Methanol has attracted increasing attention as a sustainable one-carbon feedstock because it can be produced from carbon dioxide and hydrogen or from waste biomass. Additionally, it can be economically stored and transported as a liquid. The methylotrophic yeast Komagataella phaffii is a promising host for producing valuable biochemicals from methanol, while d-lactic acid is an important chemical used for producing stereocomplex polylactic acid; microbial processes enable d-lactic acid synthesis with high optical purity. However, most microbial d-lactic acid production processes rely on sugar substrates, and efficient production from methanol remains limited. We deleted the cell wall stability-related gene PAS_chr4_0305 in d-lactic acid-producing K. phaffii. Evaluation of d-lactic acid production capacity in flask cultures with methanol as the sole carbon source revealed that the deletion strain showed an approximately 1.20-fold increase in d-lactic acid production compared with the parent strain. Furthermore, optimization of culture conditions resulted in d-lactic acid production reaching a maximum of 25.7 g/L. Transcriptome analysis results suggested that deletion of PAS_chr4_0305 induced expression changes in alcohol metabolism- and ribosome-related genes, and these changes potentially contributed to improved methanol assimilation and d-lactic acid productivity. This study demonstrates that combining the deletion of the cell wall stability-related gene with culture condition optimization can enhance d-lactic acid production from methanol in K. phaffii, providing a novel strategy for valuable biochemical production using methylotrophic yeast.

Methanol, a C1 compound that can be produced from CO₂ and waste biomass, has attracted increasing attention as a renewable feedstock alternative to petroleum. In recent years, Komagataella phaffii has emerged as a promising microbial host for the methanol-based biosynthesis of value-added compounds through metabolic engineering. Among these compounds, β-carotene—a precursor of vitamin A with antioxidant properties—is widely used in the food, pharmaceutical, and cosmetic industries. In this study, we aimed to develop a high β-carotene-producing strain of K. phaffii using methanol as the sole carbon source. To this end, gene fragments encoding three key genes involved in β-carotene biosynthesis—geranylgeranyl pyrophosphate synthase (crtE), phytoene desaturase (crtI), and lycopene cyclase/phytoene synthase (crtYB)—from Xanthophyllomyces dendrorhous were introduced into K. phaffii using an rDNA-targeted integration strategy. Following transformation, we performed iterative phenotypic screening coupled with stepwise zeocin selection to enrich candidate isolates with higher A450-derived carotenoid specific productivity. The resulting strains, GS115/crt/Z2/01 and GS115/crt/Z4/03, achieved β-carotene titers of 101.8 and 63.6 mg/L, and specific productivities of 14.4 and 17.0 mg/g dry cell weight, respectively. Our results demonstrate the effectiveness of rDNA-targeted multi-gene integration combined with A450-derived phenotypic screening and stepwise zeocin selection as a strategy to identify candidate strains with enhanced target-compound production in K. phaffii. This approach shows promise for expanding the range of valuable chemicals sustainably produced using methanol-based bioprocesses.

Methanol has attracted attention as an alternative carbon source to petroleum. Komagataella phaffii, a methanol-assimilating yeast, is a useful host for the chemical production from methanol. A previous study successfully constructed a metabolically engineered K. phaffii GS115/S8/Z3 strain capable of producing D-lactic acid from methanol. In this study, we aimed to develop a strain with improved D-lactic acid production by applying ultra-violet mutagenesis to the D-lactic acid-producing strain, GS115/S8/Z3. The resulting mutant strain DLac_Mut2_221 produced 5.38 g/L of D-lactic acid from methanol, a 1.52-fold increase compared to the parent strain GS115/S8/Z3. The transcriptome analysis of the mutant DLac_Mut2_221 identified 158 differentially expressed genes, providing insights into key mechanisms contributing to enhanced D-lactic acid production. Metabolic engineering strategies for K. phaffii based on the knowledge gained from this study will contribute to improving the productivity of various useful chemicals from methanol.

