Enhanced production of amyrin in Yarrowia lipolytica using a combinatorial protein and metabolic engineering approach.

BACKGROUND: Amyrin is an important triterpenoid and precursor to a wide range of cosmetic, pharmaceutical and nutraceutical products. In this study, we metabolically engineered the oleaginous yeast, Yarrowia lipolytica to produce α- and ß-amyrin on simple sugar and waste cooking oil. RESULTS: We first validated the in vivo enzymatic activity of a multi-functional amyrin synthase (CrMAS) from Catharanthus roseus, by expressing its codon-optimized gene in Y. lipolytica and assayed for amyrins. To increase yield, prevailing genes in the mevalonate pathway, namely HMG1, ERG20, ERG9 and ERG1, were overexpressed singly and in combination to direct flux towards amyrin biosynthesis. By means of a semi-rational protein engineering approach, we augmented the catalytic activity of CrMAS and attained ~ 10-folds higher production level on glucose. When applied together, protein engineering with enhanced precursor supplies resulted in more than 20-folds increase in total amyrins. We also investigated the effects of different fermentation conditions in flask cultures, including temperature, volumetric oxygen mass transfer coefficient and carbon source types. The optimized fermentation condition attained titers of at least 100 mg/L α-amyrin and 20 mg/L ß-amyrin. CONCLUSIONS: The design workflow demonstrated herein is simple and remarkably effective in amplifying triterpenoid biosynthesis in the yeast Y. lipolytica.

An oleaginous yeast platform for renewable 1-butanol synthesis based on a heterologous CoA-dependent pathway and an endogenous pathway.

BACKGROUND: Microbial biofuel production provides a promising sustainable alternative to fossil fuels. 1-Butanol is recognized as an advanced biofuel and is gaining attention as an ideal green replacement for gasoline. In this proof-of-principle study, the oleaginous yeast Yarrowia lipolytica was first engineered with a heterologous CoA-dependent pathway and an endogenous pathway, respectively. RESULTS: The co-overexpression of two heterologous genes ETR1 and EutE resulted in the production of 1-butanol at a concentration of 65 µg/L. Through the overexpression of multiple 1-butanol pathway genes, the titer was increased to 92 µg/L. Cofactor engineering through endogenous overexpression of a glyceraldehyde-3-phosphate dehydrogenase and a malate dehydrogenase further led to titer improvements to 121 µg/L and 110 µg/L, respectively. In addition, the presence of an endogenous 1-butanol production pathway and a gene involved in the regulation of 1-butanol production was successfully identified in Y. lipolytica. The highest titer of 123.0 mg/L was obtained through this endogenous route by combining a pathway gene overexpression strategy. CONCLUSIONS: This study represents the first report on 1-butanol biosynthesis in Y. lipolytica. The results obtained in this work lay the foundation for future engineering of the pathways to optimize 1-butanol production in Y. lipolytica.

Whole-cell biocatalytic and de novo production of alkanes from free fatty acids in Saccharomyces cerevisiae.

Rapid global industrialization in the past decades has led to extensive utilization of fossil fuels, which resulted in pressing environmental problems due to excessive carbon emission. This prompted increasing interest in developing advanced biofuels with higher energy density to substitute fossil fuels and bio-alkane has gained attention as an ideal drop-in fuel candidate. Production of alkanes in bacteria has been widely studied but studies on the utilization of the robust yeast host, Saccharomyces cerevisiae, for alkane biosynthesis have been lacking. In this proof-of-principle study, we present the unprecedented engineering of S. cerevisiae for conversion of free fatty acids to alkanes. A fatty acid α-dioxygenase from Oryza sativa (rice) was expressed in S. cerevisiae to transform C12-18 free fatty acids to C11-17 aldehydes. Co-expression of a cyanobacterial aldehyde deformylating oxygenase converted the aldehydes to the desired alkanes. We demonstrated the versatility of the pathway by performing whole-cell biocatalytic conversion of exogenous free fatty acid feedstocks into alkanes as well as introducing the pathway into a free fatty acid overproducer for de novo production of alkanes from simple sugar. The results from this work are anticipated to advance the development of yeast hosts for alkane production. Biotechnol. Bioeng. 2017;114: 232-237. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Metabolic engineering of Saccharomyces cerevisiae for the overproduction of short branched-chain fatty acids.

