Metabolic engineering of Saccharomyces cerevisiae for production of isobutanol and UV-absorbing chemical shinorine

Abstract

Saccharomyces cerevisiae is considered as promising host for production of biofuels and chemicals because it is a well-studied eukaryotic model system with high stress tolerance and robustness in harsh industrial conditions. In this dissertation, several strategies were developed and applied to produce isobutanol and shinorine in S. cerevisiae.<br/> Firstly, S. cerevisiae CEN.PK2-1C, a leucine auxotrophic strain having a LEU2 gene mutation, was engineered for the production of isobutanol and 3-methyl-1-butanol. An ALD6 encoding aldehyde dehydrogenase and BAT1 involved in valine synthesis were deleted to eliminate competing pathways. Transcription of endogenous genes in the valine and leucine biosynthetic pathways was also increased by expressing Leu3Δ601, a constitutively active form of Leu3 transcriptional activator. For the production of isobutanol, genes involved in isobutanol production (ILV2, ILV3, ILV5, ARO10, and ADH2) were additionally overexpressed in ald6Δbat1Δ strain expressing LEU3Δ601, resulting in 376.9 mg/L isobutanol production from 100 g/L glucose. To increase 3-methyl-1-butanol production, leucine biosynthetic genes were additionally overexpressed in the final isobutanol-production strain. The resulting strain overexpressing LEU2 and LEU4D578Y, a feedback inhibition-insensitive mutant of LEU4, showed a 34-fold increase in 3-methyl-1-butanol synthesis compared with CEN.PK2-1C control strain, producing 765.7 mg/L 3-methyl-1-butanol.<br/> Secondly, mitochondrial isobutanol production was improved by increasing mitochondrial pool of pyruvate, a key substrate for isobutanol production. Subcellular compartmentalization of the biosynthetic enzymes is one of the limiting factors for isobutanol production in S. cerevisiae. Previously, it has been shown that mitochondrial compartmentalization of the biosynthetic pathway through re-locating cytosolic Ehrlich pathway enzymes into the mitochondria can increase isobutanol production. Mitochondrial isobutanol biosynthetic pathway was introduced into bat1Δald6Δlpd1Δ strain, where genes involved in competing pathways were deleted, and MPC1, MPC2, and MPC3 genes encoding the subunits of mitochondrial pyruvate carrier (MPC) hetero-oligomeric complex were overexpressed with different combinations. Overexpression of Mpc1 and Mpc3 forming high-affinity MPCOX was more effective in improving isobutanol production than overexpression of Mpc1 and Mpc2 forming low-affinity MPCFERM. The final engineered strain overexpressing MPCOX produced 338.3 mg/L isobutanol from 20 g/L glucose, exhibiting about 22-fold increase in production compared with wild type. Furthermore, to increase in Ilv3 activity, Nfs1 and Isd11genes, encoding cysteine desulfurase involved in iron-sulfur cluster assembly, were overexpressed, resulting in improved isobutanol production up to 435.2 mg/L.<br/> Thirdly, isobutanol production was improved via construction of artificial cytosolic biosynthetic pathway by multi-copy integration system in S. cerevisiae. α-acetolactate synthase (ALS) is the key enzyme redirecting pyruvate flux to isobutanol production by competing with pyruvate decarboxylase (PDC) involved in ethanol production. To improve isobutanol production using the major pyruvate pool in the cytosol, cytosolic isobutanol biosynthetic pathway was constructed by overexpressing heterologous ALS (alsS) from Bacillus subtilis and Lactococcus lactis, and N-terminally truncated ILV5 (ILV5ΔN48) and ILV3 (ILV3ΔN19) lacking mitochondrial targeting signal with kozak sequence. Since overexpression of alsS from B. subtilis under the control of strong promoter promoted cell death, copper-inducible promoter, PCUP1, was used to overexpress alsS. Cytosolic isobutanol biosynthetic pathway was constructed via delta- and rDNA-integration which are powerful tools for random multi-copy gene integration in S. cerevisiae, especially coupled with antibiotic selection. Multi-copy integration of alsS was screened by using antibiotic markers and also by selecting clones showing growth defects upon alsS induction by copper. The final engineered strain (JHY43D25-4) additionally overexpressing ILV5ΔN48, ILV3ΔN19, 2-ketoacid decarboxylase (kivd), and alcohol dehydrogenase (Adh2) produced 265.5 mg/L isobutanol, exhibiting about 4.3-fold increase in production compared to control strain JHY43.<br/> Lastly, S. cerevisiae was used as a host for the heterologous production of a UV-absorbing sunscreen material shinorine. By introducing heterologous shinorine biosynthetic genes from cyanobacteria, Nostoc punctiforme and Anabaena variabilis, into S. cerevisiae, yeast strain capable of producing shinorine was successfully constructed. Furthermore, to increase the pool of sedoheptulose 7-phosphate (S7P), an intermediate in pentose phosphate pathway used for shinorine production, xylose assimilation genes, xylose reductase (XYL1), xylitol dehydrogenase (XYL2), and xylulokinase (XYL3) were introduced to use xylose as a carbon source. In a fed-batch fermentation, the engineered JHYS17-1 strain produced 64.2 mg/L shinorine with highest content (14.3 mg/gDCW) ever reported in microbes. In addition, deletion of competing pathway producing erythrose-4-phosphate and fructose-6-phosphate from S7P, and overexpression of transcriptional factor (Stb5) for genes involved in pentose phosphate pathway and transketolase (Tkl1), contributed to enhancing shinorine production.<br/>