Disruption of PHO13 improves ethanol production via the xylose isomerase pathway

Xylose is the second most abundant sugar in lignocellulosic materials and can be converted to ethanol by recombinant Saccharomyces cerevisiae yeast strains expressing heterologous genes involved in xylose assimilation pathways. Recent research demonstrated that disruption of the alkaline phosphatase gene, PHO13, enhances ethanol production from xylose by a strain expressing the xylose reductase (XR) and xylitol dehydrogenase (XDH) genes; however, the yield of ethanol is poor. In this study, PHO13 was disrupted in a recombinant strain harboring multiple copies of the xylose isomerase (XI) gene derived from Orpinomyces sp., coupled with overexpression of the endogenous xylulokinase (XK) gene and disruption of GRE3, which encodes aldose reductase. The resulting YΔGP/XK/XI strain consumed 2.08 g/L/h of xylose and produced 0.88 g/L/h of volumetric ethanol, for an 86.8 % theoretical ethanol yield, and only YΔGP/XK/XI demonstrated increase in cell concentration. Transcriptome analysis indicated that expression of genes involved in the pentose phosphate pathway (GND1, SOL3, TAL1, RKI1, and TKL1) and TCA cycle and respiratory chain (NDE1, ACO1, ACO2, SDH2, IDH1, IDH2, ATP7, ATP19, SDH4, SDH3, CMC2, and ATP15) was upregulated in the YΔGP/XK/XI strain. And the expression levels of 125 cell cycle genes were changed by deletion of PHO13. Electronic supplementary material The online version of this article (doi:10.1186/s13568-015-0175-7) contains supplementary material, which is available to authorized users.


Table S1
Down-regulated cell cycle genes after 9 h on oxygen-limited fermentation of xylose. Fold change (p < 0.05) is the ratio of expression in YΔGP/XK/XI relative to YΔG/XK/XI Gene name Fold change Annotated function TAH11 1.86 "DNA replication licensing factor, required for pre-replication complex assembly" [YJR046W] BFA1 1.62 "Component of the GTPase-activating Bfa1p-Bub2p complex involved in multiple cell cycle checkpoint pathways that control exit from mitosis" [YJR053W] CLB4 1.55 "B-type cyclin involved in cell cycle progression; activates Cdc28p to promote the G2/M transition; may be involved in DNA replication and spindle assembly; accumulates during S phase and G2, then targeted for ubiquitin-mediated degradation" [YLR210W] DOC1 1.54 "Processivity factor required for the ubiquitination activity of the anaphase promoting complex (APC), mediates the activity of the APC by contributing to substrate recognition; involved in cyclin proteolysis; contains a conserved DOC1 homology domain" [YGL240W] MET30 1.50 "F-box protein containing five copies of the WD40 motif, controls cell cycle function, sulfur metabolism, and methionine biosynthesis as part of the ubiquitin ligase complex; interacts with and regulates Met4p, localizes within the nucleus" [YIL046W] CDC20 1.47 "Cell-cycle regulated activator of anaphase-promoting complex/cyclosome (APC/C), which is required for metaphase/anaphase transition; directs ubiquitination of mitotic cyclins, Pds1p, and other anaphase inhibitors; potential Cdc28p substrate" [YGL116W] ZIP2 1.43 "Meiosis-specific protein involved in normal synaptonemal complex formation and pairing between homologous chromosomes during meiosis" [YGL249W] SPR28 1.42 "Sporulation-specific homolog of the yeast CDC3/10/11/12 family of bud neck microfilament genes; meiotic septin expressed at high levels during meiotic divisions and ascospore formation" [ 1.20 "Kinetochore protein of unknown function; associated with the essential kinetochore proteins Nnf1p and Spc24p; phosphorylated by both Clb5-Cdk1 and, to a lesser extent, Clb2-Cdk1." [YFR046C] CEF1 1.20 "Essential splicing factor; associated with Prp19p and the spliceosome, contains an N-terminal c-Myb DNA binding motif necessary for cell viability but not for Prp19p association, evolutionarily conserved and homologous to S. pombe Cdc5p" [YMR213W] RFC3 1.19 "Subunit of heteropentameric Replication factor C (RF-C), which is a DNA binding protein and ATPase that acts as a clamp loader of the proliferating cell nuclear antigen (PCNA) processivity factor for DNA polymerases delta and epsilon" [YNL290W] SCC4 1.19 "Subunit of cohesin loading factor (Scc2p-Scc4p), a complex required for the loading of cohesin complexes onto chromosomes; involved in establishing sister chromatid cohesion during double-strand break repair via phosphorylated histone H2AX" [YER147C] MCM22 1.18 "Protein involved in minichromosome maintenance; component of the kinetochore; binds to centromeric DNA in a Ctf19p-dependent manner" [YJR135C] 14 "G1 cyclin involved in regulation of the cell cycle; activates Cdc28p kinase to promote the G1 to S phase transition; late G1 specific expression depends on transcription factor complexes, MBF (Swi6p-Mbp1p) and SBF (Swi6p-Swi4p)" [YMR199W]

