Search

Abstract Dopamine is high-value compound of pharmaceutical interest, but its industrial scale production mostly focuses on chemical synthesis, possessing environment pollution. Bio-manufacturing has caused much attention for its environmental characteristic. Resting cells were employed to as biocatalysts with extraordinary advantages like offering stable surroundings, the inherent presence of expensive cofactors. In this study, whole-cell bioconversion was employed to convert dopa to dopamine. To increase the titer and yield of dopamine production through whole-cell catalysis, three kinds of aromatic amino acid transport protein, AroP, PheP and TyrP, were selected to be co-expressed. The effects of the concentration of L-dopa, pyridoxal-5’- phosphate (PLP), reaction temperature and pH were characterized for improvement of bioconversion. Under optimal conditions, dopamine titer reached 1.44 g/L with molar yield of 46.3%, which is 6.62 times than that of initial conditions. The catalysis productivity of recombinant E. coli co-expressed L-dopa decarboxylase(DDC) and AroP was further enhanced by repeated cell recycling, which maintained over 50% of its initial ability with eight consecutive catalyses. This study was the first to successfully bioconversion of dopamine by whole-cell catalysis. This research provided reference for whole-cell catalysis which is hindered by cell membrane.

Abstract Background 1,5-Diamino-2-hydroxy-pentane (2-OH-PDA), as a new type of aliphatic amino alcohol, has potential applications in the pharmaceutical, chemical, and materials industries. Currently, 2-OH-PDA production has only been realized via pure enzyme catalysis from lysine hydroxylation and decarboxylation, which faces great challenges for scale-up production. However, the use of a cell factory is very promising for the production of 2-OH-PDA for industrial applications, but the substrate transport rate, appropriate catalytic environment (pH, temperature, ions) and separation method restrict its efficient synthesis. Here, a strategy was developed to produce 2-OH-PDA via an efficient, green and sustainable biosynthetic method on an industrial scale. Results In this study, an approach was created for efficient 2-OH-PDA production from l-lysine using engineered E. coli BL21 (DE3) cell catalysis by a two-stage hydroxylation and decarboxylation process. In the hydroxylation stage, strain B14 coexpressing l-lysine 3-hydroxylase K3H and the lysine transporter CadB-argT enhanced the biosynthesis of (2S,3S)-3-hydroxylysine (hydroxylysine) compared with strain B1 overexpressing K3H. The titre of hydroxylysine synthesized by B14 was 2.1 times higher than that synthesized by B1. Then, in the decarboxylation stage, CadA showed the highest hydroxylysine activity among the four decarboxylases investigated. Based on the results from three feeding strategies, l-lysine was employed to produce 110.5 g/L hydroxylysine, which was subsequently decarboxylated to generate a 2-OH-PDA titre of 80.5 g/L with 62.6% molar yield in a 5-L fermenter. In addition, 2-OH-PDA with 95.6% purity was obtained by solid-phase extraction. Thus, the proposed two-stage whole-cell biocatalysis approach is a green and effective method for producing 2-OH-PDA on an industrial scale. Conclusions The whole-cell catalytic system showed a sufficiently high capability to convert lysine into 2-OH-PDA. Furthermore, the high titre of 2-OH-PDA is conducive to separation and possesses the prospect of industrial scale production by whole-cell catalysis.

N-acetylneuraminic acid (Neu5Ac) possesses the ability to promote mental health and enhance immunity and is widely used in both medicine and food fields as a supplement. Enzymatic production of Neu5Ac using N-acetyl-D-glucosamine (GlcNAc) as substrate was significant. However, the high-cost GlcNAc limited its development. In this study, an in vitro multi-enzyme catalysis was built to produce Neu5Ac using affordable chitin as substrate. Firstly, exochitinase SmChiA from Serratia proteamaculans and N-acetylglucosaminosidase CmNAGase from Chitinolyticbacter meiyuanensis SYBC-H1 were screened and combined to produce GlcNAc, effectively. Then, the chitinase was cascaded with N-acetylglucosamine-2-epimerase (AGE) and N-neuraminic acid aldolase (NanA) to produce Neu5Ac; the optimal conditions of the multi-enzyme catalysis system were 37°C and pH 8.5, the ratio of AGE to NanA (1:4) and addition of pyruvate (70 mM), respectively. Finally, 9.2 g/L Neu5Ac could be obtained from 20 g/L chitin within 24 h along with two supplementations with pyruvate. This work will lay a good foundation for the production of Neu5Ac from cheap chitin resources.

