A modular metabolic engineering approach for the production of 1,2-propanediol from glycerol by Saccharomyces cerevisiae

Compared to sugars, a major advantage of using glycerol as a feedstock for industrial bioprocesses is the fact that this molecule is more reduced than sugars. A compound whose biotechnological production might greatly profit from the substrate's higher reducing power is 1,2-propanediol (1,2-PDO). Here we present a novel metabolic engineering approach to produce 1,2-PDO from glycerol in S. cerevisiae. Apart from implementing the heterologous methylglyoxal (MG) pathway for 1,2-PDO formation from dihydroxyacetone phosphate (DHAP) and expressing a heterologous glycerol facilitator, the employed genetic modifications included the replacement of the native FAD-dependent glycerol catabolic pathway by the 'DHA pathway' for delivery of cytosolic NADH and the reduction of triosephosphate isomerase (TPI) activity for increased precursor (DHAP) supply. The choice of the medium had a crucial impact on both the strength of the metabolic switch towards fermentation in general (as indicated by the production of ethanol and 1,2-PDO) and on the ratio at which these two fermentation products were formed. For example, virtually no 1,2-PDO but only ethanol was formed in synthetic glycerol medium with urea as the nitrogen source. When nutrient-limited complex YG medium was used, significant amounts of 1,2-PDO were formed and it became obvious that the concerted supply of NADH and DHAP are essential for boosting 1,2-PDO production. Additionally, optimizing the flux into the MG pathway improved 1,2-PDO formation at the expense of ethanol. Cultivation of the best-performing strain in YG medium and a controlled bioreactor set-up resulted in a maximum titer of > 4 g L⁻¹ 1,2-PDO which, to the best of our knowledge, has been the highest titer of 1,2-PDO obtained in yeast so far. Surprisingly, significant 1,2-PDO production was also obtained in synthetic glycerol medium after changing the nitrogen source towards ammonium sulfate and adding a buffer.     

Genetic determinants for enhanced glycerol growth of Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae generally shows a low natural capability to utilize glycerol as the sole source of carbon, particularly when synthetic medium is used and complex supplements are omitted. Nevertheless, wild type isolates have been identified that show a moderate growth under these conditions. In the current study we made use of intraspecies diversity to identify targets suitable for reverse metabolic engineering of the non-growing laboratory strain CEN.PK113-1A. A genome-wide genetic mapping experiment using pooled-segregant whole-genome sequence analysis was conducted, and one major and several minor genetic loci were identified responsible for the superior glycerol growth phenotype of the previously selected S. cerevisiae strain CBS 6412-13A. Downscaling of the major locus by fine-mapping and reciprocal hemizygosity analysis allowed the parallel identification of two superior alleles (UBR2_{CBS 6412-13A} and SSK1_{CBS 6412-13A}). These alleles together with the previously identified GUT1_{CBS 6412-13A} allele were used to replace the corresponding alleles in the strain CEN.PK113-1A. In this way, glycerol growth could be established reaching a maximum specific growth rate of 0.08 h⁻¹. Further improvement to a maximum specific growth rate of 0.11 h⁻¹ could be achieved by heterologous expression of the glycerol facilitator FPS1 from Cyberlindnera jadinii.     

Metabolic engineering of fatty acyl-ACP reductase-dependent pathway to improve fatty alcohol production in Escherichia coli

Fatty alcohols are important components of surfactants and cosmetic products. The production of fatty alcohols from sustainable resources using microbial fermentation could reduce dependence on fossil fuels and greenhouse gas emission. However, the industrialization of this process has been hampered by the current low yield and productivity of this synthetic pathway. As a result of metabolic engineering strategies, an Escherichia coli mutant containing Synechococcus elongatus fatty acyl-ACP reductase showed improved yield and productivity. Proteomics analysis and in vitro enzymatic assays showed that endogenous E. coli AdhP is a major contributor to the reduction of fatty aldehydes to fatty alcohols. Both in vitro and in vivo results clearly demonstrated that the activity and expression level of fatty acyl-CoA/ACP reductase is the rate-limiting step in the current protocol. In 2.5-L fed-batch fermentation with glycerol as the only carbon source, the most productive E. coli mutant produced 0.75 g/L fatty alcohols (0.02 g fatty alcohol/g glycerol) with a productivity of up to 0.06 g/L/h. This investigation establishes a promising synthetic pathway for industrial microbial production of fatty alcohols.     

