Chunzhi Zhang

T7 RNA polymerase (T7RNAP) has been fused with cytosine or adenine deaminase individually, enabling in vivo C-to-T or A-to-G transitions on DNA sequence downstream of T7 promoter, and greatly accelerated directed protein evolution. However, its base conversion type is limited. In this study, we created a dual-functional system for simultaneous C-to-T and A-to-G in vivo mutagenesis, called T7-DualMuta, by fusing T7RNAP with both cytidine deaminase (PmCDA1) and a highly active adenine deaminase (TadA-8e). The C-to-T and A-to-G mutagenesis frequencies of T7-DualMuta were 4.02 × 10 ⁻³ and 1.20 × 10 ⁻² , respectively, with 24 h culturing and distributed mutations evenly across the target gene. The T7-DualMuta system was used to in vivo directed evolution of L-homoserine transporter RhtA, resulting in efficient variants that carried the four types of base conversions by T7-DualMuta. The evolved variants greatly increased the host growth rates at L-homoserine concentrations of 8 g/L, which was not previously achieved, and demonstrated the great in vivo evolution capacity. The novel molecular device T7-DualMuta efficiently provides both C/G-to-T/A and A/T-to-G/C mutagenesis on target regions, making it useful for various applications and research in Enzymology and Synthetic Biology studies. It also represents an important expansion of the base editing toolbox. Importance: A T7-DualMuta system for simultaneous C-to-T and A-to-G in vivo mutagenesis was created. The mutagenesis frequency was 4.02 × 10 ⁷ fold higher than the spontaneous mutation, which was reported to be approximately 10 ⁻¹⁰ bases per nucleotide per generation. This mutant system can be utilized for various applications and research in Enzymology and Synthetic Biology studies.

Background: Natural life systems can be significantly modified at the genomic scale by human intervention, demonstrating the great innovation capacity of genome engineering. Large epi-chromosomal DNA structures were established in Escherichia coli cells, but some of these methods were inconvenient, using heterologous systems, or relied on engineered E. coli strains. Results: The wild-type model bacterium E. coli has a single circular chromosome. In this work, a novel method was developed to split the original chromosome of wild-type E. coli. With this method, novel E. coli strains containing two chromosomes of 0.10 Mb and 4.54 Mb, and 2.28 Mb and 2.36 Mb were created respectively, designated as E. coli^0.10/4.54 and E. coli^2.28/2.36. The new chromosomal arrangement was proved by PCR amplification of joint regions as well as a combination of Nanopore and Illumina sequencing analysis. While E. coli^0.10/4.54 was quite stable, the two chromosomes of E. coli^2.28/2.36 population recombined into a new chromosome (Chr.4.64M^Mut), via recombination. Both engineered strains grew slightly slower than the wild-type, and their cell shapes were obviously elongated. Conclusion: Finally, we successfully developed a simple CRISPR-based genome engineering technique for the construction of multi-chromosomal E. coli strains with no heterologous genetic parts. This technique might be applied to other prokaryotes for synthetic biology studies and applications in the future.

The objective of our present work was to explore the possible enhanced anti-neuroinflammatory ability of Aspergillus oryzae fermented hemp seed in lipopolysaccharide (LPS)-stimulated N9 microglial cells and elucidate its underlying mechanism. The water extract of hemp seed was fermented by Aspergillus oryzae. LPS-stimulated N9 microglial cells were employed for the inflammatory cell model. The release of nitric oxide (NO) was determined by Griess assay. The cytokines and inflammatory mediator expression were measured by qPCR and ELISA. The phosphorylated key signaling proteins, including nuclear factor-κB (NF-κB), mitogen-activated protein kinases (MAPKs), and phosphatidylinositol 3-kinase (PI3K/Akt), were quantified by western blot analysis. The production of intracellular reactive oxygen species (ROS) was measured by DCFH oxidation. Fermented hemp seed (FHS) reduced NO production by downregulating inducible nitric oxide synthase (iNOS) expression in LPS-stimulated N9 microglial cells. FHS treatment decreased LPS-stimulated expression of inflammatory cytokines either on mRNA or protein levels. Moreover, FHS inhibited LPS-stimulated phosphorylation of NF-κB, MAPKs, and PI3K/Akt signaling pathways. Furthermore, FHS significantly reduced the ROS production in the cells. It was concluded that FHS exerted its anti-neuroinflammatory activities by suppressing ROS production, thus inhibiting NF-κB, MAPKs, and PI3K/Akt activation, consequently decreasing the expression levels of inflammatory mediators and cytokines.

