Metabolic Engineering of Escherichia coli for Production of 2,5-Dimethylpyrazine.

2,5-Dimethylpyrazine (2,5-DMP) is a high-value-added alkylpyrazine compound with important applications in both the food and pharmaceutical fields. In response to the increasing consumer preference for natural products over chemically synthesized ones, efforts have been made to develop efficient microbial cell factories for the production of 2,5-DMP. However, the previously reported recombinant strains have exhibited low yields and relied on expensive antibiotics and inducers. In this study, we employed metabolic engineering strategies to develop an Escherichia coli strain capable of producing 2,5-DMP at high levels without the need for inducers or antibiotics. Initially, the biosynthesis pathway of 2,5-DMP was constructed that realized 2,5-DMP production from glucose. Subsequently, efforts focused on enhancing 2,5-DMP production by improving the availability of the cofactor NAD+ and precursor l-threonine. Additionally, the supply and conversion of l-threonine were balanced by optimizing the copy number of the key gene tdh on the chromosome and by modifying the l-threonine transport system. The final engineering strain D19 produced 3.1 g/L of 2,5-DMP, which is the highest titer for fermentative production of 2,5-DMP using glucose as the carbon source up to date. The strategies used in this study lay a good foundation for the production of 2,5-DMP on a large scale.

Light irradiation changes the regulation pattern of BtCrgA on carotenogenesis in Blakeslea trispora.

CrgA has been shown to be a negative regulator of carotenogenesis in some filamentous fungi, while light irradiation is an inducible environmental factor for carotenoid biosynthesis. To clarify the relationship between CrgA and light-inducible carotenogenesis in Blakeslea trispora, the cis-acting elements of the btcrgA promoter region were investigated, followed by the analyses of correlation between the expression of btcrgA and carotenoid structural genes under different irradiation conditions. A variety of cis-acting elements associated with light response was observed in the promoter region of btcrgA, and transcription of btcrgA and carotenoid structural genes under different irradiation conditions was induced by white light with a clear correlation. Then, RNA interference and overexpression of btcrgA were performed to investigate their effects on carotenogenesis at different levels under irradiation and darkness. The analyses of transcription and enzyme activities of carotenoid structural gene, and accumulation of carotenoids among btcrgA-interfered, btcrgA-overexpressed, and wild-type strains under irradiation and darkness indicate that btcrgA negatively regulates the synthesis of carotenoid in darkness, while promotes the carotenogenesis under irradiation regardless of reduced or overexpression of btcrgA .

Conditional protein degradation in Yarrowia lipolytica using the auxin-inducible degron.

Conditional protein degradation is a powerful tool for controlled protein knockdown. The auxin-inducible degron (AID) technology uses a plant auxin to induce depletion of degron-tagged proteins, and it has been shown to be functional in several non-plant eukaryotes. In this study, we demonstrated AID-based protein knockdown in an industrially important oleaginous yeast Yarrowia lipolytica. Using the mini-IAA7 (mIAA7) degron derived from Arabidopsis IAA7, coupled with an Oryza sativa TIR1 (OsTIR1) plant auxin receptor F-box protein (expressed from the copper-inducible MT2 promoter), C-terminal degron-tagged superfolder GFP could be degraded in Yarrowia lipolytica upon addition of copper and the synthetic auxin 1-Naphthaleneacetic acid (NAA). However, leaky degradation of the degron-tagged GFP in the absence of NAA was also noted. This NAA-independent degradation was largely eliminated by replacing the wild-type OsTIR1 and NAA with the OsTIR1F74A variant and the auxin derivative 5-Ad-IAA, respectively. Degradation of the degron-tagged GFP was rapid and efficient. However, Western blot analysis revealed cellular proteolytic cleavage within the mIAA7 degron sequence, leading to the production of a GFP sub-population lacking an intact degron. The utility of the mIAA7/OsTIR1F74A system was further explored in controlled degradation of a metabolic enzyme, ß-carotene ketolase, which converts ß-carotene to canthaxanthin via echinenone. This enzyme was tagged with the mIAA7 degron and expressed in a ß-carotene producing Y. lipolytica strain that also expressed OsTIR1F74A controlled by the MT2 promoter. By adding copper and 5-Ad-IAA at the time of culture inoculation, canthaxanthin production was found to be reduced by about 50% on day five compared to the control culture without adding 5-Ad-IAA. This is the first report that demonstrates the efficacy of the AID system in Y. lipolytica. Further improvement of AID-based protein knockdown in Y. lipolytica may be achieved by preventing proteolytic removal of the mIAA7 degron tag.