Osmoregulation in Dunaliella,Part II: Photosynthesis and starch contribute carbon for glycerol synthesis during a salt stress in Dunaliella tertiolecta

In response to an osmotic stress, Dunaliella tertiolecta osmoregulates by metabolizing intracellular glycerol as compatible solute. Upon the application of a salt stress to 0.17 M or 0.7 M NaCl grown D. tertiolecta cells, rates of total glycerol synthesis were substantially higher than that arising from photosynthetic ¹⁴CO₂ fixation into glycerol. The source of this extra carbon is the reserve starch pool. The contribution of carbon from the starch breakdown to glycerol synthesis was estimated from the difference between the total glycerol synthesized and that arising from ¹⁴CO₂ fixation. The maximum observed flux of carbon from ¹⁴CO₂ to glycerol from photosynthesis was of the order of 15–20 μmol ¹⁴C-glycerol mg⁻¹ Chl h⁻¹, whereas the total glycerol synthesis reached about 70 μmol glycerol mg⁻¹ Chl h⁻¹. The contribution of products of starch breakdown to glycerol synthesis increased progressively with increasing salt stress. In light, contrary to prevailing assumptions, both the photosynthesis and the starch breakdown contribute carbon to glycerol biosynthesis. The relative contributions of these two processes in the light, while cells were actively photosynthesizing, depended on the magnitude of the salt stress. On application of dilution stress, the flux of carbon from newly photosynthetically fixed ¹⁴CO₂ into glycerol was reduced progressively with increasing dilution stress that was also accompanied by a decline in total glycerol contents of the cell. The maximum observed rate of glycerol dissimilation was about 135 μmol glycerol mg⁻¹ Chl h⁻¹.     

Production of a new exopolysaccharide (EPS) by Pseudomonas oleovorans NRRL B-14682 grown on glycerol

Pure glycerol and glycerol-rich product (GRP) obtained from the biodiesel industries were used as carbon source for the production of a new extracellular polysaccharide (EPS) by Pseudomonas oleovorans NRRL B-14682. The influence of temperature (20–40 °C) and pH (6.0–8.0) was studied. A temperature of 30 °C and pH control at 6.8 gave the maximum cell growth and EPS production. The culture attained a maximum cell dry weight (CDW) of 9.55 g l⁻¹ and an EPS concentration of 11.82 g l⁻¹ when cultivated with pure glycerol. GRP was a suitable carbon source, as shown by the slightly higher EPS concentration (12.18 g l⁻¹). The EPS productivity obtained with GRP (3.85 g l⁻¹ d⁻¹) was almost twice that obtained with pure glycerol (2.00 g l⁻¹ d⁻¹). Also, the yield on glycerol was higher for the cultivation with GRP (0.36 g g⁻¹) than for pure glycerol (0.28 g g⁻¹). The EPS was a high molecular weight heteropolysaccharide, composed by neutral sugars (37–80 wt% galactose, 2–30 wt% glucose, 0.5–25 wt% mannose and 0.5–20 wt% rhamnose) and containing acyl group substituents (pyruvil, acetyl and succinyl were identified). The EPS forms highly viscous aqueous dispersions with many potential commercial applications.     

