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Methionine is the only one of the essential amino acids that contain sulfur, widely used as a feed additive in agriculture. In this study, the availability of 5-methyl-tetrahydrofolate was confirmed as the main limitation in the complex multibranched biosynthetic pathway of L-methionine. The cycle of one-carbon units was thoroughly investigated and modified to supply 5-methyl-tetrahydrofolate for L-methionine production, such as enhancing the supply of precursor, expediting the conversion rate of the cycle, introducing exogenous serine hydroxymethyltransferase and increasing pool size of one-carbon units carrier. The final strain MYA/pAmFA-4 was able to produce 20.89 g/L L-methionine by fed-batch fermentation, which was the highest titer reported in the literatures. This study is instructive for other metabolites biosynthesized needing one-carbon units or having a complex multibranched biosynthetic pathway. [ABSTRACT FROM AUTHOR]

Methionine is the only one of the essential amino acids that contain sulfur, widely used as a feed additive in agriculture. In this study, the availability of 5-methyl-tetrahydrofolate was confirmed as the main limitation in the complex multibranched biosynthetic pathway of L-methionine. The cycle of one-carbon units was thoroughly investigated and modified to supply 5-methyl-tetrahydrofolate for L-methionine production, such as enhancing the supply of precursor, expediting the conversion rate of the cycle, introducing exogenous serine hydroxymethyltransferase and increasing pool size of one-carbon units carrier. The final strain MYA/pAmFA-4 was able to produce 20.89 g/L L-methionine by fed-batch fermentation, which was the highest titer reported in the literatures. This study is instructive for other metabolites biosynthesized needing one-carbon units or having a complex multibranched biosynthetic pathway. [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]

Objective: To evaluate whether patients with malignant biliary obstruction (MBO) benefit from balloon dilation before the placement of a self‐expanding metal stent (SEMS) for palliative biliary drainage. Methods: Consecutive patients who underwent endoscopic retrograde cholangiopancreatography with SEMS placement for palliative management of MBO were retrospectively included. Comparative analyses of serum bilirubin levels, post‐procedural adverse events, stent patency time, stent dysfunction, and patient survival were performed between the dilation and non‐dilation groups. Results: A total of 221 patients underwent palliative endoscopic SEMS implantation for MBO from January 2014 to June 2018. Dilation significantly improved the percentage of serum bilirubin improvement (37.0% vs 14.3%, P = 0.001), with a decreasing trend in the incidence of post‐procedural cholangitis (2.5% vs 7.8%, P = 0.075), while the rates of other complications such as pancreatitis and bleeding were not increased. The patency time of SEMS and patient survival did not significantly differ between patients with and without dilation. Patients had endoscopic nasobiliary drainage (ENBD) but not dilation showed similar short‐term outcomes as patients underwent dilation but without ENBD. Conclusions: Dilation with a small‐caliber balloon catheter before the placement of SEMS is a safe and effective approach for MBO. Balloon dilation may improve the short‐term efficacy of SEMS placement, while long‐term outcomes are not obviously affected. The short‐term effect of stricture dilation may be achieved by ENBD. Further studies are needed to confirm our results. [ABSTRACT FROM AUTHOR]

( S)-4-chloro-3-hydroxybutanoate (( S)-CHBE) is an important chiral intermediate to synthesize the side chain of cholesterol-lowering drug atorvastatin. To biosynthesize the ( S)-CHBE, a recombinant Escherichia coli harboring the carbonyl reductase and glucose dehydrogenase was successfully constructed. The recombinant E. coli was cultured in a 500-L fermentor; after induction and expression, the enzyme activity and cell biomass were increased to 23,661.65 U/L and 13.90 g DCW/L which was 3.24 and 2.60-folds compared with those in the 50 L fermentor. The biocatalytic process for the synthesis of ( S)-CHBE in an aqueous-organic solvent system was constructed and optimized with a substrate fed-batch strategy. The ethyl 4-chloro-3-oxobutanoate concentration reached to 1.7 M, and the ( S)-CHBE with yield of 97.2 % and enantiomeric excess ( e.e.) of 99 % was obtained after 4-h reaction in a 50-L reactor. In this study, the space-time yield and space-time yield per gram of biomass (dry cell weight, DCW) were 413.17 mM/h and 27.55 mM/h/g DCW for ( S)-CHBE production, respectively, which were the highest values as compared to previous reports. Finally, ( S)-CHBE was extracted from the reaction mixture with 82 % of yield and 95 % of purity. This study paved the foundation for the upscale production of ( S)-CHBE by biocatalysis method. [ABSTRACT FROM AUTHOR]

• PKS5 was successfully identified as one of the main precursor competing branches in Streptomyces nodosus. • The synthesis of amphotericin B was successfully increased by 40 %. • The intracellular reduction force (NADPH/NADP) was compared with the ratio of AmB/AmA. • The yield of AmB from strain D5-2M2A reached 15.6 g/L on the 7th day with fed-batch fermentation in the 5 L bioreactor. The antifungal agent amphotericin B (AmB) is a polyketide produced by Streptomyces nodosus. The synthetic precursors of the amphotericin macrolactone skeleton are acetyl-CoA, malonyl-CoA and methylmalonyl-CoA. The genome sequence of the wild type S. nodosus ATCC14899 revealed a type II polyketide synthase (PKS) competing for malonyl-CoA. The same competitive branch was sequenced and verified in a mutant named S. nodosus ZJB2016050 (S. nodosus N3) screened in our lab. The transcriptome of the secondary metabolic synthetic gene cluster comparisons suggested that type II PKS (PKS5) competition is a factor in low production. The deletion of the PKS5 gene led to the titer of AmB improved from 5.01 g/L to 6.32 g/L while the by-product amphotericin A (AmA) reduced from 0.51 g/L to 0.12 g/L. A sequence of genes including PKS amphA , acc 1, mme and mcm were overexpressed in a ΔPKS5 mutant, resulting in improved production AmB from 5.01 g/L to 7.06 g/L in shake flasks at 96 h. The yield of AmB and AmA in a 5 L bioreactor at 144 h was 15.6 g/L and 0.36 g/L, respectively. The intracellular reducibility of the wild type, mutagenesis type and genetically engineered type were detected, which was first found to be related to the by-product AmA. The increment of skeleton biosynthesis may consume more NADPH and reduces AmphC ER5 domain reduction. This study can be implemented for other polyketides in industrial production. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

( S)-4-Chloro-3-hydroxybutyronitrile [( S)-CHBN] was used as a chiral building block for the preparation of atorvastatin. In this study, ( R, S)-epichlorohydrin [( R, S)-ECH] and 1,3-dichloro-2-propanol (1,3-DCP) were investigated to prepare ( S)-CHBN by using the halohydrin dehalogenase HheC from Agrobacterium radiobacter AD1. Preparing ( S)-CHBN from ( R, S)-ECH gave a modest enantiomeric excess ( ee), whereas by using 1,3-DCP as the substrate, ( S)-CHBN was obtained with 97.3 % ee after pH optimization. However, a low ee value and low yield of ( S)-CHBN were obtained if the substrate concentration was increased to 10 g L−1. To obtain a higher ee value and yield, 16 mutants were constructed and screened. The variant W249F with improvements in activity and enantioselectivity was identified and applied at a 1,3-DCP loading of 10 g L−1, which gave ( S)-CHBN in 86 % yield with 97.5 % ee in 1 h. This is the first report of a one-step biocatalytic process for the preparation of ( S)-CHBN from prochiral 1,3-DCP. [ABSTRACT FROM AUTHOR]