Microbial production of valuable chemicals is a promising and sustainable approach, offering high energy efficiency and minimal waste generation. Production of 2,3-butanediol (2,3-BDO) by the safe industrial yeast Saccharomyces cerevisiae holds potential as a sustainable bioprocess. However, the low tolerance of 2,3-BDO in yeast remains a major challenge. In this study, we aimed to improve 2,3-BDO tolerance in S. cerevisiae by introducing DNA point and structural mutations using techniques developed in previous studies, thereby advancing the sustainable industrial production of 2,3-BDO. Through point and structural mutagenesis, we successfully obtained the mutant strain YPH499/Co58, which exhibited a 122-fold higher OD600 value than the parent strain after 96 h of cultivation in a medium containing 175 g/L 2,3-BDO. Transcriptome analysis of four mutants with particularly high 2,3-BDO tolerance suggested that the upregulation of genes related to the proteasome, peroxisome, TCA cycle, mitochondria, and transcriptional regulation was closely related to 2,3-BDO tolerance. The use of these mutant strains represents a major step toward realizing the sustainable industrial production of 2,3-BDO. Additionally, the insights gained in this study regarding 2,3-BDO tolerance may contribute to improving yeast tolerance to various stresses, including ethanol, heat, and low pH. The mutagenesis technique developed in this study holds promise for the construction of yeast strains with enhanced robustness for various applications.

Although the yeast Saccharomyces cerevisiae has been utilized for the bioproduction of various valuable substances, improving product concentration and production rate remains a challenge in its practical application. In this respect, metabolic channeling represents a potential strategy for addressing this issue. In the metabolic pathway for synthesizing a target product, closing enzymes induce substrate channeling, in which intermediates are transferred to the following enzyme to facilitate processing. To close enzymes in proximity, protein ligation is one of the solutions. However, genetic fusion often causes the generation of inactive complexes, and few techniques exist for ligating enzymes in yeast without loss of enzyme activity. Herein, we focused on sortase A, which links a short peptide tag between two target proteins. First, we demonstrated sortase A-mediated ligation in yeast using split-green fluorescent protein. Then, sortase A-mediated ligation was applied to ligate metabolic enzymes related to 3-hydroxypropionic acid, which improved 3-HP production by 2.42-fold. This strategy represents a novel approach for improving yeast bioproduction.

The yeast Saccharomyces cerevisiae is a safe microorganism with established industrial-scale culture techniques. Standard laboratory S. cerevisiae strains, such as the representative YPH499, are valuable hosts for producing proteins and chemicals through metabolic engineering. Consequently, there's a high demand for platform strains of S. cerevisiae with enhanced protein production capacity. We have previously established an efficient and straightforward technique for introducing point and structural mutations into yeast via plasmid introduction, leading to the generation of mutant strains with superior phenotypes. In this study, we aimed to develop S. cerevisiae mutants with high protein production capacity using techniques to introduce point and structural mutations. We introduced these mutations into the YPH499/pEUPGGFP strain, which expresses green fluorescent protein (GFP). Since GFP is easily detected by its fluorescence, we selected mutants based on their fluorescence intensity. Consequently, YPH499/pEUPGGFP/Mu10G39, with a GFP fluorescence intensity 2.5-fold higher than that of the parent strain, was successfully obtained. Then, a carotenoid-producing plasmid was introduced to construct YPH499Mu10G39/pEU20Beta3. YPH499Mu10G39/pEU20Beta3 produced 6.74 mg/g-dry cell carotenoids, which was 2.9-fold higher than that produced by the parent strain. Transcriptome analysis suggested that YPH499Mu10G39 exhibited improved energy production, amino acid precursor supply, ribosome function, and stress tolerance, which may have contributed to its high protein production. In conclusion, by introducing point and structural mutations, we successfully developed the platform strain, YPH499Mu10G39, which is useful for the high production of various proteins. In the future, various proteins and useful chemicals can be produced through metabolic engineering using YPH499Mu10G39 as a platform strain.

