Laboratory Evolved Biocatalysts for Stereoselective Syntheses of Substituted Benzaldehyde Cyanohydrins

Liu, Zhibin; Pscheidt, Beate; Avi, Manuela; Gaisberger, Richard; Hartner, Franz Stefan; Schuster, C.; Skranc, Wolfgang; Gruber, Karl; Glieder, Anton

doi:10.1002/cbic.200700514

Cited by 59 publications

(39 citation statements)

References 42 publications

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“…The reaction was performed under a water-based biphasic system in 4 h reaction time ( Table 6, entries 5À7). 167 5.2. Engineering of S-Selected HNLs.…”

Section: Engineering Of Hydroxynitrile Lyasesmentioning

confidence: 99%

Hydroxynitrile Lyases: Insights into Biochemistry, Discovery, and Engineering

2011

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Hydroxynitrile lyases are valuable enzymes for asymmetric synthesis of cyanohydrins. These hydroxyl and nitrileÀcontaining compounds are being used in production of very useful pharmaceuticals, agrochemicals, and other biologically active compounds using chemical or chemoenzymatic follow-up reactions in industry. Although a huge amount of information exists on the reaction parameters of these enzymes, including stability to pH and organic solvents, yield, reaction time, and valuable data on the enantiopurity of their products, cyanohydrins, there is a lack of update on the biochemistry, discovery, and engineering of the HNLs. Therefore, in the Introduction, we will have a look into these enzymes, cyanohydrins, and aldoxime-nitrile pathways. A brief view of functional groups and several examples of cyanohydrin-based chemicals and pharmaceuticals will also be described. Then we will present characteristics of many S-and R-selective HNLs with comparative tables for several enzymatic properties under biochemistry section. The methods of screening and discovery of these enzymes both from nature and a library of mutants will be described as well as their potential in the synthesis of chemicals. Cloning and expression of new HNLs will also be described under the discovery section. A pool of successful applications of protein engineering methods and the subsequent improvement in the properties of mutant HNLs will be reviewed in detail afterward.

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“…The reaction was performed under a water-based biphasic system in 4 h reaction time ( Table 6, entries 5À7). 167 5.2. Engineering of S-Selected HNLs.…”

Section: Engineering Of Hydroxynitrile Lyasesmentioning

confidence: 99%

Hydroxynitrile Lyases: Insights into Biochemistry, Discovery, and Engineering

2011

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“…In Fig. 2A, eGFP expression values for each transformant are sorted from lowest to highest, resulting in expression landscapes as typically used in the literature to present such results (40)(41)(42)(43). (59).…”

Section: Figmentioning

confidence: 99%

Effect of Plasmid Design and Type of Integration Event on Recombinant Protein Expression in Pichia pastoris

