Poly-β-hydroxybutyrate/ectoine co-production by ectoine-excreting strain Halomonas salina

The ectoine-excreting bacterial strain of Halomonas salina was employed in the co-production of poly-β-hydroxybutyrate (PHB) and ectoine (Ect) during a fermentation process (PHB/Ect co-production). An efficient PHB/Ect co-production process was carried out at low NaCl concentration (30 g L⁻¹). It was established using ¹H Nuclear Magnetic Resonance spectroscopy that H. salina produces PHB. The effects of the NaCl concentration, the initial C/N ratio, the phosphate concentration and mixed carbon sources were investigated with respect to PHB/Ect co-production. The PHB/Ect co-production system comprised growing and non-growing cell phases and was developed with NaCl concentration of 30 g L⁻¹. The optimal conditions for PHB/Ect co-production by the ectoine-excreting strain of H. salina were 30 g L⁻¹ NaCl, with an initial C/N ratio of 15, an initial phosphate concentration of 12 g L⁻¹ and mixed carbon sources of 55 g L⁻¹ glucose and 25 g L⁻¹ monosodium glutamate. Using a PHB/Ect co-production system with growing and non-growing cell phases prevents the inhibition of PHB synthesis by high concentration of NaCl and significantly reduces ectoine degradation. PHB and ectoine concentrations as high as 35.3 g L⁻¹ and 8.6 g L⁻¹, respectively, were achieved. The efficient co-production of PHB and ectoine at a low NaCl concentration has been realised.     

Metabolic engineering of Corynebacterium glutamicum for the production of itaconate

The capability of Corynebacterium glutamicum for glucose-based synthesis of itaconate was explored, which can serve as building block for production of polymers, chemicals, and fuels. C. glutamicum was highly tolerant to itaconate and did not metabolize it. Expression of the Aspergillus terreus CAD1 gene encoding cis-aconitate decarboxylase (CAD) in strain ATCC13032 led to the production of 1.4 mM itaconate in the stationary growth phase. Fusion of CAD with the Escherichia coli maltose-binding protein increased its activity and the itaconate titer more than two-fold. Nitrogen-limited growth conditions boosted CAD activity and itaconate titer about 10-fold to values of 1440 mU mg⁻¹ and 30 mM. Reduction of isocitrate dehydrogenase activity via exchange of the ATG start codon to GTG or TTG resulted in maximal itaconate titers of 60 mM (7.8 g l⁻¹), a molar yield of 0.4 mol mol⁻¹, and a volumetric productivity of 2.1 mmol l⁻¹ h⁻¹.     

Improvement of succinate production by overexpression of a cyanobacterial carbonic anhydrase in Escherichia coli

Succinate fermentation was investigated in Escherichia coli strains overexpressing cyanobacterium Anabaena sp. 7120 ecaA gene encoding carbonic anhydrase (CA). In strain BL21 (DE3) bearing ecaA, the activity of CA was 21.8 U mg⁻¹ protein, whereas non-detectable CA activity was observed in the control strain. Meanwhile, the activity of phosphoenolpyruvate carboxylase (PEPC) increased from 0.2 U mg⁻¹ protein to 1.13 U mg⁻¹ protein. The recombinant bearing ecaA reached a succinate yield of 0.39 mol mol⁻¹ glucose at the end of the fermentation. It was 2.1-fold higher than that of control strain which was just 0.19 mol mol⁻¹ glucose. EcaA gene was also introduced into E. coli DC1515, which was deficient in glucose phosphotransferase, lactate dehydrogenase and pyruvate:formate lyase. Succinate yield can be further increased to 1.26 mol mol⁻¹ glucose. It could be concluded that the enhancement of the supply of HCO₃⁻ in vivo by ecaA overexpression is an effective strategy for the improvement of succinate production in E. coli.     