Abstract Background l-Tryptophan (l-Trp) derivatives such as 5-hydroxytryptophan (5-HTP) and 5-hydroxytryptamine (5-HT), N-Acetyl-5-hydroxytryptamine and melatonin are important molecules with pharmaceutical interest. Among, 5-HT is an inhibitory neurotransmitter with proven benefits for treating the symptoms of depression. At present, 5-HT depends on plant extraction and chemical synthesis, which limits its mass production and causes environmental problems. Therefore, it is necessary to develop an efficient, green and sustainable biosynthesis method to produce 5-HT. Results Here we propose a one-pot production of 5-HT from l-Trp via two enzyme cascades for the first time. First, a chassis cell that can convert l-Trp into 5-HTP was constructed by heterologous expression of tryptophan hydroxylase from Schistosoma mansoni (SmTPH) and an artificial endogenous tetrahydrobiopterin (BH4) module. Then, dopa decarboxylase from Harminia axyridis (HaDDC), which can specifically catalyse 5-HTP to 5-HT, was used for 5-HT production. The cell factory, E. coli BL21(DE3)△tnaA/BH4/HaDDC-SmTPH, which contains SmTPH and HaDDC, was constructed for 5-HT synthesis. The highest concentration of 5-HT reached 414.5 ± 1.6 mg/L (with conversion rate of 25.9 mol%) at the optimal conditions (substrate concentration,2 g/L; induced temperature, 25℃; IPTG concentration, 0.5 mM; catalysis temperature, 30℃; catalysis time, 72 h). Conclusions This protocol provided an efficient one-pot method for converting. l-Trp into 5-HT production, which opens up possibilities for the practical biosynthesis of natural 5-HT at an industrial scale.

Cis-3-hydroxypipecolic acid (cis-3-HyPip), a key structural component of tetrapeptide antibiotic GE81112, which has attracted substantial attention for its broad antimicrobial properties and unique ability to inhibit bacterial translation initiation. In this study, a combined strategy to increase the productivity of cis-3-HyPip was investigated. First, combinatorial optimization of the ribosomal binding site (RBS) sequence was performed to tune the gene expression translation rates of the pathway enzymes. Next, in order to reduce the addition of the co-substrate α-ketoglutarate (2-OG), the major engineering strategy was to reconstitute the tricarboxylic acid (TCA) cycle of Escherichia coli to force the metabolic flux to go through GetF catalyzed reaction for 2-OG to succinate conversion, a series of engineered strains were constructed by the deletion of the relevant genes. In addition, the metabolic flux (gltA and icd) was improved and glucose concentrations were optimized to enhance the supply and catalytic efficiency of continuous 2-OG supply powered by glucose. Finally, under optimal conditions, the cis-3-HyPip titer of the best strain catalysis reached 33 mM, which was remarkably higher than previously reported.