Genomic insights into the evolutionary history and diversification of bulb traits in garlic.

BACKGROUND: Garlic is an entirely sterile crop with important value as a vegetable, condiment, and medicine. However, the evolutionary history of garlic remains largely unknown. RESULTS: Here we report a comprehensive map of garlic genomic variation, consisting of amazingly 129.4 million variations. Evolutionary analysis indicates that the garlic population diverged at least 100,000 years ago, and the two groups cultivated in China were domesticated from two independent routes. Consequently, 15.0 and 17.5% of genes underwent an expression change in two cultivated groups, causing a reshaping of their transcriptomic architecture. Furthermore, we find independent domestication leads to few overlaps of deleterious substitutions in these two groups due to separate accumulation and selection-based removal. By analysis of selective sweeps, genome-wide trait associations and associated transcriptomic analysis, we uncover differential selections for the bulb traits in these two garlic groups during their domestication. CONCLUSIONS: This study provides valuable resources for garlic genomics-based breeding, and comprehensive insights into the evolutionary history of this clonal-propagated crop.

Non-viral 2A-like sequences for protein coexpression.

Simultaneous coexpression of multiple proteins is essential for biotechnology and synthetic biology. Currently, the most popular polyprotein coexpression system utilizes the foot-and-mouth disease virus (FMDV) 2A peptide that mediates translational ribosome-skipping events. However, due to unfavorable consumer acceptance of transgenic products containing animal-virus sequences, novel non-viral 2A-like peptides from purple sea urchin (Strongylcentrotus purpuratus) and California sea slug (Aplysia californica) were investigated for polyprotein coexpression in this study. We demonstrated that these non-viral 2A sequences functioned similarly to their viral counterpart in polyprotein processing, in both plant and mammalian cells, and were successfully used to express a functional recombinant antibody. The new non-viral 2A-like sequences offer an alternative tool for engineering multigenic traits or production of protein complexes as biomedicine via coexpression of protein subunits.

Improving the bioavailability and bioactivity of garlic bioactive compounds via nanotechnology.

This review highlights main bioactive compounds and important biological functions especially anticancer effects of the garlic. In addition, we review current literature on the stability and bioavailability of garlic components. Finally, this review aims to provide a potential strategy for using nanotechnology to increase the stability and solubility of garlic components, providing guidelines for the qualities of garlic products to improve their absorption and prevent their early degradation, and extend their circulation time in the body. The application of nanotechnology to improve the bioavailability and targeting of garlic compounds are expected to provide a theoretical basis for the functional components of garlic to treat human health. We review the improvement of bioavailability and bioactivity of garlic bioactive compounds via nanotechnology, which could promisingly overcome the limitations of conventional garlic products, and would be used to prevent and treat cancer and other diseases in the near future.

Enhancing stability and anti-inflammatory properties of curcumin in ulcerative colitis therapy using liposomes mediated colon-specific drug delivery system.

Curcumin liposomes (CUR-LPs) was identified by evaluating morphology, appearance, zeta potential, particle diameter, and drug encapsulation efficiency. The results indicated that particle diameter, surface charge and polydispersity index (PDI) of curcumin (CUR)-loaded anionic liposomes were 167 nm, -34 mV and 0.09, respectively. CUR-LPs is high stable pseudo-pH-sensitive nanoparticles system which has a favorable stability in simulated gastric fluid and slower degradation rate allowing CUR sustained release for prolonged times in simulated intestinal fluid. Within 1 h, the CUR consumption was 21.82% in simulated gastric fluid (SGF) and 27.32% in simulated intestinal fluid (SIF), respectively. CUR-LPs could attenuate clinical symptoms including weight loss, diarrhea and fecal bleeding. Especially, it could also prevent dextran sulfate sodium salt (DSS)-inducedcolon tissue damage and colon shortening, and reduce the production of malondialdehyde (MDA), colonic myeloperoxidase (MPO), Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in animal model. Our study illustrated that liposomes (LPs) was a potential carrier to develop the colon-specific drug delivery system incorporating CUR for treating ulcerative colitis.

