Metabolic engineering to improve 1,5-diaminopentane production from cellobiose using β-glucosidase-secreting Corynebacterium glutamicum

Microbial production of 1,5-diaminopentane (DAP) from renewable feedstock is a promising and sustainable approach for the production of polyamides. In this study, we constructed a β-glucosidase (BGL)-secreting Corynebacterium glutamicum and successfully used this strain to produce DAP from cellobiose and glucose. First, C. glutamicum was metabolically engineered to produce l -lysine (a direct precursor of DAP), followed by the coexpression of l -lysine decarboxylase and BGL derived from Escherichia coli and Thermobifida fusca YX (Tfu0937), respectively. This new engineered C. glutamicum strain produced 27 g/L of DAP from cellobiose in CGXII minimal medium using fed-batch cultivation. The yield of DAP was 0.43 g/g glucose (1 g of cellobiose corresponds to 1.1 g of glucose), which is the highest yield reported to date. These results demonstrate the feasibility of DAP production from cellobiose or cellooligosaccharides using an engineered C. glutamicum strain.     

Engineered Escherichia coli capable of co-utilization of cellobiose and xylose

Natural ability to ferment the major sugars (glucose and xylose) of plant biomass is an advantageous feature of Escherichia coli in biofuel production. However, excess glucose completely inhibits xylose utilization in E. coli and decreases yield and productivity of fermentation due to sequential utilization of xylose after glucose. As an approach to overcome this drawback, E. coli MG1655 was engineered for simultaneous glucose (in the form of cellobiose) and xylose utilization by a combination of genetic and evolutionary engineering strategies. The recombinant E. coli was capable of utilizing approximately 6 g/L of cellobiose and 2 g/L of xylose in approximately 36 h, whereas wild-type E. coli was unable to utilize xylose completely in the presence of 6 g/L of glucose even after 75 hours. The engineered strain also co-utilized cellobiose with mannose or galactose; however, it was unable to metabolize cellobiose in the presence of arabinose and glucose. Successful cellobiose and xylose co-fermentation is a vital step for simultaneous saccharification and co-fermentation process and a promising step towards consolidated bioprocessing.     

Isobutanol production from cellobiose in Escherichia coli

Converting lignocellulosics into biofuels remains a promising route for biofuel production. To facilitate strain development for specificity and productivity of cellulosic biofuel production, a user friendly Escherichia coli host was engineered to produce isobutanol, a drop-in biofuel candidate, from cellobiose. A beta-glucosidase was expressed extracellularly by either excretion into the media, or anchoring to the cell membrane. The excretion system allowed for E. coli to grow with cellobiose as a sole carbon source at rates comparable to those with glucose. The system was then combined with isobutanol production genes in three different configurations to determine whether gene arrangement affected isobutanol production. The most productive strain converted cellobiose to isobutanol in titers of 7.64?±?0.19 g/L with a productivity of 0.16 g/L/h. These results demonstrate that efficient cellobiose degradation and isobutanol production can be achieved by a single organism, and provide insight for optimization of strains for future use in a consolidated bioprocessing system for renewable production of isobutanol.     

Metabolic engineering of Escherichia coli for the production of fumaric acid

Fumaric acid is a naturally occurring organic acid that is an intermediate of the tricarboxylic acid cycle. Fungal species belonging to Rhizopus have traditionally been employed for the production of fumaric acid. In this study, Escherichia coli was metabolically engineered for the production of fumaric acid under aerobic condition. For the aerobic production of fumaric acid, the iclR gene was deleted to redirect the carbon flux through the glyoxylate shunt. In addition, the fumA, fumB, and fumC genes were also deleted to enhance fumaric acid formation. The resulting strain was able to produce 1.45 g/L of fumaric acid from 15 g/L of glucose in flask culture. Based on in silico flux response analysis, this base strain was further engineered by plasmid‐based overexpression of the native ppc gene, encoding phosphoenolpyruvate carboxylase (PPC), from the strong tac promoter, which resulted in the production of 4.09 g/L of fumaric acid. Additionally, the arcA and ptsG genes were deleted to reinforce the oxidative TCA cycle flux, and the aspA gene was deleted to block the conversion of fumaric acid into L ‐aspartic acid. Since it is desirable to avoid the use of inducer, the lacI gene was also deleted. To increase glucose uptake rate and fumaric acid productivity, the native promoter of the galP gene was replaced with the strong trc promoter. Fed‐batch culture of the final strain CWF812 allowed production of 28.2 g/L fumaric acid in 63 h with the overall yield and productivity of 0.389 g fumaric acid/g glucose and 0.448 g/L/h, respectively. This study demonstrates the possibility for the efficient production of fumaric acid by metabolically engineered E. coli. Biotechnol. Bioeng. 2013; 110: 2025–2034. © 2013 Wiley Periodicals, Inc.     

