Zhiqiang Xiong

CRISPR-Cas9D10A Nickase-Assisted Genome Editing in Lactobacillus casei

ABSTRACT Lactobacillus casei has drawn increasing attention as a health-promoting probiotic, while effective genetic manipulation tools are often not available, e.g., the single-gene knockout in L. casei still depends on the classic homologous recombination-dependent double-crossover strategy, which is quite labor-intensive and time-consuming. In the present study, a rapid and precise genome editing plasmid, pLCNICK, was established for L. casei genome engineering based on CRISPR-Cas9D10A. In addition to the P23-Cas9D10A and Pldh-sgRNA (single guide RNA) expression cassettes, pLCNICK includes the homologous arms of the target gene as repair templates. The ability and efficiency of chromosomal engineering using pLCNICK were evaluated by in-frame deletions of four independent genes and chromosomal insertion of an enhanced green fluorescent protein (eGFP) expression cassette at the LC2W_1628 locus. The efficiencies associated with in-frame deletions and chromosomal insertion is 25 to 62%. pLCNICK has been proved to be an effective, rapid, and precise tool for genome editing in L. casei, and its potential application in other lactic acid bacteria (LAB) is also discussed in this study. IMPORTANCE The lack of efficient genetic tools has limited the investigation and biotechnological application of many LAB. The CRISPR-Cas9D10A nickase-based genome editing in Lactobacillus casei, an important food industrial microorganism, was demonstrated in this study. This genetic tool allows efficient single-gene deletion and insertion to be accomplished by one-step transformation, and the cycle time is reduced to 9 days. It facilitates a rapid and precise chromosomal manipulation in L. casei and overcomes some limitations of previous methods. This editing system can serve as a basic technological platform and offers the possibility to start a comprehensive investigation on L. casei. As a broad-host-range plasmid, pLCNICK has the potential to be adapted to other Lactobacillus species for genome editing.

Short communication: Improving the activity of bile salt hydrolases in Lactobacillus casei based on in silico molecular docking and heterologous expression

Bile salt hydrolase (BSH) plays an essential role in the cholesterol-removing effect of lactic acid bacteria, which hydrolyze conjugated bile salts to amino acid and deconjugated bile salts. However, Lactobacillus casei lacks the bsh gene, which may make it highly sensitive to bile salt stress. We wanted to improve the BSH activity of L. casei for various food-industry applications (e.g., milk fermentation). Plate assay testing indicated that Lactobacillus plantarum AR113 has the highest BSH activity. We cloned and sequenced 4 bsh genes from the genome of L. plantarum AR113. Structure modeling and molecular docking of BSH indicated that BSH1 and BSH3 could react efficiently with bile salts, so we selected BSH1 and BSH3 for heterologous expression in L. casei. Compared with single expression of BSH1 or BSH3, co-expression of both protein sequences showed the highest hydrolysis activity by HPLC analysis. Our results suggested that heterologous expression of BSH in L. casei can significantly improve host activity against bile salts, and in silico molecular docking could be an efficient method of rapid screening for BSH with high activity.

Structural characterization and rheological properties of β-D-glucan from hull-less barley (Hordeum vulgare L. var. nudum Hook. f.)

A high purity of β-D-glucan (80.8%) from hull-less barley (Hordeum vulgare L. var. nudum Hook. f.) (HBBG) was isolated by alkali extraction and multi-precipitation with ethanol. The molecular weight (Mw) of HBBG was determined as 571.4u202fkDa with a broad distribution (Mw/Mnu202f=u202f1.6) by using HPSEC. According to methylation and GC-MS analysis, HBBG was identified to be composed of (1u202f→u202f4)- and (1u202f→u202f3)-glucopyranosyl (Glcp) residues with a ratio of (3.19u202f±u202f0.01). The MALDI-TOF MS and NMR spectroscopy were further conducted to analyze the enzyme hydrolysate released by lichenase digestion on HBBG. The results suggested that HBBG possessed a typical chemical structure of cereal β-D-glucans, namely linear homopolysaccharides formed by β-D-Glcp units via (1u202f→u202f4)-linkages and occasionally single (1u202f→u202f3)-linkage. The trisaccharide and tetrasaccharide of HBBG accounted for 66.6% of total cellulosyl units, accompanying with a ratio of cellotriosyl to cellotetraosyl unitsu202f=u202f1.0, which were significant different from those reported for the other cereal β-glucans. Rheological property analysis revealed that HBBG showed a shear-thinning behavior and thermal resilience during heating-cooling process.

