Metabolic Engineering of Escherichia coli for High-Titer Biosynthesis of 3′-Sialyllactose

3′-Sialyllactose (3′-SL) is among the foremost and simplest sialylated breast milk oligosaccharides. In this study, an engineered Escherichia coli for high-titer 3′-SL biosynthesis was developed by introducing a multilevel metabolic engineering strategy, including (1) the introduction of precursor CMP-Neu5Ac synthesis pathway and high-performance α2,3-sialyltransferase (α2,3-SiaT) genes into strain BZ to achieve de novo synthesis of 3′-SL; (2) optimizing the expression of glmS-glmM-glmU involved in the UDP-GlcNAc and CMP-Neu5Ac synthesis pathways, and constructing a glutamine cycle system, balancing the precursor pools; (3) analysis of critical intermediates and inactivation of competitive pathway genes to redirect carbon flux to 3′-SL biosynthesis; and (4) enhanced catalytic performance of rate-limiting enzyme α2,3-SiaT by RBS screening, protein tag cloning. The final strain BZAPKA14 yielded 9.04 g/L 3′-SL in a shake flask. In a 3 L bioreactor, fed-batch fermentation generated 44.2 g/L 3′-SL, with an overall yield and lactose conversion of 0.53 g/(L h) and 0.55 mol 3′-SL/mol, respectively

Elucidation of Substituted Ester Group Position in Octenylsuccinic Anhydride Modified Sugary Maize Soluble Starch

The octenylsuccinic groups in esterification-modified sugary maize soluble starches with a low (0.0191) or high (0.0504) degree of substitution (DS) were investigated by amyloglucosidase hydrolysis followed by a combination of chemical and physical analysis. The results showed the zeta-potential remained at approximately the same value regardless of excessive hydrolysis. The weight-average molecular weight decreased rapidly and reached 1.22 × 10⁷ and 1.60 × 10⁷ g/mol after 120 min for low-DS and high-DS octenylsuccinic anhydride (OSA) modified starch, respectively. The pattern of <i>z</i>-average radius of gyration as well as particle size change was similar to that of <i>M</i>_w, and <i>z</i>-average radius of gyration decreased much more slowly, especially for high-DS OSA starch. Compared to native starch, two characteristic absorption peaks at 1726.76 and 1571.83 cm^–1 were observed in FT-IR spectra, and the intensity of absorption peaks increased with increasing DS. The NMR results showed that OSA starch had several additional peaks at 0.8–3.0 ppm and a shoulder at 5.56 ppm for OSA substituents, which were grafted at O-2 and O-3 positions in soluble starch. The even distribution of OSA groups in the center area of soluble starch particle has been directly shown under CLSM. Most substitutions were located near branching points of soluble starch particles for a low-DS modified starch, whereas the substituted ester groups were located near branching points as well as at the nonreducing ends in OSA starch with a high DS

Polysaccharide Modification through Green Technology: Role of Endodextranase in Improving the Physicochemical Properties of (1→3)(1→6)-α‑d‑Glucan

The structure and properties of bioengineered (1→3)(1→6)-α-d-glucan subjected to endodextranase treatment were investigated. Upon enzyme treatment, OD₂₂₀ and <i>M</i>_w decreased substantially during the first 60 min and thereafter slowed as the modification progressed. Compared to the native glucan, the modified sample solution had a lighter opalescent, bluish-white color. The morphological analysis revealed that bioengineered glucan produced quite a few small particles after hydrolysis. The molecular weight distribution curve gradually shifted to the low <i>M</i>_w region with a significant broadening distribution, and the chain hydrolysis reaction followed a combination of zeroth- and first-order processes. The NMR results showed some specific α-1,6 linkages of glucan chains were cleaved with enzyme treatment. The viscosity of modified glucan solution was markedly reduced, and the Newtonian plateaus were also observed at high shear rates (10–100 1/s). The above results suggested that the modified (1→3)(1→6)-α-d-glucan showed a tailor-made solution character similar to that of arabic gum and would be used as a novel food gum substitute in the design of artificial carbohydrate-based foods

Single nucleotide polymorphisms causing non-synonymous changes in tropoelastin.

