Kenji Okano

Biotechnological production of enantiomeric pure lactic acid from renewable resources: recent achievements, perspectives, and limits

Lactic acid (LA) is an important and versatile chemical that can be produced from renewable resources such as biomass. LA is used in the food, pharmaceutical, and polymers industries and is produced by microorganism fermentation; however, most microorganisms cannot directly utilize biomass such as starchy materials and cellulose. Here, we summarize LA production using several kinds of genetically modified microorganisms, such as LA bacteria, Escherichia coli, Corynebacterium glutamicum, and yeast. Using gene manipulation and metabolic engineering, the yield and optical purity of LA produced from biomass has been significantly improved. In this review, the drawbacks as well as improvements of LA production by fermentation is discussed.

Display of active enzymes on the cell surface of Escherichia coli using PgsA anchor protein and their application to bioconversion

We have developed a novel Escherichia coli cell surface display system by employing PgsA as an anchoring motif. In our display system, C-terminal fusion to PgsA anchor protein from Bacillus subtilis was used. The enzymes selected for display were α-amylase (AmyA) from Streptococcus bovis 148 and lipase B (CALB) from Candida antarctica. The molecular mass values of AmyA and CALB are approximately 77 and 34xa0kDa, respectively. The enzymes were displayed on the surface as a fusion protein with a FLAG peptide tag at the C terminus. Both the PgsA-AmyA-FLAG and PgsA-CALB-FLAG fusion proteins were shown to be displayed by immunofluorescence labeling using anti-FLAG antibody. The displayed enzymes were active forms, and AmyA and CALB activities reached 990xa0U/g (dry cell weight) and 4.6xa0U/g (dry cell weight), respectively. AmyA-displaying E. coli cells grew utilizing cornstarch as the sole carbon source, while CALB-displaying E. coli cells catalyzed enantioselective transesterification, indicating that they are effective whole-cell biocatalysts. Since a target enzyme with a size of 77xa0kDa and an industrially useful lipase have been successfully displayed on the cell surface of E. coli for the first time, PgsA protein is probably a useful anchoring motif to display various enzymes.

Homo-d-lactic acid production from mixed sugars using xylose-assimilating operon-integrated Lactobacillus plantarum

In order to achieve efficient d-lactic acid fermentation from a mixture of xylose and glucose, the xylose-assimilating xylAB operon from Lactobacillus pentosus (PXylAB) was introduced into an l-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum (ΔldhL1-xpk1::tkt-Δxpk2) strain in which the phosphoketolase 1 gene (xpk1) was replaced with the transketolase gene (tkt) from Lactococcus lactis, and the phosphoketolase 2 (xpk2) gene was deleted. Two copies of xylAB introduced into the genome significantly improved the xylose fermentation ability, raising it to the same level as that of ΔldhL1-xpk1::tkt-Δxpk2 harboring a xylAB operon-expressing plasmid. Using the two-copy xylAB integrated strain, successful homo-d-lactic acid production was achieved from a mixture of 25xa0g/l xylose and 75xa0g/l glucose without carbon catabolite repression. After 36-h cultivation, 74.2xa0g/l of lactic acid was produced with a high yield (0.78xa0g per gram of consumed sugar) and an optical purity of d-lactic acid of 99.5%. Finally, we successfully demonstrated homo-d-lactic acid fermentation from a mixture of three kinds of sugar: glucose, xylose, and arabinose. This is the first report that describes homo-d-lactic acid fermentation from mixed sugars without carbon catabolite repression using the xylose-assimilating pathway integrated into lactic acid bacteria.

d-lactic acid production from cellooligosaccharides and β-glucan using l-LDH gene-deficient and endoglucanase-secreting Lactobacillus plantarum

In order to achieve direct fermentation of an optically pure d-lactic acid from cellulosic materials, an endoglucanase from a Clostridium thermocellum (CelA)-secreting plasmid was introduced into an l-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum (∆ldhL1) bacterial strain. CelA expression and its degradation of β-glucan was confirmed by western blot analysis and enzyme assay, respectively. Although the CelA-secreting ∆ldhL1 assimilated cellooligosaccharides up to cellohexaose (although not cellotetraose), the main end product was acetic acid, not lactic acid, due to the conversion of lactic acid to acetic acid. Cultivation under anaerobic conditions partially suppressed this conversion resulting in the production of 1.27xa0g/l of D-lactic acid with a high optical purity of 99.5% from a medium containing 2xa0g/l of cellohexaose. Subsequently, D-lactic acid fermentation from barley β-glucan was carried out with the addition of Aspergillus aculeatus β-glucosidase produced by recombinant Aspergillus oryzae and 1.47xa0g/l of D-lactic was produced with a high optical purity of 99.7%. This is the first report of direct lactic acid fermentation from β-glucan and a cellooligosaccharide that is a more highly polymerized sugar than cellotriose.

