UV mutagenesis improves growth potential of green algae in a green algae-yeast co-culture system.

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 CO2 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 × 105 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.

Improvement of cell growth in green algae Chlamydomonas reinhardtii through co-cultivation with yeast Saccharomyces cerevisiae.

PURPOSE: 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. RESULTS: 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. CONCLUSION: 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.

Development of a metabolic engineering technology to simultaneously suppress the expression of multiple genes in yeast and application in carotenoid production.

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 ß-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.

Induction of point and structural mutations in engineered yeast Saccharomyces cerevisiae improve carotenoid production.

ß-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 ß-carotene production by applying this mutagenesis technique to S. cerevisiae strain that had been genetically engineered for ß-carotene production. Point and structural mutations were introduced into ß-carotene-producing engineered yeast. The resulting mutants showed higher ß-carotene production capacity than the parental strain. The top-performing mutant, HP100_74, produced 37.6 mg/L of ß-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 ß-carotene synthesis pathways. In contrast, expression of ERG9, which functions in the ergosterol pathway competing with ß-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.

Enhancing 3-hydroxypropionic acid production in Saccharomyces cerevisiae through enzyme localization within mitochondria.

Microbial 3-hydroxypropionic acid (3-HP) production can potentially replace petroleum-based production methods for acrylic acid. Here, we constructed a yeast strain that expressed enzymes related to 3-HP biosynthesis within the mitochondria. This approach aimed to enhance the 3-HP production by utilizing the mitochondrial acetyl-CoA, an important intermediate for synthesizing 3-HP. The strain that expressed 3-HP-producing enzymes in the mitochondria (YPH-mtA3HP) showed improved production of 3-HP compared to that shown by the strain expressing 3-HP-producing enzymes in the cytosol (YPH-cyA3HP). Additionally, cMCR was overexpressed, which regulates a rate-limiting reaction in synthesizing 3-HP. In this study, we aimed to further enhance 3-HP production by expressing multiple copies of cMCR in the mitochondria using the δ-integration strategy to optimize the expression level of cMCR (YPH-mtA3HPx*). The results of flask-scale cultivation showed that 3-HP production by cMCR δ-integration was significantly higher, exhibiting a yield of 160 mg/L in YPH-mtA3HP6* strain and 257 mg/L in YPH-mtA3HP22* strain. Notably, YPH-mtA3HP22*, exhibited the highest 3-HP titer, which was 3.2-fold higher than that of YPH-cyA3HP. Our results demonstrated the potential of utilizing the mitochondrial compartment within S. cerevisiae for enhancing 3-HP production.

Identification of genes responsible for absorbing palladium ion in Escherichia coli.

The capability of Escherichia coli BW25113 to adsorb palladium (Pd) ions in a single-gene-knockout library was investigated using high-throughput screening. The results revealed that compared to BW25113, nine strains promoted Pd ion adsorption, whereas 22 strains repressed. Although further studies are required because of the first screening results, our results will provide a new perspective for improving the biosorption.

Construction of a machine-learning model to predict the optimal gene expression level for efficient production of D-lactic acid in yeast.

The modification of gene expression is being researched in the production of useful chemicals by metabolic engineering of the yeast Saccharomyces cerevisiae. When the expression levels of many metabolic enzyme genes are modified simultaneously, the expression ratio of these genes becomes diverse; the relationship between the gene expression ratio and chemical productivity remains unclear. In other words, it is challenging to predict phenotypes from genotypes. However, the productivity of useful chemicals can be improved if this relationship is clarified. In this study, we aimed to construct a machine-learning model that can be used to clarify the relationship between gene expression levels and D-lactic acid productivity and predict the optimal gene expression level for efficient D-lactic acid production in yeast. A machine-learning model was constructed using data on D-lactate dehydrogenase and glycolytic genes expression (13 dimensions) and D-lactic acid productivity. The coefficient of determination of the completed machine-learning model was 0.6932 when using the training data and 0.6628 when using the test data. Using the constructed machine-learning model, we predicted the optimal gene expression level for high D-lactic acid production. We successfully constructed a machine-learning model to predict both D-lactic acid productivity and the suitable gene expression ratio for the production of D-lactic acid. The technique established in this study could be key for predicting phenotypes from genotypes, a problem faced by recent metabolic engineering strategies.

Bioengineering for the industrial production of 2,3-butanediol by the yeast, Saccharomyces cerevisiae.

