Yarrowia
lipolytica
is a novel microbial chassis
to upgrade renewable low-cost carbon feedstocks to high-value commodity
chemicals and natural products. In this work, we systematically characterized
and removed the rate-limiting steps of the shikimate pathway and achieved
de novo
synthesis of five aromatic chemicals in
Y. lipolytica
. We determined that eliminating amino
acids formation and engineering feedback-insensitive DAHP synthases
are critical steps to mitigate precursor competition and relieve the
feedback regulation of the shikimate pathway. Further overexpression
of heterologous phosphoketolase and deletion of pyruvate kinase provided
a sustained metabolic driving force that channels E4P (erythrose 4-phosphate)
and PEP (phosphoenolpyruvate) precursors through the shikimate pathway.
Precursor competing pathways and byproduct formation pathways were
also blocked by inactivating chromosomal genes. To demonstrate the
utility of our engineered chassis strain, three natural products,
2-phenylethanol (2-PE),
p
-coumaric acid, and violacein,
which were derived from phenylalanine, tyrosine, and tryptophan, respectively,
were chosen to test the chassis performance. We obtained 2426.22 ±
48.33 mg/L of 2-PE, 593.53 ± 28.75 mg/L of
p
-coumaric acid, 12.67 ± 2.23 mg/L of resveratrol, 366.30 ±
28.99 mg/L of violacein, and 55.12 ± 2.81 mg/L of deoxyviolacein
from glucose in a shake flask. The 2-PE production represents a 286-fold
increase over the initial strain (8.48 ± 0.50 mg/L). Specifically,
we obtained the highest 2-PE, violacein, and deoxyviolacein titer
ever reported from the
de novo
shikimate pathway
in yeast. These results set up a new stage of engineering
Y. lipolytica
as a sustainable biorefinery chassis
strain for
de novo
synthesis of aromatic compounds
with economic values.
Yarrowia lipolytica is an oleaginous yeast that has been substantially engineered for production of oleochemicals and drop-in transportation fuels. The unique acetyl-CoA/malonyl-CoA supply mode along with the versatile carbon-utilization pathways makes this yeast a superior host to upgrade low-value carbons into high-value secondary metabolites and fatty acid-based chemicals. The expanded synthetic biology toolkits enabled us to explore a large portfolio of specialized metabolism beyond fatty acids and lipid-based chemicals. In this review, we will summarize the recent advances in genetic, omics, and computational tool development that enables us to streamline the genetic or genomic modification for Y. lipolytica. We will also summarize various metabolic engineering strategies to harness the endogenous acetyl-CoA/malonyl-CoA/HMG-CoA pathway for production of complex oleochemicals, polyols, terpenes, polyketides, and commodity chemicals. We envision that Y. lipolytica will be an excellent microbial chassis to expand nature’s biosynthetic capacity to produce plant secondary metabolites, industrially relevant oleochemicals, agrochemicals, commodity, and specialty chemicals and empower us to build a sustainable biorefinery platform that contributes to the prosperity of a bio-based economy in the future.
Metabolic addiction, an organism that is metabolically addicted with a compound to maintain its growth fitness, is an underexplored area in metabolic engineering. Microbes with heavily engineered pathways or genetic circuits tend to experience metabolic burden leading to degenerated or abortive production phenotype during long-term cultivation or scale-up. A promising solution to combat metabolic instability is to tie up the end-product with an intermediary metabolite that is essential to the growth of the producing host. Here we present a simple strategy to improve both metabolic stability and pathway yield by coupling chemical addiction with negative autoregulatory genetic circuits. Naringenin and lipids compete for the same precursor malonyl-CoA with inversed pathway yield in oleaginous yeast. Negative autoregulation of the lipogenic pathways, enabled by CRISPRi and fatty acid-inducible promoters, repartitions malonyl-CoA to favor flavonoid synthesis and increased naringenin production by 74.8%. With flavonoid-sensing transcriptional activator FdeR and yeast hybrid promoters to control leucine synthesis and cell grwoth fitness, this amino acid feedforward metabolic circuit confers a flavonoid addiction phenotype that selectively enrich the naringenin-producing pupulation in the leucine auxotrophic yeast. The engineered yeast persisted 90.9% of naringenin titer up to 324 generations. Cells without flavonoid addiction regained growth fitness but lost 94.5% of the naringenin titer after cell passage beyond 300 generations. Metabolic addiction and negative autoregulation may be generalized as basic tools to eliminate metabolic heterogeneity, improve strain stability and pathway yield in long-term and large-scale bioproduction.
Efficient microbial
synthesis of chemicals requires the coordinated
supply of precursors and cofactors to maintain cell growth and product
formation. Substrates with different entry points into the metabolic
network have different energetic and redox statuses. Generally, substrate
cofeeding could bypass the lengthy and highly regulated native metabolism
and facilitates high carbon conversion rate. Aiming to efficiently
synthesize the high-value rose-smell 2-phenylethanol (2-PE) in Y. lipolytica, we analyzed the stoichiometric constraints
of the Ehrlich pathway and identified that the selectivity of the
Ehrlich pathway and the availability of 2-oxoglutarate are the rate-limiting
factors. Stepwise refactoring of the Ehrlich pathway led us to identify
the optimal catalytic modules consisting of l-phenylalanine
permease, ketoacid aminotransferase, phenylpyruvate decarboxylase,
phenylacetaldehyde reductase, and alcohol dehydrogenase. On the other
hand, mitochondrial compartmentalization of 2-oxoglutarate inherently
creates a bottleneck for efficient assimilation of l-phenylalanine,
which limits 2-PE production. To improve 2-oxoglutarate (aKG) trafficking
across the mitochondria membrane, we constructed a cytosolic aKG source
pathway by coupling a bacterial aconitase with a native isocitrate
dehydrogenase (ylIDP2). Additionally, we also engineered dicarboxylic
acid transporters to further improve the 2-oxoglutarate availability.
Furthermore, by blocking the precursor-competing pathways and mitigating
fatty acid synthesis, the engineered strain produced 2669.54 mg/L
of 2-PE in shake flasks, a 4.16-fold increase over the starting strain.
The carbon conversion yield reaches 0.702 g/g from l-phenylalanine,
95.0% of the theoretical maximal. The reported work expands our ability
to harness the Ehrlich pathway for production of high-value aromatics
in oleaginous yeast species.
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