Improvement in lactic acid production from starch using α-amylase-secreting Lactococcus lactis cells adapted to maltose or starch

To achieve direct and efficient lactic acid production from starch, a genetically modified Lactococcus lactis IL 1403 secreting α-amylase, which was obtained from Streptococcus bovis 148, was constructed. Using this strain, the fermentation of soluble starch was achieved, although its rate was far from efficient (0.09xa0g l−1 h−1 lactate). High-performance liquid chromatography revealed that maltose accumulated during fermentation, and this was thought to lead to inefficient fermentation. To accelerate maltose consumption, starch fermentation was examined using L. lactis cells adapted to maltose instead of glucose. This led to a decrease in the amount of maltose accumulation in the culture, and, as a result, a more rapid fermentation was accomplished (1.31xa0g l−1 h−1 lactate). Maximum volumetric lactate productivity was further increased (1.57xa0g l−1 h−1 lactate) using cells adapted to starch, and a high yield of lactate (0.89xa0g of lactate per gram of consumed sugar) of high optical purity (99.2% of l-lactate) was achieved. In this study, we propose a new approach to lactate production by α-amylase-secreting L. lactis that allows efficient fermentation from starch using cells adapted to maltose or starch before fermentation.

Improved homo l-lactic acid fermentation from xylose by abolishment of the phosphoketolase pathway and enhancement of the pentose phosphate pathway in genetically modified xylose-assimilating Lactococcus lactis

In order to achieve efficient homo L-lactic acid fermentation from xylose, we first carried out addition of xylose assimilation ability to Lactococcus lactis IL 1403 by introducing a plasmid carrying the xylRAB genes from L. lactis IO-1 (pXylRAB). Then modification of xylose assimilation pathway was carried out. L. lactis has two pathways for xylose assimilation called the phosphoketolase pathway (PK pathway) that produces both lactic acid and acetic acid and the pentose phosphate pathway (PP pathway) that produces only lactic acid as a final product. Thus a mutant strain that disrupted its phosphokeolase gene (ptk) was constructed. The Δptk mutant harboring pXylRAB lacked the PK pathway and produced predominantly lactic acid from xylose via the PP pathway, although its fermentation rate slightly decreased. Further introduction of the transketolase gene (tkt) to disrupted ptk locus led restoration of fermentation rate and this was attributed to enhancement of the PP pathway. As a result, ptk::tkt strain harboring pXylRAB produced 50.1xa0g/l of L-lactic acid from xylose with a high optical purity of 99.6% and a high yield of 1.58 (moles per mole xylose consumed) that is close to theoretical value of 1.67 from xylose.

Improvement of protein production in lactic acid bacteria using 5'-untranslated leader sequence of slpA from Lactobacillus acidophilus. Improvement in protein production using UTLS.

The 5′-untranslated leader sequence (UTLS) of the slpA gene from Lactobacillus acidophilus contributes to mRNA stabilization by producing a 5′ stem and loop structure, and a high-level expression system for the lactic acid bacteria was developed using the UTLS in this study. A plasmid, which expresses α-amylase under the control of the ldh promoter, was constructed by integrating the core promoter sequence with the UTLS. The role of the UTLS in increasing the copies of the α-amylase mRNA was proved by measuring α-amylase activity in the culture supernatant and the relative expression of α-amylase mRNA was determined by the quantitative real-time PCR analysis. Moreover, several expression systems were constructed by combining the core promoter sequence with the UTLS or with the partially deleted UTLS and the expression level was evaluated. The use of the UTLS led to the success in improving α-amylase expression in the two strains of Lactobacillus casei and Lactococcus lactis. The current study showed that the improvement in protein production using the UTLS could be applied to the expression system in the lactic acid bacteria.