A Chromosome-Level Genome Assembly of Garlic (Allium sativum) Provides Insights into Genome Evolution and Allicin Biosynthesis.

Garlic, an economically important vegetable, spice, and medicinal crop, produces highly enlarged bulbs and unique organosulfur compounds. Here, we report a chromosome-level genome assembly for garlic, with a total size of approximately 16.24 Gb, as well as the annotation of 57 561 predicted protein-coding genes, making garlic the first Allium species with a sequenced genome. Analysis of this garlic genome assembly reveals a recent burst of transposable elements, explaining the substantial expansion of the garlic genome. We examined the evolution of certain genes associated with the biosynthesis of allicin and inulin neoseries-type fructans, and provided new insights into the biosynthesis of these two compounds. Furthermore, a large-scale transcriptome was produced to characterize the expression patterns of garlic genes in different tissues and at various growth stages of enlarged bulbs. The reference genome and large-scale transcriptome data generated in this study provide valuable new resources for research on garlic biology and breeding.

Production and excretion of astaxanthin by engineered Yarrowia lipolytica using plant oil as both the carbon source and the biocompatible extractant.

This study aimed to develop a bioprocess using plant oil as the carbon source for lipid-assimilating yeast to produce high-value astaxanthin. Using high-oleic safflower oil as a model, efficient cell growth and astaxanthin production by the engineered Yarrowia lipolytica strain ST7403 was demonstrated, and a considerable portion of astaxanthin was found excreted into the spent oil. Astaxanthin was the predominant carotenoid in the extracellular oil phase that allowed facile in situ recovery of astaxanthin without cell lysis. Autoclaving the safflower oil medium elevated the peroxide level but it declined quickly during fermentation (reduced by 84% by day 3) and did not inhibit cell growth or astaxanthin production. In a 1.5-L fed-batch bioreactor culture with a YnB-based medium containing 20% safflower oil, and with the feeding of casamino acids, astaxanthin production reached 54 mg/L (53% excreted) in 28 days. Further improvement in astaxanthin titer and productivity was achieved by restoring leucine biosynthesis in the host, and running fed-batch fermentation using a high carbon-to-nitrogen ratio yeast extract/peptone medium containing 70% safflower oil, with feeding of additional yeast extract/peptone, to attain 167 mg/L astaxanthin (48% excreted) in 9.5 days of culture. These findings facilitate industrial microbial biorefinery development that utilizes renewable lipids as feedstocks to not only produce high-value products but also effectively extract and recover the products, including non-native ones.Key Points• Yarrowia lipolytica can use plant oil as a C-source for astaxanthin production.• Astaxanthin is excreted and accumulated in the extracellular oil phase.• Astaxanthin is the predominant carotenoid in the extracellular oil phase.• Plant oil serves as a biocompatible solvent for in situ astaxanthin extraction. Graphical abstract.

Analysis of Yarrowia lipolytica growth, catabolism, and terpenoid biosynthesis during utilization of lipid-derived feedstock.

This study employs biomass growth analyses and 13C-isotope tracing to investigate lipid feedstock utilization by Yarrowia lipolytica. Compared to glucose, oil-feedstock in the minimal medium increases the yeast's biomass yields and cell sizes, but decreases its protein content (<20% of total biomass) and enzyme abundances for product synthesis. Labeling results indicate a segregated metabolic network (the glycolysis vs. the TCA cycle) during co-catabolism of sugars (glucose or glycerol) with fatty acid substrates, which facilitates resource allocations for biosynthesis without catabolite repressions. This study has also examined the performance of a ß-carotene producing strain in different growth mediums. Canola oil-containing yeast-peptone (YP) has resulted in the best ß-carotene titer (121 ±â€¯13 mg/L), two-fold higher than the glucose based YP medium. These results highlight the potential of Y. lipolytica for the valorization of waste-derived lipid feedstock.

Intein-ubiquitin chimeric domain for coordinated protein coexpression.

