When metabolic prowess is too much of a good thing: how carbon catabolite repression and metabolic versatility impede production of esterified α,ω-diols in Pseudomonas putida KT2440

Abstract

Background Medium-chain-length α,ω-diols (mcl-diols) are important building blocks in polymer production. Recently, microbial mcl-diol production from alkanes was achieved in E. coli (albeit at low rates) using the alkane monooxygenase system AlkBGTL and esterification module Atf1. Owing to its remarkable versatility and conversion capabilities and hence potential for enabling an economically viable process, we assessed whether the industrially robust P. putida can be a suitable production organism of mcl-diols. Results AlkBGTL and Atf1 were successfully expressed as was shown by oxidation of alkanes to alkanols, and esterification to alkyl acetates. However, the conversion rate was lower than that by E. coli, and not fully to diols. The conversion was improved by using citrate instead of glucose as energy source, indicating that carbon catabolite repression plays a role. By overexpressing the activator of AlkBGTL-Atf1, AlkS and deleting Crc or CyoB, key genes in carbon catabolite repression of P. putida increased diacetoxyhexane production by 76% and 65%, respectively. Removing Crc/Hfq attachment sites of mRNAs resulted in the highest diacetoxyhexane production. When the intermediate hexyl acetate was used as substrate, hexanol was detected. This indicated that P. putida expressed esterases, hampering accumulation of the corresponding esters and diesters. Sixteen putative esterase genes present in P. putida were screened and tested. Among them, Est12/K was proven to be the dominant one. Deletion of Est12/K halted hydrolysis of hexyl acetate and diacetoxyhexane. As a result of relieving catabolite repression and preventing the hydrolysis of ester, the optimal strain produced 3.7 mM hexyl acetate from hexane and 6.9 mM 6-hydroxy hexyl acetate and diacetoxyhexane from hexyl acetate, increased by 12.7- and 4.2-fold, respectively, as compared to the starting strain. Conclusions This study shows that the metabolic versatility of P. putida, and the associated carbon catabolite repression, can hinder production of diols and related esters. Growth on mcl-alcohol and diol esters could be prevented by deleting the dominant esterase. Carbon catabolite repression could be relieved by removing the Crc/Hfq attachment sites. This strategy can be used for efficient expression of other genes regulated by Crc/Hfq in Pseudomonas and related species to steer bioconversion processes.

Pseudomonas putida as a platform for medium‐chain length α,ω‐diol production: Opportunities and challenges

“…Without the esterification step, alcohols will be overoxidized to aldehydes and acids. This has been reported in different microbial cell factories (Lu et al, 2021;van Nuland et al, 2016). The alcohol acetyltransferase Atf1 is commonly used to esterify medium-chain fatty alcohols because acetyl-CoA acts as co-substrates and is abundant in cells.…”

“…Preventing the degradation of intermediates and products P. putida was shown to degrade most of the intermediate of mcl-diols production to some extent (Lu et al, 2021(Lu et al, , 2022. Deleting est12, adhP, and pedF hampers P. putida from degrading esters and alcohols (Lu, Akwafo, et al, 2023).…”

A novel alkane monooxygenase (alkB) clade revealed by massive genomic survey and its dissemination association with IS elements

“…So far, many comprehensive studies have been carried out to investigate alk -genes, including functional characterization ( Smits, Witholt & Van Beilen, 2003 ), regulation mechanism ( Canosa et al, 2000 ), fusion and transfer ( Chakrabarty, 1973 ), heterologous expression ( Liu et al, 2011 ; Liu et al, 2010 ; Minak-Bernero et al, 2004 ; Sameshima et al, 2008 ; Smits et al, 2001 ; Wang et al, 2017 ; Whyte et al, 2002 ), promoter ( Staijen Ivo, Marcionelli & Witholt, 1999 ) and biotransformation application ( Lu et al, 2021 ; Van Nuland et al, 2017 ). Sequence analysis of alk -gene clusters had observed several insertion sequences (IS) located in its flanking regions.…”

The importance of understanding the regulation of bacterial metabolism

“…Some examples are known in which inactivating the genes responsible for CCR improved the simultaneous utilization of several compounds that would otherwise be used sequentially (Elmore et al, 2020 ; Johnson et al, 2017 ). However, once again, this solution was found not to be helpful in other situations (Lu et al, 2021 ), probably because eliminating the global regulators responsible for CCR causes imbalanced metabolism. A further possibility might be to conserve the general CCR network but detach the genes of interest from its influence.…”

“…A further possibility might be to conserve the general CCR network but detach the genes of interest from its influence. This implies engineering these genes to eliminate the elements recognized by global CCR regulators, and indeed this strategy has proved helpful in optimizing a P. putida strain engineered to generate medium-chainlength α,ω-diols (building blocks for polymer production) (Lu et al, 2021). The drawback here is that such an approach requires detailed prior knowledge of the molecular mechanisms that drive CCR in the strain of interest, including the identification of the targets recognized by the global regulators of the genes to be manipulated.…”