Enhanced production of 2’-fucosyllactose from fucose by elimination of rhamnose isomerase and arabinose isomerase in engineered Escherichia coli

2′-Fucosyllactose (2-FL), one of the most abundant oligosaccharides in human milk, has been spotlighted for its neutraceutical and pharmaceutical potentials. Microbial production of 2-FL is promising since it is efficient as compared to other production methods. In 2-FL microbial production via the salvage pathway for biosynthesis of guanosine 5′-diphosphate (GDP)-l -fucose from fucose, the conversion yield from fucose is important because of the high price of fucose. In this study, deletion of the genes (araA and rhaA) coding for arabinose isomerase (AraA) and rhamnose isomerase (RhaA) was attempted in engineered Escherichia coli for improving 2-FL production by using fucose, lactose, and glycerol. The engineered E. coli constructed previously is able to express fucokinase/GDP-l -fucose pyrophosphorylase (Fkp) from Bacteroides fragilis and the α-1,2-fucosyltransferase (FucT2) from Helicobacter pylori and deficient in β-galactosidase (LacZ), fucose isomerase (FucI), and fuculose kinase (FucK). The additional double-deletion of the araA and rhaA genes in the engineered E. coli enhanced the product yield of 2-FL to 0.52 mole 2-FL/mole fucose, and hence the concentration of 2-FL reached to 47.0 g/L, which are 44% and two-fold higher than those (23.1 g/L and 0.36 mole 2-FL/mole fucose) of the control strain in fed-batch fermentation. Elimination of sugar isomerases exhibiting promiscuous activities with fucose might be critical in the microbial production of 2-FL through the salvage pathway of GDP-l -fucose.     

Biosynthesis of the human milk oligosaccharide 3-fucosyllactose in metabolically engineered Escherichia coli via the salvage pathway through increasing GTP synthesis and β-galactosidase modification

3-Fucosyllactose (3-FL) is one of the major fucosylated oligosaccharides in human milk. Along with 2′-fucosyllactose (2′-FL), it is known for its prebiotic, immunomodulator, neonatal brain development, and antimicrobial function. Whereas the biological production of 2′-FL has been widely studied and made significant progress over the years, the biological production of 3-FL has been hampered by the low activity and insoluble expression of α-1,3-fucosyltransferase (FutA), relatively low abundance in human milk oligosaccharides compared with 2′-FL, and lower digestibility of 3-FL than 2′-FL by bifidobacteria. In this study, we report the gram-scale production of 3-FL using E. coli BL21(DE3). We previously generated the FutA quadruple mutant (mFutA) with four site mutations at S46F, A128N, H129E, Y132I, and its specific activity was increased by nearly 15 times compared with that of wild-type FutA owing to the increase in k_cat and the decrease in K_m. We overexpressed mFutA in its maximum expression level, which was achieved by the optimization of yeast extract concentration in culture media. We also overexpressed L-fucokinase/GDP- L -fucose pyrophosphorylase to increase the supply of GDP-fucose in the cytoplasm. To increase the mass of recombinant whole-cell catalysts, the host E. coli BW25113 was switched to E. coli BL21(DE3) because of the lower acetate accumulation of E. coli BL21(DE3) than that of E. coli BW25113. Finally, the lactose operon was modified by partially deleting the sequence of LacZ (lacZΔm15) for better utilization of D -lactose. Production using the lacZΔm15 mutant yielded 3-FL concentration of 4.6 g/L with the productivity of 0.076 g·L⁻¹·hr⁻¹ and the specific 3-FL yield of 0.5 g/g dry cell weight.     

Metabolic engineering strategies of de novo pathway for enhancing 2′-fucosyllactose synthesis in Escherichia coli

2′-Fucosyllactose (2′-FL), one of the most abundant human milk oligosaccharides (HMOs), is used as a promising infant formula ingredient owing to its multiple health benefits for newborns. However, limited availability and high-cost preparation have restricted its extensive use and intensive research on its potential functions. In this work, a powerful Escherichia coli cell factory was developed to ulteriorly increase 2′-FL production. Initially, a modular pathway engineering was strengthened to balance the synthesis pathway through different plasmid combinations with a resulting maximum 2′-FL titre of 1.45 g l⁻¹. To further facilitate the metabolic flux from GDP-l -fucose towards 2′-FL, the CRISPR-Cas9 system was utilized to inactivate the genes including lacZ and wcaJ, increasing the titre by 6.59-fold. Notably, the co-introduction of NADPH and GTP regeneration pathways was confirmed to be more conducive to 2′-FL formation, achieving a 2′-FL titre of 2.24 g l⁻¹. Moreover, comparisons of various exogenous α1,2-fucosyltransferase candidates revealed that futC from Helicobacter pylori generated the highest titre of 2′-FL. Finally, the viability of scaled-up production of 2′-FL was evidenced in a 3 l bioreactor with a maximum titre of 22.3 g l⁻¹ 2′-FL and a yield of 0.53 mole 2′-FL mole⁻¹ lactose.     