PCL1
6.72 "Cyclin, interacts with cyclin-dependent kinase Pho85p; member of the Pcl1,2-like subfamily, involved in the regulation of polarized growth and morphogenesis and progression through the cell cycle; localizes to sites of polarized cell growth" [YNL289W]     3.85 "G1 cyclin involved in regulation of the cell cycle; activates Cdc28p kinase to promote the G1 to S phase transition; late G1 specific expression depends on transcription factor complexes, MBF (Swi6p-Mbp1p) and SBF (Swi6p-Swi4p)" [YPL256C]

CLB6
3.28 "B-type cyclin involved in DNA replication during S phase; activates Cdc28p to promote initiation of DNA synthesis; functions in formation of mitotic spindles along with Clb3p and Clb4p; most abundant during late G1" [YGR109C]

MCD1
3.10 "Essential subunit of the cohesin complex required for sister chromatid cohesion in mitosis and meiosis; apoptosis induces cleavage and translocation of a C-terminal fragment to mitochondria; expression peaks in S phase" [YDL003W]

CDC5
2.56 "Polo-like kinase with multiple functions in mitosis and cytokinesis through substrate phosphorylation, also functions in adaptation to DNA damage during meiosis; has similarity to Xenopus Plx1 and S. 1.65 "Protein involved in bud-site selection and required for axial budding pattern; localizes with septins to bud neck in mitosis and may constitute an axial landmark for next round of budding" [YCL014W]

MOB2
1.62 "Activator of Cbk1p kinase; component of the RAM signaling network that regulates cellular polarity and morphogenesis; activation of Cbk1p facilitates the Ace2p-dependent daughter cell-specific transcription of genes involved in cell separation; similar to Mob1p" [YFL034C-B]

AMN1
1.59 "Protein required for daughter cell separation, multiple mitotic checkpoints, and chromosome stability; contains 12 degenerate leucine-rich repeat motifs; expression is induced by the Mitotic Exit Network (MEN)" [YBR158W]

RAD53
1.59 "Protein kinase, required for cell-cycle arrest in response to DNA damage; activated by trans autophosphorylation when interacting with hyperphosphorylated Rad9p; also interacts with ARS1 and plays a role in initiation of DNA replication" [YPL153C]

BUD9
1.56 "Protein involved in bud-site selection; mutant has increased aneuploidy tolerance; diploid mutants display a unipolar budding pattern instead of the wild-type bipolar pattern, and bud at the distal pole" [YGR041W]

YCG1
1.55 "Subunit of the condensin complex; required for establishment and maintenance of chromosome condensation, chromosome segregation and chromatin binding of the condensin complex; required for clustering of tRNA genes at the nucleolus" [YDR325W]

NUD1
1.53 "Component of the spindle pole body outer plaque, required for exit from mitosis" [YOR373W] PDS5 1.52 "Protein required for establishment and maintenance of sister chromatid condensation and cohesion, colocalizes with cohesin on chromosomes, may function as a protein-protein interaction scaffold; also required during meiosis" [YMR076C]

ELG1
1.52 "Subunit of an alternative replication factor C complex important for DNA replication and genome integrity; suppresses spontaneous DNA damage; involved in homologous recombination-mediated repair and telomere homeostasis" [YOR144C]

