Fusing α and β subunits of the fungal fatty acid synthase leads to improved production of fatty acids

Wernig, Florian; Born, Sandra; Boles, Eckhard; Grininger, Martin; Oreb, Mislav

doi:10.1038/s41598-020-66629-y

Cited by 11 publications

(22 citation statements)

References 29 publications

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“…To evaluate whether a limitation originates from one of these reactions, we concomitantly overexpressed ZWF1, GND1 , and SOL3 from a high copy plasmid with strong promoters (module named oxPPP [plasmid FWV171]) in FWY45. To test a possibly limiting role of FAS, we overexpressed a superior mutated FAS variant (fusFAS RK ), consisting of a fusion construct of FAS1 RK and FAS2 (Wernig et al, 2020) in addition to the oxPPP module or separately. Surprisingly, the additional expression of fusFAS RK in strain FWY45 did not influence OA production nor did the combined expression of oxPPP and fusFAS RK (Figure 6d).…”

Section: Resultsmentioning

confidence: 99%

Production of octanoic acid in Saccharomyces cerevisiae: Investigation of new precursor supply engineering strategies and intrinsic limitations

Wernig

Baumann

Boles

et al. 2021

Biotech & Bioengineering

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The eight-carbon fatty acid octanoic acid (OA) is an important platform chemical and precursor of many industrially relevant products. Its microbial biosynthesis is regarded as a promising alternative to current unsustainable production methods. In Saccharomyces cerevisiae, the production of OA had been previously achieved by rational engineering of the fatty acid synthase. For the supply of the precursor molecule acetyl-CoA and of the redox cofactor NADPH, the native pyruvate dehydrogenase bypass had been harnessed, or the cells had been additionally provided with a pathway involving a heterologous ATP-citrate lyase. Here, we redirected the flux of glucose towards the oxidative branch of the pentose phosphate pathway and overexpressed a heterologous phosphoketolase/phosphotransacetylase shunt to improve the supply of NADPH and acetyl-CoA in a strain background with abolished OA degradation. We show that these modifications lead to an increased yield of OA during the consumption of glucose by more than 60% compared to the parental strain. Furthermore, we investigated different genetic engineering targets to identify potential factors that limit the OA production in yeast. Toxicity assays performed with the engineered strains suggest that the inhibitory effects of OA on cell growth likely impose an upper limit to attainable OA yields. K E Y W O R D S acetyl-CoA, octanoic acid, phosphoketolase, phosphotransacetylase 1 | INTRODUCTION Fatty acids (FAs) with various chain lengths and their derivatives with different functional groups are important compounds in modern industry, which have numerous applications like fuels, cosmetics, pharmaceuticals, and food additives. Recently, engineering microbial FA production has attracted attention as an alternative to established methods such as petrochemistry or oil palm cultivation, which have lately been criticized for their environmental impact. In Saccharomyces cerevisiae, biosynthesis of FAs in the cytosol is catalyzed by the large multidomain fatty acid synthase (FAS) complex, which naturally generates long-chain fatty acid (LCFA, C14-C18) as building blocks of membranes or for storage lipids. The biosynthesis is initiated by cytosolic acetyl-CoA (AcCoA) and maturing FAs are elongated by AcCoA-derived malonyl-CoA until This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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Section: Resultsmentioning

confidence: 99%

Production of octanoic acid in Saccharomyces cerevisiae: Investigation of new precursor supply engineering strategies and intrinsic limitations

Wernig

Baumann

Boles

et al. 2021

Biotech & Bioengineering

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“…Later, this mutation was transferred to the FAS of S. cerevisiae (R1834K; FAS RK ) which displayed production of mainly OA in vivo (Gajewski et al, 2017). The in the scope of this study developed fusFAS RK and its enhanced expression by strong promoters increases OA production in an otherwise unmodified strain (Wernig et al, 2020b), demonstrating that FAS RK itself is a limiting factor for OA biosynthesis. The reason might be the intrinsic functionality of the FAS RK mutant, which could be operating at its maximum capacity due to reduced MalCoA loading.…”

Section: Current Challenges and Limitations In Octanoic Acid Production By Mutated Fatty Acid Synthasesmentioning

confidence: 87%

“…Because this step may be subject to cellular regulation and may be rate-limiting, the two polypeptides were fused into a single-chain construct. This fused FAS variant was investigated for OA production and showed improved performance to the split version (Wernig et al, 2020b). Furthermore, it should be investigated whether co-expression of the mutated (fused and split) FAS RK , which causes growth impairments in contrast to the wild type (WT) version, and a WT-FAS is beneficial for cell viability and OA production.…”

Section: Aim Of This Thesismentioning

confidence: 99%

“…Therefore, in one publication the engineering for AcCoA and NADPH supply for OA production were evaluated for the first time by different strategies (Wernig et al, 2021). For further improvement of OA production one publication deals with optimization of the mutated FAS by fusing the distinct β-chain (FAS1) and the α-chain (FAS2) (fusFAS RK ), which increased production compared to the separate expression of the FAS genes (FAS1 RK /FAS2) (Wernig et al, 2020b). Many FA derived compounds such as ω-HyFA are valuable industrial products (see 2.2).…”

Section: Production Of Octanoic Acid and 8-hydroxyoctanoic Acid In Saccharomyces Cerevisiaementioning

confidence: 99%

“…In this work two publications (Wernig et al, 2020b;Wernig et al, 2021) deal with increasing production of OA in S. cerevisiae. Within this thesis OA was produced by a mutated FAS (FAS RK ) by the FA biosynthesis pathway and was increased by fusing the two distinct genes of S. cerevisiae FAS (FAS1 and FAS2) into a single-chain construct (fusFAS) (Wernig et al, 2020b).…”