Spatial organization of pathway enzymes via self-assembly to improve 2′-fucosyllactose biosynthesis in engineered Escherichia coli

As one of the most abundant components in human milk oligosaccharides, 2′-fucosyllactose (2′-FL) possesses versatile beneficial health effects. Although most studies focused on overexpressing or fine-tuning the expression of pathway enzymes and achieved a striking increase of 2′-FL production, directly facilitating the metabolic flux toward the key intermediate GDP-l -fucose seems to be ignored. Here, multienzyme complexes consisting of sequential pathway enzymes were constructed by using specific peptide interaction motifs in recombinant Escherichia coli to achieve a higher titer of 2′-FL. Specifically, we first fine-tuned the expression level of pathway enzymes and balanced the metabolic flux toward 2′-FL synthesis. Then, two key enzymes (GDP-mannose 4,6-dehydratase and GDP- l -fucose synthase) were self-assembled into enzyme complexes in vivo via a short peptide interaction pair RIAD–RIDD (RI anchoring disruptor–RI dimer D/D domains), resulting in noticeable improvement of 2′-FL production. Next, to further strengthen the metabolic flux toward 2′-FL, three pathway enzymes were further aggregated into multienzyme assemblies by using another orthogonal protein interaction motif (Spycatcher–SpyTag or PDZ–PDZlig). Intracellular multienzyme assemblies remarkably enlarged the flux toward 2′-FL biosynthesis and showed a 2.1-fold increase of 2′-FL production compared with a strain expressing free-floating and unassembled enzymes. The optimally engineered strain EZJ23 accumulated 4.8 g/L 2′-FL in shake flask fermentation and was capable of producing 25.1 g/L 2′-FL by fed-batch cultivation. This work provides novel approaches for further improvement and large-scale production of 2′-FL and demonstrates the effectiveness of spatial assembly of pathway enzymes to improve the production of valuable products in the engineered host strain.     

Enhanced 2′-Fucosyllactose production by engineered Saccharomyces cerevisiae using xylose as a co-substrate

2′-Fucosyllactose (2′-FL), a human milk oligosaccharide with confirmed benefits for infant health, is a promising infant formula ingredient. Although Escherichia coli, Saccharomyces cerevisiae, Corynebacterium glutamicum, and Bacillus subtilis have been engineered to produce 2′-FL, their titers and productivities need be improved for economic production. Glucose along with lactose have been used as substrates for producing 2′-FL, but accumulation of by-products due to overflow metabolism of glucose hampered efficient production of 2′-FL regardless of a host strain. To circumvent this problem, we used xylose, which is the second most abundant sugar in plant cell wall hydrolysates and is metabolized through oxidative metabolism, for the production of 2′-FL by engineered yeast. Specifically, we modified an engineered S. cerevisiae strain capable of assimilating xylose to produce 2′-FL from a mixture of xylose and lactose. First, a lactose transporter (Lac12) from Kluyveromyces lactis was introduced. Second, a heterologous 2′-FL biosynthetic pathway consisting of enzymes Gmd, WcaG, and WbgL from Escherichia coli was introduced. Third, we adjusted expression levels of the heterologous genes to maximize 2′-FL production. The resulting engineered yeast produced 25.5 g/L of 2′-FL with a volumetric productivity of 0.35 g/L∙h in a fed-batch fermentation with lactose and xylose feeding to mitigate the glucose repression. Interestingly, the major location of produced 2′-FL by the engineered yeast can be changed using different culture media. While 72% of the produced 2′-FL was secreted when a complex medium was used, 82% of the produced 2′-FL remained inside the cells when a minimal medium was used. As yeast extract is already used as food and animal feed ingredients, 2′-FL enriched yeast extract can be produced cost-effectively using the 2′-FL-accumulating yeast cells.     