Pathway engineering of Escherichia coli for α-ketoglutaric acid production

α-Ketoglutaric acid (α-KG) is a multifunctional dicarboxylic acid in the tricarboxylic acid (TCA) cycle, but microbial engineering for α-KG production is not economically efficient, due to the intrinsic inefficiency of its biosynthetic pathway. In this study, pathway engineering was used to improve pathway efficiency for α-KG production in Escherichia coli. First, the TCA cycle was rewired for α-KG production starting from pyruvate, and the engineered strain E. coli W3110Δ4-PCAI produced 15.66 g/L α-KG. Then, the rewired TCA cycle was optimized by designing various strengths of pyruvate carboxylase and isocitrate dehydrogenase expression cassettes, resulting in a large increase in α-KG production (24.66 g/L). Furthermore, acetyl coenzyme A (acetyl-CoA) availability was improved by overexpressing acetyl-CoA synthetase, leading to α-KG production up to 28.54 g/L. Finally, the engineered strain E. coli W3110Δ4-P_(H)CAI_(H)A was able to produce 32.20 g/L α-KG in a 5-L fed-batch bioreactor. This strategy described here paves the way to the development of an efficient pathway for microbial production of α-KG.     

Co-fermentation of cellobiose and xylose using beta-glucosidase displaying diploid industrial yeast strain OC-2

The co-utilization of sugars, particularly xylose and glucose, during industrial fermentation is essential for economically feasible processes with high ethanol productivity. However, the major problem encountered during xylose/glucose co-fermentation is the lower consumption rate of xylose compared with that of glucose fermentation. Here, we therefore attempted to construct high xylose assimilation yeast by using industrial yeast strain with high β-glucosidase activity on the cell surface. We first constructed the triple auxotrophic industrial strain OC2-HUT and introduced four copies of the cell-surface-displaying β-glucosidase (BGL) gene and two copies of a xylose-assimilating gene into its genome to generate strain OC2-ABGL4Xyl2. It was confirmed that the introduction of multiple copies of the BGL gene increased the cell-surface BGL activity, which was also correlated to the observed increase in xylose-assimilating ability. The strain OC2-ABGL4Xyl2 was able to consume xylose during cellobiose/xylose co-fermentation (0.38 g/h/g-DW) more rapidly than during glucose/xylose co-fermentation (0.18 g/h/g-DW). After 48 h, 5.77% of the xylose was consumed despite the co-fermentation conditions, and the observed ethanol yield was 0.39 g-ethanol/g-total sugar. Our results demonstrate that a BGL-displaying and xylose-assimilating industrial yeast strain is capable of efficient xylose consumption during the co-fermentation with cellobiose. Due to its high performance for fermentation of mixtures of cellobiose and xylose, OC2-ABGL4Xyl2 does not require the addition of β-glucosidase and is therefore a promising yeast strain for cost-effective ethanol production from lignocellulosic biomass.     

Cloning, expression, and characterization of an aldehyde dehydrogenase from Escherichia coli K-12 that utilizes 3-Hydroxypropionaldehyde as a substrate

For efficient production of isoflavone aglycones from soybean isoflavones, we isolated three novel types of β-glucosidase (BGL1, BGL3, and BGL5) from the filamentous fungi Aspergillus oryzae. Three enzymes were independently displayed on the cell surface of a yeast Saccharomyces cerevisiae as a fusion protein with α-agglutinin. Three β-glucosidase-displaying yeast strains hydrolyzed isoflavone glycosides efficiently but exhibited different substrate specificities. Among these β-glucosidases, BGL1 exhibited the highest activity and also broad substrate specificity to isoflavone glycosides. Although glucose released from isoflavone glycosides are generally known to inhibit β-glucosidase, the residual ratio of isoflavone glycosides in the reaction mixture with BGL1-displaying yeast strain (Sc-BGL1) reached approximately 6.2%, and the glucose concentration in the reaction mixture was maintained at lower level. This result indicated that Sc-BGL1 assimilated the glucose before they inhibited the hydrolysis reaction, and efficient production of isoflavone aglycones was achieved by engineered yeast cells displaying β-glucosidase.     

