Production Of Fatty Alcohols Research Articles

Metabolic engineering of Rhodotorula toruloides IFO0880 improves C16 and C18 fatty alcohol production from synthetic media

BackgroundThe oleaginous, carotenogenic yeast Rhodotorula toruloides has been increasingly explored as a platform organism for the production of terpenoids and fatty acid derivatives. Fatty alcohols, a fatty acid derivative widely used in the production of detergents and surfactants, can be produced microbially with the expression of a heterologous fatty acyl-CoA reductase. Due to its high lipid production, R. toruloides has high potential for fatty alcohol production, and in this study several metabolic engineering approaches were investigated to improve the titer of this product.ResultsFatty acyl-CoA reductase from Marinobacter aqueolei was co-expressed with SpCas9 in R. toruloides IFO0880 and a panel of gene overexpressions and Cas9-mediated gene deletions were explored to increase the fatty alcohol production. Two overexpression targets (ACL1 and ACC1, improving cytosolic acetyl-CoA and malonyl-CoA production, respectively) and two deletion targets (the acyltransferases DGA1 and LRO1) resulted in significant (1.8 to 4.4-fold) increases to the fatty alcohol titer in culture tubes. Combinatorial exploration of these modifications in bioreactor fermentation culminated in a 3.7 g/L fatty alcohol titer in the LRO1Δ mutant. As LRO1 deletion was not found to be beneficial for fatty alcohol production in other yeasts, a lipidomic comparison of the DGA1 and LRO1 knockout mutants was performed, finding that DGA1 is the primary acyltransferase responsible for triacylglyceride production in R. toruloides, while LRO1 disruption simultaneously improved fatty alcohol production, increased diacylglyceride and triacylglyceride production, and increased glucose consumption.ConclusionsThe fatty alcohol titer of fatty acyl-CoA reductase-expressing R. toruloides was significantly improved through the deletion of LRO1, or the deletion of DGA1 combined with overexpression of ACC1 and ACL1. Disruption of LRO1 surprisingly increased both lipid and fatty alcohol production, creating a possible avenue for future study of the lipid metabolism of this yeast.

Reconfiguring workup steps in multi-cycle extractive bioconversion for sustainable fatty alcohol production: a process engineering approach

Reconfiguring workup steps between cycles of extractive bioconversion led to fatty alcohol production with improved productivity and sustainability.

Characterizing methanol metabolism-related promoters for metabolic engineering of Ogataea polymorpha.

Promoters play an important role in regulating gene expression, and construction of microbial cell factories requires multiple promoters for balancing the metabolic pathways. However, there are only a limited number of characterized promoters for gene expression in the methylotrophic yeast Ogataea polymorpha, which hampers the extensive harnessing of this important yeast toward a cell factory. Here we characterized the promoters of methanol utilization pathway, precursor supply pathway, and reactive oxygen species (ROS) defense system, by using a green fluorescence protein variant (GFPUV) as a quantification signal. Finally, the characterized promoters were used for tuning a fatty alcohol biosynthetic pathway in O. polymorpha and realized fatty alcohol production from methanol. This promoter box should be helpful for gene expression and pathway optimization in the methylotrophic yeast O. polymorpha. KEY POINTS : • 22 promoters related to methanol metabolism were characterized in O. polymorpha. • Promoter truncation resulted shorter and compact promoters. • Promoters with various strengths were used for regulating a fatty alcohol biosynthesis from methanol.

Machine learning-guided acyl-ACP reductase engineering for improved in vivo fatty alcohol production

Alcohol-forming fatty acyl reductases (FARs) catalyze the reduction of thioesters to alcohols and are key enzymes for microbial production of fatty alcohols. Many metabolic engineering strategies utilize FARs to produce fatty alcohols from intracellular acyl-CoA and acyl-ACP pools; however, enzyme activity, especially on acyl-ACPs, remains a significant bottleneck to high-flux production. Here, we engineer FARs with enhanced activity on acyl-ACP substrates by implementing a machine learning (ML)-driven approach to iteratively search the protein fitness landscape. Over the course of ten design-test-learn rounds, we engineer enzymes that produce over twofold more fatty alcohols than the starting natural sequences. We characterize the top sequence and show that it has an enhanced catalytic rate on palmitoyl-ACP. Finally, we analyze the sequence-function data to identify features, like the net charge near the substrate-binding site, that correlate with in vivo activity. This work demonstrates the power of ML to navigate the fitness landscape of traditionally difficult-to-engineer proteins.

