Heterologous Expression of Sfp-Type Phosphopantetheinyl Transferase is Indispensable in the Biosynthesis of Lipopeptide Biosurfactant

• Published in: Molecular biotechnology Vol. 61; no. 11; pp. 836 - 851
• Main Authors: Jimoh, Abdullahi Adekilekun, Lin, Johnson
• Format: Journal Article
• Language: English
• Published: New York Springer US 01-11-2019; Springer Nature B.V
• Subjects: Acetic acid; Affinity chromatography; Amino acid sequence; Amino Acid Sequence - genetics; Amino acids; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Biochemistry; Biological Techniques; Biosynthesis; Biotechnology; Carbon dioxide; Cell Biology; Chemistry; Chemistry and Materials Science; Cloning, Molecular; Cobalt; Copper; E coli; Enzymatic activity; Enzyme Stability; Escherichia coli - genetics; Esterase; Esterases - metabolism; Ethylenediaminetetraacetic acids; Fatty acids; Human Genetics; Iron; Kinetics; Lipopeptides - biosynthesis; Magnesium; Metals; Metals, Heavy - metabolism; Molecular weight; Nickel; Original Paper; p-Nitrophenylacetic acid; Paenibacillus; Paenibacillus - enzymology; Peptide Synthases - metabolism; pH effects; Phylogeny; Protein Science; Proteins; Sfp gene; Substrates; Surface tension; Surface-Active Agents - chemistry; Surface-Active Agents - metabolism; Surfactants; Transferases (Other Substituted Phosphate Groups) - chemistry; Transferases (Other Substituted Phosphate Groups) - genetics; Transferases (Other Substituted Phosphate Groups) - isolation & purification; Transferases (Other Substituted Phosphate Groups) - metabolism; Zinc; Biosurfactant; Phosphopantetheinyl transferase; sp. D9; Esterase; Cloned recombinant; Paenibacillus sp. D9
• ISSN: 1073-6085; 1559-0305
• DOI: 10.1007/s12033-019-00209-y

Phosphopantetheinyl transferases are of tremendous enthusiasm inferable from their fundamental parts in activating polyketide, fatty acid, and non-ribosomal peptide synthetase enzymes and additionally an increasing number of biotechnological applications. The present study reports the identification of
sfp
gene from the
Paenibacillus
sp. D9, which encompasses 693 bp encoding a 230-amino acid protein with a molecular weight of 25.3 kDa. The amino acid sequence
Paenibacillus
sp. D9 Sfp revealed more than 90% sequence identity to other Sfp proteins from other
Paenibacillus
. The
sfp
gene was cloned and recovered efficiently using affinity chromatography with maximal specific phosphopantetheinyl transferase activity at an optimal pH of 8.0 and temperature of 30 °C. The enzyme also exhibited stability under a wide-ranging pH and temperature. The presence of Zn
2+
, Cu
2+
, and Fe
2+
ions improved the enzymatic activity, while other metals such as Ni
2+
, Co
2+
, and Mg
2+
had inhibitory effects. The introduction of EDTA also displayed no inhibition. Kinetic parameters were obtained having values of 4.52 mg/mL, 35.33 U/mg, 3.64 s
−1
, and 0.104 mM
−1
s
−1
for
K
m
,
V
max
,
k
cat
, and
k
cat
/
K
m
, respectively. The biosurfactant synthesized by the recombinant BioSp was found to be surface active, reducing the surface tension to 33.7 mN/m on the glucose substrate after 5 days of incubation at 37 °C. The recombinant
Escherichia coli
strain also exhibited an improvement in biosurfactant yield (1.11 g/L) when contrasted with 0.52 g/L from
Paenibacillus
sp. D9. High esterase activity of 2.55 IU/mL using
p
-nitrophenyl acetate was observed on the recombinant strain, as the protein connected with the release of the biosurfactant was observed to be an esterase. The characteristics of improved biosurfactant and esterase synthesis by hyper-producing recombinant strain possess numerous values from biotechnology standpoint.