Metabolic engineering of Escherichia coli for α-farnesene production

Sesquiterpenes are important materials in pharmaceuticals and industry. Metabolic engineering has been successfully used to produce these valuable compounds in microbial hosts. However, the microbial potential of sesquiterpene production is limited by the poor heterologous expression of plant sesquiterpene synthases and the deficient FPP precursor supply. In this study, we engineered E. coli to produce α-farnesene using a codon-optimized α-farnesene synthase and an exogenous MVA pathway. Codon optimization of α-farnesene synthase improved both the synthase expression and α-farnesene production. Augmentation of the metabolic flux for FPP synthesis conferred a 1.6- to 48.0-fold increase in α-farnesene production. An additional increase in α-farnesene production was achieved by the protein fusion of FPP synthase and α-farnesene synthase. The engineered E. coli strain was able to produce 380.0 mg/L of α-farnesene, which is an approximately 317-fold increase over the initial production of 1.2 mg/L.     

解脂耶氏酵母表达调控工具的开发及天然产物合成的研究进展

解脂耶氏酵母是一种重要的产油酵母,由于其能利用多种疏水性底物,具有良好的耐酸、耐盐等胁迫耐受性,具有高通量的三羧酸循环,可提供充足的乙酰辅酶A前体等特点,被认为是生产萜类、聚酮类和黄酮类等天然产物的理想宿主,在代谢工程领域有着广泛的应用。近年来,越来越多的基因编辑、表达和调控工具被逐渐开发,这促进了解脂耶氏酵母合成各种天然产物的研究。文中综述了近年来解脂耶氏酵母中基因表达和天然产物合成方面的研究进展,并探讨了在该酵母中异源合成天然产物所面临的挑战和可能的解决方案。     

Structure of a three-domain sesquiterpene synthase: a prospective target for advanced biofuels production

The sesquiterpene bisabolene was recently identified as a biosynthetic precursor to bisabolane, an advanced biofuel with physicochemical properties similar to those of D2 diesel. High-titer microbial bisabolene production was achieved using Abies grandis α-bisabolene synthase (AgBIS). Here, we report the structure of AgBIS, a three-domain plant sesquiterpene synthase, crystallized in its apo form and bound to five different inhibitors. Structural and biochemical characterization of the AgBIS terpene synthase Class I active site leads us to propose a catalytic mechanism for the cyclization of farnesyl diphosphate into bisabolene via a bisabolyl cation intermediate. Further, we describe the nonfunctional AgBIS Class II active site whose high similarity to?bifunctional diterpene synthases makes it an important link in understanding terpene synthase evolution. Practically, the AgBIS crystal structure is important in future protein engineering efforts to increase the microbial production of bisabolene.     

Producing Biochemicals in Yarrowia lipolytica from Xylose through a Strain Mating Approach

Enabling xylose catabolism is challenging, especially for unconventional yeasts and previously engineered background strains. In this study, the efficacy of a yeast mating approach with Yarrowia lipolytica that can combine a previously engineering and evolved xylose phenotype with a metabolite overproduction phenotype is demonstrated. Specifically, several engineered Y. lipolytica strains that produce α‐linolenic acid (ALA), riboflavin, and triacetic acid lactone (TAL) with an engineered and adapted xylose‐utilizing strain to obtain three diploid strains that rapidly produce these molecules directly from xylose are mated. Titers of 0.52 g L^?1 ALA, 96.6 mg L^?1 riboflavin, and 2.9 g L^?1 TAL, are obtained from xylose in flask cultures and 1.42 g L^?1 production of ALA is obtained using bioreactor condition. This total production level is generally on par or higher than the parental strain cultivated on glucose, although specific productivities decreased as a result of improved overall cell growth by the diploid strains. In the case of ALA, this lipid content reached similar levels to that of flaxseed oil. This result showcases the first study using strain mating in Y. lipolytica for producing biomolecules from xylose, and thus demonstrates the utility of this approach as a routine tool for metabolic engineering.     

