Efficient and scalable synthesis of 1,5-diamino-2-hydroxy-pentane from L-lysine via cascade catalysis using engineered Escherichia coli.

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.

Biosynthesis of cis-3-hydroxypipecolic acid from L-lysine using an in vivo dual-enzyme cascade.

Cis-3-Hydroxypipecolic acid (cis-3-HyPip) is an important intermediate for the synthesis of GE81112 tetrapeptides, a small family of unusual nonribosomal peptide congeners with potent inhibitory activity against prokaryotic translation initiation. In this study, we constructed a microbial cell factory that can convert L-lysine into cis-3-hydroxypipecolic acid (cis-3-HyPip). Lysine cyclodeaminase SpLCD and Fe(II)/α-ketoglutarate (2-OG)-based oxygenase GetF were co-expressed in Escherichia coli. Plasmids with different copy numbers were used to balance the expression of these two enzymes, and the cell with the most appropriate balance of this kind for carrying plasmid pET-duet-getf-splcd was obtained. After determining the temperature (30 °C), pH (7.0), cell biomass, substrate concentration, Fe2+ concentration (10 mM), L-ascorbate concentration (10 mM), and TritonX-100 concentration (0.1% w/v) that were optimal for whole-cell catalysis, the yield of cis-3-HyPip reached as high as 25 mM (3.63 g/L).

Designing of an Efficient Whole-Cell Biocatalyst System for Converting L-Lysine Into Cis-3-Hydroxypipecolic Acid.

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.

High-yield production of D-1,2,4-butanetriol from lignocellulose-derived xylose by using a synthetic enzyme cascade in a cell-free system.

Approaches using metabolic engineering to produce D-1, 2, 4-butanetriol (BT) from renewable biomass in microbial systems have achieved initial success. However, due to the lack of incomplete understanding of the complex branch pathway, the efficient fermentation system for BT production was difficult to develop. Here we reconstituted a cell-free system in vitro using purified enzymes to produce BT from d-xylose. The factors that influencing the efficiency of cell-free system, including enzyme concentration, reaction buffer, pH, temperature, metal ion additives and cofactors were first identified to define optimal reaction conditions and essential components for the cascade reaction. Meanwhile, a natural cofactor recycling system was found in cell-free system. Finally, we were able to convert 18 g/L xylose to 6.1 g/L BT within 40 h with a yield of 48.0%. The feasibility of cell-free system to produce BT in corncob hydrolysates was also determined.

d-1,2,4-Butanetriol production from renewable biomass with optimization of synthetic pathway in engineered Escherichia coli.

Bio-based production of d-1,2,4-butanetriol (BT) from renewable substrates is increasingly attracting attention. Here, the BT biosynthetic pathway was constructed and optimized in Escherichia coli to produce BT from pure d-xylose or corncob hydrolysates. First, E. coli BL21(DE3) was identified as a more proper host for BT production through host screening. Then, BT pathway was systematically optimized with gene homolog screening strategy, mainly targeting three key steps from xylonic acid to BT catalyzed by d-xylonate dehydratase (XD), 2-keto acid decarboxylase (KDC) and aldehyde reductase (ALR). After screening six ALRs, four KDCs and four XDs, AdhP from E. coli, KdcA from Lactococcus lactis and XylD from Caulobacter crescentus were identified more efficiently for BT production. The co-expression of these enzymes in recombinant strain BL21-14 led to BT production of 5.1 g/L under the optimized cultivation conditions. Finally, BT production from corncob hydrolysates was achieved with a titer of 3.4 g/L.