Metabolic engineering of Clostridium acetobutylicum for butyric acid production with high butyric acid selectivity

A typical characteristic of the butyric acid-producing Clostridium is coproduction of both butyric and acetic acids. Increasing the butyric acid selectivity important for economical butyric acid production has been rather difficult in clostridia due to their complex metabolic pathways. In this work, Clostridium acetobutylicum was metabolically engineered for highly selective butyric acid production. For this purpose, the second butyrate kinase of C. acetobutylicum encoded by the bukII gene instead of butyrate kinase I encoded by the buk gene was employed. Furthermore, metabolic pathways were engineered to further enhance the NADH-driving force. Batch fermentation of the metabolically engineered C. acetobutylicum strain HCBEKW (pta⁻, buk⁻, ctfB⁻ and adhE1⁻) at pH 6.0 resulted in the production of 32.5 g/L of butyric acid with a butyric-to-acetic acid ratio (BA/AA ratio) of 31.3 g/g from 83.3 g/L of glucose. By further knocking out the hydA gene (encoding hydrogenase) in the HCBEKW strain, the butyric acid titer was not further improved in batch fermentation. However, the BA/AA ratio (28.5 g/g) obtained with the HYCBEKW strain (pta⁻, buk⁻, ctfB⁻, adhE1⁻ and hydA⁻) was 1.6 times higher than that (18.2 g/g) obtained with the HCBEKW strain at pH 5.0, while no improvement was observed at pH 6.0. These results suggested that the buk gene knockout was essential to get a high butyric acid selectivity to acetic acid in C. acetobutylicum.     

Engineering cofactor and transport mechanisms in Saccharomyces cerevisiae for enhanced acetyl-CoA and polyketide biosynthesis

Synthesis of polyketides at high titer and yield is important for producing pharmaceuticals and biorenewable chemical precursors. In this work, we engineered cofactor and transport pathways in Saccharomyces cerevisiae to increase acetyl-CoA, an important polyketide building block. The highly regulated yeast pyruvate dehydrogenase bypass pathway was supplemented by overexpressing a modified Escherichia coli pyruvate dehydrogenase complex (PDHm) that accepts NADP⁺ for acetyl-CoA production. After 24 h of cultivation, a 3.7-fold increase in NADPH/NADP⁺ ratio was observed relative to the base strain, and a 2.2-fold increase relative to introduction of the native E. coli PDH. Both E. coli pathways increased acetyl-CoA levels approximately 2-fold relative to the yeast base strain. Combining PDHm with a ZWF1 deletion to block the major yeast NADPH biosynthesis pathway resulted in a 12-fold NADPH boost and a 2.2-fold increase in acetyl-CoA. At 48 h, only this coupled approach showed increased acetyl-CoA levels, 3.0-fold higher than that of the base strain. The impact on polyketide synthesis was evaluated in a S. cerevisiae strain expressing the Gerbera hybrida 2-pyrone synthase (2-PS) for the production of the polyketide triacetic acid lactone (TAL). Titers of TAL relative to the base strain improved only 30% with the native E. coli PDH, but 3.0-fold with PDHm and 4.4-fold with PDHm in the Δzwf1 strain. Carbon was further routed toward TAL production by reducing mitochondrial transport of pyruvate and acetyl-CoA; deletions in genes POR2, MPC2, PDA1, or YAT2 each increased titer 2–3-fold over the base strain (up to 0.8 g/L), and in combination to 1.4 g/L. Combining the two approaches (NADPH-generating acetyl-CoA pathway plus reduced metabolite flux into the mitochondria) resulted in a final TAL titer of 1.6 g/L, a 6.4-fold increase over the non-engineered yeast strain, and 35% of theoretical yield (0.16 g/g glucose), the highest reported to date. These biological driving forces present new avenues for improving high-yield production of acetyl-CoA derived compounds.     

Direct bioconversion of d-xylose to 1,2,4-butanetriol in an engineered Escherichia coli

The compound 1,2,4-butanetriol (BT) is a valuable chemical used in the production of plasticizers, polymers, cationic lipids and other medical applications, and is conventionally produced via hydrogenation of malate. In this report, BT is biosynthesized by an engineered Escherichia coli from d-xylose. The pathway: d-xylose → d-xylonate → 2-keto-3-deoxy-d-xylonate → 3,4-dihydroxybutanal → BT, was constructed in E. coli by recruiting a xylose dehydrogenase and a keto acid decarboxylase from Caulobacter crescentus and Pseudomonas putida, respectively. Authentic BT was detected from cultures of the engineered strain. Further improvement on the strain was performed by blocking the native d-xylose and d-xylonate metabolic pathways which involves disruption of xylAB, yjhH and yagE genes in the host chromosome. The final construct produced 0.88 g L⁻¹ BT from 10 g L⁻¹ d-xylose with a molar yield of 12.82%. By far, this is the first report on the direct production of BT from d-xylose by a single microbial host. This may serve as a starting point for further metabolic engineering works to increase the titer of BT toward industrial scale viability.     

