Directed evolution of xylose isomerase for improved xylose catabolism and fermentation in the yeast Saccharomyces cerevisiae

The heterologous expression of a highly functional xylose isomerase pathway in Saccharomyces cerevisiae would have significant advantages for ethanol yield, since the pathway bypasses cofactor requirements found in the traditionally used oxidoreductase pathways. However, nearly all reported xylose isomerase-based pathways in S. cerevisiae suffer from poor ethanol productivity, low xylose consumption rates, and poor cell growth compared with an oxidoreductase pathway and, additionally, often require adaptive strain evolution. Here, we report on the directed evolution of the Piromyces sp. xylose isomerase (encoded by xylA) for use in yeast. After three rounds of mutagenesis and growth-based screening, we isolated a variant containing six mutations (E15D, E114G, E129D, T142S, A177T, and V433I) that exhibited a 77% increase in enzymatic activity. When expressed in a minimally engineered yeast host containing a gre3 knockout and tal1 and XKS1 overexpression, the strain expressing this mutant enzyme improved its aerobic growth rate by 61-fold and both ethanol production and xylose consumption rates by nearly 8-fold. Moreover, the mutant enzyme enabled ethanol production by these yeasts under oxygen-limited fermentation conditions, unlike the wild-type enzyme. Under microaerobic conditions, the ethanol production rates of the strain expressing the mutant xylose isomerase were considerably higher than previously reported values for yeast harboring a xylose isomerase pathway and were also comparable to those of the strains harboring an oxidoreductase pathway. Consequently, this study shows the potential to evolve a xylose isomerase pathway for more efficient xylose utilization.     

Directed evolution and the creation of enantioselective biocatalysts

Directed evolution has emerged as a key technology to generate enzymes with new or improved properties that are of major importance to the biotechnology industry. A directed evolution approach starts with the identification of a target enzyme to be optimized and the cloning of the corresponding gene. An efficient expression system is needed before the target gene is subjected to random mutagenesis and/or in vitro recombination, thereby creating molecular diversity. Subsequently, improved enzyme variants are identified, preferably after being secreted into culture medium, by screening or selection for the desired property. The genes encoding the improved enzymes are then used to parent the next round of directed evolution. Enantioselectivity is a biocatalyst property of major biotechnological importance that is, however, difficult to deal with. We discuss recent examples of creating enantioselective biocatalysts by directed evolution.     

Asymmetric synthesis of d-glyceric acid by an alditol oxidase and directed evolution for enhanced oxidative activity towards glycerol

Glycerol as a by-product of biodiesel production is an attractive precursor for producing d-glyceric acid. Here, we demonstrate the successful production of d-glyceric acid based on glycerol via glyceraldehyde in a two-step enzyme reaction with the FAD-dependent alditol oxidase from Streptomyces coelicolor A3(2). The hydrogen peroxide generated in the reaction can be used in detergent, food, and paper industry. In order to apply the alditol oxidase in industry, the enzyme was subjected to protein engineering. Different strategies were used to enhance the substrate specificity towards glycerol. Initial attempts based on rational protein design in the active site region were found unsuccessful to increase activity. However, through directed evolution, an alditol oxidase double mutant (V125M/A244T) with 1.5-fold improved activity for glycerol was found by screening 8,000 clones. Further improvement of activity was achieved by combinatorial experiments, which led to a quadruple mutant (V125M/A244T/V133M/G399R) with 2.4-fold higher specific activity towards glycerol compared to the wild-type enzyme. Through studying the effects of mutations created, we were able to understand the importance of certain amino acids in the structure of alditol oxidase, not only for conferring enzymatic structural stability but also with respect to their influence on oxidative activity.     

Pro-antibiotic Substrates for the Identification of Enantioselective Hydrolases

A new growth-based screening method for the identification of enantioselective hydrolases, such as lipases and esterases, using pro-antibiotic substrates was devised. An enantioselective hydrolase could be identified by measuring growth rates of cells in liquid media containing (R)- or (S)-2–phenylbutyric chloramphenicol esters. This method can be applied to the screening of novel enantioselective microbes and to the high-throughput screening for the directed evolution of enantioselective hydrolytic enzymes.     

