Biosynthesis of di-rhamnolipids and variations of congeners composition in genetically-engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To engineer Escherichia coli for the heterologous production of di-rhamnolipids, which are important biosurfactants but mainly produced by opportunistic pathogen Pseudomonas aeruginosa.

Results

The codon-optimized rhlAB and rhlC genes originating from P. aeruginosa and Burkholderia pseudomallei were combinatorially expressed in E. coli to produce di-rhamnolipids with varied congeners compositions. Genes involved in endogenous upstream pathways (rhamnose and fatty acids synthesis) were co-overexpressed with rhlAB–rhlC, resulting in variations of rhamnolipids production and congeners compositions. Under the shake-flask condition, co-overexpression of rfbD with rhlAB–rhlC increased rhamnolipids production (0.64 ± 0.02 g l^?1) than that in strain only expressing rhlAB–rhlC (0.446 ± 0.009 g l^?1), which was mainly composed of di-rhamnolipids congeners Rha–Rha–C₁₀–C₁₀.

Conclusion

Biosynthesis of di-rhamnolipids and variations of congeners composition in genetically engineered E. coli strains were achieved via combiniations of mono-/di-rhamnolipids synthesis modules and endogenous upstream modules.

     

Engineering the intracellular metabolism of <Emphasis Type="Italic">Escherichia coli</Emphasis> to produce gamma-aminobutyric acid by co-localization of GABA shunt enzymes

Objectives

To direct the carbon flux from Krebs cycle into the gamma-aminobutyric acid (GABA) shunt pathway for the production of GABA by protein scaffold introduction in Escherichia coli.

Results

Escherichia coli was engineered to produce GABA from glucose by the co-localization of enzymes succinate semialdehyde dehydrogenase (GadD), GABA aminotransferase (PuuE) and GABA transporter (GadC) by protein scaffold. 0.7 g GABA l^?1 was produced from 10 g glucose l^?1 while no GABA was produced in wild type E. coli. pH 6 and 30 °C were optimum for GABA production, and GABA concentration increased to 1.12 g GABA l^?1 when 20 g glucose l^?1 was used. When competing metabolic networks were inactivated, GABA increased by 24 % (0.87 g GABA l^?1).

Conclusions

The novel GABA production system was constructed by co-localization of GABA shunt enzymes.

     

Rapid evolution of hyaluronan synthase to improve hyaluronan production and molecular mass in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Objectives

To improve the production and molecular mass of the glycosaminoglycan hyaluronan (HA) in Bacillus subtilis by engineering hyaluronan synthase (HAS) from Streptococcus zooepidemicus.

Results

By mutating regions within HAS intracellular domains, five positive variants exhibiting higher HA production (from 1.22 to 2.24 g l^?1) and molecular mass values (from 1.20 to 1.36 × 10⁶ Da) were constructed and characterized. Overexpression of the V5 variant and the genes tuaD and glmU increased HA production and molecular mass to 2.8 g l^?1 and 2.4 × 10⁶ Da, respectively.

Conclusions

This study provides a novel strategy for improving HA production and its molecular mass.

     

Production of <Emphasis Type="Italic">trans</Emphasis>-10,<Emphasis Type="Italic">cis</Emphasis>-12-conjugated linoleic acid using permeabilized whole-cell biocatalyst of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Objective

To improve the production of trans-10,cis-12-conjugated linoleic acid (t10,c12-CLA) from linoleic acid in recombinant Yarrowia lipolytica.

Results

Cells of the yeast were permeabilized by freeze/thawing. The optimal conditions for t10,c12-CLA production by the permeabilized cells were at 28 °C, pH 7, 200 rpm with 1.5 g sodium acetate l^?1, 100 g wet cells l^?1, and 25 g LA l^?1. Under these conditions, the permeabilized cells produced 15.6 g t10,c12-CLA l^?1 after 40 h, with a conversion yield of 62 %. The permeabilized cells could be used repeatedly for three cycles, with the t10,c12-CLA extracellular production remaining above 10 g l^?1.

Conclusion

Synthesis of t10,c12-CLA was achieved using a novel method, and the production reported in this work is the highest value reported to date.

