Xylose fermentation efficiency and inhibitor tolerance of the recombinant industrial Saccharomyces cerevisiae strain NAPX37.

Industrial yeast strains with good xylose fermentation ability and inhibitor tolerance are important for economical lignocellulosic bioethanol production. The flocculating industrial Saccharomyces cerevisiae strain NAPX37, harboring the xylose reductase-xylitol dehydrogenase (XR-XDH)-based xylose metabolic pathway, displayed efficient xylose fermentation during batch and continuous fermentation. During batch fermentation, the xylose consumption rates at the first 36 h were similar (1.37 g/L/h) when the initial xylose concentrations were 50 and 75 g/L, indicating that xylose fermentation was not inhibited even when the xylose concentration was as high as 75 g/L. The presence of glucose, at concentrations of up to 25 g/L, did not affect xylose consumption rate at the first 36 h. Strain NAPX37 showed stable xylose fermentation capacity during continuous ethanol fermentation using xylose as the sole sugar, for almost 1 year. Fermentation remained stable at a dilution rate of 0.05/h, even though the xylose concentration in the feed was as high as 100 g/L. Aeration rate, xylose concentration, and MgSO4 concentration were found to affect xylose consumption and ethanol yield. When the xylose concentration in the feed was 75 g/L, a high xylose consumption rate of 6.62 g/L/h and an ethanol yield of 0.394 were achieved under an aeration rate of 0.1 vvm, dilution rate of 0.1/h, and 5 mM MgSO4. In addition, strain NAPX37 exhibited good tolerance to inhibitors such as weak acids, furans, and phenolics during xylose fermentation. These findings indicate that strain NAPX37 is a promising candidate for application in the industrial production of lignocellulosic bioethanol.

Synergistic effects of TAL1 over-expression and PHO13 deletion on the weak acid inhibition of xylose fermentation by industrial Saccharomyces cerevisiae strain.

In the industrial production of bioethanol from lignocellulosic biomass, a strain of Saccharomyces cerevisiae that can ferment xylose in the presence of inhibitors is of utmost importance. The recombinant, industrial-flocculating S. cerevisiae strain NAPX37, which can ferment xylose, was used as the parent to delete the gene encoding p-nitrophenylphosphatase (PHO13) and overexpress the gene encoding transaldolase (TAL1) to evaluate the synergistic effects of these two genes on xylose fermentation in the presence of weak acid inhibitors, including formic, acetic, or levulinic acids. TAL1 over-expression or PHO13 deletion improved xylose fermentation as well as the tolerance of NAPX37 to all three weak acids. The simultaneous deletion of PHO13 and the over-expression of TAL1 had synergistic effects and improved ethanol production and reduction of xylitol accumulation in the absence and presence of weak acid inhibitors.

Improvement of ethanol production from D-lactic acid by constitutive expression of lactate transporter Jen1p in Saccharomyces cerevisiae.

To improve ethanol production from D-lactate, Jen1p, a monocarboxylate-proton symporter, was constitutively expressed in Saccharomyces cerevisiae NAM34-4C. The mutant produced 2.4 g/L of ethanol, approximately 2.4 times higher than that of the wild-type strain. A monocarboxylate/proton symporter gene (JEN1) null mutant was also constructed. It produced 0.19 g/L of ethanol, 5 times lower than that of the wild-type strain.

Model-based analysis on social acceptability and feasibility of a focused protection strategy against the COVID-19 pandemic.

This paper studies the social acceptability and feasibility of a focused protection strategy against coronavirus disease 2019 (COVID-19). We propose a control scheme to develop herd immunity while satisfying the following two basic requirements for a viable policy option. The first requirement is social acceptability: the overall deaths should be minimized for social acceptance. The second is feasibility: the healthcare system should not be overwhelmed to avoid various adverse effects. To exploit the fact that the disease severity increases considerably with age and comorbidities, we assume that some focused protection measures for those high-risk individuals are implemented and the disease does not spread within the high-risk population. Because the protected population has higher severity ratios than the unprotected population by definition, the protective measure can substantially reduce mortality in the whole population and also avoid the collapse of the healthcare system. Based on a simple susceptible-infected-recovered model, social acceptability and feasibility of the proposed strategy are summarized into two easily computable conditions. The proposed framework can be applied to various populations for studying the viability of herd immunity strategies against COVID-19. For Japan, herd immunity may be developed by the proposed scheme if [Formula: see text] and the severity rates of the disease are 1/10 times smaller than the previously reported value, although as high mortality as seasonal influenza is expected.