Improvement of (R)-carbonyl reductase-mediated biosynthesis of (R)-1-phenyl-1,2-ethanediol by a novel dual-cosubstrate-coupled system for NADH recycling

An NAD(H)-dependent (R)-carbonyl reductase (RCR) from Candida parapsilosis catalyzes the asymmetric reduction of 2-hydroxyacetophenone (2-HAP) to (R)-1-phenyl-1,2-ethanediol ((R)-PED), which is a valuable chiral building block in the pharmaceutical and fine chemical industries. Biosynthesis efficiency of (R)-PED was considerably improved by a novel dual-cosubstrate-coupled system. By simultaneously employing isopropanol (10%, v v^?1) and glycerol (8%, v v^?1) as sacrificial cosubstrates, the (R)-PED product had an excellent optical purity of >99.9% and a conversion of 85.5%, which were nearly 2- and 11-fold higher than those without adding cosubstrate, respectively. Besides, the productivity was dramatically enhanced from 0.02 g L^?1 h^?1 to 5 g L^?1 h^?1, and the maximum acceptable concentration of 2-HAP was elevated to 10 g L^?1. Isopropanol was directly oxidized by RCR in the formation of NADH, while glycerol was metabolized by cellular enzymes to release NADH. Moreover, glycerol prevented cells from losing viability and alleviated the toxicity of isopropanol and acetone for cells. Interestingly, there was a cooperative interaction between isopropanol and glycerol for the improvement of biosynthesis efficiency of (R)-PED.     

Poly(3-hydroxybutyrate) production by Cupriavidus necator using waste glycerol

Polyhydroxyalkanoates (PHAs) have been recognized as good substitutes for the non-biodegradable petrochemically produced polymers. However, their high (real or estimated) current production cost limits their industrial applications. This work exploits two strategies to enhance PHAs substitution potential: the increase in PHA volumetric productivity in high density cultures and the use of waste glycerol (GRP), a by-product from the biodiesel industry, as primary carbon source for cell growth and polymer synthesis. Cupriavidus necator DSM 545 was used to accumulate poly(3-hydroxybutyrate) (P(3HB)) from GRP and from commercial glycerol (PG) as control substrate. On PG, productivities between 0.6 g_PHB L^?1 h^?1 and 1.5 g_PHB L^?1 h^?1 were attained. The maximum cell DW was 82.5 g_DW L^?1, the P(3HB) content being 62%. When GRP was used, 68.8 g_DW L^?1 with a P(3HB) accumulation of 38% resulting in a final productivity of 0.84 g_PHB L^?1 h^?1 was obtained. By decreasing the biomass concentration at which accumulation was triggered, a productivity of 1.1 g_PHB L^?1 h^?1 (50% P(3HB), w/w) was attained using GRP. P(3HB) molecular weights (M_w) ranged from 7.9 × 10⁵ to 9.6 × 10⁵ Da.     

Bioprocess strategies for mass multiplication of and metabolite synthesis by plant growth promoting pseudomonads for agronomical applications

The exponential substrate feeding (open-loop) and automated feedback substrate feeding (closed loop) strategies were developed to obtain high cell densities of fluorescent pseudomonad strains R62 and R81 and enhanced production of antifungal compound 2,4-diacetylphloroglucinol (DAPG) from glycerol as a sole carbon source. The exponential feeding strategy resulted in increased glycerol accumulation during the fed-batch cultivation when the predetermined specific growth rate (μ) was set at 0.10 or 0.20 h^?1 (<μ_m = 0.29 h^?1). Automated feeding strategies using dissolved oxygen (DO) or pH as feedback signals resulted in minimal to zero accumulation of glycerol for both the strains. In case of DO-based feeding strategy, biomass productivity of 0.24 g/(L h) and 0.29 g/(L h) was obtained for R62 and R81, respectively. Using pH-based feeding strategy, biomass productivity could be increased to a maximum of 0.51 and 0.54 g/(L h), for the strains R62 and R81, respectively, whereas the DAPG concentration was enhanced to 298 mg/L for R62 and 342 mg/L for R81 strains. These yields of DAPG are thus far the highest reported from GRAS organisms.     