Short branched-chain fatty acids (SBCFAs, C4-6) are versatile platform intermediates for the production of value-added products in the chemical industry. Currently, SBCFAs are mainly synthesized chemically, which can be costly and may cause environmental pollution. In order to develop an economical and environmentally friendly route for SBCFA production, we engineered Saccharomyces cerevisiae, a model eukaryotic microorganism of industrial significance, for the overproduction of SBCFAs. In particular, we employed a combinatorial metabolic engineering approach to optimize the native Ehrlich pathway in S. cerevisiae. First, chromosome-based combinatorial gene overexpression led to a 28.7-fold increase in the titer of SBCFAs. Second, deletion of key genes in competing pathways improved the production of SBCFAs to 387.4 mg/L, a 31.2-fold increase compared to the wild-type. Third, overexpression of the ATP-binding cassette (ABC) transporter PDR12 increased the secretion of SBCFAs. Taken together, we demonstrated that the combinatorial metabolic engineering approach used in this study effectively improved SBCFA biosynthesis in S. cerevisiae through the incorporation of a chromosome-based combinatorial gene overexpression strategy, elimination of genes in competitive pathways and overexpression of a native transporter. We envision that this strategy could also be applied to the production of other chemicals in S. cerevisiae and may be extended to other microbes for strain improvement.

Design of short membrane selective antimicrobial peptides containing tryptophan and arginine residues for improved activity, salt-resistance, and biocompatibility.

Antimicrobial peptides (AMPs) kill microbes by non-specific membrane permeabilization, making them ideal templates for designing novel peptide-based antibiotics that can combat multi-drug resistant pathogens. For maximum efficacy in vivo and in vitro, AMPs must be biocompatible, salt-tolerant and possess broad-spectrum antimicrobial activity. These attributes can be obtained by rational design of peptides guided by good understanding of peptide structure-function. Toward this end, this study investigates the influence of charge and hydrophobicity on the activity of tryptophan and arginine rich decamer peptides engineered from a salt resistant human ß-defensin-28 variant. Mechanistic investigations of the decamers with detergents mimicking the composition of bacterial and mammalian membrane, reveal a correlation between improved antibacterial activity and the increase in tryptophan and positive residue content, while keeping hemolysis low. The potent antimicrobial activity and high cell membrane selective behavior of the two most active decamers, D5 and D6, are attributed to an optimum peptide charge to hydrophobic ratio bestowed by systematic arginine and tryptophan substitution. D5 and D6 show surface localization behavior with binding constants of 1.86 × 10(8) and 2.6 × 10(8) M(-1) , respectively, as determined by isothermal calorimetry measurements. NMR derived structures of D5 and D6 in SDS detergent micelles revealed proximity of Trp and Arg residues in an extended structural scaffold. Such potential cation-π interactions may be critical in cell permeabilization of the AMPs. The fundamental characterization of the engineered decamers provided in this study improves the understanding of structure-activity relationship of short arginine tryptophan rich AMPs, which will pave the way for future de novo design of potent AMPs for therapeutic and biomedical applications.

A polycationic antimicrobial and biocompatible hydrogel with microbe membrane suctioning ability.