Analysis of metabolisms and transports of xylitol using xylose- and xylitol-assimilating Saccharomyces cerevisiae.

To clarify the relationship between NAD(P)+/NAD(P)H redox balances and the metabolisms of xylose or xylitol as carbon sources, we analyzed aerobic and anaerobic batch cultures of recombinant Saccharomyces cerevisiae in a complex medium containing 20 g/L xylose or 20 g/L xylitol at pH 5.0 and 30°C. The TDH3p-GAL2 or gal80Δ strain completely consumed the xylose within 24 h and aerobically consumed 92-100% of the xylitol within 96 h, but anaerobically consumed only 20% of the xylitol within 96 h. Cells of both strains grew well in aerobic culture. The addition of acetaldehyde (an effective oxidizer of NADH) increased the xylitol consumption by the anaerobically cultured strain. These results indicate that in anaerobic culture, NAD+ generated in the NAD(P)H-dependent xylose reductase reaction was likely needed in the NAD+-dependent xylitol dehydrogenase reaction, whereas in aerobic culture, the NAD+ generated by oxidation of NADH in the mitochondria is required in the xylitol dehydrogenase reaction. The role of Gal2 and Fps1 in importing xylitol into the cytosol and exporting it from the cells was analyzed by examining the xylitol consumption in aerobic culture and the export of xylitol metabolized from xylose in anaerobic culture, respectively. The xylitol consumptions of gal80Δ gal2Δ and gal80Δ gal2Δ fps1Δ strains were reduced by 81% and 88% respectively, relative to the gal80Δ strain. The maximum xylitol concentration accumulated by the gal80Δ, gal80Δ gal2Δ, and gal80Δ gal2Δ fps1Δ strains was 7.25 g/L, 5.30 g/L, and 4.27 g/L respectively, indicating that Gal2 and Fps1 transport xylitol both inward and outward.

Fate of transforming bacterial genome following incorporation into competent cells of Bacillus subtilis: a continuous length of incorporated DNA.

In contrast to the conventional transformation of Bacillus subtilis using purified DNA, those using DNA in lysed protoplasts have a high transformation efficiency and enable whole-genome transfer into competent B. subtilis [Akamatsu, T. and Taguchi, H., Biosci. Biotechnol. Biochem., 65, 823-829 (2001)]. Here, we examined the length of incorporated continuous DNA by analyzing the cotransfer ratio with selected and unselected markers, on the basis of a new experimental design. The cotransfer ratio of a selected marker with an unselected marker on the opposite side of the genetic map of the B. subtilis chromosome was about 5.6% and could be interpreted as congression (double transformation) ratio. In the wild-type strain, the cotransfer ratio of cysA (113 kb position on 4215 kb of B. subtilis chromosome) with metC (1384 kb) and leuB (2891 kb) was 0.77%, twice the value (5.6% x 5.6%=0.31%) calculated from the congression ratio. Moreover, in a genetic background, the cotransfer ratios of metC with cysA and leuB, and metC with cysA and arg1 (3012 kb) were 2.7% and 7.2%, respectively. These results strongly suggest that the length of continuous DNA incorporated into B. subtilis is most probably greater than 1271 kb. When the DNA from the protoplast lysate was fragmented by mixing, the cotransfer ratios of arg1 with metC, and arg1 with metC and trpC (2374 kb) were 2.8% and 0.16%, respectively. A high cotransfer ratio (2.7-7.2%) could not, therefore, be obtained using the fragmented DNA. Based on these observations, we propose a working hypothesis on the mechanism of the transformation of competent B. subtilis by DNA in protoplast lysates (LP transformation).

DNA taken into Bacillus subtilis competent cells by lysed-protoplast transformation is not ssDNA but dsDNA.