Escherichia coli HGT: Engineered for high glucose throughput even under slowly growing or resting conditions

Aerobic production-scale processes are constrained by the technical limitations of maximum oxygen transfer and heat removal. Consequently, microbial activity is often controlled via limited nutrient feeding to maintain it within technical operability. Here, we present an alternative approach based on a newly engineered Escherichia coli strain. This E. coli HGT (high glucose throughput) strain was engineered by modulating the stringent response regulation program and decreasing the activity of pyruvate dehydrogenase. The strain offers about three-fold higher rates of cell-specific glucose uptake under nitrogen-limitation (0.6 g_Glc g_CDW⁻¹ h⁻¹) compared to that of wild type, with a maximum glucose uptake rate of about 1.8 g_Glc g_CDW⁻¹ h⁻¹ already at a 0.3 h⁻¹ specific growth rate. The surplus of imported glucose is almost completely available via pyruvate and is used to fuel pyruvate and lactate formation. Thus, E. coli HGT represents a novel chassis as a host for pyruvate-derived products.     

2,3-Butanediol production using Klebsiella oxytoca ATCC 8724: Evaluation of biomass derived sugars and fed-batch fermentation process

For this study, 2,3-butanediol (BD) fermentation from pure and biomass-derived sugar were optimized in shake-flask and 5-L bioreactor levels using Klebsiella oxytoca ATCC 8724. The results showed that 70 g/L of single sugar (glucose or xylose) and 90 g/L of mixed-sugar (glucose:xylose = 2:1) were optimum concentrations for efficient 2,3-BD fermentation. At optimum sugar concentrations, 2,3-BD productivities were 1.03, 0.64 and 0.50 gL⁻¹ h⁻¹, and yields were 0.43, 0.36 and 0.35 g/g in glucose, xylose and mixed-sugar medium, respectively. The lack of simultaneous utilization of glucose and xylose led to the lowest productivity in the mixed-sugar medium. Detoxification of biomass hydrolyzates was necessary for efficient 2,3-BD fermentation when sugar concentrations in the medium was 90 g/L or higher, but not with sugar concentrations of 30 g/L or less. A fed-batch fermentation using glucose medium led to an increase 2,3-BD titer to 79.4 g/L and yields 0.47 g/g, while productivity decreased to 0.79 gL⁻¹ h⁻¹. However, the fed-batch process was inefficient using mixed-sugar and biomass hydrolyzates because of poor xylose utilization. These results indicated that appropriate biomass processing technologies must be developed to generate separate glucose and xylose streams to produce high 2,3-BD titer from biomass-derived sugar using a fed-batch process.     

Influence of specific growth rate over the secretory expression of recombinant potato carboxypeptidase inhibitor in fed-batch cultures of Escherichia coli

A high cell density cultivation protocol was developed for the secretory production of potato carboxypeptidase inhibitor (PCI) in Escherichia coli. The strain BW25113 (pIMAM3) was cultured in fed-batch mode employing minimal media and an exponential feed profile where the specific growth rate was fixed by limitation of the fed carbon source (glycerol). Plasmid loss rates were found to be proportional to the specific growth rate. Distribution of PCI along the cell compartments and the culture media was also dependent on the fixed growth rate. When specific growth rate was kept at μ = 0.10 h⁻¹, 1.4 g PCI L⁻¹ were obtained when adding the product present in periplasmic extracts and supernatant fractions, with a 50% of the total expressed protein recovered from the extracellular medium. This constituted a 1.2-fold increase compared to growth at μ = 0.15 h⁻¹, and 2.0-fold compared to μ = 0.25 h⁻¹. Last, a cell permeabilization treatment with Triton X-100 and glycine was employed to direct most of the product to the culture media, achieving over 81% of extracellular PCI. Overall, our results point out that production yields of secretory proteins in fed-batch cultures of E. coli can be improved by means of process variables, with applications to the production of small disulfide-bridged proteins. Overall, our results point out that control of the specific growth rate is a successful strategy to improve the production yields of secretory expression in fed-batch cultures of E. coli, with applications to the production of small disulfide-bridged proteins.     

