Enhanced mating-type switching and sexual hybridization in heterothallic yeast Yarrowia lipolytica.

Yarrowia lipolytica is a non-conventional, heterothallic, oleaginous yeast with wide range of industrial applications. Increasing ploidy can improve advantageous traits for industrial applications including genetic stability, stress resistance, and productivity, but the construction of knockout mutant strains from polyploid cells requires significant effort due to the increased copy numbers of target genes. The goal of this study was to evaluate the effectiveness of a mating-type switching strategy by single-step transformation without a genetic manipulation vestige, and to optimize the conventional method for increasing ploidy (mating) in Y. lipolytica. In this study, mating-type genes in haploid Y. lipolytica cells were scarlessly converted into the opposite type genes by site-specific homologous recombination, and the resulting MATB-type cells were mated at low temperature (22°C) with addition of sodium citrate with each MATA-type haploid cell to yield a MATA/MATB-type diploid strain with genetic information from both parental strains. The results of this study can be used to increase ploidy and for whole genome engineering of a yeast strain with unparalleled versatility for industrial application.

Characterization of the newly isolated ω-oxidizing yeast Candida sorbophila DS02 and its potential applications in long-chain dicarboxylic acid production.

α, ω-Dicarboxylic acids (DCAs) are multipurpose chemicals widely used in polymers, perfumes, plasticizers, lubricants, and adhesives. The biotransformation of DCAs from alkanes and fatty acids by microorganisms has attracted recent interest, since synthesis via chemical oxidation causes problems in terms of the environment and safety. We isolated an ω-oxidizing yeast from a wastewater disposal facility of a petrochemical factory by chemostat enrichment culture. The haploid strain identified as Candida sorbophila DS02 grew on glucose and dodecane, exhibiting greater cell shrinkage on the latter. In flask cultures with mixed alkanes (C10-16) and fatty acid methyl esters (C10-16), DS02 used mixed alkanes simultaneously unlike Candida tropicalis and Yarrowia lipolytica and showed high substrate resistance. In flask cultures with acrylic acid-a known inhibitor of ß-oxidation-DS02 produced 0.28 g/l dodecanedioic acid (DDDA) from dodecane, similar to wild-type C. tropicalis ATCC 20336. In fed-batch fermentation, DS02 produced 9.87 g/l DDDA, which was 5.7-fold higher than wild-type C. tropicalis. These results suggest that C. sorbophila strain DS02 has potential applications for the large-scale production of DCA.

Genome-scale metabolic modeling and in silico analysis of lipid accumulating yeast Candida tropicalis for dicarboxylic acid production.

Recently, the bio-production of α,ω-dicarboxylic acids (DCAs) has gained significant attention, which potentially leads to the replacement of the conventional petroleum-based products. In this regard, the lipid accumulating yeast Candida tropicalis, has been recognized as a promising microbial host for DCA biosynthesis: it possess the unique ω-oxidation pathway where the terminal carbon of α-fatty acids is oxidized to form DCAs with varying chain lengths. However, despite such industrial importance, its cellular physiology and lipid accumulation capability remain largely uncharacterized. Thus, it is imperative to better understand the metabolic behavior of this lipogenic yeast, which could be achieved by a systems biological approach. To this end, herein, we reconstructed the genome-scale metabolic model of C. tropicalis, iCT646, accounting for 646 unique genes, 945 metabolic reactions, and 712 metabolites. Initially, the comparative network analysis of iCT646 with other yeasts revealed several distinctive metabolic reactions, mainly within the amino acid and lipid metabolism including the ω-oxidation pathway. Constraints-based flux analysis was, then, employed to predict the in silico growth rates of C. tropicalis which are highly consistent with the cellular phenotype observed in glucose and xylose minimal media chemostat cultures. Subsequently, the lipid accumulation capability of C. tropicalis was explored in comparison with Saccharomyces cerevisiae, indicating that the formation of "citrate pyruvate cycle" is essential to the lipid accumulation in oleaginous yeasts. The in silico flux analysis also highlighted the enhanced ability of pentose phosphate pathway as NADPH source rather than malic enzyme during lipogenesis. Finally, iCT646 was successfully utilized to highlight the key directions of C. tropicalis strain design for the whole cell biotransformation application to produce long-chain DCAs from alkanes. Biotechnol. Bioeng. 2016;113: 1993-2004. © 2016 Wiley Periodicals, Inc.