Efficient coproduction of multiple proteins or their subunits is important in many facets of life sciences and biotechnology. Here, we report a novel approach that exploits the synergy between an engineered mini-intein and an ubiquitin variant to achieve coordinated coexpression of multiple proteins in eukaryotic hosts, from a single open reading frame that encodes a polyprotein precursor consisting of proteins of interest (POIs) connected by an intervening intein-ubiquitin fusion domain. The intein variant mediates highly active autocatalytic cleavage at its N-terminus, whereas the endogenous deubiquitinases cleave at ubiquitin's C-terminus, leading to the release of the POIs. Using fluorescent reporter proteins for proof-of-concept, utility of the intein-ubiquitin domain was validated in higher plants and yeast systems. Essentially complete release of the POIs was achieved as demonstrated with western blots. Proteins expressed using the intein-ubiquitin system potentially preserve their intended sequences, which is important for preventing alteration of POI function.

Magnetic Oleosome as a Functional Lipophilic Drug Carrier for Cancer Therapy.

In the present study, we fabricated magnetic oleosomes functionalized with recombinant proteins as a new carrier for oil-based lipophilic drugs for cancer treatment. The bioengineered oleosome is composed of neutral lipids surrounded by a phospholipid monolayer with embedded oleosin fusion proteins. The oleosin was genetically fused to a nanobody of a green fluorescent protein (GFP). A recombinant protein consisting of immunoglobulin-binding protein LG fused to GFP was used to couple the oleosome to an antibody for targeted delivery to breast cancer cells. The lipid core of the oleosome was loaded with magnetic nanoparticles and carmustine as the lipophilic drug. The magnetic oleosome was characterized using transmission electron microscopy and dynamic light scattering. Moreover, the specific delivery of oleosome into the target cancer cell was investigated via confocal microscopy. To examine the cell viability of the delivered oleosome, a conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was carried out. Furthermore, an animal study was conducted to confirm the effect resulting from the delivery of the anticancer drug-loaded oleosomes. Taken together, the fabricated lipophilic drug-loaded magnetic oleosome can be a powerful tool for oil-based drug delivery agent for cancer therapy.

Intein-mediated assembly of tunable scaffoldins for facile synthesis of designer cellulosomes.

In this study, extended artificial scaffoldins possessing multiple cohesin modules were created in vivo by employing split-intein-mediated protein ligation. Artificial scaffoldins having one Clostridium thermocellum cohesin (Coht), one carbohydrate binding module (CBM) from Clostridium cellulolyticum scaffolding protein CipC, and one to five cohesins (Cohc) derived from CipC, were assembled. These scaffoldins were used to assemble cellulosomal enzyme complexes for investigating the interplay among endoglucanase, exoglucanase, and scaffoldin-borne CBM, on the hydrolysis of a model microcrystalline cellulose substrate, Avicel. The cellulosomal complexes were assembled in vitro by incubating recombinant C. thermocellum endoglucanase (At) and C. cellulolyticum exoglucanase (Ec), with the various artificial scaffoldins. Under a fixed total cellulase concentration, improved hydrolysis is noted by recruiting both Ec and At on the same scaffoldin, for all scaffoldins tested, compared with free cellulases. The improvement is more profound with scaffoldins having a higher Cohc/Coht ratio (i.e., increased Ec/At ratio). Furthermore, among scaffoldins having the same Cohc/Coht ratio, highest rates of Avicel hydrolysis are noted when Coht, and hence an endoglucanase, is situated next to the CBM and not flanked by Cohc. These results point to the importance of using scaffoldins with sufficiently high numbers of cohesin units to achieve an optimal exo-/endo-glucanase ratio to create efficient designer cellulosomes. Furthermore, intein-trans-splicing is proven here to be an effective method for assembling complex scaffoldins and more intricate cellulosomes.

Valorization of papaya fruit waste through low-cost fractionation and microbial conversion of both juice and seed lipids.