Recent Progress and Trends in the Development of Microbial Biofuels from Solid Waste—A Review

This review covers the recent progress in the design and application of microbial biofuels, assessing the advancement of genetic engineering undertakings and their marketability, and lignocellulosic biomass pretreatment issues. Municipal solid waste (MSW) is a promising sustainable biofuel feedstock due to its high content of lignocellulosic fiber. In this review, we compared the production of fatty alcohols, alkanes, and n-butanol from residual biogenic waste and the environmental/economic parameters to that of conventional biofuels. New synthetic biology tools can be used to engineer fermentation pathways within micro-organisms to produce long-chain alcohols, isoprenoids, long-chain fatty acids, and esters, along with alkanes, as substitutes to petroleum-derived fuels. Biotechnological advances have struggled to address problems with bioethanol, such as lower energy density compared to gasoline and high corrosive and hygroscopic qualities that restrict its application in present infrastructure. Biofuels derived from the organic fraction of municipal solid waste (OFMSW) may have less environmental impacts compared to traditional fuel production, with the added benefit of lower production costs. Unfortunately, current advanced biofuel production suffers low production rates, which hinders commercial scaling-up efforts. Microbial-produced biofuels can address low productivity while increasing the spectrum of produced bioenergy molecules.

Selective Hydrogenation of Methyl Palmitate to Cetyl Alcohol via Ternary Synergistic Catalysis of Ni, Oxygen Vacancies, and Lewis Acid Sites under Mild Reaction Conditions

Selective hydrogenation of fatty esters into high-value-added fatty alcohols over non-noble-metal (e.g., Ni and Co) catalysts is highly desirable for the utilization of natural oils. Although these catalysts offer high hydrogenation activity and are reasonably economical, they suffer from low hydrodeoxygenation activity at low temperatures while promoting certain side reactions (e.g., C–C bond hydrogenolysis and decarbonylation/decarboxylation) at high temperatures, which are undesirable for the production of fatty alcohols. In this study, we tested and optimized the performance of Ni metal catalysts with various metal–oxide modifiers and supports for the selective hydrogenation of methyl palmitate to cetyl alcohol. The most efficient catalytic system (Ni-VOx/TiO2, Ni:V ≈ 1:1, mol/mol) achieved almost quantitative conversion with 90% selectivity for cetyl alcohol under mild conditions (220 °C and 4.0 MPa of H2), which was ascribed to the ternary synergistic action of Ni, oxygen vacancies, and Lewis acid sites. The catalytic synergy of the Ni metal and oxygen vacancies (formed by the reduction of TiO2) was primarily responsible for C–O bond cleavage in methyl palmitate to form palmitic aldehyde as the main intermediate. Furthermore, the V species engaged in metal–Lewis acid cooperation at the metal–oxide interface to efficiently promote the C═O bond hydrogenation of palmitic aldehyde and suppress the undesired C–C bond cleavage, thereby facilitating the highly selective production of cetyl alcohol. The obtained insights into the active phase of Ni-VOx/TiO2 catalysts are expected to facilitate the development of highly efficient catalysts for the selective hydrogenation of fatty esters and other biomass-derived compounds containing C═O/C–O groups.

Recombination machinery engineering facilitates metabolic engineering of the industrial yeast Pichiapastoris.

The industrial yeast Pichia pastoris has been harnessed extensively for production of proteins, and it is attracting attention as a chassis cell factory for production of chemicals. However, the lack of synthetic biology tools makes it challenging in rewiring P. pastoris metabolism. We here extensively engineered the recombination machinery by establishing a CRISPR-Cas9 based genome editing platform, which improved the homologous recombination (HR) efficiency by more than 54 times, in particular, enhanced the simultaneously assembly of multiple fragments by 13.5 times. We also found that the key HR-relating gene RAD52 of P. pastoris was largely repressed in compared to that of Saccharomyces cerevisiae. This gene editing system enabled efficient seamless gene disruption, genome integration and multiple gene assembly with positive rates of 68–90%. With this efficient genome editing platform, we characterized 46 potential genome integration sites and 18 promoters at different growth conditions. This library of neutral sites and promoters enabled two-factorial regulation of gene expression and metabolic pathways and resulted in a 30-fold range of fatty alcohol production (12.6–380 mg/l). The expanding genetic toolbox will facilitate extensive rewiring of P. pastoris for chemical production, and also shed light on engineering of other non-conventional yeasts.

