“…In our previous study, a dual-enzyme system containing SpLCD and GetF was constructed and reaction parameters were systematically optimized to biosynthesize cis-3-HyPip from L-lysine ( Hu et al, 2022 ). However, the development of this whole-cell cascade catalyst is challenged by difficulty in balancing the rates of the two main reaction steps, namely, eliminating intermediate pipecolic acid accumulation and shifting the equilibrium toward cis-3-HyPip production.…”
Section: Resultsmentioning
confidence: 99%
“…However, the high cost of substrate L-Pip restricts its practical application. To reduce the cost, we previously constructed a microbial cell factory that can convert economic L-lysine into cis-3-hydroxypipecolic acid (cis-3-HyPip) with whole-cell cascade catalysis ( Hu et al, 2022 ; Scheme 1 ). First, L-lysine was converted to L-Pip through the cyclization deamination by lysine cyclodeaminase (SpLCD), which was further hydroxylated by Fe(II)/2-OG-based oxygenase GetF to form cis-3-HyPip.…”
Section: Introductionmentioning
confidence: 99%
“…Precise control of the expression levels of multiple genes is critical in co-expression biocatalysis ( Chen et al, 2017 ), and one strategy is to use different types of vectors or co-express multiple genes through one or more vectors to avoid low expression of rate-limiting enzymes and overexpression of non-rate-limiting enzymes ( Liu et al, 2019a ; Yang et al, 2021 ; Hu et al, 2022 ). Other strategies are generally possible to precisely control certain enzyme expression levels by using different promoters and ribosomal binding sites (RBSs) ( Jiang and Fang, 2016 ; Zhang et al, 2019 ).…”
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 our previous study, a dual-enzyme system containing SpLCD and GetF was constructed and reaction parameters were systematically optimized to biosynthesize cis-3-HyPip from L-lysine ( Hu et al, 2022 ). However, the development of this whole-cell cascade catalyst is challenged by difficulty in balancing the rates of the two main reaction steps, namely, eliminating intermediate pipecolic acid accumulation and shifting the equilibrium toward cis-3-HyPip production.…”
Section: Resultsmentioning
confidence: 99%
“…However, the high cost of substrate L-Pip restricts its practical application. To reduce the cost, we previously constructed a microbial cell factory that can convert economic L-lysine into cis-3-hydroxypipecolic acid (cis-3-HyPip) with whole-cell cascade catalysis ( Hu et al, 2022 ; Scheme 1 ). First, L-lysine was converted to L-Pip through the cyclization deamination by lysine cyclodeaminase (SpLCD), which was further hydroxylated by Fe(II)/2-OG-based oxygenase GetF to form cis-3-HyPip.…”
Section: Introductionmentioning
confidence: 99%
“…Precise control of the expression levels of multiple genes is critical in co-expression biocatalysis ( Chen et al, 2017 ), and one strategy is to use different types of vectors or co-express multiple genes through one or more vectors to avoid low expression of rate-limiting enzymes and overexpression of non-rate-limiting enzymes ( Liu et al, 2019a ; Yang et al, 2021 ; Hu et al, 2022 ). Other strategies are generally possible to precisely control certain enzyme expression levels by using different promoters and ribosomal binding sites (RBSs) ( Jiang and Fang, 2016 ; Zhang et al, 2019 ).…”
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.
“…When the α-ketoglutarate/lysine ratio exceeded 1.5:1, a hydroxylysine mass concentration of 5.5% was achieved, which was 11.1% higher than that observed in the control. It was previously reported that increasing the molar ratio of ketoglutarate/lysine is conducive to producing cis-3-hydroxypipecolic acid by whole-cell catalysis [ 39 ]. Fig.…”
Section: Resultsmentioning
confidence: 99%
“…The l -lysine, hydroxylysine, and 2-OH-PDA concentrations were determined according to a previous study with modifications [ 16 , 39 ]. The reaction products were treated with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) using the precolumn derivatization method prior to HPLC analysis on an Agilent 1260 Infinity LC system (Agilent Technologies, Santa Clara, CA, USA) and liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF–MS) analysis (Additional file 1 : Fig.…”
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.
L‐Pipecolic Acid (L‐PA) is a valuable building block for the synthesis of pharmaceuticals such as anesthetics and immunosuppressants. Thus, more efficient and greener strategies are desired for its production. Herein, we have applied a previously engineered variant of the Lysine Cyclodeaminase from Streptomyces pristinaespiralis (e‐SpLCD) for the bioconversion of L‐Lysine into L‐PA. The reaction can be performed by the free e‐SpLCD reaching full conversion to 50 mM L‐PA. From a biotechnological perspective, the process scale‐up has been trialed in a SpinChem® reactor, albeit with lower conversion yields. To further enhance the biocatalyst stability, we present a detailed study of the e‐SpLCD immobilization on microparticles. This enabled the integration of the immobilized biocatalyst into a packed‐bed reactor for the continuous flow synthesis of L‐PA. The full conversion was achieved in 90 min, maintaining also high operational stability. Remarkably, the addition of exogenous cofactor was not needed for the flow reaction, although the long‐term operational stability was improved by the addition of NAD+.
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