With the advent of the post-genomic era, metabolic engineering of microorganisms plays an increasingly important role in industrial production. The genome-scale metabolic model (GSMM) integrates all known metabolic information in the organism to provide an optimal platform for global understanding of the metabolic state of the organism and rational guidance for metabolic engineering. As a model strain, Lactococcus lactis NZ9000 plays an important role in industrial fermentation, but there is still no specific genome-scale metabolic model for it. Based on genomic function annotation and comparative genomics, we constructed the first genome-scale metabolic model iWK557 of L. lactis NZ9000, which contains 557 genes, 668 metabolites, and 840 reactions, and further verified at both qualitative and quantitative levels, to provide a good tool for rationally guiding metabolic engineering.

5-Aminolevulinic acid is the key intermediate of the tetrapyrrole biosynthesis pathway in organisms and has broad application potentials. This review summarized and discussed recent progress in microbial production of 5-aminolevulinic acid, including screening, isolation and mutation of microbes to produce 5-aminolevulinic acid; microbial whole-cell transformation to synthesize 5-aminolevulinic acid depending on the C4 pathway; construction of high-yield 5-aminolevulinic acid producing strains by metabolic engineering. Finally, future research directions in microbial production of 5-aminolevulinic acid were addressed.
Aminolevulinic Acid ; metabolism ; Escherichia coli ; genetics ; metabolism ; Metabolic Engineering ; Mutation ; Rhodobacter sphaeroides ; genetics ; metabolism

Self-assembling amphipathic peptides (SAPs) have alternating hydrophilic and hydrophobic residues and can affect the thermal stabilities and catalytic properties of the fused enzymes. In this study, a novel multifunctional tag, S1vw (HNANARARHNANARARHNANARARHNARARAR) was developed to modify fused enzymes. After fusing S1vw at the enzymes/proteins N-terminus through a PT-linker, the crude enzymatic activities of polygalacturonate lyase and lipoxygenase were enhanced 3.1- and 1.89-fold, respectively, compared to the wild-type proteins. The relative fluorescence intensity of the green fluorescent protein was enhanced 16.22-fold. All the three S1vw fusions could be purified by nickel column with high purities and acceptable recovery rates. Moreover, S1vw also induced the thermostabilities enhancement of the fusions, with polygalacturonate lyase and lipoxygenase fusions exhibiting 2.16- and 3.2-fold increase compared with the corresponding wild-type, respectively. In addition, S1vw could enhance the production yield of green fluorescent protein in Escherichia coli and Bacillus subtilis while the production of GFP and its S1vw fusion changed slightly in Pichia pastoris. These results indicated that S1vw could be used as a multifunctional tag to benefit the production, thermal stability and purification of the fusion protein in prokaryotic expression system.
Escherichia coli ; Green Fluorescent Proteins ; Hydrophobic and Hydrophilic Interactions ; Peptides ; Pichia ; Recombinant Fusion Proteins ; metabolism

Diacylglycerol (DAG) is an intermediate product in lipid metabolism and plays an important physiological role in human body. It is mainly prepared by hydrolyzing lipid with lipase. However, research on the detection method of 1, 2-diacylglycerol (1, 2-DAG) and 1, 3-diacylglycerol (1, 3-DAG) and catalytic specificity of lipase was not enough, which limits its wide application. To address these challenges, an efficient quantitative detection method was first established for 1, 2-DAG (0.025-0.200 g/L) and 1, 3-DAG (0.025-0.150 g/L) by combining supercritical fluid chromatography with evaporative light scattering detector and optimizing the detection and analysis parameters. Based on the molecular docking between Thermomyces lanuginosus lipase (TLL) and triolein, five potential substrate binding sites were selected for site-specific saturation mutation to construct a mutation library for enzyme activity and position specificity screening. The specificity of sn-1, 3 of the I202V mutant was the highest in the library, which was 11.7% higher than the specificity of the wild type TLL. In summary, the position specificity of TLL was modified based on a semi-rational design, and an efficient separation and detection method of DAG isomers was also established, which provided a reference for the study of the catalytic specificity of lipase.
Humans ; Diglycerides ; Molecular Docking Simulation ; Binding Sites ; Catalysis ; Lipase/genetics*

As a typical food safety industrial model strain, Bacillus subtilis has been widely used in the field of metabolic engineering due to its non-pathogenicity, strong ability of extracellular protein secretion and no obvious codon preference. In recent years, with the rapid development of molecular biology and genetic engineering technology, a variety of research strategies and tools have been used to construct B. subtilis chassis cells for efficient synthesis of biological products. This review introduces the research progress of B. subtilis from the aspects of promoter engineering, gene editing, genetic circuit, cofactor engineering and pathway enzyme assembly. Then, we also summarized the application of B. subtilis in the production of biological products. Finally, the future research directions of B. subtilis are prospected.
Bacillus subtilis/genetics* ; Bacterial Proteins/genetics* ; Gene Editing ; Metabolic Engineering ; Promoter Regions, Genetic

A novel anaerobic fermentation process--multistage countercurrent fermentation was applied to improve the bioproduction of volatile fatty acids (VFAs) from excess municipal sludge. Results showed that the total VFAs concentration and the total VFAs yield reached (10.5 +/- 0.5) g/L and 0.20 gVFAs/gVS (Volatile solid) using this novel process. Comparing with the conventional anaerobic fermentation, the concentration and yield of total VFAs increased by 31% and by 54%, respectively. Moreover, removal ratio of organic solids also increased by 37% and it was 50% at the end of multistage countercurrent fermentation. We further investigated the mechanism of VFAs production. Results revealed that this novel process could reduce the inhibitory effect of VFAs on the acid-forming microorganisms, and the total VFAs yield and the removal ratio of organic solids respectively depended on the first stage and the third stage of this novel process. Therefore, the multistage countercurrent fermentation can efficiently improve the bioproduction of VFAs from excess municipal sludge, and relatively enhance the removal ratio of organic solids.
Anaerobiosis ; Bacteria, Anaerobic ; metabolism ; Bioreactors ; microbiology ; Fatty Acids, Volatile ; biosynthesis ; Fermentation ; Refuse Disposal ; instrumentation ; methods ; Sewage ; chemistry

