[Rational metabolic engineering of Corynebacterium glutamicum for efficient synthesis of L-glutamate].

L-glutamic acid is the world's largest bulk amino acid product that is widely used in the food, pharmaceutical and chemical industries. Using Corynebacterium glutamicum G01 as the starting strain, the fermentation by-product alanine content was firstly reduced by knocking out the gene encoding alanine aminotransferase (alaT), a major by-product related to alanine synthesis. Secondly, since the α-ketoglutarate node carbon flow plays an important role in glutamate synthesis, the ribosome-binding site (RBS) sequence optimization was used to reduce the activity of α-ketoglutarate dehydrogenase and enhance the glutamate anabolic flow. The endogenous conversion of α-ketoglutarate to glutamate was also enhanced by screening different glutamate dehydrogenase. Subsequently, the glutamate transporter was rationally desgined to improve the glutamate efflux capacity. Finally, the fermentation conditions of the strain constructed using the above strategy were optimized in 5 L fermenters by a gradient temperature increase combined with a batch replenishment strategy. The glutamic acid production reached (135.33±4.68) g/L, which was 41.2% higher than that of the original strain (96.53±2.32) g/L. The yield was 55.8%, which was 11.6% higher than that of the original strain (44.2%). The combined strategy improved the titer and the yield of glutamic acid, which provides a reference for the metabolic modification of glutamic acid producing strains.

Recent advances in the biosynthesis of isoprenoids in engineered Saccharomyces cerevisiae.

Isoprenoids, as the largest group of chemicals in the domains of life, constitute more than 50,000 members. These compounds consist of different numbers of isoprene units (C5H8), by which they are typically classified into hemiterpenoids (C5), monoterpenoids (C10), sesquiterpenoids (C15), diterpenoids (C20), triterpenoids (C30), and tetraterpenoids (C40). In recent years, isoprenoids have been employed as food additives, in the pharmaceutical industry, as advanced biofuels, and so on. To realize the sufficient and efficient production of valuable isoprenoids on an industrial scale, fermentation using engineered microorganisms is a promising strategy compared to traditional plant extraction and chemical synthesis. Due to the advantages of mature genetic manipulation, robustness and applicability to industrial bioprocesses, Saccharomyces cerevisiae has become an attractive microbial host for biochemical production, including that of various isoprenoids. In this review, we summarized the advances in the biosynthesis of isoprenoids in engineered S. cerevisiae over several decades, including synthetic pathway engineering, microbial host engineering, and central carbon pathway engineering. Furthermore, the challenges and corresponding strategies towards improving isoprenoid production in engineered S. cerevisiae were also summarized. Finally, suggestions and directions for isoprenoid production in engineered S. cerevisiae in the future are discussed.

Anti-Tumor Effects of Astaxanthin by Inhibition of the Expression of STAT3 in Prostate Cancer.

Astaxanthin is a natural product gaining increasing attention due to its safety and anti-cancer properties. In this study, we investigated the mechanisms of the anti-cancer effects of astaxanthin on prostate cancer (PCa) cell lines using aggressive PCa DU145 cells. Also an instantaneous silenced cell line (si-STAT3) derived from DU145 and a control cell line (si-NK) were used for the MTT and colony formation assays to determine the role of astaxanthin in proliferation and colony formation abilities. Flow cytometry assays were used to detect the apoptosis of tumor cells. Migration and invasion assays detected the weakening of the respective abilities. Western blot and RT-PCR tests detected the levels of STAT3 protein and mRNA. Astaxanthin resulted in suppression of the proliferation of DU145 cells and the level of STAT3. The treatment of DU145 cells with astaxanthin decreased the cloning ability, increased the apoptosis percentage and weakened the abilities of migration and invasion of the cells. Furthermore, astaxanthin reduced the expression of STAT3 at protein and mRNA levels. The effects were enhanced when astaxanthin and si-STAT3 were combined. The results of animal experiments were consistent with the results in cells. Thus, astaxanthin inhibits the proliferation of DU145 cells by reducing the expression of STAT3.