Homo-succinic acid production by metabolically engineered Mannheimia succiniciproducens

Succinic acid (SA) is a four carbon dicarboxylic acid of great industrial interest that can be produced by microbial fermentation. Here we report development of a high-yield homo-SA producing Mannheimia succiniciproducens strain by metabolic engineering. The PALFK strain (ldhA^-, pta^-, ackA^-, fruA^-) was developed based on optimization of carbon flux towards SA production while minimizing byproducts formation through the integrated application of in silico genome-scale metabolic flux analysis, omics analyses, and reconstruction of central carbon metabolism. Based on in silico simulation, utilization of sucrose would enhance the SA production and cell growth rates, while consumption of glycerol would reduce the byproduct formation rates. Thus, sucrose and glycerol were selected as dual carbon sources to improve the SA yield and productivity, while deregulation of catabolite-repression was also performed in engineered M. succiniciproducens. Fed-batch fermentations of PALFK with low- and medium-density (OD₆₀₀ of 0.4 and 9.0, respectively) inocula produced 69.2 and 78.4 g/L of homo-SA with yields of 1.56 and 1.64 mol/mol glucose equivalent and overall volumetric SA productivities of 2.50 and 6.02 g/L/h, respectively, using sucrose and glycerol as dual carbon sources. The SA productivity could be further increased to 38.6 g/L/h by employing a membrane cell recycle bioreactor system. The systems metabolic engineering strategies employed here for achieving homo-SA production with the highest overall performance indices reported to date will be generally applicable for developing superior industrial microorganisms and competitive processes for the bio-based production of other chemicals as well.     

Performance evaluation of various bioreactors for the removal of Cr(VI) and organic matter from industrial effluent

Performances of various bioreactors under different operating conditions were evaluated with respect to hexavalent chromium (Cr(VI)) reduction and COD removal. Continuous reactor studies were carried out with (i) aerobic suspended growth system, (ii) aerobic attached growth system, and (iii) anoxic attached growth system, using both synthetic and actual industrial wastewater. Arthrobacter rhombi-RE (MTCC7048), a Cr(VI) reducing strain enriched and isolated from chromium contaminated soil, was used in all the bioreactors for Cr(VI) biotransformation and COD removal. Aerobic and anoxic batch experiments were conducted to evaluate the bio-kinetic parameters. The bio-kinetic parameters for aerobic system were: μ_max = 2.34/d, K_s = 190 mg/L (as COD), K_i = 3.8 mg/L of Cr(VI), and Y_T = 0.377. These parameters for anoxic conditions were: μ_max = 0.57/d, K_s = 710 mg/L (as COD), K_i = 8.77 mg/L of Cr(VI), and Y_T = 0.13. Aerobic attached growth system, operated at a hydraulic retention time (HRT) of 24 h and an organic loading rate (OLR) of 3 kg/m³/d, performed better than aerobic suspended and the anoxic attached growth systems operated under identical conditions, while treating synthetic wastewater as well as industrial effluent.     

Selection of an endogenous 2,3-butanediol pathway in Escherichia coli by fermentative redox balance

Fermentative redox balance has long been utilized as a metabolic evolution platform to improve efficiency of NADH-dependent pathways. However, such system relies on the complete recycling of NADH and may become limited when the target pathway results in excess NADH stoichiometrically. In this study, endogenous capability of Escherichia coli for 2,3-butanediol (2,3-BD) synthesis was explored using the anaerobic selection platform based on redox balance. To address the issue of NADH excess associated with the 2,3-BD pathway, we devised a substrate-decoupled system where a pathway intermediate is externally supplied in addition to the carbon source to decouple NADH recycling ratio from the intrinsic pathway stoichiometry. In this case, feeding of the 2,3-BD precursor acetoin effectively restored anaerobic growth of the mixed-acid fermentation mutant that remained otherwise inhibited even in the presence of a functional 2,3-BD pathway. Using established 2,3-BD dehydrogenases as model enzyme, we verified that the redox-based selection system is responsive to NADPH-dependent reactions but with lower sensitivity. Based on this substrate-decoupled selection scheme, we successfully identified the glycerol/1,2-propanediol dehydrogenase (Ec-GldA) as the major enzyme responsible for the acetoin reducing activity (k_cat/K_m≈0.4 mM⁻¹ s⁻¹) observed in E. coli. Significant shift of 2,3-BD configuration upon withdrawal of the heterologous acetolactate decarboxylase revealed that the endogenous synthesis of acetoin occurs via diacetyl. Among the predicted diacetyl reductase in E. coli, Ec-UcpA displayed the most significant activity towards diacetyl reduction into acetoin (V_max≈6 U/mg). The final strain demonstrated a meso-2,3-BD production titer of 3 g/L without introduction of foreign genes. The substrate-decoupled selection system allows redox balance regardless of the pathway stoichiometry thus enables segmented optimization of different reductive pathways through enzyme bioprospecting and metabolic evolution.     