Competent Bacillus subtilis incorporates whole-genome DNA (4215 kb) from the protoplast lysate of B. subtilis subtilis [Akamatsu, T. and Taguchi, H., Biosci. Biotechnol. Biochem., 65, 823-829 (2001)]. A continuous incorporated DNA is longer than 1500 kb [J. Biosci. Bioeng., 101, 257-262 (2006)]. Whether the incorporated DNA is single-stranded (ssDNA) or double-stranded DNA (dsDNA) has been studied by examining the transforming activity of the incorporated DNA. B. subtilis BEST7027 was used as the donor strain, which has a heterologous region consisting of the 145 kb region of the Synechocystis sp. PCC6803 genome and erm gene. The donor DNA was transferred to a wild-type or a recA recipient strain (AYG2 or SYN9), and protoplast lysate was prepared from the transformants and used as the donor DNA source for the second recipient strain (AU1 or AV1). The intergenote region showed a significant transforming activity. When DNase I was added to both cells collected from the first transformation mixture and the following protoplastization, the result was similar to that obtained without DNase I. All of the observations strongly suggest that the incorporated DNA is dsDNA, and the transformation of competent B. subtilis by DNA in protoplast lysate is different from that by purified DNA taken up conventionally.

Isolation and characterization of xylitol-assimilating mutants of recombinant Saccharomyces cerevisiae.

To clarify the mechanisms of xylitol utilization, three xylitol-assimilating mutants were isolated from recombinant Saccharomyces cerevisiae strains showing highly efficient xylose-utilization. The nucleotide sequences of the mutant genomes were analyzed and compared with those of the wild-type strains and the mutation sites were identified. gal80 mutations were common to all the mutants, and recessive to the wild-type allele. Hence we constructed a gal80Δ mutant and confirmed that the gal80Δ mutant showed a xylitol-assimilation phenotype. When the constructed gal80Δ mutant was crossed with the three isolated mutants, all diploid hybrids showed xylitol assimilation, indicating that the mutations were all located in the GAL80. We analyzed the role of the galactose permease Gal2, controlled by the regulatory protein Gal80, in assimilating xylitol. A gal2Δ gal80Δ double mutant did not show xylitol assimilation, whereas expression of GAL2 under the control of the TDH3 promoter in the GAL80 strain did result in assimilation. These data indicate that Gal2 was needed for xylitol assimilation in the wild-type strain. When the gal80 mutant with an initial cell concentration of A660 = 20 was used for batch fermentation in a complex medium containing 20 g/L xylose or 20 g/L xylitol at pH 5.0 and 30°C under oxygen limitation, the gal80 mutant consumed 100% of the xylose within 12 h, but <30% of the xylitol within 100 h, indicating that xylose reductase is required for xylitol consumption in oxygen-limited conditions.

Green tea consumption and serum lipids and lipoproteins in a population of healthy workers in Japan.

PURPOSE: To examine the relation between green tea consumption and serum lipids and lipoproteins. METHODS: The subjects were 13,916 workers (8476 men and 5440 women) aged 40-69 years at over 1000 workplaces in Nagano prefecture, central Japan. They underwent health screening offered by a single medical institute between April 1995 and March 1996 and did not have morbid conditions affecting serum cholesterol levels. Serum concentrations of total cholesterol, high-density lipoprotein (HDL) cholesterol and triglycerides were measured at the screening. The consumption of green tea and other life-style characteristics were ascertained by a questionnaire. The data were analyzed with multivariate linear model. RESULTS: Daily consumption of green tea was reported by 86.7% of subjects. Green tea consumption was, statistically, significantly associated with lower levels of serum total cholesterol in both men and women while its associations with serum triglycerides and HDL cholesterol were not statistically significant. The inverse association of serum total cholesterol with green tea consumption appeared to level off at the consumption of more than 10 cups/day. Excluding the outlying subjects drinking more than 10 cups/day (0.4%), the regression analysis adjusting for age, body mass index, ethanol intake, smoking habit, coffee intake, and type of work showed that daily consumption of one cup of green tea was associated with a reduction in serum total cholesterol by 0.015 mmol/L (95% confidence interval 0.006 to 0.024, p < 0.001) in men and 0.015 mmol/L (0.004 to 0.025, p < 0.01) in women. After additional adjustment for selected dietary factors, the inverse association remained statistically significant; one cup of green tea per day was associated with a reduction in serum total cholesterol by 0.010 mmol/L (0.001 to 0.019, p = 0.03) in men and 0.012 mmol/L (0.001 to 0.022, p = 0.03) in women. CONCLUSION: Consumption of green tea was associated with lower serum concentration of total cholesterol in Japanese healthy workers age 40-69 years; however, green tea consumption was unrelated to serum HDL-cholesterol and triglycerides.