In this study, a gene encoding β-N-acetylglucosaminidase, designated NAGaseA, was cloned from Chitinibacter sp. GC72 and subsequently functional expressed in Escherichia coli BL21 (DE3). NAGaseA contains a glycoside hydrolase family 20 catalytic domain that shows low identity with the corresponding domain of the well-characterized NAGases. The recombinant NAGaseA had a molecular mass of 92 kDa. Biochemical characterization of the purified NAGaseA revealed that the optimal reaction condition was at 40°C and pH 6.5, and exhibited great pH stability in the range of pH 6.5–9.5. The Vmax, Km, kcat, and kcat/Km of NAGaseA toward p-nitrophenyl-N-acetyl glucosaminide (pNP-GlcNAc) were 3333.33 μmol min–1 l–1, 39.99 μmol l–1, 4667.07 s–1, and 116.71 ml μmol–1 s–1, respectively. Analysis of the hydrolysis products of N-acetyl chitin oligosaccharides (N-Acetyl COSs) indicated that NAGaseA was capable of converting N-acetyl COSs ((GlcNAc)2–(GlcNAc)6) into GlcNAc with hydrolysis ability order: (GlcNAc)2 > (GlcNAc)3 > (GlcNAc)4 > (GlcNAc)5 > (GlcNAc)6. Moreover, NAGaseA could generate (GlcNAc)3–(GlcNAc)6 from (GlcNAc)2–(GlcNAc)5, respectively. These results showed that NAGaseA is a multifunctional NAGase with transglycosylation activity. In addition, significantly synergistic action was observed between NAGaseA and other sources of chitinases during hydrolysis of colloid chitin. Finally, 0.759, 0.481, and 0.986 g/l of GlcNAc with a purity of 96% were obtained using three different chitinase combinations, which were 1.61-, 2.36-, and 2.69-fold that of the GlcNAc production using the single chitinase. This observation indicated that NAGaseA could be a potential candidate enzyme in commercial GlcNAc production.

A novel multifunctional chitinase (CmChi3)-encoding gene was cloned from Chitinolyticbacter meiyuanensis and actively expressed in Escherichia coli. Sequence analysis showed that CmChi3 contains two glycoside hydrolase family 18 (GH18) catalytic domains and exhibited low identity with well-characterized chitinases. The optimum pH and temperature of purified recombinant CmChi3 were 6.0 and 50°C, respectively. CmChi3 exhibited strict substrate specificity of 4.1 U/mg toward colloidal chitin (CC) and hydrolyzed it to yield N-acetyl-D-glucosamine (GlcNAc) as the sole end product. An analysis of the hydrolysis products toward N-acetyl chitooligosaccharides (N-acetyl COSs) and CC substrates revealed that CmChi3 exhibits endochitinase, N-acetyl-β-d-glucosaminidase (NAGase), and transglycosylase (TGase) activities. Further studies revealed that the N-terminal catalytic domain of CmChi3 exhibited endo-acting and NAGase activities, while the C-terminal catalytic domain showed exo-acting and TGase activities. The hydrolytic properties and favorable environmental adaptations indicate that CmChi3 holds potential for commercial GlcNAc production from chitin.

Abstract Background N-Acetyl glucosamine (GlcNAc) and N-Acetyl chitooligosaccharides (N-Acetyl COSs) exhibit many biological activities, and have been widely used in the pharmaceutical, agriculture, food, and chemical industries. Particularly, higher N-Acetyl COSs with degree of polymerization from 4 to 7 ((GlcNAc)4–(GlcNAc)7) show good antitumor and antimicrobial activity, as well as possessing strong stimulating activity toward natural killer cells. Thus, it is of great significance to discover a β-N-acetyl glucosaminidase (NAGase) that can not only produce GlcNAc, but also synthesize N-Acetyl COSs. Results The gene encoding the novel β-N-acetyl glucosaminidase, designated CmNAGase, was cloned from Chitinolyticbacter meiyuanensis SYBC-H1. The deduced amino acid sequence of CmNAGase contains a glycoside hydrolase family 20 catalytic module that shows low identity (12–35%) with the corresponding domain of most well-characterized NAGases. The CmNAGase gene was highly expressed with an active form in Escherichia coli BL21 (DE3) cells. The specific activity of purified CmNAGase toward p-nitrophenyl-N-acetyl glucosaminide (pNP-GlcNAc) was 4878.6 U/mg of protein. CmNAGase had a molecular mass of 92 kDa, and its optimum activity was at pH 5.4 and 40 °C. The V max, K m, K cat, and K cat/K m of CmNAGase for pNP-GlcNAc were 16,666.67 μmol min−1 mg−1, 0.50 μmol mL−1, 25,555.56 s−1, and 51,111.12 mL μmol−1 s−1, respectively. Analysis of the hydrolysis products of N-Acetyl COSs and colloidal chitin revealed that CmNAGase is a typical exo-acting NAGase. Particularly, CmNAGase can synthesize higher N-Acetyl COSs ((GlcNAc)3–(GlcNAc)7) from (GlcNAc)2–(GlcNAc)6, respectively, showed that it possesses transglycosylation activity. In addition, CmNAGase also has reverse hydrolysis activity toward GlcNAc, synthesizing various linked GlcNAc dimers. Conclusions The observations recorded in this study that CmNAGase is a novel NAGase with exo-acting, transglycosylation, and reverse hydrolysis activities, suggest a possible application in the production of GlcNAc or higher N-Acetyl COSs.