     

Efficient whole-cell biocatalyst for Neu5Ac production by manipulating synthetic,degradation and transmembrane pathways

Objective

To develop a strategy for producing N-acetyl-d-neuraminic acid (Neu5Ac), which is often synthesized from exogenous N-acetylglucosamine (GlcNAc) and pyruvate, but without using pyruvate.

Result

An efficient three-module whole-cell biocatalyst strategy for Neu5Ac production by utilizing intracellular phosphoenolpyruvate was established. In module I, the synthetic pathway was constructed by coexpressing GlcNAc 2-epimerase from Anabaena sp. CH1 and Neu5Ac synthase from Campylobacter jejuni in Escherichia coli. In module II, the Neu5Ac degradation pathway of E. coli was knocked out, resulting in 2.6 ± 0.06 g Neu5Ac l^?1 after 72 h in Erlenmeyer flasks. In module III, the transmembrane mode of GlcNAc was modified by disruption of GlcNAc-specific phosphotransferase system and Neu5Ac now reached 3.7 ± 0.04 g l^?1. In scale-up catalysis with a 1 l fermenter, the final Neu5Ac yield was 7.2 ± 0.08 g l^?1.

Conclusion

A three-module whole-cell biocatalyst strategy by manipulating synthetic, degradation and transmembrane pathways in E. coli was an economical method for Neu5Ac production.

     

Enhanced plasmid DNA production by enzyme-controlled glucose release and an engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To evaluate the combination of a culture medium employing glucoamylase-mediated glucose reléase from a gluco-polysaccharide and an E. coli strain engineered in its glucose transport system for improving plasmid DNA (pDNA) production.

Results

The production of pDNA was tested using E. coli DH5α grown in shake-flasks and the recently developed VH33 Δ(recA deoR)-engineered strain, which utilizes glucose more efficiently than wild type strains. Three glucoamylase concentrations for releasing glucose from the polysaccharide carbon source were used: 1, 2 and 3 U l^?1. Both strains reached similar cell densities ranging from 5 to 8.8 g l^?1 under the different conditions. The highest pDNA yields on biomass (Y_pDNA/X) for both strains were obtained when 3 U enzyme l^?1were used. Under these conditions, 35 ± 3 mgof pDNA l^?1 were produced by DH5α after 24 h of culture. Under the same conditions, the engineered strain produced 66 ± 1 mgpDNAl^?1 after 20 h. pDNA supercoiled fractionswere close to 80 % for both strains.

Conclusions

The pDNA concentration achieved by the engineered E. coli was 89 % higher than that of DH5α. The combination of the engineered strain and enzyme-controlled glucose release is an attractive alternative for pDNA production in shake-flasks.

     

Expression,purification and characterization of a thermostable leucine dehydrogenase from the halophilic thermophile <Emphasis Type="Italic">Laceyella sacchari</Emphasis>

Objective

A potential thermotolerant l-leucine dehydrogenase from Laceyella sacchari (Ls-LeuDH) was over-expressed in E. coli, purified and characterized.

Results

Ls-LeuDH had excellent thermostability with a specific activity of 183 U/mg at pH 10.5 and 25 °C. It retained a high activity in 200 mM carbonate buffer from pH 9.5 to 11. The optimal temperature for Ls-LeuDH was 60 °C.

Conclusion

It is the first time that a thermostable and highly active LeuDH originating from L. sacchari has been characterized. It may be useful for medical and pharmaceutical applications.

     

Production of tartaric acid using immobilized recominant <Emphasis Type="Italic">cis</Emphasis>-epoxysuccinate hydrolase

Objective

To investigate the expression and immobilization of recombinant cis-epoxysuccinate hydrolase (ESH), and its application in the biological production of l-(+)-tartaric acid.

Results

E. coli BL21 (DE3)/pET11a-ESH (His) was engineered to express recombinant ESH. The enzyme had an activity of 262 U mg^?1. The recombinant ESH was immobilized on agarose Ni-IDA matrix with metal ion affinity interaction to improve its thermostability and pH stability. The immobilization efficiency and activity yield were 94 and 95%, respectively. The specific catalytic efficiency of immobilized ESH was 104 mg U^?1 h^?1 during the continuous enzymatic production process.

Conclusion

ESH with a histidine tag was immobilized and used for the continuous production of l-(+)-tartaric acid.

