Rapid ethanol production at elevated temperatures by engineered thermotolerant Kluyveromyces marxianus via the NADP(H)-preferring xylose reductase-xylitol dehydrogenase pathway

Conversion of xylose to ethanol by yeasts is a challenge because of the redox imbalances under oxygen-limited conditions. The thermotolerant yeast Kluyveromyces marxianus grows well with xylose as a carbon source at elevated temperatures, but its xylose fermentation ability is weak. In this study, a combination of the NADPH-preferring xylose reductase (XR) from Neurospora crassa and the NADP⁺-preferring xylitol dehydrogenase (XDH) mutant from Scheffersomyces stipitis (Pichia stipitis) was constructed. The xylose fermentation ability and redox balance of the recombinant strains were improved significantly by over-expression of several downstream genes. The intracellular concentrations of coenzymes and the reduced coenzyme/oxidized coenzyme ratio increased significantly in these metabolic strains. The byproducts, such as glycerol and acetic acid, were significantly reduced by the disruption of glycerol-3-phosphate dehydrogenase (GPD1). The resulting engineered K. marxianus YZJ088 strain produced 44.95 g/L ethanol from 118.39 g/L xylose with a productivity of 2.49 g/L/h at 42 °C. Additionally, YZJ088 realized glucose and xylose co-fermentation and produced 51.43 g/L ethanol from a mixture of 103.97 g/L xylose and 40.96 g/L glucose with a productivity of 2.14 g/L/h at 42 °C. These promising results validate the YZJ088 strain as an excellent producer of ethanol from xylose through the synthetic xylose assimilation pathway.     

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.     

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.     

Engineering of an N-acetylneuraminic acid synthetic pathway in Escherichia coli

N-acetylneuraminic acid (NeuAc) has recently drawn much attention owing to its wide applications in many aspects. Besides extraction from natural materials, production of NeuAc was recently focused on enzymatic synthesis and whole-cell biocatalysis. In this study, we designed an artificial NeuAc biosynthetic pathway through intermediate N-acetylglucosamine 6-phosphate in Escherichia coli. In this pathway, N-acetylglucosamine 2-epimerase (slr1975) and glucosamine-6-phosphate acetyltransferase (GNA1) were heterologously introduced into E. coli from Synechocystis sp. PCC6803 and Saccharomyces cerevisiae EBY100, respectively. By derepressing the feedback inhibition of glucosamine-6-phosphate synthase, increasing the accumulation of N-acetylglucosamine and pyruvate, and blocking the catabolism of NeuAc, we were able to produce 1.62 g l^?1 NeuAc in recombinant E. coli directly from glucose. The NeuAc yield reached 7.85 g l^?1 in fed-batch fermentation. This process offered an efficient fermentative method to produce NeuAc in microorganisms using glucose as carbon source and can be optimized for further improvement.     

Metabolic engineering of Clostridium tyrobutyricum for enhanced butyric acid production from glucose and xylose

Clostridium tyrobutyricum is a promising microorganism for butyric acid production. However, its ability to utilize xylose, the second most abundant sugar found in lignocellulosic biomass, is severely impaired by glucose-mediated carbon catabolite repression (CCR). In this study, CCR in C. tyrobutyricum was eliminated by overexpressing three heterologous xylose catabolism genes (xylT, xylA and xlyB) cloned from C. acetobutylicum. Compared to the parental strain, the engineered strain Ct-pTBA produced more butyric acid (37.8 g/L vs. 19.4 g/L) from glucose and xylose simultaneously, at a higher xylose utilization rate (1.28 g/L·h vs. 0.16 g/L·h) and efficiency (94.3% vs. 13.8%), resulting in a higher butyrate productivity (0.53 g/L·h vs. 0.26 g/L·h) and yield (0.32 g/g vs. 0.28 g/g). When the initial total sugar concentration was ~120 g/L, both glucose and xylose utilization rates increased with increasing their respective concentration or ratio in the co-substrates but the total sugar utilization rate remained almost unchanged in the fermentation at pH 6.0. Decreasing the pH to 5.0 significantly decreased sugar utilization rates and butyrate productivity, but the effect was more pronounced for xylose than glucose. The addition of benzyl viologen (BV) as an artificial electron carrier facilitated the re-assimilation of acetate and increased butyrate production to a final titer of 46.4 g/L, yield of 0.43 g/g sugar consumed, productivity of 0.87 g/L·h, and acid purity of 98.3% in free-cell batch fermentation, which were the highest ever reported for butyric acid fermentation. The engineered strain with BV addition thus can provide an economical process for butyric acid production from lignocellulosic biomass.     