Vogl

Gebbie

Palfreyman

et al. 2018

Appl Environ Microbiol

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Pichia pastoris (syn. Komagataella phaffii) is one of the most common eukaryotic expression systems for heterologous protein production. Expression cassettes are typically integrated in the genome to obtain stable expression strains. In contrast to Saccharomyces cerevisiae, where short overhangs are sufficient to target highly specific integration, long overhangs are more efficient in P. pastoris and ectopic integration of foreign DNA can occur. Here, we aimed to elucidate the influence of ectopic integration by high-throughput screening of Ͼ700 transformants and whole-genome sequencing of 27 transformants. Different vector designs and linearization approaches were used to mimic the most common integration events targeted in P. pastoris. Fluorescence of an enhanced green fluorescent protein (eGFP) reporter protein was highly uniform among transformants when the expression cassettes were correctly integrated in the targeted locus. Surprisingly, most nonspecifically integrated transformants showed highly uniform expression that was comparable to specific integration, suggesting that nonspecific integration does not necessarily influence expression. However, a few clones (Ͻ10%) harboring ectopically integrated cassettes showed a greater variation spanning a 25-fold range, surpassing specifically integrated reference strains up to 6-fold. High-expression strains showed a correlation between increased gene copy numbers and high reporter protein fluorescence levels. Our results suggest that for comparing expression levels between strains, the integration locus can be neglected as long as a sufficient numbers of transformed strains are compared. For expression optimization of highly expressible proteins, increasing copy number appears to be the dominant positive influence rather than the integration locus, genomic rearrangements, deletions, or single-nucleotide polymorphisms (SNPs).IMPORTANCE Yeasts are commonly used as biotechnological production hosts for proteins and metabolites. In the yeast Saccharomyces cerevisiae, expression cassettes carrying foreign genes integrate highly specifically at the targeted sites in the genome. In contrast, cassettes often integrate at random genomic positions in nonconventional yeasts, such as Pichia pastoris (syn. Komagataella phaffii). Hence, cells from the same transformation event often behave differently, with significant clonal variation necessitating the screening of large numbers of strains. The importance of this study is that we systematically investigated the influence of integration events in more than 700 strains. Our findings provide novel insight into clonal variation in P. pastoris and, thus, how to avoid pitfalls and obtain reliable results. The underlying mechanisms may also play a role in other yeasts and hence could be generally relevant for recombinant yeast protein production strains.KEYWORDS integration, Pichia pastoris, protein expression, genome analysis T he yeast Pichia pastoris (syn. Komagataella phaffii) is widely used for heterologous protein p...

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“…200 transformants were assayed for improved conversion of 1a. Following the first law of directed evolution you get what you screen for' [19] -whose consequences we had to encounter quite recently when engineering PaHNL5 for improved (R)-2-chloromandelonitrile synthesis [18] -we searched for a method which for the first time provided the possibility to screen specifically for the cyanohydrin synthesis reaction, namely the conversion of 1a to 2 at pH 2.4 in a 96-well format. For detection, a colorimetric high-throughput screening method from Bornscheuer and co-workers [20] was Scheme 1.…”

Section: The (R)-selective Hydroxya C H T U N G T R E N N U N G Nitrimentioning

confidence: 99%

“…For saturation library generation, Pichia pastoris was transformed with linear expression cassettes made by overlap extension PCR employing the PaHNL5 gene ligated into the plasmid pGAPZA as a template. Therefore, the basic method established by Liu et al [18] was adapted for site-saturation mutagenesis. For each of the 7 targeted sites, !…”

Section: The (R)-selective Hydroxya C H T U N G T R E N N U N G Nitrimentioning

confidence: 99%

Efficient Biocatalytic Synthesis of (R)‐Pantolactone

et al. 2008

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Screening for stereoselective cyanohydrin synthesis in 96-well plates was employed in the development of an efficient, pH-stable hydroxynitrile lyase for the conversion of sterically hindered aliphatic aldehydes. Site-saturation mutagenesis (SSM) resulted in a powerful catalyst for the stereoselective conversion of hydroxypivalaldehyde and pivalaldehyde to their corresponding (R)-cyanohydrins (ee > 97%) which are used as chiral building blocks (e.g., for pantothenic acid production). Furthermore, redesigning the PaHNL5 gene and improving its expression by Pichia pastoris with the help of a new P AOX1 promoter variant and the helper protein PDI (protein disulfide isomerase) led to elevated amounts of todays most efficient biocatalyst for vitamin B 5 synthesis.

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Laboratory Evolved Biocatalysts for Stereoselective Syntheses of Substituted Benzaldehyde Cyanohydrins

Cited by 59 publications

References 42 publications

Hydroxynitrile Lyases: Insights into Biochemistry, Discovery, and Engineering

Hydroxynitrile Lyases: Insights into Biochemistry, Discovery, and Engineering

Effect of Plasmid Design and Type of Integration Event on Recombinant Protein Expression in Pichia pastoris

Efficient Biocatalytic Synthesis of (R)‐Pantolactone

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