Despite advanced sterilization and aseptic techniques, infections associated with medical implants have not been eradicated. Most present coatings cannot simultaneously fulfil the requirements of antibacterial and antifungal activity as well as biocompatibility and reusability. Here, we report an antimicrobial hydrogel based on dimethyldecylammonium chitosan (with high quaternization)-graft-poly(ethylene glycol) methacrylate (DMDC-Q-g-EM) and poly(ethylene glycol) diacrylate, which has excellent antimicrobial efficacy against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Fusarium solani. The proposed mechanism of the antimicrobial activity of the polycationic hydrogel is by attraction of sections of anionic microbial membrane into the internal nanopores of the hydrogel, like an 'anion sponge', leading to microbial membrane disruption and then microbe death. We have also demonstrated a thin uniform adherent coating of the hydrogel by simple ultraviolet immobilization. An animal study shows that DMDC-Q-g-EM hydrogel coating is biocompatible with rabbit conjunctiva and has no toxicity to the epithelial cells or the underlying stroma.

Engineering microbes to sense and eradicate Pseudomonas aeruginosa, a human pathogen.

Synthetic biology aims to systematically design and construct novel biological systems that address energy, environment, and health issues. Herein, we describe the development of a synthetic genetic system, which comprises quorum sensing, killing, and lysing devices, that enables Escherichia coli to sense and kill a pathogenic Pseudomonas aeruginosa strain through the production and release of pyocin. The sensing, killing, and lysing devices were characterized to elucidate their detection, antimicrobial and pyocin release functionalities, which subsequently aided in the construction of the final system and the verification of its designed behavior. We demonstrated that our engineered E. coli sensed and killed planktonic P. aeruginosa, evidenced by 99% reduction in the viable cells. Moreover, we showed that our engineered E. coli inhibited the formation of P. aeruginosa biofilm by close to 90%, leading to much sparser and thinner biofilm matrices. These results suggest that E. coli carrying our synthetic genetic system may provide a novel synthetic biology-driven antimicrobial strategy that could potentially be applied to fighting P. aeruginosa and other infectious pathogens.

Development of an enzyme-linked immunosorbent assay analytical platform for refolding yield determination of recombinant hepatitis B virus X (HBx) protein.

We report the development of a novel ELISA platform to quantitate hepatitis B virus X (HBx) protein refolding yields, which is critical for rational design and scaleup of aHBx bioprocess. HBx refolding yields were measured by determining the amount of HBx bound to immobilized GST-p53 on a "reduced glutathione"-functionalized maleimide surface. Refolding yields were distinguished from soluble yields, which were determined by measuring total HBx protein bound to a maleimide surface under reducing conditions. This platform is amenable to scaleup, and will expedite HBx production for structural and clinical studies, leading to the development of HBx-based therapy for liver cancer.

A new bioproduction route for a novel antimicrobial peptide.

Beta defensins are antimicrobial peptides (AMPs) with a broad spectrum antimicrobial behavior against pathogens while having minimal tendency to incur pathogen resistance. Human ß-defensin 28 (hBD28) is a strongly cationic AMP and hence hypothesized to be highly effective in permeabilizing negatively-charged pathogen membranes. However, the scarcity of hBD28 in vivo has impeded detailed structure and antimicrobial studies of hBD28. Chemical synthesis of hBD28 rendered extremely poor yields due to inefficient cysteine oxidation. In this study, a rapid and scalable production route to produce bioactive hBD28 in Escherichia coli (E. coli) is reported. The design of a dual fusion tag expression construct was pivotal in enhancing soluble expression and easing purification of hBD28. The final hBD28 (purity >95%) displayed significant antimicrobial activity against E. coli K12 and showed dose-dependent killing kinetics. Circular dichroism spectroscopy confirmed the presence of both ß-sheet and α-helix conformations in the secondary structure of hBD28.

Refolding of proteins from inclusion bodies: rational design and recipes.