Establishment of a novel gene expression method,BICES (biomass-inducible chromosome-based expression system), and its application to the production of 2,3-butanediol and acetoin

In this study, we describe a novel method for producing valuable chemicals from glucose and xylose in Escherichia coli. The notable features in our method are avoidance of plasmids and expensive inducers for foreign gene expression to reduce production costs; foreign genes are knocked into the chromosome, and their expression is induced with xylose that is present in most biomass feedstock. As loci for the gene knock-in, lacZYA and some pseudogenes are chosen to minimize unexpected effects of the knock-in on cell physiology. The promoter of xylF is inducible with xylose and is combined with the T7 RNA polymerase–T7 promoter system to ensure strong gene expression. This expression system was named BICES (biomass-inducible chromosome-based expression system). As examples of BICES application, 2,3-butanediol and acetoin were successfully produced from glucose and xylose, and the maximal concentrations reached 54 g L⁻¹ [99.6% in (R,S)-form] and 31 g L⁻¹, respectively. 2,3-Butanediol and acetoin are industrially important chemicals that are, at present, produced primarily through petrochemical processes. To demonstrate usability of BICES in practical situations, we produced these chemicals from a saccharified cedar solution. From these results, we can conclude that BICES is suitable for practical production of valuable chemicals from biomass.     

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.     

Effect of lignocellulose degradation products on microbial biomass and lipid production by the oleaginous yeast Cryptococcus curvatus

This work investigated effects of lignocellulose degradation products on cell biomass and lipid production by Cryptococcus curvatus. Furfural was found to have the strongest inhibitory effect. For the three phenolic compounds tested, vanillin was the most toxic, while PHB and syringaldehyde showed comparable inhibitions in the concentration range of 0–1.0 g/L. Generally little significant differences on the relative cell biomass and lipid contents at the same concentrations of tested compounds were observed between glucose and xylose as a sole carbon source. At 1.0 g/L of furfural, the cell biomass and lipid content decreased by 78.4% and 61.0% for glucose as well as 72.0% and 59.3% for xylose, respectively. C. curvatus ceased to grow at concentrations of PHB over 1.0 g/L or vanillin over 1.5 g/L. The strain could survive in the presence of syringaldehyde up to 2.0 g/L for glucose or 1.5 g/L for xylose. The compounds’ negative impact was reduced by an increase in inoculum size and a 10% (v/v) seed was detected to be optimal for cell biomass and lipid production. The results demonstrated C. curvatus could effectively utilize most of the dominant monosaccharides and cellobiose existing in lignocellulosic biomass hydrolysate in the presence of toxic compounds.     

Production of lactic acid from soybean stalk hydrolysate with Lactobacillus sake and Lactobacillus casei

In order to make full use of soybean stalk produced in large quantity annually in China, a process is proposed for production of lactic acid from soybean stalk hydrolysate with Lactobacillus sake and Lactobacillus casei. Experiments were conducted using the proposed process and experimental results indicate that the potential of 242 mg (g stalk)⁻¹ fermentable sugar is released from hydrolysate through enzymatic saccharication with a saccharication of 51%. The main sugar released from pretreated soybean stalk through enzymatic hydrolysis was a mixture of glucose, xylose and cellobiose at a ratio of 3.9:1.7:1. Fermentation of soybean stalk hydrolysate by L. sake and L. casei yielded the lactic acid conversion of 48% and 56%, respectively, however, lactic acid conversion increased to 71% by co-inoculation of both strains. L. sake and L. casei were able to degrade glucose, but unable to completely assimilate xylose and cellobiose. The proposed process can be used to produce lactic acid from soybean stalk hydrolysate.     