     

Improved production of carotenoid-free welan gum in a genetic-engineered <Emphasis Type="Italic">Alcaligenes</Emphasis> sp. ATCC31555

Objective

To improve the production of welan gum and obtain a carotenoid-free strain while reducing the fermentation and post-treatment costs.

Results

The vitreoscilla globin (vgb) gene combined with the β-galactosidase (lacZ) promoter was inserted into the phytoene synthase (crtB) gene region of the chromosome in Alcaligenes sp. ATCC31555. When the recombinant strain was grown in a 5 l fermentor, welan gum was produced at 24 ± 0.4 g l^?1 compared to 21 g ± 0.4 g l^?1 in the wild type. Furthermore, the carotenoid-free welan gum produced using Alcaligenes sp. ATCC31555 VHb strain was less expensive with improved properties.

Conclusions

Alcaligenes sp. ATCC31555 VHb strain was a better neutral welan-producing strain with a higher production than the wild-type strain.

     

Discovery of novel feruloyl esterase activity of BioH in <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21(DE3)

Objectives

To characterize a novel feruloyl esterase from Escherichia coli BL21 DE3.

Results

The gene encoding BioH was cloned and overexpressed in E. coli. The protein was purified and its catalytic activity was assessed. BioH exhibited feruloyl esterase activity toward a broad range of substrates, and the corresponding kinetic constants for the methyl ferulate, ethyl ferulate, and methyl p-coumarate substrates were: K _m values of 0.48, 6.3, and 1.9 mM, respectively, and k _cat /K _m values of 9.3, 3.8, and 3.8 mM^?1 s^?1, respectively.

Conclusions

Feruloyl esterase from E. coli was expressed for the first time. BioH was confirmed to be a feruloyl esterase.

     

Functional expression of a human GDP-<Emphasis Type="SmallCaps">l</Emphasis>-fucose transporter in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To investigate the translocation of nucleotide-activated sugars from the cytosol across a membrane into the endoplasmatic reticulum or the Golgi apparatus which is an important step in the synthesis of glycoproteins and glycolipids in eukaryotes.

Results

The heterologous expression of the recombinant and codon-adapted human GDP-l-fucose antiporter gene SLC35C1 (encoding an N-terminal OmpA-signal sequence) led to a functional transporter protein located in the cytoplasmic membrane of Escherichia coli. The in vitro transport was investigated using inverted membrane vesicles. SLC35C1 is an antiporter specific for GDP-l-fucose and depending on the concomitant reverse transport of GMP. The recombinant transporter FucT1 exhibited an activity for the transport of ³H-GDP-l-fucose with a V_max of 8 pmol/min mg with a K_m of 4 µM. The functional expression of SLC35C1 in GDP-l-fucose overproducing E. coli led to the export of GDP-l-fucose to the culture supernatant.

Conclusions

The export of GDP-l-fucose by E. coli provides the opportunity for the engineering of a periplasmatic fucosylation reaction in recombinant bacterial cells.

     

Conversion of glycerol to 1,3-dihydroxyacetone by glycerol dehydrogenase co-expressed with an NADH oxidase for cofactor regeneration

Objectives

To investigate the efficiency of a cofactor regeneration enzyme co-expressed with a glycerol dehydrogenase for the production of 1,3-dihydroxyacetone (DHA).

Results

In vitro biotransformation of glycerol was achieved with the cell-free extracts containing recombinant GlyDH (glycerol dehydrogenase from Escherichia coli), LDH (lactate dehydrogenase form Bacillus subtilis) or LpNox1 (NADH oxidase from Lactobacillus pentosus), giving DHA at 1.3 g l^?1 (GlyDH/LDH) and 2.2 g l^?1 (GlyDH/LpNox1) with total turnover number (TTN) of NAD⁺ recycling of 6039 and 11100, respectively. Whole cells of E. coli (GlyDH–LpNox1) co-expressing both GlyDH and LpNox1 were constructed and converted 10 g glycerol l^?1 to DHA at 0.2–0.5 g l^?1 in the presence of zero to 2 mM exogenous NAD⁺. The cell free extract of E. coli (GlyDH–LpNox) converted glycerol (2–50 g l^?1) to DHA from 0.5 to 4.0 g l^?1 (8–25 % conversion) without exogenous NAD⁺.