Owing to issues, such as the depletion of petroleum resources and price instability, the development of biorefinery related technologies that produce fuels, electric power, chemical substances, among others, from renewable resources is being actively promoted. 2,3-Butanediol (2,3-BDO) is a key compound that can be used to produce various chemical substances. In recent years, 2,3-BDO production using biological processes has attracted extensive attention for achieving a sustainable society through the production of useful compounds from renewable resources. With the development of genetic engineering, metabolic engineering, synthetic biology, and other research field, studies on 2,3-BDO production by the yeast, Saccharomyces cerevisiae, which is safe and can be fabricated using an established industrial-scale cultivation technology, have been actively conducted. In this review, we sought to describe 2,3-BDO and its derivatives; discuss 2,3-BDO production by microorganisms, in particular S. cerevisiae, whose research and development has made remarkable progress; describe a method for separating and recovering 2,3-BDO from a microbial culture medium; and propose future prospects for the industrial production of 2,3-BDO by microorganisms.

Improvement of lactic acid tolerance by cocktail δ-integration strategy and identification of the transcription factor PDR3 responsible for lactic acid tolerance in yeast Saccharomyces cerevisiae.

Although, yeast Saccharomyces cerevisiae is expected to be used as a host for lactic acid production, improvement of yeast lactic acid tolerance is required for efficient non-neutralizing fermentation. In this study, we optimized the expression levels of various transcription factors to improve the lactic acid tolerance of yeast by a previously developed cocktail δ-integration strategy. By optimizing the expression levels of various transcription factors, the maximum D-lactic acid production and yield under non-neutralizing conditions were improved by 1.2. and 1.6 times, respectively. Furthermore, overexpression of PDR3, which is known as a transcription factor involved in multi-drug resistance, effectively improved lactic acid tolerance in yeast. In addition, we clarified for the first time that high expression of PDR3 contributes to the improvement of lactic acid tolerance. PDR3 is considered to be an excellent target gene for studies on yeast stress tolerance and further researches are desired in the future.

Construction of yeast producing patchoulol by global metabolic engineering strategy.

Patchoulol is a sesquiterpene alcohol found in the leaves of the patchouli plant that can be extracted by steam distillation. Notably, patchoulol is an essential natural product frequently used in the chemical industry. However, patchouli produces an insignificant amount of patchoulol, not to mention steam distillation, and requires a lot of energy and time. Recombinant microorganisms that can be cultured in mild conditions and can produce patchoulol from renewable biomass resources may be a promising alternative. We previously developed the global metabolic engineering strategy (GMES), which produces a comprehensive metabolic modification in yeast, using the cocktail δ-integration method. In this study, we aimed to produce patchoulol by modifying engineered yeast. The expression of nine genes involved in patchoulol synthesis was modulated using GMES. Regarding patchoulol production, the resultant strain, YPH499/PAT167/MVA442, showed a concentration of 42.1 mg/L, a production rate of 8.42 mg/L/d, and a yield of 2.05 mg/g-glucose, respectably. These concentration values, production rate, and yield obtained through batch-fermentation in this study were high level when compared to previously reported recombinant microorganism studies. GMES could be used as a potential strategy for producing secondary metabolites from plants in recombinant Saccharomyces cerevisiae.

Construction of lactic acid-tolerant Saccharomyces cerevisiae by using CRISPR-Cas-mediated genome evolution for efficient D-lactic acid production.

Lactic acid (LA) is chemically synthesized or fermentatively produced using glucose as substrate, mainly using lactic acid bacteria. Polylactic acid is used as a biodegradable bioplastic for packaging materials, medical materials, and filaments for 3D printers. In this study, we aimed to construct a LA-tolerant yeast to reduce the neutralization cost in LA production. The pHLA2-51 strain was obtained through a previously developed genome evolution strategy, and transcriptome analysis revealed the gene expression profile of the mutant yeast. Furthermore, the expression of the genes associated with glycolysis and the LA synthesis pathway in the LA-tolerant yeast was comprehensively and randomly modified to construct a D-LA-producing, LA-tolerant yeast. In detail, DNA fragments expressing thirteen genes, HXT7, HXK2, PGI1, PFK1, PFK2, FBA1, TPI1, TDH3, PGK1, GPM1, ENO2, and PYK2, and D-lactate dehydrogenase (D-LDH) from Leuconostoc mesenteroides were randomly integrated into the genomic DNA in the LA-tolerant yeast. The resultant engineered yeast produced about 33.9 g/L of D-LA from 100 g/L glucose without neutralizing agents in a non-neutralized condition and 52.2 g/L of D-LA from 100 g/L glucose with 20 g/L CaCO3 in a semi-neutralized condition. Our research provides valuable insights into non-neutralized fermentative production of LA. KEY POINTS: • Lactic acid (LA) tolerance of yeast was improved by genome evolution. • The transcription levels of 751 genes were changed under LA stress. • Rapid LA production with semi-neutralization was achieved by modifying glycolysis. • A versatile yeast strain construction method based on the CRISPR system was proposed.