Model-based metabolic engineering enables high yield itaconic acid production by Escherichia coli

Itaconic acid is a high potential platform chemical which is currently industrially produced by Aspergillus terreus. Heterologous production of itaconic acid with Escherichia coli could help to overcome limitations of A. terreus regarding slow growth and high sensitivity to oxygen supply. However, the performance achieved so far with E. coli strains is still low.We introduced a plasmid (pCadCS) carrying genes for itaconic acid production into E. coli and applied a model-based approach to construct a high yield production strain. Based on the concept of minimal cut sets, we identified intervention strategies that guarantee high itaconic acid yield while still allowing growth. One cut set was selected and the corresponding genes were iteratively knocked-out. As a conceptual novelty, we pursued an adaptive approach allowing changes in the model and initially calculated intervention strategy if a genetic modification induces changes in byproduct formation. Using this approach, we iteratively implemented five interventions leading to high yield itaconic acid production in minimal medium with glucose as substrate supplemented with small amounts of glutamic acid. The derived E. coli strain (ita23: MG1655 ∆aceA ∆sucCD ∆pykA ∆pykF ∆pta ∆Picd::cam_BBa_J23115 pCadCS) synthesized 2.27 g/l itaconic acid with an excellent yield of 0.77 mol/(mol glucose). In a fed-batch cultivation, this strain produced 32 g/l itaconic acid with an overall yield of 0.68 mol/(mol glucose) and a peak productivity of 0.45 g/l/h. These values are by far the highest that have ever been achieved for heterologous itaconic acid production and indicate that realistic applications come into reach.     

Efficient oleaginous yeasts for lipid production from lignocellulosic sugars and effects of lignocellulose degradation compounds on growth and lipid production

Microbial lipid production using lignocellulosic biomass is considered an alternative for biodiesel production. In this study, 418 yeast strains were screened to find efficient oleaginous yeasts which accumulated large quantities of lipid when cultivated in lignocellulosic sugars. Preliminary screening by Nile red staining revealed that 142 strains contained many or large lipid bodies. These strains were selected for quantitative analysis of lipid accumulation by shaking flask cultivation in nitrogen-limited medium II containing 70 g/L glucose or xylose or mixture of glucose and xylose in a ratio of 2:1. Rhodosporidium fluviale DMKU-SP314 produced the highest lipid concentration of 7.9 g/L when cultivated in the mixture of glucose and xylose after 9 days of cultivation, which was 55.0% of dry biomass (14.3 g/L). The main composition of fatty acids were oleic acid (40.2%), palmitic acid (25.2%), linoleic acid (17.9%) and stearic acid (11.1%). Moreover, the strain DMKU-SP314 could grow and produce lipid in a medium containing predominantly lignocellulose degradation products, namely, acetic acid, formic acid, furfural, 5-hydroxymethylfurfural (5-HMF) and vanillin, with however, some inhibitory effects. This strain showed high tolerance to acetic acid, 5-HMF and vanillin. Therefore, R. fluviale DMKU-SP314 is a promising strain for lipid production from lignocellulosic hydrolysate.     

Xylose-based hydrolysate from eucalyptus extract as feedstock for poly(lactate-co-3-hydroxybutyrate) production in engineered Escherichia coli

Woody extract-derived hemicellulosic hydrolysate, which was obtained from dissolving pulp manufacturing, was utilized as feedstock for the production of poly(lactate-co-3-hydroxybutyrate) [P(LA-co-3HB)] in engineered Escherichia coli. The hydrolysate was composed of mainly xylose and galactose, and contained impurities mainly acetate, which was found to inhibit the polymer synthesis rather than the cell growth. Thus, acetate and other impurities were removed through active charcoal and ion-exchange columns. Using the purified hydrolysate, P(LA-co-3HB) was successfully produced (cell dry weight 8.6 g/L, polymer concentration 5.4 g/L, LA fraction 5.5 mol%, polymer content 62.4%), the amount of which was comparable to that obtained using reagent grade xylose and galactose. Therefore, the hydrolysate from woody extract is considered as an abundant, inexpensive and efficient feedstock applicable to consolidated process for P(LA-co-3HB) production, when the removal of acetic acid was satisfactorily accomplished.     