BdFAR4, a root-specific fatty acyl-coenzyme A reductase, is involved in fatty alcohol synthesis of root suberin polyester in Brachypodium distachyon.

Suberin is a complex hydrophobic polymer of aliphatic and phenolic compounds which controls the movement of gases, water, and solutes and protects plants from environmental stresses and pathogenic infection. The synthesis and regulatory pathways of suberin remain unknown in Brachypodium distachyon. Here we describe the identification of a B. distachyon gene, BdFAR4, encoding a fatty acyl-coenzyme A reductase (FAR) by a reverse genetic approach, and investigate the molecular relevance of BdFAR4 in the root suberin synthesis of B. distachyon. BdFAR4 is specifically expressed throughout root development. Heterologous expression of BdFAR4 in yeast (Saccharomyces cerevisiae) afforded the production of C20:0 and C22:0 fatty alcohols. The loss-of-function knockout of BdFAR4 by CRISPR/Cas9-mediated gene editing significantly reduced the content of C20:0 and C22:0 fatty alcohols associated with root suberin. In contrast, overexpression of BdFAR4 in B. distachyon and tomato (Solanum lycopersicum) resulted in the accumulation of root suberin-associated C20:0 and C22:0 fatty alcohols, suggesting that BdFAR4 preferentially accepts C20:0 and C22:0 fatty acyl-CoAs as substrates. The BdFAR4 protein was localized to the endoplasmic reticulum in Arabidopsis thaliana protoplasts and Nicotiana benthamiana leaf epidermal cells. BdFAR4 transcript levels can be increased by abiotic stresses and abscisic acid treatment. Furthermore, yeast one-hybrid, dual-luciferase activity, and electrophoretic mobility shift assays indicated that the R2R3-MYB transcription factor BdMYB41 directly binds to the promoter of BdFAR4. Taken together, these results imply that BdFAR4 is essential for the production of root suberin-associated fatty alcohols, especially under stress conditions, and that its activity is transcriptionally regulated by the BdMYB41 transcription factor.

Recombination machinery engineering for precise genome editing in methylotrophic yeast Ogataea polymorpha

SummaryMethanol biotransformation can expand biorefinery substrate spectrum other than biomass by using methylotrophic microbes. Ogataea (Hansenula) polymorpha, a representative methylotrophic yeast, attracts much attention due to its thermotolerance, but the low homologous recombination (HR) efficiency hinders its precise genetic manipulation during cell factory construction. Here, recombination machinery engineering (rME) is explored for enhancing HR activity together with establishing an efficient CRISPR-Cas9 system in O. polymorpha. Overexpression of HR-related proteins and down-regulation of non-homologous end joining (NHEJ) increased HR rates from 20%–30% to 60%–70%. With these recombination perturbation mutants, a competition between HR and NHEJ is observed. This HR up-regulated system has been applied for homologous integration of large fragments and in vivo assembly of multiple fragments, which enables the production of fatty alcohols in O. polymorpha. These findings will simplify genetic engineering in non-conventional yeasts and facilitate the adoption of O. polymorpha as an attractive cell factory for industrial application.

Production of Long Chain Fatty Alcohols Found in Bumblebee Pheromones by Yarrowia lipolytica.

Fatty alcohols (FA-OH) are aliphatic unbranched primary alcohols with a chain of four or more carbon atoms. Besides potential industrial applications, fatty alcohols have important biological functions as well. In nature, fatty alcohols are produced as a part of a mixture of pheromones in several insect species, such as moths, termites, bees, wasps, etc. In addition, FA-OHs have a potential for agricultural applications, for example, they may be used as a suitable substitute for commercial insecticides. The insecticides have several drawbacks associated with their preparation, and they exert a negative impact on the environment. Currently, pheromone components are prepared mainly through the catalytic hydrogenation of plant oils and petrochemicals, which is an unsustainable, ecologically unfriendly, and highly expensive process. The biotechnological production of the pheromone components using engineered microbial strains and through the expression of the enzymes participating in the biosynthesis of these components is a promising approach that ensures ecological sustenance as well. The present study was aimed at evaluating the production of FA-OHs in the oleaginous yeast, Yarrowia lipolytica, with different lengths of fatty-acyl chains by expressing the fatty acyl-CoA reductase (FAR) BlapFAR4 from B. lapidarius, producing C16:0-OH, C16:1Δ9-OH, and lower quantities of both C14:0-OH and C18:1Δ9-OH, and BlucFAR1 from B. lucorum, producing FA-OHs with a chain length of 18–26 carbon atoms, in this yeast. Among the different novel Y. lipolytica strains used in the present study, the best results were obtained with JMY7086, which carried several lipid metabolism modifications and expressed the BlucFAR1 gene under the control of a strong constitutive promoter 8UAS-pTEF. JMY7086 produced only saturated fatty alcohols with chain lengths from 18 to 24 carbon atoms. The highest titer and accumulation achieved were 166.6 mg/L and 15.6 mg/g DCW of fatty alcohols, respectively. Unlike JMY7086, the BlapFAR4-expressing strain JMY7090 produced only 16 carbon atom-long FA-OHs with a titer of 14.6 mg/L.