Simultaneous saccharification and fermentation of kraft pulp by recombinant Escherichia coli for phenyllactic acid production

Simultaneous saccharification and fermentation (SSF) of renewable cellulose for the production of 3-phenyllactic acid (PhLA) by recombinant Escherichia coli was investigated. Kraft pulp recovered from biomass fractionation processes was used as a model cellulosic feedstock and was hydrolyzed using 10–50 filter paper unit (FPU) g⁻¹ kraft pulp of a commercial cellulase mixture, which increased the glucose yield from 21% to 72% in an enzyme dose-dependent manner. PhLA fermentation of the hydrolyzed kraft pulp by a recombinant E. coli strain expressing phenylpyruvate reductase from Wickerhamia fluorescens TK1 produced 1.9 mM PhLA. The PhLA yield obtained using separate hydrolysis and fermentation was enhanced from 5.8% to 42% by process integration into SSF of kraft pulp (20 g L⁻¹) in a complex medium (pH 7.0) at 37 °C. The PhLA yield was negatively correlated with the initial glucose concentration, with a five-fold higher PhLA yield observed in culture medium containing 10 g L⁻¹ glucose compared to 100 g L⁻¹. Taken together, these results suggest that the PhLA yield from cellulose in kraft pulp can be improved by SSF under glucose-limited conditions.     

Complete genome sequence,metabolic model construction and phenotypic characterization of Geobacillus LC300, an extremely thermophilic,fast growing,xylose-utilizing bacterium

We have isolated a new extremely thermophilic fast-growing Geobacillus strain that can efficiently utilize xylose, glucose, mannose and galactose for cell growth. When grown aerobically at 72 °C, Geobacillus LC300 has a growth rate of 2.15 h⁻¹ on glucose and 1.52 h⁻¹ on xylose (doubling time less than 30 min). The corresponding specific glucose and xylose utilization rates are 5.55 g/g/h and 5.24 g/g/h, respectively. As such, Geobacillus LC300 grows 3-times faster than E. coli on glucose and xylose, and has a specific xylose utilization rate that is 3-times higher than the best metabolically engineered organism to date. To gain more insight into the metabolism of Geobacillus LC300 its genome was sequenced using PacBio׳s RS II single-molecule real-time (SMRT) sequencing platform and annotated using the RAST server. Based on the genome annotation and the measured biomass composition a core metabolic network model was constructed. To further demonstrate the biotechnological potential of this organism, Geobacillus LC300 was grown to high cell-densities in a fed-batch culture, where cells maintained a high xylose utilization rate under low dissolved oxygen concentrations. All of these characteristics make Geobacillus LC300 an attractive host for future metabolic engineering and biotechnology applications.     

Over-production of human interferon-γ by HCDC of recombinant Escherichia coli

A simple fed-batch process was developed using a modified variable specific growth rate feeding strategy for high cell density cultivation of Escherichia coli BL21 (DE3) expressing human interferon-gamma (hIFN-γ). The feeding rate was adjusted to achieve the maximum attainable specific growth rate during fed-batch cultivation. In this method, specific growth rate was changed from a maximum value of 0.55 h⁻¹ at the beginning of feeding and then it was reduced to 0.4 h⁻¹ at induction time.The final concentration of biomass and IFN-γ was reached to ∼115 g l⁻¹ (DCW) and 42.5 g(hIFN-γ) l⁻¹ after 16.5 h, also the final specific yield and overall productivity of recombinant hIFN-γ (rhIFN-γ) were obtained 0.37 g(hIFN-γ) g⁻¹ DCW and 2.57 g(hIFN-γ) l⁻¹ h⁻¹, respectively. According to available data this is the highest specific yield and productivity that has been reported for recombinant proteins production yet.     