Engineered disulfide bonds improve thermostability and activity of L-isoleucine hydroxylase for efficient 4-HIL production in Bacillus subtilis 168.

4-Hydroxyisoleucine, a promising drug, has mainly been applied in the clinical treatment of type 2 diabetes in the pharmaceutical industry. l-Isoleucine hydroxylase specifically converts l-Ile to 4-hydroxyisoleucine. However, due to its poor thermostability, the industrial production of 4-hydroxyisoleucine has been largely restricted. In the present study, the disulfide bond in l-isoleucine hydroxylase protein was rationally designed to improve its thermostability to facilitate industrial application. The half-life of variant T181C was 4.03 h at 50°C, 10.27-fold the half-life of wild type (0.39 h). The specific enzyme activity of mutant T181C was 2.42 ± 0.08 U/mg, which was 3.56-fold the specific enzyme activity of wild type 0.68 ± 0.06 U/mg. In addition, molecular dynamics simulation was performed to determine the reason for the improvement of thermostability. Based on five repeated batches of whole-cell biotransformation, Bacillus subtilis 168/pMA5-ido T181C recombinant strain produced a cumulative yield of 856.91 mM (126.11 g/L) 4-hydroxyisoleucine, which is the highest level of productivity reported based on a microbial process. The results could facilitate industrial scale production of 4-hydroxyisoleucine. Rational design of disulfide bond improved l-isoleucine hydroxylase thermostability and may be suitable for protein engineering of other hydroxylases.

LysR-Type Transcriptional Regulator MetR Controls Prodigiosin Production, Methionine Biosynthesis, Cell Motility, H2O2 Tolerance, Heat Tolerance, and Exopolysaccharide Synthesis in Serratia marcescens.

Prodigiosin, a secondary metabolite produced by Serratia marcescens, has attracted attention due to its immunosuppressive, antimicrobial, and anticancer properties. However, information on the regulatory mechanism behind prodigiosin biosynthesis in S. marcescens remains limited. In this work, a prodigiosin-hyperproducing strain with the BVG90_22495 gene disrupted (ZK66) was selected from a collection of Tn5G transposon insertion mutants. Using real-time quantitative PCR (RT-qPCR) analysis, ß-galactosidase assays, transcriptomics analysis, and electrophoretic mobility shift assays (EMSAs), the LysR-type regulator MetR encoded by the BVG90_22495 gene was found to affect prodigiosin synthesis, and this correlated with MetR directly binding to the promoter region of the prodigiosin-synthesis positive regulator PigP and hence negatively regulated the expression of the prodigiosin-associated pig operon. More analyses revealed that MetR regulated some other important cellular processes, including methionine biosynthesis, cell motility, H2O2 tolerance, heat tolerance, exopolysaccharide synthesis, and biofilm formation in S. marcescens Although MetR protein is highly conserved in many bacteria, we report here on the LysR-type regulator MetR exhibiting novel roles in negatively regulating prodigiosin synthesis and positively regulating heat tolerance, exopolysaccharide synthesis, and biofilm formation.IMPORTANCESerratia marcescens, a Gram-negative bacterium, is found in a wide range of ecological niches and can produce several secondary metabolites, including prodigiosin, althiomycin, and serratamolide. Among them, prodigiosin shows diverse functions as an immunosuppressant, antimicrobial, and anticancer agent. However, the regulatory mechanisms behind prodigiosin synthesis in S. marcescens are not completely understood. Here, we adapted a transposon mutant library to identify the genes related to prodigiosin synthesis, and the BVG90_22495 gene encoding the LysR-type regulator MetR was found to negatively regulate prodigiosin synthesis. The molecular mechanism of the metR mutant hyperproducing prodigiosin was investigated. Additionally, we provided evidence supporting new roles for MetR in regulating methionine biosynthesis, cell motility, heat tolerance, H2O2 tolerance, and exopolysaccharide synthesis in S. marcescens Collectively, this work provides novel insight into regulatory mechanisms of prodigiosin synthesis and uncovers novel roles for the highly conserved MetR protein in regulating prodigiosin synthesis, heat tolerance, exopolysaccharide (EPS) synthesis, and biofilm formation.