Using continuous directed evolution to improve enzymes for plant applications

Continuous directed evolution of enzymes and other proteins in microbial hosts is capable of outperforming classical directed evolution by executing hypermutation and selection concurrently in vivo, at scale, with minimal manual input. Provided that a target enzyme’s activity can be coupled to growth of the host cells, the activity can be improved simply by selecting for growth. Like all directed evolution, the continuous version requires no prior mechanistic knowledge of the target. Continuous directed evolution is thus a powerful way to modify plant or non-plant enzymes for use in plant metabolic research and engineering. Here, we first describe the basic features of the yeast (Saccharomyces cerevisiae) OrthoRep system for continuous directed evolution and compare it briefly with other systems. We then give a step-by-step account of three ways in which OrthoRep can be deployed to evolve primary metabolic enzymes, using a THI4 thiazole synthase as an example and illustrating the mutational outcomes obtained. We close by outlining applications of OrthoRep that serve growing demands (i) to change the characteristics of plant enzymes destined for return to plants, and (ii) to adapt (“plantize”) enzymes from prokaryotes—especially exotic prokaryotes—to function well in mild, plant-like conditions.

Continuous directed evolution using the yeast OrthoRep system is a powerful way to improve enzymes for use in plant engineering as illustrated by “plantizing” a bacterial thiamin synthesis enzyme.     

Enhanced activity of Rhizomucor miehei lipase by directed evolution with simultaneous evolution of the propeptide

Propeptides are short sequences that facilitate the folding of their associated proteins. The present study found that the propeptide of Rhizomucor miehei lipase (RML) was not proteolytically removed in Escherichia coli. Moreover, RML was not expressed if the propeptide was removed artificially during the cloning process in E. coli. This behavior in E. coli permitted the application of directed evolution to full-length RML, which included both propeptide and catalytic domain, to explore the role played by the propeptide in governing enzyme activity. The catalytic rate constant, k (cat), of the most active mutant RML protein (Q5) was increased from 10.63?±?0.80 to 71.44?±?3.20?min(-1) after four rounds of screening. Sequence analysis of the mutant displayed three mutations in the propeptide (L57V, S65A, and V67A) and two mutations in the functional region (I111T and S168P). This result showed that improved activity was obtained with essential involvement by mutations in the propeptide, meaning that the majority of mutants with enhanced activity had simultaneous mutations in propeptide and catalytic domains. This observation leads to the hypothesis that directed evolution has simultaneous and synergistic effects on both functional and propeptide domains that arise from the role played by the propeptide in the folding and maturation of the enzyme. We suggest that directed evolution of full-length proteins including their propeptides is a strategy with general validity for extending the range of conformations available to proteins, leading to the enhancement of the catalytic rates of the enzymes.     

Whole plasmid mutagenic PCR for directed protein evolution

Protein function can be engineered through iterated cycles of random mutagenesis and screening (directed evolution). Optimization of protein expression is essential for the development of sensitive and precise high throughput assays. Here we optimize the performance of a plasmid-borne Escherichia coli lacZ gene in two rounds of directed evolution. First, its promoter was "randomized" by whole plasmid polymerase chain reaction (PCR) and intra-molecular self-ligation. A genetically stable constitutive expression vector was isolated in an in vivo genetic selection. Second, the entire plasmid was randomly mutated in a slightly mutagenic long polymerase chain reaction. The PCR products were digested with a restriction enzyme, self-ligated by T4 DNA ligase and transformed into E. coli. The resulting library of beta-galactosidase (beta-gal) mutants consisted mostly ( approximately 80%) of hypomorphs, suggesting that the mutation rate was appropriate for directed evolution applications. We isolated and characterized 14 variants with increased activity in reactions with 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside (X-gal). The purified protein derived from one clone exhibited a 100-fold improvement in k(cat) over its parent in reactions with para-nitrophenyl-beta-d-galactopyranoside (pNP-gal). This latter result clearly demonstrates the utility of whole plasmid mutagenic PCR for directed protein evolution.     