The need to develop protein biomanufacturing platforms that can deliver proteins quickly and cost-effectively is ever more pressing. The rapid rate at which genomes can now be sequenced demands efficient protein production platforms for gene function identification. There is a continued need for the biotech industry to deliver new and more effective protein-based drugs to address new diseases. Bacterial production platforms have the advantage of high expression yields, but insoluble expression of many proteins necessitates the development of diverse and optimised refolding-based processes. Strategies employed to eliminate insoluble expression are reviewed, where it is concluded that inclusion bodies are difficult to eliminate for various reasons. Rational design of refolding systems and recipes are therefore needed to expedite production of recombinant proteins. This review article discusses efforts towards rational design of refolding systems and recipes, which can be guided by the development of refolding screening platforms that yield both qualitative and quantitative information on the progression of a given refolding process. The new opportunities presented by light scattering technologies for developing rational protein refolding buffer systems which in turn can be used to develop new process designs armed with better monitoring and controlling functionalities are discussed. The coupling of dynamic and static light scattering methodologies for incorporation into future bioprocess designs to ensure delivery of high-quality refolded proteins at faster rates is also discussed.

A rational design for hepatitis B virus X protein refolding and bioprocess development guided by second virial coefficient studies.

The hepatitis B virus X (HBx) protein is well known for its role in hepatitis B virus infection that often leads to hepatocellular carcinoma. Despite the clinical importance of HBx, there is little progress in anti-HBx drug development strategies due to shortage of HBx from native sources. Consistent expression of HBx as insoluble inclusion bodies within various expression systems has largely hindered HBx manufacturing via economical biosynthesis routes. Confronted by this roadblock, this study aims to quantitatively understand HBx protein behaviour in solution that will guide the rational development of a refolding-based bioprocess for HBx production. Second virial coefficient (SVC) measurements were employed to study the effects of varying physicochemical parameters on HBx intermolecular protein interaction. The SVC results suggest that covalent HBx aggregates play a key role in protein destabilisation during refolding. The use of an SVC-optimised refolding environment yielded bioactive and soluble HBx proteins from the denatured-reduced inclusion body state. This study provides new knowledge on HBx solubility behaviour in vitro, which is important in structure-function elucidation behaviour of this hydrophobic protein. Importantly, a rational refolding-based Escherichia coli bioprocess that can deliver purified and soluble HBx at large scale is successfully developed, which opens the way for rapid preparation of soluble HBx for further clinical and characterisation studies.

Control Release Coating for Urinary Catheters with Enhanced Released Profile for Sustained Antimicrobial Protection.

Catheter-associated urinary tract infections (CAUTIs) are common and pose significant costs to healthcare systems. To date, this problem is largely unsolved as commercially available antimicrobial catheters are still lacking in functionality and performance. A prior study by Lim et al. ( Biotechnol. Bioeng. 2018, 115 (8), 2000-2012) reported the development of a novel anhydrous polycaprolactone (PCL) polymer formulation with controlled-release functionality for antimicrobial peptides. In this follow-up study, we developed an improved antimicrobial peptide (AMP)-impregnated poly(ethylene glycol) (PEG)-polycaprolactone (PCL) anhydrous polymer coating for enhanced sustained controlled-release functionality to provide catheters with effective antimicrobial properties. Varying the ratio of PEG and PEG-PCL copolymers resulted in polymers with different morphologies, consequently affecting the AMP release profiles. The optimal coating, formulated with 10% (w/w) PEG-PCL in PCL, achieved a controlled AMP release rate of 31.65 ± 6.85 µg/mL daily for up to 19 days, with a moderate initial burst release. Such profile is desired for antimicrobial coating as the initial burst release acts as a sterilizer to kill the bacteria present in the urinary tract upon insertion, and the subsequent linear release functions as a prophylaxis to deter opportunistic microbial infections. As a proof-of-concept application, our optimized coating was then applied to a commercial silicone catheter for further antibacterial tests. Preliminary results revealed that our coated catheters outperformed commercial silver-based antimicrobial catheters in terms of antimicrobial performance and sustainability, lasting for 4 days. Application of the controlled-release coating also aids in retarding biofilm formation, showing a lower extent of biofilm formation at the end of seven inoculation cycles.

Novel short antibacterial and antifungal peptides with low cytotoxicity: Efficacy and action mechanisms.