Conclusions

The disadvantage of the expensive consumption of NAD⁺ for the production of DHA has been overcome.

     

Responses of the antioxidative and biotransformation enzymes in the aquatic fungus <Emphasis Type="Italic">Mucor hiemalis</Emphasis> exposed to cyanotoxins

Objectives

To investigate antioxidative and biotransformation enzyme responses in Mucor hiemalis towards cyanotoxins considering its use in mycoremediation applications.

Results

Catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GPx) in M. hiemalis maintained their activities at all tested microcystin-LR (MC-LR) exposure concentrations. Cytosolic glutathione S-transferase (GST) activity decreased with exposure to 100 µg MC-LR l^?1 while microsomal GST remained constant. Cylindrospermopsin (CYN) at 100 µg l^?1 led to an increase in CAT activity and inhibition of GR, as well as to a concentration-dependent GPx inhibition. Microsomal GST was inhibited at all concentrations tested. β-N-methylamino-l-alanine (BMAA) inhibited GR activity in a concentration-dependent manner, however, CAT, GPx, and GST remained unaffected.

Conclusions

M. hiemalis showed enhanced oxidative stress tolerance and intact biotransformation enzyme activity towards MC-LR and BMAA in comparison to CYN, confirming its applicability in bioreactor technology in terms of viability and survival in their presence.

     

Characterization of a novel <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> xanthine dehydrogenase expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objective

To characterize a novel xanthine dehydrogenase (XDH) from Acinetobacter baumannii by recombinant expression in Escherichia coli and to assess its potential for industrial applications.

Results

The XDH gene cluster was cloned from A. baumannii CICC 10254, expressed heterologously in E. coli and purified to homogeneity. The purified recombinant XDH consisted of two subunits with the respective molecular weights of 87 kDa and 56 kDa according to SDS-PAGE. XDH catalysis was optimum at pH 8.5 and 40–45 °C, was stable under alkaline conditions (pH 7–11) and the half-inactivation temperature was 60 °C. The K _m, turnover number and catalytic efficiency for xanthine were 25 μM, 69 s^?1 and 2.7 μM^?1 s^?1, respectively, which is an improvement over XDHs characterized previously. A. baumannii XDH is less than 50 % identical to previously identified XDH orthologs from other species, and is the first from the Acinetobacter genus to be characterized.

Conclusion

The novel A. baumannii enzyme was found to be among the most active, thermostable and alkaline-tolerant XDH enzymes reported to date and has potential for use in industrial applications.

     

Phosphoenolpyruvate-supply module in <Emphasis Type="Italic">Escherichia coli</Emphasis> improves <Emphasis Type="Italic">N</Emphasis>-acetyl-<Emphasis Type="SmallCaps">d</Emphasis>-neuraminic acid biocatalysis

Objectives

N-Acetyl-d-neuraminic acid (Neu5Ac) is often synthesized from exogenous N-acetylglucosamine (GlcNAc) and excess pyruvate. We have previously constructed a recombinant Escherichia coli strain for Neu5Ac production using GlcNAc and intracellular phosphoenolpyruvate (PEP) as substrates (Zhu et al. Biotechnol Lett 38:1–9, 2016).

Results

PEP synthesis-related genes, pck and ppsA, were overexpressed within different modes to construct PEP-supply modules, and their effects on Neu5Ac production were investigated. All the PEP-supply modules enhanced Neu5Ac production. For the best module, pCDF-pck-ppsA increased Neu5Ac production to 8.6 ± 0.15 g l^?1, compared with 3.6 ± 0.15 g l^?1 of the original strain. Neu5Ac production was further increased to 15 ± 0.33 g l^?1 in a 1 l fermenter.

Conclusions

The PEP-supply module can improve the intracellular PEP supply and enhance Neu5Ac production, which benefited industrial Neu5Ac production.

     

4-Vinylphenol production from glucose using recombinant <Emphasis Type="Italic">Streptomyces mobaraense</Emphasis> expressing a tyrosine ammonia lyase from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Objectives

To find a novel host for the production of 4-vinylphenol (4VPh) by screening Streptomyces species.