Production of 3‐Fucosyllactose in Engineered Escherichia coli with α‐1,3‐Fucosyltransferase from Helicobacter pylori

3‐Fucosyllactose (3‐FL), one of the major oligosaccharides in human breast milk, is produced in engineered Escherichia coli. In order to search for a good α‐1,3‐fucosyltransferase, three bacterial α‐1,3‐fucosyltransferases are expressed in engineered E. coli deficient in β‐galactosidase activity and expressing the essential enzymes for the production of guanosine 5′‐diphosphate‐l ‐fucose, the donor of fucose for 3‐FL biosynthesis. Among the three enzymes tested, the fucT gene from Helicobacter pylori National Collection of Type Cultures 11637 gives the best 3‐FL production in a simple batch fermentation process using glycerol as a carbon source and lactose as an acceptor. In order to use glucose as a carbon source, the chromosomal ptsG gene, considered the main regulator of the glucose repression mechanism, is disrupted. The resulting E. coli strain of ?LP‐YA+FT shows a much lower performance of 3‐FL production (4.50 g L^?1) than the ?L‐YA+FT strain grown in a glycerol medium (10.7 g L^?1), suggesting that glycerol is a better carbon source than glucose. Finally, the engineered E. coli ?LW‐YA+FT expressing the essential genes for 3‐FL production and blocking the colanic acid biosynthetic pathway (?wcaJ) exhibits the highest concentration (11.5 g L^?1), yield (0.39 mol mol^?1), and productivity (0.22 g L^?1 h) of 3‐FL in glycerol‐limited fed‐batch fermentation.     

Two New Native β-Glucosidases from Clavispora NRRL Y-50464 Confer Its Dual Function as Cellobiose Fermenting Ethanologenic Yeast

Yeast strain Clavispora NRRL Y-50464 is able to produce cellulosic ethanol from lignocellulosic materials without addition of external β-glucosidase by simultaneous saccharification and fermentation. A β-glucosidase BGL1 protein from this strain was recently reported supporting its cellobiose utilization capability. Here, we report two additional new β-glucosidase genes encoding enzymes designated as BGL2 and BGL3 from strain NRRL Y-50464. Quantitative gene expression was analyzed and the gene function of BGL2 and BGL3 was confirmed by heterologous expression using cellobiose as a sole carbon source. Each gene was cloned and partially purified protein obtained separately for direct enzyme assay using varied substrates. Both proteins showed the highest specific activity at pH 5 and relatively strong affinity with a K_m of 0.08 and 0.18 mM for BGL2 and BGL3, respectively. The optimum temperature was found to be 50°C for BGL2 and 55°C for BGL3. Both proteins were able to hydrolyze 1,4 oligosaccharides evaluated in this study. They also showed a strong resistance to glucose product inhibition with a K_i of 61.97 and 38.33 mM for BGL2 and BGL3, respectively. While BGL3 was sensitive showing a significantly reduced activity to 4% ethanol, BGL2 demonstrated tolerance to ethanol. Its activity was enhanced in the presence of ethanol but reduced at concentrations greater than 16%. The presence of the fermentation inhibitors furfural and HMF did not affect the enzyme activity. Our results suggest that a β-glucosidase gene family exists in Clavispora NRRL Y-50464 with at least three members in this group that validate its cellobiose hydrolysis functions for lower-cost cellulosic ethanol production. Results of this study confirmed the cellobiose hydrolysis function of strain NRRL Y-50464, and further supported this dual functional yeast as a candidate for lower-cost cellulosic ethanol production and next-generation biocatalyst development in potential industrial applications.     

