45[Noda Institute for Scientific Research GRANT]2006 Young Investigator Research Grant
Outline of Research Result

Creation and Characterization of Lactic Acid Tolerant Yeasts
for Efficient Production of Lactic Acid
Minetaka SUGIYAMA
Department of Biotechnology, Graduate School of Engineering, Osaka University

Research aim
Plant- and crop-based renewable plastics including polylactic acid have been receiving increasing attention as carbon neutral materials that contribute to reduced atmospheric CO2 emissions. Saccharomyces cerevisiae can growand survive at a pH lower than the lactic acid bacteria thatare widely used for production of lactic acid as a monomer
for polylactic acid. This offers the potential to reduce costbecause the process of desalination of lactate is costly. A
genetically-engineered S. cerevisiae strain was recentlyevaluated for efficient production of L-lactic acid and wasfound to produce high levels in the absence of pH control1).
However, it was also observed that production was lower at
pH values of 2.8. To increase production of lactic acidwithout a requirement for pH control, the acid tolerance ofS. cerevisiae must be enhanced. To this end, research wasundertaken to isolate lactic acid-tolerant strains using thefollowing approaches. i) Isolation and characterization ofmulticopy genes that enhance lactic acid tolerance, ii) evaluation of a screening method to select strains with enhanced tolerance by chromosome splitting, and iii) identification of genes required for lactic acid resistance.
Methods
A yeast genomic DNA library in a YEp13 multicopy
vector (ATCC 37323) was used to screen for genes conferring lactic acid resistance when overexpressed. Chromosome splitting was performed according to a PCR-mediatedChromosome Splitting (PCS) method2) that allows repeatedsplitting of chromosomes by a 2-step PCR procedure followed by a single transformation. Lactic acid tolerance andsensitivity were assayed by spotting cells on YPDA media
containing 6.5% and 4% (w/v) L-lactic acid, respectively.
Resultsi) Isolation and characterization of genes that enhance
lactic acid tolerance when overexpressed.
Based on the premise that specific overexpressed genes
will confer protection from damage caused by high concentrations of lactic acid, tolerant multicopy transformants of asensitive parent strain were sought. S. cerevisiae strain
BY4742 was transformed with the YEp13-based multi-copy genomic library and transformants were screened fortolerance to 6% lactic acid (pH 2.6). Of over 60,000 transformants screened, 61 tolerant strains were isolated. Three
genomic regions in plasmids were identified that conferredlactic acid tolerance. Subcloning revealed that WHI2,HAA1, and ESBP6 were responsible for the lactic acid tolerance. WHI2, HAA1, and ESBP6 encode a factor required
for full activation of the general stress response, a transcription factor for genes encoding multidrug resistance permeases, and a monocarboxylate permease-like protein, respectively.
To examine whether WHI2, HAA1, and ESBP6 are required for normal tolerance to lactic acid, we constructed
Dwhi2, Dhaa1, and Desbp6 strains and analyzed the effectof WHI2, HAA1, and ESBP6 disruptions on 5% lactic acidmedia. The disruption mutants exhibited growth defects on
5% lactic acid media although no growth defect was seen in
the absence of lactic acid. To determine if these genes also
confer increased tolerance to other stresses, multicopy
transformants were exposed to heat shock, oxidative (H2O2)stress, acetic acid, ethanol, and high salt (NaCl). Only transformants with multicopy HAA1 exhibited acetic acid tolerance, suggesting that WHI2, HAA1, and ESBP6 are specifically implicated in the acid stress response.
HAA1 and ESBP6 alleles were constructed under thecontrol of the TDH3 promoter and were expressed at theirrespective chromosomal loci. The resulting strains exhibited lactic acid tolerance, suggesting that overproduction ofproteins encoded by HAA1 and ESBP6 confer tolerance,
likely resulting from hyperactivation of the lactic acid stressresponse.
ii) Evaluation of a chromosome splitting-based screening
method to select strains highly tolerant to lactic acid.
We and others have identified gene disruption mutants
that exhibit lactic acid tolerance by screening the S. cerevisiae nonessential gene knockout collection. A chromosome splitting method was applied to evaluate the effect of
combining deletions of SED1, DSE2, EAF3, SCW11, and
IRC8 that individually confer high lactic acid tolerance.
Short chromosomal fragments (7–31 kb), containing theindividual genes noted above were converted into minichromosomes by splitting. A yeast strain having 5 minichromosomes was constructed and sub-cultured in media containing lactic acid to induce minichromosome loss based on the46

observation that chromosomes less than 50 kb exhibit highmitotic instability. Cultures were plated on 4% lactic acidmedia and clones giving rise to the largest colonies were selected and evaluated for loss events. Of the 50 clones tested
thus far, all were found to have lost only the IRC8-containing minichromosome and to exhibit modest lactic acid tolerance relative to wild-type. However, a Dsed1 Ddse2
Deaf3 Dscw11 Dirc8 quintuple disruptant constructed as acontrol strain by conventional methods was found to havehigher lactic acid tolerance than a Dirc8 single disruptant,suggesting that the other minichromosomes contained
genes required for lactic acid tolerance and thus were notlost.
iii) Identification of genes required for lactic acid resistance.
In order to identify additional genes required for lactic
acid tolerance, a S. cerevisiae nonessential gene knockoutcollection was screened for sensitivity to 4% lactic acid.
Disruption of more than 180 genes was found to result indifferential sensitivity to 4% lactic acid. Although the genes
found necessary for resistance are functionally diverse, they
were also found to function in shared processes, including
telomere maintenance, vacuole organization and acidification, vesicle-mediated transport, signal transduction, chromatin modification, and lipid metabolism. These results
suggest that these functional units comprise the lactic acidstress response pathway, and that functional enhancementof these components may further improve tolerance.
Conclusions
Three approaches were used to isolate and construct lac-tic acid tolerant strains of S. cerevisiae. i) Overexpressionof WHI2, HAA1, and ESBP6 was found to enhance lactic
acid tolerance. Stable lactic acid tolerant strains were constructed by chromosomal integration of constitutively-expressed alleles of HAA1 and ESBP6 under the control of theTDH3 promoter. Evaluation of lactic acid production by
these strains is in progress. ii) A screening method to selectthe most tolerant strains was evaluated by means of combinatorial loss of minichromosomes harboring 4 to 26 genes,including the five genes whose disruption was found to result in enhanced lactic acid tolerance. Because only one
minichromosome was found to have been lost in strains recovered in the lactic acid tolerance screen, it was presumed
that all the other minichromosomes harbored genes necessary for tolerance, indicating a quantitative trait. iii) Screening of a S. cerevisiae nonessential gene knockout collection
revealed that more than 180 genes are involved in conferring lactic acid resistance. These new findings not only provide a better understanding of the cellular response to lactic
acid, but will also contribute to efforts to breed strains withincreased tolerance for the purpose of using S. cerevisiae toproduce lactic acid.
References1)

Ishida, N., Saitoh, S., Tokuhiro, K., Nagamori, E., Matsuyama, T.,
Kitamoto, K., and Takahashi, H. Efficient production of L-lactic acidby metabolically engineered Saccharomyces cerevisiae with agenome-integrated L-lactate dehydrogenase gene. Appl. Environ. Microbiol. 71(4): 1964–1970 (2005).
2) Sugiyama, M., Ikushima, S., Nakazawa, T., Kaneko, Y., and Harashima, S. PCR-mediated repeated chromosome splitting in Saccharomyces cerevisiae. Biotechniques 38(6): 909–914 (2005).