Results

The conversion of p-coumaric acid (pHCA) to 4VPh in Streptomyces mobaraense was evaluated using a medium containing pHCA. S. mobaraense readily assimilated pHCA after 24 h of cultivation to produce 4VPh. A phenolic acid decarboxylase, derived from S. mobaraense (SmPAD), was purified following heterologous expression in Escherichia coli. SmPAD was evaluated under various conditions, and the enzyme’s k_cat/K_m value was 0.54 mM ^?1 s^?1. Using intergenetic conjugation, a gene from Rhodobacter sphaeroides encoding a tyrosine ammonia lyase, which catalyzes the conversion of l-tyrosine to p-coumaric acid, was introduced into S. mobaraense. The resulting S. mobaraense transformant produced 273 mg 4VPh l^?1 from 10 g glucose l^?1.

Conclusion

A novel strain suitable for the production of 4VPh and potentially other aromatic compounds was isolated.

     

Long-term H<Subscript>2</Subscript> photoproduction from starch by co-culture of <Emphasis Type="Italic">Clostridium butyricum</Emphasis> and <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> in a repeated batch process

Objectives

To prove the possibility of efficient starch photofermentation in co-culture of heterotrophic and phototrophic bacteria over prolonged period.

Results

Repeated batch photofermentation of starch was demonstrated in co-culture Clostridium butyricum and Rhodobacter sphaeroides under microaerobic conditions. It continued 15 months without addition of new inoculum or pH regulation when using 4–5 g starch l^?1 and 0.04 g yeast extract l^?1. The complete degradation of starch without volatile fatty acids accumulation was shown in this co-culture. The average H₂ yield of 5.2 mol/mol glucose was much higher than that in Clostridium monoculture. The species composition of co-culture was studied by q-PCR assay. The concentration of Clostridium cells in prolonged co-culture was lower than in monoculture and even in a single batch co-culture. This means that Clostridia growth was significantly limited whereas starch hydrolysis still took place.

Conclusion

The prolonged repeated batch photofermentation of starch by co-culture C. butyricum and R. sphaeroides provided efficient H₂ production without accumulation of organic acids under conditions of Clostridia limitation.

     

Isolation and characterization of a novel <Emphasis Type="SmallCaps">l</Emphasis>-glutamate oxidase with strict substrate specificity from <Emphasis Type="Italic">Streptomyces diastatochromogenes</Emphasis>

Objectives

To find an l-glutamate oxidase (LGox), to be used for the quantitative analysis of l-glutamic acid, an lgox gene encoding LGox from Streptomyces diastatochromogenes was isolated, cloned and characterized.

Results

The gene had an ORF of 1974 bp encoding a protein of 657 amino acid residues. In comparison to the LGox precursor, the proteinase K-treated enzyme exhibited improved affinity to substrate and with a K _m of 0.15 mM and V _max of 62 μmol min^?1 mg^?1. The 50% thermal inactivation temperature of the proteinase K treated enzyme was increased from 50 to 70 °C. The enzyme exhibited strict specificity for l-glutamate.

Conclusions

LGox treated by proteinase K exhibited strict specificity for l-glutamate, good thermostability and high substrate affinity.

     

Heterologous biosynthesis of triterpenoid dammarenediol-II in engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To achieve heterologous biosynthesis of dammarenediol-II, which is the precursor of dammarane-type tetracyclic ginsenosides, by reconstituting the 2,3-oxidosqualene-derived triterpenoid biosynthetic pathway in Escherichia coli.

Results

By the strategy of synthetic biology, dammarenediol-II biosynthetic pathway was reconstituted in E. coli by co-expression of squalene synthase (SS), squalene epoxidase (SE), NADPH-cytochrome P450 reductase (CPR) from Saccharomyces cerevisiae, and SE from Methylococcus capsulatus (McSE), NADPH-cytochrome P450 reductase (CPR) from Arabidopsis thaliana. Sequences of transmembrane domains were truncated if necessary in each of the genes. Different sources of SE/CPR combinations were tested, during which two CPRs were detected to be new reductase partners of McSE. When the gene encoding dammarenediol-II synthase was co-expressed with the 2,3-oxidosqualene expression modules, dammarenediol-II was detected and the production was 8.63 mg l^?1 in E. coli under the shake-flask conditions.

Conclusions

Two E. coli chassis for production of dammarenediol-II were established which could be potentially applied in other triterpenoid production in E. coli when different oxidosqualene cyclases (OSCs) introduced into the system.