Multi-level metabolic engineering of Escherichia coli for high-titer biosynthesis of 2′-fucosyllactose and 3-fucosyllactose

Fucosyllactoses (FL), including 2′-fucosyllactose (2′-FL) and 3-fucosyllactose (3-FL), have garnered considerable interest for their value in newborn formula and pharmaceuticals. In this study, an engineered Escherichia coli was developed for high-titer FL biosynthesis by introducing multi-level metabolic engineering strategies, including (1) individual construction of the 2′/3-FL-producing strains through gene combination optimization of the GDP-L-fucose module; (2) screening of rate-limiting enzymes (α-1,2-fucosyltransferase and α-1,3-fucosyltransferase); (3) analysis of critical intermediates and inactivation of competing pathways to redirect carbon fluxes to FL biosynthesis; (4) enhancement of the catalytic performance of rate-limiting enzymes by the RBS screening, fusion peptides and multi-copy gene cloning. The final strains EC49 and EM47 produced 9.36 g/L for 2′-FL and 6.28 g/L for 3-FL in shake flasks with a modified-M9CA medium. Fed-batch cultivations of the two strains generated 64.62 g/L of 2′-FL and 40.68 g/L of 3-FL in the 3-L bioreactors, with yields of 0.65 mol 2′-FL/mol lactose and 0.67 mol 3-FL/mol lactose, respectively. This research provides a viable platform for other high-value-added compounds production in microbial cell factories.     

Enhanced production of GDP-<Emphasis Type="SmallCaps">l</Emphasis>-fucose by overexpression of NADPH regenerator in recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Biosynthesis of guanosine 5′-diphosphate-l-fucose (GDP-l-fucose) requires NADPH as a reducing cofactor. In this study, endogenous NADPH regenerating enzymes such as glucose-6-phosphate dehydrogenase (G6PDH), isocitrate dehydrogenase (Icd), and NADP⁺-dependent malate dehydrogenase (MaeB) were overexpressed to increase GDP-l-fucose production in recombinant Escherichia coli. The effects of overexpression of each NADPH regenerating enzyme on GDP-l-fucose production were investigated in a series of batch and fed-batch fermentations. Batch fermentations showed that overexpression of G6PDH was the most effective for GDP-l-fucose production. However, GDP-l-fucose production was not enhanced by overexpression of G6PDH in the glucose-limited fed-batch fermentation. Hence, a glucose feeding strategy was optimized to enhance GDP-l-fucose production. Fed-batch fermentation with a pH-stat feeding mode for sufficient supply of glucose significantly enhanced GDP-l-fucose production compared with glucose-limited fed-batch fermentation. A maximum GDP-l-fucose concentration of 235.2 ± 3.3 mg l⁻¹, corresponding to a 21% enhancement in the GDP-l-fucose production compared with the control strain overexpressing GDP-l-fucose biosynthetic enzymes only, was achieved in the pH-stat fed-batch fermentation of the recombinant E. coli overexpressing G6PDH. It was concluded that sufficient glucose supply and efficient NADPH regeneration are crucial for NADPH-dependent GDP-l-fucose production in recombinant E. coli.     

Chromosome evolution of Escherichia coli Nissle 1917 for high-level production of heparosan

Heparosan is a crucial-polysaccharide precursor for the chemoenzymatic synthesis of heparin, a widely used anticoagulant drug. Presently, heparosan is mainly extracted with the potential risk of contamination from Escherichia coli strain K5, a pathogenic bacterium causing urinary tract infection. Here, a nonpathogenic probiotic, E. coli strain Nissle 1917 (EcN), was metabolically engineered to carry multiple copies of the 19-kb kps locus and produce heparosan to 9.1 g/L in fed-batch fermentation. Chromosome evolution driven by antibiotics was employed to amplify the kps locus, which governed the synthesis and export of heparosan from EcN at 21 mg L⁻¹ OD⁻¹. The average copy number of kps locus increased from 1 to 24 copies per cell, which produced up to 104 mg L^-1 OD⁻¹ of heparosan in the shaking flask cultures of engineered strains. The following in-frame deletion of recA stabilized the recombinant duplicates of chromosomal kps locus and the productivity of heparosan in continuous culture for at least 56 generations. Fed-batch fermentation of the engineered strain EcN8 was carried out to bring the yield of heparosan up to 9.1 g/L. Heparosan from the fermentation culture was further purified at a 75% overall recovery. The structure of purified heparosan was characterized and further modified by N-sulfotransferase with 3′-phosphoadenosine-5′-phosphosulfate as the sulfo-donor. The analysis of element composition showed that heparosan was N-sulfated by over 80%. These results indicated that duplicating large DNA cassettes up to 19-kb, followed by high-cell-density fermentation, was promising in the large-scale preparation of chemicals and could be adapted to engineer other industrial-interest bacteria metabolically.     

