Bio-actives: Material pool of high selectivity and specificity

• Natural products have been a prolific source and an inspiration for numerous medical agents with widely divergent
chemical structures and biological activities, including antimicrobial, immunosuppressive, anticancer, and anti-
inflammatory activities.
•
• Many of these have been developed as treatments and have potential therapeutic applications for human diseases.
• Aside from natural products, the recent development of recombinant DNA technology has sparked the
development of a wide array of biopharmaceutical products, such as recombinant proteins, offering significant
advances in treating a broad spectrum of medical illnesses and conditions.

• Structures and diverse biological activities of natural products and recombinant proteins that have been exploited
as valuable molecules in medicine, agriculture and insect control, discussed here.
• Efforts along with achievements in the development of robust and promising microorganisms as cell factories to
produce biologically active molecules.

• Multi-disciplinary and comprehensive engineering approaches directed at improving yields of microbial

production of natural products and proteins and generating novel molecules.
• Microbial-derived biologically active molecular entities and their analogs could continue to inspire the
development of new therapeutic agents in academia and industry.

Adopted from: Front. Microbiol., 20 June 2019 | https://doi.org/10.3389/fmicb.2019.01404 1

 Biological Activity of Microbial Bioactives
Since Humulin® became the first recombinant
biopharmaceutical as a treatment for diabetes (Johnson,
1983), additional FDA-approved microbial biologics have been
produced by E. coli. Somatrem (Protropin®) and somatropin
(Humatrope®) are used to treat children with growth hormone
deficiency (Baeshen et al., 2015; Sanchez-Garcia et al., 2016).

Figure: Recombinant protein production. Using recombinant DNA techniques,

the target human gene can be isolated and ligated to a vector (plasmid). The
plasmid containing the human gene is used to transform bacterial cells, which
are able to produce high amounts of the recombinant protein. Adapted with
permission from reference.

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 Biopharmaceutical Manufacturing Technology

Scheme . The biopharmaceutical manufacturing technology flowchart exemplifying the upstream and the
downstream bioprocess. Adapted with permission from reference (8).
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Brazilian J. Microbiol. 2016, 47S, 51–63
 Currently Used Microbial Hosts for Production of Bioactives

Adopted from: Front. Microbiol., 20 June 2019 | https://doi.org/10.3389/fmicb.2019.01404 4

 Currently Used Microbial Hosts for Production of Bioactives

Adopted from: Front. Microbiol., 20 June 2019 | https://doi.org/10.3389/fmicb.2019.01404

 Currently Used Microbial Hosts for Production of Bioactives

Adopted from: Front. Microbiol., 20 June 2019 | https://doi.org/10.3389/fmicb.2019.01404

 Efforts in Product Improvements and Generation of New Analogs

Strain Improvement
• By Whole-genome shuffling: It is a process that utilizes the advantages of the multi-parental crossing
allowed by DNA shuffling with the genome recombination normally associated with conventional breeding
(Zhang et al., 2002).

• Genome shuffling has been successfully improved the titers of variety of microorganisms. For example, two
strains of Streptomyces fradiae generated from two rounds of genome shuffling were able to produce up to
a ninefold increase in antibacterial tylosin production in comparison to the initial strain (Zhang et al.,
2002).

• Ribosome engineering is also a method useful in increasing secondary metabolite production titer and
productivity (Sun and Alper, 2015).

• Another study found a 37-fold increased production of avilamycin in a recombinant Streptomyces

viridochromogenes strain due to a mutation in ribosome protein S12 (rps12) acquired through a
combination of gene shuffling and ribosome engineering (Lv et al., 2013).

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Adopted from: Front. Microbiol., 20 June 2019
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| https://doi.org/10.3389/fmicb.2019.01404
 Engineering Precursor Supply

• Precursor supply is defined as the enhancement of the availability of primary metabolites or molecules derived
from primary metabolism involved in the biosynthesis of natural products (Shiba et al., 2007).

• Precursor supply engineering can be achieved by manipulating either the pathways or enzymes involved with
the precursor supply. Malonyl-CoA and methylmalonyl-CoA are the most commonly used and metabolically
available precursors for the biosynthesis of polyketides (Polyketides are a large group of secondary metabolites
which either contain alternating carbonyl and methylene groups (-CO-CH2-), or are derived from precursors
which contain such alternating groups).

• In addition, manipulating key enzymes that direct carbon flux through core biochemical pathways involved in
glucose, fatty acid, and amino acid metabolism can increase biosynthetic precursor pools. A study on the
modulation of carbon flux between the pentose phosphate pathway and the glycolysis pathway found that a
deletion of phosphofructokinase isoenzymes led to the enhanced production of antibiotics actinorhodin and
undecylprodigiosin in S. coelicolor by increasing carbon flux through the pentose phosphate pathway
(Borodina et al., 2008).

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 Pathway Engineering
• Metabolic pathway engineering can be performed in the native host through repetitive gene expression,
gene deletion, and introduction of new genes to enhance production of natural products (Pickens et al.,
2011).

• For example, overexpression of the 4–12 tandem copies of the actinorhodin cluster resulted in a 20-fold
increase in actinorhodin production in S. coelicolor (Murakami et al., 2011).

• Additionally, a S. hygroscopicus strain with 3–5 tandem copies of the 40 kb validomycin A cluster showed a
34% increase in production and a maximum titer of approximately 20 g/L (Zhou et al., 2014).

• Deletion of genes may be useful to eliminate competing pathways that may siphon off important precursors
or intermediates, or simply contribute to an unnecessary use of cellular resources which result to improve
yields of products of interest.

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 General Microbial Detection Methods and Drawbacks
Sampling Medium Method Type Details Drawbacks

Air Laser particle counter Total particulates (0.5 to 5.0 µm) Large volume is required
Active air samplers Air sampling units per cubic unit
Liquids and Membrane filtration <0.45 micron pore size filter Time-consuming
emulsions Spread-plate Neat or diluted sample
Pour-plate neat or diluted sample
solution/emulsion
Surfaces Contact-plate Useful for flat surfaces Sampling is always a challenge
Swab Useful for non-flat surfaces;
Surface rinse Useful for tanks, bioreactors, fill lines,
pipes
Tape/adhesive Useful for flat or curved surfaces
Cell suspension Flow cytometry Fluorescently stained microbes, Expensive and requires sample
generally for deal and live assays preparations
Cells or cell Electrical impedance Change in electrical impedance Requires optimization protocols
suspension
Fixed cells Fluorescence staining Imaging under microscopes Requires efficient fluorophores
Lysis of cells Nucleic acid amplification PCR techniques Very costly, time-consuming,
technologies high skill sets required
Cell suspension Light scatter Centrifuge to pellet down cells Purifications required11