Enhancement of antroquinonol production during batch fermentation using pH control coupled with an oxygen vector: Enhancing production of antroquinonol by pH control and oxygen vector

BACKGROUNDnAntroquinonol, a ubiquinone derivative that shows anticancer and anti-inflammatory activities, is produced during solid-state fermentation of Antrodia camphorata; however, it cannot be biosynthesized via conventional submerged fermentation.nnnRESULTSnA method for enhancing the biosynthesis of antroquinonol by controlling pH and adding an oxygen vector in a 7u2009L bioreactor was studied. In shake-flask experiments, a maximum antroquinonol production of 31.39xa0±xa00.78u2009mgu2009L-1 was obtained by fermentation with adding 0.2u2009gu2009L-1 coenzyme Q0 (CoQ0 ), at the 96th hour. Following kinetic analysis of the fermentation process, pH control strategies were investigated. A maximum antroquinonol production of 86.47xa0±xa03.65u2009mgu2009L-1 was achieved when the pH was maintained at 5.0, which exhibited an increase of 348.03% higher than the batch without pH regulation (19.30xa0±xa00.88u2009mgu2009L-1 ). The conversion rate of CoQ0 improved from 1.51% to 20.20%. Further research revealed that the addition of n-tetradecane could increase the production of antroquinonol to 115.62xa0±xa04.87u2009mgu2009L-1 by increasing the dissolved oxygen in the fermentation broth.nnnCONCLUSIONnThe results demonstrated that pH played an important role in antroquinonol synthesis in the presence of the effective precursor CoQ0 . It was a very effective strategy to increase the yield of antroquinonol by controlling pH and adding oxygen vector.

Lactobacillus plantarum AR501 Alleviates the Oxidative Stress of D-Galactose-Induced Aging Mice Liver by Upregulation of Nrf2-Mediated Antioxidant Enzyme Expression: LAB alleviates oxidative stress …

Lactic acid bacteria (LAB) have been used as ingredients of functional foods to promote health and prevent diseases because of their beneficial effects. This study aimed to investigate the antioxidative effects of LAB on the hepatotoxicity in D-galactose-induced aging mice. LAB were isolated from the traditional Chinese fermented foods and screened by the tolerance of hydrogen peroxide (H2 O2 ). Male ICR (Institute of Cancer Research) mice were subcutaneously injected with D-galactose for 5 weeks and then gastric gavage with LAB for 6 weeks. The results showed that Lactobacillus plantarum AR113 and AR501, and Pediococcu pentosaceus AR243 could tolerate up to 1.5 mM H2 O2 inxa0vitro, and they could live through simulated gastrointestinal tract (GIT) to colonizing the GIT of host. Inxa0vivo, oral administration of L. plantarum AR113 and AR501 improved the antioxidant status of D-galactose-induced oxidative stress mice such as alleviated liver damages and reduced abnormal activities of superoxide dismutase, glutathione peroxidase, and catalase to normal levels. In addition, L. plantarum AR501 markedly elevated the gene expression of nuclear factor erythroid-2-related factor 2 and upregulated the expressions of several antioxidant genes such as glutathione reductase, glutathione S-transferase, glutamate-cysteine ligase catalytic subunit, glutamate-cysteine ligase modifier subunit, and NAD(P)H quinone oxidoreductase 1 in the liver of an aging mice. Therefore, L. plantarum AR501 could be a good candidate for producing antiaging functional foods.

Characterization of a cryptic plasmid isolated from Lactobacillus casei CP002616 and construction of shuttle vectors based on its replicon.

The cryptic plasmid pLC2W was isolated from Lactobacillus casei CP002616. Nucleotide sequence analysis revealed that 4 putative open reading frames (ORF) were responsible for DNA replication. Four Escherichia coli-Lactobacillus shuttle vectors were constructed using different lengths of the pLC2W replicon to identify the shortest functional replicon. The length of the pLC2W replicon did not affect the stability of the plasmids. Green fluorescent protein (GFP) as a reporter was expressed successfully in several lactobacilli using our constructed vectors. The results suggested that the expression vectors pUE-F0GFP and pUE-F1GFP are potential molecular tools for heterologous gene cloning and expression in lactobacilli. Moreover, 2 plasmid-curing methods were used to eliminate pLC2W from L. casei. We detected no difference between L. casei CP002616 and L. casei CP002616 pLC2WΔ-IC (mutant strain cured by plasmid incompatibility method) in production of exopolysaccharide (EPS) or acid. However, EPS and acid production were both reduced in L. casei CP002616 pLC2WΔ-HT (mutant strain cured by high-temperature heat treatment method), demonstrating a difference between these 2 curing methods. Sequence analysis of pLC2W and plasmid curing data suggest that plasmid pLC2W is not involved in EPS synthesis.

