Improvement of heterologous protein productivity using recombinant Yarrowia lipolytica and cyclic fed-batch process strategy

A cyclic fed-batch bioprocess is designed and a significant improvement of rice alpha-amylase productivity of recombinant Yarrowia lipolytica is illustrated. A bioprocess control strategy developed and reported here entails use of a genetically stable recombinant cloned for heterologous protein, use of optimized media for cell growth and enzyme production phases, and process control strategy enabling high cell-density culture and high alpha-amylase productivity. This process control can be achieved through maintaining a constant optimal specific cell growth rate at a predetermined value (i.e., 0.1 h-1), controlling medium feed rate commensurate with the cell growth rate, and maintaining a high cell-density culture (i.e., 60-70 g/L) for high productivity of cloned heterologous protein. The volumetric enzyme productivity (1, 960 units/L. h) achieved from the cyclic fed-batch process was about 3-fold higher than that of the fed-batch culture process (630 units/L. h).     

Two-stage cultivation of recombinant Saccharomyces cerevisiae to enhance plasmid stability under non-selective conditions: experimental study and modeling

A leucine auxotroph strain of Saccharomyces cerevisiae was used to study plasmid stability and expression using a recombinant plasmid, which contained a foreign gene for firefly luciferase (luc). This recombinant yeast was tested in a series of continuous cultures in semi-defined media with varying concentrations of yeast extract in order to study its effect on stability. While the biomass concentration and luciferase activity increased with increasing concentrations of yeast extract, the plasmid stability declined. An analysis of the growth rates showed that the recombinants enjoyed a growth rate advantage over the plasmid-free cells at critically low yeast extract concentrations, possibly due to leucine starvation in the media. A two-stage cultivation strategy was designed in order to create a yeast extract limited environment so that plasmid-free cells could not grow and overtake the recombinant cells. The cells were cultivated in selective media in the first stage, and then transferred continuously to the second stage where the media was enriched by feeding yeast extract. The feed rate was kept low in order to ensure yeast extract and hence leucine starvation, thereby selecting against the plasmid-free cells. This strategy resulted in a stable existence of recombinant cells, which stabilized around 60% at steady state during the tested period of cultivation. The complex nitrogen feed helped in increasing the cell density and volumetric activity by approximately 9 and 18-fold respectively with respect to that achieved in minimal medium. The experimental data was used to formulate a mathematical model to predict cell growth and plasmid stability in two-stage cultivation, which correctly explained the experimental data.     

High-level expression of extracellular lipase Lip2 from Yarrowia lipolytica in Pichia pastoris and its purification and characterization

The extracellular lipase gene from Yarrowia lipolytica (YlLip2) was cloned into the pPICZalphaA and integrated into the genome of the methylotrophic yeast Pichia pastoris X-33. The lipase was successfully expressed and secreted with an apparent molecular weight of 39kDa using Saccharomyces cerevisiae secretion signal peptide (alpha-factor) under the control of the methanol inducible promoter of the alcohol oxidase 1 gene (AOX1). The lipase activity of 12,500,000U/l (2.10g total protein and 0.63g lipase per liter) was obtained in a fed-batch cultivation, where methanol feeding was linked to the dissolved oxygen content after initial glycerol culture. After fermentation, the supernatant was concentrated by ultrafiltration with a 10kDa cut off membrane and purified with ion exchange chromatography using Q Sepharose FF. Deglycosylation showed that the recombinant lipase is a glycoprotein which contains the same content of sugar (about 12%) as the native lipase from Y. lipolytica. The optimum temperature and pH of the recombinant lipase was 40 degrees C and 8.0, respectively. The lipase showed high activity toward long-chain fatty acid methyl esters (C12-C16).     

Heterologous protein expression and secretion in the non-conventional yeast Yarrowia lipolytica: a review

The production of heterologous proteins is a research field of high interest, with both academic and commercial applications. Yeasts offer a number of advantages as host systems, and, among them, Yarrowia lipolytica appears as one of the most attractive. This non-conventional dimorphic yeast exhibits a remarkable regularity of performance in the efficient secretion of various heterologous proteins. This review presents the main characteristics of Y. lipolytica, and the genetic and molecular tools available in this yeast. A particular emphasis is given to newly developed tools such as efficient promoters, a non-homologous integration method, and an amplification system using defective selection markers. A table recapitulates the 42 heterologous proteins produced until now in Y. lipolytica. A few relevant examples are exposed in more detail, in order to illustrate some peculiar points of the Y. lipolytica physiology, and to offer a comparison with other production systems. This amount of data demonstrates the global reliability and versatility of Y. lipolytica as a host for heterologous production.     

