Investigating strain dependency in the production of aromatic compounds in Saccharomyces cerevisiae

Abstract

Although Saccharomyces cerevisiae is the most highly domesticated yeast, strain dependency in biotechnological processes still remains as a common, yet poorly understood phenomenon. To investigate this, the entrance to the aromatic amino acid biosynthetic pathway was compared in four commonly used S. cerevisiae laboratory strains. The strains were engineered to accumulate shikimate by overexpressing a mutant version of the pentafunctional ARO1 enzyme with disrupted activity in the shikimate kinase subunit. Carbon tracing and C metabolic flux analysis combined with quantitative PCR, revealed that precursor availability and shikimate production were dramatically different in the four equally engineered strains, which were found to be correlated with the strains' capacity to deal with protein overexpression burden. By implementing a strain-dependent approach, the genetic platform was reformulated, leading to an increase in yield and titer in all strains. The highest producing strain, INVSc1-SA3, produced 358 mg L of shikimate with a yield of 17.9 mg g These results underline the importance of strain selection in developing biological manufacturing processes, demonstrate the first case of high production of shikimate in yeast, and provide an appropriate platform for strain selection for future production of aromatic compounds. Biotechnol. Bioeng. 2016;113: 2676-2685. © 2016 Wiley Periodicals, Inc.

Metabolic Engineering of the Shikimate Pathway for Production of Aromatics and Derived Compounds—Present and Future Strain Construction Strategies

“…Considering that in general the reactions of the shikimate pathway are thermodynamically favored (Averesch, 2016 ), it can be concluded that limitations are mostly kinetic and/or regulatory. This is supported by a study showing the performance of the shikimiate pathway in S. cerevisiae to be greatly dependent on the type strain (Suástegui et al, 2016 ). In reverse this means that the shikimate pathway is most likely tightly regulated, hence the greatest challenge will be to overcome this, rather than the fine tuning of individual reactions at the final step to the target product.…”

“…This was elaborated in a study combining tktA overexpression with knockout of pykA, pykF and deactivation of the PTS to increase PEP availability, thus reaching on almost 20-fold increase in carbon partitioning to the shikimate pathway (Gosset et al, 1996 ). A recent flux analysis on shikimate production in S. cerevisiae study even concluded that in yeast rather E4P is the rate limiting precursor (Suástegui et al, 2016 ). In a related study, the xylose utilizing yeast Scheffersomyces stipitis ( Pichia stipitis ) was employed, which was also further optimized for E4P formation by overexpressing TKL1 , as well as channeling flux to and accumulating carbon in shikimate (by means of ARO4 K229L and ARO1 D920A ).…”

Bioprocess Optimization for the Production of Aromatic Compounds With Metabolically Engineered Hosts: Recent Developments and Future Challenges

“…One of the main bottlenecks in the microbial production of aromatic compounds is the availability of the precursors PEP (produced during glycolysis) and E4P (derived from the pentose phosphate pathway -PPP) (Suástegui et al, 2016;Noda and Kondo, 2017;Averesch and Krömer, 2018;Wu et al, 2018). Different strategies have been described in order to engineer the central carbon metabolism into this direction (Leonard et al, 2005;Papagianni, 2012;Nielsen and Keasling, 2016).…”

“…In fact, the available fluxes of both precursors differ considerably. Suástegui et al (2016) studied the E4P and PEP flux in S. cerevisiae using metabolic flux analysis and observed that E4P was clearly the limiting precursor. Therefore, establishing a balance between the ratio of both precursors and increasing their availability appeared to be the two main strategies to follow in order to increase aromatic compounds production.…”

CRISPR–Mediated Genome Editing and Gene Repression in Scheffersomyces stipitis

“…While its previous applications were mainly demonstrated as a repository for isolating genes involved in xylose assimilation and transport, its potential as a better‐suited microbial host than Saccharomyces cerevisiae for producing compounds derived from the shikimate pathway was recently proposed . The much more active pentose phosphate pathway associated with the native xylose assimilating ability in S. stipitis renders a higher availability of the precursor erythrose 4‐phosphate (E4P), which was identified as the rate‐limiting precursor of the shikimate pathway in S. cerevisiae . Considering that in plants the downstream products of the shikimate pathway include many kinds of flavonoids and alkaloids with important pharmaceutical and nutraceutical properties, finding a well‐suited microbial chassis that can potentially synthesize these high‐value natural products at sufficient titers is very important.…”