Chitinolyticbacter meiyuanensis SYBC-H1, a bacterium capable of hydrolyzing chitin and shrimp shell to N-acetyl glucosamine (GlcNAc) as the only product, was isolated previously. Here, the hydrolysis mechanism of this novel strain toward chitin was investigated. Sequencing and analysis of the complete genome of SYBC-H1 showed that it encodes 32 putatively chitinolytic enzymes including 30 chitinases affiliated with the glycoside hydrolase (GH) families 18 (26) and 19 (4), one GH family 20 β-N-acetylglucosaminidase (NAGase), and one Auxiliary Activities (AA) family 10 lytic polysaccharide monooxygenase (LPMO). However, only eight GH18 chitinases, one AA10 LPMO, and one GH20 NAGase were detected in the culture broth of the strain, according to peptide mass fingerprinting (PMF). Of these, genes encoding chitinolytic enzymes including five GH18 chitinases (Cm711, Cm3636, Cm3638, Cm3639, and Cm3769) and one GH20 NAGase (Cm3245) were successfully expressed in active form in Escherichia coli. The hydrolysis of chitinous substrates showed that Cm711, Cm3636, Cm3638, and Cm3769 were endo-chitinases and Cm3639 was exo-chitinase. Moreover, Cm3639 and Cm3769 can convert the GlcNAc dimer and colloidal chitin (CC) into GlcNAc, which showed that they also possess NAGase activity. In addition, NAGase Cm3245 possesses a very high exo-acting activity of hydrolyzing GlcNAc dimer. These results suggest that chitinases and NAGase from SYBC-H1 both play important roles in conversion of N-acetyl chitooligosaccharides to GlcNAc, resulting in the accumulation of the final product GlcNAc. To our knowledge, this is the first report of the complete genome sequence and chitinolytic enzyme genes discovery of this strain.

A new protein immobilization and purification system has been developed based on the improved plasmid vectors, designated pETChBD-X, which contained the gene coding for two novel chitin-binding domains ChBD-AB, factor Xa cleavage site and adapted for gene fusions. The ChBD-AD from Chitinolyticbacter meiyuanensis SYBC-H1 was used as a novel affinity tag to anchor fusion proteins to chitin granules. The granules carrying the ChBD-AD fusion proteins can be isolated by a simple centrifugation step and used directly for some applications. Moreover, when required, a practically pure preparation of the soluble recombination protein can be obtained after Factor Xa cleavage. The efficiency of this system has been demonstrated by reaching 95% of protein absorbed to chitin within 30 min and recycling over 75% of interest protein after Factor Xa cleavage to separate interest protein and fusion tag. Furthermore, 65% L-glutamate oxidase with this fusion tag could be purified and immobilized within only one step and to be reused in converting L-glutamate to α-ketoglutaric acid directly, the average conversion rate kept above 65% even within four batches of enzyme conversion reaction.