Engineering the oleaginous yeast Yarrowia lipolytica for β-farnesene overproduction

β-farnesene is a sesquiterpenoid with various industrial applications which is now commercially produced by a Saccharomyces cerevisiae strain obtained by random mutagenesis and genetic engineering. We rationally designed a genetically defined Yarrowia lipolytica through recovery of L-leucine biosynthetic route, gene dosage optimization of β-farnesene synthase and disruption of the competition pathway. The resulting β-farnesene titer was improved from 8 to 345 mg L^-1. Finally, the strategy for decreasing the lipid accumulation by individually and iteratively knocking out four acyltransferases encoding genes was adopted. The result displayed that β-farnesene titer in the engineered strain CIBT6304 in which acyltransferases (DGA1 and DGA2) were deleted increased by 45% and reached 539 mg L^-1 (88 mg g^-1 DCW). Using fed-batch fermentation, CIBT6304 could produce the highest β-farnesene titer (22.8 g L^-1) among the genetically defined strains. This study will provide the foundation of engineering Y. lipolytica to produce other terpenoids more cost-efficiently.     

Microbial synthesis of wax esters

Microbial synthesis of wax esters (WE) from low-cost renewable and sustainable feedstocks is a promising path to achieve cost-effectiveness in biomanufacturing. WE are industrially high-value molecules, which are widely used for applications in chemical, pharmaceutical, and food industries. Since the natural WE resources are limited, the WE production mostly rely on chemical synthesis from rather expensive starting materials, and therefore solution are sought from development of efficient microbial cell factories. Here we report to engineer the yeast Yarrowia lipolytica and bacterium Escherichia coli to produce WE at the highest level up to date. First, the key genes encoding fatty acyl-CoA reductases and wax ester synthase from different sources were investigated, and the expression system for two different Y. lipolytica hosts were compared and optimized for enhanced WE production and the strain stability. To improve the metabolic pathway efficiency, different carbon sources including glucose, free fatty acid, soybean oil, and waste cooking oil (WCO) were compared, and the corresponding pathway engineering strategies were optimized. It was found that using a lipid substrate such as WCO to replace glucose led to a 60-fold increase in WE production. The engineered yeast was able to produce 7.6 g/L WE with a yield of 0.31 (g/g) from WCO within 120 h and the produced WE contributed to 57% of the yeast DCW. After that, E. coli BL21(DE3), with a faster growth rate than the yeast, was engineered to significantly improve the WE production rate. Optimization of the expression system and the substrate feeding strategies led to production of 3.7–4.0 g/L WE within 40 h in a 1-L bioreactor. The predominant intracellular WE produced by both Y. lipolytica and E. coli in the presence of hydrophobic substrates as sole carbon sources were C₃₆, C₃₄ and C₃₂, in an order of decreasing abundance and with a large proportion being unsaturated. This work paved the way for the biomanufacturing of WE at a large scale.     

Study of the persistence and dynamics of recombinant mCherry-producing Yarrowia lipolytica strains in the mouse intestine using fluorescence imaging

Yarrowia lipolytica is a dimorphic oleaginous non-conventional yeast widely used as a powerful host for expressing heterologous proteins, as well as a promising source of engineered cell factories for various applications. This microorganism has a documented use in Feed and Food and a GRAS (generally recognized as safe) status. Moreover, in vivo studies demonstrated a beneficial effect of this yeast on animal health. However, despite the focus on Y. lipolytica for the industrial manufacturing of heterologous proteins and for probiotic effects, its potential for oral delivery of recombinant therapeutic proteins has seldom been evaluated in mammals. As the first steps towards this aim, we engineered two Y. lipolytica strains, a dairy strain and a laboratory strain, to produce the model fluorescent protein mCherry. We demonstrated that both Y. lipolytica strains transiently persisted for at least 1 week after four daily oral administrations and they maintained the active expression of mCherry in the mouse intestine. We used confocal microscopy to image individual Y. lipolytica cells of freshly collected intestinal tissues. They were found essentially in the lumen and they were rarely in contact with epithelial cells while transiting through the ileum, caecum and colon of mice. Taken as a whole, our results have shown that fluorescent Y. lipolytica strains constitute novel tools to study the persistence and dynamics of orally administered yeasts which could be used in the future as oral delivery vectors for the secretion of active therapeutic proteins in the gut.     