The sustainable and efficient production of biofuels has generated considerable interest in the microbial synthesis of alka(e)nes, which are promising alternatives to fossil fuels. Acyl-ACP reductase (AAR) is a critical enzyme in the alka(e)ne biosynthetic pathway, and its poor solubility in Escherichia coli is a major bottleneck during the optimization of production yields. The approaches for enhancing protein solubility typically include the fusion of solubility tags at the N- or C-termini of target proteins, which can sometimes interfere with protein function or stability. The present study developed a novel strategy that leverages the regulatory potential of 3 '-untranslated regions (3 '-UTRs) by integrating the sequence coding thioredoxin (Trx), small ubiquitin-like modifier (SUMO), maltose-binding protein (MBP), or N-utilization substance protein A (NusA), into the 3 '-UTR of the AAR gene. The strategy aimed to enhance the stability of AAR mRNA for improving the solubility and expression of AAR without altering its primary structure. The findings revealed that this strategy significantly enhanced the solubility and expression levels of AAR in Escherichia coli, which markedly increased alka(e)ne production. This method has potential widespread applications in metabolic engineering and synthetic biology. The study paves the way for the development of more efficient strategies aimed at producing biofuels, and highlights the untapped potential of the 3 '-UTR engineering strategy.

Enzymatic degradation of polyethylene terephthalate (PET) has garnered attention as a new PET decomposition technology because of its progression under mild conditions. However, the practical application of these enzymes is significantly hindered by their high purification costs. To address this, we developed an Escherichia coli strain with surface-engineered capabilities for efficient PET degradation by expressing PET-degrading enzymes. Several components were considered and optimized for the efficient degradation of PET using this microorganism catalyst. When comparing the expression systems, cells displaying PETase on the surface using the arabinose-inducible system showed a 2.9-fold improvement in PET degradation compared to those using the isopropyl β-d-1-thiogalactopyranoside-inducible system. Furthermore, cells with both surface-displayed FAST-PETase and localized MHETase in the periplasm exhibited a 6.6-fold increase in PET degradation efficiency compared to the initial pETDuet system. Notably, MHETase expression shifted the product profile toward predominantly terephthalic acid (TPA) formation. These results demonstrate that incorporating MHETase into a PET degradation system predominantly yields TPA, suggesting potential applications for efficient PET upcycling into valuable chemicals.

CO2 fixation methods using green algae have attracted considerable attention because they can be applied for the fixation of dilute CO2 in the atmosphere. However, green algae generally exhibit low CO2 fixation efficiency under atmospheric conditions. Therefore, it is a challenge to improve the CO2 fixation efficiency of green algae under atmospheric conditions. Co-cultivation of certain microalgae with heterotrophic microorganisms can increase the growth potential of microalgae under atmospheric conditions. The objective of this study was to determine the culture conditions under which the growth potential of green algae Chlamydomonas reinhardtii is enhanced by co-culturing with the yeast Saccharomyces cerevisiae, and to identify the cause of the enhanced growth potential. When C. reinhardtii and S. cerevisiae were co-cultured with an initial green algae to yeast inoculum ratio of 1:3, the cell concentration of C. reinhardtii reached 133 × 105 cells/mL on day 18 of culture, which was 1.5 times higher than that of the monoculture. Transcriptome analysis revealed that the expression levels of 363 green algae and 815 yeast genes were altered through co-cultivation. These included genes responsible for ammonium transport and CO2 enrichment mechanism in green algae and the genes responsible for glycolysis and stress responses in yeast. We successfully increased C. reinhardtii growth potential by co-culturing it with S. cerevisiae. The main reasons for this are likely to be an increase in inorganic nitrogen available to green algae via yeast metabolism and an increase in energy available for green algae growth instead of CO2 enrichment.

