Xylose utilization stimulates mitochondrial production of isobutanol and 2-methyl-1-butanol in Saccharomyces cerevisiae

Abstract

Background Branched-chain higher alcohols (BCHAs), including isobutanol and 2-methyl-1-butanol, are promising advanced biofuels, superior to ethanol due to their higher energy density and better compatibility with existing gasoline infrastructure. Compartmentalizing the isobutanol biosynthetic pathway in yeast mitochondria is an effective way to produce BCHAs from glucose. However, to improve the sustainability of biofuel production, there is great interest in developing strains and processes to utilize lignocellulosic biomass, including its hemicellulose component, which is mostly composed of the pentose xylose. Results In this work, we rewired the xylose isomerase assimilation and mitochondrial isobutanol production pathways in the budding yeast Saccharomyces cerevisiae. We then increased the flux through these pathways by making gene deletions of BAT1, ALD6, and PHO13, to develop a strain (YZy197) that produces as much as 4 g/L of BCHAs (3.10 ± 0.18 g isobutanol/L and 0.91 ± 0.02 g 2-methyl-1-butanol/L) from xylose. This represents approximately a 28-fold improvement on the highest isobutanol titers obtained from xylose previously reported in yeast and the first report of 2-methyl-1-butanol produced from xylose. The yield of total BCHAs is 57.2 ± 5.2 mg/g xylose, corresponding to ~ 14% of the maximum theoretical yield. Respirometry experiments show that xylose increases mitochondrial activity by as much as 7.3-fold compared to glucose. Conclusions The enhanced levels of mitochondrial BCHA production achieved, even without disrupting ethanol byproduct formation, arise mostly from xylose activation of mitochondrial activity and are correlated with slow rates of sugar consumption.

Xylose assimilation enhances the production of isobutanol in engineered Saccharomyces cerevisiae

“…In a separate companion publication, a yeast strain engineered with a mitochondrial isobutanol production pathway that instead consumes xylose via the isomerase pathway produces as much as 3.10 ± 0.18 g/L isobutanol, as well as 0.91 ± 0.02 g/L of 2‐methyl‐1‐butanol (Y. Zhang et. al., )…”

“…In a separate companion publication, a yeast strain engineered with a mitochondrial isobutanol production pathway that instead consumes xylose via the isomerase pathway produces as much as 3.10 ± 0.18 g/L isobutanol, as well as 0.91 ± 0.02 g/L of 2-methyl-1-butanol (Y. Zhang et. al., 2019) Xylose assimilation redirects carbon flux from ethanol to isobutanol in SR8-Iso, a trend that would likely be amplified if ethanol biosynthesis was genetically disrupted.…”

Metabolic engineering considerations for the heterologous expression of xylose-catabolic pathways in Saccharomyces cerevisiae

“…MK335957) were used which are known to have the highest catalytic activities among the same group of enzymes tested [30,31]. Because acetaldehyde dehydrogenase encoded by the ALD6 gene plays a major role in acetate accumulation [32], and because acetate is detrimental to xylose metabolism of the oxidoreductase strains [3] as well as the isomerase strains [33,34], the ALD6 gene was often selected as knockout target for xylose strains [35,36]. In the present study, therefore, the xylose pathway genes, XYL1-XYL2 or xylA, were genome-integrated by replacing the ALD6 gene by a Cas9-based genome integration strategy, resulting in the XYL12 (ald6::XYL1-XYL2) and the XI (ald6::xylA) strains, respectively.…”

Genetically encoded biosensors for branched-chain amino acid metabolism to monitor mitochondrial and cytosolic production of isobutanol and isopentanol in yeast

“…We next applied the isobutanol biosensor to isolate the highest isobutanol-producers from a library of strains containing random combinations of genes from the mitochondrial isobutanol biosynthesis pathway 24 . Equal molar amounts of cassettes containing either the upstream ILV genes ( ILV2, ILV3, and ILV5 ), the downstream Ehrlich pathway (KDC and ADH), or the full isobutanol pathway, all designed to randomly integrate into YARCdelta5 δ -sites 24, 49 , were pooled and used to transform strain YZy81, containing the isobutanol biosensor in the HIS3 locus ( Supplementary Tables 1 and 2 ). The transformed population was subjected to two rounds of fluorescence activated cell sorting (FACS, see Methods) followed by fermentations with 24 randomly picked colonies from each population (unsorted, first round sorted, and second round sorted).…”

“…Deletions of BAT1 , BAT2 , ILV6 , LEU4 , LEU9 , ILV3, TMA29, PDC1 , PDC5 , PDC6 , and GAL80 were obtained using PCR-based homologous recombination. DNA fragments containing lox71- and lox66-flanked antibiotic resistance cassettes were amplified with PCR from pYZ55 (containing the hygromycin resistance gene hphMX4), pYZ17 (containing the G418 resistance gene KanMX), or pYZ84 (containing the nourseothricin resistance gene NAT1), using primers with 50-70 base pairs of homology to regions upstream and downstream of the ORF of the gene targeted for deletion 49 . Transformation of the gel-purified PCR fragments was performed using the lithium acetate method 93 .…”