1.7 GHz long-term evolution radiofrequency electromagnetic field with stable power monitoring and efficient thermal control has no effect on the proliferation of various human cell types.

Long-term evolution (LTE) radiofrequency electromagnetic field (RF-EMF) is widely used in communication technologies. Thus, the influence of RF-EMF on biological systems is a major public concern and its physiological effects remain controversial. In our previous study, we showed that continuous exposure of various human cell types to 1.7 GHz LTE RF-EMF at a specific absorption rate (SAR) of 2 W/Kg for 72 h can induce cellular senescence. To understand the precise cellular effects of LTE RF-EMF, we elaborated the 1.7 GHz RF-EMF cell exposure system used in the previous study by replacing the RF signal generator and developing a software-based feedback system to improve the exposure power stability. This refinement of the 1.7 GHz LTE RF-EMF generator facilitated the automatic regulation of RF-EMF exposure, maintaining target power levels within a 3% range and a constant temperature even during the 72-h-exposure period. With the improved experimental setup, we examined the effect of continuous exposure to 1.7 GHz LTE RF-EMF at up to SAR of 8 W/Kg in human adipose tissue-derived stem cells (ASCs), Huh7, HeLa, and rat B103 cells. Surprisingly, the proliferation of all cell types, which displayed different growth rates, did not change significantly compared with that of the unexposed controls. Also, neither DNA damage nor cell cycle perturbation was observed in the 1.7 GHz LTE RF-EMF-exposed cells. However, when the thermal control system was turned off and the subsequent temperature increase induced by the RF-EMF was not controlled during continuous exposure to SAR of 8 W/Kg LTE RF-EMF, cellular proliferation increased by 35.2% at the maximum. These observations strongly suggest that the cellular effects attributed to 1.7 GHz LTE RF-EMF exposure are primarily due to the induced thermal changes rather than the RF-EMF exposure itself.

Biosynthesis of aliphatic plastic monomers with amino residues in Yarrowia lipolytica.

Introduciton: The α,ω-diamines (NH2-(CH2)n-NH2) and ω -amino fatty acids (NH2-(CH2)n-COOH) have been widely used as building blocks in polymerindustries. Medium- to long-chain (C8 to C18) fatty acid monomers with amino residues are almost exclusively produced via chemical processes that generate hazardous waste and induce severe environmental problems, such as global warming and pollution. Here, we present the construction platformstrains of Yarrowia lipolytica a cheese-ripening yeast, for direct biotransformation of hydrocarbons into medium- to long-chain α,ω-diamines and ωamino fatty acids using metabolic engineering of endogenous fatty acid ω- and ß-oxidation pathways and introducing heterologous ω-transaminase in Y. lipolytica. Methods: We deleted six genes encoding the acyl-CoA oxidase (ACO1-6) and four fatty aldehyde dehydrogenase genes (FALDH1-4), which catalyze fatty acid ß-oxidation and downstream oxidation of fatty aldehydes in Y. lipolytica, respectively. The ω-transaminase from Chromobacterium violaceum DSM30191 was introduced into the genome of the ΔPOX ΔFALDH strain under the control of Y. lipolytica-derived EXP1 promoters. Results and Discussion: The ΔPOX ΔFALDH strains with ω-CvTA successfully accumulated the corresponding C12 αω-diamines into a shaking culture medium with dodecane or dodecanol. In addition, these strains accumulated C12 ω-amino fatty acids from dodecanoic acid. With the commercially available α,ω-diacid bioprocess, this yeast biosynthesis producing medium- and longchain α,ω-diamines and ω-amino fatty acids could complete the yeast platform technology generating all medium- and long-chain aliphatic polyamide monomers, α,ω-biofunctionalized with one or both carboxylic acid and amino residues.

