Mutation of Key Residues in β-Glycosidase LXYL-P1-2 for Improved Activity

The β-glycosidase LXYL-P1-2 identified from Lentinula edodes can be used to hydrolyze 7-β-xylosyl-10-deacetyltaxol (XDT) into 10-deacetyltaxol (DT) for the semi-synthesis of Taxol. Recent success in obtaining the high-resolution X-ray crystal of LXYL-P1-2 and resolving its three-dimensional structure has enabled us to perform molecular docking of LXYL-P1-2 with substrate XDT and investigate the roles of the three noncatalytic amino acid residues located around the active cavity in LXYL-P1-2. Site-directed mutagenesis results demonstrated that Tyr268 and Ser466 were essential for maintaining the β-glycosidase activity, and the L220G mutation exhibited a positive effect on increasing activity by enlarging the channel that facilitates the entrance of the substrate XDT into the active cavity. Moreover, introducing L220G mutation into the other LXYL-P1-2 mutant further increased the enzyme activity, and the β-d-xylosidase activity of the mutant EP2-L220G was nearly two times higher than that of LXYL-P1-2. Thus, the recombinant yeast GS115-EP2-L220G can be used for efficiently biocatalyzing XDT to DT for the semi-synthesis of Taxol. Our study provides not only the prospective candidate strain for industrial production, but also a theoretical basis for exploring the key amino acid residues in LXYL-P1-2.

Technological properties of beneficial bacteria from the dairy environment and development of a fermented milk with the beneficial strain Lactobacillus casei MRUV6

In this research paper we describe the technological properties of beneficial lactic acid bacteria (LAB) obtained from a dairy production chain and the development of a fermented milk produced with Lactobacillus casei MRUV6. Fifteen LAB isolates (Lactobacillus sp., Pediococcus sp. and Weissela sp.) presented acidifying abilities (pH ranges from 0.73 to 2.11), were able to produce diacetyl (except by 5 isolates) and exopolysaccharides, and two were proteolytic. L. casei MRUV6 was selected for producing a fermented milk, stored up to 35 d at 4 and 10°C. Counts on MRS agar with added vancomycin (10 mg/l) and MRS agar with added bile salts (1.5% w/v) ranged from 9.7 to 9.9 log CFU/g, independently of the tested conditions, indicating stability and intestinal resistance of L. casei MRUV6, despite some significant differences (P < 0.05). The study demonstrated the technological potential of a potential probiotic candidate strain, L. casei MRUV6, to be used as a starter culture in the dairy industry.

Live-attenuated Salmonella enterica serotype Choleraesuis vaccine with regulated delayed fur mutation confer protection against Streptococcus suis in mice

Background: Recombinant Salmonella enterica serotype Choleraesuis (S. Choleraesuis) vaccine vector could be used to deliver heterologous antigens to prevent and control pig diseases. We have previously shown that a live-attenuated S. Choleraesuis vaccine candidate strain rSC0011 (ΔPcrp527::TT araC PBAD crp Δpmi-2426 ΔrelA199::araC PBAD lacI TT ΔasdA33, Δ, deletion, TT, terminator) delivering SaoA, a conserved surface protein in most of S. suis serotypes, provided excellent protection against S. suis challenge, but occasionally lead to morbidity (enteritidis) in vaccinated mice (approximately 1 in every 10 mice). Thus, alternated attenuation method was sought to reduce the reactogenicity of strain rSC0011. Herein, we described another recombinant attenuated S. Choleraesuis vector, rSC0012 (ΔPfur88:: TT araC PBAD fur Δpmi-2426 ΔrelA199:: araC PBAD lacI TT ΔasdA33) with regulated delayed fur mutation to avoid inducing disease symptoms while exhibiting a high degree of immunogenicity. Results: The strain rSC0012 strain with the ΔPfur88::TT araC PBAD fur mutation induced less production of inflammatory cytokines than strain rSC0011 with the ΔPcrp527::TT araC PBAD crp mutation in mice. When delivering the same pS-SaoA plasmid, the intraperitoneal LD50 of rSC0012 was 18.2 times higher than that of rSC0011 in 3-week-old BALB/C mice. rSC0012 with either pS-SaoA or pYA3493 was cleared from spleen and liver tissues 7 days earlier than rSC0011 with same vectors after oral inoculation. The strain rSC0012 synthesizing SaoA induced high titers of anti-SaoA antibodies in both systemic (IgG in serum) and mucosal (IgA in vaginal washes) sites, as well as increased level of IL-4, the facilitator of Th2-type T cell immune response in mice. The recombinant vaccine rSC0012(pS-SaoA) conferred high percentage of protection against S. suis or S. Choleraesuis challenge in BALB/C mice.Conclusions: The live-attenuated Salmonella enterica serotype Choleraesuis vaccine rSC0012(pS-SaoA) with regulated delayed fur mutation provides a foundation for the development of a safe and effective vaccine against S. Choleraesuis and S. suis.

