scholarly journals In Vivo Evolution of Butane Oxidation by Terminal Alkane Hydroxylases AlkB and CYP153A6

2008 ◽  
Vol 75 (2) ◽  
pp. 337-344 ◽  
Author(s):  
Daniel J. Koch ◽  
Mike M. Chen ◽  
Jan B. van Beilen ◽  
Frances H. Arnold
Keyword(s):  
Directed Evolution ◽  
Chain Length ◽  
Carbon Sources ◽  
Evolution System ◽  
Alkane Hydroxylase ◽  
Medium Chain ◽  
Medium Chain Length ◽  
Selection For ◽  
Alkane Hydroxylases

ABSTRACT Enzymes of the AlkB and CYP153 families catalyze the first step in the catabolism of medium-chain-length alkanes, selective oxidation of the alkane to the 1-alkanol, and enable their host organisms to utilize alkanes as carbon sources. Small, gaseous alkanes, however, are converted to alkanols by evolutionarily unrelated methane monooxygenases. Propane and butane can be oxidized by CYP enzymes engineered in the laboratory, but these produce predominantly the 2-alkanols. Here we report the in vivo-directed evolution of two medium-chain-length terminal alkane hydroxylases, the integral membrane di-iron enzyme AlkB from Pseudomonas putida GPo1 and the class II-type soluble CYP153A6 from Mycobacterium sp. strain HXN-1500, to enhance their activity on small alkanes. We established a P. putida evolution system that enables selection for terminal alkane hydroxylase activity and used it to select propane- and butane-oxidizing enzymes based on enhanced growth complementation of an adapted P. putida GPo12(pGEc47ΔB) strain. The resulting enzymes exhibited higher rates of 1-butanol production from butane and maintained their preference for terminal hydroxylation. This in vivo evolution system could be useful for directed evolution of enzymes that function efficiently to hydroxylate small alkanes in engineered hosts.

scholarly journals Cytochrome P450 Alkane Hydroxylases of the CYP153 Family Are Common in Alkane-Degrading Eubacteria Lacking Integral Membrane Alkane Hydroxylases

2006 ◽  
Vol 72 (1) ◽  
pp. 59-65 ◽  
Author(s):  
Jan B. van Beilen ◽  
Enrico G. Funhoff ◽  
Alexander van Loon ◽  
Andrea Just ◽  
Leo Kaysser ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Pseudomonas Putida ◽  
Chain Length ◽  
Nonheme Iron ◽  
Cytochrome P450 Enzymes ◽  
Medium Chain ◽  
Ferredoxin Reductase ◽  
Medium Chain Length ◽  
Reductase Gene ◽  
Alkane Hydroxylases

ABSTRACT Several strains that grow on medium-chain-length alkanes and catalyze interesting hydroxylation and epoxidation reactions do not possess integral membrane nonheme iron alkane hydroxylases. Using PCR, we show that most of these strains possess enzymes related to CYP153A1 and CYP153A6, cytochrome P450 enzymes that were characterized as alkane hydroxylases. A vector for the polycistronic coexpression of individual CYP153 genes with a ferredoxin gene and a ferredoxin reductase gene was constructed. Seven of the 11 CYP153 genes tested allowed Pseudomonas putida GPo12 recombinants to grow well on alkanes, providing evidence that the newly cloned P450s are indeed alkane hydroxylases.

scholarly journals Characterization of Site-Specific Mutations in a Short-Chain-Length/Medium-Chain-Length Polyhydroxyalkanoate Synthase:In VivoandIn VitroStudies of Enzymatic Activity and Substrate Specificity

2013 ◽  
Vol 79 (12) ◽  
pp. 3813-3821 ◽  
Author(s):  
Jo-Ann Chuah ◽  
Satoshi Tomizawa ◽  
Miwa Yamada ◽  
Takeharu Tsuge ◽  
Yoshiharu Doi ◽  
...  
Keyword(s):  
Chain Length ◽  
Fold Increase ◽  
Short Chain ◽  
Pha Synthase ◽  
Wild Type ◽  
Short Chain Length ◽  
Medium Chain ◽  
Medium Chain Length

