This study focused on the cloning, overexpression, and characterization of the gene encoding l-asparaginase (ansZ) from a nonpathogenic strain of Bacillus subtilis B11–06. The recombinant enzyme ...showed high thermostability and low affinity to l-glutamine. The ansZ gene, encoding a putative l-asparaginase II, was amplified by PCR and expressed in B. subtilis 168 using the shuttle vector pMA5. The activity of the recombinant enzyme was 9.98 U/mL, which was significantly higher than that of B. subtilis B11–06. The recombinant enzyme was purified by a two-step procedure including ammonium sulfate fractionation and hydrophobic interaction chromatography. The optimum pH and temperature of the recombinant enzyme were 7.5 and 40 °C, respectively. The enzyme was quite stable at a pH range of 6.0–9.0 and exhibited about 14.7 and 9.0% retention of activity following 2 h incubation at 50 or 60 °C, respectively. The K m for l-asparagine was 0.43 mM, and the V max was 77.51 μM/min. Results of this study also revealed the potential industrial application of this enzyme in reducing acrylamide formation during the potato frying process.
Intermediates such as 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) have extensive clinical applications in the production of steroid pharmaceuticals. The present study explores ...the effect of two factors in the production of these intermediates in Mycobacterium neoaurum JC-12: the precursor, phytosterol and a molecule that increases AD/ADD solubility, hydroxypropyl-β-cyclodextrin (HP-β-CD). Differentially expressed proteins were separated and identified using 2D gel electrophoresis (2-DE) and matrix assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS/MS). In total, 31 proteins were identified, and improved expression levels of ten proteins involved in metabolism was induced by phytosterol and/or HP-β-CD, which strengthened the stress resistance of the strain. In the presence of phytosterol and/or HP-β-CD, five proteins involved in the synthesis of AD/ADD, acetyl-CoA acetyltransferase (AAT), alcohol dehydrogenase (ADH), enoyl-CoA hydratase (EH) and short-chain dehydrogenase 1 and 2, increased their expression levels. Reverse transcription-quantitative PCR (RT-qPCR) was used to verify the 2-DE results and the transcriptional level of these five proteins. This analysis identified AAT, ADH, EH, and electron transfer flavoprotein subunit α/β as the possible bottlenecks for AD/ADD synthesis in M. neoaurum JC-12, which therefore are suggested as targets for strain modification.
•A comprehensive proteomic analysis of AD/ADD producing M. neoaurum JC-12 at different fermentation conditions is performed.•Herein, the analysis is performed by combing 2DE analysis and mass spectrometry.•This study facilitates an understanding of global regulation mechanisms of M. neoaurum JC-12 under various conditions.•The possible bottlenecks (AAT, ADH, EH, ETF subunit α/β) for AD/ADD synthesis pathway in M. neoaurum JC-12 are identified.
ε-poly-L-lysine (ε-PL) is a naturally occurring poly(amino acid) of varying polymerization degree, which possesses excellent antimicrobial activity and has been widely used in food and pharmaceutical ...industries. To provide new perspectives from recent advances, this review compares several conventional and advanced strategies for the discovery of wild strains and development of high-producing strains, including isolation and culture-based traditional methods as well as genome mining and directed evolution. We also summarize process engineering approaches for improving production, including optimization of environmental conditions and utilization of industrial waste. Then, efficient downstream purification methods are described, including their drawbacks, followed by the brief introductions of proposed antimicrobial mechanisms of ε-PL and its recent applications. Finally, we discuss persistent challenges and future perspectives for the commercialization of ε-PL.