The effect of glycerol mixed substrate on the heterologous production of a Rhizopus oryzae lipase in Pichia pastoris system

A recombinant Rhizopus oryzae lipase producing Mut^s Pichia pastoris strain was used as a model organism to study the effect of mixed substrates (glycerol and methanol) on the specific product productivity. Different fed-batch cultivations were performed under three constant specific growth rates (0.02, 0.05 and 0.1 h⁻¹), maintaining a constant methanol concentration of 2 g l⁻¹.At the lowest μ tested (0.02 h⁻¹), the specific productivity was 1.23 and 1.61 fold higher and the specific methanol consumption rate (q_sMeOH) was 3 and 3.5 fold higher than values obtained when μ was 0.05 and 0.1 h⁻¹, respectively. This implies a relation between the q_sMeOH and the specific productivity, yielding higher specific productivities whenever the consumption of methanol is higher. Although glycerol was maintained under limiting conditions in all μ tested, when the relation between the μ_Gly and μ_MeOH was larger than 4, an important decrease on the maximal activity value was observed.Finally, a comparison under the same conditions using glycerol or sorbitol as co-substrates was also performed, obtaining better specific productivity when sorbitol was used. In addition, protease activity was detected when glycerol was used as co-substrate.     

Growth of eight Gambierdiscus (Dinophyceae) species: Effects of temperature,salinity and irradiance

Little is known about how the growth of individual Gambierdiscus species responds to environmental factors. This study examined the effects of temperature (15–34 °C), salinity (15–41) and irradiance (2–664 μmol photons m⁻² s⁻¹) on growth of Gambierdiscus: G. australes, G. belizeanus, G. caribaeus, G. carolinianus, G. carpenteri, G. pacificus and G. ruetzleri and one putative new species, Gambierdiscus ribotype 2. Depending on species, temperatures where maximum growth occurred varied between 26.5 and 31.1 °C. The upper and lower thermal limits for all species were between 31–34 °C and 15–21 °C, respectively. The shapes of the temperature vs. growth curves indicated that even small differences of 1–2 °C notably affected growth potentials. Salinities where maximum growth occurred varied between 24.7 and 35, while the lowest salinities supporting growth ranged from <14 to 20.9. These data indicated that Gambierdiscus species are more tolerant of lower salinities than is generally appreciated. Growth of all species began to decline markedly as salinities exceed 35.1–39.4. The highest salinity tested in this study (41), however, was lethal to only one species, Gambierdiscus ribotype 2. The combined salinity data indicated that differences in salinity regimes may affect relative species abundances and distributions, particularly when salinities are <20 and >35. All eight Gambierdiscus species were adapted to relatively low light conditions, exhibiting growth maxima at 50–230 μmol photons m⁻² s⁻¹ and requiring only 6–17 μmol photons m⁻² s⁻¹ to maintain growth. These low light requirements indicate that Gambierdiscus growth can occur up to 150 m depth in tropical waters, with optimal light regimes often extending to 75 m. The combined temperature, salinity and light requirements of Gambierdiscus can be used to define latitudinal ranges and species-specific habitats, as well as to inform predictive models.     