Improved phloroglucinol production by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Phloroglucinol is a valuable chemical which has been successfully produced by metabolically engineered Escherichia coli. However, the low productivity remains a bottleneck for large-scale application and cost-effective production. In the present work, we cloned the key biosynthetic gene, phlD (a type III polyketide synthase), into a bacterial expression vector to produce phloroglucinol in E. coli and developed different strategies to re-engineer the recombinant strain for robust synthesis of phloroglucinol. Overexpression of E. coli marA (multiple antibiotic resistance) gene enhanced phloroglucinol resistance and elevated phloroglucinol production to 0.27 g/g dry cell weight. Augmentation of the intracellular malonyl coenzyme A (malonyl-CoA) level through coordinated expression of four acetyl-CoA carboxylase (ACCase) subunits increased phloroglucinol production to around 0.27 g/g dry cell weight. Furthermore, the coexpression of ACCase and marA caused another marked improvement in phloroglucinol production 0.45 g/g dry cell weight, that is, 3.3-fold to the original strain. Under fed-batch conditions, this finally engineered strain accumulated phloroglucinol up to 3.8 g/L in the culture 12 h after induction, corresponding to a volumetric productivity of 0.32 g/L/h. This result was the highest phloroglucinol production to date and showed promising to make the bioprocess economically feasible.     

Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production

Limonene is a valuable monoterpene used in the production of several commodity chemicals and medicinal compounds. Among them, perillyl alcohol (POH) is a promising anti-cancer agent that can be produced by hydroxylation of limonene. We engineered E. coli with a heterologous mevalonate pathway and limonene synthase for production of limonene followed by coupling with a cytochrome P450, which specifically hydroxylates limonene to produce POH. A strain containing all mevalonate pathway genes in a single plasmid produced limonene at titers over 400 mg/L from glucose, substantially higher than has been achieved in the past. Incorporation of a cytochrome P450 to hydroxylate limonene yielded approximately 100 mg/L of POH. Further metabolic engineering of the pathway and in situ product recovery using anion exchange resins would make this engineered E. coli a potential production platform for any valuable limonene derivative.     

Synergy between methylerythritol phosphate pathway and mevalonate pathway for isoprene production in Escherichia coli

Isoprene, a key building block of synthetic rubber, is currently produced entirely from petrochemical sources. In this work, we engineered both the methylerythritol phosphate (MEP) pathway and the mevalonate (MVA) pathway for isoprene production in E. coli. The synergy between the MEP pathway and the MVA pathway was demonstrated by the production experiment, in which overexpression of both pathways improved the isoprene yield about 20-fold and 3-fold, respectively, compared to overexpression of the MEP pathway or the MVA pathway alone. The ¹³C metabolic flux analysis revealed that simultaneous utilization of the two pathways resulted in a 4.8-fold increase in the MEP pathway flux and a 1.5-fold increase in the MVA pathway flux. The synergy of the dual pathway was further verified by quantifying intracellular flux responses of the MEP pathway and the MVA pathway to fosmidomycin treatment and mevalonate supplementation. Our results strongly suggest that coupling of the complementary reducing equivalent demand and ATP requirement plays an important role in the synergy of the dual pathway. Fed-batch cultivation of the engineered strain overexpressing the dual pathway resulted in production of 24.0 g/L isoprene with a yield of 0.267 g/g of glucose. The synergy of the MEP pathway and the MVA pathway also successfully increased the lycopene productivity in E. coli, which demonstrates that it can be used to improve the production of a broad range of terpenoids in microorganisms.     

Production of β-carotene by recombinant Escherichia coli with engineered whole mevalonate pathway in batch and fed-batch cultures

Recombinant Escherichia coli engineered to contain the whole mevalonate pathway and foreign genes for β-carotene biosynthesis, was utilized for production of β-carotene in bioreactor cultures. Optimum culture conditions were established in batch and pH-stat fed-batch cultures to determine the optimal feeding strategy thereby improving production yield. The specific growth rate and volumetric productivity in batch cultures at 37°C were 1.7-fold and 2-fold higher, respectively, than those at 28°C. Glycerol was superior to glucose as a carbon source. Maximum β-carotene production (titer of 663 mg/L and overall volumetric productivity of 24.6 mg/L × h) resulted from the simultaneous addition of 500 g/L glycerol and 50 g/L yeast extract in pH-stat fed-batch culture.     