AbstractWastewater treatment using co-culture systems of microalgae and heterotrophic microorganisms is expected to be useful under atmospheric dilute carbon dioxide conditions. In this study, we investigated the combination of microalgae and heterotrophic microorganisms to improve the efficiency of wastewater treatment. Furthermore, to elucidate the cause of the changes in wastewater treatment efficiency in the co-culture system, changes in gene expression were revealed through transcriptome analysis. Three types of microalgae and five heterotrophic microorganisms were used in combination for wastewater treatment. The combination ofChlamydomonas reinhardtiiNIES-2238 andSaccharomyces cerevisiaeSH-4 showed the highest wastewater treatment efficiency. Using this combination for artificial wastewater treatment, the removal rates of TOC (Total organic carbon), PO43-, and NH4+reached 80%, 93%, and 63%, respectively, after 18 h of treatment. Transcriptome analysis revealed that the combined wastewater treatment altered the expression of 1371 and 692 genes inC. reinhardtiiandS. cerevisiae, respectively. The genes upregulated inC. reinhardtiiincluded those related to molecular and ion transport. Genes upregulated inS. cerevisiaeincluded those related to cell protection from various types of damage and stress. To the best of our knowledge, this is the first study to show that a combination of green algae and yeast improves the efficiency of wastewater treatment. As both the green algaC. reinhardtiiand the yeastS. cerevisiaeare highly safe microorganisms, the establishment of their effective combination for wastewater treatment is highly significant.

Background Currently, efficient technologies producing useful chemicals from alternative carbon resources, such as methanol, to replace petroleum are in demand. The methanol-utilizing yeast, Komagataella phaffii, is a promising microorganism to produce chemicals from methanol using environment-friendly microbial processes. In this study, to achieve efficient D-lactic acid production from methanol, we investigated a combination of D-lactate dehydrogenase (D-LDH) genes and promoters in K. phaffii. The yeast strain was constructed by integrating a gene cassette containing the identified gene and promoter into the rDNA locus of K. phaffii, followed by post-transformational gene amplification. Subsequently, D-lactic acid production from methanol was evaluated. Results Among the five D-LDH genes and eight promoters tested, the combination of LlDLDH derived from Leuconostoc lactis and CAT1 and FLD1 promoters was suitable for expression in K. phaffii. GS115_CFL/Z3/04, the best-engineered strain constructed via integration of LlDLDH linked to CAT1 and FLD1 promoters into the rDNA locus and post-transformational gene amplification, produced 5.18 g/L D-lactic acid from methanol. To the best of our knowledge, the amount of D-lactic acid from methanol produced by this engineered yeast is the highest reported value to date, including both D- and L- lactic acids. Conclusions This study demonstrated the effectiveness of combining different enzyme genes and promoters using multiple promoters with different induction and repression conditions, integrating the genes into the rDNA locus, and further amplifying the genes after transformation in K. phaffii. Using our established method, other K. phaffii strains can be engineered to produce various useful chemicals in the future.

BACKGROUND:Currently, efficient technologies producing useful chemicals from alternative carbon resources, such as methanol, to replace petroleum are in demand. The methanol-utilizing yeast, Komagataella phaffii, is a promising microorganism to produce chemicals from methanol using environment-friendly microbial processes. In this study, to achieve efficient D-lactic acid production from methanol, we investigated a combination of D-lactate dehydrogenase (D-LDH) genes and promoters in K. phaffii. The yeast strain was constructed by integrating a gene cassette containing the identified gene and promoter into the rDNA locus of K. phaffii, followed by post-transformational gene amplification. Subsequently, D-lactic acid production from methanol was evaluated. RESULTS:Among the five D-LDH genes and eight promoters tested, the combination of LlDLDH derived from Leuconostoc lactis and CAT1 and FLD1 promoters was suitable for expression in K. phaffii. GS115_CFL/Z3/04, the best-engineered strain constructed via integration of LlDLDH linked to CAT1 and FLD1 promoters into the rDNA locus and post-transformational gene amplification, produced 5.18 g/L D-lactic acid from methanol. To the best of our knowledge, the amount of D-lactic acid from methanol produced by this engineered yeast is the highest reported value to date when utilizing methanol as the sole carbon source. CONCLUSIONS:This study demonstrated the effectiveness of combining different enzyme genes and promoters using multiple promoters with different induction and repression conditions, integrating the genes into the rDNA locus, and further amplifying the genes after transformation in K. phaffii. Using our established method, other K. phaffii strains can be engineered to produce various useful chemicals in the future.

