Efficient biotransformation of l-lysine into cadaverine by strengthening pyridoxal 5’-phosphate-dependent proteins in Escherichia coli with cold shock treatment
“…In addition, developed competitive bioprocesses with high amounts of GABA titer, productivity, and yield using glucose as a sole carbon source could contribute to the establishment of the economically feasible and efficient one-step fermentative GABA production system. However, the remaining issues must be addressed to ensure the widespread adoption of the approaches proposed herein, and therefore, the following strategies will be explored in our future studies: (i) characterizing the crystal structure of the enzymes and their functions, (ii) additional expression of pyridoxal kinase without the addition of external PLP for enhanced GAD activity, , and (iii) blocking competitive pathways related to GABA degradation …”
γ-Aminobutyrate (GABA) is an important chemical
by itself
and can be further used for the production of monomer used for the
synthesis of biodegradable polyamides. Until now, GABA production
using
Corynebacterium glutamicum
harboring
glutamate decarboxylases (GADs) has been limited due to the discrepancy
between optimal pH for GAD activity (pH 4.0) and cell growth (pH 7.0).
In this study, we developed recombinant
C. glutamicum
strains expressing mutated GAD from
Escherichia coli
(EcGADmut) and GADs from
Lactococcus lactis
CICC20209 (LlGAD) and
Lactobacillus senmaizukei
(LsGAD), all of which showed enhanced pH stability and adaptability
at a pH of approximately 7.0. In shake flask cultivations, the GABA
productions of
C. glutamicum
H36EcGADmut,
C. glutamicum
H36LsGAD, and
C. glutamicum
H36LlGAD were examined at pH 5.0, 6.0, and 7.0, respectively. Finally,
C. glutamicum
H36EcGADmut (40.3 and 39.3 g L
–1
), H36LlGAD (42.5 and 41.1 g L
–1
), and H36LsGAD (41.6 and 40.2 g L
–1
) produced
improved GABA titers and yields in batch fermentation at pH 6.0 and
pH 7.0, respectively, from 100 g L
–1
glucose. The
recombinant strains developed in this study could be used for the
establishment of sustainable direct fermentative GABA production from
renewable resources under mild culture conditions, thus increasing
the availability of various GADs.
“…In addition, developed competitive bioprocesses with high amounts of GABA titer, productivity, and yield using glucose as a sole carbon source could contribute to the establishment of the economically feasible and efficient one-step fermentative GABA production system. However, the remaining issues must be addressed to ensure the widespread adoption of the approaches proposed herein, and therefore, the following strategies will be explored in our future studies: (i) characterizing the crystal structure of the enzymes and their functions, (ii) additional expression of pyridoxal kinase without the addition of external PLP for enhanced GAD activity, , and (iii) blocking competitive pathways related to GABA degradation …”
γ-Aminobutyrate (GABA) is an important chemical
by itself
and can be further used for the production of monomer used for the
synthesis of biodegradable polyamides. Until now, GABA production
using
Corynebacterium glutamicum
harboring
glutamate decarboxylases (GADs) has been limited due to the discrepancy
between optimal pH for GAD activity (pH 4.0) and cell growth (pH 7.0).
In this study, we developed recombinant
C. glutamicum
strains expressing mutated GAD from
Escherichia coli
(EcGADmut) and GADs from
Lactococcus lactis
CICC20209 (LlGAD) and
Lactobacillus senmaizukei
(LsGAD), all of which showed enhanced pH stability and adaptability
at a pH of approximately 7.0. In shake flask cultivations, the GABA
productions of
C. glutamicum
H36EcGADmut,
C. glutamicum
H36LsGAD, and
C. glutamicum
H36LlGAD were examined at pH 5.0, 6.0, and 7.0, respectively. Finally,
C. glutamicum
H36EcGADmut (40.3 and 39.3 g L
–1
), H36LlGAD (42.5 and 41.1 g L
–1
), and H36LsGAD (41.6 and 40.2 g L
–1
) produced
improved GABA titers and yields in batch fermentation at pH 6.0 and
pH 7.0, respectively, from 100 g L
–1
glucose. The
recombinant strains developed in this study could be used for the
establishment of sustainable direct fermentative GABA production from
renewable resources under mild culture conditions, thus increasing
the availability of various GADs.