Short antimicrobial peptides with nine and eleven residues were developed against several clinically important bacterial and fungal pathogens (specifically Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, and Fusarium solani). Twelve analogues of previously reported peptides BP76 (KKLFKKILKFL) and Pac-525 (KWRRWVRWI) were designed, synthesized, and tested for their antimicrobial activities. Two of our eleven amino acid peptides, P11-5 (GKLFKKILKIL) and P11-6 (KKLIKKILKIL), have very low MICs of 3.1-12.5microg ml(-1) against all five pathogens. The MICs of these two peptides against S. aureus, C. albicans and F. solani are four to ten times lower than the corresponding MICs of the reference peptide BP76. P9-4 (KWRRWIRWL), our newly designed nine-amino acid analogue, also has particularly low MICs of 3.1-6.2microg ml(-1) against four of the tested pathogens; these MICs are two to eight times lower than those reported for Pac-525 (6.2-50microg ml(-1)).These new peptides (P11-5, P11-6 and P9-4) also exhibit improved stability in the presence of salts, and have low cytotoxicity as shown by the hemolysis and MTT assays. From the results of field-emission scanning electron microscopy, membrane depolarization and dye-leakage assays, we propose that these peptides exert their action by disrupting membrane lipids. Molecular dynamics simulation studies confirm that P11-6 peptide maintains relatively stable helical structure and exerts more perturbation action on the order of acyl tail of lipid bilayer.

High potency and broad-spectrum antimicrobial peptides synthesized via ring-opening polymerization of alpha-aminoacid-N-carboxyanhydrides.

Antimicrobial peptides (AMPs), particularly those effective against methicillin-resistant Staphylococcus aureus ( S. aureus ) and antibiotic-resistant Pseudomonas aeruginosa ( P. aeruginosa ), are important alternatives to antibiotics. Typical peptide synthesis methods involving solid-phase sequential synthesis are slow and costly, which are obstacles to their more widespread application. In this paper, we synthesize peptides via ring-opening polymerization of alpha-amino acid N-carboxyanhydrides (NCA) using a transition metal initiator. This method offers high potential for inexpensive synthesis of substantial quantities of AMPs. Lysine (K) was chosen as the hydrophilic amino acid and alanine (A), phenylalanine (F), and leucine (L) as the hydrophobic amino acids. We synthesized five series of AMPs (i.e., P(KA), P(KL), P(KF), P(KAL), and P(KFL)), varied the hydrophobic amino acid content from 0 to 100%, and determined minimal inhibitory concentrations (MICs) against clinically important Gram-negative and Gram-positive bacteria and fungi (i.e., Escherichia coli ( E. coli ), P. aeruginosa , Serratia marcescens ( S. marcescens ), and Candida albicans ( C. albicans ). We found that P(K(10)F(7.5)L(7.5)) and P(K(10)F(15)) show the broadest activity against all five pathogens and have the lowest MICs against these pathogens. For P(K(10)F(7.5)L(7.5)), the MICs against E. coli , P. aeruginosa , S. marcescens , S. aureus , and C. albicans are 31 microg/mL, 31 microg/mL, 250 microg/mL, 31 microg/mL, and 62.5 microg/mL, while for P(K(10)F(15)) the respective MICs are 31 microg/mL, 31 microg/mL, 250 microg/mL, 31 microg/mL, and 125 microg/mL. These are lower than the MICs of many naturally occurring AMPs. The membrane depolarization and SEM assays confirm that the mechanism of microbe killing by P(K(10)F(7.5)L(7.5)) copeptide includes membrane disruption, which is likely to inhibit rapid induction of AMP-resistance in pathogens.

Engineering an Alcohol-Forming Fatty Acyl-CoA Reductase for Aldehyde and Hydrocarbon Biosynthesis in Saccharomyces cerevisiae.