Development of industrial yeast strain with improved acid- and thermo-tolerance through evolution under continuous fermentation conditions followed by haploidization and mating.

Continuous fermentation using the industrial Saccharomyces cerevisiae diploid strain WW was carried out under acidic or high-temperature conditions to achieve acid- or thermo-tolerant mutants. Mutants isolated at pH 2.5 and 41°C showed improved growth and fermentation ability under acidic and elevated temperature conditions. Haploid strains WW17A1 and WW17A4 obtained from the mutated diploid strain WW17A showed better growth and 4.5-6.5% higher ethanol yields at pH 2.7 than the original strains. Haploid strain WW12T4 obtained from mutated diploid strain WW12T showed 1.25-1.50 times and 2.8-4.7 times higher total cell number and cell viability, respectively, than the original strains at 42°C. Strain AT, which had significantly improved acid- and thermo-tolerance, was developed by mating strain WW17A1 with WW12T4. Batch fermentation at 41°C and pH 3.5 showed that the ethanol concentration and yield achieved during fermentation by strain AT were 55.4 g/L and 72.5%, respectively, which were 10 g/L and 13.4% higher than that of the original strain WW. The present study demonstrates that continuous cultivation followed by haploidization and mating is a powerful approach for enhancing the tolerance of industrial strains.

Potent L-lactic acid assimilation of the fermentative and heterothallic haploid yeast Saccharomyces cerevisiae NAM34-4C.

We screened an industrial thermotolerant Saccharomyces cerevisiae strain, KF7, as a potent lactic-acid-assimilating yeast. Heterothallic haploid strains KF7-5C and KF7-4B were obtained from the tetrads of the homothallic yeast strain KF7. The inefficient sporulation and poor spore viability of the haploid strains were improved by two strategies. The first strategy was as follows: (i) the KF7-5C was crossed with the laboratory strain SH6710; (ii) the progenies were backcrossed with KF7-5C three times; and (iii) the progenies were inbred three times to maintain a genetic background close to that of KF7. The NAM12 diploid between the cross of the resultant two strains, NAM11-9C and NAM11-13A, showed efficient sporulation and exhibited excellent growth in YPD medium (pH 3.5) at 35°C with 1.4-h generation time, indicating thermotolerance and acid tolerance. The second strategy was successive intrastrain crosses. The resultant two strains, KFG4-6B and KFG4-4B, showed excellent mating capacity. A spontaneous mutant of KFG4-6B, KFG4-6BD, showed a high growth rate with a generation time of 1.1 h in YPD medium (pH 3.0) at 35°C. The KFG4-6BD strain produced ascospores, which were crossed with NAM11-2C and its progeny to produce tetrads. These tetrads were crossed with KFG4-4B to produce NAM26-14A and NAM26-15A. The latter strain had a generation time of 1.6 h at 35°C in pH 2.5, thus exhibiting further thermotolerance and acid tolerance. A progeny from a cross of NAM26-14A and NAM26-15A yielded the strain NAM34-4C, which showed potent lactic acid assimilation and high transformation efficiency, better than those of a standard laboratory strain.

Draft Genome Sequence of Saccharomyces cerevisiae NAM34-4C, a Lactic Acid-Assimilating Industrial Yeast Strain.

We determined the genome sequence of industrial Saccharomyces cerevisiae strain NAM34-4C, which would be useful for bioethanol production. The approximately 11.5-Mb draft genome sequence of NAM34-4C will provide remarkable insights into metabolic engineering for effective production of bioethanol from biomass.

Isolation and characterization of a mutant recombinant Saccharomyces cerevisiae strain with high efficiency xylose utilization.