In yeast metabolic engineering, there is a need for technologies that simultaneously suppress and regulate the expression of multiple genes and improve the production of target chemicals. In this study, we aimed to develop a novel technology that simultaneously suppresses the expression of multiple genes by combining RNA interference with global metabolic engineering strategy. Furthermore, using β-carotene as the target chemical, we attempted to improve its production by using the technology. First, we developed a technology to suppress the expression of the target genes with various strengths using RNA interference. Using this technology, total carotenoid production was successfully improved by suppressing the expression of a single gene out of 10 candidate genes. Then, using this technology, RNA interference strain targeting 10 candidate genes for simultaneous suppression was constructed. The total carotenoid production of the constructed RNA interference strain was 1.7 times compared with the parental strain. In the constructed strain, the expression of eight out of the 10 candidate genes was suppressed. We developed a novel technology that can simultaneously suppress the expression of multiple genes at various intensities and succeeded in improving carotenoid production in yeast. Because this technology can suppress the expression of any gene, even essential genes, using only gene sequence information, it is considered a useful technology that can suppress the formation of by-products during the production of various target chemicals by yeast.

Enzymatic degradation of polyethylene terephthalate (PET) is attracting attention as a new technology because of its mild reaction conditions. However, the cost of purified enzymes is a major challenge for the practical application of this technology. In this study, we attempted to display the surface of the PET-degrading enzyme, PETase, onto Escherichia coli using the membrane anchor, PgsA, from Bacillus subtilis to omit the need for purification of the enzyme. Immunofluorescence staining confirmed that PETase was successfully displayed on the surface of E. coli cells when a fusion of PgsA and PETase was expressed. The surface-displaying E. coli was able to degrade 94.6% of 1 mM bis(2-hydroxyethyl) terephthalate in 60 min, and the PET films were also degraded in trace amounts. These results indicate that PgsA can be used to present active PETase on the cell surface of E. coli . This technique is expected to be applied for efficient PET degradation.

β-Carotene is an attractive compound and that its biotechnological production can be achieved by using engineered Saccharomyces cerevisiae. In a previous study, we developed a technique for the efficient establishment of diverse mutants through the introduction of point and structural mutations into the yeast genome. In this study, we aimed to improve β-carotene production by applying this mutagenesis technique to S. cerevisiae strain that had been genetically engineered for β-carotene production. Point and structural mutations were introduced into β-carotene-producing engineered yeast. The resulting mutants showed higher β-carotene production capacity than the parental strain. The top-performing mutant, HP100_74, produced 37.6 mg/L of β-carotene, a value 1.9 times higher than that of the parental strain (20.1 mg/L). Gene expression analysis confirmed an increased expression of multiple genes in the glycolysis, mevalonate, and β-carotene synthesis pathways. In contrast, expression of ERG9, which functions in the ergosterol pathway competing with β-carotene production, was decreased in the mutant strain. The introduction of point and structural mutations represents a simple yet effective method for achieving mutagenesis in yeasts. This technique is expected to be widely applied in the future to produce chemicals via metabolic engineering of S. cerevisiae.