Aldehydes are a class of highly versatile chemicals that can undergo a wide range of chemical reactions and are in high demand as starting materials for chemical manufacturing. Biologically, fatty aldehydes can be produced from fatty acyl-CoA by the action of fatty acyl-CoA reductases. The aldehydes produced can be further converted enzymatically to other valuable derivatives. Thus, metabolic engineering of microorganisms for biosynthesizing aldehydes and their derivatives could provide an economical and sustainable platform for key aldehyde precursor production and subsequent conversion to various value-added chemicals. Saccharomyces cerevisiae is an excellent host for this purpose because it is a robust organism that has been used extensively for industrial biochemical production. However, fatty acyl-CoA-dependent aldehyde-forming enzymes expressed in S. cerevisiae thus far have extremely low activities, hence limiting direct utilization of fatty acyl-CoA as substrate for aldehyde biosynthesis. Toward overcoming this challenge, we successfully engineered an alcohol-forming fatty acyl-CoA reductase for aldehyde production through rational design. We further improved aldehyde production through strain engineering by deleting competing pathways and increasing substrate availability. Subsequently, we demonstrated alkane and alkene production as one of the many possible applications of the aldehyde-producing strain. Overall, by protein engineering of a fatty acyl-CoA reductase to alter its activity and metabolic engineering of S. cerevisiae, we generated strains with the highest reported cytosolic aliphatic aldehyde and alkane/alkene production to date in S. cerevisiae from fatty acyl-CoA.

Engineering Yarrowia lipolytica towards food waste bioremediation: Production of fatty acid ethyl esters from vegetable cooking oil.

Fatty acid ethyl esters (FAEEs) can potentially be used as biodiesel, which provides a renewable alternative to petroleum-derived diesel. FAEEs are primarily produced via transesterification of vegetable oil with an alcohol catalyzed by a strong base, which raises safety concerns. Microbial production presents a more environmentally sustainable method for FAEE production, and by harnessing the ability of oleaginous yeast Yarrowia lipolytica to degrade and assimilate hydrophobic substrates, FAEE production could be coupled to food waste bioremediation. In this study, we engineered Y. lipolytica to produce FAEEs from dextrose as well as from vegetable cooking oil as a model food waste. Firstly, we introduced pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) from Zymomonas mobilis to reconstitute the heterologous pathway for ethanol production. Second, we introduced and compared two heterologous wax ester synthases ws2 and maqu_0168 from Marinobacter sp. for FAEE biosynthesis. Next, we disrupted competitive pathways to increase fatty acyl-CoA pool, and optimized carbon sources and cell density for shake-flask fermentation. The engineered strain showed a 24-fold improvement in FAEE production titer over the starting strain. Moreover, we explored the potential of the engineered strain for FAEE production from the model food waste by supplementing vegetable cooking oil to the culture medium. To the best of our knowledge, this is the first report on FAEE production with the supplementation of vegetable cooking oil in Y. lipolytica. These findings provide valuable insights into the engineering of Y. lipolytica for high-level production of FAEEs and its utilization in food waste bioremediation.

Soluble fusion expression and characterization of bioactive human beta-defensin 26 and 27.

This study reports the first successful recombinant expression of cationic antimicrobial peptides human beta-defensin-26 and human beta-defensin-27 in Escherichia coli. HBD26 and HBD27 genes were synthesized through codon optimization, and each gene was then cloned into the expression vector pET32, which feature fusion protein thioredoxin at the N-terminal. The recombinant plasmids were then transformed into E. coli BL21 (DE3) and cultured in MBL medium, which gave yields of HBD26 and HBD27 fusion proteins of up to 1.38 and 1.29 g l(-1), respectively. Affinity chromatography was used to purify the soluble fusion proteins, and the N-terminal TrxA tags were cleaved off by enterokinase. Pure HBD26 and HBD27 were then obtained by cationic exchange chromatography. The overall recovery of HBD26 was 38% and that of HBD27 reached 36%. Both variants showed salt-sensitive antimicrobial activity against gram-negative E. coli but not against gram-positive Staphylococcus aureus and Saccharomyces cerevisiae.

Production of bioactive human beta-defensin 5 and 6 in Escherichia coli by soluble fusion expression.