Coupled biosynthesis and esterification of 1,2,4‐butanetriol to simplify its separation from fermentation broth

1,2,4‐Butanetriol (BT) is a valuable chemical with versatile applications in many fields and can be produced through biosynthetic pathways. As a trihydric alcohol, BT possesses good water solubility and is very difficult to separate from fermentation broth, which does complicate the production process and increase the cost. To develop a novel method for BT separation, a biosynthetic pathway for 1,2,4‐butanetriol esters with poor water solubility was constructed. Wax ester synthase/acyl‐coenzyme A: diacylglycerol acyltransferase (Atf) from Acinetobacter baylyi, Mycobacterium smegmatis, and Escherichia coli were screened, and the acyltransferase from A. baylyi (AtfA) was found to have higher capability. The BT producing strain with AtfA overexpression produced 49.5 mg/L BT oleate in flask cultivation. Through enhancement of acyl‐CoA production by overexpression of the acyl‐CoA synthetase gene fadD and deleting the acyl coenzyme A dehydrogenase gene fadE, the production was improved to 64.4 mg/L. Under fed‐batch fermentation, the resulting strain produced up to 1.1 g/L BT oleate. This is the first time showed that engineered E. coli strains can successfully produce BT esters from xylose and free fatty acids.     

Construction of a Novel а-Agglutinin Expression System on the Surface of Wild-Type Saccharomyces cerevisiae Y5 and Genetic Immobilization of β-Glucosidase1

Saccharomyces cerevisiae Y5 is a newly developed wild-type strain demonstrating a strong bioethanol fermentation capacity. In the present study, we attempted to construct an а-agglutinin-displaying expression system for genetic immobilization β-glucosidase1 (BGL1) on a yeast cell surface in its active form. The AGA1 gene of native а-agglutinin under the control of a GAL1 promoter was integrated into the genomes of S. cerevisiae Y5. A cDNA-encoding BGL1 from the fungus Aspergillus aculeatus was fused with the gene encoding the C-terminal half of Aga2p. The multicopy plasmid containing this fusion gene was introduced into S. cerevisiae and expressed under the control of the GAL1 promoter as was the AGA1 gene. The display of the BGL1 protein on the cell surface was confirmed by immunofluorescence microscopy and Western blotting. The cells displaying BGL1 could produce 5.07 g/l ethanol from 20 g/l cellobiose as the sole carbon source. These results demonstrated that BGL1 was anchored on the cell wall in its active form.     

Combined Cell Surface Display of β‐d‐Glucosidase (BGL), Maltose Transporter (MAL11), and Overexpression of Cytosolic Xylose Reductase (XR) in Saccharomyces cerevisiae Enhance Cellobiose/Xylose Coutilization for Xylitol Bioproduction from Lignocellulosic Biomass

Xylitol is a highly valuable commodity chemical used extensively in the food and pharmaceutical industries. The production of xylitol from d ‐xylose involves a costly and polluting catalytic hydrogenation process. Biotechnological production from lignocellulosic biomass by micro‐organisms like yeasts is a promising option. In this study, xylitol is produced from lignocellulosic biomass by a recombinant strain of Saccharomyces cerevisiae (S. cerevisiae) (YPH499‐SsXR‐AaBGL) expressing cytosolic xylose reductase (Scheffersomyces stipitis xylose reductase [SsXR]), along with a β‐d ‐glucosidase (Aspergillus aculeatus β‐glucosidase 1 [AaBGL]) displayed on the cell surface. The simultaneous cofermentation of cellobiose/xylose by this strain leads to an ≈2.5‐fold increase in Yxylitol/xylose (=0.54) compared to the use of a glucose/xylose mixture as a substrate. Further improvement in the xylose uptake by the cell is achieved by a broad evaluation of several homologous and heterologous transporters. Homologous maltose transporter (ScMAL11) shows the best performance in xylose transport and is used to generate the strain YPH499‐XR‐ScMAL11‐BGL with a significantly improved xylitol production capacity from cellobiose/xylose coutilization. This report constitutes a promising proof of concept to further scale up the biorefinery industrial production of xylitol from lignocellulose by combining cell surface and metabolic engineering in S. cerevisiae.     