“…Production through bioconversion from ectoine, as demonstrated here, seems attractive to open the hydroxyectoine market. Similarly, bioconversion processes from simple precursors have helped to obtain other novel chemicals for facilitated market entry, prominent examples being the production of cadaverine [ 56 ] and 5-aminovalerate from l -lysine [ 57 ], as well as the production of γ-aminobutyric acid from l -glutamate [ 58 ]. Fig.…”
Background
Extremolytes enable microbes to withstand even the most extreme conditions in nature. Due to their unique protective properties, the small organic molecules, more and more, become high-value active ingredients for the cosmetics and the pharmaceutical industries. While ectoine, the industrial extremolyte flagship, has been successfully commercialized before, an economically viable route to its highly interesting derivative 5-hydroxyectoine (hydroxyectoine) is not existing.
Results
Here, we demonstrate high-level hydroxyectoine production, using metabolically engineered strains of C. glutamicum that express a codon-optimized, heterologous ectD gene, encoding for ectoine hydroxylase, to convert supplemented ectoine in the presence of sucrose as growth substrate into the desired derivative. Fourteen out of sixteen codon-optimized ectD variants from phylogenetically diverse bacterial and archaeal donors enabled hydroxyectoine production, showing the strategy to work almost regardless of the origin of the gene. The genes from Pseudomonas stutzeri (PST) and Mycobacterium smegmatis (MSM) worked best and enabled hydroxyectoine production up to 97% yield. Metabolic analyses revealed high enrichment of the ectoines inside the cells, which, inter alia, reduced the synthesis of other compatible solutes, including proline and trehalose. After further optimization, C. glutamicum Ptuf ectDPST achieved a titre of 74 g L−1 hydroxyectoine at 70% selectivity within 12 h, using a simple batch process. In a two-step procedure, hydroxyectoine production from ectoine, previously synthesized fermentatively with C. glutamicum ectABCopt, was successfully achieved without intermediate purification.
Conclusions
C. glutamicum is a well-known and industrially proven host, allowing the synthesis of commercial products with granted GRAS status, a great benefit for a safe production of hydroxyectoine as active ingredient for cosmetic and pharmaceutical applications. Because ectoine is already available at commercial scale, its use as precursor appears straightforward. In the future, two-step processes might provide hydroxyectoine de novo from sugar.
“…The cell density was adjusted to an OD 600 of 4 and analyzed by SDS‐PAGE, which contained 10% separating gel and 4% stacking gel. Proteins were visualized by staining with Coomassie blue R‐250 and scanned using the Image scanner (Biolab2000) (Xue et al, 2020).…”
Pyridoxal 5′‐phosphate (pyridoxal phosphate, PLP) is an essential cofactor for multiple enzymatic reactions in industry. However, cofactor engineering based on PLP regeneration and related to the performance of enzymes in chemical production has rarely been discussed. First, we found that MG1655 strain was sensitive to nitrogen source and relied on different amino acids, thus the biomass was significantly reduced when PLP excess in the medium. Then, the six KEIO collection strains were applied to find out the prominent gene in deoxyxylulose‐5‐phosphate (DXP) pathway, where pdxB was superior in controlling cell growth. Therefore, the clustered regularly interspaced short palindromic repeats interference (CRISPRi) targeted on pdxB in MG1655 was employed to establish a novel direct enzymatic evaluation platform (DEEP) as a high‐throughput tool and obtained the optimal modules for incorporating of PLP to enhance the biomass and activity of PLP‐dependent enzymes simultaneously. As a result, the biomass has increased by 55% using PlacI promoter driven pyridoxine 5′‐phosphate oxidase (PdxH) with a trace amount of precursor. When the strains incorporated DEEP and lysine decarboxylase (CadA), the cadaverine productivity was increased 32% due to the higher expression of CadA. DEEP is not only feasible for high‐throughput screening of the best chassis for PLP engineering but also practical in fine‐tuning the quantity and quality of enzymes.
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