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.     

Metabolic engineering of Escherichia coli for production of salvianic acid A via an artificial biosynthetic pathway

Salvianic acid A, a valuable derivative from L-tyrosine biosynthetic pathway of the herbal plant Salvia miltiorrhiza, is well known for its antioxidant activities and efficacious therapeutic potential on cardiovascular diseases. Salvianic acid A was traditionally isolated from plant root or synthesized by chemical methods, both of which had low efficiency. Herein, we developed an unprecedented artificial biosynthetic pathway of salvianic acid A in E. coli, enabling its production from glucose directly. In this pathway, 4-hydroxyphenylpyruvate was converted to salvianic acid A via D-lactate dehydrogenase (encoding by d-ldh from Lactobacillus pentosus) and hydroxylase complex (encoding by hpaBC from E. coli). Furthermore, we optimized the pathway by a modular engineering approach and deleting genes involved in the regulatory and competing pathways. The metabolically engineered E. coli strain achieved high productivity of salvianic acid A (7.1 g/L) with a yield of 0.47 mol/mol glucose.     

Metabolic design of a platform Escherichia coli strain producing various chorismate derivatives

A synthetic metabolic pathway suitable for the production of chorismate derivatives was designed in Escherichia coli. An L-phenylalanine-overproducing E. coli strain was engineered to enhance the availability of phosphoenolpyruvate (PEP), which is a key precursor in the biosynthesis of aromatic compounds in microbes. Two major reactions converting PEP to pyruvate were inactivated. Using this modified E.coli as a base strain, we tested our system by carrying out the production of salicylate, a high-demand aromatic chemical. The titer of salicylate reached 11.5 g/L in batch culture after 48 h cultivation in a 2-liter jar fermentor, and the yield from glucose as the sole carbon source exceeded 40% (mol/mol). In this test case, we found that pyruvate was synthesized primarily via salicylate formation and the reaction converting oxaloacetate to pyruvate. In order to demonstrate the generality of our designed strain, we employed this platform for the production of each of 7 different chorismate derivatives. Each of these industrially important chemicals was successfully produced to levels of 1–3 g/L in test tube-scale culture.     

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.     

Dihydroxyacetone production in an engineered Escherichia coli through expression of Corynebacterium glutamicum dihydroxyacetone phosphate dephosphorylase

Dihydroxyacetone (DHA) has several industrial applications such as a tanning agent in tanning lotions in the cosmetic industry; its production via microbial fermentation would present a more sustainable option for the future. Here we genetically engineered Escherichia coli (E. coli) for DHA production from glucose. Deletion of E. coli triose phosphate isomerase (tpiA) gene was carried out to accumulate dihydroxyacetone phosphate (DHAP), for use as the main intermediate or precursor for DHA production. The accumulated DHAP was then converted to DHA through the heterologous expression of Corynebacterium glutamicum DHAP dephosphorylase (cghdpA) gene. To conserve DHAP exclusively for DHA production we removed methylglyoxal synthase (mgsA) gene in the ΔtpiA strain. This drastically improved DHA production from 0.83 g/l (0.06 g DHA/g glucose) in the ΔtpiA strain bearing cghdpA to 5.84 g/l (0.41 g DHA/g glucose) in the ΔtpiAΔmgsA double mutant containing the same gene. To limit the conversion of intracellular DHA to glycerol, glycerol dehydrogenase (gldA) gene was further knocked out resulting in a ΔtpiAΔmgsAΔgldA triple mutant. This triple mutant expressing the cghdpA gene produced 6.60 g/l of DHA at 87% of the maximum theoretical yield. In summary, we demonstrated an efficient system for DHA production in genetically engineered E. coli strain.     

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.     

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.