Ribulose 5-phosphate (Ru5P) and guanosine 5′-triphosphate (GTP) are two key precursors of riboflavin, whereby Ru5P is also a precursor of GTP. Ribulose 5-phosphate 3-epimerase (Rpe) catalyzes the conversion of ribulose 5-phosphate into xylulose 5-phosphate. Inactivation of Rpe can reduce the consumption of Ru5P, enhancing the carbon flux toward riboflavin biosynthesis. Here we investigated the effect of mutation of rpe and other related genes on riboflavin production, physiological and metabolic phenotypes in Bacillus subtilis LY (BSLY). Introducing single nucleotide deletion (generated BSR) or nonsense mutation (generated BSRN) on the genomic copy of rpe, resulting in more than fivefold increase of riboflavin production over the parental strain. BSR process 62% Rpe activity, while BSRN lost the entire Rpe activity and had a growth defect compared with the parent strain. BSR and BSRN exhibited increases of the inosine and guanine titers, in addition, BSRN exhibited an increase of inosine 5′-monophosphate titer in fermentation. The transcription levels of most oxidative pentose phosphate pathway and purine synthesis genes were unchanged in BSR, except for the levels of zwf and ndk, which were higher than in BSLY. The production of riboflavin was increased to 479.90 ± 33.21 mg/L when ribA was overexpressed in BSR. The overexpression of zwf, gntZ, prs, and purF also enhanced the riboflavin production. Finally, overexpression of the rib operon by the pMX45 plasmid and mutant gnd by pHP03 plasmid in BSR led to a 3.05-fold increase of the riboflavin production (977.29 ± 63.44 mg/L), showing the potential for further engineering of this strain.

Background: Ribulose 5-phosphate (Ru5P) and guanosine 5'-triphosphate (GTP) are two key precursors of riboflavin, whereby Ru5P is also a precursor of GTP (purine de novo synthesis pathway). As a product of the oxidative pentose phosphate pathway (OPPP), Ru5P can flow back into the Embden-Meyerhof-Parnas pathway (EMP) through the non-oxidative pentose phosphate pathway (NOPPP). Major enzymes of the NOPPP include transaldolase (Tal), transketolase (Tkt), and ribulose 5-phosphate 3-epimerase (Rpe), which catalyze the conversion of Ru5P into xylulose 5-phosphate (X5P). Thus, inactivation of Rpe can reduce the consumption of Ru5P, enhancing the carbon flux toward riboflavin biosynthesis. However, there are no studies on the mutation or deletion of Rpe for improving riboflavin production. Results: We mutated the genomic copy of rpe, in the riboflavin-producing Bacillus subtilis BSLY. The resulting strain BSR produced 320.50±31.28 mg/L of riboflavin in a fermentation medium containing 40 g/L sucrose as the carbon source, representing a 5-fold increase over the parental strain. We also constructed an rpe nonsense mutation strain BSRN, which produced 365.69±27.53 mg/L riboflavin in fermentation medium. However, BSRN had a growth defect compared with BSR and BSLY, which was also present in media containing glucose, maltose, and fructose. The growth of BSRN was restored when mutant or wild-type rpe was overexpressed. Notably, BSR exhibited respective increases of the inosine and guanine titers by 163% and 40%. Furthermore, a 35.96-fold increase of inosine 5’-monophosphate (IMP) titer was detected in BSRN. The transcription levels of most OPPP, purine, and GTP synthesis genes were unchanged in BSR, except for the levels of zwf and ndk, which were respectively 49% lower and 310% higher than in BSLY. The production of riboflavin was increased to 479.90±33.21 mg/L when ribA was expressed in BSR. The expression of zwf, gntZ, prs, and purF, respectively enhanced the riboflavin production by 48%, 31%, 26%, and 35%. Finally, overexpression of the rib operon by the pMX45 plasmid and mutant gnd by pHP03 plasmid in BSR led to a 3.05-fold increase of the riboflavin production (977.29±63.44 mg/L).Conclusions: This paper describes a mutation of the ribulose 5-phosphate 3-epimerase and its influence on riboflavin production. The results of qRT-PCR and HPLC analyses indicated that the rpe mutant showed a different pattern of purine metabolism, while the cells maintained generally normal levels of the transcription of genes related to PPP and purine de novel synthesis pathways. With the increased expression of genes in the OPPP, purine, and riboflavin synthesis pathways, the production of riboflavin was effectively enhanced, showing the potential for further engineering of this strain.