Engineering Yarrowia lipolytica for Production of Fatty Alcohols with YaliBrick Vectors.

Biosynthesis of fatty alcohol holds great promise as substitute to replace petroleum-derived fatty alcohols to mitigate environmental concerns and reduce earth's carbon footprint. In this protocol, we detail the procedures of how to use the YaliBrick gene assembly platform to achieve modular assembly of fatty alcohol pathway in Y. lipolytica. To limit fatty alcohol oxidation, we will also describe the hydroxyurea-based protocols for the efficient disruption of POX1 gene, encoding the fatty acyl coenzyme A in Y. lipolytica, with the homologous arm about 500bp. We envision that this chapter would improve our ability to engineer microbial cell factories for oleochemical and fatty alcohol production in oleaginous yeast species.

Biosynthesis of Fatty Alcohols in Engineered Microbial Cell Factories: Advances and Limitations.

Concerns about climate change and environmental destruction have led to interest in technologies that can replace fossil fuels and petrochemicals with compounds derived from sustainable sources that have lower environmental impact. Fatty alcohols produced by chemical synthesis from ethylene or by chemical conversion of plant oils have a large range of industrial applications. These chemicals can be synthesized through biological routes but their free forms are produced in trace amounts naturally. This review focuses on how genetic engineering of endogenous fatty acid metabolism and heterologous expression of fatty alcohol producing enzymes have come together resulting in the current state of the field for production of fatty alcohols by microbial cell factories. We provide an overview of endogenous fatty acid synthesis, enzymatic methods of conversion to fatty alcohols and review the research to date on microbial fatty alcohol production. The primary focus is on work performed in the model microorganisms, Escherichia coli and Saccharomyces cerevisiae but advances made with cyanobacteria and oleaginous yeasts are also considered. The limitations to production of fatty alcohols by microbial cell factories are detailed along with consideration to potential research directions that may aid in achieving viable commercial scale production of fatty alcohols from renewable feedstock.

Interactions between Phytophthora cactorum, Armillaria gallica and Betula pendula Roth. Seedlings Subjected to Defoliation

The purpose of this study was to better understand the interactive impact of two soil-borne pathogens, Phytophthora cactorum and Armillaria gallica, on seedlings of silver birch (Betula pendula Roth.) subjected to stress caused by mechanical defoliation, simulating primary insect feeding. This is the first experimental confirmation of silver birch seedling root damage (and in consequence shoot mortality) caused by the additive effect of defoliation stress and P. cactorum inoculation via soil. However, the most severe damage to roots occurred after A. gallica inoculation. One year after treatments, chlorophyll fluorescence measurement, and gas chromatography coupled with mass spectrometry (GC-MS) were used to analyze the photosynthetic activity in leaves, the volatile organic compounds (VOCs) emitted by the birch leaves, and chemical compounds from the roots. The cumulative effect of the two pathogens and partial defoliation reduced photosynthetic activity, suggesting dysfunction of photosystem PSII due to the applied stresses. In summary, it seems that the main differences in photosynthetic performance could be attributed to Armillaria infection. The birch leaves in seedlings exposed to 50% defoliation, and inoculation with P.cactorum and A. gallica, emitted more aromatic carbonyls and alcohols, as well as half as much aliphatic esters, compared to controls. In infected birch roots, the production of phenols, triterpenes, and fatty alcohols increased, but fatty acids decreased. Higher levels of aromatic carbonyls and alcohols in leaves, as well as phenolic compounds in the roots of stressed birches (compared to control) suggest an activation of plant systemic acquired resistance (SAR).

Yarrowia lipolytica as a Metabolic Engineering Platform for the Production of Very-Long-Chain Wax Esters.