Metabolic engineering of Escherichia coli for the production of 2′-fucosyllactose and 3-fucosyllactose through modular pathway enhancement

Fucosyllactoses, including 2′-fucosyllactose (2′-FL) and 3-fucosyllactose (3-FL), are important oligosaccharides in human milk that are commonly used as nutritional additives in infant formula due to their biological functions, such as the promotion of bifidobacteria growth, inhibition of pathogen infection, and improvement of immune response. In this study, we developed a synthetic biology approach to promote the efficient biosynthesis of 2′-FL and 3-FL in engineered Escherichia coli. To boost the production of 2′-FL and 3-FL, multiple modular optimization strategies were applied in a plug-and-play manner. First, comparisons of various exogenous α1,2-fucosyltransferase and α1,3-fucosyltransferase candidates, as well as a series of E. coli host strains, demonstrated that futC and futA from Helicobacter pylori using BL21(DE3) as the host strain yielded the highest titers of 2′-FL and 3-FL. Subsequently, both the availability of the lactose acceptor substrate and the intracellular pool of the GDP-L-fucose donor substrate were optimized by inactivating competitive (or repressive) pathways and strengthening acceptor (or donor) availability to achieve overproduction. Moreover, the intracellular redox regeneration pathways were engineered to further enhance the production of 2′-FL and 3-FL. Finally, various culture conditions were optimized to achieve the best performance of 2′-FL and 3-FL biosynthesizing strains. The final concentrations of 2′-FL and 3-FL were 9.12 and 12.43 g/L, respectively. This work provides a platform that enables modular construction, optimization and characterization to facilitate the development of FL-producing cell factories.     

Engineering self-flocculating Halomonas campaniensis for wastewaterless open and continuous fermentation

Halomonas has been developed as a platform for the next generation industrial biotechnology allowing open and nonsterile growth without microbial contamination under a high-salt concentration and alkali pH. To reduce downstream cost associated with continuous centrifugation and salt containing wastewater treatment, Halomonas campaniensis strain LS21 was engineered to become self-flocculating by knocking out an etf operon encoding two subunits of an electron transferring flavoprotein in the predicted electron transfer chain. Self-flocculation could be attributed to the decrease of the surface charge and increase of the cellular hydrophobicity resulted from deleted etf. A wastewaterless fermentation strategy based on the self-flocculating H. campaniensis was developed for growth and the production of poly-3-hydroxybutyrate (PHB) as an example. Most microbial cells flocculated and precipitated to the bottom of the bioreactor within 1 min after stopping the aeration and agitation. The supernatant can be used again without sterilization or inoculation for the growth of the next batch after collecting the precipitated cell mass. The wastewaterless process was conducted for four runs without generating wastewater. PHB accumulation by the self-flocculent strain was enhanced via promoter and ribosome binding site optimizations, the productivities of cell dry weight and PHB were increased from 0.45 and 0.18 g·L ⁻¹·hr ⁻¹ for the batch process compared to 0.82 and 0.33 g·L ⁻¹·hr ⁻¹ for the wastewaterless continuous process, respectively. This has clearly demonstrated the advantages of the wastewaterless process in that it not only reduces wastewater but also increases cell growth and product formation efficiency in a given period of time.     

Engineering a colanic acid biosynthesis pathway in E. coli for manufacturing 2’-fucosyllactose

Human milk oligosaccharides (HMOs) are beneficial for infants’ health and growth. As one of the most abundant oligosaccharides in human milk, 2′-fucosyllactose (2′-FL) has been approved to supplement in infant formula. Microbial synthesis of 2′-FL achieved in E. coli tends to use a T7-expression system for the heterologous expression of the fucosyltransferase and/or enzymes involved in fucose metabolism. In this paper, we report a novel bioconversion route of 2′-FL by engineering a low pH triggered colanic acid (CA) synthetic pathway, found in E. coli S17−3, which supplies GDP-l-fucose for in vivo 2′-FL formation catalyzed by the heterologous α-1,2-fucosyltransferases. In medium added with 10 g/L lactose and 20 g/L glycerol, recombinant S17−3 was able to produce 0.617 g/L of 2′-FL. The concentration of 2′-FL came to 1.029 g/L when a heterologous pathway for the synthesis of polyhydroxybutyrate was additionally introduced in the engineered S17−3.     