代谢工程改造解脂耶氏酵母合成羧酸的研究进展

解脂耶氏酵母是一种具有独特生理代谢特征的非常规酵母.它具有可以利用多种廉价碳源、低pH值耐受性好、分泌能力强等优点,因此非常适合用于各种工业产品的微生物发酵.目前,解脂耶氏酵母已被证实具有高效生产多种(同源或异源)有机羧酸的能力.本文对近年来利用代谢工程及合成生物学技术改造解脂耶氏酵母生产羧酸的实例进行了总结,并重点介...     

Metabolic engineering of Yarrowia lipolytica for the production of isoprene

Yarrowia lipolytica has recently emerged as a prominent microbial host for production of terpenoids. Its robust metabolism and growth in wide range of substrates offer several advantages at industrial scale. In the present study, we investigate the metabolic potential of Y. lipolytica to produce isoprene. Sustainable production of isoprene has been attempted through engineering several microbial hosts; however, the engineering studies performed so far are challenged with low titers. Engineering of Y. lipolytica, which have inherent high acetyl-CoA flux could fuel precursors into the biosynthesis of isoprene and thus is an approach that would offer sustainable production opportunities. The present work, therefore, explores this opportunity wherein a codon-optimized IspS gene (single copy) of Pueraria montana was integrated into the Y. lipolytica genome. With no detectable isoprene level during the growth or stationary phase of modified strain, attempts were made to overexpress enzymes from MVA pathway. GC-FID analyses of gas collected during stationary phase revealed that engineered strains were able to produce detectable isoprene only after overexpressing HMGR (or tHMGR). The significant role of HMGR (tHMGR) in diverting the pathway flux toward DMAPP is thus highlighted in our study. Nevertheless, the final recombinant strains overexpressing HMGR (tHMGR) along with Erg13 and IDI showed isoprene titers of ~500 μg/L and yields of ~80 μg/g. Further characterization of the recombinant strains revealed high lipid and squalene content compared to the unmodified strain. Overall, the preliminary results of our laboratory-scale studies represent Y. lipolytica as a promising host for fermentative production of isoprene.     

Homology-independent genome integration enables rapid library construction for enzyme expression and pathway optimization in Yarrowia lipolytica

Yarrowia lipolytica is an important oleaginous industrial microorganism used to produce biofuels and other value-added compounds. Although several genetic engineering tools have been developed for Y. lipolytica, there is no efficient method for genomic integration of large DNA fragments. In addition, methods for constructing multigene expression libraries for biosynthetic pathway optimization are still lacking in Y. lipolytica. In this study, we demonstrate that multiple and large DNA fragments can be randomly and efficiently integrated into the genome of Y. lipolytica in a homology-independent manner. This homology-independent integration generates variation in the chromosomal locations of the inserted fragments and in gene copy numbers, resulting in the expression differences in the integrated genes or pathways. Because of these variations, gene expression libraries can be easily created through one-step integration. As a proof of concept, a LIP2 (producing lipase) expression library and a library of multiple genes in the β-carotene biosynthetic pathway were constructed, and high-production strains were obtained through library screening. Our work demonstrates the potential of homology-independent genome integration for library construction, especially for multivariate modular libraries for metabolic pathways in Y. lipolytica, and will facilitate pathway optimization in metabolic engineering applications.     

Biotechnological production of γ-decalactone,a peach like aroma,by <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

The request for new flavourings increases every year. Consumer perception that everything natural is better is causing an increase demand for natural aroma additives. Biotechnology has become a way to get natural products. γ-Decalactone is a peach-like aroma widely used in dairy products, beverages and others food industries. In more recent years, more and more studies and industrial processes were endorsed to cost-effect this compound production. One of the best-known methods to produce γ-decalactone is from ricinoleic acid catalyzed by Yarrowia lipolytica, a generally regarded as safe status yeast. As yet, several factors affecting γ-decalactone production remain to be fully understood and optimized. In this review, we focus on the aromatic compound γ-decalactone and its production by Y. lipolytica. The metabolic pathway of lactone production and degradation are addressed. Critical analysis of novel strategies of bioprocess engineering, metabolic and genetic engineering and other strategies for the enhancement of the aroma productivity are presented.     

