scholarly journals Engineering acetyl-CoA metabolic shortcut for eco-friendly production of polyketides triacetic acid lactone in Yarrowia lipolytica

2019 ◽  
Vol 56 ◽  
pp. 60-68 ◽  
Author(s):  
Huan Liu ◽  
Monireh Marsafari ◽  
Fang Wang ◽  
Li Deng ◽  
Peng Xu
Keyword(s):  
Yarrowia Lipolytica ◽  
Acetyl Coa ◽  
Acid Lactone ◽  
Triacetic Acid Lactone

scholarly journals Rewiring Yarrowia lipolytica toward triacetic acid lactone for materials generation

2018 ◽  
Vol 115 (9) ◽  
pp. 2096-2101 ◽  
Author(s):  
Kelly A. Markham ◽  
Claire M. Palmer ◽  
Malgorzata Chwatko ◽  
James M. Wagner ◽  
Clare Murray ◽  
...  
Keyword(s):  
Yarrowia Lipolytica ◽  
Building Blocks ◽  
Specific Productivity ◽  
Chemical Production ◽  
Conversion Yield ◽  
Acetyl Coa ◽  
Type Iii Polyketide Synthase ◽  
Chemical Catalysis ◽  
Acid Lactone ◽  
Triacetic Acid Lactone

Polyketides represent an extremely diverse class of secondary metabolites often explored for their bioactive traits. These molecules are also attractive building blocks for chemical catalysis and polymerization. However, the use of polyketides in larger scale chemistry applications is stymied by limited titers and yields from both microbial and chemical production. Here, we demonstrate that an oleaginous organism (specifically, Yarrowia lipolytica) can overcome such production limitations owing to a natural propensity for high flux through acetyl–CoA. By exploring three distinct metabolic engineering strategies for acetyl–CoA precursor formation, we demonstrate that a previously uncharacterized pyruvate bypass pathway supports increased production of the polyketide triacetic acid lactone (TAL). Ultimately, we establish a strain capable of producing over 35% of the theoretical conversion yield to TAL in an unoptimized tube culture. This strain also obtained an averaged maximum titer of 35.9 ± 3.9 g/L with an achieved maximum specific productivity of 0.21 ± 0.03 g/L/h in bioreactor fermentation. Additionally, we illustrate that a β-oxidation-related overexpression (PEX10) can support high TAL production and is capable of achieving over 43% of the theoretical conversion yield under nitrogen starvation in a test tube. Next, through use of this bioproduct, we demonstrate the utility of polyketides like TAL to modify commodity materials such as poly(epichlorohydrin), resulting in an increased molecular weight and shift in glass transition temperature. Collectively, these findings establish an engineering strategy enabling unprecedented production from a type III polyketide synthase as well as establish a route through O-functionalization for converting polyketides into new materials.

scholarly journals Engineering acetyl-CoA metabolic shortcut for eco-friendly production of polyketides triacetic acid lactone in Yarrowia lipolytica

2019 ◽  
Author(s):  
Huan Liu ◽  
Monireh Marsafari ◽  
Fang Wang ◽  
Li Deng ◽  
Peng Xu
Keyword(s):  
Acetic Acid ◽  
Yarrowia Lipolytica ◽  
Carbon Flux ◽  
Oleaginous Yeast ◽  
Low Cost ◽  
Scale Production ◽  
Acetyl Coa ◽  
Malonyl Coa ◽  
Acid Lactone ◽  
Triacetic Acid Lactone