Fermentation and purification of microbial monomer 4-amminocinnamic acid to produce ultra-high performance bioplastics

Aromatic amines are base materials for generating super-engineering plastics such as polyamides and polyimides. Recombinant Escherichia coli ferments 4-aminocinnamic acid (4ACA) from glucose, and it can be derived to plastics of biomass origin with extreme thermal properties. Here, we scaled-up 4ACA production by optimizing microbial fermentation processes. The initial fermentation of 4-aminophenylalanine (4APhe) using E. coli generated the papABC genes of Pseudomonas fluorescens that produced 4APhe with a volumetric mass transfer coefficient (k_La) of 70 h⁻¹ in 115 L of culture broth, and 334 g of 4APhe at a final concentration of 2.9 g 4APhe L⁻¹. Crude 4APhe prepared from the fermentation broth was bioconverted to 4ACA by an E. coli strain producing phenylalanine ammonia lyase of the yeast Rhodotorula glutinis. The E. coli cells cultured under optimized conditions converted 4APhe to 4ACA at a rate of 0.65 g L⁻¹ 4ACA OD₆₀₀⁻¹. These processes resulted in the final derivation of 4.1 g L⁻¹ of 4ACA from glucose. The 4ACA was purified from the reaction as a hydrochloric acid salt and photodimerized to 4,4’-diaminotruxillic acid, which was polycondensed to produce bioaromatic polyimides. Large-scale 4ACA production will facilitate investigations of the physicochemical properties of biomass-derived aromatic polymers of 4ACA origin.     

High-level production of 3-hydroxypropionic acid from glycerol as a sole carbon source using metabolically engineered Escherichia coli

As climate change is an important environmental issue, the conventional petrochemical-based processes to produce valuable chemicals are being shifted toward eco-friendly biological-based processes. In this study, 3-hydroxypropionic acid (3-HP), an industrially important three carbon (C3) chemical, was overproduced by metabolically engineered Escherichia coli using glycerol as a sole carbon source. As the first step to construct a glycerol-dependent 3-HP biosynthetic pathway, the dhaB1234 and gdrAB genes from Klebsiella pneumoniae encoding glycerol dehydratase and glycerol reactivase, respectively, were introduced into E. coli to convert glycerol into 3-hydroxypropionaldehyde (3-HPA). In addition, the ydcW gene from K. pneumoniae encoding γ-aminobutyraldehyde dehydrogenase, among five aldehyde dehydrogenases examined, was selected to further convert 3-HPA to 3-HP. Increasing the expression level of the ydcW gene enhanced 3-HP production titer and reduced 1,3-propanediol production. To enhance 3-HP production, fed-batch fermentation conditions were optimized by controlling dissolved oxygen (DO) level and employing different feeding strategies including intermittent feeding, pH-stat feeding, and continuous feeding strategies. Fed-batch culture of the final engineered E. coli strain with DO control and continuous feeding strategy produced 76.2 g/L of 3-HP with the yield and productivity of 0.457 g/g glycerol and 1.89 g·L⁻¹·h⁻¹, respectively. To the best of our knowledge, this is the highest 3-HP productivity achieved by any microorganism reported to date.     

Modulation of guanosine nucleotides biosynthetic pathways enhanced GDP-<Emphasis Type="SmallCaps">l</Emphasis>-fucose production in recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Guanosine 5′-triphosphate (GTP) is the key substrate for biosynthesis of guanosine 5′-diphosphate (GDP)-l-fucose. In this study, improvement of GDP-l-fucose production was attempted by manipulating the biosynthetic pathway for guanosine nucleotides in recombinant Escherichia coli-producing GDP-l-fucose. The effects of overexpression of inosine 5′-monophosphate (IMP) dehydrogenase, guanosine 5′-monophosphate (GMP) synthetase (GuaB and GuaA), GMP reductase (GuaC) and guanosine–inosine kinase (Gsk) on GDP-l-fucose production were investigated in a series of fed-batch fermentations. Among the enzymes tested, overexpression of Gsk led to a significant improvement of GDP-l-fucose production. Maximum GDP-l-fucose concentration of 305.5 ± 5.3 mg l⁻¹ was obtained in the pH-stat fed-batch fermentation of recombinant E. coli-overexpressing Gsk, which corresponds to a 58% enhancement in the GDP-l-fucose production compared with the control strain overexpressing GDP-l-fucose biosynthetic enzymes. Such an enhancement of GDP-l-fucose production could be due to the increase in the intracellular level of GMP.     

