[Advances in the biosynthesis of L-homoserine and its derivatives by metabolic engineering of Escherichia coli].

L-homoserine and its derivatives (O-succinyl-L-homoserine and O-acetyl-L-homoserine) are precursors for the biosynthesis of L-methionine, and various C4 compounds (isobutanol, γ-butyrolactone, 1, 4-butanediol, 2, 4-dihydroxybutyric acid) and L-phosphinothricin. Therefore, the fermentative production of L-homoserine and its derivatives became the research hotspot in recent years. However, the low fermentation yield and conversion rate, and the unclear regulation mechanism for the biosynthesis of L-homoserine and its derivatives, hamper the development of an efficient production process for L-homoserine and its derivatives. This review summarized the advances in the biosynthesis of L-homoserine and its derivatives by metabolic engineering of Escherichia coli from the aspects of substrate uptake, redirection of carbon flow at the key nodes, recycle of NADPH and export of target products. This review may facilitate subsequent metabolic engineering and biotechnological production of L-homoserine and its derivatives.

Advances in the biosynthesis of L-homoserine and its derivatives by metabolic engineering of Escherichia coli / 生物工程学报

L-homoserine and its derivatives (O-succinyl-L-homoserine and O-acetyl-L-homoserine) are precursors for the biosynthesis of L-methionine, and various C4 compounds (isobutanol, γ-butyrolactone, 1, 4-butanediol, 2, 4-dihydroxybutyric acid) and L-phosphinothricin. Therefore, the fermentative production of L-homoserine and its derivatives became the research hotspot in recent years. However, the low fermentation yield and conversion rate, and the unclear regulation mechanism for the biosynthesis of L-homoserine and its derivatives, hamper the development of an efficient production process for L-homoserine and its derivatives. This review summarized the advances in the biosynthesis of L-homoserine and its derivatives by metabolic engineering of Escherichia coli from the aspects of substrate uptake, redirection of carbon flow at the key nodes, recycle of NADPH and export of target products. This review may facilitate subsequent metabolic engineering and biotechnological production of L-homoserine and its derivatives.

Combining fermentation to produce O-succinyl-l-homoserine and enzyme catalysis for the synthesis of l-methionine in one pot.

The biosynthetic pathway of l-methionine in microorganisms was complex and regulated at multiple levels. In this study, a two-step method for l-methionine production combined fermentation and biocatalysis was realized in one pot. The O-succinyl-l-homoserine (OSH) producing strain Escherichia coli W3110(DE3) ΔIJB∗TrcmetL/pTrc-metAfbr-Trc-thrAfbr-yjeH (ΔIJB) was constructed initially. OSH in the fermentation supernatant was then converted to l-methionine in the presence of O-succinyl-l-homoserine sulfhydrylase (OSHS) and sodium methanethiol. The titer of l-methionine could reach 21.1 g/L after 88 h (84 h fermentation and 4 h catalysis) in a two-step method (process 1). In a one-pot two-strain system (process 2), two strains ΔIJB and E. coli BL21(DE3)/pET28b-OSHS-cutinase were co-cultured, and 8.24 g/L l-methionine was obtained. In another one-pot one-strain system (process 3), strain E. coli ΔIJB/pET28b-OSHS-cutinase could co-express OSHS and cutinase during ΔIJB fermentation at the same time, obtaining 13.6 g/L l-methionine in a 5 L fermentor after 84 h. By comparing the three processes for l-methionine production based on the process 1, the simplified process in process 3 provided in this study showed potent in the large-scale production of l-methionine with convenient handling and production efficiency, but further works still need to be carried out to improve the l-methionine production.

Identification and Characterization of an O-Succinyl-L-Homoserine Sulfhydrylase From Thioalkalivibrio sulfidiphilus.

L-methionine is an important natural amino acid with broad application prospects. A novel gene encoding the enzyme with the ability to catalyze O-succinyl-L-homoserine (OSH) to L-methionine was screened and characterized. The recombinant O-succinyl-L-homoserine sulfhydrylase from Thioalkalivibrio sulfidiphilus (tsOSHS) exhibited maximum activity at 35°C and pH 6.5. OSHS displayed an excellent thermostability with a half-life of 21.72 h at 30°C. Furthermore, the activity of OSHS increased 115% after Fe2+ added. L-methionine was obtained with a total yield reaching 42.63 g/L under the concentration of O-succinyl-L-homoserine 400 mM (87.6 g/L). These results indicated that OSHS is a potential candidate for applying in the large-scale bioproduction of L-methionine.

Enhanced O-succinyl-l-homoserine production by recombinant Escherichia coli ΔIJBB*TrcmetL/pTrc-metAfbr -Trc-thrAfbr -yjeH via multilevel fermentation optimization.

AIMS: Constructing a strain with high yield of O-succinyl-l-homoserine (OSH) and improving the titre through multilevel fermentation optimization. METHODS AND RESULTS: OSH high-yielding strain was first constructed by deleting the thrB gene to block the threonine biosynthesis. Single-factor experiment was carried out, where a Plackett-Burman design was used to screen out three factors (glucose, yeast and threonine) from the original 11 factors that affected the titre of OSH. The Box-Behnken response surface method was used to optimize the fermentation conditions. Through gene editing and medium optimization, the titre of OSH increased from 7·20 to 8·70 g l-1 in 500 ml flask. Furthermore, the fermentation process and fed-batch fermentation conditions including pH, temperature, feeding strategy and feeding medium were investigated and optimized. Under the optimal conditions, the titre of OSH reached 102·5 g l-1 , which is 5·6 times higher than before (15·6 g l-1 ). CONCLUSIONS: O-succinyl-l-homoserine fermentation process was established and the combination of response surface methodology and metabolic pathway analysis effectively improved the titre of OSH. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, the titre of OSH reached the needs for industrial production and the metabolic pathway of OSH was demonstrated for further optimization.