Exploring the fitness landscape of an RNA virus by using a universal barcode microarray

Studies of viral pathogenesis have relied heavily on analyses of specific clones and their genetic determinants of virulence. It is sometimes difficult to apply this reductionist approach to the study of RNA viruses, which by virtue of their very high mutation rates, exist as a complex mixture of mutants. While quasispecies theory has provided an intellectual framework for exploring the relationship between the viral population structure and phenotype, experimental studies have been limited by the relatively poor resolution of traditional sequencing-based approaches. We have addressed this problem by developing a molecular barcoding strategy in which viral subpopulations are tagged with unique 20-nucleotide sequences. The behavior of these subpopulations can be monitored using a universal barcode microarray. We demonstrate the performance of our barcode microarray platform using poliovirus, a model RNA virus. Using this platform, we explored the fitness landscape occupied by an artificial quasispecies consisting of 48 randomly mutagenized clones. We were able to rapidly derive precise fitness measurements for a majority of these clones and identified a neutral space surrounding the wild type. The experimental paradigm presented here is readily adaptable to other viral systems and can potentially be used to track thousands of variants in a cost-effective manner.     

Ultra-high-throughput screening based on cell-surface display and fluorescence-activated cell sorting for the identification of novel biocatalysts

Enzyme libraries displayed on the surface of microbial cells or microbeads can be screened with fluorogenic substrates that provide a physical linkage of the reaction product to the corresponding enzyme. Libraries exceeding 10(9) different variants can be quantitatively analysed and screened by flow cytometry at a rate of 30 000 cells/second. The promise of screening methods based on fluorescence-activated cell sorting for directed enzyme evolution is being realized and significantly improved enzymes have been reported recently.     

Directed evolution of acyl-CoA:diacylglycerol acyltransferase: Development and characterization of Brassica napus DGAT1 mutagenized libraries

Metabolic flux to triacylglycerol (TAG) may be limited by the level of acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) activity. In some species, this enzyme also appears to play a role in the channeling of specific fatty acyl moieties into TAG. The objective of this work is to implement a directed evolution approach to enhance the catalytic efficiency of type-1 DGAT from Brassica napus (BnDGAT1). We generated randomly mutagenized libraries of BnDGAT1 in a yeast expression vector using error-prone PCR. The mutagenized libraries were used to transform a Saccharomyces cerevisiae strain devoid of neutral lipid biosynthesis and analyzed using a high-throughput screening (HTS) system. The HTS, recently developed for this purpose, consisted of a positive selection of clones expressing active DGAT mutants followed by quantification of DGAT activity by fluorescence detection of TAG in yeast cells. The initial results indicated that the positive selection system efficiently eliminated DGAT mutants lacking enzyme activity. Screening of 1528 selected mutants revealed that some DGAT clones had enhanced ability to synthesize TAG in yeast. This was confirmed by analysis of individual clones that could carry mutations resulting in an increased catalytic efficiency. The directed evolution approach could lead to the development of an improved plant DGAT1 for increasing seed oil content in oleaginous crops.     

Parallel mapping of genotypes to phenotypes contributing to overall biological fitness

Laboratory selection is a powerful approach for engineering new traits in metabolic engineering applications. This approach is limited because determining the genetic basis of improved strains can be difficult using conventional methods. We have recently reported a new method that enables the measurement of fitness for all clones contained within comprehensive genomic libraries, thus enabling the genome-scale mapping of fitness altering genes. Here, we demonstrate a strategy for relating these measurements to the individual phenotypes selected for in a particular environment. We first provide a mathematical framework for decomposing fitness into selectable phenotypes. We then employed this framework to predict that single-batch selections would enrich primarily for library clones with increased growth rate, serial-batch would enrich for a broad collection of clones enhanced via a combination of increased growth rate and/or reduced lag times, and that overlap among selected clones would be minimal. We used the SCalar Analysis of Library Enrichments (SCALEs) method to test these predictions. We mapped all genomic regions for which increased copy number conferred a selective advantage to Escherichia coli when cultured via single- or serial-batch in the presence of 1-naphthol. We identified a surprisingly large collection (163 total) of tolerance regions, including all previously identified solvent tolerance genes in E. coli. We show that the majority of the identified regions were unique to the different selection strategies examined and that such differences were indeed due to differences among enriched clones in growth rate and lag times over the solvent concentrations examined. The combination of a framework for decomposing overall fitness into selectable phenotypes along with a genome-scale method for mapping genes to such phenotypes lays the groundwork for improving the rational design of laboratory selections.     

