Jung Hoon Sohn

Genome sequence of the thermotolerant yeast Kluyveromyces marxianus var. marxianus KCTC 17555.

ABSTRACT Kluyveromyces marxianus is a thermotolerant yeast that has been explored for potential use in biotechnological applications, such as production of biofuels, single-cell proteins, enzymes, and other heterologous proteins. Here, we present the high-quality draft of the 10.9-Mb genome of K. marxianus var. marxianus KCTC 17555 (= CBS 6556 = ATCC 26548).

Optimization of an acetate reduction pathway for producing cellulosic ethanol by engineered yeast

Xylose fermentation by engineered Saccharomyces cerevisiae expressing NADPH‐linked xylose reductase (XR) and NAD+‐linked xylitol dehydrogenase (XDH) suffers from redox imbalance due to cofactor difference between XR and XDH, especially under anaerobic conditions. We have demonstrated that coupling of an NADH‐dependent acetate reduction pathway with surplus NADH producing xylose metabolism enabled not only efficient xylose fermentation, but also in situ detoxification of acetate in cellulosic hydrolysate through simultaneous co‐utilization of xylose and acetate. In this study, we report the highest ethanol yield from xylose (0.463 g ethanol/g xylose) by engineered yeast with XR and XDH through optimization of the acetate reduction pathway. Specifically, we constructed engineered yeast strains exhibiting various levels of the acetylating acetaldehyde dehydrogenase (AADH) and acetyl‐CoA synthetase (ACS) activities. Engineered strains exhibiting higher activities of AADH and ACS consumed more acetate and produced more ethanol from a mixture of 20 g/L of glucose, 80 g/L of xylose, and 8 g/L of acetate. In addition, we performed environmental and genetic perturbations to further improve the acetate consumption. Glucose‐pulse feeding to continuously provide ATPs under anaerobic conditions did not affect acetate consumption. Promoter truncation of GPD1 and gene deletion of GPD2 coding for glycerol‐3‐phosphate dehydrogenase to produce surplus NADH also did not lead to improved acetate consumption. When a cellulosic hydrolysate was used, the optimized yeast strain (SR8A6S3) produced 18.4% more ethanol and 41.3% less glycerol and xylitol with consumption of 4.1 g/L of acetate than a control strain without the acetate reduction pathway. These results suggest that the major limiting factor for enhanced acetate reduction during the xylose fermentation might be the low activities of AADH and ACS, and that the redox imbalance problem of XR/XDH pathway can be exploited for in situ detoxification of acetic acid in cellulosic hydrolysate and increasing ethanol productivity and yield. Biotechnol. Bioeng. 2016;113: 2587–2596.

GroE chaperonins assisted functional expression of bacterial enzymes in Saccharomyces cerevisiae.

Rapid advances in the capabilities of reading and writing DNA along with increasing understanding of microbial metabolism at the systems‐level have paved an incredible path for metabolic engineering. Despite these advances, post‐translational tools facilitating functional expression of heterologous enzymes in model hosts have not been developed well. Some bacterial enzymes, such as Escherichia coli xylose isomerase (XI) and arabinose isomerase (AI) which are essential for utilizing cellulosic sugars, cannot be functionally expressed in Saccharomyces cerevisiae. We hypothesized and demonstrated that the mismatching of the HSP60 chaperone systems between bacterial and eukaryotic cells might be the reason these bacterial enzymes cannot be functionally expressed in yeast. The results showed that the co‐expression of E. coli GroE can facilitate the functional expression of E. coli XI and AI, as well as the Agrobacterium tumefaciens D‐psicose epimerase in S. cerevisiae. The co‐expression of bacterial chaperonins in S. cerevisiae is a promising post‐translational strategy for the functional expression of bacterial enzymes in yeast. Biotechnol. Bioeng. 2016;113: 2149–2155.