A recombinant xylose-utilizing Saccharomyces cerevisiae strain carrying one copy of heterologous XYL1 and XYL2 from Pichia stipitis and endogenous XKS1 under the control of the TDH3 promoter in the chromosomal DNA was constructed from the industrial haploid yeast strain NAM34-4C, which showed thermotolerance and acid tolerance. The recombinant S. cerevisiae strain SCB7 grew in minimal medium containing xylose as the sole carbon source, and its shortest generation time (G(short)) was 5 h. From this strain, four mutants showing rapid growth (G(short) = 2.5 h) in the minimal medium were isolated. The mutants carried four mutations that were classified into three linkage groups. Three mutations were dominant and one mutation was recessive to the wild type allele. The recessive mutation was in the PHO13 gene encoding para-nitrophenyl phosphatase. The other mutant genes were not linked to TAL1 gene encoding transaldolase. When the mutants and their parental strain were used for the batch fermentation in a complex medium at pH 4.0 containing 30 g/L xylose at 35 °C with shaking (60 rpm) and an initial cell density (Absorbance at 660 nm) of 1.0, all mutants showed efficient ethanol production and xylose consumption from the early stage of the fermentation culture. In two mutants, within 24 h, 4.8 g/L ethanol was produced, and the ethanol yield was 47%, which was 1.4 times higher than that achieved with the parental strain. The xylose concentration in the medium containing the mutant decreased linearly at a rate of 1 g/L/h until 24 h.

Ethanol production from D-lactic acid by lactic acid-assimilating Saccharomyces cerevisiae NAM34-4C.

The lactic acid-assimilating yeast Saccharomyces cerevisiae NAM34-4C grew rapidly in minimal D-lactate medium (pH 3.5) at 35°C, compared with minimal L-lactate medium. A laboratory strain, S. cerevisiae S288C, did not grow in either medium at pH 3.5. Strain NAM34-4C produced remarkably high levels of ethanol in YPDL medium at pH 3.5, but not at pH 5.5, when D-lactate was provided as the carbon source. Optimal cultivation conditions for ethanol production from D-lactate by strain NAM34-4C were as follows: shaking speed, 60 rpm; initial pH, 3.0; cultivation temperature, 35°C; yeast extract, 5 g/L; peptone, 10 g/L; and D-lactate, 30 g/L. Under these conditions, strain NAM34-4C produced 2.7 g/L ethanol, which is 18% of the theoretical maximal yield (0.51 3 initial D-lactate concentration).

Plasmid transformation of competent Bacillus subtilis by lysed protoplast DNA.

Transformation of competent Bacillus subtilis with DNA obtained from lysed protoplasts (LP transformation) was analyzed using several different plasmid vectors: pC194, pUB110, pCB1 (consisting of pC194 and pBluescript II SK+), and pAC32R2 (consisting of pUB110 derivative and pUC19). LP transformation of B. subtilis QB936 with pCB1 was 6500-fold higher than that achieved using conventional transformation with purified DNA. Greater transformation efficiencies were also obtained using pAC32R2. However, transformation frequencies using both protoplast-derived and purified pC194 were very low (1.4-2.0×10(2) transformants per µg DNA). Hence, the efficiency of transformation depends on the nucleotide sequence of the donor plasmid. The LP transformation frequency using pC194 obtained from an add5 mutant was remarkably enhanced (1.6×10(8) transformants per µg DNA), indicating that this unique form of high molecular weight DNA is likely responsible for part of the stimulatory effect. Chromosomal DNA inhibited plasmid transformation using pC194 and pUB110, but had little effect on pCB1 transformation. Conversely, pCB1 DNA did not inhibit transformation with protoplast-derived chromosomal DNA. Competence proteins under the control of transcription factor ComK were likely required for LP plasmid transformation. The DNA concentration-dependence of plasmid transformation was first order and the slope value was one.

Role of ComEA in DNA uptake during transformation of competent Bacillus subtilis.

The role of the competence protein ComEA in DNA uptake during transformation of competent Bacillus subtilis was analyzed by lysed-protoplast transformation (LP transformation). A comEA deletion mutant was constructed by a fusion polymerase chain reaction. Transformants of the mutant were obtained by LP transformation at a frequency of 1.1 × 10(2) transformants per µg DNA, representing a low relative efficiency of transformation [RET (mutant/wild type)] of 2.7 × 10(-6). This implied an important role of the protein during DNA uptake. When analyzing LP transformation of comEA with a plasmid (5.7 kb), a similar RET (mutant/wild type) of 5.6 × 10(-5) was obtained. Following addition of DNA into the comEA mutant culture, the number of transformants increased at a rate of 0.5 transformants/min, which was very low compared with the wild-type (6.9×10(4) transformants/min). However, even in the comEA mutant, DNA uptake began immediately after addition of DNA. Using co-transformation analysis of the comEA mutant, short linkages at distances of 2-156 kb could be detected, but not long linkages at distances of 671-1662 kb. Taken together, the results indicate that ComEA plays an important role in the transfer of transforming DNA into the DNA channel and in controlling the rate of DNA uptake.