ABSTRACTSaturation point mutagenesis was carried out at position 479 in the polyhydroxyalkanoate (PHA) synthase fromChromobacteriumsp. strain USM2 (PhaCCs) with specificities for short-chain-length (SCL) [(R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyvalerate (3HV)] and medium-chain-length (MCL) [(R)-3-hydroxyhexanoate (3HHx)] monomers in an effort to enhance the specificity of the enzyme for 3HHx. A maximum 4-fold increase in 3HHx incorporation and a 1.6-fold increase in PHA biosynthesis, more than the wild-type synthase, was achieved using selected mutant synthases. These increases were subsequently correlated with improved synthase activity and increased preference of PhaCCsfor 3HHx monomers. We found that substitutions with uncharged residues were beneficial, as they resulted in enhanced PHA production and/or 3HHx incorporation. Further analysis led to postulations that the size and geometry of the substrate-binding pocket are determinants of PHA accumulation, 3HHx fraction, and chain length specificity.In vitroactivities for polymerization of 3HV and 3HHx monomers were consistent within vivosubstrate specificities. Ultimately, the preference shown by wild-type and mutant synthases for either SCL (C4and C5) or MCL (C6) substrates substantiates the fundamental classification of PHA synthases.

scholarly journals Biosynthesis of medium chain length poly-3-hydroxyalkanoates by Pseudomonas guezennei from various carbon sources

2008 ◽  
Vol 68 (11) ◽  
pp. 1534-1541 ◽  
Author(s):  
Christelle Simon-Colin ◽  
Gérard Raguénès ◽  
Bernard Costa ◽  
Jean Guezennec
Keyword(s):  
Chain Length ◽  
Carbon Sources ◽  
Medium Chain ◽  
Medium Chain Length

scholarly journals Medium Chain and Long Chain Alkanes Hydroxylase Producing Whole Cell Biocatalyst From Marine Bacteria

2018 ◽  
Vol 22 (1) ◽  
pp. 12
Author(s):  
Ahmad Thontowi ◽  
Elvi Yetti ◽  
Yopi Yopi
Keyword(s):  
Chain Length ◽  
Alkane Hydroxylase ◽  
16S Rdna Gene ◽  
Short Chain Length ◽  
Medium Chain ◽  
Medium Chain Length ◽  
Degrading Bacteria ◽  
Whole Cell Biocatalyst ◽  
Test Result ◽  
Long Chain

Alkanes are  major component of crude oil that could be hydrolyzed by the enzyme of alkane hydroxylase. The are three types of alkane hydroxylase based on the chain length of alkane such as short-chain length/SCL (C2-C4), medium-chain length/MCL (C5-C17), and long-chain length/LCL (C>18). The aims of this study were to characterize and identify alkanes-degrading bacteria from these bacteria. The 30 strains from marine were grown on MCL (Pentane-C5H12, Decane-C10H22, and Pentadecane-C15H32) and LCL (n-Paraffin-C12H19C17 and branch of Pristane-C19H40). The study showed twenty-nine isolates have the ability to degrade alkanes compounds, whereas 14 isolates have grown ability on MCL and LCL medium, 11 isolates have the ability to grow on MCL and n-LCL, 3 isolates have the ability only to grow on MCL medium and 1 isolate has the ability only grow on n-LCL medium. The growth test result indicated that 29 isolates have medium-chain alkane monooxygenase and long-chain alkane hydroxylase. Based on 16S rDNA gene analysis, we obtained twenty nine of oil- degrading bacteria, namely a-proteobacteria (57 %), g-proteobacteria (30 %), Flavobacteria (7 %), Bacilli (3%) and Propionibacteriales (3 %). g-Proteobacteria and a-proteobacteria which seems to play an important role in the alkane biodegradation.

scholarly journals Draft Genome Sequence ofPseudomonas putidaCA-3, a Bacterium Capable of Styrene Degradation and Medium-Chain-Length Polyhydroxyalkanoate Synthesis

2018 ◽  
Vol 6 (4) ◽  
Author(s):  
Eduardo L. Almeida ◽  
Lekha M. Margassery ◽  
Niall O’Leary ◽  
Alan D. W. Dobson
Keyword(s):  
Pseudomonas Putida ◽  
Chain Length ◽  
Genome Sequence ◽  
Draft Genome ◽  
Carbon Sources ◽  
Draft Genome Sequence ◽  
Coding Sequences ◽  
Medium Chain ◽  
Cellular Processes ◽  
Medium Chain Length