Live-attenuated Salmonella enterica serotype Choleraesuis vaccine with regulated delayed fur mutation confer protection against Streptococcus suis in mice

Background: Recombinant Salmonella enterica serotype Choleraesuis ( S . Choleraesuis) vaccine vector could be used to deliver heterologous antigens to prevent and control pig diseases. We have previously shown that a live-attenuated S . Choleraesuis vaccine candidate strain rSC0011 (ΔP crp527 ::TT araC P BAD crp Δ pmi-2426 Δ relA199 :: araC P BAD lacI TT Δ asdA33 , Δ, deletion, TT, terminator) delivering SaoA, a conserved surface protein in most of S . suis serotypes, provided excellent protection against S. suis challenge, but occasionally lead to morbidity (enteritidis) in vaccinated mice (approximately 1 in every 10 mice). Thus, alternated attenuation method was sought to reduce the reactogenicity of strain rSC0011. Herein , we described another recombinant attenuated S. Choleraesuis vector, rSC0012 ( ΔP fur88 :: TT araC P BAD fur Δ pmi-2426 Δ relA199 :: araC P BAD lacI TT Δ asdA33 ) with regulated delayed fur mutation to avoid inducing disease symptoms while exhibiting a high degree of immunogenicity. Results: The strain rSC0012 strain with the ΔP fur88 ::TT araC P BAD fur mutation induced less production of inflammatory cytokines than strain rSC0011 with the ΔP crp527 ::TT araC P BAD crp mutation in mice. When delivering the same pS-SaoA plasmid, the intraperitoneal LD 50 of rSC0012 was 18.2 times higher than that of rSC0011 in 3-week-old BALB/C mice. rSC0012 with either pS-SaoA or pYA3493 was cleared from spleen and liver tissues 7 days earlier than rSC0011 with same vectors after oral inoculation. The strain rSC0012 synthesizing SaoA induced high titers of anti-SaoA antibodies in both systemic (IgG in serum) and mucosal (IgA in vaginal washes) sites, as well as increased level of IL-4, the facilitator of Th2-type T cell immune response in mice. The recombinant vaccine rSC0012(pS-SaoA) conferred high percentage of protection against S. suis or S . Choleraesuis challenge in BALB/C mice. Conclusions: The live-attenuated Salmonella enterica serotype Choleraesuis vaccine rSC0012(pS-SaoA) with regulated delayed fur mutation provides a foundation for the development of a safe and effective vaccine against S . Choleraesuis and S. suis . Keywords: Salmonella Choleraesuis, virulence, immunogenicity, Fur, inflammatory

Live-attenuated Salmonella enterica serotype Choleraesuis vaccine with regulated delayed fur mutation confer protection against Streptococcus suis in mice