Expression of Alt a 1 allergen from Alternaria alternata in the yeast Yarrowia lipolytica

Allergies affect almost 25% of the population in industrialized countries. Alternaria alternata is known to be a significant source of aeroallergens and sensitization to this mold is a risk factor for the development of wheezing, asthma, and atopic dermatitis. Diagnosis and treatment of allergies requires the production of large amounts of pure and well defined protein. Yarrowia lipolytica, a non-pathogenic ascomycete able to secrete high levels of enzymes that can grow in inexpensive substrates, has been considered a useful host for heterologous gene expression. In the present work, we have developed two vectors for expressing Alt a 1, the most relevant A. alternata allergen, in Y. lipolytica. One vector is autosomal and one is integrative. With both systems, rAlt a 1 was secreted into the culture medium. The immunological characteristics of the purified recombinant allergen were determined by IgE-blot using sera from 42 A. alternata-allergic patients. We have carried out ELISA-inhibition experiments using sera from four patients to compare the IgE-binding capacity of natural and recombinant allergens. Our results show that Y. lipolytica is able to produce a recombinant Alt a 1 which is immunochemically equivalent to the natural counterpart and could be used for immunotherapy and diagnostics.     

Stability studies of recombinant Saccharomyces cerevisiae in the presence of varying selection pressure

A recombinant yeast plasmid carrying the Ieu2 gene for auxotrophic complementation and a reporter gene for beta-galactosidase under the control of Gal10 promoter was studied in Saccharomyces cerevisiae. Growth, product formation, and plasmid stability were studied in defined, semi-defined, and complex media. The biomass concentration and specific activity were higher in complex medium than in defined medium, which was selective for the growth of plasmid-containing cells, leading to a 10-fold increase in volumetric activity. However, plasmid instability was very high in complex media with 50% plasmid-free cells emerging in the culture within 75 h of cultivation. In order to control instability, the growth rates of the plasmid-containing and plasmid-free cells were determined in semi-defined media, which consisted of defined medium supplemented with different concentrations of yeast extract. Below a critical concentration of yeast extract (0.05 g/L), the plasmid-containing cells had a growth rate advantage over the plasmid-free cells. This was possibly because, at this concentration of yeast extract, the availability of leucine became the rate-determining factor in the specific growth rate of plasmid-free cells. A feeding strategy was designed which maintained a low concentration of the residual yeast extract in the medium and thus continuously provided the plasmid-containing cells with a competitive advantage over the plasmid-free cells. This resulted in high stability as well as high cell density under non-selective conditions, which led to a 10-fold increase in the volumetric activity compared to that achieved in defined selective media. A simple mathematical model was formulated to verify the experimental data. The important state variables and process parameters, i.e., biomass concentration, beta-galactosidase expression, sucrose consumption, yeast extract consumption, and specific growth rates of the two cell populations, were evaluated. These variables and parameters along with the differential equations based on material balances as well as the experimental results obtained were used in a mathematical model for the fed-batch cultivation. These correctly verified the experimental data and clearly illustrated the concept behind the success of the fed-batch strategy under yeast extract starvation.     

代谢工程构建重组耶氏解脂酵母生产中长链聚羟基脂肪酸酯

中长链聚羟基脂肪酸酯(mcl-PHA)是一大类由微生物合成的天然生物聚酯,因具有可再生性和生物降解性越来越受到人们的关注。Mcl-PHA可由一些假单胞菌类利用自身的脂肪酸合成途径或β-氧化途径来合成。耶氏解脂酵母具有很好的脂/脂肪酸分解代谢能力,但是它体内缺乏PHA合成酶不能合成mcl-PHA。采用代谢工程策略构建重组解脂酵母,外源表达来自铜绿假单胞菌PAO1(Pseudomonas aeruginosa PAO1)的PHA合成酶。在PHA合成酶的C端添加PTS1过氧化物酶体定位信号序列,使其在过氧化物酶体内发挥功能,并对其编码基因PhaC1进行密码子优化得到oPhaC1。利用pINA1312载体构建表达框,借助载体上的zeta序列元件将oPhaC1基因表达框整合至酵母基因组,完成基因的稳定表达。重组菌PSOC在葡萄糖为唯一碳源的培养基中几乎不产PHA,添加0.5%的油酸时可合成占细胞干重0.67%的mcl-PHA。在含三油酸甘油酯的培养基中发酵72h产生1.51% mcl-PHA(wt%)。实验结果充分证明重组解脂酵母作为有潜力的微生物细胞工厂可以用于生产mcl-PHA,也为将来利用富含油脂和其他营养的餐厨垃圾水解液等廉价资源生产mcl-PHA打下基础。     

