Metabolic engineering of Saccharomyces cerevisiae: a key cell factory platform for future biorefineries

Abstract

Metabolic engineering is the enabling science of development of efficient cell factories for the production of fuels, chemicals, pharmaceuticals, and food ingredients through microbial fermentations. The yeast Saccharomyces cerevisiae is a key cell factory already used for the production of a wide range of industrial products, and here we review ongoing work, particularly in industry, on using this organism for the production of butanol, which can be used as biofuel, and isoprenoids, which can find a wide range of applications including as pharmaceuticals and as biodiesel. We also look into how engineering of yeast can lead to improved uptake of sugars that are present in biomass hydrolyzates, and hereby allow for utilization of biomass as feedstock in the production of fuels and chemicals employing S. cerevisiae. Finally, we discuss the perspectives of how technologies from systems biology and synthetic biology can be used to advance metabolic engineering of yeast.

Fig. 1

Illustration of the biorefinery concept and the development time of novel bioprocesses. a In a biorefinery, plant-based feed-stocks such as sugarcane, corn, wheat, or biomass are converted into sugars that are subsequently used for microbial fermentations. In the fermentation process, cell factories convert the sugars into fuels and chemicals. b The development of cell factories is the central research and development process in connection with the development of a novel bioprocess. Construction of an efficient cell factory requires large investment, in particular in connection with bringing the cell factory from proof-of-principle stage where it is producing small amounts of the desired product to a final strain that produces the product at yields, titers, and productivities that make the process financially competitive with fossil fuel-based processes

Fig. 2

Range of products and illustration of the key research problems associated with cell factory design and development. a Biotech products range from high-value-added to low-value-added products, with the latter being produced in large quantities and the former in small quantities. Examples of the different types of products are indicated. The yeast S. cerevisiae is used for the production of products in the whole spectrum. b In connection with the development of yeast for the production of different types of products using different sugars as feedstock, there is a need for an extensive platform of technologies from synthetic and systems biology

Fig. 3

Illustration of relevant substrates that have been considered for yeast fermentation. Heterologous enzymes that are currently applied are summarized for non-utilizable carbon sources in S. cerevisiae such as polymers (cellulose, starch, xylan), disaccharide (cellobiose, lactose, melibiose), pentose sugar (xylose, arabinose). In case of galactose, which is utilized slowly compared to glucose, over-expression targets of innate enzymes for improving galactose availability were screened

Fig. 4

Overview of pathways for pentose utilization covered by patent applications of DSM and Royal Nedalco. d-xylose and l-arabinose can be utilized by two pathways: (1) aldose reductase NADPH-dependent and (2) isomerase cofactors-independent. In case of the latter, requirement of cofactor balance is eliminated and enhancing activity of isomerase remains main issue. Two Dutch companies, DSM and Royal Nedalco, claim over-expression of heterologous xylose isomerases from Thermotoga maritime MSB8 and Piromyces sp. E2 (ATCC 76762), respectively. Xylulokinase and enzymes in pentose phosphate pathway were also amplified simultaneously. Arabinose genes in isomerase pathway such as l-arabinose isomerase, l-ribulokinase, and l-ribulose-5P 4-epimerase originated from Lactobacillus plantarum in DSM and Arthrobacter aurescens, Clavibacter michiganensis, Gramella forsetii in Royal Nedalco were amplified with enzymes in pentose phosphate pathways

Fig. 5

Illustration of Gevo’s strategies for n-butanol and isobutanol production in the cytosol [90, 91, 105]. a n-butanol production was attempted by amplification of heterologous genes such as the pyruvate dehydrogenase multienzyme complex (lpdA, aceE, aceF) from E. coli for increasing the cytosolic acetyl-CoA pool, and the genes in butanol synthetic pathway from Clostridia species. Moreover, the activity of pyruvate decarboxylase (PDC) was reduced. b Isobutanol was produced in the cytosol to avoid cofactor balancing in the mitochondria; all the genes in isobutanol pathway were over-expressed in cytosol. Especially, dihydroxyacid dehydratases (DHAD) from Lactococcus lactis and Neurospora crassa were used, which had specific amino sequence, P(I/L)XXXGX(I/L)XIL. Also, the transcriptional activators AFT1/AFT2 were over-expressed to increase DHAD activity

Fig. 6

Gevo’s isobutanol production strategy in the mitochondria [106]. a All enzymes involved in isobutanol synthesis were localized in the mitochondria; KIVD and ADH being in cytosol were expressed in mitochondria with signal sequence. NADPH-dependent enzymes were engineered to an NADH-dependent form, and then NADH was supplied by using an NADH shuttle concept. Acetaldehyde and ethanol produced by fermentation were transported across membranes, and alcohol dehydrogenase in the mitochondria (encoded by ADH3) provided NADH by conversion of ethanol to acetaldehyde. b Isobutyraldehyde was transferred to the cytosol from mitochondria where it is converted to isobutanol under consumption of NADH generated by glycolysis in the cytosol to make more precise cofactor balance

Fig. 7

Butamax’s strategies for isobutanol production in cytosol [102]. Four different pathways for isobutanol production were suggested: (1) pyruvate to isobutanol directly (red arrows), (2) pyruvate through valine bypass (blue arrows), (3) pyruvate through isobutyryl-CoA bypass (blue arrows), and (4) butyryl-CoA to isobutanol (blue arrows). A total of 11 enzyme reactions were considered and at least three to four heterologous enzymes in each step were claimed in patents of Butamax

Fig. 8

Butamax’s isobutanol production strategies in the mitochondria [103]. To block substrate-competing reactions BAT1, ILV1, and LEU4 were deleted and the activity of the E1 alpha subunit of the pyruvate dehydrogenase (PDH) complex (PDA1) was reduced by promoter exchange to a weak one. NADH kinase (POS5) was over-expressed to ensure sufficient supply of NADPH required by the KARI enzyme. Red arrows mean over-expression of genes

Fig. 9

Overview of Amyris metabolic engineering strategies. Industrial strain Saccharomyces cerevisiae PE-2 was used as a production host because of its higher tolerance to the industrial environment [129]. All promoters of mevalonate genes were exchanged to strong one in chromosome. Gray box means the strategies that were used in a scientific article [124] but not in the patent. Red color circles mean even higher expression than other overexpressed genes. Blue color circles mean knock-out of genes or reduction of expression level. Thick arrows mean amplified steps based on plasmids in a scientific article [20]. The dotted arrow indicates reduction of flux