Fifteen unreported compounds in Anemarrhena asphodeloides, iriflophene (3), hostaplantagineoside C (7), tuberoside G (8), spicatoside B (9), platycodin D (14), platycoside A (15), platycodin D2 (16), polygalacin D2 (17), platycodin D3 (18), isovitexin (20), vitexin (21), 3,4-dihydroxyallylbenzene-3-O-α-l-rhamnopyranosyl(1→6)-β-d-glucopyranoside (22), iryptophan (24), adenosine (25), α-d-Glucose monoallyl ether (26), together with eleven known compounds (1, 2, 4–6, 10–13, 19 and 23), were isolated from the rhizomes of Anemarrhena asphodeloides. The chemical structures of these compounds were characterized using HRMS and NMR. The anti-inflammatory activities of the compounds were evaluated by investigating their ability to inhibit LPS-induced NO production in N9 microglial cells. Timosaponin BIII (TBIII) and trans-hinokiresinol (t-HL) exhibited significant inhibitory effects on the NO production in a dose-dependent manner with IC₅₀ values of 11.91 and 39.08 μM, respectively. Immunoblotting demonstrated that TBIII and t-HL suppressed NO production by inhibiting the expressions of iNOS in LPS-stimulated N9 microglial cells. Further results revealed that pretreatment of N9 microglial cells with TBIII and t-HL attenuated the LPS-induced expression tumor necrosis factor (TNF)-α and interleukin-6 (IL-6) at mRNAs and protein levels. Moreover, the activation of nuclear factor-κB (NF-κB) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways were inhibited by TBIII and t-HL, respectively. Our findings indicate that the therapeutic implication of TBIII and t-HL for neurogenerative disease associated with neuroinflammation.

L-arabinose isomerase (l-AI) (EC 5. 3. 1. 4. l-AI) that mediates the isomerization of d-galactose to d-tagatose was isolated from Lactobacillus brevis (MF 465792), and was further purified and characterized. Pure enzyme with molecular weight of 60.1 kDa was successfully obtained after the purification using Native-PAGE gel extraction method, which was a monomer in solution. The l-AI was found to be stable at 45–75 °C, and at pH 7.0–9.0. Its optimum temperature and pH was determined as 65 °C and 7.0, respectively. Besides, we found that Ca²⁺, Cu²⁺, and Ba²⁺ ions inhibited the enzyme activity, whereas the enzyme activity was significantly enhanced in the presence of Mg²⁺, Mn²⁺, or Co²⁺ ions. The optimum concentration of Mn²⁺ and Co²⁺ was determined to be 1 mM. Furthermore, we characterized the kinetic parameters for l-AI and determined the K_m (129 mM) and the V_max (0.045 mM min^− 1) values. Notably, L. brevis l-AI exhibited a high bioconversion yield of 43% from d-galactose to d-tagatose under the optimal condition, and appeared to be a more efficient catalyst compared with other l-AIs from various organisms.

In the present study, a new strain, Bacillus cereus ZY12, producing phospholipase D (PLD) was identified. The expression of PLD in this strain was found to be induced by its substrate, phosphatidylcholine (PC), and completely silenced by other carbon sources, such as glucose, fructose, and maltose, which are generally used in microbial growth cultures, thus presenting a unique expression pattern different from other PLD-producing microorganisms. This study is the first to report on the ability of B. cereus to produce PLD, and successfully clone its PLD-coding gene and identify its function, extending the knowledge on PLD distribution and evolution in microorganisms.