This work reports the first successful recombinant expression and purification of human beta-defensin 5 (HBD5) and human beta-defensin 6 (HBD6) in Escherichia coli. HBD5 and HBD6 are cationic antimicrobial peptides with three conserved cysteine disulfide bonds. Two codon-optimized sequences coding the HBD5 gene (sHBD5) and HBD6 gene (sHBD6), respectively, were synthesized, and each gene fused with thioredoxin A (TrxA) to construct the expression vectors. The plasmids were transformed into E. coli BL21 (DE3) strains and cultured in MBL medium, which gave high volumetric productivity of HBD5 and HBD6 fusion proteins of up to 1.49 g L(-1) and 1.57 g L(-1), respectively. Soluble HBD5 and HBD6 fusion proteins account for 95.2% and 97.6% of the total fusion proteins, respectively. After cell disruption, the soluble fusion proteins were recovered by affinity chromatography and cleaved by enterokinase. Pure HBD5 and HBD6 were recovered using cationic exchange chromatography. The overall recoveries of HBD5 and HBD6 were 38% and 35%, respectively. Importantly, both HBD5 and HBD6 products showed antimicrobial activity against E. coli but not Staphylococcus aureus. Antimicrobial activity against E. coli of both HBD5 and HBD6 were suppressed by NaCl.

Synthetic biology toolkits and applications in Saccharomyces cerevisiae.

Synthetic biologists construct biological components and systems to look into biological phenomena and drive a myriad of practical applications that aim to tackle current global challenges in energy, healthcare and the environment. While most tools have been established in bacteria, particularly Escherichia coli, recent years have seen parallel developments in the model yeast strain Saccharomyces cerevisiae, one of the most well-understood eukaryotic biological system. Here, we outline the latest advances in yeast synthetic biology tools based on a framework of abstraction hierarchies of parts, circuits and genomes. In brief, the creation and characterization of biological parts are explored at the transcriptional, translational and post-translational levels. Using characterized parts as building block units, the designing of functional circuits is elaborated with examples. In addition, the status and potential applications of synthetic genomes as a genome level platform for biological system construction are also discussed. In addition to the development of a toolkit, we describe how those tools have been applied in the areas of drug production and screening, study of disease mechanisms, pollutant sensing and bioremediation. Finally, we provide a future outlook of yeast as a workhorse of eukaryotic genetics and a chosen chassis in this field.

Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production.

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic characteristics, this unconventional yeast is not only a good model for the study of the fundamental nature of fungal differentiation but is also a promising microbial platform for biochemical production and various biotechnological applications, which require extensive genetic manipulations. However, genetic manipulations of Y. lipolytica have been limited due to the lack of an efficient and stable genetic transformation system as well as very high rates of non-homologous recombination that can be mainly attributed to the KU70 gene. Here, we report an easy and rapid protocol for the efficient genetic transformation and for gene deletion in Y. lipolytica Po1g. First, a protocol for the efficient transformation of exogenous DNA into Y. lipolytica Po1g was established. Second, to achieve the enhanced double-crossover homologous recombination rate for further deletion of target genes, the KU70 gene was deleted by transforming a disruption cassette carrying 1 kb homology arms. Third, to demonstrate the enhanced gene deletion efficiency after deletion of the KU70 gene, we individually deleted 11 target genes encoding alcohol dehydrogenase and alcohol oxidase using the same procedures on the KU70 knockout platform strain. It was observed that the rate of precise homologous recombination increased substantially from less than 0.5% for deletion of the KU70 gene in Po1g to 33%-71% for the single gene deletion of the 11 target genes in Po1g KU70Δ. A replicative plasmid carrying the hygromycin B resistance marker and the Cre/LoxP system was constructed, and the selection marker gene in the yeast knockout strains was eventually removed by expression of Cre recombinase to facilitate multiple rounds of targeted genetic manipulations. The resulting single-gene deletion mutants have potential applications in biofuel and biochemical production.