Limonene
is an important plant natural product widely used in food
and cosmetics production as well as in the pharmaceutical and chemical
industries. However, low efficiency of plant extraction and high energy
consumption in chemical synthesis limit the sustainability of industrial
limonene production. Recently, the advancement of metabolic engineering
and synthetic biology has facilitated the engineering of microbes
into microbial cell factories for producing limonene. However, the
deleterious effects on cellular activity by the toxicity of limonene
is the major obstacle in achieving high-titer production of limonene
in engineered microbes. In this study, by using transcriptomics, we
identified 82 genes from the nonconventional yeast Yarrowia
lipolytica that were up-regulated when exposed to limonene.
When overexpressed, 8 of the gene candidates improved tolerance of
this yeast to exogenously added limonene. To determine whether overexpression
of these genes could also improve limonene production, we individually
coexpressed the tolerance-enhancing genes with a limonene synthase
gene. Indeed, expression of 5 of the 8 candidate genes enhanced limonene
production in Y. lipolytica. Particularly, overexpressing
YALI0F19492p led to an 8-fold improvement in product titer. Furthermore,
through short-term adaptive laboratory evolution strategy, in combination
with morphological and cytoplasmic membrane integrity analysis, we
shed light on the underlying mechanism of limonene cytotoxicity to Y. lipolytica. This study demonstrated an effective
strategy for improving limonene tolerance of Y. lipolytica and limonene titer in the host strain through the combinatorial
use of tolerance engineering and evolutionary engineering.
Metabolic engineering of yeasts for terpenoid production has mostly focused on the cytoplasm, whereas harnessing their organelles as subcellular factories has been overlooked. Herein, the farnesyl diphosphate synthetic pathway and α-bisabolene synthase were compartmentalized into the oleaginous yeast Yarrowia lipolytica's mitochondria to enable high-level α-bisabolene production. Through comprehensive metabolic engineering approaches, we exploited the potential and capability of the mitochondria as a subcellular factory to achieve 257.4 mg/L of α-bisabolene production from glucose. By combining mitochondrial and cytoplasmic engineering, we further boosted the α-bisabolene titer to 765.1 mg/L by utilizing waste cooking oil as the sole carbon source. Finally, the α-bisabolene titer of the resulting strain reached 1058.1 mg/L in a 5 L bioreactor, which is the highest titer in the engineered Y. lipolytica cell factory reported to date. Overall, our study has provided valuable insights into the mitochondrial engineering of Y. lipolytica for sustainable and green production of valuable compounds.
Itaconic acid (IA) is a high-value organic acid with a plethora of industrial applications. In this study, we seek to develop a microbial cell factory that could utilize waste cooking oil (WCO) as raw material for circular and cost-effective production of the abovementioned biochemical. Specifically, we expressed cis-aconitic acid decarboxylase (CAD) gene from Aspergillus terreus in either the cytosol or peroxisome of Yarrowia lipolytica and assayed for production of IA on WCO. To further improve production yield, the 10 genes involved in the production pathway of acetyl-CoA, an intermediate metabolite necessary for the synthesis of cis-aconitic acid, were individually overexpressed and investigated for their impact on IA production. To minimize off-target flux channeling, we had also knocked out genes related to competing pathways in the peroxisome. Impressively, IA titer up to 54.55 g/L was achieved in our engineered Y. lipolytica in a 5 L bioreactor using WCO as the sole carbon source.
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