乳糖诱导高分子量重组蛛丝蛋白发酵培养基优化

在M9培养基的基础上,以乳糖为诱导剂,对基因重组蛛丝蛋白工程菌pNSR32/BL21(DE3)的发酵培养基进行了优化。利用单因子实验和正交试验优化出表达高分子量重组蛛丝蛋白的最优培养基配方,结果表明：优化的碳源为0.3%的甘油,氮源为3%的酵母膏、0.75％的蛋白胨和0.05％(NH4)2SO4及少量的无机盐。优化培养基有利于菌体的生长和目的蛋白的表达,表达重组蛛丝蛋白达总蛋白量的20%。     

Cultivation of the producer of alpha-amylase Bacillus subtilis under batch and continuous conditions

The growth of Bacillus subtilis FU-79 and its production of alpha-amylase (EC 3.2.1.1) were studied under the conditions of batch and continuous cultivation in a semisynthetic medium. The enzyme activity fell down abruptly upon a pulse addition of either glucose or yeast extract to the chemostat culture, and remained at a low level for the following ten generations. Apparently, a double limitation of the culture growth (viz., with residual glucose and with yeast extract components) is required for the activity of alpha-amylase to be high.     

Optimum culture conditions for the production of benzonitrilase by Rhodococcus rhodochrous J1

We sought the optimum conditions for the production of benzonitrilase by Rhodococcus rhodochrous J1. The use of isovaleronitrile or isobutyronitrile as an inducer greatly enhanced benzonitrilase formation. When Rhodococcus rhodochrous J1 was cultivated at 28°C for 96 h in a medium consisting of 0.1 ml of isovaleronitrile, 0.5 g of polypeptone, 0.3 g of malt extract, 0.3 g of yeast extract and 1 g of glycerol per 100 ml of tap water (pH 7.2), and isovaleronitrile was fed twice at the concentrations of 0.1% (v/v) and 0.2% (v/v) at 55 h and 77 h, respectively, during the course of cultivation, the enzyme activity in the culture broth reached approximately 3,100-times higher than the initially obtained level.     

Construction of an Arxula adeninivorans host-vector system based on trp1 complementation

A host/vector expression system based on an Arxula adeninivorans Delta atrp1 gene disruption mutant has been constructed. For this purpose the ATRP1 gene encoding a phosphoribosyl anthranilate isomerase was isolated from the yeast A. adeninivorans and its genome locus was characterized. The Delta atrp1 mutant was generated applying an amplified DNA fragment containing the ALEU2m gene flanked by ATRP1 gene sequences of some 750 bp. The generated auxotrophic host strain was transformed with the plasmid pAL-ATRP1-amyA, which contains the ATRP1 gene as selection marker and the 25S rDNA for targeting. For expression assessment, the plasmid was equipped with an expression cassette consisting of the Bacillus amyloliquefaciens-derived amyA gene fused to the constitutive A. adeninivorans-derived TEF1 promoter and Saccharomyces cerevisiae-derived PHO5 terminator. Transformants contained a single chromosomal copy of the heterologous DNA and were found to be mitotically stable. In initial fermentation trials on a 200 ml shake flask scale maximal alpha-amylase product levels of ca. 300 nkat ml(-1) were observed after 72 h of cultivation with more than 95% of the recombinant alpha-amylase accumulated in the culture medium.     

Strong hybrid promoters and integrative expression/secretion vectors for quasi-constitutive expression of heterologous proteins in the yeast Yarrowia lipolytica

The industrial yeast Yarrowia lipolytica secretes high amounts of an alkaline extracellular protease encoded by the XPR2 gene. The industrial use of the XPR2 promoter was however hindered by its complex regulation. We designed hybrid promoters, based on tandem copies of the XPR2 promoter UAS1 region. In contrast to native XPR2 promoter, these hybrid promoters were not repressed by the preferred carbon and nitrogen sources, nor by acidic conditions, and they did not require the presence of peptones in the culture medium. They exhibited a strong quasi-constitutive activity, similar when carried on either integrative or replicative plasmids. We used these hybrid promoters to direct the production of bovine prochymosin, using XPR2 secretion signals. The production of active chymosin was several fold higher than with previously available Y. lipolytica promoters (up to 160 mg/l). Integrative vectors based on the hybrid promoters, allowing the easy insertion of a heterologous gene and its expression or expression/secretion in Y. lipolytica, were designed. We also designed new Y. lipolytica recipient strains with good secreting abilities, able to grow on sucrose, and devoid of extracellular proteases. These new tools will add to the interest of Y. lipolytica as a host for heterologous protein production.     