The oleaginous yeast Yarrowia lipolytica is an attractive cell factory platform strain and can be used for sustainable production of high-value oleochemical products. Wax esters (WEs) have a good lubricating property and are usually used as a base for the production of advanced lubricants and emollient oils. In this study, we reported the metabolic engineering of Y. lipolytica to heterologously biosynthesize high-content very-long-chain fatty acids (VLCFAs) and fatty alcohols and efficiently esterify them to obtain very-long-chain WEs. Co-expression of fatty acid elongases from different sources in Y. lipolytica could yield VLCFAs with carbon chain lengths up to 24. Combining with optimization of the central metabolic modules could further enhance the biosynthesis of VLCFAs. Furthermore, through the screening of heterologous fatty acyl reductases (FARs), we enabled high-level production of fatty alcohols. Genomic integration and heterologous expression of wax synthase (WS) and FAR in a VLCFA-producing Y. lipolytica strain yielded 95-650 mg/L WEs with carbon chain lengths from 32 to 44. Scaled-up fermentation in 5 L laboratory bioreactors significantly increased the production of WEs to 2.0 g/L, the highest content so far in yeasts. This study contributes to the further efficient biosynthesis of VLCFAs and their derivatives.

HetN and patS Mutations Enhance Accumulation of Fatty Alcohols in the hglT Mutants of Anabaena sp. PCC 7120

The heterocysts present in filamentous cyanobacteria such as Anabaena sp. PCC 7120 are known to be regulated by HetN and PatS, the repressors of heterocyst differentiation; therefore, the inactivation of these proteins will result in the formation of multiple heterocysts. To enhance the accumulation of fatty alcohols synthesized in the heterocyst, we introduced mutations of these repressors to increase heterocyst frequency. First, we isolated double mutants of hetN and patS and confirmed that the null mutation of these genes promoted higher frequencies of heterocyst formation and higher accumulation of heterocyst-specific glycolipids (Hgls) compared with its wild type. Next, we combined hetN and patS mutations with an hglT (encoding glycosyltransferase, an enzyme involved in Hgl synthesis) mutation to increase the accumulation of fatty alcohols since knockout mutation of hglT results in accumulation of very long chain fatty alcohol, the precursor of Hgl. We also observed retarded growth, lower chlorophyll content and up to a five-fold decrease in photosynthetic activity of the hetN/patS/hglT triple mutants. In contrast, the triple mutants showed three times higher heterocyst formation frequencies than the hglT single mutant and wild type. The production rate of fatty alcohol in the triple mutants attained a value 1.41 nmol/mL OD730, whereas accumulation of Hgls in the wild type was 0.90 nmol/mL OD730. Aeration of culture improved the accumulation of fatty alcohols in hetN/patS/hglT mutant cells up to 2.97 nmol/mL OD730 compared with cells cultured by rotation. Our study outlines an alternative strategy for fatty alcohol production supported by photosynthesis and nitrogen fixation.

Fatty alcohol production: an opportunity of bioprocess

AbstractFatty alcohols are widely used for various applications and are found in products ranging from detergents to cosmetics and cleaning agents. Currently, over 3 million tonnes of fatty alcohols are produced per year from bio‐based and petroleum‐based feedstocks through chemical conversion and synthetic processes. However, the environmental impact of current fatty alcohol production processes has become a major concern, and several bioprocesses have emerged as alternative methods for synthesis. These biological processes are environmentally friendly due to their metal‐free nature, low consumption of energy, and mild reaction conditions. This review overviews the current status of fatty alcohol production – both chemical and biological approaches. Our focus is on biological synthesis approaches using biocatalysis and metabolic engineering due to their proven efficacy. Potential downstream processing for these bioprocesses is also presented. © 2020 Society of Chemical Industry and John Wiley & Sons, Ltd

High titer fatty alcohol production in Lipomyces starkeyi by fed-batch fermentation

Abstract Extracellular production of long chain fatty acid-based products, such as fatty alcohols, are being investigated as intermediates amenable to separations and catalytic upgrading to hydrocarbon fuels. We previously demonstrated that the oleaginous yeast, Lipomyces starkeyi expressing the fatty acyl-CoA reductase (FAR) gene from Marinobacter aquaeolei VT8, produced up to 770 mg/L of fatty alcohols. The FAR genes expressed in L. starkeyi transformants exhibited different fatty alcohol production levels. To better understand fatty alcohol production and potentially further increase fatty alcohol titer and yields, we investigated the expression levels, copy numbers, and enzymatic activities of the expressed FAR genes. Our results showed that fatty alcohol production is dependent on the above factors for higher titers of fatty alcohols. Fatty alcohol titer was further increased in the highest producing transformant by testing different basic process configurations (batch and fed-batch) with different initial nitrogen concentrations and feed rates. In fed-batch, a maximum of 4.2 g/L fatty alcohols were produced in 5-L bioreactor. The oleaginous yeast L. starkeyi can produce high titers of fatty alcohols particularly when periodically fed glucose and nitrogen.