O-Succinyl-l-homoserine overproduction with enhancement of the precursor succinyl-CoA supply by engineered Escherichia coli.

O-Succinyl-l-homoserine (OSH) is an important platform chemical in production of C4 chemicals such as succinic acid, homoserine lactone, γ­butyrolactone, and 1,4­butanediol. The production of OSH through chemical method or the current engineering strain is difficult and not optimal, and thereby there remains a need to develop new engineering strategy. Here, we engineered an OSH overproducing Escherichia coli strain through deleting the degradation and competitive pathways, overexpressing thrA and metL to enhance the metabolic flux from l-asparate to l-homoserine. Additionally, increasing the precursor succinyl-CoA supply through simultaneously knocking out sucD and overexpressing sucA further increased the yield of OSH. The engineered strain OSH9/pTrc-metA11-yjeH with above strategies produced OSH at the concentration of 24.1 g/L (0.609 g/g glucose) in batch fermentation. To gain detailed insight into metabolism of the engineered strain, comparative metabolic profiling was performed between the engineered and wide-type strain. The metabolomics data deciphered that the carbon was directed toward the OSH biosynthesis resulting in less flexibility of the genetically modified strain than the wide-type strain.

Metabolic engineering of E. coli for the production of O-succinyl-l-homoserine with high yield.

O-succinyl-l-homoserine (OSH) is a promising platform chemical for the production of C4 chemicals with huge market potential which can be produced by fermentation from glucose. To construct a strain capable of producing OSH with high yield, the metJ (encodes transcriptional repressor) and metI (encodes a subunit of dl-methionine transporter) were deleted in Escherichia coli W3110 to obtain a strain E. coli ∆JI. Then, overexpression of metL (encodes bifunctional aspartate kinase/homoserine dehydrogenase II) and inactivation of metB (encodes cystathionine γ-synthase) were implemented in one step, and the OSH titer of the resulting strain E. coli ∆JIB* TrcmetL was dramatically increased to 7.30 g/L. The feedback regulation was further relieved by progressively overexpressing metAfbr (encodes homoserine O-succinyltransferase), yjeH (encodes l-methionine exporter), and thrAfbr (encodes bifunctional aspartate kinase/homoserine dehydrogenase I) to increase the metabolic flux from aspartate to OSH. The 100% rationally designed strain E. coli ∆JIB* TrcmetL/pTrc-metAfbr -Trc-thrAfbr -yjeH produced 9.31 g/L OSH from 20 g/L glucose (0.466 g/g glucose) in batch fermentation, which represents the highest OSH yield from glucose reported to date. The culture profiles of the newly constructed strains were recorded to investigate their productive properties. The effects of l-methionine addition on the fermentation process of the optimal strain were also studied. Our results demonstrate that tuning the expression level of metL, inactivation of metB, and attenuation of feedback resistance of the crucial enzymes in the biosynthetic pathway are the key factors that impact the OSH production in E. coli.

A role for glutamate-333 of Saccharomyces cerevisiae cystathionine γ-lyase as a determinant of specificity.

Cystathionine γ-lyase (CGL) catalyzes the hydrolysis of l-cystathionine (l-Cth), producing l-cysteine (l-Cys), α-ketobutyrate and ammonia, in the second step of the reverse transsulfuration pathway, which converts l-homocysteine (l-Hcys) to l-Cys. Site-directed variants substituting residues E48 and E333 with alanine, aspartate and glutamine were characterized to probe the roles of these acidic residues, conserved in fungal and mammalian CGL sequences, in the active-site of CGL from Saccharomyces cerevisiae (yCGL). The pH optimum of variants containing the alanine or glutamine substitutions of E333 is increased by 0.4-1.2 pH units, likely due to repositioning of the cofactor and modification of the pKa of the pyridinium nitrogen. The pH profile of yCGL-E48A/E333A resembles that of Escherichia coli cystathionine ß-lyase. The effect of substituting E48, E333 or both residues is the 1.3-3, 26-58 and 124-568-fold reduction, respectively, of the catalytic efficiency of l-Cth hydrolysis. The Km(l-Cth) of E333 substitution variants is increased ~17-fold, while Km(l-OAS) is within 2.5-fold of the wild-type enzyme, indicating that residue E333 interacts with the distal amine moiety of l-Cth, which is not present in the alternative substrate O-acetyl-l-serine. The catalytic efficiency of yCGL for α,γ-elimination of O-succinyl-l-homoserine (kcat/Km(l-OSHS)=7±2), which possesses a distal carboxylate, but lacks an amino group, is 300-fold lower than that of the physiological l-Cth substrate (kcat/Km(l-Cth)=2100±100) and 260-fold higher than that of l-Hcys (kcat/Km(l-Hcys)=0.027±0.005), which lacks both distal polar moieties. The results of this study suggest that the glutamate residue at position 333 is a determinant of specificity.