Papaya (Carica papaya) is widely cultivated in many tropical regions of the world. With an estimated 30-50% cull rate, there is a large amount of off-grade papaya produced. Here, we report very low-cost processing of culled papaya fruit waste, without needing any complex mechanized operations, to yield several products, including seed oil, sugar-rich puree, detoxified/defatted seed meal, and crude myrosinase and glucosinolates with antimicrobial and biofumigation applications. We then demonstrated that both puree and seed oil can serve as effective carbon substrates for cultivation of the oleaginous yeast Yarrowia lipolytica to produce single-cell proteins and high-value recombinant protein products. To use papaya seed oil for culturing Y. lipolytica, the concentration of the inhibitory substance benzyl isothiocyanate (BITC) in the oil needs to be minimized. If the culled fruits (and hence seeds) were stored frozen prior to drying, a very high level (>30 mM) of BITC was detected in the oil extracted from the dried seeds. However, if the seeds were not frozen prior to drying, oil from dried papaya seeds contained almost no BITC, and could support vigorous growth of Y. lipolytica, with efficient production of a functional nanobody fusion protein at a level similar to that achieved using olive oil. By using both juice and seed lipid, rather than juice alone, Y. lipolytica biomass produced per unit papaya more than doubled. As Y. lipolytica is amenable to genetic manipulation, and is known as a proficient cell factory with many industrial applications, the papaya waste valorization technology could potentially be extended to produce additional useful products such as biofuel and oleochemicals from Y. lipolytica.

Coordinated protein co-expression in plants by harnessing the synergy between an intein and a viral 2A peptide.

A novel approach is developed for coordinated expression of multiple proteins from a single transgene in plants. An Ssp DnaE mini-intein variant engineered for hyper-N-terminal autocleavage is covalently linked to the foot-and-mouth disease virus 2A (F2A) peptide with unique ribosome skipping property, via a peptide linker, to create an 'IntF2A' self-excising fusion protein domain. This IntF2A domain acts, in cis, to direct highly effective release of its flanking proteins of interest (POIs) from a 'polyprotein' precursor in plants. This is successfully demonstrated in stably transformed cultured tobacco cells as well as in different organs of transgenic tobacco plants. Highly efficient polyprotein processing mediated by the IntF2A domain was also demonstrated in lettuce and Nicotiana benthamiana based on transient expression. Protein constituents released from the polyprotein precursor displayed proper function and accumulated at similar levels inside the cells. Importantly, no C-terminal F2A extension remains on the released POIs. We demonstrated co-expression of as many as three proteins in plants without compromising expression levels when compared with those using single-protein vectors. Accurate differential cellular targeting of released POIs is also achieved. In addition, we succeeded in expressing a fully assembled and functional chimeric anti-His Tag antibody in N. benthamiana leaves. The IntF2A-based polyprotein transgene system overcomes key impediments of existing strategies for multiprotein co-expression in plants, which is particularly important for gene/trait stacking.

Carbon dioxide capture using Escherichia coli expressing carbonic anhydrase in a foam bioreactor.

The present study reports CO2 capture and conversion to bicarbonate using Escherichia coli expressing carbonic anhydrase (CA) on its cell surface in a novel foam bioreactor. The very large gas-liquid interfacial area in the foam bioreactor promoted rapid CO2 absorption while the CO2 in the aqueous phase was subsequently converted to bicarbonate ions by the CA. CO2 gas removal in air was investigated at various conditions such as gas velocity, cell density and CO2 inlet concentration. Regimes for kinetic and mass transfer limitations were defined. Very high removal rates of CO2 were observed: 9570 g CO2 m(-3) bioreactor h(-1) and a CO2 removal efficiency of 93% at 4% inlet CO2 when the gas retention time was 24 s, and cell concentration was 4 gdw L(-1). These performances are superior to earlier reports of experimental bioreactors using CA for CO2 capture. Overall, this bioreactor system has significant potential as an alternative CO2 capture technology.

A dual-intein autoprocessing domain that directs synchronized protein co-expression in both prokaryotes and eukaryotes.

Being able to coordinate co-expression of multiple proteins is necessary for a variety of important applications such as assembly of protein complexes, trait stacking, and metabolic engineering. Currently only few options are available for multiple recombinant protein co-expression, and most of them are not applicable to both prokaryotic and eukaryotic hosts. Here, we report a new polyprotein vector system that is based on a pair of self-excising mini-inteins fused in tandem, termed the dual-intein (DI) domain, to achieve synchronized co-expression of multiple proteins. The DI domain comprises an Ssp DnaE mini-intein N159A mutant and an Ssp DnaB mini-intein C1A mutant connected in tandem by a peptide linker to mediate efficient release of the flanking proteins via autocatalytic cleavage. Essentially complete release of constituent proteins, GFP and RFP (mCherry), from a polyprotein precursor, in bacterial, mammalian, and plant hosts was demonstrated. In addition, successful co-expression of GFP with chloramphenicol acetyltransferase, and thioredoxin with RFP, respectively, further substantiates the general applicability of the DI polyprotein system. Collectively, our results demonstrate the DI-based polyprotein technology as a highly valuable addition to the molecular toolbox for multi-protein co-expression which finds vast applications in biotechnology, biosciences, and biomedicine.