Acetoin and 2,3-butanediol (2,3-BD) have a large number of industrial applications. The production of acetoin and 2,3-BD has traditionally relied on oil supplies. Microbial production of acetoin and ...2,3-BD will alleviate the dependence on oil. Acetoin and 2,3-BD are neighboring metabolites in the 2,3-BD metabolic pathway of bacteria. This review summarizes metabolic engineering strategies for improvement of microbial acetoin and 2,3-BD production. We also propose enhancements to current acetoin and 2,3-BD production strategies, by offering a metabolic engineering approach that is guided by systems biology and synthetic biology.
l-arginine, a semi essential amino acid, is an important amino acid in food flavoring and pharmaceutical industries. Its production by microbial fermentation is gaining more and more attention. In ...previous work, we obtained a new l-arginine producing Corynebacterium crenatum (subspecies of Corynebacterium glutamicum) through mutation breeding. In this work, we enhanced l-arginine production through improvement of the intracellular environment. First, two NAD(P)H-dependent H2O2-forming flavin reductases Frd181 (encoded by frd1 gene) and Frd188 (encoded by frd2) in C. glutamicum were identified for the first time. Next, the roles of Frd181 and Frd188 in C. glutamicum were studied by overexpression and deletion of the encoding genes, and the results showed that the inactivation of Frd181 and Frd188 was beneficial for cell growth and l-arginine production, owing to the decreased H2O2 synthesis and intracellular reactive oxygen species (ROS) level, and increased intracellular NADH and ATP levels. Then, the ATP level was further increased by deletion of noxA (encoding NADH oxidase) and amn (encoding AMP nucleosidase), and overexpression of pgk (encoding 3-phosphoglycerate kinase) and pyk (encoding pyruvate kinase), and the l-arginine production and yield from glucose were significantly increased. In fed-batch fermentation, the l-arginine production and yield from glucose of the final strain reached 57.3g/L and 0.326g/g, respectively, which were 49.2% and 34.2% higher than those of the parent strain, respectively. ROS and ATP are important elements of the intracellular environment, and l-arginine biosynthesis requires a large amount of ATP. For the first time, we enhanced l-arginine production and yield from glucose through reducing the H2O2 synthesis and increasing the ATP supply.
•Two new H2O2-forming flavin reductases from C. glutamicum are identified.•Inactivation of the flavin reductases facilitates L-arginine production.•Increasing the ATP supply facilitates L-arginine production.
L-valine is an essential amino acid that has wide and expanding applications with a suspected growing market demand. Its applicability ranges from animal feed additive, ingredient in cosmetic and ...special nutrients in pharmaceutical and agriculture fields. Currently, fermentation with the aid of model organisms, is a major method for the production of L-valine. However, achieving the optimal production has often been limited because of the metabolic imbalance in recombinant strains. In this review, the constrains in L-valine biosynthesis are discussed first. Then, we summarize the current advances in engineering of microbial cell factories that have been developed to address and overcome major challenges in the L-valine production process. Future prospects for enhancing the current L-valine production strategies are also discussed.
Erythritol is a zero-calorie sweetener that is widely used in the food, pharmaceutical, and medical industries. Crude glycerol is the main by-product of biodiesel, and the effective utilization of ...crude glycerol will help to improve biodiesel viability. Previous studies on the production of erythritol from
using crude glycerol as a carbon source have focused on optimizing the fermentation process of the mutant
Wratislavia K1, while metabolic engineering has not been successfully applied.
To this end, we engineered the yeast
to increase the productivity of this strain. Wild strains tolerant to high concentrations of crude glycerol were screened and identified. A series of rational metabolic approaches were employed to improve erythritol production. Among them, the engineered strain Y-04, obtained by tandem overexpression of
and
, significantly increased glycerol assimilation by 33.3%, which was consistent with the results of RT-qPCR analysis. The effects of tandem overexpression of
,
,
and
on erythritol synthesis were also evaluated. The best results were obtained using a mutant that overexpressed
,
and
and knocked out
. The final Y-11 strain produced 150 g/l erythritol in a 5-L bioreactor with a yield and productivity of 0.62 g/g and 1.25 g/l/h, respectively. To the best of our knowledge, this is the highest erythritol yield and productivity from crude glycerol ever reported in
.
