again in outdoor 1000L ORP. Then, Chlorella sp. L1 and Monoraphidium dybowskii Y2 were selected due to their stronger environmental adaptability, higher lipid productivity and better biodiesel properties. Further scale up cultivation for two species with batch and semi-continuous culture was conducted. In 40,000L ORP, higher lipid productivity (5.15 versus 4.06gm(-2)d(-1) for Chlorella sp. L1, 5.35 versus 3.00gm(-2)d(-1) for M. dybowskii Y2) was achieved in semi-continuous mode. Moreover, the financial costs of 14.18$gal(-1) and 13.31$gal(-1) for crude biodiesel in two microalgae with semi-continuous mode were more economically feasible for commercial production on large scale outdoors.

Optimizing light regimes on growth and lipid accumulation in Ankistrodesmus fusiformis H1 for biodiesel production.

The aim of this study was to optimize the light regimes including initial inoculum density, photoperiod and light intensity on the growth and lipid (TAG) accumulation in Ankistrodesmus fusiformis H1 for biodiesel production. At last, the strategy of 4.47 mM urea with initial OD680-0.5, 18:6h light/dark cycle and 200 µmol photon m(-2) s(-1) regimes were optimized. The lipid productivity of 116.88 mg L(-1)d(-1) and 57.58% neutral lipid in total lipid were achieved finally. Moreover, the changes of photosynthetic activity, pigments contents and biochemical compositions revealed that more carbon flow to lipid synthesis. Therefore, A. fusiformis H1 is an ideal candidate for biodiesel production by utilizing light appropriately.

Effect of light intensity on physiological changes, carbon allocation and neutral lipid accumulation in oleaginous microalgae.

Chlorella sp. and Monoraphidium sp. were the potential microalgal species for lipid production. This study aimed to investigate different light intensities (40, 200, 400 µmol photon m(-2) s(-1)) on physiological changes, photosynthetic carbon partitioning and neutral lipid accumulation in both microalgae. Results suggested that under high light (HL, 400 µmol photon m(-2) s(-1)), chlorophyll degraded, protein and carbohydrate content decreased; more carbon allocated into lipid as well as most of intracellular space was occupied by lipid bodies. Moreover, with the lipid accumulation, Fv/Fm decreased and ROS scavenging enzyme increased. Membrane lipid reduced dramatic (29.73-37.97%) to format NL (71.66% of total lipid in Chlorella sp. L1 and 60.65% in Monoraphidium dybowskii Y2). The NL productivity under HL (51.36 and 49.71 mg L(-1) d(-1)) were more than 3 times of those under LL. Additionally, FAME profiles proved that the useful fatty acid components for biodiesel production were enhanced under HL.

Lipid accumulation by NaCl induction at different growth stages and concentrations in photoautotrophic two-step cultivation of Monoraphidium dybowskii LB50.

NaCl induction in photoautotrophic two-step cultivation is very promising, but time node and concentration are critical to the entire production. In this study Monoraphidium dybowskii LB50 was subjected to different NaCl concentrations at different growth phases. Results showed that during the initial phase (IP), fixed carbon was used for sugar and lipid under 5gL(-1) NaCl induction, as well as for protein under 10gL(-1) NaCl induction. At late-exponential growth phase (LEGP), the highest lipid productivity was obtained at 20gL(-1) NaCl. At stationary phase (SP) the highest lipid productivity was also under 20gL(-1) NaCl but lower than that of LEGP. In summary, lipid content and quality were improved at all growth phases under NaCl induction. Therefore, cultivation scale can be sued to determine the time node and dosage of the inducer, thereby realizing the economic efficiency of the fundamental guarantee in photoautotrophic two-step cultivation.

Sufficient utilization of natural fluctuating light intensity is an effective approach of promoting lipid productivity in oleaginous microalgal cultivation outdoors.

The effects of fluctuating intensity of solar radiation on biomass and lipid in oleaginous microalgae are important. However, this topic has not been the subject of studies for a long time. In this study, four oleaginous microalgae from semi-arid areas were screened and cultivated outdoors under different fluctuating intensities. Results showed that the highest lipid productivities and neutral lipid (NL) contents occurred under high fluctuating intensity (HFI), in which 13-20% of the increased NL came from glycolipid transformation without phospholipid conversion. Chlorella sp. L1 and Monoraphidium dybowskii Y2 obtained from biological soil crusts in desert had the largest biomass (137.13, 106.61mgL(-1)d(-1)) and lipid yields (35.06, 32.45mgL(-1)d(-1)) under HFI. The highest areal lipid productivities of 9.06 and 8.95gm(-2)d(-1) and better biodiesel quality were observed under HFI. Accordingly, sufficiently adopting fluctuating light intensity outdoors to culture microalgae was an economic and effective approach.