Urine nitrification with a synthetic microbial community

During long-term extra-terrestrial missions, food is limited and waste is generated. By recycling valuable nutrients from this waste via regenerative life support systems, food can be produced in space. Astronauts’ urine can, for instance, be nitrified by micro-organisms into a liquid nitrate fertilizer for plant growth in space. Due to stringent conditions in space, microbial communities need to be be defined (gnotobiotic); therefore, synthetic rather than mixed microbial communities are preferred. For urine nitrification, synthetic communities face challenges, such as from salinity, ureolysis, and organics.In this study, a synthetic microbial community containing an AOB (Nitrosomonas europaea), NOB (Nitrobacter winogradskyi), and three ureolytic heterotrophs (Pseudomonas fluorescens, Acidovorax delafieldii, and Delftia acidovorans) was compiled and evaluated for these challenges. In reactor 1, salt adaptation of the ammonium-fed AOB and NOB co-culture was possible up to 45 mS cm⁻¹, which resembled undiluted nitrified urine, while maintaining a 44 ± 10 mg NH₄⁺–N L⁻¹ d⁻¹ removal rate. In reactor 2, the nitrifiers and ureolytic heterotrophs were fed with urine and achieved a 15 ± 6 mg NO₃⁻–N L⁻¹ d⁻¹ production rate for 1% and 10% synthetic and fresh real urine, respectively. Batch activity tests with this community using fresh real urine even reached 29 ± 3 mg N L⁻¹ d⁻¹. Organics removal in the reactor (69 ± 15%) should be optimized to generate a nitrate fertilizer for future space applications.     

Optimization of biomass production with enhanced glucan and dietary fibres content by Pleurotus ostreatus ATHUM 4438 under submerged culture

This work was aimed at optimizing biomass production by the edible basidiomycete Pleurotus ostreatus ATHUM 4438 in a submerged process with enhanced glucan and dietary fibres content. β-Glucan from Pleurotus sp. (pleuran) has been used as food supplements due to its immunosuppressive activity. Like other dietary fibre components, oyster mushroom polysaccharides can stimulate the growth of colon microorganisms (probiotics), i.e. act as prebiotics. We used the FF MicroPlate for substrate utilization and growth monitoring. The pattern of substrate catabolism forms a substrate assimilation fingerprint which is useful in selecting media components for media optimization of maximum biomass production. Different carbon sources (95) were used and then 8 of them were tested in shake flask cultures. The effect of various organic and complex nitrogen sources on biomass production was also examined and response surface methodology based on central composite design was applied to explore the optimal medium composition. When the optimized culture medium was tested in a 20-L stirred tank bioreactor, using 57 g L⁻¹ xylose and 37 g L⁻¹ corn steep liquor, high yields (39.2 g L⁻¹) of dry biomass was obtained. The yield coefficients for total glucan and dietary fibres on mycelial biomass formed were 140 ± 4 and 625 ± 9 mg g⁻¹ mycelium dry weight, respectively.     

Factors controlling Zostera marina L. growth in the eastern and western Pacific Ocean: Comparisons between Korea and Oregon,USA

Zostera marina distribution is circum-global and tolerates a wide range of environmental conditions. Consequently, it is likely that populations have adapted to local environmental conditions of light, temperature and nutrient supply. We compared Z. marina growth dynamics over a 2-year period in relation to environmental characters at Jindong Bay, South Korea and Yaquina Bay, Oregon, USA. Water temperature in Jindong Bay showed stronger seasonal variation (summer–winter ΔT = 20 °C) than in Yaquina Bay (summer–winter ΔT < 5 °C). Underwater irradiance in Jindong Bay exhibited a winter maximum, while in Yaquina Bay underwater light exhibited a summer maximum. Integrated annual underwater irradiance during 2003 was 2200 and 1200 mol photons m⁻² year⁻¹ in Korea and Oregon, respectively. Z. marina shoot density, biomass and integrated production were not significantly different between the two study sites. Seasonal Z. marina growth in Jindong Bay appeared to be controlled by temperature and light, while the growth pattern in Yaquina Bay suggested light regulation. Several seagrass parameters were correlated to phosphate concentrations, even though nutrients did not appear limiting. Despite differences in environmental factors, relative growth rates and temporal growth dynamics between study sites, integrated annual leaf production was quite similar at 335 and 353 g DW m⁻² year⁻¹ in the Jindong and Yaquina Bay study sites. We suggest that Z. marina net productivity is acclimated to the local environmental conditions and may be a general characteristic of temperate seagrass populations.     