Whole-cell biocatalysis using cytochrome P450 monooxygenases for biotransformation of sustainable bioresources (fatty acids, fatty alkanes, and aromatic amino acids).

Cytochrome P450s (CYPs) are heme-thiolated enzymes that catalyze the oxidation of CH bonds in a regio and stereoselective manner. Activation of the non-activated carbon atom can be further enhanced by multistep chemo-enzymatic reactions; moreover, several useful chemicals can be synthesized to provide alternative organic synthesis routes. Given their versatile functionality, CYPs show promise in a number of biotechnological fields. Recently, various CYPs, along with their sequences and functionalities, have been identified owing to rapid developments in sequencing technology and molecular biotechnology. In addition to these discoveries, attempts have been made to utilize CYPs to industrially produce biochemicals from available and sustainable bioresources such as oil, amino acids, carbohydrates, and lignin. Here, these accomplishments, particularly those involving the use of CYP enzymes as whole-cell biocatalysts for bioresource biotransformation, will be reviewed. Further, recently developed biotransformation pathways that result in gram-scale yields of fatty acids and fatty alkanes as well as aromatic amino acids, which depend on the hosts used for CYP expression, and the nature of the multistep reactions will be discussed. These pathways are similar regardless of whether the hosts are CYP-producing or non-CYP-producing; the limitations of these methods and the ways to overcome them are reviewed here.

Development of a promising microbial platform for the production of dicarboxylic acids from biorenewable resources.

BACKGROUND: As a sustainable industrial process, the production of dicarboxylic acids (DCAs), used as precursors of polyamides, polyesters, perfumes, plasticizers, lubricants, and adhesives, from vegetable oil has continuously garnered interest. Although the yeast Candida tropicalis has been used as a host for DCA production, additional strains are continually investigated to meet productivity thresholds and industrial needs. In this regard, the yeast Wickerhamiella sorbophila, a potential candidate strain, has been screened. However, the lack of genetic and physiological information for this uncommon strain is an obstacle that merits further research. To overcome this limitation, we attempted to develop a method to facilitate genetic recombination in this strain and produce high amounts of DCAs from methyl laurate using engineered W. sorbophila. RESULTS: In the current study, we first developed efficient genetic engineering tools for the industrial application of W. sorbophila. To increase homologous recombination (HR) efficiency during transformation, the cell cycle of the yeast was synchronized to the S/G2 phase using hydroxyurea. The HR efficiency at POX1 and POX2 loci increased from 56.3% and 41.7%, respectively, to 97.9% in both cases. The original HR efficiency at URA3 and ADE2 loci was nearly 0% during the early stationary and logarithmic phases of growth, and increased to 4.8% and 25.6%, respectively. We used the developed tools to construct W. sorbophila UHP4, in which ß-oxidation was completely blocked. The strain produced 92.5 g/l of dodecanedioic acid (DDDA) from methyl laurate over 126 h in 5-l fed-batch fermentation, with a productivity of 0.83 g/l/h. CONCLUSIONS: Wickerhamiella sorbophila UHP4 produced more DDDA methyl laurate than C. tropicalis. Hence, we demonstrated that W. sorbophila is a powerful microbial platform for vegetable oil-based DCA production. In addition, by using the developed genetic engineering tools, this emerging yeast could be used for the production of a variety of fatty acid derivatives, such as fatty alcohols, fatty aldehydes, and ω-hydroxy fatty acids.

Complete genome sequence of the sulfur-oxidizing chemolithoautotrophic Sulfurovum lithotrophicum 42BKTT.

A sulfur-oxidizing chemolithoautotrophic bacterium, Sulfurovum lithotrophicum 42BKTT, isolated from hydrothermal sediments in Okinawa, Japan, has been used industrially for CO2 bio-mitigation owing to its ability to convert CO2 into C5H8NO4- at a high rate of specific mitigation (0.42 g CO2/cell/h). The genome of S. lithotrophicum 42BKTT comprised of a single chromosome of 2217,891 bp with 2217 genes, including 2146 protein-coding genes and 54 RNA genes. Here, we present its complete genome-sequence information, including information about the genes encoding enzymes involved in CO2 fixation and sulfur oxidation.