Tannin-derived bimetallic CuCo/C catalysts for an efficient in-situ hydrogenation of lauric acid in methanol-water media

Integration of aqueous-phase reforming of methanol (APRM) into the production of value-added fatty alcohols via hydrogenation of fatty acids is an attractive and efficient process in biomass conversion. Herein, we present tannin-derived carbon-supported monometallic Cu/C, Co/C and bimetallic Cu-Co/C catalysts with different Cu/Co ratios for in situ hydrogenation of lauric acid in a methanol-water hydrothermal system. The catalysts were made by a simple mixture of tannin and the metal salts, followed by pyrolysis in an H2 atmosphere. Through synergism induced by Cu-Co interactions, the bimetallic Cu-Co/C catalyst with Cu/Co ratio of 1:4 (CuCo4/C) exhibits higher catalytic activity than monometallic Cu/C catalyst towards lauric acid conversion, as well as higher selectivity to lauryl alcohol than monometallic Co/C catalyst. The CuCo4/C catalyst is composed of metallic Cu, metallic Co, and amorphous carbon, characterized by high surface area (194.5 m2/g) and hierarchical structure. The obtained lauryl alcohol yield in the methanol-water hydrothermal system is significantly higher than other alcohol-water systems, namely ethanol, 1-propanol, and isopropanol hydrothermal systems, highlighting its higher generation rate of hydrogen. Also, the effects of process parameters including the quantity of methanol, catalyst loading and reaction time on the conversion of lauric acid were systematically studied and optimized.

Exploiting nonionic surfactants to enhance fatty alcohol production in Rhodosporidium toruloides.

Fatty alcohols (FOHs) are important feedstocks in the chemical industry to produce detergents, cosmetics, and lubricants. Microbial production of FOHs has become an attractive alternative to production in plants and animals due to growing energy demands and environmental concerns. However, inhibition of cell growth caused by intracellular FOH accumulation is one major issue that limits FOH titers in microbial hosts. In addition, identification of FOH‐specific exporters remains a challenge and previous studies towards this end are limited. To alleviate the toxicity issue, we exploited nonionic surfactants to promote the export of FOHs in Rhodosporidium toruloides, an oleaginous yeast that is considered an attractive next‐generation host for the production of fatty acid‐derived chemicals. Our results showed FOH export efficiency was dramatically improved and the growth inhibition was alleviated in the presence of small amounts of tergitol and other surfactants. As a result, FOH titers increase by 4.3‐fold at bench scale to 352.6 mg/L. With further process optimization in a 2‐L bioreactor, the titer was further increased to 1.6 g/L. The method we show here can potentially be applied to other microbial hosts and may facilitate the commercialization of microbial FOH production.

Recombination Machinery Engineering for Precise Genome Editing in Methylotrophic Yeast <i>Ogataea Polymorpha</i>

Methanol biotransformation can expand biorefinary substrate spectrum other than biomass by using methylotrophic microbes. Ogataea (Hansenula) polymorpha, a representative methylotrophic yeast, attracts much attention due to its thermotolerance, but low homologous recombination (HR) efficiency hinders its precise genetic manipulation during cell factory construction. Here, recombination machinery engineering (rME) is explored for enhancing HR activity together with establishing an efficient CRISPR/Cas9 system in O. polymorpha. Overexpression of HR-related proteins and down-regulation of non-homologous end joining (NHEJ), increase HR rates from 20-30% to 60-70%. With these recombination perturbation mutants, a competition between HR and NHEJ is observed. This HR up-regulated systems has been applied for homologous integration of large fragments and in vivo assembly of multiple fragments, which enables the first production of fatty alcohols in O. polymorpha. These findings will simplify the genetic engineering in non-conventional yeasts, and facilitate the adoption of O. polymorpha as an attractive cell factory for industrial application.