A single-host fermentation process for the production of flavor lactones from non-hydroxylated fatty acids

Lactone flavors with fruity, milky, coconut, and other aromas are widely used in the food and fragrance industries. Lactones are produced by chemical synthesis or by biotransformation of plant-sourced hydroxy fatty acids. We established a novel method to produce flavor lactones from abundant non-hydroxylated fatty acids using yeast cell factories. Oleaginous yeast Yarrowia lipolytica was engineered to perform hydroxylation of fatty acids and chain-shortening via β-oxidation to preferentially twelve or ten carbons. The strains could produce γ-dodecalactone from oleic acid and δ-decalactone from linoleic acid. Through metabolic engineering, the titer was improved 4-fold, and the final strain produced 282 mg/L γ-dodecalactone in a fed-batch bioreactor. The study paves the way for the production of lactones by fermentation of abundant fatty feedstocks.     

Genome-scale model-driven strain design for dicarboxylic acid production in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Background

Recently, there have been several attempts to produce long-chain dicarboxylic acids (DCAs) in various microbial hosts. Of these, Yarrowia lipolytica has great potential due to its oleaginous characteristics and unique ability to utilize hydrophobic substrates. However, Y. lipolytica should be further engineered to make it more competitive: the current approaches are mostly intuitive and cumbersome, thus limiting its industrial application.

Results

In this study, we proposed model-guided metabolic engineering strategies for enhanced production of DCAs in Y. lipolytica. At the outset, we reconstructed genome-scale metabolic model (GSMM) of Y. lipolytica (iYLI647) by substantially expanding the previous models. Subsequently, the model was validated using three sets of published culture experiment data. It was finally exploited to identify genetic engineering targets for overexpression, knockout, and cofactor modification by applying several in silico strain design methods, which potentially give rise to high yield production of the industrially relevant long-chain DCAs, e.g., dodecanedioic acid (DDDA). The resultant targets include (1) malate dehydrogenase and malic enzyme genes and (2) glutamate dehydrogenase gene, in silico overexpression of which generated additional NADPH required for fatty acid synthesis, leading to the increased DDDA fluxes by 48% and 22% higher, respectively, compared to wild-type. We further investigated the effect of supplying branched-chain amino acids on the acetyl-CoA turn-over rate which is key metabolite for fatty acid synthesis, suggesting their significance for production of DDDA in Y. lipolytica.

Conclusion

In silico model-based strain design strategies allowed us to identify several metabolic engineering targets for overproducing DCAs in lipid accumulating yeast, Y. lipolytica. Thus, the current study can provide a methodological framework that is applicable to other oleaginous yeasts for value-added biochemical production.

     

Recent advances in metabolic engineering of Corynebacterium glutamicum for bioproduction of value-added aromatic chemicals and natural products

Recent progress in synthetic and systems metabolic engineering technologies has explored the potential of microbial cell factories for the production of industrially relevant bulk and fine chemicals from renewable biomass resources in an eco-friendly manner. Corynebacterium glutamicum, a workhorse for industrial amino acid production, has currently evolved into a promising microbial platform for bioproduction of various natural and non-natural chemicals from renewable feedstocks. Notably, it has been recently demonstrated that metabolically engineered C. glutamicum can overproduce several commercially valuable aromatic and related chemicals such as shikimate, 4-hydroxybenzoate, and 4-aminobenzoate from sugars at remarkably high titer suitable to commercial application. On the other hand, overexpression and/or extension of its endogenous metabolic pathways by integrating heterologous metabolic pathways enabled production of structurally intricate and valuable natural chemicals like plant polyphenols, carotenoids, and fatty acids. In this review, we summarize recent advances in metabolic engineering of C. glutamicum for production of those value-added aromatics and other natural products, which highlights high potential and the versatility of this microbe for bioproduction of diverse chemicals.

