Hybrid large hepatitis B surface protein composed of two viral genotypes C and D induces strongly cross-neutralizing antibodies.

Hepatitis B virus (HBV) vaccination is known to effectively decrease the risk of HBV infection. However, several issues need to be addressed in order to develop an improved HBV vaccine. Although the HBV vaccine has been shown to be effective, this vaccine needs to be more efficacious in defined groups, including non-responders (i.e., individuals who do not develop a protective response even after vaccination) and in health care workers and travelers who require rapid protection. Furthermore, it has been reported that universal HBV vaccination has accelerated the appearance of vaccine-escape mutants resulting from the accumulation of mutations altering the "a" determinant of the hepatitis B surface (HBs) protein. To address these problems, we have been focusing on the large HBs (LHBs) protein, which consists of three domains: pre-S1, pre-S2, and S (in N- to C-terminal order). To enhance the immunogenicity of LHBs, we developed a yeast-derived hybrid LHBs (hy-LHBs) antigen composed of the LHBs proteins from two distinct genotypes (Genotypes C and D). The levels of antibodies induced by hy-LHBs immunization were high not only against S, but also against the pre-S1 and pre-S2 domains. Additionally, hy-LHBs immunization induced significantly more strongly cross-reactive neutralizing antibodies than did small HBs (SHBs) or LHBs of any genotype alone. These findings suggested that hy-LHBs might serve as a candidate antigen for use in an improved prophylactic HBV vaccine.

Gene-specific amplicons from metagenomes as an alternative to directed evolution for enzyme screening: a case study using phenylacetaldehyde reductases.

Screening gene-specific amplicons from metagenomes (S-GAM) is a highly promising technique for the isolation of genes encoding enzymes for biochemical and industrial applications. From metagenomes, we isolated phenylacetaldehyde reductase (par) genes, which code for an enzyme that catalyzes the production of various Prelog's chiral alcohols. Nearly full-length par genes were amplified by PCR from metagenomic DNA, the products of which were fused with engineered par sequences at both terminal regions of the expression vector to ensure proper expression and then used to construct Escherichia coli plasmid libraries. Sequence- and activity-based screening of these libraries identified different homologous par genes, Hpar-001 to -036, which shared more than 97% amino acid sequence identity with PAR. Comparative characterization of these active homologs revealed a wide variety of enzymatic properties including activity, substrate specificity, and thermal stability. Moreover, amino acid substitutions in these genes coincided with those of Sar268 and Har1 genes, which were independently engineered by error-prone PCR to exhibit increased activity in the presence of concentrated 2-propanol. The comparative data from both approaches suggest that sequence information from homologs isolated from metagenomes is quite useful for enzyme engineering. Furthermore, by examining the GAM-based sequence dataset derived from soil metagenomes, we easily found amino acid substitutions that increase the thermal stability of PAR/PAR homologs. Thus, GAM-based approaches can provide not only useful homologous enzymes but also an alternative to directed evolution methodologies.

PCR-based amplification and heterologous expression of Pseudomonas alcohol dehydrogenase genes from the soil metagenome for biocatalysis.

The amplification of useful genes from metagenomes offers great biotechnological potential. We employed this approach to isolate alcohol dehydrogenase (adh) genes from Pseudomonas to aid in the synthesis of optically pure alcohols from various ketones. A PCR primer combination synthesized by reference to the adh sequences of known Pseudomonas genes was used to amplify full-length adh genes directly from 17 samples of DNA extracted from soil. Three such adh preparations were used to construct Escherichia coli plasmid libraries. Of the approximately 2800 colonies obtained, 240 putative adh-positive clones were identified by colony-PCR. Next, 23 functional adh genes named using the descriptors HBadh and HPadh were analyzed. The adh genes obtained via this metagenomic approach varied in their DNA and amino acid sequences. Expression of the gene products in E. coli indicated varying substrate specificity. Two representative genes, HBadh-1 and HPadh-24, expressed in E. coli and Pseudomonas putida, respectively, were purified and characterized in detail. The enzyme products of these genes were confirmed to be useful for producing anti-Prelog chiral alcohols.

Gene cloning and characterization of two NADH-dependent 3-quinuclidinone reductases from Microbacterium luteolum JCM 9174.

We used the resting-cell reaction to screen approximately 200 microorganisms for biocatalysts which reduce 3-quinuclidinone to optically pure (R)-(-)-3-quinuclidinol. Microbacterium luteolum JCM 9174 was selected as the most suitable organism. The genes encoding the protein products that reduced 3-quinuclidinone were isolated from M. luteolum JCM 9174. The bacC gene, which consists of 768 nucleotides corresponding to 255 amino acid residues and is a constituent of the bacilysin synthetic gene cluster, was amplified by PCR based on homology to known genes. The qnr gene consisted of 759 nucleotides corresponding to 252 amino acid residues. Both enzymes belong to the short-chain alcohol dehydrogenase/reductase (SDR) family. The genes were expressed in Escherichia coli as proteins which were His tagged at the N terminus, and the recombinant enzymes were purified and characterized. Both enzymes showed narrow substrate specificity and high stereoselectivity for the reduction of 3-quinuclidinone to (R)-(-)-3-quinuclidinol.

Production of (R)-3-quinuclidinol by E. coli biocatalysts possessing NADH-dependent 3-quinuclidinone reductase (QNR or bacC) from Microbacterium luteolum and Leifsonia alcohol dehydrogenase (LSADH).

We found two NADH-dependent reductases (QNR and bacC) in Microbacterium luteolum JCM 9174 (M. luteolum JCM 9174) that can reduce 3-quinuclidinone to optically pure (R)-(-)-3-quinuclidinol. Alcohol dehydrogenase from Leifsonia sp. (LSADH) was combined with these reductases to regenerate NAD+ to NADH in situ in the presence of 2-propanol as a hydrogen donor. The reductase and LSADH genes were efficiently expressed in E. coli cells. A number of constructed E. coli biocatalysts (intact or immobilized) were applied to the resting cell reaction and optimized. Under the optimized conditions, (R)-(-)-3-quinuclidinol was synthesized from 3-quinuclidinone (15% w/v, 939 mM) giving a conversion yield of 100% for immobilized QNR. The optical purity of the (R)-(-)-3-quinuclidinol produced by the enzymatic reactions was >99.9%. Thus, E. coli biocatalysis should be useful for the practical production of the pharmaceutically important intermediate, (R)-(-)-3-quinuclidinol.

Efficient synthesis of optically pure alcohols by asymmetric hydrogen-transfer biocatalysis: application of engineered enzymes in a 2-propanol-water medium.

We describe an efficient method for producing both enantiomers of chiral alcohols by asymmetric hydrogen-transfer bioreduction of ketones in a 2-propanol (IPA)-water medium with E. coli biocatalysts expressing phenylacetaldehyde reductase (PAR: wild-type and mutant enzymes) from Rhodococcus sp. ST-10 and alcohol dehydrogenase from Leifsonia sp. S749 (LSADH). We also describe the detailed properties of mutant PARs, Sar268, and HAR1, which were engineered to have high activity and productivity in media composed of polar organic solvent and water, and the construction of three-dimensional structure of PAR by homology modeling. The K(m) and V(max) values for some substrates and the substrate specificity of mutant PARs were quite different from those of wild-type PAR. The results well explained the increased productivity of engineered PARs in IPA-water medium.