RSR1
1.51 "GTP-binding protein of the ras superfamily required for bud site selection, morphological changes in response to mating pheromone, and efficient cell fusion; localized to the plasma membrane; significantly similar to mammalian Rap GTPases" 1.44 "Essential subunit of the Dam1 complex (aka DASH complex), couples kinetochores to the force produced by MT depolymerization thereby aiding in chromosome segregation; is transferred to the kinetochore prior to mitosis" [YBR233W-A] IPL1 1.44 "Aurora kinase subunit of the conserved chromosomal passenger complex (CPC; Ipl1p-Sli15p-Bir1p-Nbl1p), involved in regulating kinetochore-microtubule attachments; helps maintain condensed chromosomes during anaphase and early telophase" [YPL209C]

SHE1
1.41 "Mitotic spindle protein that interacts with components of the Dam1 (DASH) complex, its effector Sli15p, and microtubule-associated protein Bim1p; also localizes to nuclear microtubules and to the bud neck in a ring-shaped structure" [YBL031W]

SLK19
1.38 "Kinetochore-associated protein required for normal segregation of chromosomes in meiosis and mitosis; component of the FEAR regulatory network, which promotes Cdc14p release from the nucleolus during anaphase; potential Cdc28p substrate" [YOR195W]

CDC10
1.37 "Component of the septin ring that is required for cytokinesis; septins recruit proteins to the mother-bud neck and can act as a barrier to diffusion at the membrane, and they comprise the 10 nm filaments seen with EM; required for the transition from a single to double septin ring" [YCR002C]

CDC3
1.34 "Component of the septin ring of the mother-bud neck that is required for cytokinesis; septins recruit proteins to the neck and can act as a barrier to diffusion at the membrane, and they comprise the 10nm filaments seen with EM" [YLR314C]

KAR3
1.34 "Minus-end-directed microtubule motor that functions in mitosis and meiosis, localizes to the spindle pole body and localization is dependent on functional Cik1p, required for nuclear fusion during mating; potential Cdc28p substrate" [YPR141C]

CDC14
1.34 "Protein phosphatase required for mitotic exit; located in the nucleolus until liberated by the FEAR and Mitotic Exit Network in anaphase, enabling it to act on key substrates to effect a decrease in CDK/B-cyclin activity and mitotic exit; required for meiosis I spindle disassembly; released from nucleolus upon entry into anaphase I of meiosis, resequestered in metaphase II, then released again upon entry into anaphase II; maintained in nucleolus by Cdc55p in early meiosis" [YFR028C]

MCM2
1.33 "Protein involved in DNA replication; component of the Mcm2-7 hexameric complex that binds chromatin as a part of the pre-replicative complex" [YBL023C] PAP2 1.33 "Non-canonical poly(A) polymerase, involved in nuclear RNA degradation as a component of the TRAMP complex; catalyzes polyadenylation of hypomodified tRNAs, and snoRNA and rRNA precursors; overlapping but non-redundant functions with Trf5p" [YOL115W]

YCS4
1.33 "Subunit of the condensin complex; required for establishment and maintenance of chromosome condensation, chromosome segregation, chromatin binding of condensin, tRNA gene clustering at the nucleolus, and silencing at the mating type locus" [YLR272C]

RIF1
1.32 "Protein that binds to the Rap1p C-terminus and acts synergistically with Rif2p to help control telomere length and establish telomeric silencing; deletion results in telomere elongation" [YBR275C]

CLB5
1.32 "B-type cyclin involved in DNA replication during S phase; activates Cdc28p to promote initiation of DNA synthesis; functions in formation of mitotic spindles along with Clb3p and Clb4p; most abundant during late G1 phase" [YPR120C]

MMS22
1.32 "Protein that acts with Mms1p in a repair pathway that may be involved in resolving replication intermediates or preventing the damage caused by blocked replication forks; required for accurate meiotic chromosome segregation" [YLR320W]

TEM1
1.32 "GTP-binding protein of the ras superfamily involved in termination of M-phase; controls actomyosin and septin dynamics during cytokinesis" [YML064C] DSE1 1.31 "Daughter cell-specific protein, may regulate cross-talk between the mating and filamentation pathways; deletion affects cell separation after division and sensitivity to alpha-factor and drugs affecting the cell wall" [YER124C]