Chemical treatments for modification and immobilization to improve the solvent-stability of lipase.

Lipase is a lipolytic enzyme that catalyzes the hydrolysis of lipids and esterification reactions. Lipase has been utilized in industrial uses, food processing, and therapeutic applications as a biocatalyst. However, substrates of lipase are often insoluble in water, and this problem limits its utility. Lipases are also used in organic solvents where the solvent-stability of lipase is an important factor. There is a huge number of approaches that can be undertaken to improve the organic solvent-stability of lipases. For example, screening of solvent-tolerant lipase in nature and direct evolution of lipase using genetic engineering are some of the employed approaches. Here, we focus on approaches based on the chemical treatment of lipases for modification and immobilization. The solvent-stability of lipase was improved by the attachment of other molecules, such as surfactants, polymers, and carbohydrates. The immobilization of the enzyme is been known to be an effective approach for not only recycling the enzyme but also its stabilization. Several reports have demonstrated that the solvent-stability of lipase is also improved by immobilization. In this review, we provide an overview of the approaches used to improve the solvent-stability of lipase.

CRISPR system in the yeast Saccharomyces cerevisiae and its application in the bioproduction of useful chemicals.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) immune systems in bacteria have been used as tools for genome engineering. Thus far, the CRISPR-Cas system has been used in various yeast, bacterial, and mammalian cells. Saccharomyces cerevisiae is a nonpathogenic yeast, classified under "generally recognized as safe", and has long been used to produce consumables such as alcohol or bread. Additionally, recombinant cells of S. cerevisiae have been constructed and used to produce various bio-based chemicals. Some types of CRISPR-Cas system for genetic manipulation have been constructed during the early developmental stages of the CRISPR-Cas system and have been mainly used for gene knock-in and knock-out manipulations. Thereafter, these systems have been used for various novel purposes such as metabolic engineering and tolerance engineering. In this review, we have summarized different aspects of the CRISPR-Cas in the yeast S. cerevisiae, from its basic principles to various applications. This review describes the CRISPR system in S. cerevisiae based on the differences in its origin and efficiency followed by its basic applications; for example, its involvement in gene knock-in and knock-out has been outlined. Finally, advanced applications of the CRISPR system in the bioproduction of useful chemicals have been summarized.

Toward the construction of a technology platform for chemicals production from methanol: D-lactic acid production from methanol by an engineered yeast Pichia pastoris.

With the reduction in oil reserves and steady increases in the price of oil, alternative carbon sources like methanol are promising, but an efficient conversion process to fuels and other chemicals is still desired. In this study, we demonstrated for the first time the production of lactic acid from methanol using a lactate dehydrogenase copy number amplifying strategy in Pichia pastoris. We engineered methylotrophic yeast (Pichia pastoris) producing D-lactic acid by D-lactate dehydrogenase gene (d-LDH) integration into the non-transcribed spacer of the ribosomal DNA (rDNA) locus and post-transformational amplification. The resultant engineered strains GS115/S8/Z3 and GS115/S16/Z3 produced 3.48 and 3.26 g/L of D-lactic acid from methanol, respectively, in a 96-h test tube fermentation. To our knowledge, this is the first report about D-lactic acid production from methanol by an engineered P. pastoris strain. The technique of gene integration into the rDNA locus and post-transformational gene amplification could be useful for metabolic engineering in P. pastoris, and the chemical production from methanol by engineered P. pastoris represents a promising industrial technology.

Rhodium-Catalyzed C-H Activation Enabled by an Indium Metalloligand.

Rhodium complexes with an indium metalloligand were successfully synthesized by utilizing a pyridine-tethered cyclopentadienyl ligand as a support for an In-Rh bond. The indium metalloligand dramatically changes the electronic and redox properties of the rhodium metal, thereby enabling catalysis of sp2 C-H bond activation.

Rapid and stable production of 2,3-butanediol by an engineered Saccharomyces cerevisiae strain in a continuous airlift bioreactor.

Utilization of renewable feedstocks for the production of bio-based bulk chemicals, such as 2,3-butanediol (2,3-BDO), by engineered strains of the non-pathogenic yeast, Saccharomyces cerevisiae, has recently become an attractive option. In this study, to realize rapid production of 2,3-BDO, a flocculent, 2,3-BDO-producing S. cerevisiae strain YPH499/dPdAdG/BDN6-10/FLO1 was constructed from a previously developed 2,3-BDO-producing strain. Continuous 2,3-BDO fermentation was carried out by the flocculent strain in an airlift bioreactor. The strain consumed more than 90 g/L of glucose, which corresponded to 90% of the input, and stably produced more than 30 g/L of 2,3-BDO over 380 h. The maximum 2,3-BDO productivity was 7.64 g/L/h at a dilution rate of 0.200/h, which was higher than the values achieved by continuous fermentation using pathogenic bacteria in the previous reports. These results demonstrate that continuous 2,3-BDO fermentation with flocculent 2,3-BDO-producing S. cerevisiae is a promising strategy for practical 2,3-BDO production.

