S-methyl-L-cysteine sulfoxide (SMCO) is a non-protein sulfur-containing amino acid with a variety of functions. There are few reports on the enzymes catalyzing the biosynthesis of SMCO from S-methyl-L-cysteine (SMC). In this study, the flavin-containing monooxygenase gene derived from Schizosaccharomyces pombe (spfmo) was heterologously expressed in Escherichia coli BL21(DE3) and the enzymatic properties of the expressed protein were analyzed. The optimum catalytic conditions of the recombinant SpFMO were 30 ℃ and pH 8.0, under which the enzyme activity reached 72.77 U/g. An appropriate amount of Mg²⁺ improved the enzyme activity. The enzyme kinetic analysis showed that the K_m and k_cat/K_m of SpFMO on the substrate SMC were 23.89 μmol/L and 61.71 L/(min·mmol), respectively. Under the optimal reaction conditions, the yield of SMCO synthesized from SMC catalyzed by SpFMO was 12.31% within 9 h. This study provides reference for the enzymatic synthesis of SMCO.
Schizosaccharomyces/genetics* ; Escherichia coli/metabolism* ; Recombinant Proteins/metabolism* ; Cysteine/biosynthesis* ; Mixed Function Oxygenases/metabolism* ; Schizosaccharomyces pombe Proteins/metabolism* ; Oxygenases/metabolism* ; Kinetics

One of the technical bottlenecks limiting the high yield of 1,4-butanediamine is the insufficient tolerance of strains to 1,4-butanediamine. Enhancing the tolerance of strains to 1,4-butanediamine is therefore a primary challenge that needs to be addressed for the construction of strains with high yields of 1,4-butanediamine. Staphylococcus pasteuri 326180 exhibits exceptional tolerance to high-concentration 1,4-butanediamine, serving as both an ideal model for studying the mechanism underlying the 1,4-butanediamine tolerance and a novel host for constructing strains capable of efficiently producing 1,4-butanediamine. However, for both the research on the tolerance mechanism and the modification of chassis strains, gene editing of S. pasteuri needs to be carried out at the molecular level. The research objective of this paper is to establish a genetic manipulation system for S. pasteuri, laying foundation for subsequent studies on tolerance mechanism and the modification of chassis strains. This study systematically optimized the electroporation conditions, including key parameters such as the growth phase of cells, electric field strength, electroporation buffer, and recovery medium, successfully establishing an electroporation method for S. pasteuri. Additionally, we constructed the gene editing plasmid pCpfOA by replacing the resistance expression cassette, optimized the selection markers for gene editing, and finally established a CRISPR/Cpf1-based gene editing technology for S. pasteuri, achieving an editing efficiency of 90%. The genetic manipulation system of S. pasteuri established in this study provides technical support for research into the tolerance mechanism of this bacterium and the genetic modification of chassis strains.
Staphylococcus/drug effects* ; Gene Editing/methods* ; Electroporation/methods* ; Plasmids/genetics* ; CRISPR-Cas Systems ; Genetic Engineering/methods*

Dopamine is the precursor of a variety of natural antioxidant compounds. In the body, dopamine acts as a neurotransmitter that regulates a variety of physiological functions of the central nervous system. Thus, dopamine is used for the clinical treatment of various types of shock. Dopamine could be produced by engineered microbes, but with low efficiency. In this study, DOPA decarboxylase gene from Sus scrofa (Ssddc) was cloned into plasmids with different copy numbers, and transformed into a previously developed L-DOPA producing strain Escherichia coli T004. The resulted strain was capable of producing dopamine from glucose directly. To further improve the production of dopamine, a sequence-based homology alignment mining (SHAM) strategy was applied to screen more efficient DOPA decarboxylases, and five DOPA decarboxylase genes were selected from 100 candidates. In shake-flask fermentation, the DOPA decarboxylase gene from Homo sapiens (Hsddc) showed the highest dopamine production (3.33 g/L), while the DOPA decarboxylase gene from Drosophila Melanogaster (Dmddc) showed the least residual L-DOPA concentration (0.02 g/L). In 5 L fed-batch fermentations, production of dopamine by the two engineered strains reached 13.3 g/L and 16.2 g/L, respectively. The residual concentrations of L-DOPA were 0.45 g/L and 0.23 g/L, respectively. Finally, the Ssddc and Dmddc genes were integrated into the genome of E. coli T004 to obtain genetically stable dopamine-producing strains. In 5 L fed-batch fermentation, 17.7 g/L of dopamine was produced, which records the highest titer reported to date.
Animals ; Dopa Decarboxylase/genetics* ; Dopamine/biosynthesis* ; Drosophila melanogaster/genetics* ; Escherichia coli/metabolism* ; Humans ; Metabolic Engineering