The monooxygenase activity of the Pseudomonas putida cytochrome P450 system was expressed in the presence of P450cam, putidaredoxin reductase (PdR), and putidaredoxin (Pd). In turn, it has been found to catalyze the oxidation of various organic compounds. Since cytochrome P450 system substrates are often insoluble in water, reactions should be carried out at high temperatures and in the presence of organic solvents where the high stability of all of P450cam, PdR, and Pd are needed. In this study, proline was introduced to improve the stability of PdR which was estimated to have the lowest organic solvent stability compared to others. Thus, the half-life of PdR_T221P at 40°C was 1.37 times longer than that of wild-type PdR. However, the half-life of PdR_T221P in the presence of 25% (v/v) methanol was shorter than that of wild-type PdR. Furthermore, structural stabilities of PdRs using molecular dynamics (MD) simulations demonstrated these phenomena.

Acyl-acyl carrier protein (acyl-ACP) reductase (AAR) is a crucial enzyme in alka(e)ne production by recombinant Escherichia coli (E. coli). Engineered AAR expressed in E. coli holds great promise for the production of alka(e)nes, which are a valuable bio-based alternative to fossil fuels. However, its effectiveness is significantly limited by its low solubility and stability. The aim of this study is to enhance the solubility and stability of AAR to improve the production of alka(e)nes in E. coli. In this study, an integrated computational approach was employed for combining solubility prediction, aggregation propensity prediction, structural modeling, and molecular dynamics (MD) simulations. This multi-faceted approach provides new insights and tools for enzyme engineering. Through this approach, the C-terminus of AAR was identified as the sole significant hydrophobic patch and aggregation-prone regions (APR). Three strategies were evaluated experimentally: direct deletion of these hydrophobic residues; substitution of these residues with negatively charged amino acids, such as glutamic acid (Glu) or aspartic acid (Asp); and the introduction of additional negatively charged amino acids at the C-terminus to shield the hydrophobic patches. The results showed that AAR mutants with additional Glu residues at the C-terminus exhibited improved performance. Specifically, the AAR-E3 mutant, containing three consecutive Glu residues, demonstrated significantly enhanced solubility and stability, with alka(e)ne production (159.25 mg/L) being 6.3 times higher than that of the wild-type AAR (25.37 mg/L). Subsequent computational modeling and molecular dynamics simulations further validated the experimental findings. This study highlights the potential of enzyme engineering to significantly enhance biofuel production efficiency.

Wastewater treatment using the activated sludge method requires a large amount of electricity for aeration. Therefore, wastewater treatment using co-culture systems of microalgae and heterotrophic microorganisms, which do not require aeration, has attracted attention as an energy-saving alternative to the method. In this study, we investigated different combinations of microalgae and heterotrophic microorganisms to improve the efficiency of wastewater treatment. Three types of microalgae and five heterotrophic microorganisms were used in combination for wastewater treatment. The combination of Chlamydomonas reinhardtii NIES-2238 and Saccharomyces cerevisiae SH-4 showed the highest wastewater treatment efficiency. Using this combination for artificial wastewater treatment, the removal rates of total organic carbon, PO43−, and NH4+ reached 80

It is known that co-cultivation of green algae with heterotrophic microorganisms, such as yeast, improves green algae's growth potential and carbon dioxide fixation, even under low CO 2 concentration conditions such as the atmosphere. Introducing mutations into green algae is also expected to enhance their growth potential. In this study, we sought to improve the growth potential of a co-culture system of the green algae Chlamydomonas reinhardtii and the yeast Saccharomyces cerevisiae by introducing mutations into the green algae. Additionally, we performed a transcriptome analysis of the co-culture of the green algae mutant strain with yeast, discussing the interaction between the green algae mutant strain and the yeast. When the green algae mutant strain was co-cultured with yeast, the number of green algae cells reached 152 × 10 5 cells/mL after 7 days of culture. This count was 2.6 times higher than when the wild-type green algae strain was cultured alone and 1.6 times higher than when the wild-type green algae strain and yeast were co-cultured. The transcriptome analysis also indicated that the primary reason for the increased growth potential of the green algae mutant strain was its enhanced photosynthetic activity and nitrogen utilization efficiency.