Tunable nano-oleosomes derived from engineered Yarrowia lipolytica.

Oleosomes are discrete organelles filled with neutral lipids surrounded by a protein-embedded phospholipid monolayer. Their simple yet robust structure, as well as their amenability to biological, chemical, and physical processing, can be exploited for various biotechnology applications. In this study, we report facile biosynthesis of functionalized oleosomes within oleaginous yeast Yarrowia lipolytica, through expression of oleosin fusion proteins. By fusing a cDNA clone of a sesame oleosin with either the coding sequence of a red fluorescent protein mCherry or a cellulosomal scaffolding protein cohesin from Clostridium cellulolyticum, these oleosin-fusion proteins were efficiently expressed and specifically targeted to and anchored on the surface of the oleosomes within the Y. lipolytica cells. The engineered oleosomes can be easily separated from the Y. lipolytica cell extract via floating centrifugation and both mCherry and cohesin domains are shown to be functional. Upon sonication, the engineered Yarrowia oleosomes exhibit a mean diameter of 200-300 nm and are found to be highly stable. The feasibility of co-displaying multiple proteins on the Yarrowia oleosomes was demonstrated by incubating cohesin-displaying oleosomes with different dockerin-fusion proteins. Based on this strategy, engineered oleosomes with both cell-targeting and reporting activities were created and shown to be functional. Taken together, the Yarrowia oleosome surface display system in which oleosin serves as an efficient membrane anchor motif shows great promise as a simple platform for creating tunable nanoparticles.

Self-assembled amyloid-like oligomeric-cohesin Scaffoldin for augmented protein display on the saccharomyces cerevisiae cell surface.

In this study, a molecular self-assembly strategy to develop a novel protein scaffold for amplifying the extent and variety of proteins displayed on the surface of Saccharomyces cerevisiae is presented. The cellulosomal scaffolding protein cohesin and its upstream hydrophilic domain (HD) were genetically fused with the yeast Ure2p N-terminal fibrillogenic domain consisting of residues 1 to 80 (Ure2p(1-80)). The resulting Ure2p(1-80)-HD-cohesin fusion protein was successfully expressed in Escherichia coli to produce self-assembled supramolecular nanofibrils that serve as a novel protein scaffold displaying multiple copies of functional cohesin domains. The amyloid-like property of the nanofibrils was confirmed via thioflavin T staining and atomic force microscopy. These cohesin nanofibrils attached themselves, via a green fluorescent protein (GFP)-dockerin fusion protein, to the cell surface of S. cerevisiae engineered to display a GFP-nanobody. The excess cohesin units on the nanofibrils provide ample sites for binding to dockerin fusion proteins, as exemplified using an mCherry-dockerin fusion protein as well as the Clostridium cellulolyticum CelA endoglucanase. More than a 24-fold increase in mCherry fluorescence and an 8-fold increase in CelA activity were noted when the cohesin nanofibril scaffold-mediated yeast display was used, compared to using yeast display with GFP-cohesin that contains only a single copy of cohesin. Self-assembled supramolecular cohesin nanofibrils created by fusion with the yeast Ure2p fibrillogenic domain provide a versatile protein scaffold that expands the utility of yeast cell surface display.

Combination of site-directed mutagenesis and yeast surface display enhances Rhizomucor miehei lipase esterification activity in organic solvent.

To increase the activity of Rhizomucor miehei lipase (RML) in organic solvent, multiple sequence alignments and rational site-directed mutagenesis were used to create RML variants. The obtained proteins were surface-displayed on Pichia pastoris by fusion to Flo1p as an anchor protein. The synthetic activity of four variants showed from 1.1- to 5-fold the activity of native lipase in an esterification reaction in heptane with alcohol and caproic acid as substrates. The increase in esterification activity may be attributed to the four mutations changing the flexibility of RML or facilitating the reaction. In conclusion, this method demonstrated that multiple sequence alignments and rational site-directed mutagenesis combined with yeast display technology is a faster and more effective means of obtaining high-efficiency esterification lipase variants compared with previous similar methods.