cis, cis-muconic acid (MA) is an important platform chemical. Now, majority of reported engineered strains are genetically instable, the exogenous genes are expressed under the control of expensive inducer and the components of their fermentation medium are complex, thus large-scale microbial production of MA is limited due to the lack of suitable strains. Hence, it is still necessary to construct novel high-performance strain that is genetically stable, no induction and grows in simple inorganic fermentation medium. In this study, after 3 exogenous genes (aroZ, aroY, catA) for biosynthesis of MA were integrated into previously constructed 3-hydroshikimate producing Escherichia coli WJ060 strain and combinatorially regulated with 3 constitutive promoters with different strengths, 27 engineered strains were constructed. The best engineered strain, E. coli MA30 could produce 1.7 g/L MA in the simple inorganic fermentation medium without induction. To further enhance the production capacity of MA, the mutant library of E. coli MA30 was constructed by genome replication engineering and screened via high-throughput assay. After two-round screening, the new strain, E. coli MA30-G2 with improved production of MA was obtained, and the titer of MA increased more than 8%. Under the condition of 5 L fed-batch fermentation, E. coli MA30-G2 could produce about 11.5 g/L MA. Combinatorial regulation and high-throughput screening provide important reference to microbial production of other bio-based chemicals.
Escherichia coli ; metabolism ; Fermentation ; Industrial Microbiology ; Metabolic Engineering ; Microorganisms, Genetically-Modified ; Promoter Regions, Genetic ; Sorbic Acid ; analogs & derivatives ; metabolism

To indicate the relationship between structure and function of loops from Bacillus thuringiensis insecticidal crystal protein Cry1Ba, and the influence of amino acids mutation on toxicity against diamond back moth Plutella xylostella, five mutations at the loops of Cry1Ba were constructed by overlapping primer PCR, and expressed in E. coli BL21 (DE3). Bioassay results showed that the toxicity of mutation M1 (loop1: 340WSNTR344-deletion), compared with that of Cry1Ba (LC50 0.96 microg/mL), decreased significantly with LC50 35.51 microg/mL. And the toxicity of mutation M2 (402Y-G), M3 (400GIYLEP405-PSAV), M4 (400GIYLEPIH407-ILGS) was also reduced to some extent respectively. Only M5 (mutation at loop3: 472LQSRV476 - AGAVYTL) showed slightly increased activity against P. xylostella, but not significantly (LC50 0.81 microg/mL). Referring to the structures of Cry1Ba which was predicted using Swiss-Model software, and bioassay data, we can conclude that loop1 and loop2 play a important role on determining the activity of Cry1Ba against P. xylostella.
Animals ; Bacillus thuringiensis ; genetics ; metabolism ; Bacterial Proteins ; chemistry ; genetics ; Endotoxins ; chemistry ; genetics ; Escherichia coli ; genetics ; metabolism ; Hemolysin Proteins ; chemistry ; genetics ; Models, Molecular ; Moths ; microbiology ; Mutation ; Protein Structure, Secondary ; Structure-Activity Relationship