Reconstruction of tricarboxylic acid cycle in Corynebacterium glutamicum with a genome-scale metabolic network model for trans-4-hydroxyproline production

trans-4-Hydroxy- l -proline (Hyp) is an abundant component of mammalian collagen and functions as a chiral synthon for the syntheses of anti-inflammatory drugs in the pharmaceutical industry. Proline 4-hydroxylase (P4H) can catalyze the conversion of l -proline to Hyp; however, it is still challenging for the fermentative production of Hyp from glucose using P4H due to the low yield and productivity. Here, we report the metabolic engineering of Corynebacterium glutamicum for the fermentative production of Hyp by reconstructing tricarboxylic acid (TCA) cycle together with heterologously expressing the p4h gene from Dactylosporangium sp. strain RH1. In silico model-based simulation showed that α-ketoglutarate was redirected from the TCA cycle toward Hyp synthetic pathway driven by P4H when the carbon flux from succinyl-CoA to succinate descended to zero. The interruption of the TCA cycle by the deletion of sucCD-encoding the succinyl-CoA synthetase (SUCOAS) led to a 60% increase in Hyp production and had no obvious impact on the growth rate. Fine-tuning of plasmid-borne ProB* and P4H abundances led to a significant increase in the yield of Hyp on glucose. The final engineered Hyp-7 strain produced up to 21.72 g/L Hyp with a yield of 0.27 mol/mol (Hyp/glucose) and a volumetric productivity of 0.36 g·L ⁻¹·hr ⁻¹ in the shake flask fermentation. To our knowledge, this is the highest yield and productivity achieved by microbial fermentation in a glucose-minimal medium for Hyp production. This strategy provides new insights into engineering C. glutamicum by flux coupling for the fermentative production of Hyp and related products.     

Enhancing acid tolerance of Escherichia coli via viroporin-mediated export of protons and its application for efficient whole-cell biotransformation

Escherichia coli-based whole-cell biocatalysts are widely used for the sustainable production of value-added chemicals. However, weak acids present as substrates and/or products obstruct the growth and fermentation capability of E. coli. Here, we show that a viroporin consisting of the influenza A matrix-2 (M2) protein, is activated by low pH and has proton channel activity in E. coli. The heterologous expression of the M2 protein in E. coli resulted in a significant increase in the intracellular pH and cell viability in the presence of various weak acids with different lengths of carbon chains. In addition, the feasibility of developing a robust and efficient E. coli-based whole-cell biocatalyst via introduction of the proton-selective viroporin was explored by employing (Z)-11-(heptanolyoxy)undec-9-enoic acid (ester) and 2-fucosyllactose (2′-FL) as model products, whose production is hampered by cytosolic acidification. The engineered E. coli strains containing the proton-selective viroporin exhibited approximately 80% and 230% higher concentrations of the ester and 2′-FL, respectively, than the control strains without the M2 protein. The simple and powerful strategy developed in this study can be applied to produce other valuable chemicals whose production involves substrates and/or products that cause cytosolic acidification.     

CRISPR/Cas9编辑大肠杆菌工程菌生产氨基葡萄糖

【背景】氨基葡萄糖(glucosamine, GlcN)及其衍生物N-乙酰氨基葡萄糖(N-acetylglucosamine,GlcNAc)是合成糖胺聚糖的重要前体物质,在医药、化妆品和保健品领域具有广泛的应用价值。传统的生产方式存在诸多弊端,如环境污染、原料限制、不适于海鲜易过敏人群等问题,因此利用微生物发酵法生产GlcN和GlcNAc越来越受到青睐。【目的】利用微生物发酵生产并提高N-乙酰氨基葡萄糖的产量,探索分子改造及发酵条件优化策略。【方法】以大肠杆菌MG1655为出发菌株,首先利用表达载体共表达大肠杆菌来源的glmS和酿酒酵母来源的gna1,构建GlcNAc的生物合成路径,然后利用CRISPR/Cas9技术敲除GlcNAc的分解代谢与转运途径,以提高GlcNAc的产量,最后结合发酵条件优化使GlcNAc的产量得到进一步提升。【结果】通过分子改造得到一株产GlcNAc菌株RY-5,发酵20 h后GlcNAc的产量达到了2.36 g/L,相较于初始构建的菌株RY-1提高了29倍,进一步对装液量和诱导剂IPTG的添加时间等条件进行发酵优化,GlcNAc产量达到了7.74g/L,与优...     