Background: Recombinant Salmonella enterica serotype Choleraesuis ( S . Choleraesuis) vaccine vector could be used to deliver heterologous antigens to prevent and control pig diseases. We have previously shown that a live-attenuated S . Choleraesuis vaccine candidate strain rSC0011 (ΔP crp527 ::TT araC P BAD crp Δ pmi-2426 Δ relA199 :: araC P BAD lacI TT Δ asdA33 , Δ, deletion, TT, terminator) delivering SaoA, a conserved surface protein in most of S . suis serotypes, provided excellent protection against S. suis challenge, but occasionally lead to morbidity (enteritidis) in vaccinated mice (approximately 1 in every 10 mice). Thus, alternated attenuation method was sought to reduce the reactogenicity of strain rSC0011. Herein , we described another recombinant attenuated S. Choleraesuis vector, rSC0012 ( ΔP fur88 :: TT araC P BAD fur Δ pmi-2426 Δ relA199 :: araC P BAD lacI TT Δ asdA33 ) with regulated delayed fur mutation to avoid inducing disease symptoms while exhibiting a high degree of immunogenicity. Results: The strain rSC0012 strain with the ΔP fur88 ::TT araC P BAD fur mutation induced less production of inflammatory cytokines than strain rSC0011 with the ΔP crp527 ::TT araC P BAD crp mutation in mice. When delivering the same pS-SaoA plasmid, the intraperitoneal LD 50 of rSC0012 was 18.2 times higher than that of rSC0011 in 3-week-old BALB/C mice. rSC0012 with either pS-SaoA or pYA3493 was cleared from spleen and liver tissues 7 days earlier than rSC0011 with same vectors after oral inoculation. The strain rSC0012 synthesizing SaoA induced high titers of anti-SaoA antibodies in both systemic (IgG in serum) and mucosal (IgA in vaginal washes) sites, as well as increased level of IL-4, the facilitator of Th2-type T cell immune response in mice. The recombinant vaccine rSC0012(pS-SaoA) conferred high percentage of protection against S. suis or S . Choleraesuis challenge in BALB/C mice. Conclusions: The live-attenuated Salmonella enterica serotype Choleraesuis vaccine rSC0012(pS-SaoA) with regulated delayed fur mutation provides a foundation for the development of a safe and effective vaccine against S . Choleraesuis and S. suis .

Influence of a cholesterol-lowering strain Lactobacillus plantarum LP3 isolated from traditional fermented yak milk on gut bacterial microbiota and metabolome of rats fed with a high-fat diet

L. plantarum LP3 isolated from traditinal fermented Tibetan yak milk has been identified as a potential probiotic candidate strain with high cholesterol-lowering activity.

Engineered Thermoanaerobacterium aotearoense with nfnAB knockout for improved hydrogen production from lignocellulose hydrolysates

Background As a renewable and clean energy carrier, the production of biohydrogen from low-value feedstock such as lignocellulose has increasingly garnered interest. The NADH-dependent reduced ferredoxin:NADP+ oxidoreductase (NfnAB) complex catalyzes electron transfer between reduced ferredoxin and NAD(P)+, which is critical for production of NAD(P)H-dependent products such as hydrogen and ethanol. In this study, the effects on end-product formation of deletion of nfnAB from Thermoanaerobacterium aotearoense SCUT27 were investigated. Results Compared with the parental strain, the NADH/NAD+ ratio in the ∆nfnAB mutant was increased. The concentration of hydrogen and ethanol produced increased by (41.1 ± 2.37)% (p < 0.01) and (13.24 ± 1.12)% (p < 0.01), respectively, while the lactic acid concentration decreased by (11.88 ± 0.96)% (p < 0.01) when the ∆nfnAB mutant used glucose as sole carbon source. No obvious inhibition effect was observed for either SCUT27 or SCUT27/∆nfnAB when six types of lignocellulose hydrolysate pretreated with dilute acid were used for hydrogen production. Notably, the SCUT27/∆nfnAB mutant produced 190.63–209.31 mmol/L hydrogen, with a yield of 1.66–1.77 mol/mol and productivity of 12.71–13.95 mmol/L h from nonsterilized rice straw and corn cob hydrolysates pretreated with dilute acid. Conclusions The T. aotearoense SCUT27/∆nfnAB mutant showed higher hydrogen yield and productivity compared with those of the parental strain. Hence, we demonstrate that deletion of nfnAB from T. aotearoense SCUT27 is an effective approach to improve hydrogen production by redirecting the electron flux, and SCUT27/∆nfnAB is a promising candidate strain for efficient biohydrogen production from lignocellulosic hydrolysates.