AbstractAcetyl-CoA is the central metabolic node connecting glycolysis, Krebs cycle and fatty acids synthase. Plant-derived polyketides, are assembled from acetyl-CoA and malonyl-CoA, represent a large family of biological compounds with diversified bioactivity. Harnessing microbial bioconversion is considered as a feasible approach to large-scale production of polyketides from renewable feedstocks. Most of the current polyketide production platform relied on the lengthy glycolytic steps to provide acetyl-CoA, which inherently suffers from complex regulation with metabolically-costly cofactor/ATP requirements. Using the simplest polyketide triacetic acid lactone (TAL) as a target molecule, we demonstrate that acetate uptake pathway in oleaginous yeast (Yarrowia lipolytica) could function as an acetyl-CoA shortcut to achieve metabolic optimality in producing polyketides. We identified the metabolic bottlenecks to rewire acetate utilization for efficient TAL production in Y. lipolytica, including generation of the driving force for acetyl-CoA, malonyl-CoA and NADPH. The engineered strain, with the overexpression of endogenous acetyl-CoA carboxylase (ACC1), malic enzyme (MAE1) and a bacteria-derived cytosolic pyruvate dehydrogenase (PDH), affords robust TAL production with titer up to 4.76 g/L from industrial glacier acetic acid in shake flasks, representing 8.5-times improvement over the parental strain. The acetate-to-TAL conversion ratio (0.149 g/g) reaches 31.9% of the theoretical maximum yield. The carbon flux through this acetyl-CoA metabolic shortcut exceeds the carbon flux afforded by the native acetyl-CoA pathways. Potentially, acetic acid could be manufactured in large-quantity at low-cost from Syngas fermentation or heterogenous catalysis (methanol carbonylation). This alternative carbon sources present a metabolic advantage over glucose to unleash intrinsic pathway limitations and achieve high carbon conversion efficiency and cost-efficiency. This work also highlights that low-cost acetic acid could be sustainably upgraded to high-value polyketides by oleaginous yeast species in an eco-friendly and cost-efficient manner.

scholarly journals Purification and properties of 6-methylsalicylic acid synthase from Penicillium patulum

1992 ◽  
Vol 288 (3) ◽  
pp. 839-846 ◽  
Author(s):  
J B Spencer ◽  
P M Jordan
Keyword(s):  
Fatty Acid ◽  
Proteinase Inhibitors ◽  
Type I ◽  
Acetyl Coa ◽  
Purification Method ◽  
Methylsalicylic Acid Synthase ◽  
Acid Lactone ◽  
Penicillium Patulum ◽  
Triacetic Acid Lactone

6-Methylsalicylic acid synthase has been isolated in homogeneous form from Penicillium patulum grown in liquid culture from a spore inoculum. The enzyme is highly susceptible to proteolytic degradation in vivo and in vitro, but may be stabilized during purification by incorporating proteinase inhibitors in the buffers. The enzyme exists as a homotetramer of M(r) 750,000, with a subunit M(r) of 180,000. 6-Methylsalicyclic acid synthase also accepts acetoacetyl-CoA as an alternative starter molecule to acetyl-CoA. The enzyme also catalyses the formation of small amounts of triacetic acid lactone as an oligatory by-product of the reaction. In the absence of NADPH, triacetic acid lactone is the exclusive enzymic product, being formed at 10% of the rate of 6-methylsalicylic acid. The enzyme is inactivated by 1,3-dibromopropan-2-one, leading to the formation of cross-linked dimers similar to that observed with type I fatty acid synthases. Acetyl-CoA protects the enzyme against the inactivation and inhibits dimer formation. An adaptation of the purification method for 6-methylsalicylic acid synthase may be used for the isolation of fatty acid sythase from Penicillium patulum.