Loss of Serine-Type D-Ala-D-Ala Carboxypeptidase DacA Enhances Prodigiosin Production in Serratia marcescens.

Serratia marcescens, a gram-negative bacterium, found in a wide range of ecological niches can produce several high-value products, including prodigiosin, althiomycin, and serratamolide. Among them, prodigiosin has attracted attention due to its immunosuppressive, antimicrobial, and anticancer properties. However, the regulatory mechanisms behind prodigiosin synthesis in Serratia marcescens remains limited. Here, a transposon mutant library was constructed to identify the genes related to prodigiosin synthesis, and BVG90_02415 gene encoding a peptidoglycan synthesizing enzyme D-Ala-D-Ala carboxypeptidase DacA was found to negatively regulates prodigiosin synthesis. Quantitative measurements revealed that disruption of dacA increased prodigiosin production 1.46-fold that of the wild-type strain JNB5-1 in fermentation medium. By comparing differences in cell growth, pigA gene expression level, cell morphology, membrane permeability, and intracellular prodigiosin concentration between wild-type strain JNB5-1 and dacA mutant SK4-72, results revealed that the mechanism for hyper-producing of prodigiosin by the dacA mutant was probably that dacA disruption enhanced prodigiosin leakage, which in turn alleviated feedback inhibition of prodigiosin and increased expression of pig gene cluster. Collectively, this work provides a novel insight into regulatory mechanisms of prodigiosin synthesis and uncovers new roles of DacA protein in regulating cell growth, cell morphology, and membrane permeability in Serratia marcescens. Finally, this study offers a new strategy for improving production of high-value compounds in Serratia marcescens.

Intracellular Environment Improvement of Mycobacterium neoaurum for Enhancing Androst-1,4-Diene-3,17-Dione Production by Manipulating NADH and Reactive Oxygen Species Levels.

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, Mycobacterium neoaurum 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 Bacillus subtilis to balance the intracellular NAD+ availability in order to strengthen the ADD yield. Then, the catalase gene from M. neoaurum was also over-expressed to simultaneously scavenge the generated H2O2 and eliminate its toxic effects on cell growth and sterol transformation. Finally, using a 5 L fermentor, the recombinant strain JC-12yodC-katA 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.

Synthetic engineering of Corynebacterium crenatum to selectively produce acetoin or 2,3-butanediol by one step bioconversion method.

BACKGROUND: Acetoin (AC) and 2,3-butanediol (2,3-BD) as highly promising bio-based platform chemicals have received more attentions due to their wide range of applications. However, the non-efficient substrate conversion and mutually transition between AC and 2,3-BD in their natural producing strains not only led to a low selectivity but also increase the difficulty of downstream purification. Therefore, synthetic engineering of more suitable strains should be a reliable strategy to selectively produce AC and 2,3-BD, respectively. RESULTS: In this study, the respective AC (alsS and alsD) and 2,3-BD biosynthesis pathway genes (alsS, alsD, and bdhA) derived from Bacillus subtilis 168 were successfully expressed in non-natural AC and 2,3-BD producing Corynebacterium crenatum, and generated recombinant strains, C. crenatum SD and C. crenatum SDA, were proved to produce 9.86 g L-1 of AC and 17.08 g L-1 of 2,3-BD, respectively. To further increase AC and 2,3-BD selectivity, the AC reducing gene (butA) and lactic acid dehydrogenase gene (ldh) in C. crenatum were then deleted. Finally, C. crenatumΔbutAΔldh SD produced 76.93 g L-1 AC in one-step biocatalysis with the yield of 0.67 mol mol-1. Meanwhile, after eliminating the lactic acid production and enhancing 2,3-butanediol dehydrogenase activity, C. crenatumΔldh SDA synthesized 88.83 g L-1 of 2,3-BD with the yield of 0.80 mol mol-1. CONCLUSIONS: The synthetically engineered C. crenatumΔbutAΔldh SD and C. crenatumΔldh SDA in this study were proved as an efficient microbial cell factory for selective AC and 2,3-BD production. Based on the insights from this study, further synthetic engineering of C. crenatum for AC and 2,3-BD production is suggested.