Increased production of L-serine in Escherichia coli through Adaptive Laboratory Evolution

L-serine is a promising building block biochemical with a high theoretical production yield from glucose. Toxicity of L-serine is however prohibitive for high-titer production in E. coli. Here, E. coli lacking L-serine degradation pathways was evolved for improved tolerance by gradually increasing L-serine concentration from 3 to 100 g/L using adaptive laboratory evolution (ALE). Genome sequencing of isolated clones revealed multiplication of genetic regions, as well as mutations in thrA, thereby showing a potential mechanism of serine inhibition. Additional mutations were evaluated by MAGE combined with amplicon sequencing, revealing role of rho, lrp, pykF, eno, and rpoB on tolerance and fitness in minimal medium. Production using the tolerant strains resulted in 37 g/L of L-serine with a 24% mass yield. The resulting titer is similar to the highest production reported for any organism thereby highlighting the potential of ALE for industrial biotechnology.     

Over expression of GroESL in Cupriavidus necator for heterotrophic and autotrophic isopropanol production

We previously reported a metabolic engineering strategy to develop an isopropanol producing strain of Cupriavidus necator leading to production of 3.4 g L⁻¹ isopropanol. In order to reach higher titers, isopropanol toxicity to the cells has to be considered. A toxic effect of isopropanol on the growth of C. necator has been indeed observed above a critical value of 15 g L⁻¹. GroESL chaperones were first searched and identified in the genome of C. necator. Native groEL and groES genes from C. necator were over-expressed in a strain deleted for PHA synthesis. We demonstrated that over-expressing groESL genes led to a better tolerance of the strain towards exogenous isopropanol. GroESL genes were then over-expressed within the best engineered isopropanol producing strain. A final isopropanol concentration of 9.8 g L⁻¹ was achieved in fed-batch culture on fructose as the sole carbon source (equivalent to 16 g L⁻¹ after taking into account evaporation). Cell viability was slightly improved by the chaperone over-expression, particularly at the end of the fermentation when the isopropanol concentration was the highest. Moreover, the strain over-expressing the chaperones showed higher enzyme activity levels of the 2 heterologous enzymes (acetoacetate carboxylase and alcohol dehydrogenase) of the isopropanol synthetic operon, translating to a higher specific production rate of isopropanol at the expense of the specific production rate of acetone. Over-expressing the native chaperones led to a 9–18% increase in the isopropanol yield on fructose.     

Galactose-limited fed-batch cultivation of Escherichia coli for the production of lacto-N-tetraose

Lacto-N-tetraose (Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc) is one of the most abundant oligosaccharide structures in human milk. We recently described the synthesis of lacto-N-tetraose by a whole-cell biotransformation with recombinant Escherichia coli cells. However, only about 5% of the lactose was converted into lacto-N-tetraose by this approach. The major product obtained was the intermediate lacto-N-triose II (GlcNAc(β1-3)Gal(β1-4)Glc).In order to improve the bioconversion of lactose to lacto-N-tetraose, we have investigated the influence of the carbon source on the formation of lacto-N-tetraose and on the intracellular availability of the glycosyltransferase substrates, UDP-N-acetylglucosamine and UDP-galactose. By growth of the recombinant E. coli cells on D-galactose, the yield of lacto-N-tetraose (810.8 mg L⁻¹ culture) was 3.6-times higher compared to cultivation on D-glucose.Using fed-batch cultivation with galactose as sole energy and carbon source, a large-scale synthesis of lacto-N-tetraose was demonstrated. During the 26 h feeding phase the growth rate (μ = 0.05) was maintained by an exponential galactose feed. In total, 16 g L⁻¹ lactose were fed and resulted in final yields of 12.72 ± 0.21 g L⁻¹ lacto-N-tetraose and 13.70 ± 0.10 g L⁻¹ lacto-N-triose II. In total, 173 g of lacto-N-tetraose were produced with a space-time yield of 0.37 g L⁻¹ h⁻¹.     

Survivability of indigenous microflora and a Salmonella typhimurium marker strain in poultry mash treated with buffered propionic acid

Buffered propionic acid (BPA) was evaluated as a potential treatment for the elimination of Salmonella spp. in poultry mash. A primary poultry isolate marker strain of Salmonella typhimurium was added as either a broth or in a dry chalk carrier form to poultry mash containing soybean meal as a protein supplement. The mash was supplemented with buffered propionic acid at 2, 4, 6, 8, 10, 20, 30, 50 and 100 g kg⁻¹ diet and samples were enumerated for indigenous aerobic bacteria, fungi and the S. typhimurium marker strain. Total indigenous aerobic bacteria and fungal populations were generally decreased by addition of more than 20 g BPA kg⁻¹, but an addition of 100 g BPA kg⁻¹ mash was usually required to achieve reductions of approximately 90% of indigenous aerobic bacteria and 99% of indigenous fungi. After 7 days of storage, 8 g BPA kg⁻¹ mash also reduced S. typhimurium populations by more than 90% in mash inoculated via chalk, while at least 50 g BPA kg⁻¹ mash was required to provide the same level of reduction in mash inoculated with a liquid culture of S. typhimurium. Although BPA does not appear to be an overly effective antimicrobial agent with respect to indigenous aerobic bacterial populations in animal feed, higher concentrations may have the potential for reducing fungal and Salmonella spp. contamination in poultry mash.     