Directed evolution of an endoinulinase from Talaromyces purpureogenus toward efficient production of inulooligosaccharides

Inulinases are fructofuranosyl hydrolases that target the β‐2,1 linkage of inulin and hydrolyze it into fructose, glucose and inulooligosaccharides (IOS), the latter are of growing interest as dietary fibers. Inulinases from various microorganisms have been purified, characterized and produced for industrial applications. However, there remains a need for inulinases with increased catalytic activity and better production yields to improve the hydrolysis process and fulfill the growing industrial demands for specific fibers. In this study, we used directed enzyme evolution to increase the yield and activity of an endoinulinase enzyme originated from the filamentous fungus Talaromyces purpureogenus (Penicillium purpureogenum ATCC4713). Our directed evolution approach yielded variants showing up to fivefold improvements in soluble enzyme production compared to the starting point which enabled high‐yield production of highly purified recombinant enzyme. The distribution of the enzymatic reaction products demonstrated that after 24 h of incubation, the main product (57%) had a degree of polymerization of 3 (DP3). To the best of our knowledge, this is the first application of directed enzyme evolution to improve inulooligosaccharide production. The approach enabled the screening of large genetic libraries within short time frames and facilitated screening for improved enzymatic activities and properties, such as substrate specificity, product range, thermostability and pH optimum. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:868–877, 2018     

Combinatorial engineering to enhance amylosucrase performance: construction, selection, and screening of variant libraries for increased activity

Amylosucrase is a glucosyltransferase belonging to family 13 of glycoside hydrolases and catalyses the formation of an amylose-type polymer from sucrose. Its potential use as an industrial tool for the synthesis or the modification of polysaccharides, however, is limited by its low catalytic efficiency on sucrose alone, its low stability, and its side reactions resulting in sucrose isomer formation. Therefore, combinatorial engineering of the enzyme through random mutagenesis, gene shuffling, and selective screening (directed evolution) was started, in order to generate more efficient variants of the enzyme. A convenient zero background expression cloning strategy was developed. Mutant gene libraries were generated by error-prone polymerase chain reaction (PCR), using Taq polymerase with unbalanced dNTPs or Mutazyme™, followed by recombination of the PCR products by DNA shuffling. A selection method was developed to allow only the growth of amylosucrase active clones on solid mineral medium containing sucrose as the sole carbon source. Automated protocols were designed to screen amylosucrase activity from mini-cultures using dinitrosalicylic acid staining of reducing sugars and iodine staining of amylose-like polymer. A pilot experiment using the described mutagenesis, selection, and screening methods yielded two variants with significantly increased activity (five-fold under the screening conditions). Sequence analysis of these variants revealed mutations in amino acid residues which would not be considered for rational design of improved amylosucrase variants. A method for the characterisation of amylosucrase action on sucrose, consisting of accurate measurement of glucose and fructose concentrations, was introduced. This allows discrimination between hydrolysis and transglucosylation, enabling a more detailed comparison between wild-type and mutant enzymes.     

Cultural transmission and biological markets

Active cultural transmission of fitness-enhancing behavior (sometimes called “teaching”) can be seen as a costly strategy: one for which its evolutionary stability poses a Darwinian puzzle. In this article, we offer a biological market model of cultural transmission that substitutes or complements existing kin selection-based proposals for the evolution of cultural capacities. We demonstrate how a biological market can account for the evolution of teaching when individual learners are the exclusive focus of social learning (such as in a fast-changing environment). We also show how this biological market can affect the dynamics of cumulative culture. The model works best when it is difficult to have access to the observation of the behavior without the help of the actor. However, in contrast to previous non-mathematical hypotheses for the evolution of teaching, we show how teaching evolves, even when innovations are insufficiently opaque and therefore vulnerable to acquisition by emulators via inadvertent transmission. Furthermore, teaching in a biological market is an important precondition for enhancing individual learning abilities.     