Mycotoxins - Their Biosynthesis in Fungi: Patulin and Related Carcinogenic Lactones

1979 ◽  
Vol 42 (10) ◽  
pp. 810-814 ◽  
Author(s):  
G. MAURICE GAUCHER
Keyword(s):  
Mycophenolic Acid ◽  
Biosynthetic Pathway ◽  
Current Status ◽  
Penicillic Acid ◽  
Acetyl Coa ◽  
Acid Lactone ◽  
Triacetic Acid Lactone

A brief introduction to the mycotoxin, patulin is followed by a summary of the early contributions which demonstrated that patulin and its precursor, 6-methylsalicylic acid were derived from acetyl-CoA and hence are polyketides. The polyketide lactones, triacetic acid lactone, mycophenolic acid, patulin, penicillic acid and multicolic acid are compared with respect to their biosynthetic origins and the obscured sequence of acetate units in the latter three lactones is discussed. The current status of the patulin biosynthetic pathway is described with special emphasis on recent findings which have altered the late or post-gentisaldehyde portion of the pathway. This new appreciation of the patulin pathway is then extrapolated to the closely related γ-lactones, penicillic acid and multicolic acid. Pathways analogous to that of patulin are proposed for these two lactones and are discussed. These biosynthetic relationships are then related to the co-occurrence of some of these lactones in cultures of the same fungus.

scholarly journals Complete and efficient conversion of plant cell wall hemicellulose into high-value bioproducts by engineered yeast

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Liang Sun ◽  
Jae Won Lee ◽  
Sangdo Yook ◽  
Stephan Lane ◽  
Ziqiao Sun ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Cellulosic Biomass ◽  
Hemicellulose Hydrolysate ◽  
Acetyl Coa ◽  
Engineered Yeast ◽  
Fermentation Inhibitor ◽  
Acid Lactone

AbstractPlant cell wall hydrolysates contain not only sugars but also substantial amounts of acetate, a fermentation inhibitor that hinders bioconversion of lignocellulose. Despite the toxic and non-consumable nature of acetate during glucose metabolism, we demonstrate that acetate can be rapidly co-consumed with xylose by engineered Saccharomyces cerevisiae. The co-consumption leads to a metabolic re-configuration that boosts the synthesis of acetyl-CoA derived bioproducts, including triacetic acid lactone (TAL) and vitamin A, in engineered strains. Notably, by co-feeding xylose and acetate, an enginered strain produces 23.91 g/L TAL with a productivity of 0.29 g/L/h in bioreactor fermentation. This strain also completely converts a hemicellulose hydrolysate of switchgrass into 3.55 g/L TAL. These findings establish a versatile strategy that not only transforms an inhibitor into a valuable substrate but also expands the capacity of acetyl-CoA supply in S. cerevisiae for efficient bioconversion of cellulosic biomass.

scholarly journals Engineering sensitivity and specificity of AraC-based biosensors responsive to triacetic acid lactone and orsellinic acid

2020 ◽  
Vol 33 ◽  
Author(s):  
Zhiqing Wang ◽  
Aarti Doshi ◽  
Ratul Chowdhury ◽  
Yixi Wang ◽  
Costas D Maranas ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Negative Selection ◽  
Regulatory Protein ◽  
Polyketide Synthases ◽  
Computational Framework ◽  
Orsellinic Acid ◽  
Acid Lactone ◽  
Triacetic Acid Lactone ◽  
Protein Redesign ◽  
Background Expression

Abstract We previously described the design of triacetic acid lactone (TAL) biosensor ‘AraC-TAL1’, based on the AraC regulatory protein. Although useful as a tool to screen for enhanced TAL biosynthesis, this variant shows elevated background (leaky) expression, poor sensitivity and relaxed inducer specificity, including responsiveness to orsellinic acid (OA). More sensitive biosensors specific to either TAL or OA can aid in the study and engineering of polyketide synthases that produce these and similar compounds. In this work, we employed a TetA-based dual-selection to isolate new TAL-responsive AraC variants showing reduced background expression and improved TAL sensitivity. To improve TAL specificity, OA was included as a ‘decoy’ ligand during negative selection, resulting in the isolation of a TAL biosensor that is inhibited by OA. Finally, to engineer OA-specific AraC variants, the iterative protein redesign and optimization computational framework was employed, followed by 2 rounds of directed evolution, resulting in a biosensor with 24-fold improved OA/TAL specificity, relative to AraC-TAL1.

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