Search

L‐Methionine is the unique sulfur‐containing amino acid essential for humans and animals, which is widely used in pharmaceutical, food and feed industries. Its green and efficient production has attracted a great deal of attention. Fermentation‐enzymatic coupling route is regarded to be promising for L‐methionine biosynthesis, while O‐succinyl‐L‐homoserine mercaptotransferase (MetZ) is the key biocatalyst. Exploring and engineering of MetZ with ideal catalytic properties is highly desired. Herein, the MetZ from Chromobacterium violaceum (CvMetZ) was screened and through in silico analyses, potential beneficial amino acid residues both at the access channel and around the oxygen anion holes were anchored for site‐saturation mutagenesis. The positive mutants were obtained with improved activity for L‐methionine biosynthesis using O‐succinyl‐L‐homoserine (OSH) and methyl mercaptan as substrate. The best mutant was further applied for L‐methionine production and supply of methyl mercaptan was optimized in a fed‐batch approach. The conversion of 100 g/L OSH reached 92% with L‐methionine yield of 62.6 g/L, which was well coupled with the OSH fermentation level. [ABSTRACT FROM AUTHOR]

Objective: l-methionine is an important sulfur-containing amino acid essential for humans and animals. Its biosynthesis pathway is complex and highly regulated. This study aims to explore the bottleneck limiting the improvement of l-methionine productivity and apply efficient strategies to increase l-methionine production in engineered E. coli. Results: The enzyme O-succinylhomoserine sulfhydrylase involved in thiolation of OSH to form homocysteine was overexpressed in the engineered strain E. coli W3110 IJAHFEBC/PAm, resulting in l-methionine production increased from 2.8 to 3.22 g/L in shake flask cultivation. By exogenous addition of l-glycine as the precursor of one carbon unit, the titer of l-methionine was increased to 3.68 g/L. The glycine cleavage system was further strengthened for the efficient one carbon unit supply and a l-methionine titer of 3.96 g/L was obtained, which was increased by 42% compared with that of the original strain. Conclusions: Insufficient supply of one carbon unit was found to be the issue limiting the improvement of l-methionine productivity and its up-regulation significantly promoted the l-methionine production in the engineered E. coli. [ABSTRACT FROM AUTHOR]

l -Methionine biosynthesis is through multilevel regulated and multibranched biosynthetic pathway (MRMBP). Because of the complex regulatory mechanism and the imbalanced metabolic flux between branched pathways, microbial production of l -methionine has not been commercialized. In this study, local metabolic response in MRMBP of l -methionine was investigated and various crucial genes in branched pathways were determined. In l -serine pathway, the crucial gene was serABC. In O -succinyl homoserine (OSH) pathway, which was the C4 backbone of l -methionine, metB and metL controlled the metabolic flux jointly. In l -cysteine pathway, the crucial gene cysE fbr could disturb the flux distribution of local network in l -methionine biosynthesis. However, no crucial gene for l -methionine production in 5-methyl tetrahydrofolate (CH 3 -THF) pathway was found. The relation between these pathways was also researched. l -Serine pathway, as the upstream pathway of l -cysteine and CH 3 -THF, played a crucial role in l -methionine biosynthesis. l -Cysteine pathway showed the strongest controlling force of the metabolic flux, and OSH pathway was second to l -cysteine pathway. In contrast, CH 3 -THF pathway was the weakest, which was probably the mainly limited steps at present and had great potential in further research. In addition, constructed W3110 IJAHFEBC/pA∗HAmL was able to produce 2.62 g/L l -methionine in flask. This study is instructive for l -methionine biosynthesis and provides a new research method of biosynthesizing other metabolic products in MRMBPs. [Display omitted] • Multilevel regulated and multibranched biosynthetic pathways (MRMBPs) for l -methionine biosynthesis were analyzed. • Various crucial genes in MRMBPs for l -methionine biosynthesis were determined. • The balance between MRMBPs for l -methionine biosynthesis was clarified. [ABSTRACT FROM AUTHOR]

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∗Trc metL /pTrc- metA fbr-Trc- thrA fbr- 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. • High-yield O-succinyl- l -homoserine (OSH) producing strain was constructed in Escherichia coli W3110(DE3) (ΔIJB). • Enzyme O-succinyl- l -homoserine sulfhydrylase and cutinase were co-expressed in E. coli BL21(DE3) (OSHS–Cut). • A one-pot two-strain system was established for l -methionine production with co-cultivation of ΔIJB and OSHS–Cut. • A system combining of OSH production and enzymatic catalysis in one pot was established. [ABSTRACT FROM AUTHOR]

• Pathway engineering for OSH production from glucose using E. coli W3110. • Increase succiny-CoA supply for succinylation of l -homoserine further increased the yield of OSH. • The metabolomics data revealed the less flexibility of engineered strain for OSH biosynthesis. • The maximum OSH titer is 24.1 g/L (0.609 g/g glucose) in batch fermentation. O -Succinyl- l -homoserine (OSH) is an important platform chemical in production of C 4 chemicals such as succinic acid, homoserine lactone, γ‑butyrolactone, and 1,4‑butanediol. 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- metA 11- 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. [ABSTRACT FROM AUTHOR]