ABSTRACTPseudomonas putidastrain CA-3 is an industrial bioreactor isolate capable of synthesizing biodegradable polyhydroxyalkanoate polymers via the metabolism of styrene and other unrelated carbon sources. The pathways involved are subject to regulation by global cellular processes. The draft genome sequence is 6,177,154 bp long and contains 5,608 predicted coding sequences.

scholarly journals Development of a New Strategy for Production of Medium-Chain-Length Polyhydroxyalkanoates by Recombinant Escherichia coli via Inexpensive Non-Fatty Acid Feedstocks

2011 ◽  
Vol 78 (2) ◽  
pp. 519-527 ◽  
Author(s):  
Qin Wang ◽  
Ryan C. Tappel ◽  
Chengjun Zhu ◽  
Christopher T. Nomura
Keyword(s):  
Escherichia Coli ◽  
Fatty Acid ◽  
Chain Length ◽  
Acyl Carrier Protein ◽  
Carbon Sources ◽  
Content Type ◽  
Medium Chain ◽  
Medium Chain Length ◽  
Coa Transferase ◽  
Fatty Acid Carbon

ABSTRACTPseudomonas putidaKT2440 is capable of producing medium-chain-length polyhydroxyalkanoates (MCL-PHAs) when grown on unrelated carbon sources during nutrient limitation. Transcription levels of genes putatively involved in PHA biosynthesis were assessed by quantitative real-time PCR (qRT-PCR) inP. putidagrown on glycerol as a sole carbon source. The results showed that two genes,phaGand the PP0763 gene, were highly upregulated among genes potentially involved in the biosynthesis of MCL-PHAs from unrelated carbon sources. Previous studies have describedphaGas a 3-hydroxyacyl-acyl carrier protein (ACP)-coenzyme A (CoA) transferase, and based on homology, the PP0763 gene was predicted to encode a medium-chain-fatty-acid CoA ligase. High expression levels of these genes during PHA production inP. putidaled to the hypothesis that these two genes are involved in PHA biosynthesis from non-fatty acid carbon sources, such as glucose and glycerol. ThephaGppand PP0763 genes fromP. putidawere cloned and coexpressed with the engineeredPseudomonassp. 61-3 PHA synthase genephaCl(STQK)psin recombinantEscherichia coli. Up to 400 mg liter−1MCL-PHAs was successfully produced from glucose. This study has produced the largest amount of MCL-PHAs reported from non-fatty acid carbon sources in recombinantE. colito date and opens up the possibility of using inexpensive feedstocks to produce MCL-PHA polymers.

scholarly journals Identification of an Amino Acid Position That Determines the Substrate Range of Integral Membrane Alkane Hydroxylases

2005 ◽  
Vol 187 (1) ◽  
pp. 85-91 ◽  
Author(s):  
Jan B. van Beilen ◽  
Theo H. M. Smits ◽  
Franz F. Roos ◽  
Tobias Brunner ◽  
Stefanie B. Balada ◽  
...  
Keyword(s):  
Amino Acid ◽  
Chain Length ◽  
Amino Acid Position ◽  
Site Directed Mutagenesis ◽  
Structure Model ◽  
Medium Chain Length ◽  
Selection Experiments ◽  
Selection For ◽  
Alkane Hydroxylases ◽  
Subsequent Selection

ABSTRACT Selection experiments and protein engineering were used to identify an amino acid position in integral membrane alkane hydroxylases (AHs) that determines whether long-chain-length alkanes can be hydroxylated by these enzymes. First, substrate range mutants of the Pseudomonas putida GPo1 and Alcanivorax borkumensis AP1 medium-chain-length AHs were obtained by selection experiments with a specially constructed host. In all mutants able to oxidize alkanes longer than C13, W55 (in the case of P. putida AlkB) or W58 (in the case of A. borkumensis AlkB1) had changed to a much less bulky amino acid, usually serine or cysteine. The corresponding position in AHs from other bacteria that oxidize alkanes longer than C13 is occupied by a less bulky hydrophobic residue (A, V, L, or I). Site-directed mutagenesis of this position in the Mycobacterium tuberculosis H37Rv AH, which oxidizes C10 to C16 alkanes, to introduce more bulky amino acids changed the substrate range in the opposite direction; L69F and L69W mutants oxidized only C10 and C11 alkanes. Subsequent selection for growth on longer alkanes restored the leucine codon. A structure model of AHs based on these results is discussed.

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