PMID- 33990309
OWN - NLM
STAT- MEDLINE
DCOM- 20210823
LR  - 20211226
IS  - 1098-5336 (Electronic)
IS  - 0099-2240 (Print)
IS  - 0099-2240 (Linking)
VI  - 87
IP  - 14
DP  - 2021 Jun 25
TI  - In Vitro Production of Coenzyme A Using Thermophilic Enzymes.
PG  - e0054121
LID - 10.1128/AEM.00541-21 [doi]
LID - e00541-21
AB  - Coenzyme A (CoA) is an essential cofactor present in all domains of life and is 
      involved in numerous metabolic pathways, including fatty acid metabolism, 
      pyruvate oxidation through the tricarboxylic acid (TCA) cycle, and the production 
      of secondary metabolites. This characteristic makes CoA a commercially valuable 
      compound in the pharmaceutical, cosmetic, and clinical industries. However, CoA 
      is difficult to accumulate in living cells at a high level, since it is consumed 
      in multiple metabolic pathways, hampering its manufacturing by typical cell 
      cultivation and extraction approaches. The feedback inhibition by CoA to a 
      biosynthetic enzyme, pantothenate kinase (PanK), is also a serious obstacle for 
      the high-titer production of CoA. To overcome this challenge, in vitro production 
      of CoA, in which the CoA biosynthetic pathway was reconstructed outside cells 
      using recombinant thermophilic enzymes, was performed. The in vitro pathway was 
      designed to be insensitive to the feedback inhibition of CoA using 
      CoA-insensitive type III PanK from the thermophilic bacterium Thermus 
      thermophilus. Furthermore, a statistical approach using design of experiments 
      (DOE) was employed to rationally determine the enzyme loading ratio to maximize 
      the CoA production rate. Consequently, 0.94 mM CoA could be produced from 2 mM 
      d-pantetheine through the designed pathway. We hypothesized that the insufficient 
      conversion yield is attributed to the high K(m) value of T. thermophilus PanK 
      toward ATP. Based on these observations, possible CoA regulation mechanisms in T. 
      thermophilus and approaches to improve the feasibility of CoA production through 
      the in vitro pathway have been investigated. IMPORTANCE The biosynthesis of 
      coenzyme A (CoA) in bacteria and eukaryotes is regulated by feedback inhibition 
      targeting type I and type II pantothenate kinase (PanK). Type III PanK is found 
      only in bacteria and is generally insensitive to CoA. Previously, type III PanK 
      from the hyperthermophilic bacterium Thermotoga maritima was shown to defy this 
      typical characteristic and instead shows inhibition toward CoA. In the present 
      study, phylogenetic analysis combined with functional analysis of type III PanK 
      from thermophiles revealed that the CoA-sensitive behavior of type III PanK from 
      T. maritima is uncommon. We cloned type III PanKs from Thermus thermophilus and 
      Geobacillus sp. strain 30 and showed that neither enzyme's activities were 
      inhibited by CoA. Furthermore, we utilized type III PanK for a one-pot cascade 
      reaction to produce CoA.
FAU - Suryatin Alim, Gladwin
AU  - Suryatin Alim G
AD  - International Center for Biotechnology, Osaka University, Suita, Osaka, Japan.
FAU - Iwatani, Tomoka
AU  - Iwatani T
AD  - International Center for Biotechnology, Osaka University, Suita, Osaka, Japan.
FAU - Okano, Kenji
AU  - Okano K
AD  - International Center for Biotechnology, Osaka University, Suita, Osaka, Japan.
AD  - Industrial Biotechnology Initiative Division, Institute for Open and 
      Transdisciplinary Research Initiatives, Osaka University, Suita, Osaka, Japan.
FAU - Kitani, Shigeru
AU  - Kitani S
AD  - International Center for Biotechnology, Osaka University, Suita, Osaka, Japan.
AD  - Industrial Biotechnology Initiative Division, Institute for Open and 
      Transdisciplinary Research Initiatives, Osaka University, Suita, Osaka, Japan.
FAU - Honda, Kohsuke
AU  - Honda K
AD  - International Center for Biotechnology, Osaka University, Suita, Osaka, Japan.
AD  - Industrial Biotechnology Initiative Division, Institute for Open and 
      Transdisciplinary Research Initiatives, Osaka University, Suita, Osaka, Japan.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20210625
PL  - United States
TA  - Appl Environ Microbiol
JT  - Applied and environmental microbiology
JID - 7605801
RN  - 0 (Bacterial Proteins)
RN  - EC 2.7.1.- (Phosphotransferases (Alcohol Group Acceptor))
RN  - EC 2.7.1.33 (pantothenate kinase)
RN  - SAA04E81UX (Coenzyme A)
SB  - IM
MH  - Bacterial Proteins/*metabolism
MH  - Coenzyme A/*biosynthesis
MH  - Phosphotransferases (Alcohol Group Acceptor)/*metabolism
MH  - Thermus thermophilus/*enzymology
PMC - PMC8231720
OTO - NOTNLM
OT  - coenzyme A
OT  - in vitro metabolic engineering
OT  - pantothenate kinase
OT  - thermophiles
EDAT- 2021/05/16 06:00
MHDA- 2021/08/24 06:00
PMCR- 2021/12/25
CRDT- 2021/05/15 05:36
PHST- 2021/05/16 06:00 [pubmed]
PHST- 2021/08/24 06:00 [medline]
PHST- 2021/05/15 05:36 [entrez]
PHST- 2021/12/25 00:00 [pmc-release]
AID - AEM.00541-21 [pii]
AID - 00541-21 [pii]
AID - 10.1128/AEM.00541-21 [doi]
PST - ppublish
SO  - Appl Environ Microbiol. 2021 Jun 25;87(14):e0054121. doi: 10.1128/AEM.00541-21. 
      Epub 2021 Jun 25.