A Novel 3-Phytosterone-9α-Hydroxylase Oxygenation Component and Its Application in Bioconversion of 4-Androstene-3,17-Dione to 9α-Hydroxy-4-Androstene-3,17-Dione Coupling with A NADH Regeneration Formate Dehydrogenase.

9α-Hydroxy-4-androstene-3,17-dione (9-OH-AD) is one of the significant intermediates for the preparation of ß-methasone, dexamethasone, and other steroids. In general, the key enzyme that enables the biotransformation of 4-androstene-3,17-dione (AD) to 9-OH-AD is 3-phytosterone-9α-hydroxylase (KSH), which consists of two components: a terminal oxygenase (KshA) and ferredoxin reductase (KshB). The reaction is carried out with the concomitant oxidation of NADH to NAD+. In this study, the more efficient 3-phytosterone-9α-hydroxylase oxygenase (KshC) from the Mycobacterium sp. strain VKM Ac-1817D was confirmed and compared with reported KshA. To evaluate the function of KshC on the bioconversion of AD to 9-OH-AD, the characterization of KshC and the compounded system of KshB, KshC, and NADH was constructed. The optimum ratio of KSH oxygenase to reductase content was 1.5:1. An NADH regeneration system was designed by introducing a formate dehydrogenase, further confirming that a more economical process for biological transformation from AD to 9-OH-AD was established. A total of 7.78 g of 9-OH-AD per liter was achieved through a fed-batch process with a 92.11% conversion rate (mol/mol). This enzyme-mediated hydroxylation method provides an environmentally friendly and economical strategy for the production of 9-OH-AD.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production by Haloarchaeon Halogranum amylolyticum.

Haloarchaea is an important group of polyhydroxyalkanoate (PHA)-accumulating organisms. However, few promising haloarchaeal species for economical and efficient PHA production have been reported. Here, we first discovered that Halogranum amylolyticum TNN58 could efficiently accumulate poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with a high 3-hydroxyvalerate (3HV) fraction using glucose as carbon source. Briefly, transmission electron microscopy (TEM) analysis revealed the presence of a large number of PHA granules in the cells. Gas chromatography-mass spectrometry (GC-MS) and proton nuclear magnetic resonance ((1)H NMR) analyses showed that PHAs synthesized from glucose was PHBV. Moreover, the 3HV content reached 20.1 mol%, which is the highest 3HV fraction thus far reported, as for PHBV produced by the wild-type strains grown on unrelated carbon courses. Fermentation experiments suggested that nitrogen-limited MG medium was better than nutrient-rich NOMG and AS168 medium for PHBV production. Additionally, glucose was the most suitable carbon source among the tested carbon sources. Interestingly, PHBV accumulation was almost paralleled by cell growth and glucose consumption. By applying the fed-batch process in fermentor, the PHBV production and cell dry weight were increased by approximately eight and four times, respectively, as compared with those of the batch process in shaking flasks. The classical PHA synthase genes were successfully cloned via consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) and high-efficiency thermal asymmetric interlaced (hiTAIL) PCR methods. This finding suggested that H. amylolyticum shows promising potential in the low-cost biotechnological production of PHBV after further process optimization.