Transporter engineering in biomass utilization by yeast.

Biomass resources are attractive carbon sources for bioproduction because of their sustainability. Many studies have been performed using biomass resources to produce sugars as carbon sources for cell factories. Expression of biomass hydrolyzing enzymes in cell factories is an important approach for constructing biomass-utilizing bioprocesses because external addition of these enzymes is expensive. In particular, yeasts have been extensively engineered to be cell factories that directly utilize biomass because of their manageable responses to many genetic engineering tools, such as gene expression, deletion and editing. Biomass utilizing bioprocesses have also been developed using these genetic engineering tools to construct metabolic pathways. However, sugar input and product output from these cells are critical factors for improving bioproduction along with biomass utilization and metabolic pathways. Transporters are key components for efficient input and output activities. In this review, we focus on transporter engineering in yeast to enhance bioproduction from biomass resources.

Enhanced d-lactic acid production by recombinant Saccharomyces cerevisiae following optimization of the global metabolic pathway.

Utilization of renewable feedstocks for the production of bio-based chemicals such as d-lactic acid by engineering metabolic pathways in the yeast Saccharomyces cerevisiae has recently become an attractive option. In this study, to realize efficient d-lactic acid production by S. cerevisiae, the expression of 12 glycolysis-related genes and the Leuconostoc mesenteroides d-LDH gene was optimized using a previously developed global metabolic engineering strategy, and repeated batch fermentation was carried out using the resultant strain YPH499/dPdA3-34/DLDH/1-18. Stable d-lactic acid production through 10 repeated batch fermentations was achieved using YPH499/dPdA3-34/DLDH/1-18. The average d-lactic acid production, productivity, and yield with 10 repeated batch fermentations were 60.3 g/L, 2.80 g/L/h, and 0.646, respectively. The present study is the first report of the application of a global metabolic engineering strategy for bio-based chemical production, and it shows the potential for efficient production of such chemicals by global metabolic engineering of the yeast S. cerevisiae. Biotechnol. Bioeng. 2017;114: 2075-2084. © 2017 Wiley Periodicals, Inc.

Improvement of lipid production by the oleaginous yeast Rhodosporidium toruloides through UV mutagenesis.

Oleaginous yeasts are considered a promising alternative lipid source for biodiesel fuel production. In this study, we attempted to improve the lipid productivity of the oleaginous yeast Rhodosporidium toruloides through UV irradiation mutagenesis and selection based on ethanol and H2O2 tolerance or cerulenin, a fatty acid synthetase inhibitor. Glucose consumption, cell growth, and lipid production of mutants were evaluated. The transcription level of genes involved in lipid production was also evaluated in mutants. The ethanol and H2O2 tolerant strain 8766 2-31M and the cerulenin resistant strain 8766 3-11C were generated by UV mutagenesis. The 8766 2-31M mutant showed a higher lipid production rate, and the 8766 3-11C mutant produced a larger amount of lipid and had a higher lipid production rate than the wild type strain. Transcriptional analysis revealed that, similar to the wild type strain, the ACL1 and GND1 genes were expressed at significantly low levels, whereas IDP1 and ME1 were highly expressed. In conclusion, lipid productivity in the oleaginous yeast R. toruloides was successfully improved via UV mutagenesis and selection. The study also identified target genes for improving lipid productivity through gene recombination.

Eudistomin C, an Antitumor and Antiviral Natural Product, Targets 40S Ribosome and Inhibits Protein Translation.

Eudistomin C (EudiC), a natural product, shows potent antitumor and antiviral activities, but the target molecule and the mechanism of action remain to be revealed. Here, we show that the 40S ribosome is the target in EudiC cytotoxicity. We isolated EudiC-resistant mutants from a multidrug-sensitive yeast strain, and a genetic analysis classified these YER (yeast EudiC resistance) mutants into three complementation groups. A genome-wide study revealed that the YER1-6 mutation is in the uS11 gene (RPS14A). Biotinylated EudiC pulled down Rps14p-containing complexes from 40S and 80S ribosomes, but not from the 60S ribosome. EudiC strongly inhibited translation of the wild-type strain but not of YER1-6 in cells and in vitro. These results indicate that EudiC is a protein synthesis inhibitor targeting the uS11-containing ribosomal subunit, and shows cytotoxicity by inhibiting protein translation.