Functional analysis and heterologous expression of bifunctional glutathione synthetase from Lactobacillus

Bifunctional glutathione synthetase (GshF) has recently been reported to simultaneously catalyze the 2-step ATP-dependent biosynthesis of reduced glutathione (GSH). In this work, 19 putative gshF were mined from the complete sequenced genome of 20 representative Lactobacillus species. To functionally analyze these putative GshF, GshF from Lactobacillus plantarum and Lactobacillus casei were selected and successfully expressed in Escherichia coli. Compared with the control without expressing GshF, GSH titers were enhanced significantly in E. coli with overexpression of GshF, demonstrating that putative GshF from Lactobacillus have functional activities on GSH biosynthesis. Moreover, with the expression of GshF from L. plantarum in E. coli as a paradigm, GSH yield (286.5 μM) was strongly improved by 177.9% with optimized induced conditions and precursor concentration compared with the control under unoptimized conditions. Transcriptional analysis showed that key genes of endogenous GSH metabolism and precursor biosynthesis were remarkably suppressed by GshF expression, indicating that the increase of GSH titer was attributed to heterologous expression of GshF. Overall, our results suggested that gshF is enriched in Lactobacillus and that heterologous expression of GshF is an efficient strategy for improving GSH biosynthesis.

Carrageenan polysaccharides and oligosaccharides with distinct immunomodulatory activities in murine microglia BV-2 cells

This study was to investigate the anti-inflammatory activities in vitro of various carrageenans (Car) fractions (κ-, ι-, and λ-types) with well characterized molecular properties, using murine microglia BV-2 cell line treated with lipopolysaccharide (LPS) as model. It is indicated that pretreatments with the oligosaccharide fractions from κ- or ι-carrageenan acid hydrolysates (κ- and ι-CarAOS, respectively) at 125-500u202fμg/mL significantly and dose-dependently decreased the levels of tumor necrosis factor α (TNF-α) secreted from LPS-treated BV-2 cells, showing promisingly anti-inflammatory effects. Differently, pretreatments of most of polymeric carrageenans at 250-500u202fμg/mL significantly increased the TNF-α level, implying the co-inflammatory effects with LPS. The co-inflammatory effectiveness of pure carrageenans at 125u202fμg/mL was notable for λ-Car, followed by ι-Car, and insignificantly for κ-Car. Generally, cytokine TNF-α was a more sensitive biomarker to the presence of carrageenans than was the IL-6. The TNF-α level varied greatly at a low carrageenan concentration (125u202fμg/mL) and high polymer percentage (e.g. purified κ- and ι-Car). Conclusively, the anti-inflammatory effects on LPS-treated BV-2 cells could be attenuated by pretreatments with κ- and ι-CarAOS at 125-500u202fμg/mL.

Relationship Between Putative eps Genes and Production of Exopolysaccharide in Lactobacillus casei LC2W

Lactobacillus casei LC2W, a probiotic strain, can produce exopolysaccharide (EPS) with anti-hypertensive bioactivity. The relationship between eps genes and EPS synthesis in LC2W due to unclear regulation mechanism of EPS biosynthesis was investigated. The several relevant genes in EPS biosynthetic gene cluster were deleted, overexpressed and complemented. The results suggested that glucose-1-phosphate thymidyltranseferase gene (LC2W_2179), uncharacterized EPS biosynthesis protein (LC2W_2188), and EPS biosynthesis protein (LC2W_2189) were related to EPS biosynthesis. EPS titer decreased 15, 13, and 21% when the three genes were deleted, respectively. When they were overexpressed, EPS titer increased 16, 10, and 18%. When they were complemented, EPS titer was similar to the wild-type strain. This work showed the three eps genes from LC2W played important roles on EPS production.

Improvement of flavor profiles in Chinese rice wine by creating fermenting yeast with superior ethanol tolerance and fermentation activity

Producing alcoholic beverages with novel flavor are desirable for winemakers. We created fermenting yeast with superior ethanol tolerance and fermentation activity to improve the flavor profiles of Chinese rice wine. Strategies of ethanol domestication, ultraviolet mutagenesis (UV) and protoplast fusion were conducted to create yeast hybrids with excellent oenological characteristic. The obtained diploid hybrid F23 showed a cell viability of 6.2% under 25% ethanol, whereas its diploid parental strains could not survive under 20% ethanol. During Chinese rice wine-making, compared to diploid parents, F23 produced 7.07%-12.44% higher yield of ethanol. Flavor analysis indicated that the total content of flavor compounds in F23 wine was 19.99-26.55% higher than that of parent wines. Specifically, F23 exhibited higher capacity in producing 2-phenylethanol, short-chain and long-chain fatty-acid ethyl-ester than diploid parents. Compared to diploid parents, F23 introduced more flavor contributors with odor activity values (OAVs) above one to Chinese rice wine, and those contributors were found with higher OAVs. Based on principal component analysis (PCA), the flavor characteristic of F23 wine was similar to each of parent wine. Additionally, sensory evaluation showed that F23 wine was highly assessed for its intensive levels in fruit-aroma, alcohol-aroma and mouthfeel. Hybrid F23 not only displayed superior flavor production and oenological performance in making Chinese rice wine, but also could act as potential mixed-like starter to enrich wine style and differentiation.