     

γ-decalactone production by Yarrowia lipolytica and Lindnera saturnus in crude glycerol

Flavor compounds are commonly obtained from chemical synthesis or extracted from plants. These sources have disadvantages, such as racemic mixture generation, more steps to yield the final product, low yield, and high cost, making the microbial fermentation an alternative and potential way to obtain flavor compounds. The most important lactone for flavor application is γ-decalactone, which has an aroma of peach and can be obtained by ricinoleic acid biotransformation through yeast peroxisomal β-oxidation. The aim of this work was to use crude glycerol, a residual biodiesel industry, for the production of bioaroma from two different yeasts. Yarrowia lipolytica CCMA 0357 and Lindnera saturnus CCMA 0243 were grown at different concentrations (10, 20, and 30% w/v) of substrates (castor oil and crude glycerol) for γ-decalactone production. L. saturnus CCMA 0243 produced higher concentration of y-decalactone (5.8?g/L) in crude glycerol, whereas Y. lipolytica CCMA 0357 showed a maximum production in castor oil (3.5?g/L). Crude glycerol showed better results for γ-decalactone production when compared to castor oil. L. saturnus CCMA 0243 has been shown to have a high potential for γ-decalactone production from crude glycerol.     

In silico and in vivo analysis of signal peptides effect on recombinant glucose oxidase production in nonconventional yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Signal peptide (SP) is an important factor and biobrick in the production and secretion of recombinant proteins. The aim of this study was in silico and in vivo analysis of SPs effect on the production of recombinant glucose oxidase (GOX) in Yarrowia lipolytica. Several in silico softwares, namely SignalP4, Signal-CF, Phobius, WolfPsort 0.2, SOLpro and ProtParam, were used to analyse the potential of 15 endogenous and exogenous SPs for the secretion of recombinant GOX in Y. lipolytica. According to in silico results, the SP of GOX was predicted as suitable in terms of high secretory potential and of protein solubility and stability which is chosen for in vivo analysis. The recombinant Y. lipolytica strain produced 280 U/L of extracellular GOX after 7 days in YPD medium. The results show that the SP of GOX can be applied to efficient production of extracellular heterologous proteins and metabolic engineering in Y. lipolytica.     

Production of β-carotene by expressing a heterologous multifunctional carotene synthase in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Objectives

To obtain functional expression of a heterologous multifunctional carotene synthase containing phytoene synthase, phytoene dehydrogenase, and lycopene β-cyclase activities encoded by carS from Schizochytrium sp. in order to allow Yarrowia lipolytica to produce β-carotene.

Results

To increase the integration efficiency of a 3.8 kb carS under the control of P _GPD promoter with a 2 kb selection marker, ura3, along with a geranylgeranyl diphosphate synthase (GGS1) expression cassette (~10 kb in total), was inserted into the Y. lipolytica chromosome, and the DNA assembler method was combined with double chromosomal deletions of ku70 and ku80. This method resulted in a 13.4-fold increase in integration efficiency compared with the original method, reaching 63% (10/16). The resulting recombinant Y. lipolytica produced 0.41 mg β-carotene per g dry cell weight, while the wild type did not produce any indicating the functionality of the multifunctional carotene synthase in Y. lipolytica.

Conclusion

Expression of GGS1 and a multifunctional carotene synthase from Schizochytrium sp. in Y. lipolytica led to β-carotene production. DNA assembler efficiency was greatly increased by the deletion of ku70 and ku80, which resulted in decreased in vivo nonhomologous end-joining (NHEJ) in Y. lipolytica.

     

High‐yield lipid production from lignocellulosic biomass using engineered xylose‐utilizing Yarrowia lipolytica

Lignocellulosic biomass shows high potential as a renewable feedstock for use in biodiesel production via microbial fermentation. Yarrowia lipolytica, an emerging oleaginous yeast, has been engineered to efficiently convert xylose, the second most abundant sugar in lignocellulosic biomass, into lipids for lignocellulosic biodiesel production. Yet, the lipid yield from xylose or lignocellulosic biomass remains far lower than that from glucose. Here we developed an efficient xylose‐utilizing Y. lipolytica strain, expressing an isomerase‐based pathway, to achieve high‐yield lipid production from lignocellulosic biomass. The newly developed xylose‐utilizing Y. lipolytica, YSXID, produced 12.01 g/L lipids with a maximum yield of 0.16 g/g, the highest ever reported, from lignocellulosic hydrolysates. Consequently, this study shows the potential of isomerase‐based xylose‐utilizing Y. lipolytica for economical and sustainable production of biodiesel and oleochemicals from lignocellulosic biomass.