Production of the postharvest biocontrol agent Bacillus subtilis CPA-8 using low cost commercial products and by-products

The aim of this research was to identify a low cost medium based on commercial products and by-products that provided maximum Bacillus subtilis CPA-8 growth and maintained biocontrol efficacy. Low cost media combining economical nitrogen and carbon sources such as yeast extract, peptone, soy products, sucrose, maltose and molasses were tested. Tests were carried out in 250-ml flasks containing 50 ml of each tested medium. Maximum cell growth (>3 × 10⁹ CFU ml^?1) was obtained in defatted soy flour 44% combined with sucrose or molasses media. Second, CPA-8 production was scaled up in a 5-l fermenter and CPA-8 population dynamics, pH and oxygen consumption in the optimized medium (defatted soy flour 44% – molasses) was recorded. In these tests, there was a 5-h lag phase before growth, after which exponential growth occurred and maximum production was 3 × 10⁹ CFU ml^?1 after 20 h. Fruit trials with cells and cell free supernatants from CPA-8 grown in optimized medium maintained biocontrol efficacy against Monilinia fructicola on peaches, resulting in disease reductions up to 95%. CPA-8 populations survived in wounds on inoculated peaches, regardless of the culture media used. The results show that B. subtilis CPA-8 can be produced in a low cost medium combining inexpensive nitrogen and carbon sources (40 g l^?1 defatted soy flour 44%, 5 g l^?1 molasses plus mineral trace supplements) in shake flasks and a laboratory fermenter (5 l). The results could be used to provide a reliable basis for scaling up the fermentation process to an industrial level.     

Bio-based succinate from sucrose: High-resolution 13C metabolic flux analysis and metabolic engineering of the rumen bacterium Basfia succiniciproducens

Succinic acid is a platform chemical of recognized industrial value and accordingly faces a continuous challenge to enable manufacturing from most attractive raw materials. It is mainly produced from glucose, using microbial fermentation. Here, we explore and optimize succinate production from sucrose, a globally applied substrate in biotechnology, using the rumen bacterium Basfia succiniciproducens DD1. As basis of the strain optimization, the yet unknown sucrose metabolism of the microbe was studied, using ¹³C metabolic flux analyses. When grown in batch culture on sucrose, the bacterium exhibited a high succinate yield of 1 mol mol⁻¹ and a by-product spectrum, which did not match the expected PTS-mediated sucrose catabolism. This led to the discovery of a fructokinase, involved in sucrose catabolism. The flux approach unraveled that the fructokinase and the fructose PTS both contribute to phosphorylation of the fructose part of sucrose. The contribution of the fructokinase reduces the undesired loss of the succinate precursor PEP into pyruvate and into pyruvate-derived by-products and enables increased succinate production, exclusively via the reductive TCA cycle branch. These findings were used to design superior producers. Mutants, which (i) overexpress the beneficial fructokinase, (II) lack the competing fructose PTS, and (iii) combine both traits, produce significantly more succinate. In a fed-batch process, B. succiniciproducens ΔfruA achieved a titer of 71 g L⁻¹ succinate and a yield of 2.5 mol mol⁻¹ from sucrose.     

Recycling biodiesel-derived glycerol by the oleaginous yeast Rhodosporidium toruloides Y4 through the two-stage lipid production process

Direct utilization of crude glycerol, a major byproduct in biodiesel industry, becomes imperative, because its production has outpaced the demand recently. We demonstrated that the oleaginous yeast Rhodosporidium toruloides Y4 had a great capacity to convert glycerol into lipids with high yield using the two-stage production process. Significantly higher cell mass and lipid yield were observed when the media were made with synthetic crude glycerol than pure glycerol. The process achieved a lipid yield of 0.22 g g⁻¹ glycerol, which was comparable with the lipid yield using glucose as the substrate. Lipid samples showed similar fatty acid compositional profiles to those of vegetable oils, suggesting that such microbial lipids were potential feedstock for biodiesel production. Our data provided an attractive route to integrate biodiesel production with microbial lipid technology for better resource efficiency and economical viability.