<p>Tropoelastin protein sequence corresponds to RefSeq, variant 1 (NM_000501). This variant does not include exons 22 or 26a (an extension of exon 26). Exons are boxed, with exon numbers above. The positions of mutations and substituted amino acids are indicated within the shaded boxes, minor allele is indicated second.</p

Mechanical properties of elastin-like peptides (ELPs) containing G to S substitutions.

<p>The four bar graphs indicate the means and standard errors for tensile mechanical properties of sheets of materials constructed with reference ELP, EP20–24–24, and ELPs containing the single G to S substitution. Materials constructed from ELPs containing the triple G to S substitution were too fragile to generate meaningful values. The number of replicates for each experiment (n) is indicated. ** indicates a significant difference between the two materials (ANOVA with Bonferoni correction, p<0.01).</p

Domain Sequences Represented in Elastin-Like Polypeptides.

<p>Mutated amino acids are indicated in bold</p

Effect of introducing select amino acid substitutions on the secondary structure of elastin-like peptides (ELPs).

<p>(A) CD spectra comparing reference ELP, EP20–24–24, with ELPs containing single and triple G to S substitutions. (B) CD spectra comparing reference ELP, EP20–24–24, with ELPs containing single and double K to R substitutions. Compared to the reference polypeptide, the introduction of the substitutions does not appear to result in any major changes in conformation of these ELPs.</p

Coacervation characteristics of select elastin-like peptides (ELPs).

<p>(A) Coacervation (temperature-induce phase separation) of reference ELP, EP20–24–24, and ELPs containing single and triple G to S substitutions. Time 0 corresponds to 20°C, and temperature was raised at a rate of 1°C/min. (B) Coacervation of reference ELP, EP20–24–24, and ELPs containing single and double K to R substitutions. Time 0 corresponds to 15°C, and temperature was raised at a rate of 1°C/min. Coacervation was followed by turbidity as measured by absorbance at 440 nm. Curves represent means for three replicate experiments. Note the curves for the K to R substitutions are shifted to the left indicating that coacervation is initiated at a lower temperature.</p

Single nucleotide polymorphisms identified in human tropelastin.

<p>1. SNPs identified through dbSNP are indicated with an appropriate SNP reference. EST indicates that the polymorphism was identified through EST libraries.</p><p>2. Tropoelastin RefSeq variant 1 (NM_000501) was used for numbering mRNA and amino acid positions, counting from the initiator methionine. Exons 22 and 26a (an extension of exon 26) are not present in this variant and are not included in the position count.</p><p>3. T/C and Leu/Pro designate Major/Minor allele respectively e.g. from T to C or Leu to Pro, etc.</p><p>4. Minor allele amino acid is indicated in square brackets</p><p>5. For SNPs detected through ESTs, minor allele frequency (MAF) indicates the proportion of ESTs sequences coding the minor allele. For SNPs obtained from dbSNP, MAF indicates the range across populations provided through dbSNP.</p><p>6. Because of phase 1 intron/exon borders, although the mutation site is in the last base of exon 11 the mutated amino acid is the first amino acid coded by exon 12.</p

Mechanical properties of full-length human tropoelastin containing G to S substitutions.

<p>The four bar graphs indicate the means and standard errors for tensile mechanical properties of sheets of materials constructed with full-length human tropoelastin (hTE) and hTE variants containing either a single or triple G to S substitution in domain 20. The number of replicates for each experiment (n) is indicated. *, ** and *** indicate a significant difference between hTE and hTE with the single G to S substitution (ANOVA with Bonferoni correction, p<0.05, p<0.01 and p<0.001 respectively). † and ††† indicate a significant difference between hTE with the single G to S substitution and hTE with the triple G to S substitution (ANOVA with Bonferoni correction, p<0.05, p<0.001 respectively).</p