Live-attenuated Salmonella enterica serotype Choleraesuis vaccine with regulated delayed fur mutation confer protection against Streptococcus suis in mice

Background: Recombinant Salmonella enterica serotype Choleraesuis (S. Choleraesuis) vaccine vector could be used to deliver heterologous antigens to prevent and control pig diseases. We have previously shown that a live-attenuated S. Choleraesuis vaccine candidate strain rSC0011 (ΔPcrp527::TT araC PBAD crp Δpmi-2426 ΔrelA199::araC PBAD lacI TT ΔasdA33, Δ,deletion, TT, terminator) delivering SaoA, a conserved surface protein that is present in many S. suis serotypes, provided excellent protection against S. suis challenge, but occasionally lead to morbidity (enteritidis) in vaccinated mice (approximately 1 in every 10 mice). Thus, alternated attenuation method was sought to reduce the reactogenicity of strain rSC0011. Herein, we described another recombinant attenuated S. Choleraesuis vector, rSC0012 (ΔPfur88:: TT araC PBAD fur Δpmi-2426 ΔrelA199:: araC PBAD lacI TT ΔasdA33) with regulated delayed fur mutation to avoid inducing disease symptoms while exhibiting a high degree of immunogenicity. Results:The strain rSC0012 strain with the ΔPfur88::TT araC PBAD fur mutation induced less production of inflammatory cytokines than strain rSC0011 with the ΔPcrp527::TT araC PBAD crp mutation in mice. When delivering the same pS-SaoA plasmid, the intraperitoneal LD50 of rSC0012 was 18.2 times higher than that of rSC0011 in 3-week-old BALB/C mice. rSC0012 with either pS-SaoA or pYA3493 was cleared from spleen and liver tissues 7 days earlier than rSC0011 with same vectors after oral inoculation. The strain rSC0012 synthesizing SaoA induced high titers of anti-SaoA antibodies in both systemic (IgG in serum) and mucosal (IgA in vaginal washes) sites, as well as increased level of IL-4, the facilitator of Th2-type T cell immune response in mice. The recombinant vaccine rSC0012(pS-SaoA) conferred high percentage of protection against S. suis or S. Choleraesuis challenge in BALB/C mice. Conclusions: The live-attenuated Salmonella enterica serotype Choleraesuis vaccine rSC0012(pS-SaoA) with regulated delayed fur mutation provides a foundation for the development of a safe and effective vaccine against S. Choleraesuis and S. suis. Keywords: Salmonella Choleraesuis, virulence, immunogenicity, Fur, inflammatory

Selection of Tempoyak Lactic Acid Bacteria As Candidate Strain for Yoghurt Starter Culture

Selection of bacteria in yoghurt fermentation is important to produce yoghurt with good quality. Tempoyak lactic acid bacteria is potential to be yoghurt starter culture becouse tempoyak fermentation has similarities in producing lactic acid such as yoghurt. This study aimed to isolate and identify the lactic acid bacteria (LAB) from tempoyak which will be used as a yoghurt starter culture. The methods used in this study included isolation and selection of acid-producing bacteria, lactase and protease activity test, identification of morphology and biochemistry as well as testing the quality of the yoghurt. The results of the study obtained 32 isolates of the LAB with the same characteristic colony, include the round shape, cream-coloured with convex elevation and, smooth surface and entire edge. Selection of acid-producing bacteria obtained 12 isolates with the ability to produce clear zones on MRSA + CaCO3 media ≥ 0.7 cm. Selection of lactase-producing LAB obtained six strains and the protease test obtained two superior strains. Two superior strains namely Tp 12 and Tp 28 have characteristics of coccus, gram-positive, negative catalase, non-endospore and non-motile forms. The organoleptic and several quality tests showed yoghurt using Tp 12 as starter has higher acceptability, the highest levels of lactic acid and lactose levels with values respectively 4.25, 0.84% and 24.53%. This study obtained the LAB strain which can be used as yoghurt starter culture. Tp 12 strain can be used to improve the quality of yoghurt and become a commercial starter that can be applied to various fermented products.