Section: Current Challenges and Limitations In Octanoic Acid Production By Mutated Fatty Acid Synthasesmentioning

confidence: 99%

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Metabolic engineering of Saccharomyces cerevisiae for production of medium-chain fatty acids and their derivatives

Wernig¹

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The oleochemical and petrochemical industries provide diverse chemicals used in personal care products, food and pharmaceutical industries or as fuels, oils, polymers and others. However, fossil resources are dwindling and concerns about these conventional production methods have risen due to their strong negative impact on the environment and contribution to climate change. Therefore, alternative, sustainable and environmentally friendly production methods for oleochemical compounds such as fatty acids, fatty alcohols, hydroxy fatty acids and dicarboxylic acids are desired. The biotechnological production by engineered microorganism could fulfill these requirements. The concept of metabolic engineering, which is the modification of metabolic pathways of a host organism for increased production of a target compound, is a widely used strategy in biotechnology to generate cell factories or chassis strains for robust, efficient and high production. In this work, the versatile model and industrial yeast Saccharomyces cerevisiae was manipulated by metabolic engineering strategies for increased production of the medium-chain fatty acid octanoic acid and de novo production the derived 8-hydroxyoctanoic acid. Octanoic acid production was enabled by the fatty acid biosynthesis pathway by use of a mutated fatty acid synthase (FASRK) in a wild type FAS deficient strain. The yeast fatty acid synthase (FAS) consists of two polypeptides, α and β, which assemble to a α6β6 complex in a co-translational manner by interaction of the subunits. Because this step might be subject to cellular regulation, the α- and β- subunits of fatty acid synthase were fused to form a single-chain construct (fusFASRK), which displayed superior octanoic acid production compared with split FASRK. Thus, FASRK expression was identified as a limiting step of octanoic acid production. But the strains that produce octanoic acid have a severe growth defect that is undesirable for biotechnological applications and could lead to lower production titers. One reason is the strong inhibitory effect of octanoic acid. Another possibility is that the mutant FAS no longer produces enough essential long-chain fatty acids. To compensate for this, the mutated split and fused FAS variants were co-expressed individually in a strain harboring genomic wild type FAS alleles. In addition, mutant and wild type variants of fused and split FAS were co-expressed together in a FAS deficient strain. However, both cases resulted in decreased octanoic acid titers potentially by physical and/or metabolic crosstalk of the FAS variants. The fatty acid biosynthesis relies on cytosolic acetyl-CoA for initiation and derived malonyl-CoA for elongation and requires NADPH for reductive power. To increase production of octanoic acid, engineering strategies for increased acetyl-CoA and NADHP supply were investigated. First, the flux through the native cytosolic acetyl-CoA and NADPH providing pyruvate dehydrogenase bypass was enhanced by overexpression of the target genes ADH2, ALD6 and ACSL461P from Salmonella enterica in combination or individually. Next, the acety-CoA forming heterologous phosphoketolase/phosphotransacetylase pathway was expressed and NADPH formation was increased by redirecting the flux of glucose-6-phosphate into the NADPH producing oxidative branch of the pentose phosphate pathway. In particular, the flux through glycolysis and pyruvate dehydrogenase bypass was reduced by downregulating the expression of the phosphoglucose isomerase PGI1 and deleting the acetaldehyde dehydrogenase ALD6. Glucose-6-phosphate was guided into the pentose phosphate pathway by overexpressing the glucose-6-phosphate dehydrogenase ZWF1. The first approach did not influence octanoic acid production but the latter increased yields in the glucose consumption phase by 65 %. However, combining the superior fusFASRK with acetyl-CoA and NADPH supply engineering strategies did not result in additive production effects, indicating that other limitations hinder high octanoic acid accumulation. Limitations could be caused in particular by the strong inhibitory effects of octanoic acid or by intrinsic limitations of the FASRK mutant. To enlarge the octanoic acid production platform towards other derived valuable oleochemical compounds the de novo production of 8-hydroxyoctanoic acid was targeted. Since short- and medium-chain fatty acids have a strong inhibitory effect on Saccharomyces cerevisiae, the inhibitory effect of hydroxy fatty acid and dicarboxylic with eight or ten carbon atoms were compared and revealed only little or no growth impairment. Subsequently, the formation of 8-hydroxyoctanoic acid was targeted by a terminal hydroxylation of externally supplied octanoic acid in a bioconversion. For that, three heterologous genes, encoding for cytochromes P450 enzymes and their cognate cytochrome P450 reductases were expressed and 8-hydroxyoctanoic acid production was compared. In addition, the use of different carbon sources was compared. ...

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Functional genome annotation and transcriptome analysis of Pseudozyma hubeiensis BOT-O, an oleaginous yeast that utilizes glucose and xylose at equal rates

Mierke¹,

Brink²,

Norbeck³

et al. 2023

Fungal Genetics and Biology

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Fusing α and β subunits of the fungal fatty acid synthase leads to improved production of fatty acids

Cited by 11 publications

References 29 publications

Production of octanoic acid in Saccharomyces cerevisiae: Investigation of new precursor supply engineering strategies and intrinsic limitations

Production of octanoic acid in Saccharomyces cerevisiae: Investigation of new precursor supply engineering strategies and intrinsic limitations

Metabolic engineering of Saccharomyces cerevisiae for production of medium-chain fatty acids and their derivatives

Functional genome annotation and transcriptome analysis of Pseudozyma hubeiensis BOT-O, an oleaginous yeast that utilizes glucose and xylose at equal rates

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