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.     

Production of an anti-MUC1 C595 dbFv antibody fragment in recombinant Escherichia coli

The production of C595 diabody fragment (dbFv) in Escherichia coli (E. coli) HB2151 clone has been explored. The comparison of fermentation processes mode demonstrated that a higher biomass inoculum operation enhanced C595 dbFv production. It was demonstrated that a concentration of 12.1 mg l⁻¹ broth of dbFv and a cell concentration of 23.6 g l⁻¹ broth were achieved at the end of 75 l fermentation.     

Ectoine improves yield of biodiesel catalyzed by immobilized lipase

To improve the production of biodiesel by enzymatic conversion of triglycerides in cottonseed oil, compatible solutes were added to the solvent-free methanolysis system to prevent competitive methanol inhibition on the immobilized lipase (Novozym^® 435). The results indicated that the addition of ectoine increased biodiesel synthesis using a three-step methanol addition process. The concentration of methyl ester (ME) reached a maximum of 95.0% in the presence of 1.1 mmol/l ectoine, an increase of 20.9% compared to that in the absence of ectoine. On the other hand, excess ectoine decreased the ME concentration. Ectoine was also shown to enhance reuse of the immobilized lipase, significantly improving ME concentrations in each recycling test. Total concentrations of ME with added ectoine were about 1.5 times that without ectoine during five recycling tests (molar ratio of cottonseed oil to methanol, 1:4). Enzymatic reaction kinetics showed, in the concentration ranges of 0.8–1.14 mol/l and 0.03–8 mol/l for triglyceride and methanol, respectively, that ectoine had no effect on the initial reaction rates when methanol concentrations were below 0.5 mol/l. When methanol concentration exceeded 0.5 mol/l, the addition of 0.8 mmol/l ectoine increased the initial reaction rates, and the lipase exhibited a lower affinity for methanol and higher affinity for triglyceride (kinetic parameters of K_mA increase, K_mTG decrease). However, the initial reaction rates decreased significantly when 8 mmol/l ectoine was added, with the lipase having higher affinity for methanol and lower affinity for triglyceride (K_mA decrease, K_mTG increase). The supplementation of ectoine provided a new method for the purpose of improving yield of biodiesel catalyzed by enzyme.     

Metabolic engineering of Escherichia coli for the production of 5-aminovalerate and glutarate as C5 platform chemicals

5-Aminovalerate (5AVA) is the precursor of valerolactam, a potential building block for producing nylon 5, and is a C5 platform chemical for synthesizing 5-hydroxyvalerate, glutarate, and 1,5-pentanediol. Escherichia coli was metabolically engineered for the production of 5-aminovalerate (5AVA) and glutarate. When the recombinant E. coli WL3110 strain expressing the Pseudomonas putida davAB genes encoding delta-aminovaleramidase and lysine 2-monooxygenase, respectively, were cultured in a medium containing 20 g/L of glucose and 10 g/L of l-lysine, 3.6 g/L of 5AVA was produced by converting 7 g/L of l-lysine. When the davAB genes were introduced into recombinant E. coli strainXQ56allowing enhanced l-lysine synthesis, 0.27 and 0.5 g/L of 5AVA were produced directly from glucose by batch and fed-batch cultures, respectively. Further conversion of 5AVA into glutarate could be demonstrated by expression of the P. putida gabTD genes encoding 5AVA aminotransferase and glutarate semialdehyde dehydrogenase. When recombinant E. coli WL3110 strain expressing the davAB and gabTD genes was cultured in a medium containing 20 g/L glucose, 10 g/L l-lysine and 10 g/L α-ketoglutarate, 1.7 g/L of glutarate was produced.