Cutinase (EC 3.1.1.74) is a kind of hydrolase capable of catalyzing the cleavage of ester bonds of cutin to release fatty acids. Cutinase displayed hydrolytic activity not only toward cutin but also a variety of soluble synthetic esters, insoluble triglycerides and polyesters. Besides its hydrolytic activity, cutinase also showed synthetic activity and transester activity. Therefore, cutinase was evaluated as a versatile lipolytic enzyme used in food and chemical industry. Recently, it is found that cutinase has potential use in cotton bio-scouring and synthetic fibers modification. Cutinase is the most important enzyme in clean production of textile industry.
Amino Acid Sequence ; Carboxylic Ester Hydrolases ; chemistry ; genetics ; metabolism ; Catalysis ; Environmental Pollution ; prevention & control ; Molecular Sequence Data ; Textile Industry

We reviewed the microbial production of alkaline polygalacturonate lyase (PGL) and its application in the clean production of textile industry. Currently PGL is mainly produced by microbial fermentation and Bacillus sp. is an ideal wild strain for PGL production. Microbial PGL production was affected by many factors including the concentration and feeding mode of substrate, cell concentration, agitation speed, aeration rate, pH and temperature. Constructing the recombinant strain provided an effective alternative for PGL production, and the concentration of PGL produced by the recombinant Pichia pastoris reached 1305 U/mL in 10 m3 fermentor. The recombinant Pichia pastoris had the potential to reach the industrial production of PGL. PGL can be applied in bio-scouring process in the pre-treatment of cotton. Compared with the traditional alkaline cooking process, the application of PGL can protect fiber, improve the bio-scouring efficiency, decrease energy consumption and alleviate the environmental pollution. The future research focus will be the molecular directed evolution of PGL to make PGL more suitable for the application of PGL in bio-scouring process to realize the clean production of textile industry.
Alkalies ; Bacillus subtilis ; metabolism ; Environmental Pollution ; prevention & control ; Fermentation ; Industrial Microbiology ; Pichia ; genetics ; metabolism ; Polysaccharide-Lyases ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Textile Industry

Microbial catalase is an important industrial enzyme that catalyzes the decomposition of hydrogen peroxide to water and oxygen. This enzyme has great potential of application in food, textile and pharmaceutical industries. The production of microbial catalase has been significantly improved thanks to advances in bioprocess engineering and genetic engineering. In this paper, we review the progresses in fermentation production of microbial catalase and its application in textile industry. Among these progresses, we will highlight strain isolation, substrate and environment optimization, enzyme induction, construction of engineering strains and application process optimization. Meanwhile, we also address future research trends for microbial catalase production and its application in textile industry. Molecular modification (site-directed mutagenesis and directed revolution) will endue catalase with high pH and temperature stabilities. Improvement of catalase production, based on the understanding of induction mechanism and the process control of recombinant stain fermentation, will further accelerate the application of catalase in textile industry.
Bacillus subtilis ; genetics ; metabolism ; Bacteria, Aerobic ; enzymology ; genetics ; Catalase ; biosynthesis ; genetics ; Fermentation ; Genetic Engineering ; methods ; Industrial Microbiology ; Recombinant Proteins ; biosynthesis ; genetics ; Textile Industry ; methods

Glucose oxidase catalyzes the oxidation of β-D-glucose to gluconic acid and its derivatives, thus shows a great potential in the development of antibiotic-free feed. However, its production and processing still have the problem of poor thermal stability of enzyme activity. In this study, fusion of amphiphilic peptide technology was used to improve the stability of glucose oxidase. Herein, eight self-assembling peptides with different amino acid lengths and Linkers were fused to the N terminus of the glucose oxidase, yielding eight chimeric fusions SAP1-GS-GOD, SAP1-PT-GOD, SAP2-PT-GOD, SAP3-PT-GOD, SAP4-PT-GOD, SAP5-PT-GOD, SAP6-PT-GOD and SAP7-PT-GOD. Then, the 8 recombinant proteins were expressed in P. pastoris GS115. After separation and purification, the stability of glucose oxidase at 60 ℃was determined. The relative enzyme activities of the PT Linker-linked fusion enzyme incubated at 60 ℃ for 60 min were higher than those of the original enzyme, and the relative activity of SAP5-PT-GOD was 67% at 60 ℃ for 30 min, which was 10.9 times higher than that of the initial enzyme with the same treatment. Among them, the Kcat/Km value of SAP1-PT-GOD, SAP2-PT-GOD, SAP3-PT-GOD and SAP5-PT-GOD of the fusion enzyme was further improved than that of the initial enzyme. Through the analysis of the intramolecular force of the fusion enzyme, the increase of the thermal stability of the fusion enzyme is mainly due to the increase of the hydrogen bond. In summary, the study indicates that translational fusion of self-assembling peptides with PT Linker was able to augment the thermo-stability of glucose oxidase, which has certain potential in the production and application of glucose oxidase. The glucose oxidase with improved thermostability obtained in the above study and the related mechanism will play an important role in improving the activity of related enzymes in the proceeding of processing and application.