Metabolic Engineering of Probiotic Saccharomyces boulardii

ABSTRACT Saccharomyces boulardii is a probiotic yeast that has been used for promoting gut health as well as preventing diarrheal diseases. This yeast not only exhibits beneficial phenotypes for gut health but also can stay longer in the gut than Saccharomyces cerevisiae. Therefore, S. boulardii is an attractive host for metabolic engineering to produce biomolecules of interest in the gut. However, the lack of auxotrophic strains with defined genetic backgrounds has hampered the use of this strain for metabolic engineering. Here, we report the development of well-defined auxotrophic mutants (leu2, ura3, his3, and trp1) through clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9-based genome editing. The resulting auxotrophic mutants can be used as a host for introducing various genetic perturbations, such as overexpression or deletion of a target gene, using existing genetic tools for S. cerevisiae. We demonstrated the overexpression of a heterologous gene (lacZ), the correct localization of a target protein (red fluorescent protein) into mitochondria by using a protein localization signal, and the introduction of a heterologous metabolic pathway (xylose-assimilating pathway) in the genome of S. boulardii. We further demonstrated that human lysozyme, which is beneficial for human gut health, could be secreted by S. boulardii. Our results suggest that more sophisticated genetic perturbations to improve S. boulardii can be performed without using a drug resistance marker, which is a prerequisite for in vivo applications using engineered S. boulardii.

Direct fermentation of Jerusalem artichoke tuber powder for production of l-lactic acid and d-lactic acid by metabolically engineered Kluyveromyces marxianus

An efficient production system for optically pure l- and d-lactic acid (LA) from Jerusalem artichoke tuber powder (JAP) was developed by metabolic engineering of Kluyveromyces marxianus. To construct LA-producing strains, the ethanol fermentation pathway of K. marxianus was redirected to LA production by disruption of KmPDC1 and expression of l- and d-lactate dehydrogenase (LDH) genes derived from Lactobacillus plantarum under the control of the K. marxianus translation elongation factor 1α promoter. To further increase the LA titer, the l-LA and d-LA consumption pathway of host strains was blocked by deletion of the oxidative LDH genes KmCYB2 and KmDLD1. The recombinant strains produced 130g/L l-LA and 122g/L d-LA by direct fermentation from 230g/L JAP containing 140g/L inulin, without pretreatment or nutrient supplementation. The conversion efficiency and optical purity were ≫>95% and ≫>99%, respectively. This system using JAP and the inulin-assimilating yeast K. marxianus could lead to a cost-effective process for the production of LA.

Low-pH production of d-lactic acid using newly isolated acid tolerant yeast Pichia kudriavzevii NG7: PARK et al.

Lactic acid is a platform chemical for the sustainable production of various materials. To develop a robust yeast platform for low‐pH production of d‐lactic acid (LA), an acid‐tolerant yeast strain was isolated from grape skins and named Pichia kudriavzevii NG7 by ribosomal RNA sequencing. This strain could grow at pH 2.0 and 50°C. For the commercial application of P. kudriavzevii NG7 as a lactic acid producer, the ethanol fermentation pathway was redirected to lactic acid by replacing the pyruvate decarboxylase 1 gene (PDC1) with the d‐lactate dehydrogenase gene (d‐LDH) derived from Lactobacillus plantarum. To enhance lactic acid tolerance, this engineered strain was adapted to high lactic acid concentrations, and a new transcriptional regulator, PAR1, responsible for acid tolerance, was identified by whole‐genome resequencing. The final engineered strain produced 135 g/L and 154 g/L of d‐LA with productivity over 3.66 g/L/hr at pH 3.6 and 4.16 g/L/hr at pH 4.7, respectively.

Draft genome sequence of an acid-tolerant yeast, Candida zemplinina NP2, a potential producer of organic acids

ABSTRACT Here, we report the draft genome sequence of the acid-tolerant yeast Candida zemplinina NP2, which was isolated from peach peels. This genome sequence will aid in the understanding of the organism’s physiological properties as a potential producer of organic acids in acidic environments.