A simple assay for 6-aminohexanoate-oligomer-hydrolase using N-(4-nitrophenyl)-6-aminohexanamide.

We synthesized N-(4-nitrophenyl)-6-aminohexanamide (AHpNA) and used it as a substrate in a kinetic study of 6-aminohexanoate-hydrolase (NylB), a nylon oligomer-hydrolyzing enzyme. NylBs derived from Arthrobacter sp. KI72 and Pseudomonas sp. NK87 hydrolyzed AHpNA as well as a 6-aminohexanoic acid dimer, a known substrate for NylB. The K(m) values of the NylB from Arthrobacter sp. KI72 and Pseudomonas sp. NK87 for AHpNA were 0.5 mM and 2.0 mM, respectively.

Essential involvement of the Bacillus subtilis ABC transporter, EcsB, in genetic transformation of purified DNA but not native DNA from protoplast lysates.

Involvement of the Bacillus subtilis ABC transporter EcsB in genetic transformation with native DNA from protoplast lysate (LP transformation) was investigated using an ecsB deletion mutant constructed by fusion polymerase chain reaction. In these experiments, the non-transformability phenotype of the ecsB mutant was reversed and high numbers of transformants generated (1.5×10(5)/µg DNA). The relative efficiency of transformation (RET) of ecsB to wild type (1.2×10(-2)) was a thousand times higher using native chromosomal DNA than the RET obtained from purified DNA (<8.6×10(-6)). Similar transformation efficiencies were observed using native plasmid DNA. These results rule out a primary role for EcsB as a competence gene regulator. DNA-binding proteins attached to native DNA are not present in purified DNA preparations, and it is possible that such proteins could account for the transformability of the ecsB mutant. Because EcsB may play a role in protein(s) export, we tested exogenous proteins to identify functional replacements. We found that bovine serum albumin (fraction V) partially suppressed the phenotype of the ecsB mutation, leading to transformability with purified DNA. Linkage analysis of the ecsB mutant by LP co-transformation produced a higher co-transformation ratio (42% and 20%) at a distance of 34kb and 121kb in the ecsB mutant, compared to the wild-type strain, AYG2 (30.5% and 12.3%). The stimulatory linkage effect observed could be derived from a regulating gene involved in homologous recombination.

Role of ComFA in controlling the DNA uptake rate during transformation of competent Bacillus subtilis.

The roles of ComFA and ComEC in DNA uptake by competent Bacillus subtilis were analyzed by transformation with DNA in protoplast lysates (LP transformation). Deletion mutants of comFA and comEC and putative Walker A mutants (K152N, K152Q, K152E) of comFA were constructed by fusion polymerase chain reaction. Transformants of comEC mutant with purified DNA and DNA in protoplast lysate were not obtained, which shows a lack of transformation ability and backwards recombination of the mutant. Transformants of the comFA mutant were obtained by LP transformation (1.8 × 10(4) transformants/µg DNA). Low relative efficiency of transformation (RET) of comFA compared to wild type (4.3 × 10(-4)) showed an important role for comFA in DNA uptake. Walker A mutants showed 1.8-19 × 10(-4) RET, suggesting a dependence on ATPase activity for transformation. Co-transformation between short linkages was only detected in comFA mutants. The results demonstrated that ComFA controlled the DNA uptake rate. The interpretation was further supported by analyzing the plasmid used in LP transformation of the comFA mutant. The RET of comFA compared to the wild type was 2.7 × 10(-2), 60-fold higher than that with chromosomal DNA (4.3 × 10(-4)). Following addition of DNA into comFA culture, transformants were obtained after 15 min, with the number of transformants increasing over time. The kinetics strongly suggested that in comFA mutants, formation of another DNA uptake complex without ComFA would be a lengthy process.

Functional replacement of yeast flavocytochrome b(2) with bacterial L-lactate dehydrogenase.

Using yeast genetic complementation, we show here that Rhodobacter sphaeroidesl-lactate dehydrogenase can functionally replace flavocytochrome b(2) in Saccharomyces cerevisiae, only when a matrix-targeting signal of flavocytochrome b(2) is fused with the bacterial enzyme. The recombinant l-lactate dehydrogenase may add alternative route of mitochondrial electron transport other than flavocytochrome b(2).