Lysine decarboxylase (CadA) can directly convert L-lysine to cadaverine, which is an important platform chemical that can be used to produce polyamides. However, the non-recyclable and the poor pH tolerance of pure CadA hampered its practical application. Herein, a one-step purification and immobilization procedure of CadA was established to investigate the cadaverine production from L-lysine. Renewable biomass chitin was used as a carrier for lysine decarboxylase (CadA) immobilization via fusion of a chitin-binding domain (ChBD). Scanning electron microscopy, laser scanning confocal microscopy, fourier transform infrared spectra, elemental analysis, and thermal gravimetric analysis proved that the fusion protein ChBD-CadA can be adsorbed on chitin effectively. Furthermore, the fusion protein (ChBD-CadA) existed better pH stability compared to wild CadA, and kept over 73% of the highest activity at pH 8.0. Meanwhile, the ChBD-CadA showed high specificity toward chitin and reached 93% immobilization yield within 10 min under the optimum conditions. The immobilized ChBD-CadA (I-ChBD-CadA) could efficiently converted L-lysine at 200.0 g/L to cadaverine at 135.6 g/L in a batch conversion within 120 min, achieving a 97% molar yield of the substrate L-lysine. In addition, the I-ChBD-CadA was able to be reused under a high concentration of L-lysine and retained over 57% of its original activity after four cycles of use without acid addition to maintain pH. These results demonstrate that immobilization of CadA using chitin-binding domain has the potential in cadaverine production on an industrial scale.

Abstract Background N-acetyl-d-glucosamine (GlcNAc) possesses many bioactivities that have been used widely in many fields. The enzymatic production of GlcNAc is eco-friendly, with high yields and a mild production process compared with the traditional chemical process. Therefore, it is crucial to discover a better chitinase for GlcNAc production from chitin. Results A novel chitinase gene (Cmchi1) cloned from Chitinolyticbacter meiyuanensis SYBC-H1 and expressed in Escherichia coli BL21(DE3) cells. The recombinant enzyme (CmChi1) contains a glycosyl hydrolase family 18 catalytic module that shows low identity (12–27%) with the corresponding domain of the well-characterized chitinases. CmChi1 was purified with a recovery yield of 89% by colloidal chitin affinity chromatography, whereupon it had a specific activity of up to 15.3 U/mg. CmChi1 had an approximate molecular mass of 70 kDa after the sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its optimum activity for colloidal chitin (CC) hydrolysis occurred at pH 5.2 and 50 °C. Furthermore, CmChi1 exhibited k cat/K m values of 7.8 ± 0.11 mL/s/mg and 239.1 ± 2.6 mL/s/μmol toward CC and 4-nitrophenol N,N′-diacetyl-β-d-chitobioside [p-NP-(GlcNAc)2], respectively. Analysis of the hydrolysis products revealed that CmChi1 exhibits exo-acting, endo-acting and N-acetyl-β-d-glucosaminidase activities toward N-acetyl chitooligosaccharides (N-acetyl CHOS) and CC substrates, behavior that makes it different from typical reported chitinases. As a result, GlcNAc could be produced by hydrolyzing CC using recombinant CmChi1 alone with a yield of nearly 100% and separated simply from the hydrolysate with a high purity of 98%. Conclusion The hydrolytic properties and good environmental adaptions indicate that CmChi1 has excellent potential in commercial GlcNAc production. This is the first report on exo-acting, endo-acting and N-acetyl-β-d-glucosaminidase activities from Chitinolyticbacter species.

N-acetylneuraminic acid (Neu5Ac) possesses the ability to promote mental health and enhance immunity and is widely used in both medicine and food fields as a supplement. Enzymatic production of Neu5Ac using N-acetyl-D-glucosamine (GlcNAc) as substrate was significant. However, the high-cost GlcNAc limited its development. In this study, an in vitro multi-enzyme catalysis was built to produce Neu5Ac using affordable chitin as substrate. Firstly, exochitinase SmChiA from Serratia proteamaculans and N-acetylglucosaminosidase CmNAGase from Chitinolyticbacter meiyuanensis SYBC-H1 were screened and combined to produce GlcNAc, effectively. Then, the chitinase was cascaded with N-acetylglucosamine-2-epimerase (AGE) and N-neuraminic acid aldolase (NanA) to produce Neu5Ac; the optimal conditions of the multi-enzyme catalysis system were 37°C and pH 8.5, the ratio of AGE to NanA (1:4) and addition of pyruvate (70 mM), respectively. Finally, 9.2 g/L Neu5Ac could be obtained from 20 g/L chitin within 24 h along with two supplementations with pyruvate. This work will lay a good foundation for the production of Neu5Ac from cheap chitin resources. [ABSTRACT FROM AUTHOR]