Muconic acid is a
potential platform chemical for the production
of nylon, polyurethanes, and terephthalic acid. It is also an attractive
functional copolymer in plastics due to its two double bonds. At this
time, no economically viable process for the production of muconic
acid exists. To harness novel genetic targets for improved production
of
cis,cis
-muconic acid (CCM) in the yeast
Saccharomyces cerevisiae
, we employed a CCM-biosensor coupled
to GFP expression with a broad dynamic response to screen UV-mutagenesis
libraries of CCM-producing yeast. Via fluorescence activated cell
sorting we identified a clone Mut131 with a 49.7% higher CCM titer
and 164% higher titer of biosynthetic intermediate–protocatechuic
acid (PCA). Genome resequencing of the Mut131 and reverse engineering
identified seven causal missense mutations of the native genes (
PWP2
,
EST2
,
ATG1
,
DIT1
,
CDC15
,
CTS2
, and
MNE1
) and a duplication of two CCM biosynthetic genes, encoding
dehydroshikimate dehydratase and catechol 1,2-dioxygenase, which were
not recognized as flux controlling before. The Mut131 strain was further
rationally engineered by overexpression of the genes encoding for
PCA decarboxylase and AROM protein without shikimate dehydrogenase
domain (Aro1p
ΔE
), and by restoring
URA3
prototrophy. The resulting engineered strain produced 20.8 g/L CCM
in controlled fed-batch fermentation, with a yield of 66.2 mg/g glucose
and a productivity of 139 mg/L/h, representing the highest reported
performance metrics in a yeast for
de novo
CCM production
to date and the highest production of an aromatic compound in yeast.
The study illustrates the benefit of biosensor-based selection and
brings closer the prospect of biobased muconic acid.
Biofilms can cause severe problems to human health due to the high tolerance to antimicrobials; consequently, biofilm science and technology constitutes an important research field. Growing a relevant biofilm in the laboratory provides insights into the basic understanding of the biofilm life cycle including responses to antibiotic therapies. Therefore, the selection of an appropriate biofilm reactor is a critical decision, necessary to obtain reproducible and reliable in vitro results. A reactor should be chosen based upon the study goals and a balance between the pros and cons associated with its use and operational conditions that are as similar as possible to the clinical setting. However, standardization in biofilm studies is rare. This review will focus on the four reactors (Calgary biofilm device, Center for Disease Control biofilm reactor, drip flow biofilm reactor, and rotating disk reactor) approved by a standard setting organization (ASTM International) for biofilm experiments and how researchers have modified these standardized reactors and associated protocols to improve the study and understanding of medical biofilms.
Enzymes are considered an innovative and environmentally friendly approach for biofilm control due to their lytic and dispersal activities. In this study, four enzymes (β-glucanase, α-amylase, lipase and protease) were tested separately and in combination with the quaternary ammonium compound cetyltrimethylammonium bromide (CTAB) to control flow-generated biofilms of Pseudomonas fluorescens. The four enzymes caused modest reduction of biofilm colony forming units (CFU). Protease, β-glucanase and α-amylase also caused modest biofilm removal. CTAB combined with either β-glucanase or α-amylase increased biofilm removal. Its combination with either β-glucanase or protease increased CFU reduction. However, CTAB-protease combination was antagonist in biofilm removal. Long-term effects in biofilm mass reduction were observed after protease exposure. In contrast, biofilms treated with β-glucanase were able to regrow significantly after exposure. Moreover, short-term respirometry tests with planktonic cells were performed to understand the effects of enzymes and their combination with CTAB on P. fluorescens viability. Protease and lipase demonstrated antimicrobial action, while α-amylase increased bacterial metabolic activity. The combination of CTAB with either protease or α-amylase was antagonistic, decreasing the antimicrobial action of CTAB. The overall results demonstrate a modest effect of the selected enzymes in biofilm control, either when applied alone or each one in combination with CTAB. Total biofilm removal or CFU reduction was not achieved and, in some cases, the use of enzymes antagonized the effects of CTAB. The results also propose that complementary tests, to characterize biofilm integrity and microbial viability, are required when someone is trying to assess the role of novel biocide - enzyme mixtures for effective biofilm control.
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