Metabolic engineering of Candida utilis for isopropanol production

A genetically-engineered strain of the yeast Candida utilis harboring genes encoding (1) an acetoacetyl-CoA transferase from Clostridium acetobutylicum ATCC 824, (2) an acetoacetate decarboxylase, and (3) a primary–secondary alcohol dehydrogenase derived from Clostridium beijerinckii NRRL B593 produced up to 0.21 g/L of isopropanol. Because the engineered strain accumulated acetate, isopropanol titer was improved to 1.2 g/L under neutralized fermentation conditions. Optimization of isopropanol production was attempted by the overexpression and disruption of several endogenous genes. Simultaneous overexpression of two genes encoding acetyl-CoA synthetase and acetyl-CoA acetyltransferase increased isopropanol titer to 9.5 g/L. Moreover, in fed-batch cultivation, the resultant recombinant strain produced 27.2 g/L of isopropanol from glucose with a yield of 41.5 % (mol/mol). This is the first demonstration of the production of isopropanol by genetically engineered yeast.     

Metabolic engineering of Escherichia coli for the production of cadaverine: A five carbon diamine

A five carbon linear chain diamine, cadaverine (1,5‐diaminopentane), is an important platform chemical having many applications in chemical industry. Bio‐based production of cadaverine from renewable feedstock is a promising and sustainable alternative to the petroleum‐based chemical synthesis. Here, we report development of a metabolically engineered strain of Escherichia coli that overproduces cadaverine in glucose mineral salts medium. First, cadaverine degradation and utilization pathways were inactivated. Next, L ‐lysine decarboxylase, which converts L ‐lysine directly to cadaverine, was amplified by plasmid‐based overexpression of the cadA gene under the strong tac promoter. Furthermore, the L ‐lysine biosynthetic pool was increased by the overexpression of the dapA gene encoding dihydrodipicolinate synthase through the replacement of the native promoter with the strong trc promoter in the genome. The final engineered strain was able to produce 9.61 g L⁻¹ of cadaverine with a productivity of 0.32 g L⁻¹ h⁻¹ by fed‐batch cultivation. The strategy reported here should be useful for the bio‐based production of cadaverine from renewable resources. Biotechnol. Bioeng. 2011; 108:93–103. © 2010 Wiley Periodicals, Inc.     

In silico and in vivo stability analysis of a heterologous biosynthetic pathway for 1,4-butanediol production in metabolically engineered E. coli

Recently, several approaches have been published in order to develop a functional biosynthesis route for the non-natural compound 1,4-butanediol (BDO) in E. coli using glucose as a sole carbon source or starting from xylose. Among these studies, there was reported as high as 18 g/L product concentration achieved by industrial strains, however BDO production varies greatly in case of the reviewed studies. Our motivation was to build a simple heterologous pathway for this compound in E. coli and to design an appropriate cellular chassis based on a systemic biology approach, using constraint-based flux balance analysis and bi-level optimization for gene knock-out prediction. Thus, the present study reports, at the “proof-of concept” level, our findings related to model-driven development of a metabolically engineered E. coli strain lacking key genes for ethanol, lactate and formate production (ΔpflB, ΔldhA and ΔadhE), with a three-step biosynthetic pathway. We found this strain to produce a limited quantity of 1,4-BDO (.89 mg/L BDO under microaerobic conditions and .82 mg/L under anaerobic conditions). Using glycerol as carbon source, an approach, which to our knowledge has not been tackled before, our results suggest that further metabolic optimization is needed (gene-introductions or knock-outs, promoter fine-tuning) to address the redox potential imbalance problem and to achieve development of an industrially sustainable strain. Our experimental data on culture conditions, growth dynamics and fermentation parameters can consist a base for ongoing research on gene expression profiles and genetic stability of such metabolically engineered E. coli strains.     

Production of phloroglucinol by Escherichia coli using a stationary-phase promoter

Escherichia coli was metabolically engineered using a new host-vector system to produce phloroglucinol. The key biosynthetic gene phlD (encoding a type III polyketide synthase) from Pseudomonas fluorescens was expressed in E. coli using the stationary-phase promoter of the fic gene and a high-copy plasmid. In shake-flasks, the engineered strain produced phloroglucinol up to 0.28 g/l with a productivity of 0.014 g/l h. About 9.2% of the glucose consumed was converted to phloroglucinol after 20 h. Compared with the widely used inducible T7 promoter system, this strain did not require IPTG induction and the final titer of phloroglucinol was 22% higher.