Improving the quality of industrially important enzymes by directed evolution

Directed evolution is a new process for developing industrially viable biocatalysts. This technique does not require a comprehensive knowledge of the relationships between sequence structure and function of proteins as required by protein engineering. It mimics the process of Darwinian evolution in a test tube combining random mutagenesis and recombination with screening or selection for enzyme variants that have the desired properties. Directed evolution helps in enhancing the enzyme performance both in natural and synthetic environments. This article reviews the process of directed evolution and its application to improve substrate specificity, activity, enantioselectivity and thermal stability.     

Directed evolution of LuxI for enhanced OHHL production

Quorum sensing is a common mechanism used by bacteria to coordinate population behavior, and is involved in a variety of biological processes, such as bioluminescence, virulence factor synthesis, antibiotic production, and biofilm formation. To engineer the LuxI enzyme of the LuxI-LuxR quorum-sensing system, we developed a high throughput genetic selection to identify LuxI mutants with improved OHHL (3-oxo-hexanoyl homoserine lactone) synthesis in E. coli. Using this genetic selection, we created LuxI mutants with improved OHHL synthesis rates and yields through directed evolution, identifying three LuxI mutants after two generations. An in vivo semi-quantitative method allowed for verification of the genetic screen and OHHL yields were quantified using HPLC-MS/MS, revealing an 80-fold increase in a mutant culture compared to the wildtype culture. In addition to OHHL, the yields of C6HSL (hexanoyl homoserine lactone) and C8HSL (octanoyl homoserine lactone) were also improved, and a slight change in substrate specificity towards C6HSL production was observed. Based on alignment with the crystal structure of EsaI, a homolog of LuxI, two mutations are most likely involved in enhancing the interactions between the enzyme and the substrates. The high throughput genetic selection and the semi-quantitative method can be conveniently modified for the directed evolution of LuxI homologs. The identification of these LuxI mutants has implications in synthetic biology, where they can be used for the construction of artificial genetic circuits. In addition, development of drugs that specifically target quorum sensing to attenuate the pathogenesis of gram-negative infectious bacteria might also benefit from the insights into the molecular mechanism of quorum sensing revealed by the amino acid substitutions.     

一种高效的多点组合突变克隆构建方法

高质量的突变方法和高效的筛选方法相结合可以提高酶定向进化的效率。文中开发了一种高效的多点组合突变(Multi-points combinatorial mutagenesis,MCM)的克隆方法。MCM方法通过引入DNA组装、融合PCR和杂交技术,实现高效多点组合突变。应用优化后的方法定向进化改造苯甲酰甲酸脱羧酶(Benzoylformate decarboxylase,BFD)来测试MCM方法的效率。通过电转至大肠杆菌感受态Escherichia coli TreliefTM 5α所获得的单菌落数量(Colony-formingunits,CFUs)超过106 CFUs/μgDNA。经验证90/100单菌落精确组装;5个位点L109、L110、H281、Q282和A460同时组合突变的效率达到88%。最后,筛选到一种kcat/Km提高10倍的突变酶(L109Y、L110D、H281G、Q282V和A460M)。因此,应用该方法可以有效地创建突变体库,促进酶的定向进化技术的快速发展。     

Construction of diverse adeno-associated viral libraries for directed evolution of enhanced gene delivery vehicles

Rational design of improved gene delivery vehicles is a challenging and potentially time-consuming process. As an alternative approach, directed evolution can provide a rapid and efficient means for identifying novel proteins with improved function. Here we describe a methodology for generating very large, random adeno-associated viral (AAV) libraries that can be selected for a desired function. First, the AAV2 cap gene is amplified in an error-prone PCR reaction and further diversified through a staggered extension process. The resulting PCR product is then cloned into pSub2 to generate a diverse (>10(6)) AAV2 plasmid library. Finally, the AAV2 plasmid library is used to package a diverse pool of mutant AAV2 virions, such that particles are composed of a mutant AAV genome surrounded by the capsid proteins encoded in that genome, which can be used for functional screening and evolution. This procedure can be performed in approximately 2 weeks.