This work demonstrated that overexpression of
,
and
and knockdown of
could be used to improve crude glycerol utilization and erythritol synthesis in
. The process parameters such as erythritol yield and productivity were significantly elevated, which is valuable for industrial applications. Crude glycerol, as a carbon source, could efficiently restrict the synthesis of by-products while enhancing the generation of erythritol, compared to glucose. This indicates considerable potential for synthesizing value-added products from crude glycerol by
.
During the production of L-arginine through high dissolved oxygen and nitrogen supply fermentation, the industrial workhorse Corynebacterium glutamicum is exposed to oxidative stress. This generates ...reactive oxygen species (ROS) and reactive nitrogen species (RNS), which are harmful to the bacteria. To address the issue and to maintain redox homeostasis during fermentation, the flavohaemoprotein (Hmp) was employed. The results showed that the overexpression of Hmp led to a decrease in ROS and RNS content by 9.4% and 22.7%, respectively, and improved the survivability of strains. When the strains were treated with H.sub.2O.sub.2 and NaNO.sub.2, the RT-qPCR analysis indicated an up-regulation of ammonium absorption and transporter genes amtB and glnD. Conversely, the deletion of hmp gives rise to the up-regulation of eight oxidative stress-related genes. These findings suggested that hmp is associated with oxidative stress and intracellular nitrogen metabolism genes. Finally, we released the inhibitory effect of ArnR on hmp. The Cc-DELAarnR-hmp strain produced 48.4 g/L L-arginine during batch-feeding fermentation, 34.3% higher than the original strain. This report revealed the influence of dissolved oxygen and nitrogen concentration on reactive species of Corynebacterium glutamicum and the role of the Hmp in coping with oxidative stress. The Hmp first demonstrates related to redox homeostasis and nitrite metabolism, providing a feasible strategy for improving the robustness of strains.
Prodigiosin (PG), a red linear tripyrrole pigment produced by
Serratia marcescens
, has attracted attention due to its immunosuppressive, antimicrobial, and anticancer properties. Although many ...studies have been used to dissect the biosynthetic pathways and regulatory network of prodigiosin production in
S. marcescens
, few studies have been focused on improving prodigiosin production through metabolic engineering in this strain. In this study, transcription factor engineering and promoter engineering was used to promote the production of prodigiosin in
S. marcescens
JNB5-1. Firstly, through construing of a Tn5G transposon insertion library of strain JNB5-1, it was found that the DNA-binding response regulator BVG89_19895 (OmpR) can promote prodigiosin synthesis in this strain. Then, using RNA-Seq analysis, reporter green fluorescent protein analysis and RT-qPCR analysis, the promoter P17 (P
RplJ
) was found to be a strong constitutive promoter in strain JNB5-1. Finally, the promoter P17 was used for overexpressing of prodigiosin synthesis activator OmpR and PsrA in strain JNB5-1 and a recombinant strain PG-6 was obtained. Shake flask analysis showed that the prodigiosin titer of this strain was increased to 10.25 g/L, which was 1.62-times that of the original strain JNB5-1 (6.33 g/L). Taken together, this is the first well-characterized constitutive promoter library from
S. marcescens
, and the transcription factor engineering and promoter engineering can be also useful strategies to improve the production of other high value-added products in
S. marcescens
.
As one of the most significant steroid hormone precursors, androst-1,4-diene-3,17-dione (ADD) could be used to synthesize many valuable hormone drugs. The microbial transformation of sterols to ADD ...has received extensive attention in recent years. In a previous study,
JC-12 was isolated and converted sterols to the major product, ADD. In this work, we enhanced ADD yield by improving the cell intracellular environment. First, we introduced a nicotinamide adenine dinucleotide (NADH) oxidase from
to balance the intracellular NAD
availability in order to strengthen the ADD yield. Then, the catalase gene from
was also over-expressed to simultaneously scavenge the generated H
O
and eliminate its toxic effects on cell growth and sterol transformation. Finally, using a 5 L fermentor, the recombinant strain JC-12
produced 9.66 g/L ADD, which increased by 80% when compared with the parent strain. This work shows a promising way to increase the sterol transformation efficiency by regulating the intracellular environment.
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