Engineering high-gravity fermentations for ethanol production at elevated temperature with Saccharomyces cerevisiae

Thermal damage, high osmolarity, and ethanol toxicity in the yeast Saccharomyces cerevisiae limit titer and productivity in fermentation to produce ethanol. We show that long-term adaptive laboratory evolution at 39.5°C generates thermotolerant yeast strains, which increased ethanol yield and productivity by 10% and 70%, in 2% glucose fermentations. From these strains, which also tolerate elevated-osmolarity, we selected a stable one, namely a strain lacking chromosomal duplications. This strain (TTY23) showed reduced mitochondrial metabolism and high proton efflux, and therefore lower ethanol tolerance. This maladaptation was bolstered by reestablishing proton homeostasis through increasing fermentation pH from 5 to 6 and/or adding potassium to the media. This change allowed the TTY23 strain to produce 1.3–1.6 times more ethanol than the parental strain in fermentations at 40°C with glucose concentrations ~300 g/L. Furthermore, ethanol titers and productivities up to 93.1 and 3.87 g·L ⁻¹·hr ⁻¹ were obtained from fermentations with 200 g/L glucose in potassium-containing media at 40°C. Albeit the complexity of cellular responses to heat, ethanol, and high osmolarity, in this study we overcome such limitations by an inverse metabolic engineering approach.     

Production of GDP-l-fucose, l-fucose donor for fucosyloligosaccharide synthesis, in recombinant Escherichia coli

A recombinant Escherichia coli strain was developed to produce guanosine 5′-diphosphate (GDP)-l-fucose, donor of l-fucose, which is an essential substrate for the synthesis of fucosyloligosaccharides. GDP-d-mannose-4, 6-dehydratase (GMD) and GDP-4-keto-6-deoxymannose 3, 5-epimerase 4-reductase (WcaG), the two crucial enzymes for the de novo GDP-l-fucose biosynthesis, were overexpressed in recombinant E. coli by constructing inducible overexpression vectors. Optimum expression conditions for GMD and WcaG in recombinant E. coli BL21(DE3) were 25°C and 0.1 mM isopropyl-β-d-thioglucopyranoside. Maximum GDP-l-fucose concentration of 38.9 ± 0.6 mg l⁻¹ was obtained in a glucose-limited fed-batch cultivation, and it was enhanced further by co-expression of NADPH-regenerating glucose-6-phosphate dehydrogenase encoded by the zwf gene to achieve 55.2 ± 0.5 mg l⁻¹ GDP-l-fucose under the same cultivation condition.     

Chassis engineering of Escherichia coli for trans-4-hydroxy-l-proline production

Microbial production of trans-4-hydroxy-l -proline (Hyp) offers significant advantages over conventional chemical extraction. However, it is still challenging for industrial production of Hyp due to its low production efficiency. Here, chassis engineering was used for tailoring Escherichia coli cellular metabolism to enhance enzymatic production of Hyp. Specifically, four proline 4-hydroxylases (P4H) were selected to convert l -proline to Hyp, and the recombinant strain overexpressing DsP4H produced 32.5 g l⁻¹ Hyp with α-ketoglutarate addition. To produce Hyp without α-ketoglutarate addition, α-ketoglutarate supply was enhanced by rewiring the TCA cycle and l -proline degradation pathway, and oxygen transfer was improved by fine-tuning heterologous haemoglobin expression. In a 5-l fermenter, the engineered strain E. coliΔsucCDΔputA-VHb_(L)-DsP4H showed a significant increase in Hyp titre, conversion rate and productivity up to 49.8 g l⁻¹, 87.4% and 1.38 g l⁻¹ h⁻¹ respectively. This strategy described here provides an efficient method for production of Hyp, and it has a great potential in industrial application.