From shake flasks to bioreactors: survival of E. coli cells harboring pGST-hPTH through auto-induction by controlling initial content of yeast extract

A high content of yeast extract in complex media can cause auto-induction of phage T7 RNA polymerase and the consequent expression of recombinant protein in Escherichia coli BL21(DE3) during long-term cultivation. Our study demonstrated that the auto-induction of recombinant protein varied in different vectors harboring heterologous genes. Trx, GST, and their fusion proteins such as GST–human parathyroid hormone (hPTH), expressed by pET32a (+), were easily auto-induced by media containing a high content of yeast extract; however, rtPA was not easily auto-induced when using pET22b (+), although both pET systems were under the control of T7lac promoter. Furthermore, the auto-induction of GST–hPTH may start within 1–2 h after inoculation in bioreactors, which is a deficiency in the scale-up from shake flasks to bioreactors. Our results indicated that too much yeast extract in bioreactor cultivations may be responsible for the early auto-induction of target proteins and consequent loss of cell viability and plasmid instability. To achieve a satisfactory yield, host cells with both high cell viability and plasmid stability were necessary for the starter cultures in shake flasks and pre-induction cultures in bioreactors. This could be achieved simply by controlling the initial content of yeast extract and its subsequent supplementation.     

The expression of the Cuphea palustris thioesterase CpFatB2 in Yarrowia lipolytica triggers oleic acid accumulation

The conversion of industrial by‐products into high‐value added compounds is a challenging issue. Crude glycerol, a by‐product of the biodiesel production chain, could represent an alternative carbon source for the cultivation of oleaginous yeasts. Here, we developed five minimal synthetic glycerol‐based media, with different C/N ratios, and we analyzed the production of biomass and fatty acids by Yarrowia lipolytica Po1g strain. We identified two media at the expense of which Y. lipolytica was able to accumulate ～5 g L^?1 of biomass and 0.8 g L^?1 of fatty acids (0.16 g of fatty acids per g of dry weight). These optimized media contained 0.5 g L^?1 of urea or ammonium sulfate and 20 g L^?1 of glycerol, and were devoid of yeast extract. Moreover, Y. lipolytica was engineered by inserting the FatB2 gene, coding for the CpFatB2 thioesterase from Cuphea palustris, in order to modify the fatty acid composition towards the accumulation of medium‐chain fatty acids. Contrary to the expected, the expression of the heterologous gene increased the production of oleic acid, and concomitantly decreased the level of saturated fatty acids. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:26–35, 2016     

Production of salmosin, a snake venom-derived disintegrin, in recombinant Pichia pastoris using high cell density fed-batch fermentation

Salmosin, a snake venom-derived disintegrin, was successfully expressed in the methylotrophic yeast Pichia pastoris and secreted into the culture supernatant, as a 6 kDa protein. High-cell density fermentation of recombinant P. pastoris was optimized for the mass production of salmosin. In a 5 L jar fermentor, recombinant P. pastoris was fermented in growth medium containing 5% (w/v) glycerol at the controlled pH of 5.0. After culturing for 21 h, glycerol feeding medium was fed at one time into the culture broth. After 7 h (a total of 28 h), induction medium that contained methanol was increasingly added until the culture time totaled 75 h. Finally, these optimized culture conditions produced a high cell density of recombinant P. pastoris (dry cell weight of 113.38 g/L) and led to the mass production of salmosin (a total protein concentration of 369.2 mg/L). The culture supernatant containing salmosin inhibited platelet aggregation, resulting in a platelet aggregation of 9% compared to that of 94% in the control experiment, without culture supernatant. These results demonstrate that recombinant salmosin in culture supernatant from high cell density fed-batch fermentation can serve as a platelet aggregation inhibitor.     

Recombinant shrimp (Litopenaeus vannamei) trypsinogen production in Pichia pastoris

Shrimp (Litopenaeus vannamei) trypsinogen has never been isolated from its natural source. To assess the production of L. vannamei trypsinogen, we engineered Pichia pastoris strains and evaluated two culture approaches with three induction culture media, to produce recombinant shrimp trypsinogen for the first time. The trypsinogen II cDNA was fused to the signal sequence of the Saccharomyces cerevisiae alpha mating factor, placed under the control of the P. pastoris AOX1 promoter, and integrated into the genome of P. pastoris host strain GS115. Using standard culture conditions for heterologous gene induction of a GS115 strain in shake flasks, recombinant shrimp trypsinogen was not detected by SDS‐PAGE and Western blot analysis. Growth kinetics revealed a toxicity of recombinant shrimp trypsinogen or its activated form over the cell host. Thus, a different culture approach was tested for the induction step, involving the use of high cell density cultures, a higher frequency of methanol feeding (every 12 h), and a buffered minimal methanol medium supplemented with sorbitol or alanine; alanine supplemented medium was found to be more efficient. After 96 h of induction with alanine supplemented medium, a 29‐kDa band from the cell‐free culture medium was clearly observed by SDS‐PAGE, and confirmed by Western blot to be shrimp trypsinogen, at a concentration of 14 μg/mL. Our results demonstrate that high density cell cultures with alanine in the induction medium allow the production of recombinant shrimp trypsinogen using the P. pastoris expression system, because of improved cell viability and greater stability of the recombinant trypsinogen. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009